The Research Of Inhibitory Effect On Laryngeal Cancer Through Apoptin Which Was Mediated By Attenuated Salmonella

Based on our previous study, here we using attenuated Salmonellavector system, construct recombinant attenuated Salmonella withtumor-specific proliferation and tumor-specific inhibition capacity. Objective:To investigate the anti-tumor effect recombinant attenuated Salmonella onHep-2human laryngeal carcinoma cells cultured in vitro, study its inhibitoryeffect on nude mice model bearing human laryngeal cancer. The researchmay lay a sound foundation of developing medicine candidates in order to getnew type gene that can be safety, targeted, high efficiency and without drugresistance theoretically and physically, and provide basic and applied researchin gene therapy of accumulated experience laryngeal, and accumulateexperience for fundamental and applied research of Laryngeal cancer genetherapy.Method:Salmonella were transformed by power and selected byantibiotic, we get recombinant attenuated Salmonella which can produceapoptin，named rC-Apoptin. Hep-2cells were cultured in vitro and infectedby rC-Apoptin. The expression of exogenous gene Apoptin was verified byWestern bolt, RT-PCR and other methods. The anti-tumor effects on Hep-2cells in vitro was measured through Methylthiazolyl-tertrazolium bromide(MTT)staining to detect. AO/EB staining was done to test and verifyrC-Apoptin can induce Hep-2cells apoptosis. A syngeneic nude murine tumor model was used to examine the anti-tumor effect of the recombinantbacteria in vivo. Recombinant bacteria were introduced into the mice bodyby tail vein injection. By observing tumor inhibition rate, survival rate,growth trend to detect the anti-tumor effect of the recombinant bacteria invivo. Solid tumors were isolated from the nude murine human laryngealmodels,using an optical microscope observe tissue sections of tumors tolearn rC-Apoptin’s inhibitory effect on solid tumors. Pathologicalexamination and bacterial distribution analysis were used to learn how theattenuated Salmonella distributed in the mice body and influence on otherorgans.Results: The exogenous gene (Apoptin) carried by recombinantattenuated salmonella can be effectively expressed in Hep-2cells.Recombinant salmonella can inhibit Hep-2cells cultured in vitro, and thedose of bacteria has certain effects on inhibition rate, Between differentinfectious dose, high dose of recombinant attenuated Salmonella show abetter inhibition rate on Hep-2cells. The rC-Apoptin can effectively inhibitthe growth of laryngeal cancer in vivo. Systemic injection of the recombinantbacteria caused significant tumor growth delay, thereby extending hostsurvival. With re-dosing of Apoptin-expressing bacteria significantlyincreased the tumor doubling time (TDT) and lagged tumor growth delay(TGD). Repeated injections of recombinant attenuated salmonella canobviously prolong the tumor doubling time (TDT, tumor doubling time) to extend the time of tumor hysteresis (TGD, tumor growth delay). Systemicinoculation Apoptin-expressing bacteria resulted in the preferentialcolonization of bacteria within the tumor at a1,000-fold increase comparedto liver tissue, and the recombinant attenuated salmonella has no adverseimpact on organs such as lung and kidney.Conclusions: We successfully construct recombinant attenuatedSalmonella with tumor-specific proliferation and tumor-specific inhibitioncapacity. The attenuated salmonella-mediated Apoptin can induce Hep-2cells apoptosis in vitro. The rC-Apoptin can effectively inhibit the growth oflaryngeal cancer in vivo and extending host survival. Our findings indicatedthat the use of recombinant Salmonella as an Apoptin expression vector haspotential Laryngeal cancer therapeutic benefits.