An insight into the mechanism of cytotoxicity of ricin to hepatoma cell: roles of Bcl-2 family proteins, caspases, Ca(2+)-dependent proteases and protein kinase C.
The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.
Hu
R
R
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 20031, China.
Zhai
Q
Q
Liu
W
W
Liu
X
X
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002154:Calpain / Q000187:drug effects* / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000201:enzymology; D020169:Caspases / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004357:Drug Synergism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D011493:Protein Kinase C / Q000187:drug effects* / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D012276:Ricin / Q000378:metabolism / Q000494:pharmacology*
D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012276:Ricin; D003513:Cycloheximide; D011493:Protein Kinase C; D002154:Calpain; D020169:Caspases