Analysis The Mechanisms Of Autophagy And The Transition Of Autophagy/apoptosis In Cells That Induced By Polychlorinated Biphenyls

Polychlorinated biphenyls(PCBs)are some kind of Organic pollutants,which are ubiquitous in nature.Although PCBs was banned in the 1970 s for its endocrine toxicity,immune toxicity and neurotoxicity.PCBs have still been found in various environment include in Aquatic systems and Land systems.Autophagy is an Self digestion progress that transport the damaged and caducity organelles to lysosomal,where the organelles are degraded.The feature of this progress is the appearance of autophagosome.So the autophagy was known as a self protective progress.The main point of this research was to analysis the specific mechanism of how PCB29-pQ activate the autophagy,as well as the conversion relationship between autophagy and apoptosis.Part ?: In this part we research the molecular mechanism of how the autophagy happened that induced through the mTOR/p70S6 k signal path by PCB29-pQ.In the research we found out the autophagy induced by PCB29-PQ have no difference between HepG2 and MDA-MB-231 cell,which means this progress may have no specific cell effect.At first,cells were deal with 5 ?M PCB29-pQ for 24 h before the detection.Then ultrastructure feature of autophagic vacuole of these two kinds of cells were detected by Transmission electron microscopy(TEM),what's more the formation of autophagic vacuole were obviously obeserved after dyed by AO,MDC,from which we can prove the cell autophagy can be induced by PCB29-pQ.After that,the molecular mechanisms of the autophagy induction were analyzed through the three phases of the formation of autophagy.In the first stage,the expression of the negative regulation switch protein mTOR and p70S6 k were reduced both in time gradient and concentration gradient in the effect of PCB29-pQ,which means the autophagy was activated.In the second stage,the expression of the ATG5,ATG12,LC3 were detected,what's more the mRNA level of LC3 were analyzed by RT-PCR.Compared to control groups,the autophagy specific gene that deal with PCB29-pQ were obviously improved,the peak value were observed after deal by 5 ?M PCB29-pQ for 24 h.Meanwhile the level of autophagic substrate p62 were reduced in time gradient,the lowest value were observed after deal by 5 ?M PCB29-pQ for 24 h.The result suggest that the formation of autophagy and the degradation substrate can be activited by PCB29-pQ.In the third stage,the turnover assay were used to investigate LC3B-II levels in the absence and presence of CQ.The result suggested that PCB29-pQ provoked LC3B-II netflux.Interestingly,the most effective concentration for the upregulation of LC3B-II netflux is 5 ?M.Moreover,we transfected GFP-LC3,by AVs,lysosomes which colocalized in HepG2 and MDA-MB-231 cells.We clearly demonstrated that PCB29-pQ provoked autophagosome-lysosome fusion and enhanced autophagic flux in both cell lines.Then,we explored the role of autophagy in PCB29-p Q-induced cytotoxicity.CCK8 assay,flow cytometry and the expression of caspase3 were used,after incubate with autophagy inhibitors 3-MA and CQ.The result suggest that inhibition of autophagy enhanced the cytotoxicity of PCB29-pQ in these cells.Finally,we evaluated the effect of NAC(a scavenger of ROS)on autophagy,the findings presented here strongly support a role for ROS in PCB29-pQ-induced autophagy.According to the whole research,these results revealed that PCB29-pQ activates the process of autophagy in both HepG2 and MDA-MB-231 cells,and they were mediated by ROS production.Its showed that mTOR/p70S6 k and ATG5/ATG12/LC3 signaling is involved in PCB29-pQ mediated autophagy,suggesting that autophagy serves as pro-survival machinery that plays a protective role in the early stage of PCB29-pQ-induced insult.Part ?: From our research,we can see that a lower concentration of PCB29-pQ induced autophagy and a higher concentration of PCB29-pQ induced apoptosis in HepG2 cells.So whether the activity of calpain is effectivein this process or not.First,we investigatedthe mitochondrial membrane potential(MMP)detected by JC-1 fluorescence probe.Western blot was used to detect the expressions of caspase9/caspase3.TUNEL to detect the apoptosis.Immunofluorescence analysis of LC3.The results showed that the level of apoptosis was concentration-dependent,and the concentration of autophagy was the highest at 5 ?M PCB29-pQ treatment compared with the control group.Then,flow cytometry was used to detect changes of theintracellular calcium level and spectrofluorometer was used to detect the calpain activityinduced by PCB29-pQ.The results showed that PCB29-pQ induced intracellular calcium accumulation and calpain activity,whichwere significantly inhibited by the pretreatment of BAPTA-AM(Ca2+chelator).According to the research,Atg5 and Beclin1 could be cleavaged by calpain,turn to tAtg5 and Beclin1-c,they can move to mitochondria and induce apoptosis,which were represents a molecular switch between autophagy and apoptosis.In our study,we can not find this phenomenon.From the whole research,PCB29-pQ induced autophagy at a lower concentration and induced apoptosis at a higher concentration in HepG2 cells,at the same time,we can find the elevation of intracellular Ca2+ levels and calpain activity.But the calpain activity can not cause ATG5,Beclin1 produced cleavage to transmit mitochondria,which switchthe signal from autophagy to apoptosis.Part ?:To further explored the mechanism of the switching of PCB29-pQinduced autophagy to apoptosisin HepG2 cells,we analyzed the p53/HMGB1 protein which were regulated autophagy and apoptosis.Western Blot and immunofluorescence assay showed that p53/HMGB1 translocated to cytosolic in HepG2 cell that induced by PCB29-pQ.We found increased complex formation between p53 and HMGB1 following 5 ?M PCB29-pQ in nuclear by immunoprecipitation assay,and the interaction following 15 ?M PCB29-pQ in cytosol.p53/HMGB1 can be used as nuclear transcription factor to regulate the expression of target genes,which were affecting autophagy and apoptosis.we conducted the expression of DRAM?ULK1?Bax in p53 knockdown HepG2 cells,and the expression of HSPB1 in HMGB1 knockdown cells.The results showed that as inhibiting p53/HMGB1 as inhibiting the expression of the target gene regulated by the protein.To determine the interaction between p53 and HMGB1 response to cell autophagy and apoptosis,we inhibited p53/HMGB1 tested.Western Blot showed that p53 siRNA cells had increased levels of HMGB1 in the cytosol,the cell viability assay showed that p53 siRNA inhibited PCB29-pQ-induced apoptosis,immunoprecipitation assay's LC3 focus showed that p53 siRNA promoted PCB29-pQ-induced autophagy,while adding HMGB1 inhibitor EP,it was increased the level of autophagy.These results suggested that translocation to the cytosol of HMGB1 plays a role in inhibiting apoptosis and promoting autophagy.The same experimental method was used to demonstrate that HMGB1 siRNA promoted PCB29-pQ-induced p53 translocation to cytosol,and promoted PCB29-pQ-induced apoptosis,and inhibited PCB29-pQ-induced autophagy.While adding p53 inhibitor PFT-?,the level of apoptosis was decreased,the level of autophagy was increased.These results suggest that translocation into the cytosol of p53 plays a role in promoting apoptosis and inhibiting autophagy.The research revealed that PCB29-pQ enhanced p53/HMGB1 complexes which regulate the cytoplasmic localization of the p53 and HMGB1 binding reciprocal protein and subsequent levels of autophagy and apoptosis.