Attenuated Salmonella Carrying The Plasmid Co-expressed Endostatin And SiRNA-Stat3for Glioma Therapy In Vivo

Glioma is the most common type of primary intracranial tumor. It has a poor prognosisand limited therapeutic options. Thus, new effective treatments are urgently needed and genetherapy may be a novel option. Glioma is a hypervascular tumor. Since the theory of tumorgrowth depending on angiogenesis proposed Folkman, many scientists have tried to restrainthe growth of glioma by inhibiting angiogenesis. Since the mechanism of tumorangiogenesis is very complicated, and the interaction will form a wide range of network, it’simpossible to inhibit the tumor growth only by a single factor treatment.Endostatin, an endogenous inhibitor of angiogenesis, is a20kDa C-terminal fragmentof collagen XVIII. It is considered to be one of the strongest inhibitor of angiogenesis untilrecently. Several studies have shown that endostatin has a good effect of inhibiting glioma.Therefore, we combined endostatin withSi-Stat3to treat glioma.Stat3(Signal transducer and activator of transcription3) is a kind of protein which hasdouble functions not only as a signal transduction, but also as a transcriptional activation.Many researches have shown that there are abnormal expression or activity enhancement ofStat3in a wide variety of human cancer tissues and tumor cell lines. Stat3is a joint of manytyrosine kinase signal pathways associated with carcinogenesis. Blocking Stat3-mediatedsignaling pathway can promote tumor cell apoptosis and inhibit tumor cell proliferation,which show it can be used as a therapeutic target. Several studies have shown thatdown-regulating Stat3expression obvious inhibit the growth of glioma in vivo.Gene vector is the transporter of therapeutic genes into tumor cells. Looking a genevector with high safety targeted and transduction efficiency is a research focus in recentyears. In the research, we used Salmonella phoP/phoQ mutant, which was processed bygenetic engineering, as a transporter carrying the plasmid co-expressed endostatin andsi-stat3to treat subcutaneous glioma exnograft in mice. The results showed that theattenuated salmonella able to aggregate in tumor, and the appling Endostatin andSiRNA-Stat3treatment has a synergistic effect to inhibit glioma.Thus we think using the attenuated salmonella as a vector to transport the plasmidco-expressed Endostatin and siRNA-stat3may become a novel treatment for glioma. Objective:To study the effect of the attenuated salmonella carrying the plasmid co-expressedEndostatin and siRNA-stat3on glioma exnograft in mice and explore the mechanism.Methods:1、We choose male athymic BALB/c nude mice, aged6weeks, as the experimentalanimals to established a mouse tumor model. C6glioma cells were subcutaneously injectedinto the lateral back of2mice. When the tumor grew to about1cm in diameter, we cut thetumor into2×2×2mm~3, and implanted it into the lateral back of the mice.2、When the tumor grew to about4mm in diameter, the mice were randomly dividedinto six groups (five mice/group). The mice were treated by intravenous administration asfollows:(1) PBS buffer;(2) PQ(Attenuated Salmonella);(3) Scramble (attenuatedSalmonella carrying pSi-Scramble);(4) Endostatin (attenuated Salmonella carryingp-endostatin).(5) Si-Stat3(attenuated Salmonella carrying Si-Stat3);(6) ES (attenuatedSalmonella carrying Endostatin and Si-Stat3). Tumor volumes were measured bytridimensional calliper measurements (length and width) performed every other day，and thegrowth curve was drawn.3、RT-PCR, Western Blot and Enzyme-Linked Immuno Sorbent Assay analyses wereused to determine the expression of related genes on protein and mRNA level respectively.Results:1、The average tumor volume in Endostatin, SiRNA-Stat3and ES group decreased insome extent compared with control group. However, that in the ES group decreased moreobviously.2、The results from RT-PCR and ELISA demonstrated that both of the plasmidscontaining endostatin could increase endostatin expression. The results from RT-PCTshowed that both of the plasmids containing SiRNA-Stat3could reduce Stat3expression atmRNA level. However, the results from Western Blot assay showed that all of the threeplasmids containing Endostatin, SiRNA-Stat3and ES could reduce Stat3expression atprotein level.3、The results from Western Blot assay showed that all of the three plasmids containingEndostatin, SiRNA-Stat3and ES could increase Caspase3expression; Both of the plasmidscontaining SiRNA-Stat3could inhibit the expression of C-Myc.Conclusion:The sduty demonstrated that the combined-gene therapy with co-expressed Endostatin and SiRNA-Stat3was carried by attenuated salmonella showed a synergistic suppressioneffect on subcutaneously gliomas in a nude mice model.