Studies On Relationship And Its Molecular Mechanisms Of Abnormal Autophagy To Apoptosis/Survival In The Cells

The present study,using cellular and molecular biology methods and techniques including cell culture,fluorescence staining and imaging analysis,RNA interference,protein fluorescent labeling,Western blotting,cell counting,cell viability detection,etc.,was divided into three parts.In part 1,PC12,SH-SY5Y cells and mouse primary neurons were used as experimental objects.We systematically studied the relationship of cadmium(Cd)-induced blockade of autophagic flux and accumulation of autophagosomes to apoptosis in neuronal cells,dissected the role of Cd-induced ROS in blocking autophagic flux contributing to neuronal apoptosis,and further clarified the role and mechanisms of PP2A/PP5-JNK pathway in the above events.In part 2,employing Raji and mouse primary B cells as experimental objects,we revealed the role and its regulatory mechanisms of autophagy in hsBAFF-stimulated B-cell proliferation and survival.The third part's experimental objects were C2C12 and L6 cells.We explored the relationship between autophagic flux impairment and apoptosis in skeletal muscle cells induced by maduromycin(Mad),and explained the mechanism of Mad-caused damage in the cells from the perspective of autophagy.The detailed results were summarized as follows:1 Studies on molecular mechanisms of Cd-induced autophagic flux blockage contributing to accumulation of autophagosomes-dependent apoptosis in neuronal cells1.1 Cd-induced ROS results in autophagic flux blockade and apoptosis in neuronal cellsPC12,SH-SY5Y cells and/or mouse primary neurons were treated with Cd(0,10 or 20 ?M)for 8 or 24 h following pretreatment with NAC for 1 h.The expressions of LC3-II,p62,cleaved-caspase-3 and cleaved-PARP proteins were analyzed by Western blot.The number of autophagosomes was labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.ROS levels were evaluated by fluorescent images using CM-H2DCFDA probe.The results showed that Cd induced elevation of ROS and increases of autophagosomes,and significantly increased the expressions of LC3-?,p62,Cleaved-caspase-3 and Cleaved-PARP in neuronal cells.However,ROS scavenger NAC potently attenuated the above events caused by Cd,suggesting that Cd-induced ROS contributes to blockade of autophagic flux and apoptosis in neuronal cells.1.2 Cd-induced ROS blocks autophagic flux leading to accumulation of autophagosomes-dependent apoptosis in neuronal cellsPC 12 cells and mouse primary neurons,or PC 12 cells infected with lentiviral shRNA to Atg5,LC3-?/? or GFP were treated with additive drugs.The changes of autophagy-and apoptosis-related proteins were evaluated by Western blot.The number of autophagosomes was labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.Trypan blue and DAPI staining were used to assess cell viability and apoptosis.ROS levels were evaluated by fluorescent images using CM-H2DCFDA probe.The results showed that downregulation of Atg5 inhibition of autophagy by silencing Atg5 significantly declined the number of autophagosomes caused by Cd and simultaneously obviously resisted Cd-induced cell viability reduction and apoptosis.Inhibition of autophagy and autophagosomes by 3-MA significantly decreased cell apoptosis evoked by Cd.NAC potentiated the effects of 3-MA.Knockdown of LC3-?/? attenuated Cd-induced cell viability reduction and apoptosis in neuronal cells,which was further strengthened by NAC.Additionally,NAC markedly decreased the number of autophagosomes,whereas inhibition of autophagosomes by 3-MA or silencing LC3-?/? significantly attenuated Cd-elicited ROS,indicating that there exists a mutual feedback effect between ROS production and autophagosomes' accumulation in the cells induced by Cd.Taken together,these findings clearly demonstrated that Cd-induced ROS blocks autophagic flux leading to accumulation of autophagosomes-dependent apoptosis in neuronal cells.1.3 Cd-hindered fusion of autophagosomes and lysosomals blocks autophagic flux leading to apoptosis in neuronal cellsPC 12 cells and mouse primary neurons were pretreated with/without rapamycin for 48 h,followed by treatment with/without Cd for 8 or 24 h or with/without 0.4 ?M bafilomycin A1 for 3 or 12 h.The changes of autophagy-and apoptosis-related proteins were evaluated by Western blot.DAPI staining was used to assess cell apoptosis.Co-localization of autophagosomes and lysosomes was analyzed by showing fluorescence of infecting Ad-GFP-LC3 and LysoTracker in the cells.Lysosomal enzyme CTSL activity and lysosomal pH were also detected.The results showed that rapamycin significantly reduced the expressions of Cd-induced LC3-II,p62 and Cleaved-caspase-3 proteins,and potently inhibited Cd-induced apoptosis.Rapamycin promoted fusion of autophagosomes with lysosomes hindered by Cd,and Cd did not alter the lysosomal enzyme CTSL activity and lysosomal pH.The results imply that Cd-hindered fusion of autophagosomes and lysosomals blocks autophagic flux leading to apoptosis in neuronal cells.1.4 Cd-induced ROS blocks autophagic flux contributing to apoptosis by activating JNK pathway in neuronal cellsPC 12 cells and mouse primary neurons,PC 12 cells infected with Ad-dn-c-Jun or Ad-lacZ,or PC 12 cells infected with lentiviral shRNA to c-Jun or GFP were pretreated with/without NAC and/or JNK inhibitor SP600125 for 1 h and then exposed to Cd for 8 or 24 h.The changes of autophagy-and apoptosis-related proteins were evaluated by Western blot.The number of autophagosomes was labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.Trypan blue and DAPI staining were used to assess cell viability and apoptosis.ROS levels were evaluated by fluorescent images using CM-H2DCFDA probe.The results showed that Cd-induced increases of LC3-II,p62,Cleaved-caspase-3,ROS levels,number of autophagosomes,cell apoptosis and reductive viability were alleviated by SP600125.NAC enhanced the effects of SP600125.Similar findings were observed in the cell of silencing c-Jun or expressing dn-c-Jun.The results demonstrate that Cd-induces ROS blocks autophagic flux contributing to apoptosis by activating JNK pathway in neuronal cells.1.5 Cd-induced ROS blocks autophagic flux contributing to apoptosis by PP2A/PP5-JNK signaling in neuronal cellsPC 12 cells and mouse primary neurons,or PC 12 cells infected with Ad-PP5-wt,Ad-PP2A-wt,Ad-dn-PP2A or Ad-lacZ(as control)were pretreated with/without NAC and/or SP600125,OA for 1 h,followed by treatment with Cd for 8 or 24 h.The changes of autophagy-and apoptosis-related proteins were evaluated by Western blot.The number of autophagosomes was labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.DAPI staining was used to assess cell apoptosis.ROS levels were evaluated by fluorescent images using CM-H2DCFDA probe.The results showed that Cd inhibited activity of PP2A and PP5,which was reversed by NAC.Over-expression of PP2A and PP5 resisted Cd-induced activation of JNK pathway,increases of LC3-?,p62 and Cleaved-caspase-3,and simultaneously attenuated Cd-increased the number of autophagosomes,ROS levels and apoptosis in the cells.On the contrary,inhibition of PP2A or expression of dn-PP2A potentiated Cd neurotoxicity.These results suggest that Cd-induces ROS blocks autophagic flux contributing to apoptosis by PP2A/PP5-JNK signaling in neuronal cells.1.6 Cd induction of NOX2-derived ROS blocks autophagic flux contributing to apoptosis via PP2A/PP5-JNK signaling in neuronal cellsPC 12 cells and mouse primary neurons,or PC 12 cells infected with lentiviral shRNA to NOX2 or GFP(as control)were pretreated with/without NAC or apocinin for 1 h and then treated with/without Cd for 8 or 24 h.The changes of autophagy-and apoptosis-related proteins were evaluated by Western blot.The number of autophagosomes was labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.Trypan blue and DAPI staining were used to assess cell apoptosis.ROS levels were evaluated by fluorescent images using CM-H2DCFDA probe.The results showed that inhibition of NOX2 by apocinin or down-regulation of NOX2 by RNA interference significantly declined Cd-evoked ROS production in neuronal cells.Cd-induced inhibition of PP2A and PP5 activity,activation of JNK and c-Jun,and increase expression of LC3-?,p62 and Cleaved-caspase-3 were reversed in the cells as well.Inhibition or down-regulation of NOX2 obviously ameliorated Cd-elicited accumulation of autophagosomes,cell viability reduction and apoptosis.The results suggest that Cd induction of NOX2-derived ROS blocks autophagic flux contributing to apoptosis via PP2A/PP5-JNK signaling in neuronal cells.2 Studies on molecular mechanisms of hsBAFF-inhibited autophagy promoting proliferation and survival in B cells2.1 hsBAFF inhibits autophagy promoting proliferation and survival by activating mTOR pathway in B cellsRaji and mouse primary B cells were treated with different concentrations of hsBAFF(0,0.5,1,1.5,2.5,5 ?g/ml)for 12 or 48 h,or with/without hsBAFF(2.5?g/ml)for different times(0,4,8,12,24,48 h),or pretreated with/without mTORC1 inhibitor rapamycin(100 ng/ml)for 2 h,or with/without Akt inhibitor X(20 ?M)or mTORCl/2 inhibitor PP242(1 ?M)for 1 h and then treated with/without hsBAFF(2.5 ?g/ml)for 12 or 48 h.Expressions of Atg5,LC3-?,p-Akt(Ser473),p-S6K1(Thr389),p-4E-BP1(Thr70),p-ULK1(Ser757)and survivin proteins were analyzed by Western Blot.Cell proliferation and viability were evaluated by cell counting and MTS assay,respectively.The results showed that hsBAFF inhibited Atg5 expression and promoted proliferation and survival in B cells in a concentration-and time-dependent manner.Rapamycin,Akt inhibitor X and PP242 remarkably inhibited hsBAFF-increased p-ULK1,p-Akt,p-S6K,p-4E-BP1 and survivin,and reversed hsBAFF-restrained LC3-? and hsBAFF-stimulated proliferation and survival in the cells.Our findings demonstrate that hsBAFF inhibits autophagy promoting proliferation and survival by activating mTOR pathway in B cells.2.2 Atg5/LC3 plays a vital role in hsBAFF-promoted proliferation and survival in B cellsRaji cells infected with lentiviral shRNA to Atg5,LC3 or GFP were treated with hsBAFF(0,1,2.5 ?g/ml)for 12 or 48 h.Expressions of Atg5,LC3-?,p-Akt(Ser473),p-S6K1(Thr389),p-4E-BP1(Thr70),and survivin proteins were analyzed by Western Blot.The number of autophagosomes was assayed by MDC staining and labeled and counted by showing fluorescence of infecting Ad-GFP-LC3 in the cells.Cell proliferation and viability were evaluated by cell counting and MTS assay,respectively.The results showed that down-regulation of Atg5 or LC3 inhibited hsBAFF-increased p-Akt(Ser473)?p-S6K1(Thr389)?p-4E-BP1(Thr70)and surviving,and attenuated hsBAFF-stimulated proliferation and survival in the cells.The results suggest that Atg5/LC3 plays a vital role in hsBAFF-promoted proliferation and survival in B cells.3 Studies on molecular mechanisms of Mad-impaired autophagic flux contributing to apoptosis in skeletal muscle cells3.1 Mad impairs autophagic flux leading to apoptosis in skeletal muscle cellsC2C12 and L6 cells were treated with different concentrations of maduramycin(Mad,0,0.05,0.1,0.2,0.5,1 ?M)for 24 h,or pretreated with rapamycin(0.2 ?g/ml)for 24 h and then treated with Mad(0,0.5,1 ?M)for 24 h.Expressions of cleaved-caspase-3 and cleaved-PARP were detected by Western Blot.Caspase-3/7 activity was determined.Cell apoptosis was evaluated by DAPI/TUNEL staining.The status of autophagic flux was assayed by labeled and counted by showing fluorescence of infecting Ad-GFP-p62 in the cells.The results showed that Mad induced apoptosis in C2C12 and L6 cells in a concentration-dependent manner.Regulation of autophagic flux by rapamycin effectively ameliorated apoptosis in the cells.The results suggest that Mad impairs autophagic flux leading to apoptosis in skeletal muscle cells.3.2 Mad impairs autophagic flux leading to apoptosis by Ca2+-CaMKII pathway in skeletal muscle cellsC2C12 and L6 cells were pretreated with/without BAPTA/AM,2-APB,EGTA for 1 h and then treated with/without Mad for 24 h,or pretreated with/without KN93 and then treated with Mad for 24 h.Expressions of of p-CaMKII,LC3-II,p62 and cleaved-caspase-3 were detected by Western Blot.The state of accumulation of autophagosomes and cell apoptosis were assessed by GFP-LC3 fluorescent labeling and DAPI staining,respectively.The results showed that inhibition of[Ca2+]i level by BAPTA/AM,EGTA or 2-APB or inhibition of CaMKII by KN93 significantly diminished Mad-induced increased LC3-?,p62,cleaved-caspase-3,autophagosome accumulation,and apoptosis in the cells.Our results reveal that Mad impairs autophagic flux leading to apoptosis by Ca2+-CaMKII pathway in skeletal muscle cells.