Anti-tumor Effects Of Stat3-siRNA And Endostatin Combined Therapies, Delivered By Attenuated Salmonella, On Orthotopically-implanted Hepatocarcinoma

Background: HCC, which accounts for85–90%of all primary liver cancers, is one ofthe most aggressive malignancy and is a highly lethal complication of chronic liver disease.Curative treatment options chiefly comprise surgical resection or liver transplantation. Othertreatment, including systemic chemotherapy, ethanol injection, arterial chemoembolizationand radiotherapy etc, are only fractionally effective in controlling localized tumors, usuallyaccompanying some side effects. In spite of many scientific advances, prognosis for HCCremains poor due to absence of early disease symptoms, rapid tumor progression andinvasion-related spreading during early tumor stages. As a result of these factors, only10-20%of patients are considered eligible for surgical treatment. Therefor, development ofnovel therapeutic tactics targeting HCC, without remarkable toxicity, is of high priority.Signal transducer and activator of transcription3(Stat3) is a highly studied member ofthe STAT family. Constitutively activated Stat3is oncogenic and has been detected in a widevariety of tumors, including HCC. Aberrantly activated Stat3contributes to carcinogenesisby promoting tumor cell proliferation and angiogenesis together with suppressing apoptosisand host tumor immunity. Inhibition of Stat3signaling has been found to suppress tumorgrowth and improve survival, providing a molecular target for cancer therapy.Furthermore, HCC is a hypervascular tumor and angiogenesis plays a crucial role intumor growth and metastasis. Thus, anti-angiogenic therapy, aimed at blocking the tumorblood supply, combined with inhibition of Stat3may be an effective approach to combatHCC. Endostatin, a20KDa C-terminal fragment of collagen XVIII, was isolated and purifiedfrom murine hemangioendothelioma by O'Reilly in1997. It has been shown to be one of themost powerful angiogenesis inhibitors by specific inhibition of endothelial proliferation.Several studies have exhibited the antiangiogenic properties of Endostatin in a wide varietyof established tumor models of mouse, rat and human origins accompanied by low toxicityand lacking acquired drug resistance.Choosing an efficient gene delivery system which should selectively target tumors anddeliver the anticancer therapeutic agents into tumors while reducing damage to normal tissuehas been a major concern. S. Typhimurium, a facultative anaerobe, offers particular promise as an anticancer vector over other vectors currently used. They can preferentially amplifyand reside within solid tumors (>1,000fold greater in tumors than in normal tissues), whileminimizing toxicity to normal tissues. More recently, these bacteria have been engineered toexpress numerous genes, including interleukin-2, interleukin-12and MDR1siRNA etc.Additionally, previous studies demonstrated that Stat3-specific siRNA carried by attenuatedS. Typhimurium showed protection against prostate cancer in our laboratory. In view of thecomplicated biologic heterogeneity of tumors, a novel combination approach would beexpected to enhance the effect of either single therapeutic approach. We have exploited theuse of attenuated S. Typhimurium (a phoP/phoQ null mutant) to deliver the eukaryoticco-expression plasmid vector encoding both Stat3-specific small interfering-RNA andEndostatin, as combined tumor-targeted anticancer agents to examine their anti-tumor effectsin an orthotopic hepatoma mouse model and investigate the mechanism underlying.Objective: To test the effect that the combination therapy consisting of endostatin (apowerful angiogenesis inhibitor) and Stat3-specific siRNA, using a DNA vector delivered byattenuated S. Typhimurium, on an orthotopic HCC model in C57BL/6mice and explore themechanism underlying..Method: H22, a mouse hepatoma cell line, was employed to establish an orthotopicHCC model in C57BL/6mice. Next, H22-bearing mice were treated intraperitoneally withPBS or different recombinant attenuated S. Typhimurium therapeutic strains. For toxicityanalysis, the animals were monitored everyday with their activity behavior. The effects ofcombination treatment on tumor growth and survival rate were evaluated. We also exploredthe molecular mechanisms underlying anti-tumor effects including the apoptosis,proliferation, angiogenesis and immune response. At various time points after treatment, themice in each group were chosen randomly and sacrificed, the blood was collected and theorgans including heart, liver, spleen, lung, kidney and tumor of mice were isolated. Thebacteria distribution in every organ was screened from LB plates. The tumors were weightedand performed the cell cycle analysis, Annexin V, TUNEL and immunohistochemistry (IHC)experiments respectively. The expression of PCNA, CD34, CD4and CD8in tumor tissueswas deteced by IHC. Stat3, Endostatin mRNA and protein expression were analyzed usingreal-time quantitative, western blot and ELISA respectively. The spleens were weighted andthe spleen index was calculated. Then the spleens were homogenized and splenocytes wereevaluated for immune cells expression using flow cytometer. The NK cell and T lymphocytefunction analysis were detected via lymphocyte proliferation measurement and NK cytotoxicity assay respectively. The serum cytokines level of TNF-α, TGF-β1and IFN-γwere tested via ELISA.Result: Although anti-tumor effects were observed with either single therapeutictreatment, the combination therapy provided superior anti-tumor effects. The attenuatedsalmonella preferentially accumulated in tumors and the mice did not show any commonlyobserved sign of toxicity. Correlated with this findings, the combination treatment resulted insignificant alteration of Stat3and Endostatin levels. IHC experiment demonstrated that thePCNA and CD34in tumor were decreased and infiltration of CD4+, CD8+T cellslymphocytes in tumor was increased after pEndo-Si-Stat3treatment. Cell cycle assayillustrated the cell cycle block in combined treatment group. Annexin V and TUNELanalysises showed that combined therapy significantly induced cell apoptosis. NKcytotoxicity assay and lymphocyte proliferation measurement indicated that the fuction ofNK cell and T lymphocyte were enhanced after combined treatment. The expression of CD8+Tlymphocytes was elevated and the expression of Tregcells was reduced after combinationtreatment. The pEndo-Si-Stat3therapy also increased the secretion of IFN-γ, TNF-α(inflammatory cytokines) and decreased the secretion of TGF-β1(immune-supressing factor).Altogether, our data showed that combination therapy result in tumor regression and survivalrate improvement. The antitumor effect appear to stem from essential changed in severalparameters, which included cell apoptosis induced, cell proliferation inhibition, angiogenesisdecreasement and immune response partly restored.Conclusion: We have demonstrated that Salmonella-delivered combination therapy (i.e.Endostatin plus siRNA-Stat3) provides superior anti-tumor effects over either singletherapeutic approach. In summary, these studies demonstrate, in a mouse orthotopic implanttumor model, the enhanced value of combined anti-tumor gene therapies in the treatment ofthe aggressive HCC and verify safety and tumor-targeting ability of the attenuatedSalmonella as a delivery system.