The Investigation Of The Mechanisms Involved In The Apoptosis Induced By Trichosanthin In Human Leukemia Cell Lines K562 And HL-60

Trichosanthin (TCS), a 27kDa ribosome-inactivating protein (RIP) extracted from the root tuber of a traditional Chinese medicinal herb Trichosanthes kirilowiii, has multiple biological and pharmacological activities, including termination of mid- pregnancy, immunoregulation, anti-tumor and anti-virus. Recent studies indicated that TCS has the cytotoxicity on many cancer cell lines by the induction of apoptosis, while the mechanisms remain obscure.TCS had selective cytotoxicity on tumor cell lines, and TCS had more cytotoxicity on human leukemia cell lines K562 and HL-60. TCS treatment induced apoptosis in K562 and HL-60 cells in a time- and dose-dependent manner examined by Hoechst 33258 staining, Annexin V-FITC/PI staining, flow cytometry assay and DNA fragmentation assay. Further investigation indicated that TCS treatment induced a transient intracellular calcium elevation in K562 cells, while it had no significant effect on HL-60 cells. Actually, calcium chelators pretreatment had no significant inhibitory effect on TCS-induced apoptosis in both cell lines, which suggested that calcium might not be involved in the apoptosis. Neither calpain inhibitors nor antioxidants had the significant inhibitory activity on the apoptosis, suggesting that calpain and ROS signaling pathways might not be involved in the apoptosis.Caspase-8, -9 and–3 were activated and the inhibitors pretreatment, respectively, could partly block TCS-induced apoptosis, suggesting caspase-8, -9 and–3 were involved in the apoptosis. However, pretreatment with caspase-9 and–8 inhibitors could not block completely the apoptosis, indicating that some other pathway was involved in the apoptosis. TCS treatment activated caspase-4, increased ER stress chaperons GRP78/BiP and GADD153/CHOP, and caspase-4 inhibitors pretreatment blocked the apoptosis, suggesting that ER stress was involved in the apoptosis. Caspase-8 was activated in a caspase-9 and–4 dependent manner, while Fas/Fas ligand pathway was not involved in the apoptosis. TCS treatment decreased the mitochondrial membrane potential, released cytochrome c and smac from mitochondria to cytosol and activated caspase-9, suggesting that mitochondrial pathway was involved in the apoptosis. Further investigation showed that caspase-9, -4 and–8 acted upstream to the activation of caspase-3. Taken together, our results suggested that TCS-induced apoptosis in K562 and HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways, leading to the activation of a caspase cascade.Further investigation in K562 cells indicated that TCS partly inhibited PKC activity. Indeed, PKC activator, PMA, inhibited while PKC inhibitor, cal C, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitor prevented cal c-enhanced apoptosis induced by TCS. In summary, we concluded that TCS induced apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation. And there is an unknown protein on K562 plasma membrane, which was purified with affinity chromatography. There are three major results in the study: 1) Calcium-dependent ROS pathways was not involved in the TCS-induced apoptosis in K562 and HL-60 cells; 2)TCS induced apoptosis in K562 and HL-60 cells via ER stress and mitochondrial signaling pathway; 3) PKC inhibition was involved in TCS-induced apoptosis in K562 cells. Among them, the study delineated for the first time the involvement of three major apoptotic pathways in TCS-induced apoptosis in cancer cells. And ER stress pathways or PKC inhibition was involved in the apoptosis induced by TCS, a type I RIP, which was the first report about the apoptotic mechanism induced by RIPs. All the results might provide some new clues to the study of mechanism involved in TCS and RIPs-induced apoptosis.