Piperlongumine Upregulation The Anti-tumor Activity Of PD-1 Inhibitor Via Immunogenic Cell Death Pathway In Prostate Cancer

Background:Prostate cancer is the second most frequent cancer diagnosis made in men and the fifth leading cause of death worldwide.Castration-resistant prostate cancer(CRPC)which is characterized by poor prognosis and high lethality is a main cause of deaths.Treatment include hormonal agents,immunotherapy with Sipuleucel T,chemotherapy,and radi?m-223.However,benefits are rarely durable,and none of these therapies are curative.More effective treatment is urgently needed.Cancer immunotherapy has greatly advanced in recent years,and PD-1/PD-L1 blocking therapy has become a major pillar of immunotherapy.Successful clinical trials of PD-1/PD-L1blocking therapies in cancer treatments have benefited many patients,but it shown largely disappointing results in patients with CRPC?Because the cytotoxic T cell have been excluded by components of the prostate tumor microenvironment.Immune T cells have been unable to penetrate such tumors.Immunogenic cell death(ICD)could increase the number of T lymphocytes as well as an increased ratio of cytotoxic CD8~+T lymphocytes within the tumor after some special chemotherapy therapy.Combination therapy with ICD and PD-1 antibody may provide improvements in efficacy by enhancing adaptive immunity,altering the tumor immune microenvironment to promote anti-cancer immune responses through T cell activation and tumor-antigen generation manipulating the immune system to turn CRPC into immunoresponsive cancers could prove to be an alternative strategy.This could enable the promise of immunotherapy to produce long term durable responses and extend overall survival in CRPC to be realized.Part one--Piperlongumine Induced the Apoptosis of Human Prostate Cancer Cells Through the Induction of ROS--ER stress PathwayObjective:The purpose of this study was to find out the anti-tumor effect and mechanisms of Piperlongumine on prostate cancer cells in vitro.Method:1.The prostate cancer cell lines PC3,DU145,RM-1 were treated with different doses of PL(100--3?M),MTT assay and colony formation were used to investigate the effect of PL on cell proliferation inhibition.The flow cytometry,fluorescent probe and Western Blot assays were applied to measure cell-mediated cytotoxicity.2.The prostate cancer cells were treated with both NAC and PL,either individually or in combination,Reactive oxygen species(ROS)generation and activation of caspase-3were detected by fluorescent probe or flow cytometry,cleaved caspase 3 analyzed by Western Blot,to measure the role of ROS in anti-proliferation of PL.3.Western Blot was used to detect the p-EIF2??ATF4?chop expression on cells lysate induced by PL.PC3 cells were pretreated with Chop siRNA or NAC and cultured with PL,flow cytometry,Western Blot and fluorescent probe assays were used to analysis apoptosis,Western Blot were used to investigate the p-EIF2??ATF4?chop expression,the aim was to determine the ROS-endoplasmic reticulum(ER)stress pathway in PL treated prostate cancer cells.Results:1.PL significantly inhibited prostate cancer cells proliferation and colony formation in a dose dependent manner,via induction of apoptosis.2.PL induced cells apoptosis though upregulating the induction of ROS.3.PL induced ER stress though EIF2?–ATF4--Chop pathway and ROS-ER stress pathway to induce the cells apoptotic cell death.Conclusion:PL mainly activated the ER-stress pathway by inducing oxidative stress in prostate cancer cells and caused apoptosis by up-regulating the expression of chop protein.Part ?—Mechanism of PL enhancing the anticancer effect of PD-1 inhibitor Objective:The aim of this study was to investigate the mechanisms and immunogenicity of PL induced cell death through in vivo and in vitro experiments,and to evaluate whether PL can improve the anticancer effect of PD-1 inhibitors in prostate cancer in vivo experiments.Method:1.The different doses of PL on human DU145,PC3 and mice RM-1 prostate cancer cells were examined in cell culture,and the cell surface expression of calreticulin was analyzed by flow cytometry,the extracellular ATP and HMGB1 concentration measured by fluorescent probe and enzyme-linked immunosorbent assay(ELISA),to determine the DAMP expression.The PC3 cells,pretreated with NAC or CHOP siRNA,were treated with or without PL and then examined for CRT,ATP and HMGB1 expression,to determine the role of ER stress and ROS in mediating induction of ICD by PL.2.The murine RM-1 cell model of prostate cancer was used to examine the efficacy of PL in mouse vaccine experiment.21 male C57BL/6 mice random separated into 3groups,7 mice per group.Group 1:Injection the tumor cell vaccine pretreated with PL.Group 2:Injection the tumor cells pretreated with 5-fu.Group 3:PBS.They were injected subcutaneously in the shaved left flank of each mouse with different treated3x10~5 RM-1 cells.One-week postvaccination,2X10~6 living RM-1 cells suspension were injected subcutaneously in the shaved right flank of each mouse.To do so,measure RIGHT tumor size every 3 days using a caliper.Each mouse had been euthanized when the tumor volume reaching 2000mm~3,to graph tumor-free survival,tumor growth.3.To determine the anti-tumor effect of PL in vivo.RM-1 cells were injected subcutaneous into male 6--8 weeks C57BL/6 and male NSG mice.Fourteen days post-injection,when the tumor volume reaching 50 mm~3,12 C57BL/6 mice random allocate to 2 groups.Group 1:animals received PL,Group 2:PBS as negative control.6 NSG mice as Group 3:animals received PL as comparison.When the tumor volume of one mouse reached 2000 mm3,all of mice euthanized together and graphed tumor growth curve.We performed flow cytometric and immunofluorescence to analysis of the CD4,CD8,DC,Macrophages cells isolated from fresh spleen tissues,tumor tissue obtained from every mouse.4.After 14 days of RM-1 tumor cells implantation,20 C57BL/6 mice were randomly separated into 4 groups,5 mice per group:Group 1:mice were injected with IgG isotype i.p.once per week,totally 3 weeks;Group 2:PL,twice per week,intratumorally injection,totally 3 weeks;Group 3:anti-mouse PD-1.i.p.once per week,totally 3 weeks;Group 4:PL combined with antimouse PD-1.Each mouse will be euthanized when reaching ethical endpoint,to graph overall survival.Results:1.PL inhibited cell proliferation and enhanced calreticulin,ATP,HMGB1 expression on PC3 DU145,RM-1 cells in a dose-dependent manner,mainly via activating ROS-dependent ER stress pathways.2.Antitumor vaccines based on prostate cancer cells RM-1 pretreated with PL also exhibited good anticancer ability at the tumor cells rechallenge experiment.The tumor volume in vaccine of RM-1 cells pretreated with PL group regressed 73%and in 5-fu pretreated group regressed 9%than control(PBS)group,there were 3 mice tumor free in totally 7 mice in PL vaccine group.3.PL administered on C57BL/6 mice regression tumor burden by 69%,as compared to 24%in NSG animals.The number of CD8+T-lymphocytes was significantly higher in the mice tumor and spleen that received PL-treated than PBS group.The number of DC cells was significantly higher in the mice that received PL-treated than PBS group.4.Mice treated with anti-PD-1 alone exhibited significant reduction 50%in tumor volume without obvious improvement in overall survival,PL treatment alone had strong anti-tumor effect but not on survival too.In contrast,combination treatment with anti-PD-1 plus PL significantly slowed tumor growth 89%and there were 2 mice tumor free in totally 5 mice in combination group.The combination treatment prolonged survival compared to either vehicle or single treatment groups.Conclusion:PL can boost therapeutic efficacy of anti-PD-1 immunotherapy against prostate cancer through ICD pathway.It is a promising treatment.