Advances in Cancer Biology - Metastasis

Advances in Cancer Biology - Metastasis 4 (2022) 100022
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Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Virtual screening, molecular docking, molecular dynamics simulations and
free energy calculations to discover potential DDX3 inhibitors

Shailima Rampogu a,*, Mary Rampogu Lemuel b, Keun Woo Lee a,**

a Division of Life Sciences, Division of Applied Life Science (BK21 Plus), Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), 501 Jinju-
daero, Jinju, 52828, South Korea
b West Thames College, London, TW7 4HS, UK

ARTICLE INFO ABSTRACT

Keywords: DEAD-box RNA helicase 3 (DDX3) is a versatile target that is elevated in several cancer cases besides being a
DDX3 validated target for viral infections. RK-33 is a well-known compound that has been used to target DDX3. In the
RK-33 current investigation, we have used several computational methods to discover RK-33 like compounds with
Molecular dynamic simulation greater affinity towards DDX3. Correspondingly, 95 compounds were obtained from PubChem and were subjected
Cancer to molecular docking studies with DDX3 target (PDB code: 2I4I). The resultant two compounds were subjected to
Antiviral agents molecular dynamics simulation (MDS) studies to investigate the stabilities of the complex, performed for 100 ns in
triplicates (100 ns x 3 ¼ 300 ns). The MDS results have shown that the identified compounds have established
stable results during the evolution of the simulation across the triplicates, read according to root mean square
deviation (RMSD), radius of gyration (Rg) and root mean square fluctuations (RMSF). Taken together we propose
two compounds as alternatives to RK-33 with better binding affinity, stable MDS results and acceptable ADMET
properties.

1. Introduction N-terminal of DDX3 holds the ATP-binding pocket that associates with
ATP in RNA-stimulated fashion [1]. DDX3 has AMP binding at this pocket
DEAD-box RNA helicase 3 (DDX3) belongs to a subfamily of DEAD- that demonstrates the open conformation [1]. Furthermore, DDX3 can
box proteins and in humans two DDX3 homologs are present, they are exhibit varied conformations due to the P-loop flexibility. A closed
the DDX3X and DDX3Y [1]. DDX3 has a role in wide range of cellular conformation could be obtained when the domain 2 is roughly rotated to
biogenesis processes including apoptosis and cell survival [1]. Notably, about 180 relative to domain 1 and supplementing with RNA binding
the role of DDX3 is widely associated with cancer and is overexpressed in and ATP [1,5]. This ATP binding pocket is exploited to discover and
liver, breast, colon, oral and lung cancer demonstrating the tumor sup- screen potential inhibitors.
pressor and oncogenic role [1]. Correspondingly, DDX3 is a beneficial
target to counter cancer. Not only in cancer, DDX3 is also a potential Mounting reports disclose several inhibitors specifically reported for
target to treat viral infections. Mounting reports disclosed that DDX3 is DDX3. A recent study reported 21 new DDX3 inhibitors that have
vital for the replication of viruses, such as, CMV (Herpesviridae), HIV-1 demonstrated antiviral activity towards human immunodeficiency virus-
(Retroviridae), HCV, Japanese Encephalitis virus, Dengue virus 1 (HIV-1) wild type and HIV-1 resistant strains [6]. Another study
(DENV), West Nile virus (WNV; Flaviviridae), Vaccinia virus (Poxvir- showed the use of natural compound curcumin against DDX3 and also in
idae), and Norovirus (Caliciviridae) [2], suggesting that DDX3 could a combination with exemestane in cancer cell lines [7]. Reportedly, the
potential viral target. initial development of DDX3 drugs were discovered against HIV-1 [8]
employing high throughput screening techniques [9] and docking
Almost all the organisms manifest DEAD-box helicases and knock- methods [10] from commercial databases using the structure of DDX3 in
down of the helicases are embryonically lethal [1,3]. The target of in- complex with AMP. These structures were further optimized with a few
terest, DDX3 consists of 661 or 662 amino acid residues [4].Structurally, manifesting the repression of replication of viruses and tumor aggression
DDX3 has two recA-like domains and 12 conserved motifs. The [11]. The compounds that were active towards DDX3 are the derivatives

* Corresponding author.
** Corresponding author.;

E-mail addresses: [email protected] (S. Rampogu), [email protected] (K.W. Lee).

https://doi.org/10.1016/j.adcanc.2021.100022
Received 25 October 2021; Received in revised form 15 December 2021; Accepted 16 December 2021
Available online 20 December 2021
2667-3940/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
).nc-nd/4.0/

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of pyrazolo-, or diarylurea, naphthyl-, ring-expanded nucleoside and 2.3. Target selection and preparation
rhodanine and triazine to name a few [11].
The target for the current study is retrieved from protein data bank
One of the inhibitors with high regard as DDX3 targeting agent is the (PDB) bearing the code 2I4I [36]. The protein was then subjected to
(tricyclic diimidazodiazepine analogue) RK-33, a ring expanded nucle- ‘Prepare protein’ module available with the DS that standardizes atom
oside (REN) analogue [9]. This compound has shown remarkable anti- names, removes alternate conformations (disorder), and inserting
viral and anticancer activity [12–20]. The molecule, RK-33 is believed to missing main and side-chain atoms and the structure was minimized.
target the DDX3 and blocks the helicase activity [17]. It is evidenced that Furthermore, the heteroatoms and the water molecule were removed.
RK-33 can bring about the G1 cell cycle arrest, impair Wingless-related The active site was chosen around the cocrystallized ligand [36] for all
integration site (Wnt) signalling, promotes apoptosis, decreasing mito- the residues and atoms that fall within radius of 12 Å.
chondrial translation and impairing tumor proliferation [17].
2.4. Binding affinity studies
In general the process of drug discovery takes 10–15 years and in-
volves huge money and time [21]. The computer aided drug design For the binding affinity studies between the target protein and the
(CADD) appears as the best alternative [22]. CADD has gained popularity small molecules, the LibDock [37] available with the DS was used. This is
among the researchers due to reduced time and results can be obtained a high-throughput algorithm for docking ligands into an active receptor
quickly in comparison with the traditional experimental methods [22]. site. Mechanistically, LibDock utilized protein binding site features called
The discovery of the drug sildenafil and thalidomide are instances to the the HotSpots which can be divided into two types the polar and the
success of CADD [22]. Besides this, the CADD/in silico approaches can apolar hospots. Typically, a polar Hotspot is chosen by a polar ligand
discover novel targets for the known drugs, and can foresee the presence atom and an apolar HotSpot is selected by an apolar atom [37]. The static
of side effects [22]. Recently, a lot of research has been in progress using (rigid) ligand poses are stationed at the active site and HotSpots are
different CADD approaches [23–28] and identification of inhibitors such compared as triplets. The poses are then pruned and a concluding opti-
as the identification of cyclin-dependent kinase 2 inhibitors [29]. A mization step is performed prior to the poses are scored. In brief, the
report was published on Aurora-A-TPX2 complex [30], natural molecules ligand conformations are aligned to polar and apolar receptor sites of
as γ-aminobutyric acid receptor antagonist [31] and identification of interaction (hotspots) and the best scoring poses are put forth. For the
inhibitors for checkpoint kinase 1 [32]. current study the number of HotSpots were selected as 500, docking
tolerance was chosen as 0.25 with high quality docking preference. The
With an aim to discover the compounds with high affinity towards ‘FAST’ conformation method was chosen with maximum conformations
DDX3, in the current investigation, we have used computational methods as 255 and energy threshold as 20. The sp2-sp2 rotation and the Grid
to obtain a candidate compound with higher binding affinity than RK-33. Scoring were selected as ‘True’. The best poses were chosen from the
Owing to the beneficial therapeutic applications of RK-33, in the current largest cluster correlated by the key residue interactions. The selected
investigation we attempted to discover similar compounds from Pub- complexes were escalated to molecular dynamics simulation (MDS)
Chem. Initially the compounds were retrieved from PubChem and were studies to gain insights into the atomistic interactions.
correspondingly subjected to molecular docking, and triplicate molecular
dynamics simulations for robust analysis. To this end, we identified two 2.5. Molecular dynamics simulation analysis
compounds with better binding affinity complemented by stable in-
teractions with the target DDX3. In order to understand the in-depth mechanism that is undergoing in
the interactions between the protein and the ligands, the MDS was per-
2. Materials and methods formed using the GROningen MAchine for Chemical Simulations (GRO-
MACS) v2016.6, using CHARMM all atom force field [38]. The ligand
2.1. Collection of the small molecules and their preparation topologies were obtained from SwissParam [39]. A dodecahedron water
box was generated and subsequently solvated, which was then followed
The parent or the starting structure for the present study is the RK-33 by the addition of counter ions. The system was energy minimized using
compound and correspondingly the similar compounds were down- the steepest descent minimization algorithm. The ligand and the protein
loaded from the PubChem [33] database. A total of 95 compounds were were coupled followed by the equilibration process. The first equilibra-
downloaded in the 2D format and were subsequently upgraded to the tion was conducted with the (constant number of particles, volume, and
Discovery Studio v18 (henceforth referred to as DS) to obtain their 3D temperature) NVT ensembles with V-rescale thermostat for 1 ns and the
structures. The small molecules were subsequently checked for the du- second equilibration was conducted with (constant number of particles,
plicates and were minimized using the ‘Full Minimization’ module pressure, and temperature) NPT ensembles for 1 ns with
available in the DS using Chemistry at HARvard Macromolecular Me- Parrinello-Rahman barostat. During the simulation run, the backbone of
chanics (CHARMm). the protein was restrained and the solvent molecules and the
counter-ions were allowed to move. The NPT ensembles were proceeded
2.2. Drug-like dataset preparation to MD run for 100 ns in triplicates under periodic boundary condition.
The results were analysed according to root mean square deviation
The prepared compounds were assessed for their drug-like capability. (RMSD) [40], radius of gyration (Rg) [41,42], root mean square fluctu-
Accordingly, Lipinski's rule of 5 was assessed by initiating the ‘Filter by ation (RMSF), interaction energies (IE), binding mode analysis and
Lipinski and Veber Rule’ module [34,35]. The resultant ligands are comprehensive intermolecular interactions.
presumed to have greater probability of good oral bioavailability. This
was followed by the assessment of Absorption, Distribution, Metabolism, 3. Results
Excretion, and Toxicity (ADMET) predictions using the ‘ADMET De-
scriptors’ accessible on the DS. The selected limits for the process were as 3.1. Drug-like dataset preparation
follows: the absorption as 0 (good) and 1 (moderate), solubility 2 (low), 3
(good) and 4 (optimal). The Blood Brain Barrier (BBB) was kept at The 95 compounds obtained from the PubChem were subjected to
1(high), 2(medium) and 3(low). The CYP2D6 and Hepatotoxicity were
selected as False. The resultant compounds were forwarded for evalu-
ating the binding affinity studies against the target.

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Lipinski's Rule of 5 that has yielded 78 compounds. When ADMET was 3.6. RMSF
applied the compound number was further reduced to 6. These six
compounds were upgraded to molecular docking studies to evaluate the The RMSF profiles are instrumental in analysing the residue specific
binding affinity with the protein at the active site of the target. fluctuations/flexibility [46] for each residue during the progression of
the MDS. The target protein has 167–580 residues that have demon-
3.2. Binding affinity studies strated a remarkable stability during the MDS. The RK-33 was highly
rigid in comparison with the selected compounds, which were marginally
Binding affinity studies were conducted by molecular docking. flexible (Fig. 2B), notable at three different time periods. The first peak
Generally, molecular docking occurs in dual levels, that includes the was observed with the Arg199 residue, the second with Arg311 and third
ligand conformation prediction and the binding affinity assessment [43]. with Arg333 residues. Interestingly, all the Arg residues were responsible
For superior sampling method, we have used RK-33 as the reference for the three fluctuations. Nonetheless, these residues do not lie in the
molecule. Two compounds have demonstrated a better dock score than vicinity of the binding pocket. However, a higher peak in the fluctuations
the reference compound (Table 1) complemented by largest cluster and was noticed with Ser410 of 71524060 with 0.6 nm. Since this residue is
displaying the key residue interactions. Additionally, these compounds much away for the binding pocket and therefore, we speculate that this
have obeyed to the aforementioned drug-like assessment parameters has no major impact on binding. Notably the residues from the binding
(Table 1). It is noteworthy that the selected molecules have predicted pocket was substantially rigid (Fig. 2B).
favourable result. These compounds were upgraded to molecular dy-
namics studies to gain insights into the atomistic interactions between 3.7. Interaction energy (IE)
the protein and the ligands and behaviour of the small molecules at the
active site of the target. The IE imparts knowledge on the strength of the binding between
protein and the ligand. Accordingly, the Coulombic short-range inter-
3.3. Molecular dynamics simulation (MDS) studies action energy (CIE) and the short-range Lennard-Jones short range (LJ-
SR) were calculated. The results have communicated that the CIE was
MDS analysis serves as a superlative approach in understanding the similar for three systems and ranged between À10 and À150 kJ/mol
nature of protein and ligand when in complex. Here, we pursued the MDS (Fig. 3A) with an average of À76.56 kJ/mol for RK-33, -39.50 kJ/mol for
to evaluate the stabilities of the small molecule, at the binding pocket of 71524060 and -45.50 for 71523969 kJ/mol, respectively. Likewise, the
the target protein using the RMSD, Rg, RMSF, IE, binding mode analysis LJ-SR was computed for the three systems that ranged between À10 kJ/
and comprehensive intermolecular interactions. mol to À240 kJ/mol (Fig. 3B), with an average of À188.68 kJ/mol,
À124.23 kJ/mol and À115.05 kJ/mol for RK-33, 71524060 and
3.4. The ligands were stable at the binding pocket 71523969, respectively. These results infer that the strength of the
identified Hits is comparable with RK-33.
The RMSD facilitates the stability analysis during the evolution of the
MD run. Mechanistically, the RMSD computes the deviations existing in 3.8. Compounds occupy the binding pocket of the cocrystallized ligand
the protein backbone from the initial to the final conformation. This
states that the protein is more stable with smaller deviations [44]. In the From the last 5 ns simulation run, the representative structures were
current study, the RMSD was conducted for the protein backbone (bb) extracted and were superimposed onto the X-ray structure. It was
and the protein ligand complex (com). Both the RMSD profiles have revealed that the ligands and the RK-33 has occupied the binding pocket
demonstrated a similar kind of plots during the MDS demonstrating that in the similar fashion as that of the cocrystallized ligand (Fig. 4), held by
the system was highly stable (Fig. 1A and B). However, 71524060 in several intermolecular interactions.
complex has displayed a minute surge between 91000 ps and 95000 ps
and then subsided quickly (Fig. 1B). Apart from this, the other two sys- Upon inspecting the intermolecular interactions, it was noticed that
tems have demonstrated a relatively stable RMSD profiles. Interestingly, the residue Glu413 has formed a hydrogen bond interaction with RK-33
the other 100 ns þ 100 ns simulation runs has also demonstrated a (Fig. 5A). The ring B and ring D are held by the key residues Try200 and
similar result and are detailed in the discussion section. Gly227, respectively. The key residue Tyr200 is noticed to bind with ring
B and ring D thereby stabilizing the ligand at the binding pocket. The
3.5. Rg oxygen atom from the anisole group (ring A and ring E) of RK-33 has
prompted carbon hydrogen bonds with the residues His527 and Val535.
Rg governs the compactness of the protein [45]. The Rg was The nitrogen atoms from ring B and ring D have gained carbon hydrogen
measured for the backbone atoms for three systems which were highly bond with the residue Gly229, and the residue Thr411 has formed a
compact. A marginal bump was noticed with 71523969 during the initial π-donor hydrogen bond with ring E of the ligand (Fig. 6A). Furthermore,
run below 10 ns that has plateaued swiftly and remained stable the nitrogen atom of ring C has interacted with Ser412 forming a carbon
throughout the simulation run (Fig. 2A). The 71524060 and RK-33 have hydrogen bonds (Fig. 6A). Upon viewing at the carbon hydrogen bonds,
demonstrated a stable compact Rg from the beginning of the MDS it can be stated that these interactions have contributed greatly towards
inferring their high compactness (Fig. 2A). holding the RK-33 at the binding pocket of the target. Several other
residues have held the ligand firmly at the binding pocket of the target
protein by van der Waals interactions (Fig. 6A).

The 71524060 has formed hydrogen bonds with the residues Gln207
and Ala232 (Fig. 5B). The key residue Tyr200 contributed a π-π stacked

Table 1
Binding affinity scores and pharmacokinetic properties of the selected compounds.

PubChem Lib solubility BBB CYP2D6 Hepatotoxicity Absorption H H Mol Rotatable Molecular polar
Id DockScore acceptors donors weight bonds surface area

71524060 122.759 3 3X X 0 10 0 421.452 6 96.91
71523969 117.188 2 2X X 0 8 0 466.415 6 84.44
46184988 67.800 2 3X true 0 9 0 428.443 6 93.67

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Fig. 1. MD derived stability analysis according to RMSD. A) RMSD of bb. B) RMSD of com.

interaction with ring B and ring D accommodating the ligand at the which is an application of principal component analysis (PCA) [47]. The
binding pocket of the protein (Fig. 6B). The residue Ala232 has inter- PCA determines the conformational variations of the backbone of the
acted via the π-alkyl interaction with ring B and ring D. The oxygen atom protein [48]. Here, the eigenvector 1 and eigenvector 2 were used for the
of Arg202 has generated two carbon hydrogen bonds with H45 and H46 PC profiles using the g_covar. The PCA has shown that the protein –
atoms of the ligand. The oxygen atom from the anisole group (ring A) has reference was highly stable when compared to the Hits (Fig. 7). In here,
established a carbon hydrogen bond with His527 (Fig. 6B). Another the eigenvalues are considered to calculate the PC. The 71524060-pro-
carbon hydrogen bond was formed with the oxygen group from ring D tein complex has also demonstrated a lower movement than
and the Thr231 residue in addition to Glu285 (Fig. 6B). A host of other 71523969, and has shown to travel via the PC2 during the simulation
key residues have produced the van der Waals interactions adhering the (Fig. 7). The 71523969-protein has shown a wide range of movement
compound at the binding pocket (Fig. 6B). through PC1 and PC2 before interacting with the small molecule (Fig. 7).
This result was in agreement with the structural analysis results such as
The 71523969 has formed the highest number of hydrogen bond RMSD and Rg. To further explore the low-energy basin (minima), the 2D
interactions with the pivotal residues, Thr231, Ala232, Gly229 and FEL was plotted with PC1 and PC2. Notably, each protein-ligand complex
Lys230 (Fig. 5C). The Gly229 residue is also involved in the generation of has demonstrated a unique pattern of FEL profiles (Fig. 8). The red spots
carbon hydrogen bond with the oxygen atom of ring B (Fig. 6C). The represent the favourable confirmations, while the cyan spots corresponds
residue Lys230 interacts with the ligand via the hydrophobic alkyl to the unfavourable conformations (Fig. 8).
interaction. The key residue Tyr200 has created a π-π stacked interaction
assisting to hold the ligand at the active site of the target protein 4. Discussion
(Fig. 6C). The other key residues have contributed for the stabilizing of
the ligand at the binding pocket via the van der Waals interactions RK-33 is one of the main compounds available currently that is
(Fig. 6C). effectively targeting DDX3 [14,17]. This drug has been used to treat
different cancers [14,49–54] and is also regarded as a broad spectrum
3.9. Essential dynamics (ED) and free energy landscape (FEL) antiviral drug [17]. Notably, DDX3 is essential for replication in viruses
such as hepatitis C (HCV) [55], dengue virus (DENV) [17] and human
The ED is one of the remarkable techniques to understand the dy- immunodeficiency virus type 1 (HIV-1) [6,17,56]. In the current inves-
namic movements of the protein. In general, to retrieve the biologically tigation we attempted to screen RK-33 like compounds with better
significant navigation from the trajectories of the protein the ED is used,

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Fig. 2. MD computed compactness and fluctuations. A) Compactness of the bb during the simulation run. B) Residue based fluctuations during the MD. The results
show that the systems were largely stable.

affinity towards the target DDX3. In this pursuit, several computational state (Table 2 and Fig. 10). These results infer that the compounds have
methods has been employed for selecting the best alternatives. not induced any abnormal behaviour in the complex system. The first MD
run was detailed in the manuscript. The Rg plots also have additionally
From the 95 compounds obtained from the PubChem, two com- proved that the protein remained compact during the progression of the
pounds have shown better binding affinity as rendered by the dock score simulation. Delineating on the average recorded findings (Table 3 and
towards the target protein. The compounds were subsequently upgraded Fig. 11), it can be deduced that the protein backbone was highly
to MDS to gauge on the atomistic interactions between the target and the compact. The residue fluctuations were monitored during the simula-
small molecules. The best poses from the binding affinity and the MD tions to understand the behaviour of the residues during the simulation
derived poses have shown that the small molecules have been occupying run. Interestingly, the first run showed that the residues were rigid with
the active site during the simulation run with a minute change in their minimal fluctuations, while in the second run, 71523969 seemed to show
conformation (Fig. 9). These results suggest that the compounds have fluctuation and in the third run RK-33 residues were observed to be
displayed strong affinity towards the target. wobbling. With respect to both the cases, residues until 350 appeared to
fluctuate (Table 3 and Fig. 12).
Manual inspection of the interactions have revealed interesting
findings. Each of the compounds have 5 rings and ring D has generated a We then made an effort to analyse the structure activity relationship
π-π stacked interactions with the benzene ring of the key residue Tyr200 of the selected compounds in comparison with RK-33. The structural
(according to the generated 2D interactions) (Fig. 6). A similar kind of difference between RK-33 and the selected compounds is noticed in the
interaction was noticed with the crystal structure [36] and was reported ring E (Fig. 13). In case of RK-33, ring E has formed two carbon hydrogen
with inhibitor earlier [7]. This finding demonstrates that the selected bonds with the residues Thr411 and His527, respectively, while in the
compounds might work in par with RK-33. 71524060 the ring E hasn't produced hydrogen bond or prominent in-
teractions. Nevertheless, the 2D interactions display Lys288 in close
Furthermore, MD results have shown stable profiles during the evo- proximity with it. Interestingly, in 71523969, the fluorine atoms from the
lution of the simulation run. In order to ensure our results, we have modified group have prompted hydrogen bond interactions with the
performed triplicate MD runs. The three MD results have projected Gly229 and Lys230 along with π-alkyl interaction, apparently leading to
similar outcome supporting our findings. The backbone and the complex demonstrate higher binding affinity (Fig. 13).
of the MD run 2 and run 3 have generated a stable profiles, however, the
RK-33 has shown a higher RMSD value in run 3 while retaining the stable

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Fig. 3. Interaction energy between the protein and the ligand. A) Coulombic short range interaction energy (CIE). B) The short-range Lennard-Jones energy (LJ-SR).

Fig. 4. Binding mode analysis of the small molecules at the binding pocket of the protein. All the compounds occupy the same site as that of the cocrystallized ligand.
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Fig. 5. Intermolecular hydrogen bond interactions between the protein and the ligands. A) RK-33 B) 71524060C) 71523969.

Fig. 6. Comprehensive intermolecular interactions between the protein and the ligands. A) RK-33 B) 71524060C) 71523969.
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Fig. 7. PC analysis of the three systems.

Fig. 8. Free energy landscape of the three systems.
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Fig. 9. Conformational comparison between A) the docked pose and B) the MD pose. The ligands are accommodated within the binding pocket.

Table 2 DDX3 expression is noticed to be elevated in distant breast cancer
Average results of RMSD for three MD runs. metastases, notably the brain [57]. The identified Hits have demon-
strated an ability to cross the BBB with 71524060 and 71523969
Compound Run1 Run2 Run3 showing the predicted value as 3 (low) and 2(medium), respectively.
Logically, these results infer that the selected compounds can be used for
bb com bb com bb com breast cancer metastases to brain. The remaining results of the selected

RK-33 0.54 0.58 0.62 0.70 1.69 1.77
71524060 0.37 0.44 0.72 0.78 0.70 0.74
71523969 0.82 0.87 0.52 0.56 0.78 0.82

Fig. 10. Stability analysis projected according to RMSD for MD run 2 and MD run 3. A) MD run 2. B) MD run 3.
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Table 3
Average results of Rg and RMSF for three MD runs.

MD structural analysis RK-33 71524060 71523969

Run1 Run2 Run3 Run1 Run2 Run3 Run1 Run2 Run3

Rg 2.4 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.5
RMSF 0.56 0.29 0.21 0.40 0.25 0.40 0.22
0.16 0.24

Fig. 11. Compactness analysis projected according to Rg for MD run 2 and MD run 3. A) MD run 2. B) MD run 3.

compounds were comparable to that of RK-33. These predictions show compounds (PubChem Ids: 71524060, 71523969). These two com-
that the discovered compounds might be promising inhibitors targeting pounds have demonstrated higher binding affinity than the RK-33. These
DDX3. compounds have displayed stable MD results and have accommodated
within the binding pocket throughout the simulation run. We therefore
The novelty of the compounds was determined by extracting their present these two compounds as the plausible DDX3 inhibitors that may
SMILES id and giving it as an input in ChemSpider [58]. The results have serve as scaffolds for designing new compounds.
shown that these Hits have not been tested for DDX3. From the detailed
analysis, we assume that the Hits could be potential DDX3 inhibitors Funding
assisting as an anticancer and antiviral agent. The overall work flow is
illustrated in Fig. 14. This research was supported by the Bio & Medical Technology
Development Program of the National Research Foundation (NRF) &
5. Conclusion funded by the Korean government (MSIT) (No. NRF-
2018M3A9A7057263).
In pursuit of finding the RK-33 analogues, the current study proceeds
using several computational techniques that have yielded two

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Fig. 12. Residue specific fluctuation analysis projected according to RMSF for MD run 2 and MD run 3. A) MD run 2. B) MD run 3.

Fig. 13. Structural difference between the discovered compounds from RK-33 and the interacting residues.
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Fig. 14. Infographic illustration of the overall workflow.

Declaration of interests [4] J. Mo, H. Liang, C. Su, P. Li, J. Chen, B. Zhang, DDX3X: structure, physiologic
functions and cancer, Mol. Cancer 20 (2021) 38, https://doi.org/10.1186/s12943-
The authors declare that they have no known competing financial 021-01325-7.
interests or personal relationships that could have appeared to influence
the work reported in this paper. [5] D. Sharma, E. Jankowsky, The Ded1/DDX3 subfamily of DEAD-box RNA helicases,
Crit. Rev. Biochem. Mol. Biol. 49 (2014) 343–360.
Appendix A. Supplementary data
[6] A. Brai, V. Riva, F. Saladini, C. Zamperini, C.I. Trivisani, A. Garbelli, C. Pennisi,
Supplementary data to this article can be found online at https A. Giannini, A. Boccuto, F. Bugli, M. Martini, M. Sanguinetti, M. Zazzi, E. Dreassi,
://doi.org/10.1016/j.adcanc.2021.100022. M. Botta, G. Maga, DDX3X inhibitors, an effective way to overcome HIV-1
resistance targeting host proteins, Eur. J. Med. Chem. 200 (2020) 112319, https://
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Advances in Cancer Biology - Metastasis 4 (2022) 100025
Contents lists available at ScienceDirect

Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

In vitro anticancer activity of “Methanolic extract of papaya blackseeds”
(MPB) in Hep G2 cell lines and its effect in the regulation of bcl-2, caspase-3
and p53 gene expression

Akshay Anilkumar, Alisha Bhanu P A *

Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Mannuthy, Kerala, 680651, India

ARTICLE INFO ABSTRACT

Keywords: Papaya (Carica papaya) is a fruit that grows mainly in tropical areas with significant commercial worth due to its
Papaya extraordinary nutritional and therapeutic value. Its fruit, leaves, and seeds are used as a traditional medicine in
Seeds India for various ailments, including cancer. Papaya fruit was always a treatment choice among traditional ay-
Anticancer urvedic practitioners for various ailments. The usage of papaya leaf extract for curing dengue fever had gained
Relative gene expression popularity at the time of severe dengue outbreak in various Asian countries, including India. Studies had proved
Apoptosis that papaya leaf extract can increase platelet count and improve dengue patients' health conditions. However, the
usage of papaya seeds for treatment has not gained much popularity even though it has been widely recom-
mended in ancient Ayurvedic texts. In this study, we have tried to assess the anticancer potential of papaya black
seeds, which are present in the ripe papaya fruits. The “methanolic extract of papaya black seeds” (MPB) 1have
been studied for the in vitro cytotoxicity against the human liver cancer Hep G2 cell lines. The half maximal
inhibitory concentration (IC50) value was quantified using the 3-(4,5-dimethylthiazoline-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay. Along with this, the apoptotic changes on the cancer cells induced by MPB have
been assessed with the help of Acridine Orange-Ethidium Bromide (AO-EB) staining. The regulation of gene
expression has been evaluated by targeting B cell Lymphoma -2 (Bcl-2), p53, and Caspase-3 genes by relative gene
expression studies via quantitative Real Time Polymerase Chain Reaction (qRT PCR). From this study, we have
concluded that papaya black seeds may be a prospective therapeutic agent in liver cancer therapy with an IC50
value of 24.35 μg/mL along with the potential to induce apoptotic changes by downregulating the Bcl-2 and
upregulating p53 and Caspase-3 genes.

1. Introduction p53 has sparked much interest in the process [3]. The Bcl-2 gene is the
most well-known member of genes that regulate cell homeostatic func-
A healthy individual's body comprising 30 trillion cells coexist in a tions during development and adulthood. Some members of the BCL2
complicated, interdependent condominium, with each cell controlling family (Bcl-2 alpha, Bcl-xL) suppress apoptosis, while others stimulate it
the growth of the others. Normal cells, in fact, only multiply when they (Bax, Bclxs) [4]. According to new research, the Bcl-2 gene shields tumor
are told to by other cells nearby. As a result of this constant collaboration, cells against apoptosis triggered by a range of agents, including ionizing
each tissue maintains size and architecture suited for the body's demands radiation, and is therefore linked to resistance towards treatments that
[1]. Most varieties of cancer are defined by the presence of certain ge- damage DNA [5].
netic abnormalities that can cause cell proliferation. This type of cancer
usually affects different organs and cell types [2]. Apoptosis occurs With the increasing number of cancer patients, and elevated expenses
naturally in malignant tumors, slowing their development significantly, of allopathic treatments, the practice of traditional herbal medicines is
and it is amplified in cancers that react to irradiation, cytotoxic chemo- becoming more prevalent. Carica papaya seems to be a tropical and
therapy, heating, and hormone ablation. However, the discovery that it subtropical fruit that is used to treat a number of ailments in traditional
may be controlled by proto-oncogenes and the tumor suppressor gene medicine. In Ayurveda, the seeds, fruits, roots, barks, latex, flowers, and
leaves of Carica papaya are used for various therapeutic purposes [6].

* Corresponding author.
E-mail address: [email protected] (A. Bhanu P A).

https://doi.org/10.1016/j.adcanc.2021.100025
Received 1 December 2021; Received in revised form 18 December 2021; Accepted 21 December 2021
Available online 23 December 2021
2667-3940/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
).nc-nd/4.0/

A. Anilkumar, A. Bhanu P A Advances in Cancer Biology - Metastasis 4 (2022) 100025

People on Australia's Gold Coast consume Carica papaya leaf juice for its amphotericin B. Cells were incubated at 37 Celsius in a humidified at-
alleged anticancer properties, with some tales of successful instances mosphere with 5.0% CO2.
appearing in various media. Carica papaya leaf extracts have been used as
an aboriginal medicine for a variety of diseases, including cancer and 2.3. Preparation of MPBs
viral infections [7]. Papaya seeds, according to studies, contain a sig-
nificant amount of chemicals with anticancer effects. It has been To extract the seeds, the papaya collected from the vegetable garden
demonstrated to inhibit the proliferation of certain cancer cell lines in an of the College of Veterinary and Animal Sciences Mannuthy, was cleaned
in vitro study. Isothiocyanate, a chemical present in papaya seeds, has with distilled water. It was wiped dry with a tissue paper, then sliced in
been proven to have anticancer characteristics, preventing tumor for- half. The seeds were scraped and rinsed in distilled water three times.
mation in the colon, lung, leukemia, breast, and prostate [8–10]. Fatty The cleaned seeds were distributed on plastic trays and dried until they
acids, crude protein, crude fiber, papaya oil, carpaine, caricin, gluco- attained a constant weight in a chemical hood. An electric pulverizer was
tropaeolin, benzyl glucosinolates, benzyl thiourea, hentriacontane, used to grind the dried seeds, and the powder was extracted with
ß-sitosterol, caressing, and the enzyme myrosin are also contained in methanol using a Soxhlet extraction device. The methanol extract was
papaya seeds. Carica papaya seeds and pulp contain benzyl glucosinolate, then concentrated in a rotary vacuum evaporator at 40 Celsius under
which myrosinase may hydrolyze to create benzyl isothiocyanate. Seed lowered pressure and temperature. After total evaporation, the extract
extracts have a high level of bactericidal activity. Benzyl isothiocyanate, yield was estimated and preserved in an airtight container at À20 C.
a sulfur-containing molecule that works as a germicide and pesticide, is
abundant in unripe fruit seeds. These compounds are necessary for the 2.4. Cytotoxicity studies in Hep G2 cell line
natural defensive systems of plants [11]. A perusal of the literature
revealed that the antineoplastic activity and the apoptotic changes 2.4.1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
induced by papaya black seeds had not been analysed on Hep G2 cell assay
lines by targeting Bcl-2, Caspase-3, and p53 genes yet. Hence, the current
study performed relative gene expression investigations to assess the MTT assay was conducted to analyze the viability of cells and
antineoplastic activity of papaya black seeds and their impact on cell calculation of the half maximal inhibitory concentration (IC50) of the
apoptosis by targeting Bcl-2, Caspase-3, and p53 genes. MPB [12]. The MTT colorimetric test, which detects live cells by reducing
the yellow tetrazolium salt to purple formazan, evaluated cell viability. In
2. Materials and methods 200 microliters of media, the Hep G2 liver cancer cell line was planted at
a density of 1 x 105 cells per well and left to adhere overnight in a CO2
2.1. Chemicals and reagents incubator. For a period of 48 h, cells were treated with MPB at doses of
1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 μg/mL. The media was
Acridine orange,3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenylte- removed after treatment with MPB, and 10 μL of MTT (5 mg/mL) in 100
trazolium bromide (MTT), Agarose, Antibiotic antimycotic solution μL medium was added and incubated at 37 C for 4 h. The
(100X), Dimethylsulphoxide (DMSO), 0.25% Trypsin-EDTA, Dulbecco's MTT-containing medium was then withdrawn, and the purple formazan
Phosphate-Buffered Saline (D-PBS), Ethidium bromide, nucleotide crystals generated were dissolved in 200 μL DMSO and the absorbance
primers for GAPDH, Bcl-2, Caspase-3, and p53, Doxorubicin, horseradish was measured at 570 nm on an ELISA plate reader (Varioskan Flash,
peroxidase-conjugated secondary anti-goat antibody, primary anti Bcl-2 Thermo Fischer Scientific, Finland).
antibody, primary anti-caspase-3 antibody, primary anti-p53 antibody,
and primary anti beta actin antibody were purchased from Sigma-Aldrich By comparing the vitality of treated cells to that of control cells, the
Chemical Co. (St. Louis, M O, USA). Dulbecco's Modified Eagle's Medium effect of test substances on cell viability was determined. To calculate the
percent cell viability and percent cell inhibition, the following formulae
were used:

Percent cell viability ¼ ðAverage absorbance of treated cells = Average absorbance of untreated cellsÞ Â 100 (1)

(DMEM), L-glutamine, fetal bovine serum (FBS), and sodium pyruvate for Percent cell inhibition ¼ 100 À percent cell viability (2)
cell maintenance were purchased from GIBCO, India. DNA Synthesis kit,
Real-Time PCR master mix, gel loading dye, and TRI Reagent for RNA The total absorbance from control wells was assumed to be 100%
isolation were purchased from Fermentas Thermo Fischer Scientific, viable. Graph pad prism was used to compute the IC50 value from the
USA. percentage of inhibition.

2.2. Cell line culture 2.4.2. Microscopic studies using acridine orange/ethidium bromide (AO/EB)
staining
The human liver cancer cell lines Hep G2 was purchased from the
National Centre for Cell Sciences (NCCS) (Pune, India). The cell culture 1 x 105 DLA cells were seeded into a six-well cell culture plate and
flasks were examined under inverted microscopes for cell degeneration
and development, pH, and turbidity. Subculturing was performed when treated for 24 h with the IC50, half, and double concentration of the MPB.
the cells had acquired 80% confluency. The living, apoptotic, and necrotic cells were identified using the acridine
orange ethidium bromide (AO/EB) staining technique. Twenty-five mi-
The Hep G2 cell lines were altered to grow in Dulbecco's Modified croliters of treated or untreated cells were stained with five microlitres of
Eagle's Medium (DMEM), which included 2.0 mM L-glutamine, 10.0% acridine orange (10g/mL) and ethidium bromide (10g/mL) and analysed
foetal bovine serum (FBS),1.0 mM sodium pyruvate, and a combination at 20Â magnification using a Trinocular Research fluorescence micro-
of 10.0 mg/mL streptomycin, 10000 units/mL penicillin, and 25 g/mL scope, DM 2000 LED, Leica, with blue excitation (488nm) and

2

A. Anilkumar, A. Bhanu P A Advances in Cancer Biology - Metastasis 4 (2022) 100025

emission(550nm) filters [13].

2.5. Evaluation of the apoptotic effect of the MPB in Hep G2 cell lines by
targeting Bcl-2, Caspase-3, and p53 genes

2.5.1. Relative gene expression via quantitative RT-PCR Fig. 1. Graph depicting the percent cell viability of Hep G2 cells upon the dose-
The IC50, half, and double of the MPB were incubated in cells for 24 h. dependent treatment of MPB. The data is presented as mean Æ SD with ‘n’ ¼
3 replicates.
According to the manufacturer's instructions, total RNA was obtained
separately using the ‘TRIzol’ reagent (Thermo Scientific) RNA extraction with the IC50 (24.35 μg/mL) of MPB showed both green and orange red
technique [14]. Then, total RNA was reverse transcribed for each sample, cells (late apoptotic). In the cells that had reached late apoptotic phase,
and the cDNA samples were evaluated using SYBR Green PCR Master mix cellular alterations such as nucleus condensation and fragmentation,
through quantitative real-time PCR (qRT-PCR). The expression of the nuclear marginalization, and the production of apoptotic bodies were
genes of interest was estimated for each sample through the relative gene observed. When treated with the double of IC50 of the extract at 48.7 μg/
expression study using the comparative threshold cycle number with the mL, most of the cells died.
2ÀΔΔCt method [15]. Bcl-2, caspase-3, and p53 were the genes analysed.
To acquire reliable results, the glyceraldehyde-3-phosphate dehydroge- 3.4. Evaluation of the apoptotic effect of the MPB in Hep G2 cell lines by
nase (GAPDH) housekeeping gene was employed as a benchmark. targeting Bcl-2, Caspase-3, and p53 genes

2.5.2. Western blot analysis 3.4.1. Relative gene expression study
To study the expression of Bcl-2, caspase-3 and p53 protein expres- Total RNA values in all the samples were found to be between 200

sion, western blot analysis was performed [16]. The cells were collected and 500 ng/μL. The samples displaying A260/A280 and A260/230 ratios in
after 24 h of treatment with IC50 half, IC50, and double of IC50 of MPB. the 1.8–2.5 range, implying good purity, were utilized for cDNA syn-
Sodium dodecyl sulphated polyacrylamide gel electrophoresis (SDS- thesis (Table 2). Primer designs used in this study is presented in Table 3.
PAGE) was used to separate proteins. The gel was submerged in transfer The optimal annealing temperature for the primers used to amplify the
buffer for 15 min after electrophoresis. The proteins were then trans- Bcl-2, Caspase-3, p53, and GAPDH genes was determined using gradient
ferred to a 0.45 μm polyvinylidene diflouride (PVDF) membrane using a PCR. The annealing temperature that yielded the best results for ampli-
Hoefer semi-dry transfer equipment, as directed by the manufacturer. fication by gradient PCR was used for real-time PCR (Table 4).
The transmission was done for 1 h at 350 mA. After that, the PVDF
membrane was removed and immunoblotting was used to identify pro- The relative gene expression of Bcl-2, Caspase-3, and p53 in Hep G2
tein. β actin was employed as an internal control to guarantee equal cell lines in response to dose dependent treatment of the MPB compared
protein loading. The Image J Density Measurement tool was used to to control, using GAPDH as the reference gene is indicated in Fig. 3. In
measure the western blotting band strength [17]. control cells, the expression of all genes was normalized to unity. Bcl-2
gene expression was downregulated in all treatments compared to the
2.6. Statistical analysis control, but Caspase-3 and p53 gene expression was elevated.

All results were presented as Mean Æ SE with ‘n’ equal to the number 3.4.2. Western blot analysis
of replicates. Graphpad Prism Version 5 was used to compute the IC50 In the Table 5, the relative expression of Bcl-2, caspase-3, and p53
value of the MPB. Using SPSS software version 22, the intergroup com-
parison was analysed using one-way analysis of variance (ANOVA) proteins in Hep G2 cells in response to MPB IC50 half, IC50, and double of
accompanied by Duncan Multiple Range Test (DMRT) for paired analysis. IC50 concentrations is compared to untreated control. Western blot pic-
tures of β actin, bcl-2, caspase-3, and p53 protein in Hep G2 cells are
3. Results shown in Fig.4. In control cells, the expression of all proteins was

3.1. Preparation of MPB Table 1
inhibition of the MPB in Hep G2 cell lines. The data is presented as mean Æ SD
3.1.1. Yield of methanolic extract of papaya black seed with ‘n’ ¼ 3 replicates. All significant differences compared to control (0 μg/mL)
The MPB yielded 12.5% about starting dry material. were reported at *P < 0.05.

3.2. Cytotoxicity studies: 3-(4,5-dimethylthiazol-2-yl)-2,5- Concentration of MPB (μg/mL) % Inhibition
diphenyltetrazolium bromide (MTT)assay
00
3.2.1. Analysing the effect of MPB on Hep G2 cell viability and
determination of IC50 7.8 19.883577 Æ .5264010*
15.6 31.784612 Æ .1905468*
The viability of cells exposed to various doses of extract decreased 31.3 59.488608 Æ .0744129*
dose-dependently, with the survival rate lowest at 1000 μg/mL (Fig. 1). 62.5 76.135021 Æ .2758832*
Percentage of inhibition calculated from the MTT assay (Table 1) 125 81.493206 Æ .1523154*
revealed that the IC50 of MPB in Hep G2 cell line was 24.35 μg/mL. 250 86.416344 Æ .0369783*
500 90.666462 Æ .0840992*
3.3. Microscopic studies 1000 95.766505 Æ .0401445*

3.3.1. Acridine orange/ethidium bromide (AO/EB) staining
The findings of AO/EB staining of apoptotic phases after treatment of

cells with MPB are shown in Fig.2. Control cells emitted uniform green
fluorescence with a circular nucleus in the center, but the cells treated
with the half of IC50 (12.18 μg/mL) of MPB emitted yellow-green fluo-
rescence with a crescent-shaped nucleus (early apoptotic). Cells treated

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A. Anilkumar, A. Bhanu P A Advances in Cancer Biology - Metastasis 4 (2022) 100025

Fig. 2. Cells treated with the MPB after AO/EB staining. A. Control cells. B. Cells treated with half of IC50 (12.18 μg/mL). C. Cells treated with the IC50 (24.35 μg/mL).
D. Cells treated with double the concentration of IC50 (48.7 μg/mL).

Table 2 normalized to unity. Bcl-2 protein expression was downregulated in all
Concentration and purity of RNA samples selected for gene expression studies. treatments compared to the control, but Caspase-3 and p53 protein
expression was elevated.
RNA samples Concentration (ng/ A260/ A260/
μL) 4. Discussion
Untreated cells 487.5 280 230
Cells treated with half of IC50 (12.18 289.7 Because of the exceptional nutritional and therapeutic characteristics,
2.1 2.3 papaya is considered a highly nutritious fruit [18]. Carotenoids (fruits
μg/mL) 200.5 1.9 2.1 and seeds), enzymes (latex), alkaloids (leaves), phenolics (shoots, fruits,
Cells treated with the IC50 (24.35 μg/ and leaves), and glucosinolates (fruits and seeds) are some of the phy-
198.7 2.2 2.4 tochemicals found in Carica papaya [19]. Papaya extracts from various
mL) sections have shown anticancer properties. Hep G2 liver cancer cells
Cells treated with double of IC50 (48.7 1.9 2.0 were substantially inhibited by papaya juice extracts and pure lycopene
having IC50 values of about 20 μg/mL and 22.8 μg/mL, respectively [20].
μg/mL) On acute promyelotic leukemia HL-60 cells, papaya seed extracts had
substantial anticancer action at the half maximal inhibitory concentra-
Table 3 tion (IC50) of 20 μg/mL [7]. PC-3 prostate cancer cells were inhibited by
Description of primers used. papaya seed extracts at 25 μg/mL efficiently [21]. The CT-26 colorectal
cancer cell line's viability and migration were also drastically reduced by
Gene Primer sequence (500- 300) Product size (bp) papaya black seed extracts [22].
65
GAPDH F: TCT CCA CTT TGC CAC TGC AA 148 Although the seeds make up just 7% of the papaya's weight, they are
Bcl-2 R: GAA CGG ATT TGG CCG TAT TG 115 frequently thrown away. This study aimed to explore how papaya seed
Caspase-3 F: GTC CCG CCT CTT CAC CTT TCA G 89 extracts affected the human liver cancer cells Hep G2. We observed
p53 R: GAT TCT GGT GTT TCC CCG TTG G 24.35 μg/mL as the IC50 value of the MPB in Hep G2 cell lines. These
F: GGA GCT TGG AAC GGT ACG CTA A results are in the same concentration range as those found in prior in-
R: CCA CTG ACT TGC TCC CAT GTA vestigations [7,20–22]. The percentage of inhibition had increased
F: GAG TAT TTC ACC CTC AAG AT significantly (P<0.05) as the concentration of the extract was increased.
R: GCA TGG GCA TCC TTT AAC TC Benzyl isothiocyanate (BITC), which is the kind of isothiocyanates pre-
sent in papaya black seeds, are capable of suppressing cancer cell for-
Table 4 mation and growth through a multitude of paths and mechanisms. The
qRT PCR conditions for Bcl-2, Caspase-3, and p53 genes. lethal effects of BITC on cancer cells were stronger in proliferating cells
than in quiescent ones. In addition, specific investigations showed that
Steps Temperature Time papaya black seed extracts containing 1.25 and 2.5 μM BITC have anti-
3min cancer cell migratory properties [22]. Hence, we consider BITC as the
Initial denaturation 95 C 30s primary anticancer agent in papaya black seeds, which helped to inhibit
94 C 30 s the viability of Hep G2 cell lines.
30 cycles of Denaturation Bcl-2
Caspase-3 35 s Fluorescence light microscopy with selective absorption of DNA
Annealing p53 63 C
GAPDH 62 C
72 C 59 C
61 C

Extension

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A. Anilkumar, A. Bhanu P A Advances in Cancer Biology - Metastasis 4 (2022) 100025

Fig. 3. Graph representing the relative gene expression study. A. Downregulation of Bcl-2 in Hep G2 cell lines in response to dose-dependent treatment of the MPB
compared to control, using GAPDH as the reference gene. B. Upregulation of p53 genes in Hep G2 cell lines in response to dose-dependent treatment of the MPB
compared to control, using GAPDH as the reference gene. C. Upregulation of Caspase-3 genes in Hep G2 cell lines in response to dose-dependent treatment of the MPB
compared to control, using GAPDH as the reference gene. Comparison of all significant differences to control (0 μg/mL) were reported at **P < 0.01.

Table 5 dependent manner. Cells treated with half of the IC50 value
Relative protein expression of Bcl-2, caspase-3 and p53 upon treatment with (12.18μg/mL) were in the early apoptotic stage. In contrast, the ones
various concentrations of MPB analysed through western blotting compared to treated with the IC50 (24.35 μg/mL) had both early apoptotic and cells in
control. The data is presented as mean Æ SD with ‘n’ ¼ 3 replicates. All significant late apoptotic phase, and wells that were treated with the 2x concen-
differences compared to control (0 μg/mL) were reported at **P < 0.01. tration of IC50 (48.7μg/mL) mainly showed dead cells. This suggests that
apoptosis is the cell death type generated by papaya seed extract, and it is
Relative protein expression dosage dependent [8].

Control Bcl-2 p53 Caspase-3 Uncontrolled cell growth produced by a mismatch between tumor
growth and programmed cell death (apoptosis) defines the cancer con-
1 11 dition. Overexpression or under expression of particular genes has been
identified to contribute to carcinogenesis by reducing apoptosis in cancer
MPB 12.18 μg/mL 0.015 Æ 0.001** 4.16 Æ 0.33** 4.56 Æ 0.009** cells. Benzyl isothiocyanate (BITC) contained in papaya black seeds
MPB 24.35 μg/mL 0.0033 Æ 15.94 Æ 12.44 Æ possesses many cell-cycle-dependent functions, such as phosphorylation
0.0002** 0.83** 0.008** of Bcl-2 in the G2/M phase, which leads to apoptosis by its down-
(IC50) 0.0039 Æ 34.99 Æ 1.5** 53.55 Æ 0.08** regulation. The upregulation of p53 by BITC in papaya black seeds is also
MPB 48.7 μg/mL 0.0002** recognised to play a vital role in tumorigenesis prevention [24]. BITC
triggered apoptosis by increasing the protein level of caspase -3 which is
Fig. 4. Western blot pictures of β actin, bcl-2, caspase-3, and p53. an effector caspase in the apoptotic pathway [25]. A flavonoid named
quercetin derived from the aqueous leaf extract of C. papaya, has been
binding fluorescent dyes acridine orange (AO) and ethidium bromide shown to promote apoptosis in MCF-7 breast cancer cell lines by inhib-
(EB) is an effective approach to detect apoptotic changes [23]. In our iting Bcl-2 protein expression [26]. By upregulating p53 protein
study, when Hep G2 cells were treated with the IC50, its half and double expression, ribosome-inactivating proteins (RIPs) isolated from C. papaya
concentrations of MPB, the apoptotic changes were also visible in a dose leaves caused apoptosis in breast cancer cell lines [27]. In Hep G2 cells,
treating them with papaya peel extracts increased caspase-3 activity and
produced DNA fragmentation, showing that papaya peel extracts trig-
gered apoptosis [28].

In the present study, the expression of Bcl-2 (antiapoptotic), p53
(tumor suppressor), and Caspase -3 (apoptotic effector caspases/tumor
suppressors) genes in Hep G2 cell line to dose-dependent treatments of
MPB as compared to control using GAPDH as the reference gene was
determined by relative gene expression study. The protein expression of
all these genes were studied through western blot analysis. There is a
significant difference at P value less than 0.01, in the downregulation of
Bcl-2 and the upregulation of p53 and Caspase-3 induced by the MPB in a
dose dependent manner compared to control. However, there was no

5

A. Anilkumar, A. Bhanu P A Advances in Cancer Biology - Metastasis 4 (2022) 100025

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7

Advances in Cancer Biology - Metastasis 4 (2022) 100018
Contents lists available at ScienceDirect

Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Comparing transcriptomic profiles from seven cell lines to elucidate liver

metastatic potential

Lindsay R. Dresang a,*, Christian A. Van Scoyk a,b,1, Kirby J. Kuehn a,1, Taylor A. Tauber a,c,2,
Arthur R. Tondin a,d,2, Morgan A. Broske a,e,2, Cody J. Schreiner a,f,2

a University of Wisconsin-Stevens Point, USA
b Pharmaceutical Product Development, USA
c Arizona State University, USA
d University of Miami, USA
e West Virginia School of Osteopathic Medicine, USA
f Medical College of Wisconsin-Central Wisconsin, USA

ARTICLE INFO ABSTRACT

Keywords: Overview: The liver is a vital organ, performing over 500 functions. Metastasis to the liver can disrupt these
Oncology functions, resulting in poor prognoses. It is not always clear why liver metastasis arises in one case but not another
Cancer cell lines involving the same cancer type. We sought to understand which transcripts and cellular pathways are dysregu-
Liver metastases lated in cell lines shown to metastasize substantially to the liver in a NOD-Scid-Gamma (NSG) mouse-xenograft
Bioinformatics model. Cancer cell lines of the same type not observed to metastasize to the liver were used for comparison,
Transcriptomic profiles reducing cell type-specific changes or general pathways associated with cancer not linked to liver metastasis.
Gene ontology Three metastatic versus non-metastatic pairs of diverse origin–Merkel cell, colorectal, and pancreatic
carcinomas–as well as a normal fibroblast control were used for deep sequencing and transcriptome analysis with
subsequent pathway identification.
Results: Dysregulated pathways involve cell adhesion, proliferation, and motility (among others), which are
consistent with increased malignant potential in the cell lines that support liver metastasis. In addition, dysre-
gulated peroxisome proliferating activated receptor (PPAR) signaling and lipid metabolic / trafficking pathways
are candidates for fostering homing to the liver. A surprise was a significant drop in AGR2 expression in cells
favoring liver metastasis, while still remaining elevated relative to normal fibroblast controls. Newer clinical data
revealed declining levels of AGR2 correlate with higher grade lesions and poorer prognoses in patients with
various cancer types. Decreased expression of FOXA2 similarly correlates with clinical data as a prognostic factor.
A drop in FOXA2 expression was observed in cell lines favoring liver metastasis, as well as a cell line generated
from an NSG-xenograft liver metastasis, which may also explain the liver site preference of select cancer cell lines.
Both genes correlate with PPAR signaling dysregulation and either directly or indirectly link to such pathways.
Meanwhile, LOXL2 is lower in the cancer cell lines supporting liver metastasis compared to normal fibroblasts, but
is substantially elevated relative to paired cancer cell lines which did not metastasize to the liver. LOXL2 is a gene
involved in epithelial-mesenchymal transition (EMT), which is expressed at high levels in both normal and cancer-
associated fibroblasts.
Conclusions: Using only a normal fibroblast control for comparison, or only comparing cancer cells as separate
pairs, would have masked several potential candidate genes and pathways linked to liver metastasis. Our findings
correlate well with newer clinical data and reinforce biomarkers of disease progression. The dysregulated genes
and pathways highlight potential targets to slow disease progression.

* Corresponding author. University of Wisconsin-Stevens Point, Dept. of Biology, CBB Bldg. Rm. 313, 2101 Fourth Ave., Stevens Point, WI, 54481-3897, USA.
E-mail address: [email protected] (L.R. Dresang).

1 Contributed equally to this work.
2 Contributed equally to this work.

https://doi.org/10.1016/j.adcanc.2021.100018
Received 27 September 2021; Received in revised form 29 November 2021; Accepted 30 November 2021
Available online 7 December 2021
2667-3940/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
).nc-nd/4.0/

L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Abbreviations MAPK Mitogen Activated Protein Kinase
MCC Merkel Cell Carcinomas
*All Cell Lines BJ, Caco2, Capan2, Hct-116, HeLa, Jurkat, K562, MCPyV Merkel Cell Polyomavirus
MiaPaca2, MKL-1, MS-1, MX-1, SKNSH, and WM35 are cell MYBBP1A MYB Binding Protein 1A
lines with names derived from their cell type, cancer type, MYC Myelocytomatosis (gene)
patient/doctor identifier, and/or their sequence of NSG/NOG Non-obese diabetic, Severely immuno-compromised,
isolation.
interleukin-2-Gamma-receptor-null (mice)
ABCA1 ATP Binding Cassette subfamily A member 1 (gene) PBS Phosphate-Buffered Saline
AGR2 Anterior Gradient 2 (gene) PGC-1β Peroxisome gamma coactivator-1β
AKT Ak transforming (gene) PHACTR3 Phosphatase and Actin Regulator 3 (gene)
APOA1 Apolipoprotein A1 (gene) PI3K Phosphoinositide 3-Kinase
ATCCR American Type Culture Collection (registered) PKB Protein Kinase B
Ct Cycle threshold PPAR Peroxisome Proliferator-Activated Receptors
DAVID Database for Annotation, Visualization, and Integrated PPARGC1A PPAR-Gamma Coactivator 1 Alpha (gene)
PURPL p53 Upregulated Regulator of p53 Levels
Discovery Rap2 Ras related Protein 2
DICE Database of Immune Cell Expression, Expression RARRES1/TIG1 Retinoic Acid Receptor Responder 1/tazarotene-

quantitative trait loci and Epigenomics induced gene 1
ECM Extra-Cellular Matrix RasGEF1C Ras Guanine-nucleotide Exchange Factor 1C
EMT Epithelial-Mesenchymal Transition Rb Retinoblastoma
ERK Extracellular signal-Regulated Kinase RET Rearranged During Transfection (gene)
EZH2 Enhancer of Zeste Homolog 2 (gene) RIN RNA Integrity Number
FDR False Discovery Rate RT-qPCR Reverse Transcription quantitative Polymerase Chain
FOXA2 Forkhead box A2 (gene)
GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase (gene) Reaction
G.I. Gastrointestinal SEC14L4/hTAP3 Secretory-14 like Lipid binding protein-4/human
GO Gene Ontology
IGF2BP1 Insulin like Growth Factor 2 mRNA Binding Protein 1 Tocopherol Associated Protein 3
KEGG Kyoto Encyclopedia of Genes & Genomes SEER Surveillance, Epidemiology, and End Results
lincRNA Long Intergenic Non-Coding RNA sT small Tumor (antigen)
lncRNA Long Non-Coding RNA TNF tumor necrosis factor
LOXL2 Lysyl Oxidase Like 2 (gene) ULK4P2 Unc-51 Like Kinase 4 Pseudogene 2
LT Large Tumor (antigen) ZNF469 Zinc Finger protein 469

1. Introduction expression an important area of research.
While capillary bed filtration explains some target organs along
1.1. Why are we interested in metastasis, and specifically liver metastasis?
metastatic pathways, several other cancer types exhibit metastatic
Hundreds of different cancer types exist because there are as many growth to specific organs not readily explained by physical connections.
differentiated cells in the human body. However, all cancers are evalu- For example, in addition to lymph nodes, lungs, and liver, breast cancer
ated using the same four-stage standard, whereby stage IV metastatic can also metastasize to bone and brain tissue, as can melanomas and
disease has the worst overall prognosis. In >90% of cancer deaths, esophageal cancers, the latter of which also metastasizes to the adrenal
metastasis is the ultimate cause of mortality [53,63,71], as it results in glands [15,19,75]. The reason for apparent metastatic site preference can
the shutdown of our organs’ essential physiological processes. Therefore, be enigmatic, although select, active cellular pathways in both the pri-
understanding pathways involved in metastasis has broad implications mary tumor and the preferred metastatic niche are emerging as impor-
toward disease progression for many cancer types. tant determinants in this specificity [15,19,71,75]. We chose to assess the
properties of select cancer cell lines which specifically favor substantial
Some of the most common metastatic sites may relate to filtration metastasis to the liver in previous mouse-xenograft model studies.
barriers along bodily fluid conduits, such as lymph nodes along
lymphatic drainage, the lungs along pulmonary circulation, and the liver The liver represents a dangerous site for metastatic lesions to occur, as
along portal venous flow [15,19,74]. In the latter case, circulating tumor it performs over 500 vital functions. Disruption of normal liver physi-
cells detected in portal venous blood are correlated with high rates of ology is particularly associated with poor prognosis in cancer patients.
pancreatic metastatic disease to the liver [74]. Circulating tumor cells For example, breast cancer survival to 5 years is generally high at over
entering portal venous blood from gastrointestinal (G.I.) organs is also 99%, whereas breast cancer with liver metastasis results in a prognosis of
much higher than in peripheral venous blood, as the liver represents the only 3–15 months [22,82]. Analysis of Surveillance, Epidemiology, and
first narrow, capillary bed downstream of many G.I. cancer sites [74]. End Results (SEER) data evaluating cancer incidences across all reported
The colon is connected to both portal venous blood flow and peripheral cancer types from 2010 to 2015 revealed that individuals with liver
venous blood flow through portal/systemic anastomosis along the metastasis had a 1-year survival rate at 15% compared to a 1-year sur-
rectum, and also interfaces with the peritoneal cavity [61]. More than vival rate of 24% in individuals with metastasis at any other site [22].
90% of stage IV colorectal carcinomas will present with distant metas- Researchers further identified a median 4-month survival in individuals
tases to the liver, lung, brain, and the peritoneum, often with distinct with de novo liver metastasis upon diagnosis, which was observed in ~5%
metastases and transcriptional profile differences between primary tu- of all cases [22]. Understanding the events supporting liver metastasis
mors and their metastatic disease [61]. The evolutionary progression of may help elucidate new cancer targets. Preventing this progressive state
metastatic disease is highly varied from one case to the next [61], making may not serve as a cure, but serve to stabilize the disease and prolong
the identification of metastatic pathways and changes to transcriptional survival.

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

1.2. What was our overall approach to understand liver metastasis better? in the patient or subsequently after decades in cell culture could be of
interest. Such mutations can also involve random pathways since they
Prior to this research, an observation was made when studying can arise with greater frequency than normal cells. Since MCCs, colo-
human tumor viruses and a pre-clinical trial involving Merkel cell car- rectal carcinomas, and pancreatic carcinomas have very different cell
cinomas [13]. Merkel cell carcinomas (MCCs) are a non-melanoma skin origins, any transcriptional dysregulation or altered cellular pathways
cancer of neuroendocrine cell origin, which are causally-associated with shared by MKL-1, Hct-116, and MiaPaca2 cell lines should be more
Merkel cell polyomavirus (MCPyV). MCPyV is clonally integrated in over narrowly related to liver metastasis. However, if each cell line contains
80% of all MCCs in a manner which disrupts the normal viral life cycle, the same dysregulated pathway but through separate mutations
but maintains expression of the viral oncoproteins small tumor (sT) and involving different genes, a simple transcriptional profile comparison
large tumor (LT) antigens [9,16]. These viral proteins drive early initi- may miss pertinent targets. Meanwhile, including all transcriptional
ating and promoting events in MCCs by inhibiting cell checkpoint protein changes across these cell lines would broaden identification of cellular
Rb (retinoblastoma), activating pro-survival AKT (Ak transforming) and pathways important for favoring liver metastasis, but it would also
survivin signaling pathways, and either through sT and LT or other elevate background pathways related to their three distinct cell origins.
mutational pathways have inhibited p53 and/or p53 regulatory pathway
disruption [6,9,23,57]. Therefore, we sought to elucidate what the more metastatic cell lines
have in common relative to their paired counterparts which did not favor
YM155, an inhibitor of survivin, was assessed for MCC chemothera- liver metastasis. Specifically, we identified transcripts differentially
peutic potential by testing optimal dosage and duration parameters after regulated in cell lines with greater liver metastatic potential relative to
first establishing MCCs as xenografts in immunocompromised-mice [13]. cell lines of paired cancer type which did not metastasize to the liver
Surprisingly, one of these MCC cell lines, MKL-1, metastasized to the liver using the same mouse-xenograft model. Utilizing paired metastatic
in up to a quarter of mice surviving past one-month with prolonged versus non-metastatic cell lines of diverse origin, including MCCs (MKL-1
treatment [13]. Liver metastasis could be substantial, whereby overall vs. MS-1), colorectal carcinomas (Hct-116 vs. Caco2), and pancreatic
liver weight accounted for up to 30% of total mouse body weight; a carcinomas (MiaPaca2 vs. Capan2) affords a broad transcriptional
healthy liver would normally account for 5–6% body weight in these repertoire to identify candidate transcripts responsible for increased
mice [13]. Some observed cases with liver metastasis still occurred metastatic potential. These broad cross-comparisons should also reduce
despite full primary tumor regression. None of the other MCC cell lines transcriptional noise from cancer and cell type-specific changes not
tested (MS-1, WaGa, or MKL-2) metastasized to the liver in this study related to the support of liver metastasis. However, the evolutionary path
[13]. MCCs have been estimated to metastasize to lymph nodes in 60% of for each cancer type is still heavily swayed by cell origin connotation,
cases, the liver and the lungs each in 30% of cases, as well as other sites from differences in route of carcinogen exposure (e.g., ingested com-
[38]. Additionally, case studies of MCC and substantial liver metastasis pounds interacting with colorectal epithelium), to specific acquired
have been reported involving infiltration of over 60% of parenchymal mutations (e.g., mutant p53 in MiaPaca2), and even exposure to viral
tissue [68]. infections (e.g., MCPyV in MKL-1 & MS-1). Therefore, the collective
pathways, not just individual genes, altered in these cell lines may be
MKL-1 was originally isolated in 1987 from a metastatic lesion within necessary to elucidate cellular signaling events important for liver met-
an axillary lymph node [64]. MS-1 and WaGa cell lines were also astatic potential.
developed from metastatic sites [18,24] (MKL-2's location of origin is
unknown [76]), but only MKL-1 underwent liver metastasis in the 2. Materials & methods
mouse-xenograft model [13]. Xenograft studies conducted by other
research groups using the same mouse model (Non-obese diabetic, 2.1. Biosafety
Severely immuno-compromised, interleukin-2-Gamma-receptor-null, or
NSG/NOG mice), have also characterized subsets of specific colorectal The experimental protocols for this research were approved by the
cancers [20] and pancreatic cancers [73] which either substantially University of Wisconsin-Stevens Point Institutional Biosafety Committee.
metastasized to the liver (or not) in a comparable manner. Specifically, All researchers conducting these experiments met biosafety training
colorectal carcinoma cell lines which metastasized to the liver in 100% of standards required before beginning these research activities.
mouse-xenografts included Hct-116; zero metastasis to the liver corre-
sponded to Caco2 [20]. Meanwhile, pancreatic carcinoma cell lines 2.2. Cell culture
which metastasized to the liver between 70% and 100% of the time
included MiaPaca2; zero metastasis to the liver corresponded to Capan2 MKL-1 [64], MS-1 [18,24], MX-1 [13] and low passage BJ fibroblasts
[73]. [5] were provided by the lab of Dr. Yuan Chang and Dr. Patrick S. Moore
and the lab of Dr. Masa Shuda from the University of Pittsburgh Cancer
Cancers arising in abdominal organs drained by the portal venous Institute. Hct-116 [7], Caco2 [21], Capan2 [40], and MiaPaca2 [88] were
system may have an intuitive link toward liver metastasis [19]. However, purchased from the American Type Culture Collection, or ATCCR. These
this neither accounts for the excessive metastatic nature of Hct-116 and cell lines were passaged several times over a period of several weeks to
MiaPaca2 cell lines, nor does it account for the lack of liver metastasis months post-thaw, in order to ensure proper recovery prior to expansion
observed for Caco2 or Capan2 cell lines [20,73]. Additionally, subcu- for various downstream experiments. All cell lines were incubated with
taneous injection of MKL-1 cell lines along the right hind flank of mice 5% CO2 at 37 C within a humidified atmosphere (Heracell Vios 160i
would not have a direct conduit to explain liver metastasis physically. Incubator, Thermo Scientific). Growth media varied per cell line ac-
Enhanced malignant phenotypes (e.g., changes in adhesion, motility, and cording to which medium had previously been identified to best support
extracellular matrix remodeling) of the metastasis-supporting cell lines each cell line independently (i.e., suspension cells in RPMI 1640, Hct-116
must play a role in epithelial-mesenchymal transition (EMT) and subse- & Capan2 in McCoy's 5A, BJ fibroblasts & MiaPaca2 in DMEM, and Caco2
quent intravasation during early stages of metastasis. Cell-to-cell in- in MEM/EBSS). All growth media were supplemented with 10% fetal
teractions, cell communication, and cell signaling between extravasating bovine serum (Hyclone™ SH30910.03, filter sterilized) & antibiotics
cancer cells and the variety of cells encompassing the liver must also play (200U/mL penicillin and 200 μg/mL streptomycin, Gibco #15140122).
an important role in supporting liver-specific metastasis. Cells were grown to ~70–90% confluency (or near high density for cells
in suspension) prior to collection for RNA harvests to ensure transcrip-
Since cancer cells accrue substantial mutations given their acceler- tional patterns of over- or under-growth would not influence the exper-
ated growth rates and compromised checkpoints, they are capable of imental expression profiles. Trypsin (0.05%, Hyclone™ SH30236.01)
undergoing independent evolutionary processes, even within the same
individual. Identifying cell lines capable of frequently metastasizing to
the liver suggests that whatever evolutionary pressures arose previously

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

was used 1:20-1:10 for adherent cells when reseeding, except for Capan-2 2.6. Sequencing analysis
which required 1:5-1:4 trypsin treatment and prolonged incubation for
detachment. Transcriptional reads were generated by NovaSeq analysis for seven
cell lines, in triplicate. Raw data files were retrieved from the UW-
2.3. Wound healing assays Madison Biotechnology & Gene Expression Center's online repository
and transferred to the Galaxy platform (https://usegalaxy.org/) using the
Wound healing assays, or migration scratch assays, were performed in public server to analyze data;[1,2,30] file types were manipulated as
quadruplicate using the adherent cell lines BJ fibroblasts, MiaPaca2, fastqsanger.gz files with database key reference GRCh38/hg38 (Human
Capan2, Hct-116, and Caco2. MS-1, MX-1, and MKL-1 are non-adherent, Genome, December 2013). Sample profiles were then assessed using
thus they were not assayed. Cells were grown to confluency in 24-well FastQC, version 0.72þgalaxy1 [11], followed by MultiQC, version
treated tissue culture plates and then scratched using a pipette tip. Im- 1.8þgalaxy0 [14]. Initial quality control identified between ~2.5–6%
ages were captured on day zero and at various time-points up through adapter sequence contamination at read ends (Fig. S1). Leftover adapter
wound closure (only days 0, 1, an 2 are reported). Percent wound healing sequences were removed using Trim Galore!, version 0.6.3 [39]. Trim-
was calculated along the widest point of the scratch mid-well and ming datasets removed adapter sequences to a threshold less than 0.1%.
normalized to the day-0 width to eliminate variations in initial scratch Sample profiles were then reassessed using FastQC, followed by MultiQC
width well-to-well. (Figs. S2–S3). Total read counts exceeded the minimum threshold of 20
million sequence reads, ranging from ~29 million reads to just under 100
2.4. RNA isolation million reads per sample (Fig. S2). Sequence reads were ~150 bases long
on average, with Phred scores at ~36 from read end-to-end, suggesting a
Cell harvests were performed in triplicate, often on separate dates in base calling rate >99.9% accuracy (Fig. S3).
addition to separate cultured flasks for true biological replicates. Sepa-
rate individuals were also responsible for different cell lines, helping to Each sample's read sets were aligned to the human genome (build
prevent cross-contamination and/or swapping samples. During harvests, hg38) using HiSat2, version 2.1.0þgalaxy7 [37]; this reference build was
media was removed, cells were washed with Phosphate-Buffered Saline augmented to also contain human viral genomic sequences for a separate
(PBS, Hyclone™ SH30256.FS), and PBS was then aspirated before alignment, provided by Mitch Hayes at the University of
application of TRIzol™ (Invitrogen #15596026) for cell lysis and sub- Wisconsin-Madison. Viral genome transcript read counts were subse-
cellular suspension. Total RNA was isolated using an RNA-Direct-ZolTM quently identified using Samtools Idxstats, version 2.0.3 [42–44] on
(Zymo Research R2050) silica-based column with in-column DNase I HiSat2 alignments to validate viral transcript detection in MCCs (and lack
(Zymo Research E1010) digestion per manufacturer's instructions. Initial of detection in other samples). Feature Counts, version 1.6.4þgalaxy1
total RNA quality and concentrations were determined using a NanoDrop [46], were obtained for all cellular genome aligned reads to generate
2000 spectrophotometer (Thermo Scientific, ND-2000). All total RNA transcriptomic profiles for each sample. Raw feature counts for all cell
samples submitted for library generation and sequencing analysis had lines, in triplicate are reported in Table S1. Transcriptomic profiles were
260 nm/280 nm absorbance ratios between 1.8 and 2.0, with 100–1000 cross-compared using DESeq2, version 2.11.40.6þgalaxy1 [49].
ng of RNA in dilutions between 20 and 500 ng/μL, per sequencing facility Cross-comparisons were generated with multiple groupings, including all
recommendations. cancer samples relative to normal samples, a single replicate of all seven
cell lines per group of three cross-compared groups, and cell lines sup-
2.5. Total RNA library preparation & deep sequencing porting liver metastasis relative to those which do not (with or without
normal BJ fibroblasts in the latter group). An additional analysis
Total RNA was submitted to the University of Wisconsin-Madison involving only paired comparisons of MCCs, then colorectal carcinomas,
Biotechnology & Gene Expression Center. Samples were first run on an and then pancreatic carcinomas with post-processing comparison was
Agilent 2100 Bioanalyzer to assess overall sample quality. Each sample's considered briefly; this analysis lacked proper group-to-group normali-
combined RNA distribution (including 28S and 18S rRNA as a part of this zation for accurate comparisons and resulted in over- and under-inflated
analysis) was assessed to estimate their overall RNA Integrity Number candidate lists (without and with required overlap, respectively) as dis-
[69]. Samples with confirmed RIN scores !7 (Table 1) were used for cussed in the results.
cDNA library construction (with polyA enrichment) and subsequent
paired-end Illumina NovaSeq6000 amplification and sequencing anal- 2.7. Analysis of candidate genes & cellular pathways
ysis. Communication regarding sample replicate preparation and RNA
quality was consulted with and relayed by the Core Director, Sandra Cross-comparison of transcriptomic profiles included a normalized
Splinter BonDurant. table output. Multiple cross-comparisons were generated and assessed,
however the focus of this study involved grouping transcriptomic profiles
Table 1 from cell lines supporting liver metastasis in a mouse-xenograft model
RNA Quality: RNA integrity number, or RIN, was determined using several (i.e., MKL-1, Hct-116, and MiaPaca2) to paired sets which did not
points of RNA distribution and signal intensity as determined using a bio- metastasize to the liver in the same mouse model (i.e., MS-1, Caco2, and
analyzer. Ratios between 7 and 10 are considered optimal for subsequent tran- Capan2). Transcript feature counts were consolidated to counts based
scriptomic profiling. upon individual gene names [72]. Group-to-group comparisons gener-
ated output base means of sequence read counts, adjusted P-values for
Cell Line RIN Values Per Replicate group consistency, and overall fold changes relative to the normalized
group (as well as some additional statistical information). Genes were not
MKL-1 A%7 B ¼ 8.1 C ¼ 8.3 considered as differentially-expressed candidates if they had average
MS-1 D%7 E ¼ 8.4 F ¼ 8.2 base means less than 0.5, adjusted P-values greater than 0.05 (Fig. S4), or
Hct-116 G ¼ 10 H ¼ 9.6 I ¼ 9.1 fold-changes between Æ5-fold when normalized. Candidate gene lists
Caco2 J ¼ 9.8 K ¼ 9.6 L ¼ 9.9 were first generated for all genes meeting these criteria, even if an in-
MiaPaca2 M ¼ 9.5 O ¼ 9.6 P ¼ 9.5 dividual gene did not meet these criteria across all three cell lines which
Capan2 Q ¼ 9.4 R ¼ 9.3 S ¼ 9.3 favored liver metastasis (in other words, overlap for all three cell lines
BJ Fibroblasts U ¼ 9.1 V ¼ 9.7 W ¼ 9.5 was not required). The full list of differentially-expressed genes is re-
ported in Table S2.
RIN ¼ RNA Integrity Number
Candidate gene lists were used for subsequent GO term and pathway

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

analysis. Individual candidate cellular pathways were elucidated using for 15 s, 54 C for 10 s, and 62 C for 1 min and 30 s. Products were
the Database for Annotation, Visualization and Integrated Discovery, or amplified using a QuantStudio 3 thermocycler (Applied Biosystems).
DAVID (version 6.8; https://david.ncifcrf.gov/) online bioinformatics Overall expression was assessed using the delta-delta Ct method (2^-
database [26–28], specifically using Kyoto Encyclopedia of Genes and (Cttarget – Ctcontrol)) using GAPDH (glyceraldehyde 3-phosphate dehy-
Genomes (KEGG) pathway database reports [32–34]. Interconnectedness drogenase) as the housekeeping gene and comparing expression of
of cellular pathways based upon these genes was further assessed using FOXA2 (forkhead box A2), AGR2 (anterior gradient 2), and LOXL2 (lysyl
GOnet (https://tools.dice-database.org/GOnet/) [60] from the oxidase like 2) across cell lines. Data are reported first using BJ fibro-
DICE-Tools online database (Database of Immune Cell Expression, blasts as the control for delta-delta Ct, and then using the paired cell lines
Expression quantitative trait loci and Epigenomics). Reported pathway not observed to metastasize to the liver in a mouse-xenograft model as
connections were restricted to those with false-discovery-rate (FDR) the control.
adjusted P-values <0.0001. A more narrow set of candidate cellular
pathways was also identified using a more restricted candidate gene list 3. Results
involving similar changes across all three cell lines favoring liver
metastasis (in other words, overlap in all three cell lines was required). 3.1. Graphical overview
This narrow list of differentially expressed genes, consistent in their
changes across all three cell lines favoring liver metastasis is reported in An overview of the experiments carried out in this study is repre-
Table S3. sented as a flow chart in Fig. 1 and the Graphical Abstract (online).
Further details are elaborated upon in the subsequent subsections.
2.8. Single-step reverse transcription, quantitative-polymerase chain
reaction 3.2. Cell morphology & cell culture observations

The Luna® Universal One-Step RT-qPCR (reverse transcriptase, The inherent properties of the three paired sets of cell lines discussed
quantitative-polymerase chain reaction) Kit (New England Biological, relative to normal cells like BJ fibroblasts vary substantially, from overall
E3005) was used to reassess transcript levels across cell lines. Primers morphology (Fig. 2) to growth rates (data not shown) and adhesion or
used in these reactions are reported in Table 2. suspension in treated cell culture flasks (Table 3). However, none of the
observed patterns coincided solely with the cell lines supporting liver
The manufacturer's protocol was generally followed with some metastasis relative to their paired cell lines which did not favor liver
modifications. The complementary DNA (cDNA) synthesis reaction was metastasis. All cell types exhibited anticipated morphology (Fig. 2 &
carried out at 50 C for 30 min. Since some PCR products were over 100 Table 3) and grew at rates described by ATCCR or by labs previously
base pairs, the amplification stages were also modified as follows: 95 C working with these cell lines under similar conditions [5,7,13,18,21,40,
64,88].
Table 2
Primers Used for RT-qPCR Analysis: The forward and reverse primers for 3.3. Overall cross-comparison of transcriptomic profiles
FOXA2, AGR2, LOXL2, and the housekeeping gene GAPDH are reported 5-prime
to 3-prime, along with their intended product sizes (in base pairs, bp) and cita- NovaSeq was performed using total RNA from the seven cell lines–BJ
tions corresponding to prior use of these sequences. fibroblasts, MCCs MKL-1 and MS-1, colorectal carcinomas Hct-116 and
Caco2, and pancreatic carcinomas MiaPaca2 and Capan2. Bioinformatic
Gene Forward (50→30) Reverse (50→30) Product Citation analysis was conducted using the generated sequence reads as outlined in
Fig. 1. Total read counts per human transcript were assessed and sub-
FOXA2 GGA GCA GCT ACT CGT GTT CAT GCC 83 bp [10] sequently used to cross-compare transcriptomic profiles of the seven cell
AGR2 ATG CAG AGC GTT CAT CC lines. Multiple comparisons (described in the materials and methods)
LOXL2 ATG AGT GCC CAC GGA CAT ACT GGC 141 bp [54] were made to verify that the twenty-one transcriptomic profiles clustered
GAPDH ACA GTC AA CAT CAG GA in the same way both in terms of dual-principal component variance
TGC ACA GGC AAT GGT TTC TGA GCA 456 bp [4] (Fig. 3A) and hierarchy (Fig. 3B) regardless of how groups were orga-
GAG AAG TC GGC AAC TC nized. The transcriptomic profiles of replicate samples from the same cell
TCA CCC ACA CTG TGC TGA GGT AGT CAG 78 bp [59] line clustered closest to each other, as expected. Normal BJ fibroblast
CCA TCT ACG A TCA GGT CCC G

bp ¼ base pairs; 50 ¼ 5-prime; 30 ¼ 3-prime

Fig. 1. Experimental Flow Chart: Each step sum-
marizes what experimental or bioinformatic manipu-
lation was needed to reach the final datasets and
results. A) Dark gray boxes indicate initial wet-lab
experiments performed at the University of
Wisconsin-Stevens Point. B) Medium gray boxes
indicate sample processing and sequencing performed
at the University of Wisconsin-Madison Biotechnology
& Gene Expression Center. C) Light gray boxes indi-
cate bioinformatics analyses performed using various
programs consolidated on the “UseGalaxy.org” public
server platform. D) White boxes indicate final
compiled genes of interest supporting liver metastasis,
as well as additional online bioinformatic analyses
identifying cellular pathways of interest using DAVID,
KEGG Pathways, and interconnected pathways using
GOnet. E) The last gradient box indicates the final
wet-lab experiments performed to follow up some of
the identified cellular pathways and cell transcripts
potentially related to liver metastasis.

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Fig. 2. General Morphology & Cell Culture Prop-
erties of Seven Cell Lines: The seven cell lines
assessed in transcriptomic profiling studies are
depicted, with each panel containing a 100-μm scale
bar and two individual cells with a green dashed
outline. A color-coding legend is also provided for this
figure, which is used consistently in other figures as
well. A) Normal BJ fibroblasts (passage 12) are
depicted. They are adherent and the only cells shown
which are contact-inhibited. Carcinoma cell lines in
the middle column were highly metastatic to the liver
in a mouse-xenograft model; carcinoma cell lines on
the right were not. Merkel cell carcinomas B) MKL-1
and C) MS-1 were grown in suspension. Colorectal
carcinomas D) Hct-116 and E) Caco2, as well as
pancreatic carcinomas G) MiaPaca2 and H) Capan2
are adherent.

profiles clustered farthest from all other samples (the six cancer cell lines) scratched with a pipette tip and imaged over two days. The percent of
both by dual-principal component variance and hierarchy. Profiles from each gap being filled in was calculated. Normal BJ fibroblasts reached
Merkel cell carcinomas, MKL-1 and MS-1, clustered separately from the full gap closure in all four repeated experiments. All but Caco2 would be
other cancer cell lines’ profiles (Hct-116, Caco2, MiaPaca2, and Capan2). observed to fill in these gaps in some of the replicate assays. Capan2 fairly
Profiles from the metastasis-supporting cell lines Hct-116 and MiaPaca2 quickly filled in the gap, however, the extremely high re-adherence as
clustered closer together than profiles from their paired colorectal and described in materials and methods (even in separate cell passaging
pancreatic carcinoma counterparts, Caco2 and Capan2, respectively. manipulation with high trypsin treatment) will influence this apparent
wound healing not necessarily linked to growth rate or migration. Mia-
3.4. Cellular pathway dysregulation & gene ontology interconnections that Paca2 also quickly filled in the gap, but would also quickly overgrow the
support liver metastasis wells due to their rapid growth rates generating off-target toxicity as
partially seen in Fig. 4, panel L. Hct-116 filled in the gap much faster than
Genes whose expression changed Æ5-fold or more were uploaded to Caco2, the former of which also had a faster growth rate. Thus, while
DAVID [26–28]. These genes were then analyzed for functional clus- wound healing assays are helpful in assessing the ability of cells to
tering by GO terms and pathway maps generated through KEGG analysis migrate into an artificial wound, proliferate, and make contacts to
[32–34]. Table 4 reports the top 30 cellular pathways with matched adjacent cells and the treated plate, they are limited in assessing these
dysregulated cellular genes from cell lines favoring liver metastasis; these pathways separately and are also limited based upon overall cell growth
pathways were sorted by number of candidate genes matched to the properties in culture.
pathways indicated. The full list of pathways identified is reported in
Table S4. 3.6. Gene dysregulation supporting liver metastasis & a final, restricted
pathway analysis
Since several of these identified pathways are functionally related,
further assessment of pathway interconnectedness was warranted. This Each sample's read sets were aligned to the human genome; a sepa-
analysis was performed using the DICE-Tools GO-database, GOnet [60]. rate, parallel alignment included the human genome and the genomes of
Over 150 GOnet interconnected pathways were identified using Æ5-fold known or suspected human tumor viruses. Transcriptional reads were
changes and a highly restricted P-value of <0.0001; zero GOnet path- mapped to MCPyV in MCCs. Specifically, ~16,700 average reads were
ways are identified if candidate genes are restricted to meeting narrow detected in MKL-1 and ~9,990 average reads were detected in MS-1.
criteria across all three cell lines supporting liver metastasis even with a MCPyV reads were not detected in any of the other cell lines, as ex-
relaxed P-value of 0.05. Fig. S5 shows groups of pathway in- pected. Detection of MCPyV transcript reads in MCCs but not in colo-
terconnections and a short summary of what functional groups were rectal carcinomas, pancreatic carcinomas, or normal fibroblasts
observed. Table 5 shows a subset of specific GO terms corresponding to highlights the specificity of the transcriptional mapping; detection of
clusters shown in panels A-K of Fig. S5. The full list of GO terms with a MCPyV transcripts to several thousand reads also indicates sensitivity of
false-discovery rate P-value adjusted to <0.0001 is reported in Table S5. the sequencing results since only a small number of viral transcripts are
expressed in MCCs. Viral reads were not assessed further than this
3.5. Comparing dysrergulated cellular pathways & observations of wound- sensitivity and specificity assessment.
healing
Normalized cross-comparisons of cellular transcriptomic profiles
Several of the dysregulated cellular pathways identified are pathways were generated in several different ways as discussed in the materials and
also exploited in artificial wound-healing assays, or migration scratch methods. The focus of this study was to remove background genes related
assays, in cell culture (Fig. 4). Adherent cells grown to confluency were to cell origin and cancer pathways not specifically related to favoring

6

L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Fig. 3. Clustering of Transcriptomic Profiles: The twenty-one transcriptomic profiles—from seven cell lines, in triplicate—were cross-compared multiple times in
different ways as outlined in Materials and Methods. A) Dual-principal component variance plots and B) hierarchical clustering were reproduced with each analysis.
Normal BJ fibroblast transcriptomic profiles clustered farthest from all other, cancer-associated transcriptomic profiles. Profiles from Merkel cell carcinomas, MKL-1
and MS-1, clustered separately from the other cancer cell lines' profiles. Profiles from the metastasis-supporting cell lines Hct-116 and MiaPaca2 clustered closer
together than profiles from their paired colorectal and pancreatic carcinoma counterparts, Caco2 and Capan2, respectively. These images were made using DESeq2
on Galaxy.

liver metastasis. If instead comparisons relied on individual pairs which 3.7. Validating transcriptional levels of FOXA2, AGR2, and LOXL2
were then reconsolidated, then 13,806 genes would require sorting for
cellular pathway analysis and yet only 386 genes would have been found The narrow list of cellular pathways identified peroxisome
overlapping all three cell lines favoring liver metastasis. Meanwhile, proliferator-activated receptor (PPAR) signaling, a pathway linked to
generating normalized comparisons of the three cell lines favoring liver lipid metabolism vs. lipid biogenesis [25,55,70,84] and also linked to the
metastasis (MKL-1, Hct-116, and MiaPaca2) relative to the group of cell genes FOXA2 [90], the latter of which can regulate AGR2 [8], both re-
lines which did not metastasize to the liver in that same mouse-xenograft ported in Table 6. Meanwhile the cellular pathways identified in the full
model (MS-1, Caco2, and Capan2) identified 2,488 genes whose list of genes whose expression changed Æ5-fold or more in any of the
expression changed Æ5-fold or more with 565 genes in the overlap. This three cell lines which favored liver metastasis identified many pathways
comparison also better controls for differences in sample-to-sample favoring malignancy, proliferation, and EMT. LOXL2 is a gene related to
sequencing read differences given the group-based normalization. The EMT and other pathways associated with malignancy [4,35,52,71,81]
full list of genes whose expression changed Æ5-fold or more in any of the and is also reported in Table 6. These three genes were assessed via
three cell lines favoring liver metastasis is reported in Table S2. The full reverse transcription quantitative-polymerase chain reaction (RT-qPCR)
list of genes whose expression changed Æ5-fold or more in all the three relative to the housekeeping gene GAPDH. The transcriptional levels
cell lines favoring liver metastasis is reported in Table S3. The top & were determined using a ΔΔCt method as reported in Fig. 5. If tran-
bottom 20 genes whose expression increased or decreased across all three scriptional levels were compared using GAPDH from normal BJ fibro-
cell lines which favored liver metastasis is shown in Table 6. blasts (Fig. 5A), it is apparent that FOXA2 and AGR2 are elevated in the
different cancer cells, while LOXL2 is depressed. Both FOXA2 and AGR2
The list of 565 genes whose expression changed Æ5-fold or more are generally elevated in cancer cells, meanwhile LOXL2 is generally
across all three cell lines favoring liver metastasis was also used in a more elevated in fibroblasts. Thus, there are limits to using cellular controls of
restricted analysis of cellular pathways. Only seven cellular pathways different cell origin. If instead the cell line which did not favor liver
were identified using this narrow gene list, which are reported in Table 7; metastasis of paired cancer type served as the control for the cell lines
zero GOnet interconnetions were found using this restricted gene list. which did metastasize to the liver (Fig. 5B), then the relative changes in
Since cellular pathways are identified with significant enrichment given these transcripts correlate with the data reported in the transcriptional
the total number of gene matches to the pathway relative to the number profiling analysis for colorectal and pancreatic carcinomas for all three
of genes input, the cellular pathways using the narrow set of genes are not genes assayed. The Merkel cell carcinomas matched data for FOXA2 and
the same as those identified in Table 4. to a lesser extent LOXL2; AGR2 expression did not correlate well with the

7

Table 3
Cell Line Culture Characteristics & Media: The seven cell lines used in transcriptional profiling are indicated,
are indicated, along with whether or not they metastasized to the liver in the NSG mouse-xenograft model. Gene
year) are also provided.

Cell Line Type & Information on Original Biopsy Site aMetastatic in NSG Contac
mice? Inhibi

MKL-1 Merkel cell carcinoma (neuroendocrine), from an axillary Yes No
MS-1 lymph node metastasis No No
cMX-1 Merkel cell carcinoma (neuroendocrine), from an adrenal Not Tested No
Hct-116 gland metastasis Yes No
Re-established cell line from metastasis of MKL-1 to Liver in
mouse-xenograft
Colorectal carcinoma, from the primary tumor

8 Caco2 Colorectal (adeno)carcinoma No No

MiaPaca2 Pancreatic carcinoma, from the primary tumor Yes No

Capan2 Pancreatic (adeno)carcinoma, from a primary tumor No No
BJ Fibroblast beginning to invade the duodenal wall Not Tested Yes
Normal, neonatal foreskin fibroblasts

F/M ¼ Female or Male, respectively; ATCCR ¼ American Type Culture Collection (Registered); TM ¼ Trade M

DMEM ¼ Dulbecco's Modified Eagle's Medium, Hyclone™ SH30022FS

RPMI 1640 ¼ Roswell Park Memorial Institute 1640 Medium, Hyclone™ SH30027.FS

MEM/EBSS ¼ Minimum Essential Medium with Earle's Balanced Salts, Hyclone™ SH30265.FS (Eagle's MEM

McCoy's 5A ¼ McCoy's 5A Modified Medium, Hyclone™ SH30200.FS
a Metastasized substantially to the liver in a Non-obese diabetic, Severely immune-compromised, interleuki
b All media were supplemented with 10% fetal bovine serum (Hyclone TM SH30910.03, filter sterilized), 20
c Not used in the transcriptional profiling studies.

L.R. Dresang et al.

, along with a cell line used in follow-up validation assays. Their cell origins, cancer types, and biopsy locations
eral properties regarding cell characteristics, growth in culture, ATCCR availability, and original citations (with

ct Sex (F/M) bMedium Culture ATCCR # Citation & Year
ited? (HycloneTM) Condition
M RPMI 1640 Suspension N/A [64]
F RPMI 1640 Suspension N/A 1987
M RPMI 1640 Suspension N/A [18,24]
M McCoy's 5A Adherent CCL-247 2010
M MEM/EBSS Adherent HTB-37TM [13]
M DMEM Adherent CRL-1420 2013
M McCoy's 5A Adherent HTB-80TM [7]
M DMEM Adherent CRL-2522 1981
[21]
1989
[88]
1977
[40]
1986
[5] 1998 (repeatedly
isolated)

Marked; N/A ¼ Not Applicable

can be substituted) Advances in Cancer Biology - Metastasis 4 (2022) 100018

in-2-Gamma-receptor-null (NSG) mouse-xenograft model.
00U/mL penicillin, and 200 μg/mL streptomycin (Gibco #15140122).

L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Table 4 may account for this difference, although other factors cannot be ruled
Top 30 Dysregulated Pathways Linked to Liver Metastasis: (overlap across the out as an explanation.
three liver metastasis-favoring cell lines not required) Genes dysregulated comparing
cell lines supporting liver metastasis (i.e., MKL-1, Hct-116, and MiaPaca2) and 4.2. Cellular pathway dysregulation & gene ontology interconnections that
cell lines not supporting liver metastasis (i.e., MS-1, Caco2, and Capan2) were support liver metastasis
used to identify candidate cellular pathways. The top 30 cellular pathways are
shown; the full list of pathways is available in Table S4. Pathways are sorted by The identification of the PPAR signaling pathway using a narrow gene
the greatest number of total matched genes, which correlates with the fraction of list is of particular significance given the number of differentially-
the pathway's genes being matched. The overall P-values assigned to these expressed genes of interest already discussed and additional genes and
matches are also indicated. pathways to be discussed further. This pathway is enhanced when also
taking into account the role of PI3K signaling (phosphoinositide 3-ki-
KEGG Pathway Term Count % P-Value nase) interconnected to this pathway. Additional pathways known to
be dysregulated with malignancy are readily identified, including PI3K-
Pathways in cancer 63 3.4 2.90E-06 AKT signaling, proliferative MAPK (mitogen activated protein kinase)
Neuroactive ligand-receptor interaction 51 2.8 5.30E-07 and Ras signaling, and pathways involving focal adhesion, regulation of
PI3K-AKT signaling pathway 49 2.7 8.50E-04 the actin cytoskeleton, cell adhesion molecules, and extra-cellular matrix
Cytokine-cytokine receptor interaction 45 2.5 2.40E-06 (ECM) receptor interactions. The top pathway identified using the full
MAPK signaling pathway 35 1.9 8.00E-03 gene list was “Pathways in Cancer”, shown in Fig. S6. Here many genes
Calcium signaling pathway 33 1.8 7.50E-05 are identified in particular carcinogenic signaling pathways of interest.
Focal adhesion 33 1.8 1.00E-03
Regulation of actin cytoskeleton 29 1.6 1.70E-02 GO-term identification and interconnected networks found a sub-
Ras signaling pathway 28 1.5 6.20E-02 stantial enrichment of pathways known to favor malignancy (Table 5).
cAMP signaling pathway 27 1.5 2.50E-02 Overall functional pathway connections can largely be grouped as fol-
Proteoglycans in cancer 27 1.5 2.80E-02 lows: 1) cell movement, motility, localization, and locomotion, 2)
Chemokine signaling pathway 25 1.4 3.60E-02 extracellular matrix regulation, response to wound healing, and adhe-
Serotonergic synapse 24 1.3 7.80E-05 sion, 3) cell signaling, signal transduction, and communication, 4) pro-
Complement and coagulation cascades 23 1.3 4.70E-08 liferation and ERK signaling (extracellular signal-regulated kinase, a.k.a.
Hematopoietic cell lineage 23 1.3 4.00E-06 MAPK1, downstream effector of Ras/MAPK pathways), 5) regulation of
Cell adhesion molecules 23 1.3 6.10E-03 phosphorylation, protein modification, and other regulatory enzymes,
ECM-receptor interaction 22 1.2 1.40E-05 and 6) development, morphogenesis, and differentiation. Altogether
Protein digestion and absorption 22 1.2 1.70E-05 these pathways and functional gene enrichment have clear links to favor
Oxytocin signaling pathway 22 1.2 2.20E-02 malignant and metastatic potential, as dysregulation of cell movement
Influenza A 22 1.2 8.50E-02 processes as well as connections to surrounding cells and extracellular
Amoebiasis 21 1.1 8.10E-04 matrix are critical in the ability to breach adjacent tissues and distant
Transcriptional misregulation in cancer 21 1.1 9.60E-02 organs. These cellular pathways would not have been identified using a
Vascular smooth muscle contraction 20 1.1 6.30E-03 narrow list of genes. Meanwhile, too many pathways were instead
Hippo signaling pathway 20 1.1 7.10E-02 identified using only separate pairs of cell lines which favored liver
Metabolism of xenobiotics by cytochrome P450 19 1.0 5.20E-05 metastasis or not. Likewise, analysis purely related to wound healing
Chemical carcinogenesis 19 1.0 1.50E-04 analysis does not parse different pathways involved in these physiolog-
Dilated cardiomyopathy 19 1.0 3.00E-04 ical processes, potentially masking some of these pathways.
Retrograde endocannabinoid signaling 19 1.0 2.80E-03
Hypertrophic cardiomyopathy 18 1.0 3.50E-04 4.3. Genes of interest with higher expression in cell lines supporting liver
Drug metabolism–cytochrome P450 17 0.9 2.10E-04 metastasis

KEGG ¼ Kyoto Encyclopedia of Genes & Genomes; % ¼ percentage of the full There were several genes of interest with elevated overall expression
pathway with matched genes provided in the upload list in metastatic cell lines in Table 6. The most highly expressed gene,
SEC14L4/hTAP3 (secretory-14 like lipid binding protein-4/human
transcriptional profiling in MCCs. Finally, the cell line MX-1, derived tocopherol associated protein 3, respectively), is involved in secretion,
from the liver metastasis in an NSG mouse MKL-1-xenograft previously lipid transport, and a linked pathway involving PI3K, protein kinase B
reported [13], was also assessed. MX-1 displayed decreased expression in (PKB), & PPAR signaling (PI3K/PKB/PPAR) [36]. Importantly, dysre-
FOXA2 and AGR2 relative to both MS-1 and MKL-1; it was elevated in gulation of different arms of PPAR signaling pathways are linked to he-
LOXL2 expression relative to MS-1, but not significantly. patocellular carcinomas and liver metastases from colorectal cancer,
albeit with highly variable inhibitory and stimulatory effects comparing
4. Discussion PPAR-alpha, -gamma, and -delta homolog signaling [25,55,70,84].
SEC14L4 has recently been shown to promote lipid uptake in select
4.1. Overall transcriptional profile comparisons, human and viral tocopherol-based PI3K signaling [89], and has been indicated as a
biomarker of thymomas [48], but otherwise this gene is poorly charac-
Detection of viral reads validated the read depth of the NovaSeq terized in humans. Additionally, the long intergenic non-coding RNA, or
analysis. MCC transcriptomic profiles from MKL-1 and MS-1 clustered lincRNA ULK4P2 (unc-51 like kinase 4 pseudogene 2; also shown on
separately from all other cancer cell line transcriptomic profiles (Fig. 3). Table 6) is elevated in hepatitis B-virus associated hepatocellular carci-
Viral oncogenesis, combined with DNA-damage from ultraviolet radia- nomas relative to paired non-cancerous tissue of the same patients. It is
tion will influence transcriptional changes in a distinct way relative to the proposed to work with enhancer of zeste homolog 2 (EZH2) to alter
carcinogenic events which occur in the large intestines and pancreas [9, PI3K-PKB signaling and other pathways involved in proliferation and
41,63]. metastasis [87].

The pancreatic and colorectal carcinomas loosely grouped together The proto-oncogene RET (“rearranged-transfection” gene) is a tyro-
(Fig. 3). However, the colorectal and pancreatic carcinomas supporting sine kinase receptor with known mutation and activation in thyroid
liver metastasis in the mouse-xenograft model (Hct-116 and MiaPaca2) cancer [58]. Tyrosine kinases are common components of MAPK
had transcriptomic profiles which clustered closer to each other than to
profiles of their paired non-metastatic counterparts (Caco2 and Capan2,
respectively). This closer transcriptomic clustering of Hct-116 and Mia-
Paca2 relative to their non-metastatic pairs suggests a greater relatedness
of carcinogenic events in their disease evolution. Further de-
differentiation of MiaPaca2 and Hct-116 relative to Capan2 and Caco2

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Table 5
Select Gene Ontology Terms: This table reports a subset of enriched GO terms in each of the interconnected pathways graphically depicted in Fig. S5, sorted as clusters
in panels A–K. The full list of identified, interconnected GO terms is provided in Table S5. The GO term identifiers, the false-discovery rate adjusted P-values (<0.0001),
and total number of dysregulated genes associated with these interconnected GO terms are also provided.

Cluster Gene Ontology Term GO Term ID FDR #
Description (GOnet) P-Value

A tissue development 0009888 0Eþ00 237

anatomical structure development 0048856 0Eþ00 579

animal organ morphogenesis 0009887 0Eþ00 145

cell differentiation 0030154 6.94E-08 391

angiogenesis 0001525 5.85E-07 57

B cell migration 0016477 6.00E-10 140

Locomotion 0040011 9.00E-10 176

localization of cell 0051674 2.30E-09 150

cell motility 0048870 2.30E-09 150

wound healing 0042060 4.81E-07 77

cell surface receptor signaling pathway 0007166 6.51E-07 268

cell communication 0007154 5.38E-06 532

signal transduction 0007165 6.51E-05 485

C positive regulation of developmental process 0051094 0Eþ00 186

regulation of wound healing 0061041 3.80E-07 34

regulation of cell differentiation 0045595 1.15E-06 210

D regulation of localization 0032879 0Eþ00 329

regulation of locomotion 0040012 3.30E-09 139

regulation of cell motility 2000145 3.40E-09 131

regulation of cell migration 0030334 1.90E-08 122

E positive regulation of protein phosphorylation 0001934 2.89E-06 130

positive regulation of phosphate metabolic process 0045937 3.29E-06 142

regulation of ERK1 and ERK2 cascade 0070372 8.91E-06 51

positive regulation of ERK1 and ERK2 cascade 0070374 2.10E-05 40

positive regulation of protein modification process 0031401 6.48E-05 145

F regulation of peptidase activity 0052547 3.43E-06 70

negative regulation of proteolysis 0045861 1.02E-05 58

negative regulation of hydrolase activity 0051346 2.85E-05 68

G regulation of cell adhesion 0030155 0Eþ00 117

positive regulation of cell-cell adhesion 0022409 4.86E-06 47

positive regulation of cell-substrate adhesion 0010811 7.54E-05 27

H regulation of cell population proliferation 0042127 0Eþ00 222

regulation of epithelial cell proliferation 0050678 3.80E-07 60

I biological adhesion 0022610 0Eþ00 151

cell adhesion 0007155 0Eþ00 149

cell-cell adhesion 0098609 3.80E-07 80

J extracellular structure organization 0043062 3.45E-24 102

extracellular matrix organization 0030198 5.97E-22 91

K regulation of endothelial cell apoptotic process 2000351 7.98E-05 16

GO Term ID ¼ Gene Ontology Term Identifier; FDR ¼ False Discovery Rate; # ¼ gene count matched to GO term

signalling cascades which trigger G1 exit, and further downstream p53. It creates a feedback loop by inhibiting MYBBP1A (MYB Binding
S-phase entry (pending activation of select checkpoint proteins). RET Protein 1A), a stabilizer of p53 [45]. This lncRNA was expressed 42-fold
targets Ras, a specific activator in the MAPK signaling pathway [58]. more in cell lines supporting liver metastasis. There was only a single
Ras-GEF1C is a guanine-nucleotide exchange factor of Ras-related pro- read in three out of nine non-metastatic cell line replicate samples by
tein 2, or Rap2. It activates this protein to enhance endothelial barrier comparison, accounting for the relatively low normalized base mean of
permeability [56]. LOXL2 reorganizes extracellular matrix by targeting ~11 (~18 average raw read counts, Table S1). Interestingly, raw total
collagens, fibronectin, and cytokines, promoting migrational capacity, read counts of PURPL are much lower in the cell lines supporting
angiogenesis, and metastasis [4,71]. LOXL2 is highly expressed in normal metastasis compared to normal BJ fibroblasts (~1,580 average raw read
fibroblasts and cancer-associated fibroblasts where it is involved in EMT counts, Table S1), however total reads in these normal cell lines were also
[35,52,81]. While LOXL2 expression in these reported cancer cell lines is greatest amongst the seven cell lines. Additionally, TP53 mRNA expres-
lower relative to a normal fibroblast control (Fig. 5A), its expression was sion is ~6-fold higher in the cell lines supporting liver metastasis
significantly increased in cancer cell lines favoring liver metastasis (Tables S2–S3).
relative to paired cancer cell lines which did not metastasize to the liver
(Fig. 5B). This difference suggests that the enhanced metastatic pathways This apparent low yet comparatively elevated expression of PURPL
might be missed when using an unmatched cellular control. Zinc-Finger and disconnect to p53 levels is explained by distinct p53 dysregulation
protein 469 (ZNF469) is also thought to regulate collagens as a tran- events in the different cell lines assessed. MiaPaca2 expresses mutant
scription factor in brittle cornea syndrome, although it has also been p53, the dominant negative form of the protein which disrupts wild-type
reported as a biomarker of tumor immune cell infiltration in esophageal copy downstream signaling, and also exhibits oncogenic gain of function
cancer [77]. Increased proliferation, extracellular matrix remodeling, [83]. Meanwhile, p53 signaling is either blocked by virally-encoded
and vascular permeability changes would all favor components of EMT tumor-antigens or other non-viral mechanisms in MCCs [6,23,57]. In
and intravasation stages of metastasis. Caco2, both copies of p53 are truncated [78], thus PURPL transcription
would be inhibited. PURPL had previously been characterized in
PURPL is a long non-coding RNA (lncRNA), aptly named the p53 Hct-116 cells as a pro-survival lncRNA when p53 activation is enhanced
upregulated regulator of p53 levels, previously shown to be upregulated through DNA damage [45]. Thus, PURPL is elevated in other cancers, and
in colorectal and liver cancers [17,45]. This lncRNA is transcribed by is slightly elevated in this cross-comparative analysis, but its role in liver

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Fig. 4. Wound-Healing Assays: The overall ability to proliferate, migrate, and form cell connections to other cells and substrates are all components in a wound-
healing assay (a.k.a. migration scratch assay). Adherent cells were assayed over a two-day period in quadruplicate; MCC cell lines are excluded because they are
not adherent in culture. Green dashed lines are indicated in each of these panels to highlight the edges along the initial scratch through to when it closed. Normal BJ
fibroblasts were assessed in row 1 (A, B, & C); Hct-116 was assessed in row 2 (D, E, & F); Caco2 was assessed in row 3 (G, H, & I); MiaPaca2 was assessed in row 4 (J, K,
& L); Capan2 was assessed in row 5 (M, N, & O). Percent wound-healing was calculated where the widest point along the initial scratch was set to 100% to eliminate
initial well-to-well scratch size variation. P) These percentages are represented graphically and Q) in table format with reported averages, standard deviations, and
color-coding reference. a.k.a. ¼ also known as.

metastasis specifically remains unclear when p53 aberrative signaling is RARRES1, retinoic acid receptor responder 1 (a.k.a., TIG1, or
prevalent. tazarotene-induced gene 1) acts downstream of anti-oxidant activity, is
involved in cell differentiation control, and apoptosis signaling (among
4.4. Genes of interest with lower expression in cell lines supporting liver others). It is a possible tumor suppressor gene with decreased expression
metastasis observed in malignant prostate cancer [31]. RARRES1 also has decreased
expression through promotor hypermethylation as detected in tumors of
There were also several genes of interest with decreased overall breast cancer, colon cancer, head and neck cancers, acute myeloid leu-
expression in the metastatic cell lines seen in Table 6. Substantial kemia, and chronic myeloid leukemia, in addition to many cancer cell
decrease in the levels of AGR2, a gene involved in cell migration, lines [86]. RARRES1 is also induced by PPAR-alpha signaling and plays a
transformation, metastasis, and inhibition of TP53 was initially surpris- role in triggering lipid metabolism and beta-fatty-acid oxidation [50], a
ing. AGR2 levels were first characterized in association with early pathway typically not employed by cancer cells for ATP generation. Lipid
developmental pathways and have been detected at high levels in various metabolism tends to be lower in cancer cells which favor aerobic
tumor types (reviewed in Ref. [8]). However, AGR2 decreased expression glycolysis (i.e., the Warburg effect) and require lipid biosynthesis for
was more recently observed in high grade ovarian serous carcinomas, rapid growth and cell membrane expansion needs (reviewed in
whereby declining levels of AGR2 and p53 aberrant expression were Ref. [85]). Similarly, we observe decreased expression of PPARGC1A
associated with more metastatic phenotypes and lower disease-free sur- (PPAR-gamma coactivator 1-alpha; Table S2), which correlates with
vival [3]. More recent identification of AGR2 downregulation in various levels of RARRES1 in other studies as well [50]. We further see a decrease
patient tumors of several cancer types–prostate, ovarian, colorectal, and in apolipoprotein A1 (APOA1; Table 6), a protein secreted by the liver
pancreatic carcinomas–revealed that high levels of AGR2 in tumors through ABCA1 (ATP binding cassette subfamily A member 1) trans-
correlated with lower grade lesions and better prognosis, while low levels porters upon PPAR signaling, which is involved in high-density lipo-
of AGR2 correlated with higher grade lesions and poor prognosis [3,51, protein formation, cholesterol excretion, and lipid metabolism [65].
54,62,79]. Expression levels of AGR2 mRNA matched the expression Given the relatively high expression of PPAR signaling and lipid regu-
levels of the colorectal and pancreatic cancer cell lines also used in our lation in the liver with abundant lipid efflux, and the shutoff of lipid
study [54,62], indicating our observations match the findings of several metabolism, increase in lipid uptake and high lipid demands in these
other groups. This consistency also highlights how our analyses, which metastatic cells, it is worth further exploring what role lipid transfer from
utilized cancer cell lines instead of patient tumors, still successfully the liver to extravasating cancer cells plays in liver-site preference.
identified dysregulated genes correlated with current clinical data and
validated newer biomarkers of disease severity. Since AGR2 levels can be Altered regulation of actin polymerization impacts cellular motility,
regulated by FOXA2 [8], which in turn is linked to PPAR signalling [90], locomotion, and related pathways favoring EMT and intravasation as
it is worth exploring the link between this gene and PPAR signalling in well. An actin regulator, PHACTR3 (phosphatase and actin regulator 3,
future studies. also known as scapinin), is also not detected in several cancer cell lines of
various origin (i.e., HeLa cervical carcinoma, K562 leukemia, Jurkat

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Table 6
Genes Dysregulated with Liver Metastasis: These genes had the greatest fold-changes comparing normalized read counts from cell lines supporting liver metastasis in
the NSG mouse-xenograft model (i.e., MKL-1, Hct-116, and MiaPaca2) relative to read counts from cell lines which did not metastasize to the liver (i.e., MS-1, Caco2, and
Capan2). The upper rows indicate the top 20 positive-fold changes, indicating much higher expression in the liver metastasis-supporting cell lines, while the bottom
rows indicate the bottom 20 negative-fold changes, indicating much lower expression in the liver metastasis-supporting cell lines. The actual fold changes are reported in
order (from either the greatest positive-fold change or the greatest negative-fold change), along with the normalized base means and their adjusted P-values. All raw
counts, including a cross-reference to normal BJ fibroblasts, are reported in Table S1. All dysregulated genes with 5-fold or higher expression and -5-fold or lower
expression determined are reported in Table S2.

Gene Names aFull Gene Names Fold Change Base Means Adjusted
P-Value

Top 20, Increased Expression

SEC14L4 SEC14 (secretory 14)-like lipid binding 4, a.k.a. hTAP3 (human tocopherol associated protein 3) 249.15 115.59 3.72E-11
105.10 218.17 3.83E-35
RET ret (rearranged during transfection) proto-oncogene 102.94 143.34 2.22E-12
71.39 2552.64 1.50E-13
GABRD gamma-aminobutyric acid type A receptor delta subunit 66.85 305.03 5.12E-12
42.09 10.77 5.90E-07
LOXL2 lysyl oxidase like 2 41.09 436.16 3.43E-10
40.26 23.31 2.70E-08
AC090197.1 lncRNA, a.k.a. LOC100507156 38.21 26.52 2.27E-07
34.54 36.73 9.84E-15
PURPL p53 upregulated regulator of p53 levels (a lncRNA) 32.52 6.47 2.79E-04
29.03 3.83 7.00E-05
DOC2B double C2 domain beta 27.34 171.83 1.01E-09
21.98 131.74 2.54E-07
AC243562.2 golgin A6 family like 9 pseudogene 21.91 5.91 9.46E-05
21.93 14.92 7.45E-09
TMEM132B transmembrane protein 132B 21.81 371.70 4.13E-39
20.58 7.47 1.28E-03
RASGEF1C RasGEF (rat-sarcoma, guanine-nucleotide exchange factor) domain family member 1C 20.49 9.00 2.46E-06
19.65 180.08 2.96E-36
BX649567.1 (no data)
-3196.68 4704.07 8.07E-19
ULK4P3 ULK4 pseudogene 3 -1410.55 389.52 1.20E-19
-1296.96 8854.71 3.25E-17
SLC47A1 solute carrier family 47 member 1 -1104.15 8856.41 1.33E-12
-892.46 157.41 7.24E-38
ZNF469 zinc finger protein 469 -705.05 467.63 1.30E-11
-686.99 645.32 4.22E-15
AL139339.1 a lncRNA, a.k.a. ENSG00000234699 -671.22 465.39 1.24E-11
-607.81 285.19 2.60E-19
GOLGA8K golgin A8 family member K -580.41 9952.97 8.30E-18
-515.76 663.87 2.55E-09
SELENOM selenoprotein M -504.68 2033.02 2.99E-12
-469.81 240.72 9.87E-13
P2RX7 purinergic receptor P2X 7 -464.23 245.89 2.77E-15
-453.30 326.09 1.06E-11
ULK4P2 ULK4 (unc-51 like kinase 4) pseudogene 2 (a lincRNA) -416.08 4918.14 3.64E-13
-411.10 245.74 6.07E-09
SLC16A14 solute carrier family 16 member 14 -387.06 156.64 6.82E-13
-381.31 91.03 1.63E-12
Bottom 20, Decreased Expression -354.63 315.64 4.43E-08

AGR2 anterior gradient 2 (protein disulphide isomerase family member)

THNSL2 threonine synthase like 2

RARRES1 retinoic acid receptor responder 1, a.k.a. TIG1 (tazarotene-induced gene 1)

APOA1 apolipoprotein A1

LINC01291 lincRNA 1291

GJB1 gap junction protein beta 1

DZIP1 DAZ (deleted in azoospermia) interacting zinc finger protein 1

GYG2 glycogenin 2

AC006058.1 lncRNA, a.k.a. ENSG00000261786

SLC7A7 solute carrier family 7 member 7

PHACTR3 phosphatase and actin regulator 3, a.k.a. scapinin

CDH17 cadherin 17

DRD2 dopamine receptor D2

FOXA2 forkhead box A2

AC245041.2 lncRNA, a.k.a. ENSG00000276850

IGF2BP1 insulin like growth factor 2 mRNA binding protein 1, a.k.a. CRD-BP (coding region determinant-binding protein)

HOXC12 homeobox C12

PSG5 pregnancy specific beta-1-glycoprotein 5

AC006058.4 uncategorized, a.k.a. ENSG00000280435

SLCO1A2 solute carrier organic anion transporter family member 1A2

a.k.a. ¼ also known as; lncRNA ¼ long non-coding RNA; lincRNA ¼ long intergenic non-coding RNA
a Gene Cards (Registered), The Human Gene Database: https://www.genecards.org.

lymphoma, WM35 melanoma, or SKNSH neuroblastoma) [67]. Para- suppress FOXA2 and trigger tumorigenesis through NOTCH signaling
doxically, this gene has been proposed to induce cell spreading and [47]. Select components of the tumor necrosis factor (TNF)-alpha
motility when re-expressed in HeLa cells [66], but has also been identi- pathway are dysregulated (data not shown) with ~5-fold increased
fied as frequently mutated with MYC (myelocytomatosis gene) in expression of NOTCH4 (Table S2). The hepatocellular carcinoma study
non-small cell lung cancers [12] and is readily detected in normal brain demonstrated that low levels of FOXA2 expression correlated with
tissue [80]. Meanwhile, a translational regulator of beta-actin mRNA, greater rates of recurrent disease and increased markers of EMT [10].
insulin-like growth factor mRNA, and MYC mRNA called IGF2BP1 (in- Additionally, a PPAR regulatory protein PGC-1β (peroxisome gamma
sulin like growth factor 2 mRNA binding protein 1) [29], is also coactivator-1β) has recently been shown to turn off FOXA2 in breast
decreased in expression (Table 6). On average the expression of this gene cancer cell lines [90]. Inhibition of PGC-1β resulted in overexpression in
remains elevated in the cancer cell lines relative to normal BJ fibroblasts, FOXA2 in these breast cancer cell lines, which then inhibited cell pro-
as are MYC, MYC-L, and MYC-N with some cell line variability (Table S2). liferation and migration [90]. FOXA2 in our studies was decreased in the
The role of IGF2BP1, therefore, may be important for carcinogenesis but liver metastasis cell line developed from an MKL-1 NSG mouse-xenograft
not liver metastasis specifically. called MX-1 [13], relative to both MS-1 and MKL-1, further highlighting
additional downregulation from cells developed directly from the met-
FOXA2 is a hepatocyte differentiation transcription factor highly astatic niche. Decreased expression of FOXA2 in the
expressed in normal liver; it has recently been identified as a target of metastasis-supporting cell lines again identifies a potential biomarker
EZH2-lncRNA inhibition in hepatocellular carcinomas [10]. Signaling correlated with clinical data regarding metastatic potential and poor
through the tumor necrosis factor-alpha pathway has also been shown to

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L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

Table 7
Dysregulated Pathways Linked to Liver Metastasis: (overlap across the three
liver metastasis-favoring cell lines was required) Genes dysregulated comparing cell
lines supporting liver metastasis (i.e., MKL-1, Hct-116, and MiaPaca2) and cell
lines not supporting liver metastasis (i.e., MS-1, Caco2, and Capan2) were used to
identify candidate cellular pathways. Pathways were generated only using genes
dysregulated across all three cell lines which favored liver metastasis. Pathways
are sorted by the greatest number of total matched genes, which correlates with
the fraction of the pathway's genes being matched. The overall P-values assigned
to these matches are also indicated.

KEGG Pathway Term Count % P-Value

Neuroactive ligand-receptor interaction 12 3 1.20E-02
Gastric acid secretion 8 2 3.30E-04
Calcium signaling pathway 8 2 4.40E-02
PPAR signaling pathway 6 1.5 7.20E-03
Ovarian steroidogenesis 4 1 5.90E-02
Legionellosis 4 1 7.40E-02
Maturity onset diabetes of the young 3 0.8 8.00E-02

KEGG ¼ Kyoto Encyclopedia of Genes & Genomes; % ¼ percentage of the full
pathway with matched genes provided in the upload list

prognosis in patients. Many additional genes of interest are present in
these lists and warrant further analysis.

5. Conclusions Fig. 5. Validating Transcriptional Levels: Bioinformatic analysis identified
FOXA2, AGR2, and LOXL2 as transcripts whose expression changed in cell lines
5.1. Correlation to experimental & clinical data favoring liver metastasis. Their transcriptional levels were validated by con-
ducting RT-qPCR. The housekeeping gene GAPDH was used as a control for
Analysis of cancer cell lines in a cell culture system will not always calculating ΔΔCt. A) Using BJ fibroblasts for comparison as a normal control
reflect clinical data, due to inherent variability across cancer cases, reveals a dramatically different profile of transcriptional levels across samples
evolution within a culture system over time, and limited capacity to relative to B) using the paired cell lines which did not metastasize to the liver as
recapitulate interactions with extracellular matrices and other cells the control groups. Both profiles provide information about relative transcrip-
within this artificial system. Thus, it was important to compare our tional levels in cancer cells, as well as their changes in transcriptional expression
findings with those in the literature evaluating primary clinical data. in those cells favoring liver metastasis compared to paired cancers of the same
Reviewing the transcriptomic profiles of ~500 metastases across many cell origin which did not metastasize to the liver. RT-qPCR ¼ reverse tran-
biopsied sites (conducted by Robinson et al in 2017) [63], liver metas- scription, quantitative-polymerase chain reaction.
tases were the only organ site with a relatively segregated transcriptomic
profile, suggesting that hallmark pathways regulated at the level of proliferation and growth pathways like MAPK and PI3K signaling.
transcription are likely. However, even with supplemental evaluation of Meanwhile, the liver as a metastatic niche is naturally lending to
meta-signatures in metastatic liver biopsies, many of the GO terms are extravasation due to the leakiness of liver sinusoids. Interestingly, this
dominated by general carcinogenic pathways. niche also provides a rich lipid source whereby newly micro-
metastasizing cells are primed to take up these lipids while also inter-
An advantage normalizing transcriptomic profiles of cancer cell lines nally shutting off lipid metabolism to meet membrane expansion with
supporting liver metastasis to paired cancer types which did not metas- rapid growth demands. Conversely, if the cancer cells themselves secrete
tasize to the liver in the same mouse model is that cancer cell-origin dysregulated factors which lower FOXA2 endogenously, they may then
specific pathways would be reduced. Therefore, our results reflect liver target FOXA2 in the surrounding hepatocytes. FOXA2, which controls
site preference and metastasis-support more narrowly, and global hepatocyte differentiation, may then be dysregulated locally in the liver
changes for carcinogenesis could be minimized. Focusing in on specific to favor EMT and promote new micro-metastasis formation from newly
dysregulated genes also permitted potential biomarker identification. extravasating cancer cells. The role of potential downstream loss of AGR2
Specifically, decreased detection of AGR2 and FOXA2 correlate well with remains unknown. Follow-up studies are warranted to verify the effects
clinical data also detecting low levels of these proteins with more of directed gene shutoff and/or gene reconstitution in in vivo cell culture
aggressive forms of a variety of cancers [3,10,51,54,62,79]. Thus, our assays with migration/wound healing assessment. Additional studies
observed data correlate well with multiple studies, both broadly assess-
ing transcriptomic profiles from metastatic biopsies and evaluating in-
dividual biomarkers of progressive disease.

5.2. Where do we go from here?

These analyses highlight both broad pathways and genes related to
metastatic potential like proliferation and migration pathways and the
LOXL2 gene. These analyses also highlight select genes and pathways
related to potential liver site preference, like PPAR signaling, lipid traf-
ficking, and the FOXA2 gene. The broad metastatic-supporting pathways
include logical changes in cell migration and locomotion related to
dysregulated cell adhesion, extracellular matrix remodeling, and actin
polymerization control. Greater aggressive properties likely needed
during the EMT and intravasation stages of metastasis are reflected by

13

L.R. Dresang et al. Advances in Cancer Biology - Metastasis 4 (2022) 100018

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Advances in Cancer Biology - Metastasis 4 (2022) 100027
Contents lists available at ScienceDirect

Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Root extract of Plumbago zeylanica L. induces cytotoxicity, inhibits cell
migration and induces S-phase cell cycle arrest through down regulation of
EGFR in HeLa cervical cancer cells

Shubhasmita Mohapatra a, Jasmine Mohanty b, Sarita Pani a, Sunitee Hansdah a,
Anil Kumar Biswal b, Atish Kumar Sahoo c, Priya Ranjan Debata a,*

a P. G. Department of Zoology, North Orissa University, Takatpur, Baripada, Mayurbhanj, Odisha, 757003, India
b P. G. Department of Botany, North Orissa University, Takatpur, Baripada, Mayurbhanj, Odisha, 757003, India
c Regional Plant Resource Centre, Dept. of Forest & Environment, Govt. of Odisha, Nayapalli, Bhubaneswar, 751015, India

ARTICLE INFO ABSTRACT

Keywords: Plumbago zeylanica has been used in the traditional system of medicine from thousands of years owing to its
Plumbago zeylanica potential therapeutic properties, however the anticancer and anti-metastatic effects are largely unknown against
HeLa cells cervical cancer cells. In this study we demonstrated the cytotoxicity activity, inhibition of cell migration, in-
MTT assay duction of S-phase cell cycle arrest and the down regulation of expression level of epidermal growth factor re-
Cell migration ceptor (EGFR) by ethanol extract of the root of P. zeylanica (ERPZ). The cytotoxicity effects were analyzed by MTT
S phase arrest assay the IC50 was determined to be 10.49 μg/ml after 72 h of incubation. Besides, strong inhibition of wound
HRLC-MS plant Extract healing activity was observed at 20 μg/ml concentration, where the wound size was reduced to 33%. The ethanol
root extract of P. zeylanica also showed significant S-phase cell cycle arrest of 68.9% at 20 μg/ml concentration in
HeLa cells which was accompanied by the down regulation of EGFR. Ethanol extracts of the root of P. zeylanica
(ERPZ) treatment inhibited the growth of cervical cancer cells. The ERPZ arrested cells at the S-phase of the cell
cycle. Besides the HRLC-MS analysis of ERPZ identified 15 compounds and among which the five major com-
pounds such as 7,8-dihydroxy-4-methylcoumarin, neodiosmin, diosmetin, hispidulin, and formononetin were
reported to possess antioxidant and anticancer activities, could plausibly induce cell death in HeLa cells.

1. Introduction important resources of alternative medicine. Several plant-derived nat-
ural compounds are currently used as frontline treatments in many dis-
Cervical cancer is one of the leading causes of morbidity and mor- eases including cancer. The molecular mechanism of many of these
tality worldwide. The viral oncoproteins such as E6 and E7 degrade the compounds are known and many of them are under investigations.
two major tumor suppressor proteins namely p53 and pRb which are Natural compounds like Vinca alkaloids destabilizes the microtubules
considered as key events in the initiation of carcinogenesis [1]. Besides thus inhibiting cell division [5]. Similarly, the compounds such as
these, the Epidermal Growth Factor Receptor (EGFR) signaling leads to Docetaxel, Cabazitaxel, Larotaxel, and Tesetaxel originated from Pacli-
the alternation in the cell cycle has been reported both in cervical cancer taxel stabilize the microtubules with higher [6]. Compounds like
tissues and dispersed cells [2]. Noscapine, Combretastatin, and Podophyllotoxin, etc. are the microtu-
bule targeting compounds that have been discovered on a large scale.
Although several treatment options available like surgery, radio- These compounds target the processes of mitosis i.e. cell division [7]. Cell
therapy, and chemotherapy but none of them provide optimum results. cycle progression is a highly ordered and tightly regulated process that
The frequent failure of cancer treatment is mainly due to the develop- involves multiple checkpoints such as extracellular growth signals, cell
ment of resistance to chemotherapy [3] and the cancer stem cells [4]. size, and DNA integrity [8]. The deregulation of the cell cycle is the major
Herbal medicine has been used in many parts of the world as comple- event in the process of carcinogenesis. The cell cycle progression is very
mentary and alternate medicine. Particularly traditional Chinese medi- rapid in cancer cells. Several compounds from both natural and synthetic
cine (TCM) and Ayurveda, traditional Indian medicine (TIM) are sources have been characterized which interfere with the cell cycle

* Corresponding author.
E-mail address: [email protected] (P.R. Debata).

https://doi.org/10.1016/j.adcanc.2022.100027
Received 18 October 2021; Received in revised form 31 December 2021; Accepted 7 January 2022
Available online 11 January 2022
2667-3940/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
).nc-nd/4.0/

S. Mohapatra et al. Advances in Cancer Biology - Metastasis 4 (2022) 100027

Abbreviations branches spreading; leaves 3.5-8.5x2-4cm, crowded at apex; flowers in
elongate, terminal and panicle racemes, white, rachis glandular; capsules
DMEM Dulbecco's Modified Eagle Medium with 5 furrows. The voucher specimens were collected from different
DMSO Dimethyl sulphoxide localities in around Baripada town, Odisha (Field nos. NOU -1539, 1557,
EDTA Ethylene diamine tetra acetic acid 2465 and 3678) and live plants are preserved in Botanical Garden, North
EGFR Epidermal Growth Factor Receptor Orissa University.
FBS Fetal Bovine Serum
ITS Insulin Transferrin Selenium The root samples were excised randomly from plants collected from
MTT 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium eight different sites adjacent to Baripada, Odisha, India. The roots were
bromide thoroughly cleaned with tap water, air-dried in shade for 10 days fol-
PBS Phosphate Buffer Saline lowed by oven drying at 60 C for further 2 days before pulverization.
PI Propidium iodide The powder form of root (500 gm) was subjected to Soxhlet extraction
PS Penicillin-Streptomycin successively with petroleum ether and hexane for defeating and then,
extracted with ethanol for 72 h. The ethanol was then removed under
process. Such isolated phytocompounds are isothiocyanates, indoles, vacuum using a rotary evaporator to obtain ethanol extracts of the root of
flavonoids, isoflavonoids, dithiolthiones, coumarines, isoprenoids and P. zeylanica (ERPZ) and, stored in cryovial at 4 C for further use. For the
organosulphides etc. [9]. study, ERPZ was dissolved in dimethyl sulfoxide (DMSO) and stored at
-20 C for further experimental use.
In this study, we evaluated the anticancer properties of Plumbago
zeylanica L in HeLa cervical cancer cells, which is a medicinal herb used 2.3. Cell culture
for treatment in several diseases. The bioactivities include leishmanici-
dal, anti-inflammatory, trypanocidal, antimalarial, antiviral, anticarci- HeLa cells were purchased from National Centre for Cell Sciences
nogenic, antibacterial, antifertility, anticandidal, anti-allergic activities (NCCS), Pune, India and were cultured in DMEM (Dulbecco's modified
in the Indian traditional system of medicine [10]. The roots of the herb Eagle's medium) with 10% fetal bovine serum and 1% Penicillin Strep-
are also being used by tribal people of India for termination of pregnancy tomycin in cell culture. The cells were cultured in an incubator with 5%
at an early stage. In Ayurveda, the root of P. zeylanica or Chitrak is CO2 at 37 C in a humidified atmosphere. The cells were grown to about
commonly used in the treatment of cancer [11]. The important active 70–80% confluence and then were sub cultured in a 10 cm plate.
compound Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a
naphthoquinone compound isolated from roots of P.zeylanica L., has a lot 2.4. Determination of cytotoxicity by MTT assay
of pharmacological properties including antidiabetic and anti-cancer
[12]. Plumbagin induces cell death through a copper-redox cycle The cytotoxicity effect of ethanol extracts of the root of P. zeylanica
mechanism in Human skin carcinoma A-431 cells [13]. Plumbagin in- (ERPZ) was determined by 3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphe-
duces cell death in BG1 ovarian cancer cells which show superior anti- nyltetrazolium bromide (MTT) assay in HeLa cells [17]. In brief, the cells
cancer activities than known chemotherapeutic agents like doxorubicin, were seeded with a density of 2000 cells/well in a 96 well plate. After 24
tamoxifen, cisplatin [14]. Plumbagin also inhibits tumor angiogenesis h, the cells were treated with various concentrations of ERPZ prepared in
and tumor growth through the Ras signaling pathway by activation of the DMSO and solubilized in DMEM for 72 h. The photographs of each well
VEGF receptor-2 [15]. P. zeylanica exhibits proapoptotic and were taken. For the MTT assay, the cells were incubated with 50 μl MTT
growth-inhibitory effects on human gastric cancer cells via suppression of (5 mg/ml) solution for 4 h. Then the MTT solution was replaced with
signal transducer and activator of transcription 3 and protein kinase B DMSO solution (80 μl) to solubilize the formazan crystals. Absorbance
[16]. Our study identified several bioactive compounds in the ethanol was measured at 595 nm using a microplate reader (Bio-Rad iMark
extract of the root of P. zeylanica through HRLCMS analysis and also Microplate Absorbance Reader Version 1.02.01).
revealed that the ethanol extract is cytotoxic, downregulates EGFR
expression, inhibits cell migration, and arrests cell cycle at S-phase in 2.5. Wound healing assay
HeLa cervical cancer cells.
The effect of ethanol extracts of the root of P. zeylanica (ERPZ) on cell
2. Materials and methods migration was carried out using a wound-healing assay in HeLa cells.
Cells were maintained up to 90–100% confluence before scratched and
2.1. Reagents seeded in 12 well plates with a seeding density of 1 Â 105 cells per well
and allowed to adhere at 37 C with 5% CO2 for overnight. After 24 h, the
Dulbecco's Modified Eagle Medium (DMEM Himedia, India), Fetal plate was taken out and artificial wounds were created in the monolayer
Bovine Serum (FBS, Cell clone, USA), Penicillin-Streptomycin (Himedia, using a 200 μl pipette tip. Any cellular debris created from the scratch
India), Phosphate Buffer Saline (PBS), Propidium Iodide (PI, Sigma), ITS- was removed by gently washing the wells with PBS. The cells were then
Insulin Transferrin Selenium (Gibco,USA), 0.25% Trypsin (Cell clone, treated with 10 and 20 μg/ml of various concentrations for 48 h. DMEM
USA), Trypan Blue (SRL, India), MTT-3-(4, 5- dimethylthiazol-2-yl)-2, 5- containing the desired concentration of DMSO was used as the control.
diphenyltetrazolium bromide (Himedia, India), DMSO-Dimethyl sulph- The photographs of the scratch were taken using a digital camera
oxide (Merck), 96,12 well plate (Nest, Tarsons, India), Primary Antibody mounted on an inverted microscope at the time of wounding and also
(EGFR Santacruz Biotechnology, USA), Secondary antibody (Goat anti- after 48 h of the treatment. Open areas not containing cells after scratch
rabbit, Santacruz Biotechnology, USA), Ethanol (Merck), EDTA- were analyzed using the software TScratch [18]. The migration rate is
Ethylenediaminetetraacetic acid (Fisher scientific)are the main re- expressed as the percentage of area reduction of the wound, which in-
agents used in the study. creases as cells migrate over time [19].

2.2. Preparation of plant extracts 2.6. Cell cycle analysis by flow cytometry

P. zeylanica L, a perennial erect, under shrubs, up to 1.5 m high, The sub-confluent cells (4 Â 105 no. of cells) were incubated in 6 mm
cluster plates for 24 h, and then treated with both carrier and ethanol
extracts of the root of P. zeylanica (ERPZ) (10 and 20 μg/ml) for 24 h
[20]. The cells were harvested with trypsin/EDTA, centrifuged to pellet

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S. Mohapatra et al. Advances in Cancer Biology - Metastasis 4 (2022) 100027

down for obtaining the pellets. 75% of ethanol was added to the cells for 3. Results
preservation. The cells were finally resuspended in propidium iodide (PI)
solution (50 μl/ml) and RNAase (10 mg/ml) and incubated for 30 min at 3.1. Cytotoxic effect of ERPZ on HeLa cervical cancer cells
25 C. Finally, the distribution of cells in various phases of the cell cycle
was estimated using flow cytometry of a population of 10,000 cells (BD HeLa cells were seeded in 96 well cluster plates at a density of 2 Â
Accuri C6 plus, US). 103 cells/well. Cells were treated with ERPZ (5, 10, 20, 25, 30 μg/ml) for
72 h. The dose-dependent cytotoxic effect was observed. Photographs
2.7. Immunocytochemistry were taken in an inverted microscope attached with a camera (Helmut
Hund, Gmbh, Wilovert Standard 30, Germany). The morphology of the
Cells were grown on poly-L-lysine coated coverslips and treated with HeLa cells was altered with the irregularity of shape, cellular detach-
ethanol extracts of the root of P. zeylanica (ERPZ) for 24 h. The cells were ment, and a bright circle around the nucleus (Fig. 1A). The cell viability
fixed by 4% paraformaldehyde in PBS for overnight at 4 C, per- was determined by MTT based assay. The data obtained from three trails
meabilized using 0.25% Triton X-100, washed twice with ice-cold PBS, with triplicate wells were converted to % control, the nonlinear regres-
blocked with 1% BSA in PBST for 30 min [21]. Then the cells were sion analysis was carried out using GraphPad prism program (Version
incubated with primary antibody (1:300) overnight at 4 C. The next day 6.0) and the IC50 value was calculated and indicated in the graph
the cells were washed thrice with PBS and incubated with diluted sec- (Fig. 1B). Results were expressed as the mean Æ standard error.
ondary antibody (1:300) for 2 h in dark. The nuclei were stained with
0.1–1 mg/ml Hoechst (Sigma). Images were taken using an Inverted 3.2. Inhibition of cell migration and invasion by ERPZ
Fluorescence Microscope both at UV and 488 nm wavelengths separately.
The intensity was measured using the ImageJ program (NIH, USA). Cell migration was checked using a wound-healing assay for both
carriers (DMSO) and ethanol extracts of the root of P. zeylanica (ERPZ)
2.8. HRLC-MS analysis of ethanol extracts of the root of P. zeylanica treated cells. Images were taken at 0, 24, and 48 h. In carrier treated cells,
(ERPZ) the wound size was reduced to 70% but in ethanol extracts of the root of
P. zeylanica (ERPZ) (10 and 20 μg/ml) wound size reduced to15 and 33%,
The High-Resolution Liquid chromatography-Mass spectroscopy respectively (Fig. 2A). The results demonstrated that ERPZ significantly
(HRLC-MS) analysis was carried out by passing the sample through a ((p < 0.05) suppressed the migration of HeLa cells in a dose-dependent
reverse-phase column (Q-Exactive Plus Biopharma, Thermo Scientific) manner (Fig. 2B) and this is due to the presence of bioactive com-
attached to mass spectrometer (Q Exactive plus-orbitrap MS) [22]. The pounds in ERPZ.
compounds were separated by gradient elution using water and aceto-
nitrile, each containing 0.1% formic acid in Milli-Q water, at a flow rate 3.3. ERPZ induces S-phase cell cycle arrest in HeLa cervical cancer cells
of 0.2 ml/min, and were characterized by a conventional method. The
MS conditions were maintained as follows: time 30 min, polarity posi- The distribution of various phases of cell cycle both in carrier and
tive, resolution 17,500. Mass spectra were analyzed using Exactive ERPZ treated HeLa cells were analyzed using propidium iodide (PI)
Plus-Orbitrap MS software (Thermo Scientific Xcalibur, Version staining and flow cytometry (Fig. 3A). In the carrier treated cells, the
4.2.28.14). population of different phases of the cells was G1 (79.4%), S (8.9%), and
G2/M (8.8%). Whereas the ERPZ treated (10 μg/ml) cells, the different
2.9. Statistical analysis phases of cells include G1 (14.5%), S (52.9%), and G2/M (16.8%) and, at
higher concentration (20 μg/ml) showed the population G1 (6.8%), S
The significance between the means of more than one data with P (68.9%), and G2/M (12.1%) (Fig. 3B). The finding clearly indicates that
values less than 0.05 was analyzed using ANOVA (Graphpad prism, USA) ERPZ inhibits cell cycle at S-phase. The % of cells with various phases of
Data was expressed as the mean Æ standard deviations. the cell cycle is represented in the graph (Fig. 3B).

3.4. Downregulation of EGFR expression by ERPZ on HeLa cells

EGFR expression in both carrier and ERPZ treated cells were analyzed

Fig. 1. Plumbago zeylanica root extract inhibit cell viability. A. Morphology of HeLa cells treated with various concentrations of P.zeylanica for 72 h. Repre-
sentative images of carrier, 5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml and 30 μg/ml are shown. B. Graph represents a dose dependent cytotoxic effect as determined by
MTT cell viability assay. The data is the mean from three independent experiments and expressed as the mean Æ standard error.

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S. Mohapatra et al. Advances in Cancer Biology - Metastasis 4 (2022) 100027

Fig. 2. Plumbago zeylanica root extract
inhibits cell migration. A. Wound healing
assay to determine HeLa cell migration
treated with P. zeylanica extract. Represen-
tative images of cells treated with 10 and 20
μg/ml for 24 and 48 h. The wound area of
carrier treated cells (between the dotted
lines) decreases significantly from 0 to 48 h
but in P. zeylanica extract treated cells both in
10 μg/ml and 20 μg/ml the wound area was
not altered significantly. B. Graph represents
the measurement of wound area for carrier,
10 μg/ml and 20 μg/ml treated cells at 0 h,
24 h and 48 h respectively. Statistical anal-
ysis was carried out using ANOVA and the p
< 0.05.

Fig. 3. Plumbago zeylanica root extract arrest cell cycle at S phase in HeLa cells.
A. Cell cycle analysis of HeLa cells treated with either carrier or P.zeylanica extract (10 μg/ml and 20 μg/ml) for 24 h and analyzed by flow cytometry using propidium
iodide staining. In the carrier treated cells majority of cells are in G1 phase while P. zeylanica treated cells majority of cells are arrested in S phase. B. Distribution of
HeLa cells at various phases of cell cycle for both carrier and P. zeylanica treated cells.

by immunocytochemistry (Fig. 4A, upper panel). Hoechst 33342 was significantly reduced in ERPZ treated cells. Statistical analysis was car-
used to stain the nucleus (Fig. 4A, lower panel). Images were quantified ried out using Student's ‘t’ test comparing control vs. treatment with p-
using the Image J program and graphs were plotted using GraphPad value less than 0.05.
Prism (Version 6.0) (Fig. 4 B). The data shows the expression of EGFR is

Fig. 4. Down regulation of EGFR expression by Plumbago zeylanica root extraction HeLa cells. A. EGFR immunoflourscence analysis of P. zeylanica treated HeLa
cells. The nuclei are stained by Hoechst 33342. B. The images were quantified by using image J program, the EGFR expression significantly down regulated in
P. zeylanica treated cells. Statistical analysis was carried out by Graphpad prism using student's‘t’ test comparing control vs treatment with p value less than 0.05.

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Table 1
HRLC-MS analysis of ethanol extract of Plumbago zelynica.

Sl. RT Name of compound Molecular Molecular Bioactivities Reference
no. formula mass
129.07 Antioxidant and Jin et al., 2015
1 1.02 Nipecotic acid C6H11NO2 157.07 anticancer
2 2.73 Acetyl proline C7H11NO3 282.01 Dong et al., 2017
3 6.21 Methyl (2Z)-3-iodo-2-octenoate C9H15IO2 192.04 Antioxidant
4 7.11 7,8-dihydroxy-4-methylcoumarin C10H8O4 Liao et al., 2014
740.12 Antioxidant and Patel and Patel,
5 8.78 (4E)-1,1,1-trichloro-7-{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-6-({4- C28H36Cl3F9O4Si anticancer 2017
[(4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy]benzyl}oxy)-4-octen-3-one 288.02 Antioxidant and Rufer and
C7H13O10P 319.11 anticancer Kulling, 2017
6 9.07 3-deoxy-D-arabino-heptulosonic acid 7-phosphate C14H17N5O2S Antioxidant and
7 12.06 2-[(1,3-dimethyl-1H-pyrazol-4-yl)carbonyl]-N-(2-methoxyphenyl) 594.15 anticancer
C27H30O15
hydrazinecarbothioamide 608.13
8 12.26 4-(3,4-dihydroxyphenyl)-7-methoxy-2-oxo-2H-chromen-5-yl-6-O-[3,4- C28H32O15 416.04
C24H17BrO2 234.16
dihydroxy-4-(hydroxymethyl) tetrahydro-2-furanyl]hexopyranoside C15H24O3 127.06
9 13.69 Neodiosmin C6H9NO2 300.06
10 15.08 1,1'-[9-(4-bromobenzylidene)-9H-fluorene-2,7-diyl] diethanone C16H12O6
11 17.58 2-(8-hydroxy-4a,8-dimethyldecahydro-2-naphthalenyl)acrylic acid 300.06
12 20.19 Guvacine C16H12O6
13 20.37 Diosmetin 268.07
C16H12O4
14 20.81 Hispidulin

15 22.89 Formononetin

Fig. 5. HRLC–MS analysis of ethanol extract of Plumbago zeylanica
A. HRLC-MS analysis of ethanol extract of P.zeylanica B. Structure of identified compounds having antioxidant and anticancer activities.

3.5. HRLC-MS analysis of ERPZ for presence of bioactive compounds 9.07/min), 2-[(1,3-dimethyl-1H-pyrazol-4-yl)carbonyl]-N-(2-methoxy-
phenyl) hydrazine carbothioamide (RT-12.06/min), 4-(3,4-dihydrox-
The ERPZ was analyzed by HRLC-MS and compounds were identified yphenyl)-7-methoxy-2-oxo-2H-chromen-5-yl-6-O-[3,4-dihydroxy-4-
with the support of “Compound Discoverer 2.1” software. HRLC-MS (hydroxymethyl) tetrahydro-2-furanyl]hexopyranoside (RT-12.26/min),
chromatograms identified a total of 15 no. of compounds and were neodiosmin (RT-13.69/min), 1,1'-[9-(4-bromobenzylidene)-9H-fluo-
represented with peak number, retention time (RT), name of the com- rene-2,7-diyl] diethanone (RT-15.08/min), 2-(8-hydroxy-4a,8-dime-
pound, molecular formula, molecular mass and bioactivities. The iden- thyldecahydro-2-naphthalenyl)acrylic acid (RT-17.58/min), guvacine
tified compounds were nipecotic acid (RT-1.02/min), acetyl proline(RT- (RT-20.19/min), diosmetin (RT-20.37/min), hispidulin (RT-20.81/min),
2.73/min), methyl (2Z)-3-iodo-2-octenoate (RT-6.21/min), 7,8-dihy- and formononetin (RT-22.89/min) (Table 1; Fig. 5B).
droxy-4-methylcoumarin (RT-7.11/min), (4E)-1,1,1-trichloro-7-
{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-6-({4- 4. Discussion
[(4,4,5,5,6,6,7,7,7nonafluoroheptyl)oxy]benzyl}oxy)-4-octen-3-one
(RT-8.78/min), 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (RT- From time immemorial, the plants serve as an important source of

5

S. Mohapatra et al. Advances in Cancer Biology - Metastasis 4 (2022) 100027

therapeutics for the treatment of various diseases. The derivatives of PI3K/AKT signaling pathway [39]. We checked the role of P. zeylanica
potential medicinal plants are extremely useful in combination with root extract in the inhibition of cell migration through in vitro wound
modern medicines for the treatment of cancer. Moreover, herbal medi- healing assay. Our result reveals it inhibits cell migration in a dose
cines exhibit beneficial effects on cancer chemotherapy side effects [23]. dependent manner. This strongly suggests that active ingredients of
With the widespread phytotherapeutic uses for the treatment of cancer, P. zeylanica are potential candidates for cervical cancer treatment.
studies on the discovery of novel compounds having cytotoxic activities
gained momentum among the researchers. We have screened several 5. Conclusion
locally available medicinal plants for anticancer and anti -HPV activities.
In this report we demonstrated the anticancer activities of the ethanol Ethanol extract of P. zeylanica root inhibits proliferation and migra-
extract of root of P. zeylanica in HeLa cervical cancer cells. The root of this tion of HeLa cervical cancer cells. The extract also down regulates
plant has been reported to have potent hepatoprotective, expression of oncoproteins EGFR. The cell cycle analysis shows the
anti-inflammatory, anti-diabetic, anti-cancer and anti-hyperlipidemic P. zeylanica treated cells were arrested in the S phase. The HRLC-MS
activities. A methanol extract of P. zeylanica has been reported against analysis identified several active compounds. The cytotoxic effect of
many Human and agricultural pathogens [24]. P. zeylanica, commonly the root extract may be due to presence of potential bioactive compounds
known as “leadwort” which protective effect against along with their synergistic activities. Further work is being carried out
cyclophosphamide-induced genotoxicity and oxidative stress [25]. to explore the details of the molecular mechanisms associated with cell
Compounds from P. zeylanica have been shown to kill malarial vector death in response to the extract of P. zeylanica.
Anopheles stephensi Liston (Diptera: auicidae) [26]. The HRLC-MS anal-
ysis identified several bioactive compounds from the root extract of Ethical approval and consent to participate
P. zeylanica. Among the 15 no. of identified compounds, only five com-
pounds such as 7, 8-dihydroxy-4-methylcoumarin, neodiosmin [27], Not applicable.
diosmetin, hispidulin, and formononetin were found to possess antioxi-
dant and anticancer activities. 7, 8-dihydroxy-4-methylcoumarin deplete Human and animal rights
the generation of reactive oxygen species in ischemic brain injury and its
long alkyl chain improve anticancer activity in K562 (human chronic No animals/humans were used for studies that are the basis of this
myelogenous leukemia), LS180 (human colon adenocarcinoma) and research.
MCF-7 (human breast adenocarcinoma) cell [28,29]. Diosmetin, a
flavonoid, attenuated intracellular antioxidant effect in AAPH induced Consent for publication
oxidative stress by scavenging reactive oxygen species in human eryth-
rocytes. This effect was attributed to the presence of hydroxyl group and Not applicable.
conjugated double bond in the structure [30]. It possesses cytotoxicity
properties against various cancer cells including hepatocarcinoma, breast Availability of data and materials
and colorectal cancer. It is a potential p53 activator and performs an
anticancer effect by regulating cell cycle and cell proliferation in HepG2 The data that support the findings of this study are available from the
cells. It also inhibits tumor development and blocks tumor angiogenesis corresponding author, PRD, upon reasonable request.
in B16F10 melanoma cancer cells [31]. The Neodiosmin (5, 7, 3-
trihydroxy-40- methoxyflavone 7 β-neohesperidoside) is a flavones Funding
glycoside has been isolated and identified from the leaves of Citrus aur-
antium which has anti edematogenic activity [32]. Hispidulin, showed The study was supported by grants received from Department of
microsomal lipid peroxidation in in vitro and in vivo model and also in- Science and Technology, Govt.of Odisha, No. 5012/ST and Department
hibits the growth of gastric cancer cells through induced G1/S phase of Biotechnology Govt of India sponsored Ramalingaswami Re-entry
arrest and apoptosis in time- and concentration-dependent manners [33]. Scheme award no. BT/RLF/41/2012.
Formononetin, an O-methylated isoflavone, showed ability to scavenge
peroxyl radicals ORAC assay [34]. Formononetin is one of the bioactive Declaration of competing interest
isoflavones isolated from different plants e.g.Trifolium pretense, Glycine
max, Sophora flavescens, Pycnanthus angolensis and Astragalus mem- The authors declare that they have no known competing financial
branaceus. It is widely used as anticancer drug against several types of interests or personal relationships that could have appeared to influence
cancer such as breast, colon, glioma, osteosarcoma, multiple myeloma, the work reported in this paper.
adrenal medulla, nasopharyngeal, prostate, bladder, laryngeal, lung, and
cervical cancer. It shows apoptosis by inhibiting cell cycle at G0/G1 Acknowledgments
phase in ES2, OV90 and HepG2 cancerous cells. It down-regulates cyclin
D1 and arrests the cell cycle at G0/G1 phase in HCT-116 cells. It alters the The work was supported by funding from Ramalingaswami fellow-
expression of p21, cyclin A, and cyclin D and causes cell cycle arrest at G1 ship project, Department of Biotechnology, Government of India. The
phase of lung cancer. It can arrest cell cycle at the phase of G0/G1 by authors greatly acknowledge the service provided by the flow cytometry
reducing the expression of AKT/cyclin D1/CDK4 in PC-3 and DU145 cells facilities of the Institute of Life Sciences, Bhubaneswar and Sophisticated
[35]. Various plant-derived compounds as well as plant extract has been Analytical Instrument Facility (SAIF) IIT Bombay for HRLC-MS analysis
reported to alter cell cycle in cancer cells. The crude extract of Calystegia of plant extract.
soldanella arrests cell cycle in G1 and S phase in HepG2 cells [36]. Dorema
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7

Advances in Cancer Biology - Metastasis 4 (2022) 100029
Contents lists available at ScienceDirect

Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Ehrlich Ascites carcinoma mice model for studying liver inflammation
and fibrosis

Nirmala G. Sannappa Gowda a, Varsha D. Shiragannavar a, Samudyata C. Prabhuswamimath b,
Sunanda Tuladhar c,d, Saravana Babu Chidambaram c,d, Prasanna K. Santhekadur a,*

a Department of Biochemistry, Center of Excellence in Molecular Biology & Regenerative Medicine, JSS Medical College, JSS Academy of Higher Education and Research,
Mysore, India
b Department of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, 570 015, Karnataka, India
c Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru, India
d Centre for Experimental Pharmacology & Toxicology, Central Animal Facility, JSS Academy of Higher Education & Research, Mysuru, India

ARTICLE INFO ABSTRACT

Keywords: Hepatic inflammation and fibrosis are the most common pathological conditions of chronic liver diseases such as
Erhlich ascites carcinoma obesity associated non-alcoholic fatty liver disease (NAFLD), Alcohol associated liver disease (ALD), viral hepatitis
Hepatitis and which can further progress to Hepatocellular carcinoma (HCC). The role of hepatic angiogenesis in the
Tumor angiogenesis progression of inflammation, fibrosis, tumor development and metastasis are known from long time. However, the
Inflammation role of breast cancer associated tumor angiogenesis in liver inflammation and fibrosis is not well studied and still
Fibrosis elusive. Therefore, in this study, we established a mouse model (EAC) to study the role of breast cancer associated
tumor angiogenesis in liver inflammation and fibrosis. We used EAC cells to induce liquid tumor in Swiss albino
mice and studied their effect on liver functions. We noticed a considerable rise in body weight and liver weight in
EAC tumor bearing mice along with increased peritoneal neo-angiogenesis. Further, EAC tumor bearing mice
revealed the increased expression of liver enzymes and elevated glucose level which are involved in the cause of
liver inflammation, which is evident from our immunohistochemistry data. Further, we validated our in vivo data
with various bioinformatics tools and compared the expression of TNF-α and TGF-β with liver inflammation and
fibrosis and VEGF and MTDH with angiogenesis. Based on our multiapproach study, we suggest that EAC induced
tumor angiogenesis can be used as a suitable model to discover new and more potential therapeutic targets for the
treatment of inflammation associated hepatic injury, especially, in patients suffering from cancer associated
hepatitis.

1. Introduction numerous growth factors, inflammatory cytokines and also physiological
hypoxic condition [3]. Tumor angiogenesis is the development of new
The liver is the largest and one of the most vital organs in our body blood vessels to supply nutrients to rapidly dividing tumor cells, and it is
which acts as a master regulator organ for various biochemical and a critical component of tumor metastasis pathways in almost all cancers
physiological processes. The liver aids in various functions such as [4]. There are many signalling pathways involved in inflammation
macronutrient metabolism, lipid and glucose homeostasis, endocrine mediated liver injury and fibrosis. Most of these reports suggest that
control of various signalling pathways, xenobiotic metabolism and, it tumor angiogenesis is one of the hallmarks of cancer which is involved in
also regulates the immune system and its function [1]. Angiogenesis is the hepatic inflammation and fibrosis in chronic liver diseases and
one of the fundamental physiological and pathological processes further aids in the development of hepatocellular carcinoma (HCC) [5].
involved in the promotion of tumorigenesis as well as in progression and However, the understanding of the association between tumor angio-
metastasis as ascertained by various convincing reports [2]. Angiogenesis genesis and hepatitis is still unclear. Consequently, a comprehensive
is a highly complex physiological process and tightly regulated by understanding of the association between tumor angiogenesis and in-
flammatory conditions of the liver is essential for the identification of

* Corresponding author. Department of Biochemistry, Center of Excellence in Molecular Biology and Regenerative Medicine, JSS Medical College, JSS Academy of
Higher Education and Research, Sri Shivarathreeshwara Nagar, Mysore, 570015, Karnataka, India.

E-mail address: [email protected] (P.K. Santhekadur).

https://doi.org/10.1016/j.adcanc.2022.100029
Received 15 September 2021; Received in revised form 31 December 2021; Accepted 19 January 2022
Available online 23 January 2022
2667-3940/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
).nc-nd/4.0/

N.G. Sannappa Gowda et al. Advances in Cancer Biology - Metastasis 4 (2022) 100029

Abbreviations many inflammatory cytokines also play a prominent role in hepatic
inflammation and fibrosis [13]. Therefore, targeting this tumor angio-
AFLD Alcoholic Fatty Liver Disease genesis will be a promising therapeutic option to treat patients with
ALP Alkaline Phosphatase tumor associated hepatic inflammation and fibrosis [14].
ALT Alanine Aminotransferase
AST Aspartate Aminotransferase Breast cancer is among the one of the deadliest malignancies in
EAC- Erhlich Ascites Carcinoma women all over the world [15]. Various reports advocate that number of
GEPIA Gene Expression Profiling Interactive Analysis deaths in breast cancer patients are due to the adverse effects of liver
GOD-POD Glucose oxidase-peroxidase inflammation and fibrosis [16]. Exploration and elucidation of the mo-
HCC Hepatocellular Carcinoma lecular mechanism of tumor angiogenesis induced hepatitis in breast
HIF-1α Hypoxia-inducible factor 1-alpha cancer patients is an urgent need of the hour [17]. Therefore, in this
HSC Hepatic Stellate Cells investigation, we have tried to establish a functional and molecular
IHC Immunohistochemistry relationship between the development of liver inflammation and pro-
MTDH Metadherin gression of breast cancer by using EAC mouse model [18]. Erhlich Ascites
mRNA microRNA mammary carcinoma is a spontaneous murine mammary adenocarci-
NAFLD Non-alcoholic Fatty Liver Disease noma and it is one of the well-established models in cancer biology [19].
PIGF Placental Growth Factor However, the breast cancer metastasis to various other organs like the
TGF-β Tumor Growth Factor-beta heart, lung, brain and bone is well studied [20]. Based on the pre-existing
TNF-α Tumor Necrosis Factor-alpha concepts, here we have tried to reveal the basic mechanism and events
VEGF Vascular Endothelial Growth Factor associated with tumor associated angiogenesis and hepatitis.

new therapeutic strategies for tumor associated hepatitis [6,7]. Various reports including our own study suggested that angiogenesis
There are several growth factors and cytokines are involved in this is one of the key features of various cancers including HCC [21–23]. In
normal physiological condition, angiogenesis supplies nutrition and ox-
fatal illness associated malady [8]. Some of the landmark studies ygen as well as aids in movement of immune cells from one organ to other
revealed that hypoxia is also one of the important inducers which pro- also helps in healing of tissue damage and leading to the restoration of
motes the new capillaries growth by activating multiple pro-angiogenic damaged tissue during tissue homeostasis [24]. However, due to the
pathways which secrete various angiogenic growth factors including rapid EAC cell division in the peritoneal cavity increased overall tumor
well studied vascular endothelial growth factor (VEGF), placental growth cell number and tumor volume and which led to the hypoxic environ-
factor (PIGF), fibrosis associated transforming growth factor-beta ment in the adjacent micro environment. This, in turn, causes the release
(TGF-β) and inflammation associated tumor necrosis growth factor-α of numerous angiogenic growth factors and cytokines such as VEGF,
(TNF-α) via the master regulator of hypoxia called Hypoxia Inducible PIGF, TNF-α and TGF-β which activates endothelial cells [25,26]. These
Factor-1 alpha (HIF-1α), a well-established transcription factor [9–11]. factors were shown for their prominent role in HCC associated angio-
Moreover, there are studies which revealed the direct role of Metadherin genesis [27,28]. The current findings suggest that EAC tumor induced
(MTDH), a well-studied oncogene in tumor angiogenesis. However, in peritoneal angiogenesis and the newly formed capillaries may transport
detail molecular action and the mechanism of MTDH associated angio- the angiogenic and inflammatory markers from the peritoneal region to
genesis is still not clear [12]. Along with these angiogenic growth factors, the liver and potentially promoting the activation of liver associated
stellate cells, Kupffer cells and mast cells which may trigger inflammation
associated signalling [29]. Our previous reports suggests that, the pro-
gression of hepatic inflammation, fibrosis and HCC are critical for
angiogenesis pathways [30].

Fig. 1. EAC tumor transplantation bearing mice develops hepatitis features. A) Schematic representation of the model proposed to study the breast cancer
induced hepatitis. B) Picture representing the development of EAC liquid tumor in EAC mice. C) Graph representing the change in the body weight of EAC tumor
bearing mice and control mice. n ¼ 5 mice per group. D) Graph illustrate the change in liver weight in EAC tumor bearing and control mice. n ¼ 5 mice per group. E)
Representative images show the change in liver weight. n ¼ 5 mice per group.

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Fig. 2. EAC tumor cells induces increase in serum liver enzymes and glucose level. (A–C) Graph showing the changes in serum ALT, AST, ALP levels in EAC
tumor bearing mice. n ¼ 5 mice per group. D) Graph representing the changes in blood glucose level in EAC tumor bearing mice and control mice. n ¼ 5 mice
per group.

Fig. 3. EAC tumor cells induces peritoneal angiogenesis in mice. A) Graph representing the change in total ascites volume till 13th day. B) Representative images
and comparisons of the peritoneal tumor blood vessels in EAC implanted mice model. n ¼ 5 mice per group.

Therefore, this novel in vivo study sheds new light on the role of tumor prior to all the in vivo experiments. All the animals were housed in
angiogenesis in hepatitis using EAC mouse model. EACs secrete various polypropylene cages (5–6/cage) and maintained at 25 Æ 3 C, the relative
growth factors and cytokines to the blood stream and these secreted humidity of the room was 45–55% with 12 h light/12 h dark cycle
angiogenesis and inflammation associated markers activate hepatic (artificial photoperiod). The animals were fed with rodent chow diet
inflammation and fibrosis [31]. These angiogenic and inflammatory were procured from M/s. Adita Biosys and mineral water ad libitum.
factors mainly act through their cognate and specific receptors present on
the liver [32]. In addition to hepatocytes and hepatic stellate cells 2.2. Transplantation of tumor
(HSCs), mast cells also may get activated by damage related stimuli
which promote the causes of hepatic inflammation and fibrosis and Tumor transplantation in animals was performed as described else-
finally leading to chronic liver diseases and HCC [33]. Therefore, our where [35]. A total 1.5 million (100 μl) EAC cells were injected to
experimental evidence-based study shows that the EAC induces the peritoneal cavity of each mouse. Following EAC cells inoculation it took
hepatitis in mice by secreting various growth factors and cytokines [34]. 3–5 days for tumor development and to show the significant body weight
differences. So, the body weight of all the animals was measured from the
2. Methods and materials 5th day of tumor inoculation. The weight measurement was carried out
every alternative day for a period of 13 days. Non-tumor bearing control
2.1. Animals mice (without EAC) and tumor bearing (EAC) mice were sacrificed after
13th day and all the livers were collected, weighed and preserved in
Animal experimental protocol was approved by the Institutional formalin buffer and processed for histopathological analysis. Blood was
Animal Ethics Committee (JSSAHER/CPT/IAEC/012/202), JSS Medical collected through retro orbital puncture (under ketamine anaesthesia)
College, JSS AHER, Mysore. Female Swiss albino mice (18–22 g) were and processed for biochemical investigations.
procured from M/s. Adita Biosys, Tumakuru, and acclimatized for 7 days

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Fig. 4. Human breast cancer patients express increased level of angiogenesis and inflammatory markers. A) Analyse the expression of TNF-alpha in the BRCA
gene profile, and expression was significantly higher in breast cancer (1.17 (Transcripts Per Million (TPM)) than in normal (0.54 (Transcripts Per Million (TPM)). B)
Analyse the expression of TNF-alpha in the BRCA gene profile and expression was significantly higher in breast cancer (27.89 (Transcripts Per Million (TPM)) than in
normal (23.06 (Transcripts Per Million (TPM)). Data summary images were obtained from human protein atlas via the following links (http://gepia.cancer-pku.cn/de
tail.php?gene¼TNF) and (http://gepia.cancer-pku.cn/detail.php?gene¼TGFþbeta).

2.3. Biochemical estimation expression pattern using GEPIA (Gene Expression Profiling Interactive
Analysis) and human protein atlas tools as previously described [36,37].
Serum AST, ALT, and ALP activity were measured and evaluated The GEPIA, http://gepia.cancer-pku.cn/is an informational net server for
using a semi-autoanalyzer according to the manufacturer's procedure normal and cancer gene expression profiling and interactive analysis.
(Agappe diagnostics Ltd, India). Serum glucose level was determined by With the help of these databases, we also compared with the some of the
conventional GOD-POD method. gene (TNF-α and TGF-β) expression between breast cancer tissue and
their adjacent normal counterpart tissue. The human protein atlas is one
2.4. Histopathological estimation of the openly available databases at https://www.proteinatlas.org/. It
provides easily accessible immunohistochemistry data for general public.
Control mice and EAC bearing mice were sacrificed after 13th day of By using these databases, we analysed the VEGF and MTDH genes
injection, a portion of collected liver tissues were fixed immediately in a expression in breast cancer patients using Immunohistochemistry (IHC)
fixative (neutral buffered formalin) and stored at room temperature until data.
further process. Five micrometre thick section of liver tissue were
embedded in paraffin blocks and were used for further analysis. Hae- 2.6. Data analysis
matoxylin and Eosin staining was performed to assess the inflammation.
Trichome Masson's staining was conducted to visualise fibrosis in liver All the experimental data were analysed and graphs were plotted
tissue. using a statistical software (graph pad prism version 5.0). Data analysis
was performed by two-way ANOVA, where * signifies p value less than
2.5. Bio informatics analysis 0.05. The microscopic images were taken from stereo zoom microscope
with CCD Olympus camera (cellSens Dimension 1.12).
We explored various liver inflammation and fibrosis associated gene

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Fig. 5. Human breast cancer patients express increased level of angiogenesis and inflammatory markers. A) VEGF protein expression were significantly
increased in breast cancer patients compared to normal. Data summary images were obtained from human protein atlas via the following links
https://www.proteinatlas.org/ENSG00000112715-VEGFA/pathology/breastþcancer#img (Breast cancer tissue) and https://www.proteinatlas.org/
ENSG00000112715-VEGFA/tissue/breast#img (Normal tissue) B) MTDH protein expression were significantly increased in breast cancer patients compared to
normal. Data summary images were obtained from human protein atlas via the following links https://www.proteinatlas.org/ENSG00000147649-MTDH/path
ology/liverþcancer#img (Breast cancer tissue) and https://www.proteinatlas.org/ENSG00000147649-MTDH/tissue/breast#img (Normal tissue).

3. Results elevation of these enzymes in serum of EAC mice. Biochemical estimation
showed a significant increase in the enzyme ALT (P < 0.0001), AST (P <
3.1. EAC tumor transplantation bearing mice develops hepatitis features 0.0001), ALP (P < 0.0005) levels in serum of EAC bearing mice as
compared control mice (Fig. 2A–C). Collectively our findings suggest that
In this analysis, we explored the association between EAC tumor EAC tumor burden increased the secretion of liver enzymes as well as
growth and liver physiology. We were interested to determine the glucose (P < 0.0002) level as compared to control mice (Fig. 2 D).
pathophysiological effects of EAC tumor cells on the liver. We used EAC
cells to induce tumor in Swiss albino mice and further, we checked the 3.3. EAC tumor cells induces peritoneal angiogenesis in mice
comorbid effects of these cancer cells on the liver function [38]. From the
day of injection, we alternatively measured the body weight and found Since it is very well established and known that tumor-bearing EAC
that there was a slight and moderate increase in the body weight of EAC mice shows higher expression of angiogenesis associated markers and
bearing mice when compared with non-EAC-bearing control mice. (Fig. 1 our experimental evidence showed significant increase in liver enzymes.
A-C). Further, we validated whether there are any changes in the liver To further validate our findings, we observed the peritoneal angiogenesis
weight in tumor bearing mice compared to non-EAC-bearing mice (Fig. 1 of EAC and non EAC bearing mice [Fig. 3 B].
D-E). In that contrast tumor bearing, mice exhibited a significant (P <
0.0001) increase in the body weight as well as the liver weight (P < 3.4. Human breast cancer patients express increased level of angiogenesis
0.0065) and it is one of the important early characteristic features of liver and inflammation markers
inflammation (as in NAFLD, AFLD). We also used ImageJ software to
evaluate the length of the liver in EAC-bearing mice and non-EAC bearing Our bioinformatics analysis of human breast cancer patients tissue
mice (Supplementary Fig. 1). To analyse the volume of tumor and ascites, array and mRNA expression using online GEPIA tool and Human Protein
and to count the total number of tumor cells, we collected the ascites Atlas showed significantly increased expression of TNF-α, TGF-β, VEGF
from EAC-bearing mice. We measured the total ascites volume as well and MTDH in human breast cancer tissue samples in comparison with
calculated the total number of cells at the end of the thirteenth day by healthy counterparts [Figs. 4 and 5].
using haemocytometer (Fig. 3 A). We compared the EAC tumor pro-
gression with that of liver inflammation. These data suggest that EAC 3.5. EAC tumor cells induces hepatic inflammation and fibrosis in liver
tumor can affect the functions of the liver by increasing the body weight
and liver weight when compared with normal physiological mice. Here we evaluated that whether the EAC tumor cell induces inflam-
mation and fibrotic changes in liver tissue of Swiss albino mice. So, we
3.2. EAC tumor cells induces increase in serum liver enzymes and glucose performed haematoxylin and eosin (H&E) staining and we found the
level inflammatory cells infiltration in the liver tissue section of EAC tumor
bearing mice but not in the liver of control mice [Fig. 6A]. Further, we
Elevation of liver enzymes such as alanine transfereases (ALT), performed Masson's trichome staining to evaluate liver fibrosis and we
aspartate transaminase (AST), alkaline phosphatase (ALP) is very com- found out that intense level of collagen fibres formation in liver tissue
mon in liver diseases and has been reported in liver dysfunction condi- section of EAC mice compared to non EAC mice [Fig. 6B].
tion [39,40]. Therefore, we got an interest to test whether there is an

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Fig. 6. EAC tumor cells induces hepatic inflammation and fibrosis in liver. A) Representative histological images of liver, following haematoxylin and eosin
staining of EAC tumor bearing mice and control mice demonstrating inflammatory cells infiltration. n ¼ 5 mice per groups. B) Liver sections of EAC tumor bearing and
control mice stained with Masson's trichome stain showing liver fibrosis. n ¼ 5 mice per groups.

Fig. 7. Schematic representation of the mechanism of breast cancer induced inflammation and fibrosis through angiogenesis. Angiogenic and inflammatory
growth factors and cytokines like VEGF, MTDH, TNF-α, and TGF-β are released by breast cancer primary tumor cells into the circulation and binds to their specific
receptors on the liver. These angiogenic growth factors activates hepatic stellate cells, Kupffer cells and mast cells resulting in hepatic inflammation and fibrosis.

4. Discussion hepatitis using EAC mouse model. Therefore, this model can be used to
explore the novel signalling mechanisms and it also sheds more light on
In humans and mouse models, breast cancer induced hepatitis may how cancer induces hepatitis and associated liver diseases.
cause changes in the body and liver weights [41,42]. In our EAC model,
we discovered that tumor-bearing mice have increased peritoneal In support of our results few other studies also have shown that EAC
angiogenesis. Based on this experimental observation we have described tumor metastasises to various organs and it might be due to the activation
the novel molecular mechanism involving the breast cancer induced of angiogenesis pathways and this might result in the cause of hepatic
inflammation and fibrosis by secreting various angiogenic growth factors

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