Studies On The Regulatory Mechanisms Of Oridonin-and Fas Agonistic Antibody CH11-Induced Autophagy And Apoptosis In Tumor Cells

In this dissertation,we investigated the different regulatory mechanisms of oridonin-induced autophagy and apoptosis in human cervical carcinoma HeLa cell and human fibrosarcoma HT 1080 cell.The molecular mechanisms involving CH11(one of the agonistic antibodies of Fas)-induced autophagy and apoptosis were also examined.Here,we found that autophagy and apoptosis were simultaneously induced by oridonin both in HeLa and HT1080 cells.When treated with oridonin,the typical apoptotic features including membrane blebbing,apoptotic bodies as well as nuclear condensation,were observed both in these two cell lines,and the nuclear damage and the granular apoptotic bodies were also identified by Hoechst 33258 staining.Oridonin could induce autophagy in HeLa and HT1080 cells at the same time,which were characterized by increases of MDC recruitment and the enhancements of the expression level of Beclin 1 and the conversion from LC3-Ⅰto LC3-Ⅱ.However,the relationship between oridonin-induced autophagy and apoptosis and their regulatory mechanisms were different in these two cell lines.Oridonin-induced autophagy was a protective mechanism against apoptosis in HeLa cell.Treatment with 3-MA(a specific autophagy inhibitor) prior to the addition of oridonin significantly increased the cell growth inhibitory ratio and the apoptotic ratio compared with the group treated with oridonin alone.PKC signal pathways played an anti-apoptotic effect through promoting autophagy in this process.We found that PKC inhibitor staurosporine significantly reduced oridonin-induced autophagy whereas markedly increased apoptosis,while pretreatment with PKC activator PMA caused opposite results.Subsequently,the oridonin-induced autophagy was also suppressed by Raf-1 inhibitor GW5074 or JNK MAPK inhibitor SP600125 accompanied by the increase of apoptosis,but it was not affected by ERK or p38 MAPK inhibition.In addition,oridonin-induced protein levels of Raf-1,JNK and p-JNK were sharply downregulated by staurosporine,and they were enhanced by PMA,but the decreases of ERK,p-ERK,p38 and p-p38 levels induced by oridonin did not change by staurosporine.Taken together,these results demonstrated that PKC enhanced oridonin-induced autophagy against apoptosis through regulating its downstream factors Raf-1 and JNK in HeLa cells.Moreover,oridonin induced the generation of ROS in HeLa cells,and ROS promoted oridonin-induced autophagy and apoptosis.Pretreatment with ROS scavenger NAC significantly decreased oridonin-induced autophagy as well as apoptosis,and oridonin-induced changes of apoptotic and autophagic related protein expression levels such as Bax,Bcl-2,caspase-3 and caspase-8 were also reversed by NAC.Our results from flowcytometric analysis showed that significant generation of ROS was observed initially at 1 h after oridonin administration,and it was persistently increased,indicating that ROS mediated oridonin-induced cell death.Then,we found that inhibition of Ras and PARP increased whereas inhibition of NF-κB reduced oridonin-induced autophagy and apoptosis.More interestingly,p53 played an autophagy-promoting role to against cell death in this circumstance.Pretreatment with pan-caspase,caspase-8 and caspase-9 inhibitor all significantly enhanced oridonin-induced autophagy while inhibited apoptosis.And Fas pathway had no influence on autophagy and apoptosis in this system.Autophagy and apoptosis acted in synergy to mediate cell death in oridonin-treated HT 1080 cells,and inhibition of autophagy by 3-MA decreased apoptosis.In this system, Ras,Raf-1,ERK,JNK and p38 all educed as protective factors to against oridonin-induced cell death,and inhibition of them by specific inhibitors respectively suppressed oridonin-induced cell growth inhibition and apoptosis.As to autophagy,Ras, ERK and p38 played suppressing role whereas Raf-1 and JNK had no effect on it.Our results from Western blot showed that oridonin induced time-dependent decreases in expression levels of all the proteins above.And pretreatment with manumycin A(a Ras inhibitor),sharply intensified oridonin-induced down-regulation of Raf-1,ERK and p-ERK while had no influence on the changes of JNK and p38.All these results indicated that Ras played its anti-apoptotic and anti-autophagic role through regulating ERK in oridonin-treated HT1080 cells.In addition,treatment with oridonin caused an increase in NF-κB and p53 activities in a time-dependent manner.Inhibition of NF-κB or p53 activation by its specific inhibitor PDTC or pifithrin-αrespectively,significantly reduced both oridonin-induced apoptosis and autophagy.Moreover,further experiments confirmed that oridonin-induced p53 activation was reduced by the PDTC whereas the activation of NF-κB was not changed by p53 inhibition.Taken together,these results demonstrated that NF-κB promoted oridonin-induced apoptotic and autophagic cell death through regulating p53 activation in HT 1080 cells.Fas agonistic antibody CH11 induced autophagy in HeLa cells,and inhibition of autophagy by 3-MA increased CH11-induced apoptosis,suggesting autophagy played a protective role in CH11-induced cell death.A Fas antagonistic antibody UB2 suppressed both CH11-induced autophagy and apoptosis,which indicated that autophagy and apoptosis were mediated by the activation of Fas.In addition,the CH11-induced autophagy was blocked by JNK inhibitor(SP600125),but it was not affected by caspase 8 inhibitor(Z-IETD);whereas the CH11-induced apoptosis was increased by SP600125,and it was suppressed by Z-IETD.Further experiments confirmed that JNK was activated by CH11 dose-dependently,and the activation was suppressed when autophagy was blocked by 3-MA.Together,our results suggested that JNK,but not caspase 8,involved in Fas-mediated CH11-induced autophagy in HeLa cells.In summary,we demonstrated the regulatory mechanisms of oridonin- and CH11-induced autophagy and apoptosis,and found the signal transduction pathway which regulated both autophagy and apoptosis.Our results might be useful as bases of research on common signal pathways existing between apoptosis and autophagy.