The Mechanism Of Wp1066 On Inhibiting Pancreatic Cancer By Targeting JAK2/STAT3

Objective To investigate the effects of JAK2/STAT3 inhibitor WP1066 on inhibiting the proliferation of pancreatic cancer cells,and explore the corresponding molecular and cell mechanisms,providing a new experimental and theoretical basis for the clinical treatment of pancreatic cancer.Methods First,the expression level of p-STAT3 in clinical human pancreatic cancer tissues and normal pancreatic tissues were detected by immunohistochemistry.Then,the effect of WP1066 on cell proliferation was evaluated by CCK-8 assay.The migration and invasion of pancreatic cancer cells were measured by scratch test and transwell.Cell apoptosis,cell cycle and MHC molecules of pancreatic cancer cells were detected with flow cytometry.RT-q PCR and western blot were used to check the genes and proteins involved in cell apoptosis,cell cycle and migration.Confocal fluorescence microscope was hired to examined the expression of Ki 67 and p-STAT3 in pancreatic cancer cells.To evaluate the potency of WP1066 on anti-pancreatic cancer in vivo,the pancreatic tumor bearing model mice were sacrificed after intraperitoneal injection of WP1066(40 mg/Kg)or equal concentration DMSO for 10 times.The tumor,liver and kidney tissues were harvested for HE staining.The peripheral blood of tumor-bearing mice was collected to test liver function in order to judge the toxicity and side effects of WP1066.The expression of MHC,PD-L1 on the surface of pancreatic cancer cells and the percentage of tumor-associated immune cells infiltrated in the tumor tissue were investigated by flow cytometry.The number of TECs cell subsets,ETPs in thymus and T cell subsets in spleen of pancreatic tumor-bearing mice treated by WP1066 were also probed by flow cytometry.Furthermore,lymphocytes isolated from spleen of mice in different groups were co-cultured with PAN02 cells for 48 h.The expression of Ki 67 in CD4~+and CD8~+T lymphocytes and the cytokines IFN-γ,IL-2 and TGF-βsecreted by lymphocytes were examined by flow cytometry to judge the potency of improving the anti-tumor immunity of WP1066.Results The expression of p-STAT3 in clinical pancreatic cancer samples was higher than that in normal pancreatic tissues(P<0.01).The proliferation of pancreatic cancer cells could be inhibited in dose-dependent after treated with WP1066 at 0μM,2.5μM,5.0μM,7.5μM and 10μM for 24 h,48 h and 72 h(P<0.01).The relatived scratch healing areas of pancreatic cancer cells were decreased(P<0.01).Meanwhile,WP1066 could inhibit the invasion of pancreatic cancer cells(P<0.01).The apoptosis of WP1066 treated pancreatic cancer cells for 24 h,48 h was increased,and the number of survival cells was decreased(P<0.05).The G2/M phase cells’number in total PANC-1 cells was increased,arresting PANC-1 cells in G2/M phase with WP1066 treated for 48 h(P<0.001).While the G0/G1 phase cells’number in total PAN02 was increased,arresting PAN02 cells in G0/G1 phase with WP1066 treated for 48 h(P<0.05).The m RNA expression levels of Mcl-1,Bcl-2,Kras,Cyclin B1,Cyclin D1 and Cyclin E1 were reduced,while the m RNA expression levels of Caspase-3 and Bax in the WP1066 48 h-treated group became higer than that in the control group(P<0.05).The total protein levels of JAK2 and STAT3 in pancreatic cancer cells treated by WP1066 had no significantly change,while the expressions of p-JAK2 and p-STAT3 were lowered,accompanied by decreased expression of Cyclin A1,Cyclin B1,Cyclin D1,Cyclin E1,Kras,Mcl-1,Bcl-2,MMP-9 and increased expression of Caspase-3,cleaved caspase-3,Bax(P<0.05).The same results about the genes and the proteins above were shown us in the transplanted tumor tissue of the tumor bearing model mice treated by WP1066.Other results of the tumor model mice after WP1066 treatment were as the following.The expression of MHCⅠand MHCⅡmolecules of tumor cells in the transplanted tumor tissue was heightened(P<0.05),while the expression of PD-L1 was lowered(P<0.05).In addition,the percentages of infiltrating CD8~+T cells,NK cells and NKT cells were increased,while the percentages of MDSCs and Treg cells were decreased(P<0.05).Furthermore,WP1066 enhanced the proliferation of TECs,c TECs and ETPs in thymus(P<0.05),and increased the number of CD4~+T cells,CD8~+T cells,CD4~+Naive T cells,CD8~+Naive T cells,CD4~+RTEs,CD8~+RTEs in spleen(P<0.05).WP1066 facilitated the expression of Ki67 in tumor specific T cells and IFN-γ,IL-2 involved in anti-tumor immune response,while inhibited the expression of inhibitory cytokines TGF-β(P<0.05).Conclusion WP1066 inhibits the proliferation of PANC-1 and PAN02 cells by blocking the phosphorylation of JAK2 and STAT3,regulating the expression of downstream genes,arresting cell cycle and inducing apoptosis.Meanwhile,it is capable of promoting the cellular immune response of anti-tumor by enhancing tumor immunogenicity and thymic function,facilitating the infiltration of anti-tumor immune cells into tumor tissue and regulating the cytokine secretion of T cells.Taken together,our data indicate that WP1066 can be a potential small molecule inhibitor against pancreatic cancer.