- Research article
- Open Access
- Open Peer Review
High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model
- Anna Zajakina†1Email author,
- Jelena Vasilevska†1,
- Dmitry Zhulenkovs1,
- Dace Skrastina1,
- Artjoms Spaks2,
- Aiva Plotniece3 and
- Tatjana Kozlovska1
© Zajakina et al.; licensee BioMed Central Ltd. 2014
- Received: 23 December 2013
- Accepted: 17 June 2014
- Published: 20 June 2014
The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).
Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.
Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.
Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.
- Semliki Forest virus
- Cytotoxic effect
- Combined cancer treatment
- 4 T1 tumor
Several preclinical studies in recent years have demonstrated therapeutic synergy between viral vectors and chemotherapy [1, 2]. As reported previously, chemical compounds might be acting as adjuvants for the applied genetic vaccines  and/or could enhance the infectivity and gene transfer efficiency of the viral vector . Among the potential therapeutic viruses, alphaviral vectors are good candidates for cancer therapy because of the high level of transgene expression and their ability to mediate strong cytotoxic effects through the induction of p53-independent apoptosis [5, 6]. The advantages of alphaviral vectors also include a low specific immune response against the vector itself, the absence of vector pre-immunity and a high level of biosafety [7, 8].
Alphaviruses are enveloped viruses that belong to the Togaviridae family and contain a positive-strand RNA genome. The classic vectors for the expression of heterologous genes were developed primarily based on Semliki Forest virus (SFV) and Sindbis virus (SIN) replicons. In these vectors, a heterologous insert replaces the structural genes under the control of the 26S viral subgenomic promoter [9, 10]. The vector RNA can be packaged into recombinant alphaviral particles in cells via co-transfection with a helper RNA encoding structural genes (capsid and envelope). Upon infection, the vector RNA replicates and generates a high level of expression of the heterologous gene. The vector cannot propagate because it lacks the genes encoding the required viral structural proteins. Replication of the recombinant alphaviral genome, which occurs on the cytoplasmic membrane, causes cellular apoptosis, even in the absence of viral structural gene expression .
Due to the rapid induction of apoptosis in infected cells, treatment with natural oncolytic alphaviral vectors results in tumor regression [12–15]. Administration of replication-deficient vectors encoding reporter or immunomodulator genes, such as cytokines or growth factors, has also been demonstrated. This leads to successful tumor inhibition or complete regression in animal models [16–19]. Nevertheless, the application of alphaviral immunogene therapy in a clinical study using Venezuelan equine encephalitis (VEE) virus (VEE/CEA) in phase I/II demonstrated insufficient anti-tumor efficacy in patients, most likely due to the inefficient induction of anti-tumor immune responses in patients with end-stage disease . Moreover, the alphaviral vectors were administered to patients after standard treatment (usually chemotherapy), which may significantly reduce the efficiency of alphavirus infection and transgene expression. Remarkably, the majority of the successful preclinical studies using alphaviral vectors were performed in animal cancer models that did not involve pretreatment with chemical drugs. Therefore, the effect of combined chemotherapy and alphaviral therapy has not been comprehensively studied.
The efficacy of virotherapy depends on specific tumor targeting and the level of viral replication . It has been reported that the application of classical chemical drugs, e.g., 5-fluorouracil (5-FU) and gemcitabine, in combination with oncolytic herpes or adenoviral vectors make cancer cells more prone to virus infection and replication [4, 22], thereby enhancing the therapeutic effects of the viral vector. Alternatively, the viruses may improve the chemotherapy outcomes. For example, Newcastle disease virus has been shown to assist in overcoming cisplatin resistance in a lung cancer mouse model . Moreover, the use of herpes simplex virus following doxorubicin treatment was demonstrated to eradicate chemoresistant cancer stem cells in a murine breast cancer model . Also co-administration of reovirus with docetaxel synergistically enhanced chemotherapy in a human prostate cancer model , allowing reduced doses of chemotherapeutics to be used. Furthermore, the combination of an asymptomatic low dose of 5-FU with recombinant adenoviruses produces a synergistic effect in various cell lines and in vivo tumor models [26–30]. Although the detailed molecular mechanism underlying the therapeutic benefits of the combined treatment remains unknown, such a treatment has already demonstrated promising results in a clinical setting [31, 32].
Whether the synergistic anti-tumor effect can be achieved using a drug combination that includes alphaviral vectors has been poorly investigated. One study showed that application of a Sindbis vector with oncolytic properties in combination with the topoisomerase inhibitor irinotecan in SCID mice bearing human ovarian cancer resulted in prolonged animal survival . The authors highlight the role of natural killer cells in the induction of the anti-cancer effect by the combined treatment. Targeting of different anti-cancer mechanisms involving immune cell activation could lead to effective combinatorial therapies, though these would have to be evaluated in immunocompetent tumor models.
Using a 4 T1 mouse mammary tumor model, we investigated the efficiency of combined 5-FU and SFV vector treatment. We focused on the inhibition of cell proliferation and efficiency of transgene delivery under combined treatment in vitro and in vivo.
Cell lines and animals
BHK-21 (baby hamster kidney cells) and 4 T1 cells (metastasizing mammary carcinoma from BALB/c mice) were obtained from the American Type Culture Collection (ATCC/LGC Prochem, Boras, Sweden). BHK-21 cells were propagated in BHK - Glasgow MEM (GIBCO/Invitrogen, Paisley, UK) supplemented with 5% fetal bovine serum (FBS), 10% tryptose phosphate broth, 2 mM L-glutamine, 20 mM HEPES, streptomycin 100 mg ml−1 and penicillin 100 U ml−1. The 4 T1 cell line was cultured in Dulbecco’s minimal essential medium (GIBCO/Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, streptomycin 100 mg ml−1 and penicillin 100 U ml−1. Specific pathogen-free 4- to 6-week-old female BALB/c mice were obtained from Latvian Experimental Animal Laboratory of Riga Stradin’s University and maintained under pathogen-free conditions in accordance with the principles and guidelines of the Latvian and European Community laws. All experiments were approved by the local Animal Protection Ethical Committee of the Latvian Food and Veterinary Service (permission for animal experiments no. 32/23.12.2010).
Production of SFV (SFV/EGFP, SFV/DS-Red, SFV/EnhLuc) and SIN (SIN/EGFP) recombinant virus particles
The pSFV1  and pSinRep5  vectors were used in this study. The enhanced green fluorescent protein (EGFP) gene was introduced into both vectors under the 26S subgenomic promoter. The EGFP gene was cut out of the pEGFP-C1 plasmid (Clontech, CA, USA) with NheI and HpaI restriction endonucleases, treated with T4 DNA polymerase (Thermo Scientific, Lithuania) to blunt the DNA ends and ligated with the pSFV1 and pSinRep5 vectors, which were cleaved with SmaI and PmlI, respectively. Additionally, a pSFV1/DS-Red construct carrying the red fluorescent protein gene (DS-Red)  was generated. The DS-Red gene was amplified by PCR (primers: 5′-ATTAGGATCCACCGGTCGCCACCATG-3′ and 5′-TATCCCGGGCTACAGGAACAGGTGGTG-3′) using the pDsRed-Monomer-C1 plasmid as a template (Clontech, CA, USA). The PCR fragment was cleaved with BamHI and SmaI and ligated into a pSFV1 vector cleaved with the same enzymes. An SFV vector carrying the firefly luciferase gene was used for the in vivo experiments .
The resulting plasmids were used to produce recombinant virus particles as previously described . pSFV-Helper  and pSIN-DH-EB helper  were used to produce the SFV and SIN particles, respectively. The DNA template was removed by digestion with RNase-free DNase (Fermentas, Lithuania). The viral titers (infectious units per ml, iu ml−1) were quantified by infecting BHK-21 cells with serial dilutions of viral stock and analyzing EGFP or DS-Red expression via fluorescence microscopy on a Leica DM IL microscope (Leica Microsystems Wetzlar GmbH, Germany). For the in vivo application, SFV/EnhLuc viral particles (v.p.) were concentrated, and the viral titer was quantified by Real-time PCR as previously described .
Infection of cell lines with recombinant virus particles
Cells were cultivated in 24-well plates at a density of 2 × 105 cells per well in a humidified 5% CO2 incubator at 37°C. For transduction, the cells were washed twice with PBS containing Mg2+ and Ca2+ (Invitrogen, UK). Next, 0.3 ml of the solution containing the virus particles was added. The SFV/EGFP, SFV/DS-Red and SIN/EGFP virus particles were diluted in PBS (containing Mg2+ and Ca2+) to achieve a multiplicity of infection (MOI) of 10. The cells were incubated for 1 h in a humidified 5% CO2 incubator at 37°C. The control cells (uninfected) were incubated with PBS (containing Mg2+ and Ca2+). After incubation, the solution containing the virus was replaced with 0.5 ml of growth medium. The cells were gently washed with PBS and transferred to fresh medium every day.
MTT cell proliferation assay
The cytotoxicity was quantified using the MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based cell viability assay. Cells were infected in 24-well plates as described above, and proliferation was analyzed 0, 1, 2, 3, 4 and 5 days after infection. The medium was replaced with 0.3 ml of solution containing 0.5 mg ml−1 MTT (Affymetrix, Cleveland, USA) dissolved in D-MEM without phenol red (GIBCO/Invitrogen, UK) supplemented with 5% FBS. The cells were incubated for 2 h in a humidified 5% CO2 incubator at 37°C. After incubation, the formazan crystals were dissolved by adding 0.3 ml of MTT solubilization solution consisting of 10% Triton X-100 and 0.1 N HCl in anhydrous isopropanol. The absorbance was measured using a microplate spectrophotometer (BioTek Instruments, Winooski, USA) at a test wavelength of 570 nm and a reference wavelength of 620 nm. Cell viability (%) was obtained using the following equation: Percent cell viability = (test 570 nm – 620 nm)/(control 570 nm – 620 nm) × 100, where the control is the value obtained from uninfected cells (the standard error of the control was less than 3% for days 0–3 and less than 6% for days 4–5 in three independent experiments).
Fluorescence-activated cell sorting (FACS) analysis
Cells were infected on 6-well plates with SFV/EGFP and SIN/EGFP virus particles at an MOI of 10 as described above (1 ml of virus-containing solution was used for the infection). The infected cells were harvested 24 h after infection. Detached cells were harvested from the cell medium by centrifugation, and attached cells were trypsinized. The collected cells (approximately 106) were washed with PBS and resuspended in 1 ml of PBS. For propidium iodide (PI) staining, the cells were incubated with 10 μl of 50 μg ml−1 PI solution (Becton Dickinson Biosciences, San Jose, California, USA) and immediately processed for FACS analysis. EGFP and PI fluorescence was measured using a FACSAria II (Becton Dickinson Biosciences, San Jose, California, USA). The FACS data were analyzed by BD FACSDiva 6.1.2 software. Uninfected cells were used as a negative control for both the PI and EGFP FACS analysis and contained approximately 1-2% PI-positive cells in 4 T1 culture.
Fluorometry of infected/reinfected cells
Cells were seeded on 24-well plates and infected with SFV/EGFP as described above. After 24, 48 and 72 h, the infected cells were reinfected with the SFV/DS-Red virus. DS-Red fluorescence was measured 24 h after each reinfection using a fluorometric plate reader (Tecan Infinite M 200, Austria) with an excitation wavelength of 535 nm and an emission wavelength of 590 nm. The fluorometry data were expressed as the percentage of the reinfected cell fluorescence units relative to the fluorescence units obtained from the control cells infected with SFV/DS-Red alone (positive control, 100%). The experiments were performed in triplicate.
Treatment of cells with 5-FU
5-FU powder (Sigma, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 70 mg ml−1 and further diluted in filtered water to 7 mg ml−1. 4 T1 cells were seeded in a 24-well plate (2 × 105 cells per well). The next day, the cells were treated with medium containing 5-FU at 13, 26, 65 or 130 μg ml−1. Every day for 5 days, the cells were gently washed with PBS to remove dead and detached cells, and fresh medium containing 5-FU was added. The control cells were not treated with 5-FU. The MTT cell proliferation assay was performed 0, 1, 2, 3, 4 and 5 days after the start of 5-FU treatment. The presence of DMSO traces did not affect 4 T1 cell proliferation.
Induction of tumor nodules
A 4 T1 mouse mammary tumor model was established as previously described . Briefly, 4 T1 tumor cells were resuspended in PBS at a final concentration of 2.5 × 106 cells ml−1. Two hundred microliters of the 4 T1 cell suspension were subcutaneously injected above the right shoulder blade of the mice. After 10 days, the obtained tumor volumes reached at least 1000 mm3.
5-FU treatment and SFV/EnhLuc injection in vivo
5-FU powder (Sigma, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 300 mg ml−1 and then diluted in filtered water to 30 mg ml−1. 4 T1 tumor-bearing mice (n ≥ 5) were treated with 5-FU at different doses (40, 150 or 400 mg kg−1) via peroral administration 4 times over a period of 8 days (every other day). One hour after the last 5-FU treatment, the mice were inoculated either i.t. (intratumoral) or i.p. (intraperitoneal) with 200 μl (4 injections of approximately 50 μl each) or 300 μl of SFV1/EnhLuc particle-containing stocks (6 × 109 v.p. ml−1), respectively. As a control, 4 T1 tumor-bearing mice not treated with 5-FU were i.t or i.p. inoculated with the same dose and volume of SFV1/EnhLuc.
Analysis of luciferase gene expression in mouse organs and tumors
The Luc gene expression level was estimated by measuring luciferase enzymatic activity in tissue homogenates 24 h after SFV/EnhLuc virus administration. The tumors and organs were excised and manually homogenized in a 1x concentration of ice-cold lysis buffer (Cell Culture Lysis buffer, Promega) containing a protease inhibitor cocktail (10 μl per 1 ml of lysis buffer) (Sigma, St. Louis, MO, USA). After homogenization, the samples were centrifuged for 10 min at 9000 × g, and the protein concentration was determined in tissue lysates using the BCA Protein Assay Kit (Pierce™ BCA Protein Assay Kit, Thermo Scientific, UK). Luciferase activity was measured by adding 100 μl of freshly reconstituted luciferase assay buffer to 20 μl of the tissue homogenate (Luciferase Assay System, Promega, USA) and then was quantified as relative light units (RLUs) using a luminometer (Luminoskan Ascent, Thermo Scientific, UK). The RLU values were expressed per mg of protein in the lysates. As a negative control, 4 T1 tumor-bearing mice were inoculated with PBS, and the maximal negative values were subtracted from the presented results.
The efficacy index of the 5-FU and SFV combined treatment was calculated using the formula (RLU in 5-FU treated mice/RLU in 5-FU non-treated mice)/(tumor weight in 5-FU treated mice/tumor weight in 5-FU non-treated mice). For example: the efficacy index = (3497925.0/1397062.5)/(681.3/690.9) = 2.5. The efficacy index thus reflects the level of SFV expression (increase in RLU) and the effect of the 5-FU treatment (reduction in tumor weight).
Analysis of FITC-dextran accumulation
The first group of 4 T1 tumor-bearing mice (n = 3) was treated with 150 mg kg−1 5-FU as described above and the second group (n = 3) was untreated with 5-FU. Next day after the last 5-FU treatment the mice from both groups were inoculated i.v. with 120 μl of FITC-dextran 2000 kDa solution (40 mg ml−1 in PBS) (Sigma). Two hours later tumors were collected and incubated overnight in 4% paraformaldehyde. After cryoprotection in 20% sucrose tumors were frozen in OCT compound (Sigma). Cryosections (10 μm) were prepared and the intensity of FITC-dextran leakage was visualized by fluorescent microscopy. Pixels of images were measured by ImageJ software.
Analysis of IFN-alpha in tumor lysates
Two groups of 4 T1 tumor-bearing mice (n = 6 each) were either treated or non-treated with 150 mg kg−1 5-FU as described above. One hour after the last 5-FU treatment, three mice from each group (n = 3) were inoculated i.t. with 200 μl (4 injections of approximately 50 μl each) of SFV1/EnhLuc particle-containing stocks (6 × 109 v.p. ml−1). 18 hours after the virus administration, 4 T1 tumors were isolated and frozen in liquid nitrogen. Frozen tumors were manually homogenized with homogenization hammer and tissue powders were resuspended in 500 μl of PBS. To provide better tumors homogenization, two freeze-thaw cycles were performed. After homogenization, samples were centrifuged for 10 min at 5000 × g and the protein concentration was equalized in all tissue lysates using the BCA Protein Assay Kit (Pierce™ BCA Protein Assay Kit, Thermo Scientific, UK). Expression of IFN-alpha in 4 T1 lisates was determined using ELISA Kit for Interferon Alpha (Uscn Life Science Inc., China), according provided protocol. The obtained data (pg/ml) were expressed in % relative to lysates non-treated with both the 5-FU and the virus.
The cell viability and RLU results are presented as the means ± standard error of 3 independent experiments. The statistical analysis of the results was performed using Microsoft Excel and Statistica7 (StatSoft, Tulsa, OK, USA). Statistically significant differences were determined using Student’s t-test (P < 0.05).
Transduction efficiency and cytotoxicity of alphaviral vectors in 4 T1 cells
Repeated infections were next tested as a means of enhancing the infectivity and cytotoxicity of the alphavirus. Remarkably, repeated infection of surviving cell culture with the same or a different alphaviral vector (SFV or SIN, respectively) did not produce a significant enhancement of transgene production or prolongation of cytotoxicity. As shown in Figure 1b, the 4 T1 cell culture infected with SFV/EGFP were less susceptible to repeated infection with SFV/DS-Red particles encoding the DS-Red fluorescent protein . Only a very small number of EGFP-negative cells (which did not express the transgene after the first infection) were able to express the DS-Red gene, indicating that the cells could not be doubly infected by both alphaviruses. Similar results were obtained with the SIN vector and with other combinations of SFV/SIN and SIN/SFV reinfection (not shown). Moreover, an MTT cell viability analysis did not reveal a difference in the cell proliferation patterns of singly and doubly-infected cells (not shown). We conclude that the repeated application of alphaviral vectors is not an efficient strategy to achieve complete inhibition of cancer cell proliferation. This effect may be attributable to the overall cellular protein synthesis down regulation  and strong induction of an anti-viral response [36, 37] that makes the repeated application of the vector inefficient.
The SFV vector was selected for further cytotoxicity analysis in combination with 5-FU.
Combined treatment of 4 T1 cells with SFV and 5-FU
The low efficiency of oncolytic virotherapy in preclinical studies might be associated with anti-vector immunity or the resistance of tumors to repeated infections. Recently, multiple strategies involving the combination of oncolytic vectors with classic cytotoxic drugs have proven to be advantageous for certain types of cancer (for review, see Wennier et al. 2012) . Here, we analyzed whether the combination of the SFV alphaviral vector and 5-FU exerts a synergetic effect on cancer cell proliferation.
The notion that recombinant alphaviruses expressing, e.g., anti-tumor genes and/or inducing anti-tumor immune responses must be applied prior to chemical drug treatment is rational. Therefore, we first tested whether 5-FU could inhibit the proliferation of cells previously infected with SFV. As shown in Figure 2b, 4 T1 cells were infected with SFV/EGFP 2 days prior to treatment with 5-FU. The kinetics of 4 T1 cell proliferation in the combined treatment approach (SFV plus 5-FU) was similar to those of infected 4 T1 cells. The SFV infection of 4 T1 cells alone resulted in 55% of cell viability on day 5 after infection (Figure 1a, MTT-test, SFV). In the case of combined treatment, the cell viability was not significantly changed and resulted in 50% and 40% viability after treatment with 13 μg and 130 μg of 5-FU on day 5, respectively (Figure 2b). Therefore, the application of 5-FU after SFV did not significantly influence the survival of the 4 T1 cell culture, even at the high drug dose (130 μg ml−1), providing the evidence for infected cell culture resistance to further treatment with cytotoxic agent.
The effect of 5-FU treatment on SFV expression in 4 T1 tumor-bearing mice
Because the low dose improved transgene expression and had no signs of toxicity, this dose was used to evaluate the tumor targeting and biodistribution of SFV particles upon intraperitoneal (i.p. 1.8 × 109 v.p.) administration in combination with 5-FU. As presented in Figure 5c, the highest levels of Luc gene expression were detected in the tumors and hearts of mice treated with 40 mg kg−1 5-FU. Although significantly lower total Luc expression was observed with i.p. inoculation compared with the i.t. route, the Luc level in the tumors was still 2.1-fold higher (p < 0.05) in i.p. inoculated mice relative to 5-FU untreated mice. Among the other organs, only the heart showed an increase in Luc expression after 5-FU treatment (1.4-fold; not significant). Remarkably, there were no significant changes in vector biodistribution observed in the case of i.t. administration (not shown). The i.t. inoculation provided no further distribution of the vector to organs in both 5-FU treated and untreated mice, confirming therefore the enhancement of vector expression specifically in tumor of 5-FU treated animals.
One strategy to enhance cancer virotherapy is to apply viral vectors in combination with standard and well-studied chemical drugs to promote synergistic actions and potentially lead to effective therapy outcomes. Classic alphaviral vectors based on SFV and SIN replicons have been used for in vitro and in vivo cancer gene therapy experiments and have shown promising results in different cancer models [39, 40]. Nevertheless, the problems of tumor recovery and the inefficiency of repeated vector administration remain to be solved. In this study, we explored the efficiency of SFV-mediated gene transfer in combination with 5-FU and the possibility of a synergistic cytotoxic effect of the combined treatment in the highly proliferative 4 T1 mouse breast cancer model.
5-FU is an antitumor drug typically included in breast carcinoma chemotherapeutic regimens [41, 42]. The cytotoxic effect of 5-FU occurs through the inhibition of the synthesis and functioning of DNA and RNA. Although the general mechanism of 5-FU action as an anti-metabolite has been investigated , little is known about the intracellular molecular changes that lead to apoptosis in the presence of 5-FU. Protein kinase R (PKR) has been shown to be a molecular target of 5-FU-induced apoptosis , suggesting that 5-FU might induce apoptosis via a mechanism similar to that of alphaviruses: the double-stranded RNA intermediates made during alphavirus genome/subgenome replication also activate PKR, which contributes to the inhibition of protein synthesis . PKR has also been shown to play an important role in the induction of apoptosis by other drugs, such as doxorubicin and etoposide [46, 47], which have been successfully used in combination with other viruses [48, 49]. Therefore, the combined treatment with alphavirus and 5-FU presented herein could potentially produce a synergistic effect due to the targeting of similar pathways that may work together to enhance cytotoxicity in cancer cells. Nevertheless, this combined treatment showed poor efficiency in 4 T1 cells in vitro. Neither SFV infection with subsequent 5-FU treatment (Figure 2b) nor the opposite strategy of pretreatment with 5-FU and later infection with SFV (Figure 4) produced a more efficient inhibition of cell proliferation compared with SFV or 5-FU alone (Figures 1a and 2a). Moreover, pretreatment of cells with 5-FU significantly inhibited SFV infection and transgene expression (Figure 3).
The basis for the resistance of the surviving cell population to high 5-FU doses and SFV infection in the combined treatment remains unclear. Cabrele et al.  and others demonstrated stimulation of adenoviral vector infection via 2 h of low-dose pretreatment with 5-FU in human colon carcinoma cell lines. In contrast to adenoviruses, RNA containing alphaviruses replicate their genome in the cytoplasm. The extremely efficient alphaviral RNA replication is regulated by the virus-encoded replicase complex and the specific secondary structure of the RNA genome . As previously described, incorporation of 5-FU metabolites into RNA may change RNA structure and/or affect tRNA and rRNA function . It is thus possible that a similar incorporation of 5-FU metabolites into alphaviral genomic and subgenomic RNAs may likewise alter RNA secondary structure and inhibit its replication and translation. The presence of 5-FU and its metabolites could also inhibit the viral replicase in a similar manner to that observed in the inhibition of the active center of thymidylate synthetase by 5-fluorodeoxyuridine monophosphate . We conclude that this combined treatment produces no synergy in the induction of apoptosis but rather inhibits alphaviral replication and transgene production.
Several oncolytic viruses have been applied in combined treatments in mouse tumor models . However, less is known about the efficiency of infection or the kinetics of virus persistence under combined treatment in mice because most studies focused on the significant therapeutic effects and tumor growth inhibition. The fact that multiple different combinations of viruses (enveloped, unenveloped, dsDNA, RNA, ssDNA) and cytotoxic chemical drugs (antimetabolites, antibiotics) all produce synergistic therapeutic effects implies a common non-specific mechanism underlying such a benefit. Here, we observed a significant enhancement of intratumoral SFV-mediated transgene expression in mice treated with 5-FU (Figure 5). The low dose (suboptimal) of 5-FU provoked a 3.6-fold increase in Luc gene expression, whereas the high dose (400 mg kg−1, which is close to the maximum-tolerated dose of chemotherapy regimens) yielded a 50-fold increase. This positive correlation between 5-FU dose and the level of Luc expression contradicts the in vitro results; however, this correlation is in line with the promising results obtained using other viruses in combination with 5-FU in mouse models [52–54].
Besides the changes in tumor vascular permeability mediated by 5-FU treatment, an antiviral immune response has to be considered as a factor which affects the infection. At the early step of infection alphaviruses are sensitive to type I IFN production [60, 61]. We have examined the intratumoral level of IFN-alpha in 5-FU treated and untreated tumor bearing mice as a response to i.t. administrated SFV (Figure 6c). The results indicate a significant inhibition of IFN-alpha antiviral response in 5-FU treated tumors, evidencing the innate immunity inhibition by 5-FU that at the same time might lead to enhanced virus replication.
Therefore, we propose that pretreatment with a cytotoxic drug may enhance the efficiency of alphaviral-mediated transgene delivery through the EPR effect and the inhibition of antiviral IFN-alpha response. Here we have demonstrated a significant 3.6-50.0 fold increase in Luc transgene expression that can be regulated by 5-FU dose. Although we did not observe any differences in tumor growth and survival rates (not shown) between the groups of animals treated with 5-FU and treated with combination of 5-FU and SFV/EnhLuc, the observed enhancement of intratumoral virus expression mediated by 5-FU pretreatment has a potential to advance the alphavirus-driven transgene delivery field. The insertion of proinflammatory transgenes into the vector instead of reporter luc gene could be promising for further optimization of SFV-based virotherapy of cancer to enhance the effect of chemotherapy and to prevent tumor recurrence and metastasis.
Although the combined treatment did not show a synergistic anti-proliferative effect in vitro due to the strong inhibition of SFV replication by 5-FU, the significant increase observed in intratumoral SFV expression (even at a low drug dose) might enhance the transgene delivery of alphaviral vectors and their general therapeutic potential.
We thank I. Shestakova and her group at the Latvian Institute of Organic Synthesis for useful suggestions during the work. We also acknowledge Prof. P. Pumpens and his lab members for helpful discussions and excellent technical assistance. This study was supported by The Latvian National Research Program 2010–2013, “BIOMEDICINE” and bilateral Latvia-Belarus project 2014–2015.
- Wennier ST, Liu J, McFadden G: Bugs and drugs: oncolytic virotherapy in combination with chemotherapy. Curr Pharm Biotechnol. 2012, 13: 1817-1833.View ArticlePubMedPubMed CentralGoogle Scholar
- Naik JD, Twelves CJ, Selby PJ, Vile RG, Chester JD: Immune recruitment and therapeutic synergy: keys to optimizing oncolytic viral therapy?. Clin Cancer Res. 2011, 17: 4214-4224.View ArticlePubMedPubMed CentralGoogle Scholar
- Eralp Y, Wang X, Wang JP, Maughan MF, Polo JM, Lachman LB: Doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against HER 2/neu in a murine mammary carcinoma model. Breast Cancer Res. 2004, 6: R275-R283.View ArticlePubMedPubMed CentralGoogle Scholar
- Cabrele C, Vogel M, Piso P, Rentsch M, Schroder J, Jauch KW, Schlitt HJ, Beham A: 5-Fluorouracil-related enhancement of adenoviral infection is Coxsackievirus-adenovirus receptor independent and associated with morphological changes in lipid membranes. World J Gastroenterol. 2006, 12: 5168-5174.PubMedPubMed CentralGoogle Scholar
- Glasgow GM, McGee MM, Tarbatt CJ, Mooney DA, Sheahan BJ, Atkins GJ: The Semliki Forest virus vector induces p53-independent apoptosis. J Gen Virol. 1998, 79 (Pt 10): 2405-2410.View ArticlePubMedGoogle Scholar
- Venticinque L, Meruelo D: Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak. Mol Cancer. 2010, 9: 37-View ArticlePubMedPubMed CentralGoogle Scholar
- Lundstrom K: Alphaviruses in gene therapy. Viruses. 2009, 1: 13-25.View ArticlePubMedPubMed CentralGoogle Scholar
- Riezebos-Brilman A, De MA, Bungener L, Huckriede A, Wilschut J, Daemen T: Recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy. J Clin Virol. 2006, 35: 233-243.View ArticlePubMedGoogle Scholar
- Liljestrom P, Garoff H: A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y). 1991, 9: 1356-1361.View ArticleGoogle Scholar
- Bredenbeek PJ, Frolov I, Rice CM, Schlesinger S: Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol. 1993, 67: 6439-6446.PubMedPubMed CentralGoogle Scholar
- Urban C, Rheme C, Maerz S, Berg B, Pick R, Nitschke R, Borner C: Apoptosis induced by Semliki Forest virus is RNA replication dependent and mediated via Bak. Cell Death Differ. 2008, 15: 1396-1407.View ArticlePubMedGoogle Scholar
- Maatta AM, Makinen K, Ketola A, Liimatainen T, Yongabi FN, Vaha-Koskela M, Pirinen R, Rautsi O, Pellinen R, Hinkkanen A, Wahlfors J: Replication competent Semliki Forest virus prolongs survival in experimental lung cancer. Int J Cancer. 2008, 123: 1704-1711.View ArticlePubMedGoogle Scholar
- Ketola A, Hinkkanen A, Yongabi F, Furu P, Maatta AM, Liimatainen T, Pirinen R, Björn M, Hakkarainen T, Mäkinen K, Wahlfors J, Pellinen R: Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma. Cancer Res. 2008, 68: 8342-8350.View ArticlePubMedGoogle Scholar
- Maatta AM, Liimatainen T, Wahlfors T, Wirth T, Vaha-Koskela M, Jansson L, Valonen P, Häkkinen K, Rautsi O, Pellinen R, Mäkinen K, Hakumäki J, Hinkkanen A, Wahlfors J: Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models. Int J Cancer. 2007, 121: 863-870.View ArticlePubMedGoogle Scholar
- Zhang YQ, Tsai YC, Monie A, Wu TC, Hung CF: Enhancing the therapeutic effect against ovarian cancer through a combination of viral oncolysis and antigen-specific immunotherapy. Mol Ther. 2010, 18: 692-699.View ArticlePubMedPubMed CentralGoogle Scholar
- Asselin-Paturel C, Lassau N, Guinebretiere JM, Zhang J, Gay F, Bex F, Hallez S, Leclere J, Peronneau P, Mami-Chouaib F, Chouaib S: Transfer of the murine interleukin-12 gene in vivo by a Semliki Forest virus vector induces B16 tumor regression through inhibition of tumor blood vessel formation monitored by Doppler ultrasonography. Gene Ther. 1999, 6: 606-615.View ArticlePubMedGoogle Scholar
- Chikkanna-Gowda CP, Sheahan BJ, Fleeton MN, Atkins GJ: Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12. Gene Ther. 2005, 12: 1253-1263.View ArticlePubMedGoogle Scholar
- Rodriguez-Madoz JR, Prieto J, Smerdou C: Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas. Mol Ther. 2005, 12: 153-163.View ArticlePubMedGoogle Scholar
- Lyons JA, Sheahan BJ, Galbraith SE, Mehra R, Atkins GJ, Fleeton MN: Inhibition of angiogenesis by a Semliki Forest virus vector expressing VEGFR-2 reduces tumour growth and metastasis in mice. Gene Ther. 2007, 14: 503-513.View ArticlePubMedGoogle Scholar
- Morse MA, Hobeika AC, Osada T, Berglund P, Hubby B, Negri S, Niedzwiecki D, Devi GR, Burnett BK, Clay TM, Smith J, Lyerly HK: An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer. J Clin Invest. 2010, 120: 3234-3241.View ArticlePubMedPubMed CentralGoogle Scholar
- Vacchelli E, Eggermont A, Sautes-Fridman C, Galon J, Zitvogel L, Kroemer G, Galluzzi L: Trial watch: Oncolytic viruses for cancer therapy. Oncoimmunology. 2013, 2: e24612-View ArticlePubMedPubMed CentralGoogle Scholar
- Eisenberg DP, Adusumilli PS, Hendershott KJ, Yu Z, Mullerad M, Chan MK, Chou TC, Fong Y: 5-fluorouracil and gemcitabine potentiate the efficacy of oncolytic herpes viral gene therapy in the treatment of pancreatic cancer. J Gastrointest Surg. 2005, 9: 1068-1077.View ArticlePubMedPubMed CentralGoogle Scholar
- Meng S, Zhou Z, Chen F, Kong X, Liu H, Jiang K, Liu W, Hu M, Zhang X, Ding C, Wu Y: Newcastle disease virus induces apoptosis in cisplatin-resistant human lung adenocarcinoma A549 cells in vitro and in vivo. Cancer Lett. 2012, 317: 56-64.View ArticlePubMedGoogle Scholar
- Zhuang X, Zhang W, Chen Y, Han X, Li J, Zhang Y, Zhang Y, Zhang S, Liu B: Doxorubicin-enriched, ALDH(br) mouse breast cancer stem cells are treatable to oncolytic herpes simplex virus type 1. BMC Cancer. 2012, 12: 549-View ArticlePubMedPubMed CentralGoogle Scholar
- Heinemann L, Simpson GR, Boxall A, Kottke T, Relph KL, Vile R, Melcher A, Prestwich R, Harrington KJ, Morgan R, Pandha HS: Synergistic effects of oncolytic reovirus and docetaxel chemotherapy in prostate cancer. BMC Cancer. 2011, 11: 221-View ArticlePubMedPubMed CentralGoogle Scholar
- Halloran CM, Ghaneh P, Shore S, Greenhalf W, Zumstein L, Wilson D, Neoptolemos JP, Costello E: 5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells. J Gene Med. 2004, 6: 514-525.View ArticlePubMedGoogle Scholar
- Hallden G: Optimisation of replication-selective oncolytic adenoviral mutants in combination with chemotherapeutics. J BUON. 2009, 14 (Suppl 1): S61-S67.PubMedGoogle Scholar
- Qiu S, Ruan H, Pei Z, Hu B, Lan P, Wang J, Zhang Z, Gu J, Sun L, Qian C, Liu X, Qi Y: Combination of Targeting Gene-ViroTherapy with 5-FU enhances antitumor efficacy in malignant colorectal carcinoma. J Interferon Cytokine Res. 2004, 24: 219-230.View ArticlePubMedGoogle Scholar
- Uchida H, Shinoura N, Kitayama J, Watanabe T, Nagawa H, Hamada H: 5-Fluorouracil efficiently enhanced apoptosis induced by adenovirus-mediated transfer of caspase-8 in DLD-1 colon cancer cells. J Gene Med. 2003, 5: 287-299.View ArticlePubMedGoogle Scholar
- Kadota K, Huang CL, Liu D, Yokomise H, Haba R, Wada H: Combined therapy with a thymidylate synthase-inhibiting vector and S-1 has effective antitumor activity against 5-FU-resistant tumors. Int J Oncol. 2011, 38: 355-363.PubMedGoogle Scholar
- Karapanagiotou EM, Roulstone V, Twigger K, Ball M, Tanay M, Nutting C, Newbold K, Gore ME, Larkin J, Syrigos KN, Coffey M, Thompson B, Mettinger K, Vile RG, Pandha HS, Hall GD, Melcher AA, Chester J, Harrington KJ: Phase I/II trial of carboplatin and paclitaxel chemotherapy in combination with intravenous oncolytic reovirus in patients with advanced malignancies. Clin Cancer Res. 2012, 18: 2080-2089.View ArticlePubMedGoogle Scholar
- Hecht JR, Farrell JJ, Senzer N, Nemunaitis J, Rosemurgy A, Chung T, Hanna N, Chang KJ, Javle M, Posner M, Waxman I, Reid A, Erickson R, Canto M, Chak A, Blatner G, Kovacevic M, Thornton M: EUS or percutaneously guided intratumoral TNFerade biologic with 5-fluorouracil and radiotherapy for first-line treatment of locally advanced pancreatic cancer: a phase I/II study. Gastrointest Endosc. 2012, 75: 332-338.View ArticlePubMedPubMed CentralGoogle Scholar
- Granot T, Meruelo D: The role of natural killer cells in combinatorial anti-cancer therapy using Sindbis viral vectors and irinotecan. Cancer Gene Ther. 2012, 19: 588-591.View ArticlePubMedGoogle Scholar
- Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA: Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 1999, 17: 969-973.View ArticlePubMedGoogle Scholar
- Vasilevska J, Skrastina D, Spunde K, Garoff H, Kozlovska T, Zajakina A: Semliki Forest virus biodistribution in tumor-free and 4 T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon. Cancer Gene Ther. 2012, 19: 579-587.View ArticlePubMedGoogle Scholar
- Akhrymuk I, Kulemzin SV, Frolova EI: Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II. J Virol. 2012, 86: 7180-7191.View ArticlePubMedPubMed CentralGoogle Scholar
- Burke CW, Gardner CL, Steffan JJ, Ryman KD, Klimstra WB: Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses. Virology. 2009, 395: 121-132.View ArticlePubMedPubMed CentralGoogle Scholar
- Egami T, Ohuchida K, Miyoshi K, Mizumoto K, Onimaru M, Toma H, Sato N, Matsumoto K, Tanaka M: Chemotherapeutic agents potentiate adenoviral gene therapy for pancreatic cancer. Cancer Sci. 2009, 100: 722-729.View ArticlePubMedGoogle Scholar
- Quetglas JI, Ruiz-Guillen M, Aranda A, Casales E, Bezunartea J, Smerdou C: Alphavirus vectors for cancer therapy. Virus Res. 2010, 153: 179-196.View ArticlePubMedGoogle Scholar
- Osada T, Morse MA, Hobeika A, Lyerly HK: Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy. Semin Oncol. 2012, 39: 305-310.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun S, Wang LP, Zhang J, Yang XY, Zhang QL, Jia Z, Hu XC, Wang BY: Phase II study of oxaliplatin plus leucovorin and 5-fluorouracil in heavily pretreated metastatic breast cancer patients. Med Oncol. 2012, 29: 418-424.View ArticlePubMedGoogle Scholar
- Kantelhardt EJ, Vetter M, Schmidt M, Veyret C, Augustin D, Hanf V, Meisner C, Paepke D, Schmitt M, Sweep F, von Minckwitz G, Martin PM, Jaenicke F, Thomssen C, Harbeck N: Prospective evaluation of prognostic factors uPA/PAI-1 in node-negative breast cancer: phase III NNBC3-Europe trial (AGO, GBG, EORTC-PBG) comparing 6xFEC versus 3xFEC/3xDocetaxel. BMC Cancer. 2011, 11: 140-View ArticlePubMedPubMed CentralGoogle Scholar
- Longley DB, Harkin DP, Johnston PG: 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003, 3: 330-338.View ArticlePubMedGoogle Scholar
- Garcia MA, Carrasco E, Aguilera M, Alvarez P, Rivas C, Campos JM, Prados JC, Calleja MA, Esteban M, Marchal JA, Aránega A: The chemotherapeutic drug 5-fluorouracil promotes PKR-mediated apoptosis in a p53-independent manner in colon and breast cancer cells. PLoS One. 2011, 6: e23887-View ArticlePubMedPubMed CentralGoogle Scholar
- Gorchakov R, Frolova E, Williams BR, Rice CM, Frolov I: PKR-dependent and -independent mechanisms are involved in translational shutoff during Sindbis virus infection. J Virol. 2004, 78: 8455-8467.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoon CH, Lee ES, Lim DS, Bae YS: PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53. Proc Natl Acad Sci U S A. 2009, 106: 7852-7857.View ArticlePubMedPubMed CentralGoogle Scholar
- Peidis P, Papadakis AI, Muaddi H, Richard S, Koromilas AE: Doxorubicin bypasses the cytoprotective effects of eIF2alpha phosphorylation and promotes PKR-mediated cell death. Cell Death Differ. 2011, 18: 145-154.View ArticlePubMedGoogle Scholar
- Zhao Q, Zhang W, Ning Z, Zhuang X, Lu H, Liang J, Li J, Zhang Y, Dong Y, Zhang Y, Zhang S, Liu S, Liu B: A novel oncolytic herpes simplex virus type 2 has potent anti-tumor activity. PLoS One. 2014, 9 (3): e93103-View ArticlePubMedPubMed CentralGoogle Scholar
- Cheema TA, Kanai R, Kim GW, Wakimoto H, Passer B, Rabkin SD, Martuza RL: Enhanced antitumor efficacy of low-dose Etoposide with oncolytic herpes simplex virus in human glioblastoma stem cell xenografts. Clin Cancer Res. 2011, 17: 7383-7393.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim DY, Firth AE, Atasheva S, Frolova EI, Frolov I: Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution. J Virol. 2011, 85: 8022-8036.View ArticlePubMedPubMed CentralGoogle Scholar
- Carreras CW, Santi DV: The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995, 64: 721-762.View ArticlePubMedGoogle Scholar
- Bhattacharyya M, Francis J, Eddouadi A, Lemoine NR, Hallden G: An oncolytic adenovirus defective in pRb-binding (dl922-947) can efficiently eliminate pancreatic cancer cells and tumors in vivo in combination with 5-FU or gemcitabine. Cancer Gene Ther. 2011, 18: 734-743.View ArticlePubMedGoogle Scholar
- Tu SP, Cui JT, Liston P, Huajiang X, Xu R, Lin MC, Zhu YB, Zou B, Ng SS, Jiang SH, Xia HH, Wong WM, Chan AO, Yuen MF, Lam SK, Kung HF, Wong BC: Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant. Gastroenterology. 2005, 128: 361-375.View ArticlePubMedGoogle Scholar
- Leveille S, Samuel S, Goulet ML, Hiscott J: Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy. Cancer Gene Ther. 2011, 18: 435-443.View ArticlePubMedGoogle Scholar
- Diasio RB, Harris BE: Clinical pharmacology of 5-fluorouracil. Clin Pharmacokinet. 1989, 16: 215-237.View ArticlePubMedGoogle Scholar
- Greish K: Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. J Drug Target. 2007, 15: 457-464.View ArticlePubMedGoogle Scholar
- Maeda H, Fang J, Inutsuka T, Kitamoto Y: Vascular permeability enhancement in solid tumor: various factors, mechanisms involved and its implications. Int Immunopharmacol. 2003, 3: 319-328.View ArticlePubMedGoogle Scholar
- Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A: Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst. 2006, 98: 335-344.View ArticlePubMedGoogle Scholar
- Tseng JC, Granot T, DiGiacomo V, Levin B, Meruelo D: Enhanced specific delivery and targeting of oncolytic Sindbis viral vectors by modulating vascular leakiness in tumor. Cancer Gene Ther. 2010, 17: 244-255.View ArticlePubMedGoogle Scholar
- Huang PY, Guo JH, Hwang LH: Oncolytic Sindbis virus targets tumors defective in the interferon response and induces significant bystander antitumor immunity in vivo. Mol Ther. 2012, 20: 298-305.View ArticlePubMedGoogle Scholar
- Zhang Y, Burke CW, Ryman KD, Klimstra WB: Identification and characterization of interferon-induced proteins that inhibit alphavirus replication. J Virol. 2007, 81: 11246-11255.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/460/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.