Celastrol-induced Apoptosis Potentiated By M1 Macrophage Products In Human Cancer Cells

Introduction Macrophages are essential cellular components in tumor microenvironment,which commonly termed tumor-associated macrophages(TAMs)and play critical roles in tumor progression,like angiogenesis,invasion,metastasis and immunosuppression.In recent years,more and more studies have shown that due to the different types of treatment and cancer model,TAMs can either promote or resist the anti-tumor effect of radiotherapy,chemotherapy and immune therapeutic agents.TAMs also mediate repair in cancers after radiotherapy or treatment by vascular targeting agents.Classically activated macrophage or M1 macrophage,which mediated by Th1 cytokines(e.g.,IFN-γ),exogenous TNF-α or endogenous TNF-α inducer(e.g.,LPS),and subsequently secreted varieties of proinflammatory factors(e.g.,IL-6,IL-12,IL-1,TNF-α),chemokines(e.g.,CCL2,CCL3)and chemokine ligands(e.g.,CXCL9,CXCL10).Therefore,M1 macrophage,as a positive effector cell can kill tumor cells and eliminate infections pathogens,and it’s also as an effective inflammatory mediator involved in the inflammatory response.Celastrol(Cel)is a natural compound with anti-inflammatory,anti-tumor and immunosuppressive pharmacological activities,which suppress angiogenesis and tumor growth by the inhibition of AKT/m TOR/P70S6 K pathway,and it also inhibit cancer cells proliferation and induce apoptosis by regulation of survival signaling pathways,such as Notch or NF-κB.Cel,as a natural proteasome inhibitor,also mediate cancer cells apoptosis via suppression of 20 S proteasomal activity,and then inhibit tumor growth.However,the role in regulation of cytotoxic chemotherapy is still not clear.This study explored the effects of lipoplysaccharides(LPS)-stimulated macrophage products(LSMP)/M1 macrophage products combined with Cel on the apoptosis of human breast carcinoma cells MDA-MB-231 and prostate carcinoma cells PC-3 and the underlying mechanisms.Cells were treated with various concentrations of LSMP(10%,30%,50%,100%)with or without Cel(1μM、1.5μM)for 0.5h-48 h.MTT assay,clonogenic assay and wound-healing assay were performed to detect the effect of LSMP and Cel on the proliferation and migration of MDA-MB-231 cells and PC-3 cells.Cells apoptosis were examined by flow cytometry.The expression levels of apoptosis-related proteins were assessed by Western blotting and q RT-PCR.Our results confirmed that Cel-induced apoptosis may be potentiated by LSMP in human breast carcinoma cells MDA-MB-231 and prostate carcinoma cells PC-3,the possible mechanisms might be closely related to up-regulation of caspase-3,-8 and-9 activities,inactivation of NF-κB signaling pathway,and subsequently down-regulation of the expression of XIAP and c IAP1/2.These findings indicate that in tumor microenvironment,macrophages can sensitize tumor cells response to cytotoxic chemotherapy.Thus,targeting macrophages will provide an effective strategy for the anti-tumor effect of cytotoxic agents.Methods 1.Cells were treated with Cel(1μM,1.5μM)or different concentrations of LSMP(10%,30%,50%,100%)alone or in combination,and MTT assay,clonogenic assay and scratch wound-healing assay were used to detect proliferation and migration of breast carcinoma cells MDA-MB-231 and prostate carcinoma cells PC-3.2.Cells were treated with Cel(1.5μM)or different concentrations of LSMP(0%,50%,100%)alone or in combination,and cells apoptosis were tested by flow cytometry.3.Cells were treated with Cel(1.5μM)or different concentrations of LSMP(30%,50%,100%)alone or in combination.Subsequently,the protein levels of PARP,pro-caspase-3,pro-caspase-8,pro-caspase-9 were determined by Western blotting at indicated times.4.Cells were treated with Cel(1.5μM)or different concentrations of LSMP(30%,50%,100%)alone or in combination.The protein levels of Ub-prs,IκB,TNF-α,cytoplasmic and nuclear transcription factor κB P65(NF-κB-P65)were determined by Western blotting at indicated times. 5.Cells were treated with Cel(1.5μM)or different concentrations of LSMP(30%,50%,100%)alone or in combination.The protein levels of XIAP and c IAP1/2 were determined by Western blotting at different times.6.NF-κB-P65 was knocked down by small interfering RNA(si RNA),the m RNA and protein levels of P65,XIAP and c IAP1/2 were measured by q RT-PCR and Western blotting respectively.Results 1.Compared with Cel treatment,LSMP combined with Cel significantly increased tumor cells apoptosis,and suppressed cells proliferation and migration.2.Compared with Cel treatment,LSMP combined with Cel promoted degradation of PARP,pro-caspase-3,pro-caspase-8 and pro-caspase-9.3.Compared with Cel treatment,LSMP combined with Cel had no effect on proteasomal inhibition.4.Compared with Cel treatment,LSMP combined with Cel significantly suppressed the expression of XIAP and c IAP1/2.5.Compared with Cel treatment,LSMP combined with Cel suppressed the degradation of IκB,nuclear translocation of NF-κB-P65,and inhibited the activation of NF-κB signaling pathway,as well as decreased the level of TNF-α.6.Compared with LSMP+Vector si RNA treatment,LSMP combined with Cel significantly suppressed the m RNA and protein levels of NF-κB-P65,XIAP and c IAP1/2.Conclusions In the present study,all of these results suggested that the suppressing effects of Cel on the cell proliferation,survival and migration were promoted by LSMP in human breast cancer cells MDA-MB-231 and prostate cancer cells PC-3.LSMP had little effect on Cel-mediated proteasomal inhibition and Cel-induced apoptosis was enhanced by LSMP.The underlying mechanism appears to be through inhibiting the degradation of IκBα,nuclear translocation of NF-κB-P65,as well as specifically suppressing the activation of NF-κB signaling pathway and subsequently down-regulating the expression of inhibitor of apoptosis protein XIAP and c IAP1/2.Simultaneously,promoted degradation of PARP,pro-caspase-3,pro-caspase-8 and pro-caspase-9 and activated apoptotic pathway,ultimately induced apoptosis.