Effect And Mechanism Of IAP Antagonists On Autophagy In Tumor Cells

Induction of tumor cell apoptosis is considered to be an important therapeutic strategy for cancer.In recent years,induction of autophagy and necroptosis has become a new direction in anti-tumor drug development.However,the relationship between apoptosis and autophagy is complex and controversial.Although they have some common regulators,the regulatory mechanism is still dim.Therefore,exploring the interaction between apoptosis and autophagy induced by anti-tumor drugs is of great value for clarifying their regulatory mechanism and discovering potent anti-tumor drugs.Inhibitor of apoptosis proteins(IAPs)are closely related to tumor development as important inhibitors of cellular apoptosis.IAP antagonists have entered into different phases of clinical researches as anti-tumor drugs.A large number of basic and preclinical studies have demonstrated that IAP antagonists selectively bind to the BIR3 domain of XIAP,cIAP1,and cIAP2,and activate endogenous and exogenous apoptosis pathways to induce tumor cell apoptosis.It is worth noting that in recent years,some studies found that several IAP antagonists and their target proteins XIAP and cIAP1/2 were involved in the regulation of autophagy in tumor cells,but the regulatory mechanism is quite different and undefined.As early as 2003,our group found that apart from inducing apoptosis in tumor cells,IAP antagonists also exhibited regulatory activity on autophagy,and showed tumor cell specificity.Based on the unique role of IAP antagonists in inducing apoptosis and their newly-found participation in autophagy regulation,this study systematically explored the autophagy regulatory mechanism of IAP antagonists in order to further elucidate the anti-tumor mechanism of IAP antagonists,reveal the relationship between apoptosis and autophagy,and provide a theoretical basis for the development of related anti-cancer drugs.This study was divided into two parts.The first part of this study mainly focused on the novel IAP antagonist WX20120108(an analogue of GDC-0152),which was synthesized in our institute and exhibited strong anti-tumor activity and potent autophagy regulatory activity in our preliminary screening.At first,we utilized virtual molecular docking technique,IAPs affinity and function assay,NF-?B pathway activation assay,and apoptosis-inducing activity assay to confirm that WX20120108 is a novel IAP antagonist with the biological characteristics of typical IAP antagonists.Then,the role and mechanism of WX20120108 in regulating tumor cell autophagy were explored on HeLa and MDA-MB-231 cells.Imaging-based HCA,Western blot assay and transmission electron microscopy observation revealed that WX20120108 dose-(1-30 ?M)and time-(6-48 h)dependently increased LC3 B puncta or LC3B-II formation and formed well-defined autophagosomes in cancer cells,the expression of p62 protein and mRNA increased as well.CQ co-incubation significantly enhanced LC3B-II levels after both short-term and long-term WX20120108 exposure.In addition,WX20120108 promoted the fusion of autophagosomes with lysosomes and significantly increased the activity of lysosomal cathepsin B(CTSB)and CTSD.The screening results of the twelve EGFP-tagged reporter cell lines stably expressing autophagy initiation-associated signaling proteins revealed that WX20120108 selectively activated Foxo3 in a dose-dependent manner and upregulated the expression of Foxo3 target genes,Bnip3,Pik3c3,Atg4 b,and Atg5,which are key genes in autophagy induction.Further screening of regulatory factors for Foxo3 activation revealed that WX20120108 dose-dependently promoted cellular ROS release,and catalase(a known ROS scavenger)suppressed Foxo3 activation and LC3B-II formation induced by WX20120108.In addition,transfection with siRNA against cIAP1/2 and not against XIAP partially impaired WX20120108-induced autophagy,but silencing XIAP and cIAP1/2 affected neither ROS release nor Foxo3 activation induced by WX20120108.This study also found that cell count declined with the increase of WX20120108-induced autophagy levels,which was not affected by Z-VAD-FMK,a pan-caspase inhibitor.But Z-VAD-FMK rescued cell loss caused by WX20120108.Taken together,as a novel IAP antagonist,WX20120108 induced autophagy in HeLa and MDA-MB-231 cells,and its autophagy regulatory effect depended partly on cIAP1/2.IAPs-independent ROS-Foxo3 pathway was also involved in the tumor cell autophagy induced by WX20120108.Besides,WX20120108 induced autophagic cell death ahead of apoptosis and it still needs further exploration of the way autophagy promotes apoptosis.The second part of this study mainly concerned on the autophagy regulatory activity and mechanism of the monovalent IAP antagonist GDC-0152 and the bivalent IAP antagonist TL32711,both of which have entered into clinical trials.GDC-0152 and TL32711 were found to have autophagy regulatory activity in HeLa and MDA-MB-231 cells via immunofluorescent HCA,Western blot assay and transmission electron microscopy observation.Further,the results of autophagic flux assay,detection of autophagic substrate p62 protein and mRNA levels,autophagosome and lysosomal co-localization analysis,and lysosomal enzyme activity assay demonstrated that GDC-0152 and TL32711 induced autophagic flux as autophagy inducers.And TL32711 had a stronger activity in inducing LC3B-II formation than GDC-0152 when co-incubated with CQ.Autophagy induced by GDC-0152 was greatly inhibited when cIAP1/2 were interfered via RNAi technique,while knocking down either XIAP or cIAP1/2 impaired the autophagy-inducing activity of TL32711.The parallel dynamic observations on cellular autophagy regulatory level and cell survival showed that as the concentration of IAP antagonists increased or the incubation time prolonged,number of cells decreased accompanied with the increase of cellular LC3 B level and there was an evident ‘switch' from autophagy to cell death.Patterns of cell death induced by GDC-0152 and TL32711 were evaluated by Flow cytometry,PI/Hoechst 33342 fluorescent staining HCA and cellular lactate dehydrogenase release assay,in combination with pan-caspase inhibitor Z-VAD-FMK,necroptosis inhibitor necrostatin-1,and autophagy inhibitor 3-methyladenine.GDC-0152 and TL32711 induced caspase-dependent apoptosis and necroptosis,and autophagy regulatory activity of GDC-0152 and TL32711 was not affected by their apoptosis inducing activity.Given that GDC-0152 and TL32711 increased autophagic flux without decreasing p62,p62 mRNA levels,co-localization of p62 and lysosomes,co-immunoprecipitation analysis of p62 and RIP1 were further tested.The results revealed that GDC-0152 and TL32711 increased p62 mRNA levels to different extent,TL32711 promoted the co-localization of p62 with lysosomes,and GDC-0152 induced the interaction of p62 and RIP1.In summary,the above results showed that monovalent IAP antagonist GDC-0152 and bivalent IAP antagonist TL32711 induced autophagy in a cIAP1/2-dependent and a XIAP-and cIAP1/2-dependent manner,respectively.They induced apoptosis via promoting autophagy,and induced a certain degree of necroptosis as well.GDC-0152 promoted the formation of necrosome and induced the occurrence of necroptosis by mediating the interaction of p62 with RIP1 protein.This study systematically explored the autophagy regulatory effect and mechanism of different IAP antagonists for the first time.In summary,the major conclusions are as follows:1)IAP antagonists induced autophagy in tumor cells of their clinical indications;2)IAP antagonists-induced autophagy in tumor cells involved both target-dependent and target-independent mechanisms,which are determined by the chemical structures of IAP antagonists;3)continuous autophagy induced by IAP antagonists promoted apoptosis;in addition,necroptosis induced by IAP antagonists was closely related to p62.