Critical role of Bid and Bax in indirubin-3′-monoxime-induced apoptosis in human cancer cells

Indirubin-T-monoxime (13M) is a derivative of indirubin, an active component from a Chinese medicinal recipe with known anti-cancer function. 13M has been well established as a cyclin- dependent kinase (CDK) inhibitor, while the molecular mechanism underlying 13M-induced apoptosis has not been fully elucidated. In this study, we focused on the critical role of the pro-apoptosis Bcl-2 family members in 13M-induced apoptosis. We first observed 13M-induced apoptosis in a time- and dose-dependent manner in three different types of human cancer cells-cervical cancer HeLa, hepatoma HepG2 and colon cancer HCT116. Induction of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9 and -3 activation. Initiation of the death receptor pathway started with enhanced surface expression of DR4 and DR5, as well as increased total protein level, which correlated with the up-regulation of p53 and its transcriptional activity. Importantly, we found in HeLa cells that caspase-8 activation resulted in Bid cleavage, followed by Bax conformational change and hence the amplification of the apoptotic signals through the mitochondrial pathway. Consistently, stable knockdown of Bid abrogated 13M-induced Bax conformational change and cell death. Moreover, ectopic expression of a viral caspase inhibitor (CrmA) or Bcl-2 partially protected 13M-induced apoptosis. In conclusion, our results indicate that 13M mainly elicites apoptosis through extrinsic pathway with type II response mediated by the pro-apoptotic Bcl-2 family members (Bid and Bax). (C) 2008 Elsevier Inc. All rights reserved.