Anti-tumor Effects And The Mechanism Of The Combination Of VNP20009-DNase I Engineering Stain And Triptolide

Background and objective:Attenuated Salmonella typhimurium VNP20009 is a mutant stain with targeting and suppressing tumor effects.However,studies indicated that tumor hypoxia microenvironment could significantly reduce the survival of VNP20009 following the inhibition of tumor,resulting in attenuation of the anti-tumor effect of VNP20009.In addition,the human immunity and relative strong toxicity of attenuated VNP20009 severely limited its clinical application in tumor suppression.Eukaryotic expressed VNP20009-DNase I engineering strain was constructed and the combined with triptolide(TPL)in order to enhance the colonization and its anti-tumor activity.This strategy of the combination application should provide an experimental evidence for its clinical application.Methods:1.The recombinant gene DNase I was introduced into VNP20009 by electroporation,and then the activity of cleavage DNA was verified by DNA digestion experiment;2.MTT assay was used to detect the proliferation ability of melanoma cells induced by VNP20009-DNase I,TPL and the combination of VNP20009-DNase I with TPL,and cell scratch test was used to detect wound healing ability.3.Annexin V and PI double standard flow cytometry was used to test the apoptosis ability.4.DAPI staining was used to detect the apoptosis of the cells.5.Mouse melanoma model was prepared for evaluating the effects of VNP20009-DNase I combined with TPL on tumor size and survival of mice.6.Western blot was used to detect the death-and inflammation-related proteins in tumors.7.Inflammatory factors of IL-1? and TNF-? in serum were detected by ELISA.8.Inflammatory factors of IL-1?,TNF-a and IL-6 in tumor tissues were detected by q-PCR.Results:1.DNA digestion experiments showed that VNP20009-DNase I could completely cleave DNA.2.Both VNP20009-DNase I and TPL could significantly inhibit the growth and migration of B16F10 cells.3.TPL could markedly enhance the apoptosis of B16F10 cells induced by VNP20009-DNase I.4.TPL treatment significantly improved the VNP20009-DNase I colonization ability in tumor.5.VNP20009-DNase I combined with TPL significantly inhibited tumor volume,prolonged mouse life-span,and enlarged the necrotic area of melanoma.6.The combined therapy could up-regulate the expression of Bax/Bcl-2 and Caspase-3 and down-regulate the expression of p-AKT/AKT in TLR4/NF-?B signaling pathway.7.TPL also down-regulated the expressions of the pro-inflammatory factors induced by VNP20009-DNase I.Conclusion:1.VNP20009-DNase I significantly inhibited the growth,infiltration and migration of mouse melanoma tumor compared with wild-type VNP20009.2.TPL significantly increased the colonization ability of VNP20009-DNase I in tumor tissues.3.The combination of VNP20009-DNase I and TPL enhanced the anti-tumor activity compared with the efficiency of each used alone,and the possible mechanisms might be related with upregulating the expressions of Bax/Bcl-2 and Caspase-3,downregulating the expressions of p-AKT/AKT,and inhibited the expressions of the pro-inflammatory factors induced by VNP20009-DNase I.