The Mechanism Of Autophagic Cell Death And Autophagy-dependent Apoptosis In Perfluorooctane Sulfonate–treated HepG2 Cells

Perfluorooctane sulfonate(PFOS)has been found to be predominant,and the most extensively distributed member of Perfluorinated compounds.Several studies have revealed that PFOS accumulates in both humans and wildlife species,due to the widespread use and its chemical property characterized by resistance to environmental and metabolic degradation.Pharmacokinetic studies have shown that once PFOS is absorbed,it distributes and accumulates in the serum and liver,but is poorly eliminated.Due to its bioaccumulative nature and potential toxicities,PFOS was added among persistent organic pollutants(POPs)listed under the Stockholm Convention in 2009.The accumulation of PFOS in human tissues led to the rising concern about its possible adverse effects on human health.PFOS was reported to cause hepatotoxicity in monkeys and SD rats,inducing their hepatocellular hypertrophy and lipid vacuolation.A multivariate linear regression found evidence of associations of biomarkers of liver function with PFOS at levels found in the general U.S.population.However,the molecular mechanisms for PFOS-induced hepatotoxicity have not been fully elucidated.PFOS is one kind of agonist of peroxisome proliferator-activated receptor-?(PPAR?)?PPAR? could activate autophagy in the liver of mice.However,few studies were carried to investigate whether PFOS is able to activate autophagy and the underlying mechanism.It was reported that a significant reduction of lysosomal membrane stability was observed in the thicklip grey mullets Chelon labrosus exposed to PFOS.Since lysosomal-dependent degradation is an integral part of the autophagic process,it was thus another interest for us to investigate whether PFOS could influence the stability of lysosomal membrane and the relationship of PFOS-induced alteration of lysosomal membrane stability with autophagy.PFOS was reported to induce apoptosis in human hepatoma Hep G2 cells.It is revealed that there are extensive crosstalk between apoptosis(type I programmed cell death)and autophagic cell death(type II programmed cell death).Autophagy is a highly regulated intracellular degradative process by which cells remove cytosolic long-lived proteins and damaged organelles.When autophagy is initiated,cytoplasmic constituents are sequestered into the autophagosome.The autophagosome eventually fused with a lysosome,forming an autolysosome where the contents are degraded and recycled for protein synthesis.Although autophagy is regarded as a regular degradative process maintaining cellular homeostasis,this process also represents one mode of programmed cell death.Aiming at further clarifying the molecular mechanism of PFOS-induced hepatotoxicity,we investigated whether autophagy and lysosomal membrane permeabilization were involved in cytotoxicity induced by PFOS.The purpose of this study was to investigate the relationship of PFOS-induced alteration in autophagy,apoptosis and lysosomal membrane stability.This would throw new light for understanding of the molecular mechanism and provide clues for effective prevention and treatment of PFOS-induced hepatic disease.The objectives of this study:1.To investigate whether PFOS altered autophagic process and which role autophagy served,i.e.,cytoprotective or pro-death,in human liver cells.2.To investigate whether PFOS altered lysosomal membrane stability in human liver cells.3.To investigate the relationship of PFOS-induced alteration in autophagy,apoptosis and lysosomal membrane stability,to find the specific mediator,and to provide clues for effective prevention and treatment of PFOS-induced hepatic disease.The results of this study:1.PFOS increased autophagosome formation at the early stage of treatmentWe utilized transmission electron microscopy to observe the ultrastructure of Hep G2 cells treated with 200 ?M PFOS for 6 h.Quantification of the autophagic vesicles numbers per viable cell demonstrated that PFOS increased autophagic vesicles number significantly in Hep G2 cells.Consistently,the level of LC3B-II increased in a concentration dependent manner after Hep G2 cells were treated with 100-200 ?M PFOS for 6 h as shown in Western blot assay.Microtubule-associated protein light chain3(LC3)-II is considered the most reliable biochemical markers of autophagy,and P62 is a known substrate of autophagic degradation.In this study,activation of autophagy was observed in Hep G2 cells after treatment with PFOS for 6 h,as indicated by an increase in LC3-II,and a decrease in P62.To investigate the lysosomal turnover of LC3-II,we used chloroquine to prevent autophagosome-lysosome fusion.The turnover assay allows the estimation of autophagy flux.The chloroquine-induced LC3-II accumulation was further elevated by treatment with PFOS for 6 h in Hep G2 cells.These data indicated that the increased autophagosome level was caused by an increase in autophagosome formation rather than impairment of autophagosome clearance.2.PFOS impaired autophagosome degradation at the late stage of treatmentAfter Hep G2 cells were treated with 200 ?M PFOS for 24 h,quantification of the autophagic vesicles numbers per viable cell demonstrated that PFOS increased autophagic vesicles number significantly in a time-dependent manner.LC3B-II increased dramatically in a concentration dependent manner after Hep G2 cells were treated with 150 and 200 ?M PFOS for 24 h as shown in Western blot assay.To investigate the lysosomal turnover of LC3,we used chloroquine to prevent autophagosome-lysosome fusion.The amount of LC3-II was not further increased in the presence of chloroquine compared with that in the absence of chloroquine after cells were treated with 200 ?M PFOS for 24 h.It indicated that the increased autophagosome was caused by impaired autophagosome clearance rather than increased formation.Consistently,after Hep G2 cells were treated with 150-200 ?M PFOS for 24 h,autophagic substrate P62 increased significantly in Hep G2 cells.3.PFOS caused autophagic cell death in Hep G2 cellsTo further uncover the role of autophagic cell death in cytotoxicity induced by PFOS,Hep G2 cells were pretreated respectively with autophagosome fomation inhibitor 3-MA,autophagosome formation stimulator rapamycin,cathepsin D inhibitor pepstatin A,and cysteine protease inhibitor E64 to observe the change of cell viability.MTT assay and lactate dehydrogenase release assay showed that PFOS-induced cytotoxicity was strongly reversed by 3-MA,even after Hep G2 cells were treated with300 ?M PFOS.Rapamycin further decreased the cell viability of PFOS-treated Hep G2 cells,knockdown of Atg5 with Atg5 si RNA increased the cell viability of CIT-treated Hep G2 cells.Pepstatin A and E64 did not influence the viability of PFOS-treated Hep G2 cells.These results indicated that PFOS-induced cytotoxicity was related with autophagic cell death,and independent of lysosomal cell death.4.PFOS-induced lysosomal membrane permeabilization was autophagy dependentWe performed acridine orange(AO)staining to examine the lysosome membrane stability.After treatment with 200 ?M PFOS for 12 h,the number of red puncta(intact lysosome)decreased significantly compared with the control,indicating PFOS caused significant LMP in Hep G2 cells.To evaluate the role of autophagy in the PFOS-induced LMP,Hep G2 cells were pretreated with the autophagosome formation inhibitor 3-MA before the treatment with PFOS.The pretreatment of Hep G2 cells with 1 m M 3-MA for2 h relieved the LMP observed in cells treated with 200 ?M PFOS for 12 h.After treatment with 200 ?M PFOS for 6 h,the intensity of red fluorescence did not change significantly compared with the control.It suggested that PFOS-activated autophagosome formation preceded the LMP.5.PFOS induced release of cathepsin DLysosomal release of cathepsin D is a causative event,leading to apoptosis.After treatment with 200 ?M PFOS for 12 h,the cathepsin D level in the cytosolic extracts increased significantly compared with the control.The pretreatment of Hep G2 cells with 1 m M 3-MA for 2 h relieved the PFOS-induced release of cathepsin D,indicating the release of cathepsin D was autophagy dependent.After treatment with 200 ?M PFOS for 6 h,the cytosolic cathepsin D level did not change significantly compared with the control,it was consistent with the result of PFOS-induced LMP.6.PFOS induced collapse of mitochondrial transmembrane potentialWe performed JC-1 staining to examine the mitochondrial transmembrane potential(??m).To evaluate the role of autophagy in the PFOS-induced collapse of??m,Hep G2 cells were pretreated with the autophagosome formation inhibitor 3-MA before the treatment with PFOS.The pretreatment of Hep G2 cells with 1 m M 3-MA for2 h was able to relieve the PFOS-induced collapse of ??m.It indicated that the PFOS-induced collapse of ??m was autophagy dependent.After Hep G2 cells were treated with 200 ?M PFOS for 12 h,the ??m did not change significantly compared with the control.It suggested that PFOS-induced LMP and subsequent release of cathepsin D preceded the collapse of ??m.7.PFOS impaired mitophagy in Hep G2 cellsAfter treatment of Hep G2 cells with 200 ?M PFOS for 24 h,the accumulation of aberrant mitochondria was observed.These round and swollen mitochondria were characterized with altered mitochondrial morphology.These aberrant mitochondria showed scarce or disordered cristae.The percentage of cells with giant mitochondria(larger than 1 ?m in width)increased significantly after the Hep G2 cells were treated with 200 ?M PFOS for 24 h.These results were indicative of impaired mitophagy.8.PFOS induced autophagy-dependent apoptosis in Hep G2 cellsWe performed hoechst 33342 and annexin V/ propidium iodide(AV/PI)staining to examine apoptosis.PFOS was shown to induce condensation of chromatin in Hep G2 cells after hoechst staining.The autophagosome formation inhibitor 3-MA was able to relieve PFOS-induced condensation of chromatin.In AV/PI staining,PFOS induced externalization of phosphatidylserine,a hallmark of the early apoptosis.Pretreatment of cells with 3-MA relieved PFOS-induced apoptosis,which was proved by AV(+),PI(-)%cells.9.Construction of Spinster 1 expression plasmidWe proposed that Spinster 1 might mediate PFOS-induced LMP,therefore we investigated the effect of Spinster 1 through the over-expression of Spinster 1 in this study.Human spinster 1 m RNA has 5 transcript variant.The beginning sequence and the terminal sequence of transcript variant 1,2,4 and 5 are identical.The beginning sequence of transcript variant 3 is different from other transcript variant,the terminal sequence of transcript variant 3 is the same with other transcript variant.However,which transcript variant expressed in Hep G2 cells is unclear.Therefore,we designed two set of PCR primers.One set of primer was for transcript variant 1,2,4,and 5:upstream primer(Spin-1),downstream primer(Spin-2).The other set of primers was for transcript variant 3: upstream primer(Spin-3),downstream primer(Spin-2).In the spinster 1 RT-PCR reaction,the primer of Spinster 1 transcript variant 1,2,4 and 5could not produce any product.However,the primer of transcript variant 3(Spin-3 and Spin-2)could amplify the product with the same length as transcript variant 3.PCR products were cloned to p JET plasmid to form p JET-spinseter 1 vector.The p JET-spinster 1 cutting sequence with restriction enzyme was as expectation,and the nucleotide sequence aligement was the same with transcript variant 3.The molecular size of the Spinster 1 in the Hep G2 cells shown in the Western blot assay was the same with Spinster 1 isoform 2,the transcription product of Spinster 1 transcript variant 3.All of these data indicated that the predominant Spinster 1 transcript variant was transcript variant 3 in Hep G2 cells.Spinster 1 was cloned to p HTN Halo Tag? CMV-neo Vector to form p HTN-spinster 1 expression vector.After transfection with p HTN-spinster 1expression vector into Hep G2 cells for 48 h,Spinster 1 was over-expressed in Hep G2 cells as shown in Western blot assay.10.Spinster 1 mediated PFOS-induced LMPAfter transfection with si RNA against Spinster 1 into Hep G2 cells,the result of acridine orange staining indicated that inhibition the expression of Spinster 1 could alleviate PFOS-induced LMP.After transfection with p HTN-spinster 1 exprssion vector into Hep G2 cells,the results of acridine orange staining indicated that over-expression of Spinster 1 could aggravate PFOS-induced LMP.Conclusions:1.PFOS increased autophagosome formation at the early stage of treatment(6 h).2.PFOS impaired autophagosome degradation at the late stage of treatment(24 h).3.PFOS cytotoxicity was related with autophagic cell death in Hep G2 cells.4.PFOS-induced lysosomal membrane permeabilization was autophagy dependent5.PFOS induced autophagy-dependent apoptosis in Hep G2 cells through lysosomal-mitochondrial axis and impaired mitophagy.6.The predominant spinster 1 transcript variant was transcript variant 3 in Hep G2 cells.7.The results from both over-expression of spinster 1 gene and down-regulation of spinster 1 gene confirmed that Spinster 1 mediated PFOS-induced LMP.Further studies:1.Liver is the main metabolic organ in humans.Liver disease is related with lipid metabolism,obesity,diabetes and cardiovascular disease.It is reported that PFOS is related with insulin resistance in general human population.Therefore,we will investigate the molecular mechanism of PFOS-related insulin resistance.2.We will investigate the autophagy related factor binding directly with Spinster 1using pull-down method or co-immunoprecipitation.3.We will investigate the toxicological mechanism of PFOS with in vivo test.