Therapeutic potential of antiviral drugs targeting chemorefractory colorectal adenocarcinoma cells overexpressing endogenous retroviral elements

Cell cultures and patient samples

HCT8 colon carcinoma cells employed in this study were obtained from the cell and
tumor bank of the University of Duisburg-Essen, Medical School. Mononuclear cells
(MNC) were isolated from whole blood using Ficoll (Sigma-Aldrich, Missouri, USA) gradient
following the manufacturer’s instructions. CD34+ cells were isolated using magnetic
bead kits (Milteny, Cologne, Germany) following the kit instructions.

Patient samples

Ethical considerations

This study was reviewed and approved by the Committee on Ethics of the Ruhr-University
of Bochum, Medical School (register numbers: 4042-11 and 5235-15). Written informed
consent was obtained from each participant. Informed written consent regarding eligible
subjects below 18 years was obtained from parents. Samples were anonymised, coded
and accessible only by research staff. All patient samples were gathered by the division
of visceral surgery, Marienhospital Herne, Germany. The histopathological of samples
were performed by the institute of pathology of the Ruhr-University of Bochum, Medical
School.

IC
₅₀
values and induction of etoposide resistance

IC
₅₀
values were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] proliferation assay as described previously, and reported as the mean of
three independent experiments. Briefly, cells in exponential growth phase were harvested,
washed with medium, and seeded in 96-well plates at appropriate densities according
to their growth kinetics. After a conditioning period of 24 hours, cells were exposed
to increasing concentrations of cytostatics for 72 hours. The cultures were then incubated
with MTT (Sigma-Aldrich, Munich, Germany) dissolved in PBS at a final concentration
of 1 mg/ml for 4 hours. Supernatants were aspirated and the purple formazan crystals
dissolved in 100 ?l of solubilization solution (10 % SDS in DMSO, Sigma-Aldrich, Munich,
Germany). The absorbance was measured in a microtiter plate reader (Infinite F200
Tecan, Berlin, Germany) at 570 nm. Both methods were formerly described 30], 31].

Resistance to etoposide in HCT8 cells was induced in the same form previously described
1], 30], 32]. Briefly, IC
₅₀
values for cytostatics were determined by MTT assay. Exponentially growing cells were
then exposed to 2× IC
₅₀
for 24 hours. For recovery, cells were washed and incubated with drug-free culture
medium until new colonies had formed. This procedure was repeated several times, each
time doubling the original IC
₅₀
until 64× IC
₅₀
was reached. The surviving cells were subjected to a resistance selection by incubation
with increasing concentrations of the respective drugs (16× to 512× IC
₅₀
) for 24 hours. Cells which proliferated at higher drug concentrations (128×) within
one week were considered chemotherapy refractory. Resistant colonies were then expanded
in the continuous presence of cytostatics and used for molecular-biological analysis,
in particular for studying the expression of CSC features. The resistance factor (RF)
was determined by MTT proliferation assay and reported as the IC
₅₀
iCSCs/ IC
₅₀
parental ratio. Using etoposide as chemoresistance-inducer it is feasible to induce
a wide HCT8 subpopulation of cells (HCT8
^RETO
) with cancer stem cell features (CSCs) in a very short time. HCT8
^WT/RETO
cells were cultured in DMEM medium (Biochrom, Berlin, Germany) containing 10 % heat-inactivated
fetal calf serum (FCS) and 15 ?g/ml Ciprobay (Bayer AG, Wuppertal, Germany).

Studies on the expression of human endogenous retrovirus elements (HERVs) in colorectal
carcinomas (CRCs)

We analyzed the expression of HERV-
_WE1
and HERV-
_FRD1
in patient samples as well in HCT8
^WT/RETO
colon carcinoma cell line using both immunocytochemical (ICC) and immunohistochemical
(IHC) staining.

ICC and IHC staining was performed according to standard protocols 1]. Briefly, ICC cells were grown in chamber slides to appropriate densities, washed
with 1× PBS, fixed with 4 % formaldehyde in PBS for 20 minutes, rinsed twice with
1× PBS for 5 minutes, and blocked with 10 % normal goat serum (AbD Serotec, London,
UK) at room temperature for 60 minutes. For IHC, tissue samples were fixed with 4 %
formaldehyde in PBS and embedded in paraffin. Paraffin tissue sections of 4 ?m thickness
were baked overnight at 60 °C to firmly attach the sections to the slides. After baking,
the sections were deparaffinized in 2 changes of xylene-substitute (Thermo Scientific,
London, UK) solution for 10-15 min and rehydrated in a series of graded ethanol solutions
(100 %, 100 %, 95 %, 70 %, 50 %) for 3 minutes each. HE staining was performed using
conventional techniques. For IHC, antigens were retrieved by heating the sections
for 30 minutes in 10 mM sodium citrate buffer pH 9.0 at 95 °C in a domestic vegetable
steamer. The slides were washed twice in 1 × PBS for 5 minutes and blocked for 60 minutes
with 10 % normal goat serum at room temperature. Primary antibodies (Bioss Antibodies,
Woburn, USA and Biorbyt, Cambridge, England) were applied overnight according to the
manufacturers’ recommendations. On the next day, the slides were washed 3 times in
PBST (PBS/0.05 % Tween 20) for 5 minutes each and rinsed in 1 × PBS for another 5 minutes.
Conjugated secondary antibodies (Cell signaling, Cambridge, UK) diluted in PBS/0.05 %
Tween 20/2.5 % goat serum were incubated for 120 minutes at room temperature according
to the manufacturers’ recommendations. Next, the samples were stained for 15 minutes
with 1 ?g/ml Hoechst 33258 diluted in PBS in order to visualize the nuclei. The slides
were then washed 3 times in PBST (PBS/0.05 % Tween 20) for 5 minutes each and rinsed
in 1 × PBS for another 5 minutes. Tissue specimens were mounted in Faramound Mounting
medium (Dako) for visualization.

Differential expression of HERV transcripts in HCT8
^WT/RETO
colon carcinoma cells

RNA purification and cDNA synthesis

Total RNA was extracted with Trizol® (Life Technologies, California, USA). To eliminate
genomic DNA contamination, the eluted RNA containing 10 IU RNase inhibitor was treated
with 7 Kunitz units of RNase-free DNase I (Qiagen, Hilden, Germany) in the appropriate
buffer and incubated at 25 °C for 20 minutes. The RNA samples were then purified further
on RNeasy mini columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
RNA integrity was ascertained by agarose gel electrophoresis and densitometric analysis.
1 ?g of pure and intact RNA was used for first-strand cDNA synthesis using the cDNA
Reverse Transcription Kit from Life Technologies, following the kit instructions.

qPCR

HERV expression was monitored by qPCR with validated primers and probes from Life
Technologies (Cat. Nr.: 18S Hs99999901_s1, HERV WE1 Hs01926764_u1, HERV-FRD1 Hs01942443_s1,
HERV3-1 Hs 04184598_s1 and HERV-V1 Hs00708335_s1), using the Taqman PCR core reagents
according to the manufacturer’s recommendations. In addition, the expression of these
HERV-elements was confirmed using specific primers purchased from Biomol (Hamburg,
Germany). The primers details are reflected in Table 1. The amplification of 25 ng of RNA was performed in triplicate in a CFX96TM Real-Time
System (Biorad Laboratories, California, USA). Results were analyzed with CFX-ManagerTM
Software Version 3.1 (Biorad Laboratories, California, USA). The evaluation of HERV
relative expression was determined using the Ct comparative method.

Table 1. Real Time PCR primers used for the detection of HERVs. The accession, region, sequence,
polarity and product size for the primers used are reflected

Analysis of the simultanean interaction of antiviral and cytostatic drugs

Amantadine, ribavirin, pleconaril, lamivudine, and doxorubicin were purchased from
Sigma-Aldrich, acyclovir and ganciclovir from HEXAL AG, Holzkirchen, Germany. Retrovir
was obtained from ViiV Healthcare, London, UK, Foscavir from Clinigen Healthcare,
Staffordshire, UK and brivudine from Berlin Chemie, Germany. Etoposide and cisplatin
were purchased from TEVA GmbH and 5FU from Medac, both Hamburg, Germany.

The simultaneous effect of antiviral drugs and cytostatics was analyzed by the isobologram
method (50 % isodose) as described previously 30]. Briefly, the IC
₅₀
for both substances were first determined using the MTT proliferation assay. Applying
fixed percentages of the IC
₅₀
for the first drug (20, 40, 60, 80 and 100 %) and varying the concentration of the
second drug from 0.1 to 50 ?M, the variation in the resulting IC
₅₀
was determined for every percentage. The same procedure was carried out inversely
for the second drug. Dose-response curves were then plotted and evaluated.

Protein isolation and Western blot analysis

To evaluate the direct effect of antiviral drugs on the expression of HERV proteins
we exposure HCT8 cells to amantadine, pleconaril and ribavirin alone or simultaneously
at 1-fold their respective IC
₅₀
-values for 24 hours.

3?×?10
⁶
HCT8
^WT/RETO
cells growing exponentially in 75 cm
²
TC flasks were incubated in medium containing the respective IC
₅₀
of amantadine, pleconaril, and ribavirin alone or with all drugs simultaneously for
24 hours. Medium was then removed and the cells washed twice with cold PBS. Protein
extraction was performed using RIPA buffer as previously described 1]. Briefly, pellets were lysed in RIPA buffer [150 mM NaCl, 1 mM EDTA, 1 % Triton X-100,
1 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris-HCl pH 7.4] in the presence of a proteinase
inhibitor cocktail according to the manufacturer’s instructions (Roche Diagnostics
GmbH, Mannheim, Germany) for 30 minutes on ice and then centrifuged for 20 minutes
at 14 000 g, 4 °C. The homogenates were measured for protein content using Bradford
and normalized to the same protein concentration. Protein extracts (30 ?g) were resolved
by SDS-PAGE in a 4–12 % gradient gel (Invitrogen, Karlsruhe, Germany) using Tris-glycine
(0.025 M Tris-HCl, 0.192 M glycine pH 8.5) buffer, and transferred overnight to 0.2 ?m
nitrocellulose membrane (Pierce Protein, Thermo Scientific Inc., MA, USA). Blots were
blocked with 5 % BSA or non-fat milk taking into consideration the recommendations
of the manufacturers of the primary and secondary antibodies. Primary antibodies were
purchased from Bioss Antibodies, Woburn, USA. Conjugated secondary antibodies were
obtained from Cell Signaling and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).
Immunoblots were developed by Western Lightning® Plus-ECL (Perkin Elmer, CA, USA)
using a ChemiDoc XRS+ system with Image Lab Version 2.0.1 software (Biorad, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

Differential HERV expression and its repression by antiviral drugs were monitored
using an indirect ELISA method. In brief, 96-well microtiter plates (Greiner Bio-One
GmbH, Frickenhausen, Germany) were coated with protein homogenates (5 ?g/100 ?l) overnight
at 4 °C. Well contents were aspirated and the wells washed 3 times with washing buffer
(PBS/0.05 % Tween 20). The wells were then incubated with 300 ?l blocking buffer [PBS/0.05 %
Tween 20/1 % bovine serum albumin (BSA)] each at 37 °C for 1 h and then washed 3 times.
Primary antibodies diluted 100 ?l in blocking buffer 1:500 were added, followed by
incubation at 37 °C for 1 h. The wells were aspirated and washed three times followed
by incubation with an HRP-conjugated secondary antibody (Sigma-Aldrich) in 100 ?l
at 37 °C for 1 h, dilution 1:2000. The wells were washed 3 times and incubated at
37 °C for 30 min with 100 ?l of fresh 0.4 mg/ml o-phenylenediamine and 0.4 mg/ml urea/H
₂
O
₂
dissolved in 0.05 M Na
₂
HPO
₄
/0.05 M citric acid adjusted to pH 5. The color reaction was stopped with 50 ?l of
1 M HCl per well, and the optical density measured after 1 h at 492 nm (OD
₄₉₂
) on an Infinite M200 microtiter plate reader (Tecan, Maennedorf, Switzerland). Results
were normalized using beta-actin as control and presented as percent of expression.

Statistical analysis

Experiments were performed at least in triplicate and the data given as means?±?standard
error of means (SEM), unless stated otherwise. Student’s t-test with four degrees
of freedom was used to compare independent groups. The statistical analyses were performed
with Sigma Plot 12 (Systat Software Inc., California, USA). A probability (p) value
was considered *: significant (p??0.05); **: very significant (p??0.01); ***: highly significant (p??0.001). ICC and IHC microscopy studies were descriptive and therefore not analyzed
statistically; the results shown are representative of at least n?=?3 independent experiments.