Blockage Of Autophagy Pathway Enhance Tumor-targeting Salmonella Mediated Cancer Cell Killing

Cancer has become a severe disease that seriously threat human health. To date, the main treatments of tumors are surgical operation, chemotherapy, radiotherapy. But many treatments such as radiotherapy, chemotherapy, often cause serious side effects because of their poor tumor targeting. As innovative anticancer strategy, tumor-targeting bacteria have many advantages such as high degree of tumor targeting, efficient replication in tumor and suppress the growth of tumor. Tumor-targeting salmonella, Al-R and VNP20009, as the classic strains of tumor-targeting salmonella, not only can replicate in large solid tumors, but also can grow in small metastases, and inhibit the tumor growth. Previous studies show that tumor-targeting bacteria significantly suppress the growth of tumors, but rarely cure tumors, indicating that tumor itself possesses resistance mechanisms to the bacteria treatment.Autophagy is a type of cellular catabolic degradation response to nutrient starvation or metabolic stress. The main function of autophagy is to maintain intracellular metabolic homeostasis through degradation of unfolded or aggregated proteins and organelles. Studies find that some chemotherapy of cancer induced autophagy, and autophagy led to the drug resistance. Furthermore, inhibition of autophagy can restore the chemosensitivity of tumor and enhance tumor cell killing. Therefore, blockage of protective autophagy in tumor represents a new tumor combination therapy and chemosensitization strategy.In this study, we evaluate the role of autophagy in the process of tumor-targeting salmonella killing the tumor cells, and then explore the new strategy that enhance tumor-targeting salmonella mediated anticancer therapy through regulating autophagy pathway.Tumor-targeting Salmonella infection in tumor cells induces autophagyTo access tumor-targeting salmonella infection in tumor cells can induce autophagy, we use salmonella Al-R and VNP20009infected human liver cancer cells Hep G2and Huh7, and gastric carcinoma cells AGS. The infection of Al-R and VNP20009induces the accumulation of LC3II (LC3II/LC3I ration increase, which is one of the classical hallmarks of autophagy). Under fluorescence microscope, salmonella infection in tumor cells can induce the accumulation of autophagic punctuative distribution of membrane-associated lapidated LC3II in tumor cells. In addition, infection of AIR and VNP20009leads to the proportion of cells containing acidic vesicular organelle (AVO) increased, another classical hallmark of autophagy. These above results show that Al-R and VNP20009infection induces autophagy in human cancer cell lines.Autophagy inhibit the replication of tumor-targeting Salmonella in infected-cells To illustrate the role of autophagy to the replication of tumor-targeting salmonella, we block the autophagy pathway via siRNA to decrease the expression of autophagy related gene Atg5and Beclin1in human liver cancer cells Hep G2and Huh7. The blockage of autophagy pathways increase the titer of the intracellular bacteria, suggesting autophagy inhibits the replication of tumor-targeting salmonella. Moreover, a pair of mouse embryonic fibroblast cells, autophagy-complete MEF-Atg5(+/+) and autophagy-deficient MEF-Atg5(-/-) were employed to validate the role of autophagy. Finally we found autophagy-deficient MEF-Atg5(-/-) showed greater intracellular Al-R and VNP20009burden compared with autophagy-complete MEF-Atg5(+/+). Both experiments indicate that autophagy inhibits the replication of salmonella AIR and VNP20009in infected cells.Blockage of autophagy pathway enhanced tumor-targeting Salmonella mediated cencer cell killingAccording to the discovery of blockage of autophagy pathways can significantly enhance the intracellular replication of tumor-targeting salmonella, we propose that blockage of autophagy pathway can enhance tumor-targeting salmonella mediated cancer cell killing. To verify this hypothesis, we block the autophagy pathway of human cancer cells Hep G2and Huh7via siRNA to decrease the expression of autophagy related gene Atg5and Beclin1. We found that blockage of autophagy via RNA interference enhances tumor-targeting salmonella mediated cancer cell killing. Finally, we also found blockage of autophagy pathway via autophagy inhibitors chloroquine, bafilomycin A1can enhance tumor-targeting salmonella mediated cancer cell killing.In summary, the induction of autophagy response serves as a new interaction model of tumor-targeting salmonella and tumor cells, as well as a general resistance mechanism to the tumor-targeting bacteria treatment. And then this study preliminarily confirmed blockage of autophagy pathway can enhance tumor-targeting salmonella mediated anticancer therapy.