Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines.
In this study, we investigated the signaling pathways implicated in SSa-induced apoptosis of human colon carcinoma (HCC) cell lines. SSa-induced apoptosis of HCC cells was associated with proteolytic activation of caspase-9, caspase-3, and PARP cleavages and decreased levels of IAP family members, such as XIAP and c-IAP-2, but not of survivin. The fluorescence intensity of DiOC6 was significantly reduced after SSa treatment. CsA significantly inhibited SSa-induced loss of mitochondrial transmembrane potential and moderately inhibited SSa-induced cell death. SSa treatment also enhanced the activities of caspase-2 and caspase-8, Bid cleavage, and the conformational activation of Bax. Additionally, SSa-induced apoptosis was inhibited by both the selective caspase-2 inhibitor z-VDVAD-fmk and the selective caspase-8 inhibitor z-IETD-fmk and also by si-RNAs against caspase-2 and caspase-8. The selective caspase-9 inhibitor, z-LEHD-fmk, also inhibited SSa-induced apoptosis, albeit to a lesser extent compared to z-VDVAD-fmk and z-IETD-fmk, indicating that both mitochondria-dependent and mitochondria-independent pathways are associated with SSa-induced apoptosis. Both z-VDVAD-fmk and z-IETD-fmk significantly attenuated the colony-inhibiting effect of SSa. Moreover, inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk, or by knockdown of protein levels using a si-RNA, suppressed SSa-induced caspase-8 activation, Bid cleavage, and the conformational activation of Bax. Although caspase-8 is an initiator caspase like caspase-2, the inhibition of caspase-8 activation by knockdown using a si-RNA did not suppress SSa-induced caspase-2 activation. Altogether, our results suggest that sequential activation of caspase-2 and caspase-8 is a critical step in SSa-induced apoptosis.
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-Gu, Seoul 139-706, Korea.
Byeong Mo
BM
Kim
Sung Hee
SH
Hong
D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D015153:Blotting, Western; D053143:Caspase 2 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms; D016572:Cyclosporine / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012503:Saponins / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction; D000077022:Survivin; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D009842:Oligopeptides; D034741:RNA, Small Interfering; D012503:Saponins; D000077022:Survivin; D051028:bcl-2-Associated X Protein; C427308:benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D009828:Oleanolic Acid; D016572:Cyclosporine; D011065:Poly(ADP-ribose) Polymerases; D053143:Caspase 2; D053181:Caspase 8; D053453:Caspase 9; C025759:saikosaponin D