The Role And Mechanism Of Plumbagin In Pancreatic Cancer And Differentiation Of Myeloid-Derived Suppressor Cells

Background&Objective:Plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone;PLB),is a naturally occurring naphthoquinone isolated from the roots of Plumbaginaceae plants.It has been reported that Plumbagin exhibits powerful anticarcinogenic,anti-inflammatory,analgesic and antimicrobial activities in vitro and in vivo.Plumbagin shows anticarcinogenic effects via multi-channeled molecular mechanisms,including the induction of apoptosis and autophagy,the disruption of cell cycle,the inhibition of invasion and metastasis,and anti-angiogenesis.But the effect of Plumbagin on tumor immune microenvironment is not fully understood.In the tumor microenvironment,tumor cells produce various cytokines advantageous to the recruitment of immune inhibitory cells.The immune inhibitory cells can inhibit the body's immune response,make cancer cells evade the host immune system attack.Myeloid-derived suppressor cells(MDSCs)play a critical role in promoting immune tolerance and lesion growth,and contribute to tumor immune escape.In this study we investigated whether Plumbagin can modulate the differentiation and function of MDSCs directly,and then modulate the tumor immune microenvironment especially on MDSCs.The molecular mechanisms that mediate this regulation are also studied.Methods:We use inducement and differentiation model of peripheral blood mononuclear cell(PBMC)to investigate the influence of different concentrations of Plumbagin on MDSCs.Then,in the in vivo study,Panc02 cells were orthotropic implanted in C57BL/6 mice for model of pancreatic cancer in mice.Following teated with Plumbagin by intraperitoneal injection,the inhibitory effects of Plumbagin on the development of pancreatic cancer were evaluated by the volume and weight of tumor and ascites.The immune cell sub-population(CD4~+T,CD8~+T,NKT,??T)and myeloid-derived suppressor cells(MDSCs) infiltrated in murine spleen,bone marrow and tumor tissues were detected by flow cytometry to explore the regulation of Plumbagin on tumor immune microenvironment.The analysis of MDSCs cell surface markers,inflammatory cytokine srcretion and the migration ability were conducted by flow cytometry and Transwell.STAT3,the downstream effector of IL-6,plays an important role in the process of MDSCs differentiation,we detect the effect of Plumbagin on the phosphorylation of STAT3 in pancreatic cancer cells and MDSCs by Western Blot.Results:In the differentiation model of peripheral blood mononuclear cell(PBMC),compared with the positive control group,Plumbagin can inhibit the differentiation of MDSCs in a dose-dependent manner.Meanwhile,Plumbagin inhibits the expression of ARG1 and NO in MDSCs,and reduces the inhibitory effect of MDSCs on T cells.In the model of in situ pancreatic cancer in mice,Plumbagin can significantly inhibit the growth of pancreatic tumor,promote the anti-tumor immune cell sub-population(CD4~+T,CD8~+T,NKT,??T)infiltrated in tumor tissues and inhibit the recruitment of myeloid-derived suppressor cells(MDSCs)in murine spleen,bone marrow and tumor tissues contrast with control.We also found Plumbagin can inhibit the expression of CCL2 in human pancreatic cancer cells(Panc-1,BxPC3)and murine pancreatic cancer cells(Panc02).Plumbagin inhibits MDSCs expressing inflammatory cytokines and inhibits the expression of CCL2 to further inhibit the migration ability of MDSCs.We also used Western Blot to show that the level of phosphorylation of STAT3 in pancreatic cancer cells and MDSCs cells was down-regulated by Plumbagin.Conclusion:Plumbagin exhibits suppressive effects on pancreatic cancer developmentthroughinhibitingthedifferentiationandfunctionof immunosuppressive cells MDSCs,promoting the infiltration of anti-tumor immune cell subpopulation in tumor immune microenvirenment and inhibiting the recruitment of MDSCs to tumor site.Plumbagin is able to regulate MDSCs directly or inhibit the expression of inflammatory cytokines such as CCL2 and IL-6 accordingly inhibit the differentiation and function of MDSCs indirectly.These results suggest that Plumbagin may be used as a new drug for pancreatic cancer and also provide new scientific basis for immunotherapy of pancreatic cancer.