Suppression of Cancer Growth by Nonviral Gene Therapy Based on a Novel Reactive Oxygen Species-responsive Promoter
Authors: Lucía L Policastro, Irene L Ibañez, Hebe A Durán, Gastón Soria, Vanesa Gottifredi, Osvaldo L Podhajcer
Journal: Molecular Therapy (2009)

Increased reactive oxygen species (ROS) production has been reported as a distinctive feature of different pathologies including cancer. Therefore, we assessed whether increased ROS production in the cancer microenvironment could be selectively exploited to develop a selective anticancer therapy. For this purpose, we constructed a novel chimeric promoter, based on a ROS-response motif located in the VEGF gene promoter placed, in turn, downstream of a second ROS-response motif obtained from the early growth response 1 (Egr-1) gene promoter. The activity of the chimeric promoter was largely dependent on variations in intracellular ROS levels and showed a high inducible response to exogenous H2O2. Transient expression of the thymidine kinase (TK) gene driven by the chimeric promoter, followed by gancyclovir (GCV) administration, inhibited human colorectal cancer and melanoma cell growth in vitro and in vivo. Moreover, electrotransfer of the TK gene followed by GCV administration exerted a potent therapeutic effect on established tumors. This response was improved when combined with chemotherapeutic drugs. Thus, we show for the first time that a distinctive pro-oxidant state can be used to develop new selective gene therapeutics for cancer. Increased reactive oxygen species (ROS) production has been reported as a distinctive feature of different pathologies including cancer. Therefore, we assessed whether increased ROS production in the cancer microenvironment could be selectively exploited to develop a selective anticancer therapy. For this purpose, we constructed a novel chimeric promoter, based on a ROS-response motif located in the VEGF gene promoter placed, in turn, downstream of a second ROS-response motif obtained from the early growth response 1 (Egr-1) gene promoter. The activity of the chimeric promoter was largely dependent on variations in intracellular ROS levels and showed a high inducible response to exogenous H2O2. Transient expression of the thymidine kinase (TK) gene driven by the chimeric promoter, followed by gancyclovir (GCV) administration, inhibited human colorectal cancer and melanoma cell growth in vitro and in vivo. Moreover, electrotransfer of the TK gene followed by GCV administration exerted a potent therapeutic effect on established tumors. This response was improved when combined with chemotherapeutic drugs. Thus, we show for the first time that a distinctive pro-oxidant state can be used to develop new selective gene therapeutics for cancer. IntroductionIncreased reactive oxygen species (ROS) levels have been associated with numerous pathological conditions, including atherosclerosis, cardiovascular diseases, rheumatoid arthritis, neurodegenerative disorders, and cancer.1Valko M Leibfritz D Moncol J Cronin MT Mazur M Telser J Free radicals and antioxidants in normal physiological functions and human disease.Int J Biochem Cell Biol. 2007; 39: 44-84Crossref PubMed Scopus (9751) Google Scholar ROS play a role in tumor development as DNA-damaging agents increasing mutation rates, leading to malignant transformation.2Jackson AL Loeb LA The contribution of endogenous sources of DNA damage to the multiple mutations in cancer.Mutat Res. 2001; 477: 7-21Crossref PubMed Scopus (507) Google Scholar,3Halliwel B Oxidative stress and cancer: have we moved forward?.Biochem J. 2007; 401: 1-11Crossref PubMed Scopus (1013) Google Scholar Moreover, ROS act as mediators of signal transduction pathways related to cell proliferation,4Irani K Xia Y Zweier JL Sollott SJ Der CJ Fearon ER et al.Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1421) Google Scholar,5Arnold RS Shi J Murad E Whalen AM Sun CQ Polavarapu R et al.Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1.Proc Natl Acad Sci USA. 2001; 98: 5550-5555Crossref PubMed Scopus (420) Google Scholar,6Veal EA Day AM Morgan BA Hydrogen peroxide sensing and signaling.Mol Cell. 2007; 26: 1-14Abstract Full Text Full Text PDF PubMed Scopus (1222) Google Scholar,7Laurent A Nicco C Chéreau C Goulvestre C Alexandre J Alves A et al.Controlling tumor growth by modulating endogenous production of reactive oxygen species.Cancer Res. 2007; 65: 948-956Google Scholar angiogenesis,8Shono T Ono M Izumi H Jimi SI Matsushima K Okamoto T et al.Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.Mol Cell Biol. 1996; 16: 4231-4239Crossref PubMed Google Scholar,9Kuroki M Voest E Amano S Beerepoot L Takashima S Tolentino M et al.Reactive oxygen intermediates increased vascular endothelial growth factor expression in vitro and in vivo.J Clin Invest. 1996; 98: 1466-1471Crossref Scopus (412) Google Scholar,10Ushio-Fukai M Redox signaling in angiogenesis: role of NADPH oxidase.Cardiovasc Res. 2006; 71: 226-235Crossref PubMed Scopus (401) Google Scholar and cell migration.11Wu WS The signaling mechanism of ROS in tumor progression.Cancer Metastasis Rev. 2006; 25: 695-705Crossref PubMed Scopus (616) Google Scholar,12Radisky DC Levy DD Littlepage LE Liu H Nelson CM Fata JE et al.Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability.Nature. 2005; 436: 123-127Crossref PubMed Scopus (994) Google Scholar,13Nelson KK Ranganathan AC Mansouri J Rodriguez AM Providence KM Rutter JL et al.Elevated sod2 activity augments matrix metalloproteinase expression: evidence for the involvement of endogenous hydrogen peroxide in regulating metastasis.Clin Cancer Res. 2003; 9: 424-432PubMed Google Scholar Evidence from the literature indicates that a shift in cellular redox status may be a crucial event in the appearance of the malignant phenotype.3Halliwel B Oxidative stress and cancer: have we moved forward?.Biochem J. 2007; 401: 1-11Crossref PubMed Scopus (1013) Google Scholar Indeed, several studies have revealed higher levels of ROS in different types of human cancer tissues compared with their noncancerous counterparts.14Szatrowski TP Nathan CF Production of large amounts of hydrogen peroxide by human tumor cells.Cancer Res. 1991; 51: 794-798PubMed Google Scholar,15Policastro L Molinari B Larcher F Blanco P Podhajcer OL Costa CS et al.Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide.Mol Carcinog. 2004; 39: 103-113Crossref PubMed Scopus (71) Google Scholar,16Toyokuni S Okamoto K Yodoi J Hiai H Persistent oxidative stress in cancer.FEBS Lett. 1995; 358: 1-3Abstract Full Text PDF PubMed Scopus (1003) Google Scholar,17Evans MD Dizdaroglu M Cooke MS Oxidative DNA damage and disease: induction, repair and significance.Mutat Res. 2004; 567: 1-61Crossref PubMed Scopus (1019) Google Scholar,18Al-Gayyar MM Eissa LA Rabie AM El-Gayar AM Measurements of oxidative stress status and antioxidant activity in chronic leukaemia patients.J Pharm Pharmacol. 2007; 59: 409-417Crossref PubMed Scopus (43) Google Scholar It is, therefore, plausible that the persistent oxidative stress of cancer cells is a differential feature of the tumor environment that may be exploited to develop a selective anticancer therapy.Gene therapy is a relatively new strategy in clinical terms, and most clinical trials are still in early phases (www.wiley.co.uk/genetherapy/clinical/). More than 60% of clinical trials target cancers, and many obstacles remain to be overcome before cancer gene therapy becomes a routine procedure. Recently, it was shown that conditional targeting of a therapeutic gene using cancer-specific promoters to drive gene expression might become a useful strategy toward this end.19Ko D Hawkins L Yu DC Development of transcriptionally regulated oncolytic adenoviruses.Oncogene. 2005; 24: 7763-7774Crossref PubMed Scopus (68) Google Scholar The high levels of heterogeneity in gene expression from cell to cell, and from tumor to tumor, limit the potential use of a promoter obtained from a tumor-associated gene. Therefore, different groups aim to specifically target the tumor mass by taking advantage of defined microenvironmental differences between cancer tissues and normal cells. For instance, hypoxia can be used for the selective expression of therapeutic genes driven by hypoxia-response elements in cancer tissues.20Ido A Uto H Moriuchi A Nagata K Onaga Y Onaga M et al.Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter.Cancer Res. 2001; 61: 3016-3021PubMed Google Scholar,21Ballinger JR Imaging hypoxia in tumor.Semin Nucl Med. 2001; 31: 321-329Abstract Full Text PDF PubMed Scopus (109) Google Scholar Thus, the conditional targeting of a cancer tissue by taking advantage of the particular characteristics of the tumor remains possible both for cancer gene therapy and to attack other diseases. DNA sequences responsive to ROS are present in the promoters of several redox-regulated genes.22Liu H Colavitti R Rovira II Finkel T Redox-dependent transcriptional regulation.Circ Res. 2005; 97: 967-974Crossref PubMed Scopus (347) Google Scholar Particularly, oxidative stress was shown to regulate VEGF-A gene expression in gastric cancer cells.23Schäfer G Cramer T Suske G Kemmner W Wiedenmann B Höcker M Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements.J Biol Chem. 2003; 278: 8190-8198Crossref PubMed Scopus (169) Google Scholar Additional examples of oxidative stress-responsive DNA sequences include a region present in the promoter of the early growth response 1 (Egr-1) gene.24Datta R Taneja N Sukhatme VP Qureshi SA Weichselbaum R Kufe DW Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.Proc Natl Acad Sci USA. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar Interestingly, no sequence consensus has been found among the different response sequences defined to date.We propose a new strategy that makes use of the increased levels of ROS in cancer cells to enhance therapeutic selectivity. Here, we demonstrate that the expression of the Herpes Simplex virus thymidine kinase (TK) gene, driven by a ROS-response chimeric promoter, was effective in inhibiting tumor cell growth in vitro and in vivo. Electrotransfer of this nonviral construct was able to strongly inhibit established human colorectal carcinoma and melanoma growth in a nude mouse model. The therapeutic effect of this construct was enhanced when combined with chemotherapeutic drugs.ResultsA ROS-response chimeric promoter is active in malignant cellsWe initially explored the activities of ROS-response elements from different promoters, in an effort to develop novel selective gene therapeutics. We selected three DNA motifs that were previously described as redox-response DNA elements: a CG-rich motif located in the VEGF-A promoter (named VE),23Schäfer G Cramer T Suske G Kemmner W Wiedenmann B Höcker M Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements.J Biol Chem. 2003; 278: 8190-8198Crossref PubMed Scopus (169) Google Scholar six-repeated CArG motives from the Egr-1 promoter (named E6),24Datta R Taneja N Sukhatme VP Qureshi SA Weichselbaum R Kufe DW Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.Proc Natl Acad Sci USA. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar and a −2002, −1546 fragment corresponding to the matrix metalloproteinase-1 promoter (named MMP-1).13Nelson KK Ranganathan AC Mansouri J Rodriguez AM Providence KM Rutter JL et al.Elevated sod2 activity augments matrix metalloproteinase expression: evidence for the involvement of endogenous hydrogen peroxide in regulating metastasis.Clin Cancer Res. 2003; 9: 424-432PubMed Google Scholar The different fragments were cloned into the minimal cytomegalovirus (CMV) plasmid upstream of the luciferase reporter gene (Figure 1a).To evaluate the responsiveness of the different constructs to oxidative stress, A375N melanoma and LoVo colorectal cancer cells were transiently transfected with each plasmid and exposed to H2O2 generated by the glucose/glucose oxidase system.25Minotti G Menna P Salvatorelli E Cairo G Gianni L Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.Pharmacol Rev. 2004; 56: 185-229Crossref PubMed Scopus (2836) Google Scholar The VE element exhibited the highest basal activity, and inducible response, to H2O2, although all the elements tested were active to some extent (Figure 1b). These responses were redox-specific, as shown by their complete reversal upon addition of exogenous catalase (Figure 1b). As additional copies of the VE motif did not increase ROS response (data not shown), we decided to construct a chimeric promoter that will include the combination of different motives. In addition to the VE motif, we selected E6 because of it shorter length, and its slightly higher response to increased H2O2 levels, compared to MMP-1. Moreover, E6 can be additionally activated by ionizing radiation.24Datta R Taneja N Sukhatme VP Qureshi SA Weichselbaum R Kufe DW Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.Proc Natl Acad Sci USA. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar The E6 and VE motifs were placed at different positions relative to the luciferase gene (Figure 1c). The 5′-E6(6)VE-3′ [hereafter termed E6(6)VE] chimera showed a basal and inducible activity higher than that of the 5′-VE(6)E6-3′ chimera (Figure 1d), but lower than that of VE alone, when the motifs were assessed in parallel (compare Figure 1d with 1b), suggesting that steric hindrance might hamper the activity of the chimeric promoter. In efforts to improve E6(6)VE activity, we moved the E6 and VE motifs either 20 or 40 bp apart, by introducing DNA spacers. The E6(40)VE chimera showed the best basal, and inducible, activities in the presence of exogenous H2O2 (Figure 1d). Notably, the E6(40)VE response to exogenous H2O2 was redox-specific, as shown by the complete reversal of inducible luciferase activity in the presence of catalase (Figure 1d). The basal luciferase activity driven by E6(40)VE was as strong as or higher than the activity of a SV40 promoter in various cell lines, including melanoma and colorectal cancer cells (Supplementary Figure S1).Variation in endogenous ROS levels modulates E6(40)VE activityWe next compared the basal activity of E6(40)VE in normal and malignant cells in response to ROS endogenous levels. First, we confirmed that malignant cells exhibited higher intracellular ROS levels than their normal counterparts. Indeed, quantification of dichlorofluorescein staining showed a two- to fivefold increase in intracellular ROS levels in malignant LoVo and A375N cells as well as in MCF-7 breast cancer cells, and in transformed WI-38VA fibroblasts compared to their normal counterparts (Figure 2a). Next, we transiently transfected normal and malignant cells lines with E6(40)VE-LUC, where luciferase activity was driven by the chimeric promoter. As expected, ROS dependent-luciferase activity was higher in malignant cells than in their respective normal counterparts (Figure 2b). We were unable to transfect primary cultures of melanocytes (data not shown).Figure 2Variation in endogenous reactive oxygen species (ROS) levels modulates E6(40)VE activity. (a) Quantification of endogenous ROS in different cell types as assessed by DCF. Data represent the means ± SEMs of fluorescence levels of ∼50 cells (*P < 0.01). Confocal microscopy photographs showing ROS levels in different cell types. Bar represent 20 µm. (b) Luciferase activity in cells transfected with E6(40)VE-LUC and the respective controls. Luciferase activity was normalized and quantified as described in Materials and Methods. (*P < 0.001). (c) Luciferase activity in LoVo (black) and A375N (white) cells transfected with E6(40)VE-LUC and treated with NAC (#P < 0.05, *P < 0.01) (d) ROS levels in NAC-treated cells measured by the DCF assay (*P < 0.01). (e) LoVo (black) and A375N (white) cells co-transfected with E6(40)VE-LUC and p-Cat, exposed or not to G/GO. Luciferase activity was expressed as -fold induction over control cells. (*P < 0.01). Data show the mean ± SD of three independent experiments. (f) Levels of ROS in LoVo SOD+(F10) and LoVo SOD−G9 cells as assessed by DCF followed by fluorescence-activated cell sorting analysis. Data show a representative result from three independent experiments. (g) Luciferase activity in stable LoVo SOD+(F10) and LoVo SOD−(G9) cell clones obtained from LoVo cells (see Results section for details). Cell clones were transfected with E6(40)VE-LUC. Luciferase activity was normalized as described and further quantified as a -fold induction over control G9 cells. Data are expressed as means ± SD values of three independent experiments (*P < 0.014). G/GO, glucose/glucose oxidase; SOD, superoxide dismutase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we evaluated whether E6(40)VE activity was dependent on intracellular redox status. First, we transfected LoVo and A375N cells with E6(40)VE-LUC and treated cells with the ROS scavenger N-acetylcysteine. Under these conditions N-acetylcysteine reduced luciferase activity and intracellular ROS levels in a dose-dependent manner (Figure 2c,d). Further confirmation that E6(40)VE activity was driven by intracellular ROS levels was obtained when luciferase expression driven by the chimeric motif was strongly reduced when cells were co-transfected with a plasmid encoding for human catalase (Figure 2e). Finally, the increase in luciferase activity induced by cell treatment with glucose/glucose oxidase system that augments H2O2 levels was strongly inhibited when cells were co-transfected with the plasmid encoding for human catalase (Figure 2e). Furthermore, we stably transfected LoVo cells with a construct containing a cDNA coding for the human CuZn-superoxide dismutase (SOD), as previous data demonstrated increased ROS levels in cells constitutively expressing SOD.13Nelson KK Ranganathan AC Mansouri J Rodriguez AM Providence KM Rutter JL et al.Elevated sod2 activity augments matrix metalloproteinase expression: evidence for the involvement of endogenous hydrogen peroxide in regulating metastasis.Clin Cancer Res. 2003; 9: 424-432PubMed Google Scholar The stable blasticidin-resistant clone LoVo SOD+(F10) showed the highest intracellular ROS levels compared to LoVo SOD−(G9) cells that were stably transfected with an empty vector (Supplementary Figure S2a,b). We confirmed by dichlorofluorescein quantification that LoVo SOD+(F10) produced higher levels of H2O2 than LoVo SOD−(G9) cells (Figure 2f). LoVo SOD+(F10) cells exhibited 2.5-fold increased luciferase activity after transfection with E6(40)VE-LUC, compared to LoVo SOD−(G9) control cells, further demonstrating that E6(40)VE activity correlated with increased intracellular ROS levels (Figure 2g). The sum of the evidence clearly demonstrates that the activity of the chimeric promoter was ROS-dependent.Expression of TK driven by the ROS-responsive promoter inhibited the in vitro and in vivo proliferation of malignant cellsBased on the evidence mentioned earlier, we next determined if E6(40)VE might drive the expression of a therapeutic gene. As a proof of concept we cloned the TK gene downstream of E6(40)VE to generate E6(40)VE-TK. Transient expression of E6(40)VE-TK led to a significant inhibition of LoVo and A375N cell proliferation in the presence of gancyclovir (GCV) (Figure 3a). No significant inhibition was observed in control cells transfected with E6(40)VE-LUC, whether or not these cells were exposed to GCV, or when TK was expressed in the absence of GCV (Figure 3a). Moreover, normal CCD-481 human colonic cells were completely resistant to TK expression driven by E6(40)VE, as almost no death was observed in transfected cells in the presence of GCV, compared to a massive death of malignant cells under the same conditions (Supplementary Figure S3 and Supplementary Materials and Methods).Figure 3TK expression driven by the reactive oxygen species-responsive chimeric promoter, followed by GCV, inhibits in vitro and in vivo cell growth. (a) In vitro growth inhibition of LoVo and A375N cells transiently transfected with E6(40)VE-TK plasmid, and exposed to GCV. “Growth fraction” means the percentage of surviving cells, compared to control cells. Transfection efficiencies were 40–60%. Data show the mean ± SD values of three independent experiments (*P < 0.0001). (b) Spheroids made of LoVo or A375N cells transiently transfected with E6(40)VE-LUC and E6(40)VE-TK plasmids. Spheroids were grown in the presence or absence of 50 µmol/l GCV. Data represent the mean ± SD of measurements from three to eight spheroids, corresponding to one of two independent experiments. Inset: photomicrographs (×25) of spheroids taken after 20 days of GCV treatment (*P < 0.01). (c) In vivo tumorigenicity of LoVo and A375N cells transiently transfected with E6(40)VE-TK. Mice were treated intraperitoneal with GCV (50 mg/kg), or vehicle, every day during the first 15 days after cell inoculation (n = 5–7 mice per group); (*P < 0.0002 for the melanoma model). Inset: photographs of mice taken at 60 days, showing the presence of tumors in control mice only. GCV, gancyclovir; PBS, phosphate buffered saline; TK, thymidine kinase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)E6(40)VE was also able to drive TK expression in multicellular spheroids. Indeed, we observed a strong reduction in the growth capacity of spheroids made of LoVo or A375N cells previously transfected with E6(40)VE-TK, when exposed to GCV, compared to the same spheroids without GCV, or compared to spheroids made of cells transfected with E6(40)VE-LUC followed by GCV (Figure 3b). This effect was observed immediately, after only 2 days of culture (Supplementary Figure S4).To evaluate the capacity of E6(40)VE to drive TK expression in vivo, LoVo and A375N cells were transiently transfected with E6(40)VE-TK and injected subcutaneously into nude mice. No mouse injected with LoVo cells expressing TK, and treated with GCV, developed a tumor (Figure 3c). In addition, mice injected with A375N, and receiving the same treatment as animals receiving LoVo cells, showed significant tumor growth delay compared to control mice (Figure 3c). It can be concluded that transient expression of TK driven by E6(40)VE, followed by GCV, was sufficient to strongly inhibit the in vivo growth of colorectal cancer and melanoma cells.The combination of E6(40)VE-TK with γ irradiation, bleomycin, or doxorubicin, enhanced the inhibition of tumor cell growth in vitro, in cell monolayers and spheroidsIonizing radiation, and some chemotherapeutic drugs such as bleomycin (Bleo) and doxorubicin (Dx), are associated with the formation of reactive oxygen intermediates, and direct damage to DNA.24Datta R Taneja N Sukhatme VP Qureshi SA Weichselbaum R Kufe DW Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.Proc Natl Acad Sci USA. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar,25Minotti G Menna P Salvatorelli E Cairo G Gianni L Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.Pharmacol Rev. 2004; 56: 185-229Crossref PubMed Scopus (2836) Google Scholar,26Chen J Stubbe J Bleomycins: towards better therapeutics.Nat Rev Cancer. 2005; 5: 102-112Crossref PubMed Scopus (484) Google Scholar We hypothesized that the combination with chemotherapeutic drugs or ionizing radiation might enhance the therapeutic potential of the ROS-responsive plasmid in a setting that resembles a clinical situation. Using luciferase expression as reporter gene, we confirmed that the activity of E6(40)VE was stimulated by increasing doses of ionizing radiation (Supplementary Figure S5). Next, we performed in vitro studies in which LoVo and A375N cells were transiently transfected with E6(40)VE-TK, followed by GCV combined either with γ irradiation, Bleo, or Dx. The combination of E6(40)VE-TK/GCV with γ irradiation enhanced the inhibition of LoVo cell proliferation compared to the proliferation levels seen with any other treatment, and the optimum inhibition occurred at 2Gy γ irradiation (Figure 4a). In addition, the combination of γ irradiation and E6(40)VE-TK/GCV treatment was slightly more effective on A375N melanoma cells than any other treatment, when E6(40)VE-TK/GCV was combined with 5Gy γ irradiation (Figure 4a). Moreover, the combination of E6(40)VE-TK/GCV treatment with either Bleo or Dx enhanced cell growth inhibition, compared to the inhibitions noted when each treatment was applied separately (Figure 4b). Apoptosis was the main form of cell death induced by Dx or E6(40)VE-TK/GCV as single agent or in combination (Supplementary Figure S6 and Supplementary Materials and Methods). On the other hand, no enhancement of cell growth inhibition was observed when LoVo and A375N cells were transfected with E6(40)VE-LUC, followed by GCV in combination with either γ irradiation, Bleo, or Dx (Supplementary Figure S7).Figure 4A combination of TK expression driven by the reactive oxygen species-responsive chimeric promoter, and GCV, with g radiation, Bleo, or Dx, enhances in vitro cell growth inhibition. (a) LoVo and A375N cells were transiently transfected with E6(40)VE-TK and exposed to g radiation in the presence or absence of 10 µmol/l GCV (***P < 0.001, **P < 0.01). (b) LoVo and A375N cells were transiently transfected with E6(40)VE-TK and exposed to Dx (0.5 µmol/l), or Bleo (20 µmol/l), in the presence or absence of 10 or 50 µmol/l GCV. Growth inhibition was measured by the MTT assay and “growth fraction” refers to the inhibition seen in comparison with untreated cells (***P < 0.001). (c) Spheroids made of LoVo or A375N cells, transiently transfected with E6(40)VE-TK, were exposed to GCV (50 µmol/l), Dx (0.5 µmol/l), or both. Spheroid growth was measured using the MTT assay. Data show the mean ± SD of three to five measurements within a representative experiment of two experiments. Each measurement includes one spheroid of A375N cells and a pool of three spheroids for LoVo cells. Inset: photomicrographs (×25) of spheroids taken at day 20 (*P < 0.05 and **P < 0.01). Data show the means ± SD values of three independent experiments and lines over the bars indicate the groups that were compared. Bleo, bleomycin; C, control; Dx, doxorubicin; GCV, gancyclovir; PBS, phosphate buffered saline; TK, thymidine kinase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We further evaluated the effect of the combination of E6(40)VE-TK/GCV treatment and Dx on spheroid growth. Spheroids made of cells transiently transfected with E6(40)VE-TK were exposed to GCV and Dx treatments and the viabilities of the spheroids were assessed using the MTT assay. We observed enhanced inhibition of spheroid growth of both cell types after the combination treatment, compared to the inhibitions noted when each treatment was applied separately (Figure 4c). Overall, these series of experiments demonstrate that E6(40)VE-TK and GCV can be combined with γ irradiation, and chemotherapeutic drugs, to enhance antitumor effects.Electrotransfer of E6(40)VE-TK, followed by GCV, with or without chemotherapy, inhibited the in vivo growth of established colorectal cancer and melanomaWe finally decided to assess whether E6(40)VE was sufficiently potent to drive TK expression, and thus to exert a therapeutic effect, on established tumors. For this purpose, mice harboring LoVo or A375N tumors (150–200 mm3 average diameter) received six intratumor administrations of E6(40)VE-TK followed by local electroporation, once every 2 days. Starting on the day of the first electroporation, control mice received intraperitoneal (i.p.) injections of phosphate buffered saline for 15 consecutive days. A second group received GCV i.p. for 15 consecutive days. A third group received Dx i.p. on three occasions, once every 4 days, starting at the second round of electroporation. A fourth group received both GCV and Dx (Figure 5a,b).Figure 5Combination of TK expression driven by the reactive oxygen species-responsive chimeric promoter, and GCV, with or without chemotherapy, inhibited the in vivo growth of established tumors. (a,b) Nude mice harboring established LoVo and A375N tumors were distributed into four groups (n = 5 per group): control (C), Dx, GCV, and GCV+Dx. All mice received six intratumoral injections of 50 µg of naked plasmid by electroporation, every 2 days. GCV (50 mg/kg) or PBS was administrated i.p from day 0 to 15, as indicated. Dx (5 mg/kg) was administrated i.p 1 hour prior to electroporation, when indicated. Gr