Recent studies have revealed the positive antiproliferative and cytotoxic effects of antiviral agents in cancer treatment. The real effect of adjuvant antiviral therapy is still controversial due to the lack of studies in biochemical mechanisms. Here, we studied the effect of the antiviral agent acyclovir on morphometric and migratory features of the MCF7 breast cancer cell line. Molecular levels of various proteins have also been examined.
These results are encouraging and demonstrate the possibility of partial suppression of cancer cell proliferation using an antiviral agent. Acyclovir antiviral agents have a great potential as an adjuvant therapy in the cancer treatment. However, more research is necessary to identify relevant biochemical mechanisms by which acyclovir induces a potent anti-cancer effect.
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Namba et al. demonstrated the use of zidovudine, an antiviral drug, in combination with gemcitabine, a chemotherapeutic agent - in an attempt to overcome a gemcitabine resistance for the pancreatic cancer treatment. In this type of malignancy, the gemcitabine resistance is associated with a decreased level of human equilibrative nucleoside transporter 1 (hENT1) and acquisition of epithelial-to-mesenchymal transition (EMT) - like phenotype. The zidovudine adjunct therapy was shown to reverse both events in this study [9]. Furthermore, authors demonstrated that activation of Akt-GSK3β-Snail mechanism, one of the major signaling pathways during gemcitabine resistance, is inhibited by zidovudine so that gemcitabine-resistant cancer cells were resensitized.
Although there is a plethora of evidence suggesting the beneficial effect of the antiviral agents in cancer treatment, the therapeutic benefit of their use in cancer treatment remains a grey area due to the lack of studies of the biochemical mechanisms. Antiviral agents such as acyclovir and ribavirin have been reported to have a suppressive effect on the proliferation and ability to increase an apoptosis in various cancers [7, 8]. Acyclovir was discovered 40 years ago and remains one of the main existing therapies for herpes simplex virus (HSV) infections. This drug is a highly potent inhibitor of this virus and commonly used for the treatment of the infections caused by the herpes viruses, CMV and EBV. It also has a low toxicity for the normal cells [10].
Based on our results, we conclude that ACV as an antiviral agent has a potential suppressive effect on MCF7 breast cancer cells. ACV does not affect viability of non-cancerous breast epithelial cells, while showing a decrease of the viability of MCF7 breast cancer cells. Observed morphological changes and apoptosis analysis demonstrated the ability of ACV to affect the process of programmed cell death of MCF7 cells. The mechanism of apoptosis requires a number of proteins that regulate a proper cell death. One of these proteins is caspase-3 which is included in a family of cysteine proteases [28]. An upregulated level of the apoptosis associated cytokine Caspase-3 was detected in ACV treated cells, correlating with the higher number of apoptotic cells and decreased rate of the cancer cell proliferation. Previously, it was reported that zidovudine treatment combined with a chemotherapeutic agent cisplatin has increased the apoptosis level of head and neck cancer cells [29]. This synergistic strategy of zidovudine and cisplatin was shown to trigger abnormal regulation of the mitochondria, increase of oxidative stress response and cause a significant cytotoxic effect on the cancer cells through the inhibition of a thiol metabolism [29]. Quantitative analysis revealed a moderate effect of acyclovir with a slight increase of the apoptotic cells.
A study by Curiel et al. reported that ACV had an inhibitory effect on one of the immune system components as T-regulatory cells (Treg) in glioblastomas through the suppression of indoleamine 2, 3-dioxygenase activity [35]. Another antiviral agent - ribavirin was also reported as an immune response inducer in the renal cell carcinoma lines through the downregulation of IL-10 expression and the upregulation of TGF-β expression [14]. The mechanisms by which ACV enables its anticancer effects might involve an enhanced immune response of the cancer cells and further study is required in this area.
There are several limitations in this study. All experiments were performed in vitro only on one cell line. Future research should focus on an adjuvant strategy of different antiviral agents to determine whether a combinatorial effect exists, and if so, which pathways are affected during the mechanism. Additionally, an examination of epigenetic modifications might serve as a platform for understanding the molecular mechanism underlying the antiviral therapy.
Twenty colorectal carcinoma patient samples were scrutinized for HERV-WE1 and HERV-FRD1 endoretroviruses using immunohistochemical approaches. In order to search for differential expression of these elements in chemotherapy refractory cells, a resistant HCT8 colon carcinoma subline was developed by serial etoposide exposure. Endoretroviral elements were detected by immunocytochemical staining, qPCR and ELISA. IC50-values of antiviral and cytostatic drugs in HCT8 cells were determined by MTT proliferation assay. The antivirals-cytostatics interaction was evaluated by the isobologram method.
In this work, we show for the first time that HERV-WE1, HERV-FRD1, HERV-31, and HERV-V1 are a) simultaneously expressed in treatment-naïve colon carcinoma cells and b) upregulated after cytostatic exposure, suggesting that these retroviral elements are intimately related to chemotherapy resistance. We found a number of antiviral drugs to have cytotoxic activity and the ability to force the downregulation of HERV proteins in vitro. We also demonstrate that the use of different antiviral compounds alone or in combination with anticancer agents results in a synergistic antiproliferative effect and downregulation of different endoretroviral elements in highly chemotherapy-resistant colorectal tumor cells.
Enhanced HERV-expression is associated with chemoresistance in colon carcinomas which can be overcome by antiviral drugs alone or in combination with anticancer drugs. Therefore, the introduction of antiviral compounds to the current chemotherapy regimens potentially improves patient outcomes.
Here, we show that cytostatic stress induces the development of highly resistant, HERV-overexpressing tumor cells. We determine the cytotoxic activity of different antiviral agents and highlight their capacity to shut down HERV expression. Finally, we demonstrate that the combination of antiviral compounds and antitumoral drugs reflects synergistic antiproliferative effects in highly resistant, HERV-overexpressing colorectal tumor cells.
The simultaneous effect of antiviral drugs and cytostatics was analyzed by the isobologram method (50 % isodose) as described previously [30]. Briefly, the IC50 for both substances were first determined using the MTT proliferation assay. Applying fixed percentages of the IC50 for the first drug (20, 40, 60, 80 and 100 %) and varying the concentration of the second drug from 0.1 to 50 μM, the variation in the resulting IC50 was determined for every percentage. The same procedure was carried out inversely for the second drug. Dose-response curves were then plotted and evaluated.
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 IC50-values for 24 hours.
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 μg/100 μl) 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 μl 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 μl 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 μl 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 μl of fresh 0.4 mg/ml o-phenylenediamine and 0.4 mg/ml urea/H2O2 dissolved in 0.05 M Na2HPO4/0.05 M citric acid adjusted to pH 5. The color reaction was stopped with 50 μl of 1 M HCl per well, and the optical density measured after 1 h at 492 nm (OD492) on an Infinite M200 microtiter plate reader (Tecan, Maennedorf, Switzerland). Results were normalized using beta-actin as control and presented as percent of expression.
To get a first hint on the antiproliferative activity of antiviral compounds in colon carcinoma cells, we analyzed antiviral drugs like amantadine, ribavirin, pleconaril, lamivudine, acyclovir, ganciclovir, AZT, foscavir and brivudine in HCT8WT/RETO cells. Of these, only three compounds showed some degree of antiproliferative effect, as defined by their IC50 values: amantadine, ribavirin and pleconaril (Fig. 4). All other drugs did not show detectable activity at concentrations up to 200 μg/ml (data not shown) and therefore were excluded from further analysis. In general, amantadine and pleconaril were the most active compounds in the HCT8RETO cells (Fig. 4).
We next questioned the potential interaction between antiviral compounds and classical, clinically used anticancer drugs in vitro. In this work results were only shown for amantadine, but similar data were also achieved for pleconaril and ribavirin, respectively.
In general, using the chemo-sensitive HCT8WT cell line (Fig. 5, panels a, c and e), interaction between amantadine and doxorubicin was slightly synergistic (panel a), between amantadine and cisplatin (CP) additive (panel c), and remarkable antagonistic activity was observed between amantadine and 5FU (panel e), respectively. In contrast, in the chemoresistant HCT8RETO cell line (Fig. 5, panels b, d and f), synergistic interaction between amantadine and all three anticancer drugs was shown, although synergism was less pronounced using 5-fluorouracil (5FU) (panel f). These results indicate that antiviral compounds like amantadine do not only exhibited substantial cytotoxic activity in chemoresistant tumor cells by itself, but also enhance the cytotoxic efficacy of anticancer drugs in vitro. 2ff7e9595c
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