Cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing uracil phosphoribosyltransferase (UPRT) in immunocompetent Syrian hamsters

Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing uracil phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5‐fluorouracil (5‐FU), in immunodeficient, Ad‐nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5‐FU therapy and of its combination with CP for syngenic HaP‐T1 pancreatic cancers in immunocompetent, Ad‐permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5‐FU in HaP‐T1 cells in vitro. I.t. AxE1CAUP/5‐FU treatment inhibited the growth of subcutaneous HaP‐T1 allografts. The combination with high‐dose CP inhibited serum Ad‐neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5‐FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically‐approved, intermediate‐dose CP also enhanced the efficacy of i.t. AxE1CAUP/5‐FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad‐permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV‐mediated gene therapy.     

Suppression of metastasis of human pancreatic cancer to the liver by transportal injection of recombinant adenoviral NK4 in nude mice

NK4, a 4-kringle fragment of hepatocyte growth factor (HGF), is an HGF antagonist that also acts as an angiogenesis inhibitor. NK4 strongly inhibits the infiltration, metastasis, and tumor growth of pancreatic cancer. The aim of our study was to evaluate the antitumor effect of adenovirus-mediated NK4 gene transfer to the liver on hepatic metastasis of pancreatic cancer in vivo. We constructed recombinant adenoviral NK4 (Ad-NK4), which encodes a secreted form of human NK4. Intrasplenic injection of Ad-NK4 induced high and relatively maintained expression of NK4 protein in the liver and suppressed the number and growth of metastatic foci in the liver in a nude mouse model. Microscopically, central necrosis was found even in small metastatic foci in Ad-NK4 treated mice. Immunohistochemical analysis of metastatic tumors showed a remarkable decrease in microvessel density and an increase in the number of apoptotic tumor cells after treatment with Ad-NK4. These results indicate that intraportal injection of Ad-NK4 may be a useful therapeutic modality for the clinical control of hepatic metastasis in pancreatic cancer.     

Adenoviral-mediated transfer of Escherichia coli uracil phosphoribosyltransferase (UPRT) gene to modulate the sensitivity of the human colon cancer cells to 5-fluorouracil

5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coli UPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines were used (HT29, KM12 and SW1116). The intracellular levels of 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-FU incorporated into RNA after 5-FU treatment in cells infected with adenovirus containing the UPRT gene (AdCA-UPRT) were significantly higher than those of non-infected cells. This was accompanied by marked inhibition of thymidylate synthase (TS) in all cell lines. Furthermore, HT29, KM12 and SW1116 infected with AdCA-UPRT were, respectively, 13.1-, 30.2- and 70.5-fold more sensitive to 5-FU than non-infected cells. Most importantly, treatment with AdCA-UPRT and 5-FU effectively inhibited the growth of HT29-xenografted subcutaneous tumours in nude mice. Therefore, AdCA-UPRT/5-FU treatment had the potential to enhance the actions of 5-FU at both the DNA and RNA levels. Treatment augmented the sensitivity of human colon cancer cells to 5-FU both in vitro and in vivo. We conclude that adenoviral-mediated transfer of the E. coli UPRT gene into colon cancer cells can achieve biochemical modulation of 5-FU and this provides a new approach in the treatment of colorectal cancer.     

EpCAM- and EGFR-targeted selective gene therapy for biliary cancers using Z33-fiber-modified adenovirus

A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by β-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.     

Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and connexin26 genes in pancreatic cells results in variable efficiency on the bystander killing: implications for gene therapy.

Currently, there is no effective treatment for pancreatic cancer and prodrug-activating gene therapy with the herpes simplex virus thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) has been suggested as a candidate approach against this disease. In the present study, we have evaluated the efficacy of the HSV-tk/GCV treatment in a panel of pancreatic tumor cells (NP-9, NP-18, NP-31) and the potentiation of the cytotoxic effect in combination with the overexpression of the connexin 26 gene (Cx26). Pancreatic cells transduced with a retrovirus containing the HSV-tk gene showed different sensitivities to GCV that seemed to be independent of HSV-tk expression levels. The extent of the bystander effect also varied among the pancreatic tumor cells and correlated with the level of gap junction intercellular communication (GJIC). Transduction of the pancreatic tumor cells with a retrovirus carrying the connexin 26 gene resulted in high levels of connexin 26 expression and in an increase in the GJIC that correlated to an extent in the bystander effect in both NP-9Cx26 and NP-18Cx26 cells. Neither an increment in GJIC nor an increase in the bystander killing was detected in NP-31Cx26. The bystander effect in NP-18 Cx26 cells was also prevented by the long term inhibitor of GJIC, 18-alpha-glycyrrhetinic acid (AGA). Together, these results demonstrate that pancreatic tumor cells are highly different as regards the susceptibility to HSV-tk/GCV treatment. Moreover, they indicate that overexpression of the Cx26 gene does not always correspond to an increase in GJIC although they clearly suggest the role of GJIC in mediating the bystander effect.     

Gene therapy for bladder cancer using E1B-55 kD-deleted adenovirus in combination with adenoviral vector encoding plasminogen kringles 1-5

Mutations or loss of heterozygosity of p53 are detected in approximately 50% of bladder cancers. E1B-55 kD-deleted adenovirus has been shown to kill tumour cells with defective p53 function while sparing normal cells. Here, we examined the cytolytic effect and replication of E1B-55 kD-deleted adenovirus, designated Ad5WS1, on human bladder cancer cell lines with various p53 status. Ad5WS1 caused more severe cytolytic effect and replicated more efficiently in J82 and TCC-SUP bladder cancer cells carrying mutant p53 compared with TSGH-8301 and BFTC-905 bladder cancer cells retaining wild-type p53. Introduction of dominant negative p53 into BFTC-905 cells rendered them more susceptible to Ad5WS1-induced cytolysis. Furthermore, cells susceptible to lysis caused by Ad5WS1 were not attributable to their greater infectability by adenovirus. Finally, Ad5WS1 suppressed the growth of TCC-SUP bladder tumour xenografts, which could be augmented when combined with replication-defective adenoviral vector encoding kringles 1-5 of plasminogen (K1-5), an angiogenic inhibitor. Taken together, our results show that E1B-55 kD-deleted adenovirus replicates and hence lyses bladder cancer cells with mutant p53 much more efficient than those with wild-type p53. Thus, E1B-deleted adenovirus may have therapeutic potential, especially in combination with adenoviral vector expressing K1-5, for the treatment of bladder cancer.     

Gene therapy with SOCS1 for gastric cancer induces G2/M arrest and has an antitumour effect on peritoneal carcinomatosis

Background:

Suppressor of cytokine signaling1 (SOCS1) is a negative regulator of various cytokines. Recently, it was investigated as a therapeutic target in various cancers. However, the observed antitumour effects of SOCS1 cannot not be fully explained without taking inhibition of proliferation signalling into account. Our aim was to discover a new mechanism of antitumour effects of SOCS1 for gastric cancer (GC).

Methods:

We analysed the mechanism of antitumour effect of SOCS1 in vitro. In addition, we evaluated antitumour effect for GC using a xenograft peritoneal carcinomatosis mouse model in preclinical setting.

Results:

We confirmed that SOCS1 suppressed proliferation in four out of five GC cell lines. SOCS1 appeared to block proliferation by a new mechanism that involves cell cycle regulation at the G2/M checkpoint. We showed that SOCS1 influenced cell cycle-associated molecules through its interaction with ataxia telangiectasia and Rad3-related protein. The significant difference in therapeutic effects was noted in terms of the post-treatment weight and total photon count of the intra-abdominal tumours.

Conclusion:

Forced expression of SOCS1 revealed a heretofore-unknown mechanism for regulating the cell cycle and may represent a novel therapeutic approach for the treatment of peritoneal carcinomatosis of GC.     

Preclinical study of a "tailor-made" combination of NK4-expressing gene therapy and gefitinib (ZD1839, Iressa) for disseminated peritoneal scirrhous gastric cancer

We evaluated the effect of a "tailor-made" chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailor-made approach, we first selected gefitinib (epidermal growth factor receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric fibroblasts, NF22, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between fibroblasts and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n = 8), 11.9; treated (n = 8), 17.3; p = 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p = 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p = 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, 22; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination.     

Suppression of Tregs by anti‐glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon‐α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3⁺ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4⁺ and CD8⁺ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.     

Double‐targeted and double‐enhanced suicide gene therapy mediated by generation 5 polyamidoamine dendrimers for prostate cancer

Herpes simplex virus (HSV)‐thymidine kinase (TK)/ganciclovir (GCV) system is one of the most widely used and efficient suicide gene therapy for prostate cancer, but the lack of favorable gene vector and target limits its application. In this study, we established a novel system using nonviral gene vector G5‐PAMAM‐D to express HSV‐TK and connexin43 (Cx43) gene driven by prostate‐specific membrane antigen (PSMA) promoter, and evaluated the anti‐tumor effect of this system. G5‐PAMAM‐D delivered PSMAe/p‐TK‐Cx43 showed expression of TK and Cx43 only in LNCaP cells, but not in PC‐3 and other cells. The transfection efficiency of this system was comparable to lipofectamine 2000 by propidium iodide staining assay. With gemcitabine, folate‐G5‐PAMAM‐D delivered PSMAe/p‐TK‐Cx43 (folate‐G5‐PAMAM‐D/PSMAe/p‐TK‐Cx43) significantly decreased prostate cancer LNCaP cell proliferation and promoted apoptosis in vitro. With gemcitabine, the systemic deliver of folate‐G5‐PAMAM‐D/PSMAe/p‐TK‐Cx43 significantly inhibited tumor growth in the LNCaP xenograft animal model. Our study demonstrates that this double‐targeted and double‐enhanced system is effective in inducing cell growth inhibition and apoptosis in vitro and suppressing tumor growth in vivo. In conclusion, Cx43 and gemcitabine combined with HSV‐TK/GCV gene therapy using nonviral vector G5‐PAMAM‐D hold great potential as a novel approach for the gene therapy of prostate cancer. © 2011 Wiley Periodicals, Inc.