Molecular Mechanism Of Tripchlorolide (TC) Induced Apoptosis Of Tumor Cells

Recent studies indicate that the control of cell cycle is tightly correlated with theregulation of apoptosis, in which several molecules like pRB, p53 and survivin, playimportant roles. In the present study, we focused on the contribution of pRB, p53 andsurvivin in the compound from Chinese herb induced apoptosis of tumor cells and theinterconnections amongst these molecules.The potential antitumor drug TC is a component purified from Chinese herbTripterygium Wilfordii Hook. f.. However, its mechanism is still unclear. Here, we useseveral tumor cell lines to dissect the anti-tumor mechanisms of TC. In the study ofTC-induced apoptosis of human cervical carcinoma cell line HeLa cells, our presentworks showed that the expression of E6/E7 oncogenes of human papillomavirus inHeLa cells was inhibited in the presence of TC, which resulted in the up-regulation ofp53 in HeLa cells. The TC induced apoptosis was p53-dependent. With further analysisthe relationship of cell cycle and apoptosis, our results displayed that under the sameTC-treatment, the cells at the early S-phase were more susceptible to apoptosis thanthose at the middle S-phase although p53 protein was stabilized to the same level inboth situations. Significant difference was exhibited between the two specifiedexpression profiles. Further analysis demonstrated that anti-apoptotic gene survivin wasup-regulated by p53 in the TC-treated middle-S cells, whereas it was down-regulated byp53 in the TC-treated early-S cells. Taken together, the present study indicated that thedifferential p53-regulated expression of survivin at different stages of the cell cycleresulted in different cellular outputs under the same apoptosis-inducer.Long-duration treatment of TC caused apoptosis of human gastric adenocarcinomacell line AGS cells. In the present study, the degradation of pRB was observed inapoptotic gastric tumor cells treated with TC. The inhibition of pRB degradation by ageneral cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells.Furthermore, the survival of the gastric tumor cells under the TC treatment wasenhanced by an over-expression of exogenous pRB. These results suggest that the pRBdegradation of the gastric tumor cells under the TC treatment involves in the apoptoticprogression. By using protease inhibitors and overexpression of an uncleavable versionof RB, we concluded that two different mechanisms involved in the pRB degradationprogression: one required the activity of caspase(s), and the other required the activityof unknown cysteine protease. In addition, the same extent of TC-inducedpRB-degradation was detected in the gastric tumor cells containing a p53dominant-negative construct, indicating that this kind of pRB degradation isp53-independent.The CDK inhibitor p21 is transcriptionally regulated by p53. Generally, p21 isupregulated by p53, which can be activated by DNA damage signaling. However, weobserved the downregulation of p21 when p53 protein was activated by TC in gastrictumor cell line AGS. Result from chromatin immunoprecipitation showed that affinityof the active p53 binding to p21 promoter was reduced. The result using p53 dominantnegative indicated that the downregulation of p21 induced by TC was p53-dependent.