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  current news   Press   selected story    
     
  7th April  
  Chelerythrine Induces Apoptosis through a Bax/Bak-independent Mitochondrial Mechanism
 
 




Authors
Kah Fei Wan1, Shing-Leng Chan2, Sunil Kumar Sukumaran1, Mei-Chin Lee1 and Victor C. YU1

1 Institute of Molecular and Cell Biology, Proteos, Singapore.
2 Oncology Research Institute, National University of Singapore, 28 Medical Drive, Centre For Life Sciences, Cancer Program, Singapore.

Correspondence should be addressed to Victor C. YU (mcbyuck@imcb.a-star.edu.sg).


Abstract
Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (ΔΨm) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in theDKOMEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.


Figure & Legend: The ability of chelerythrine in triggering cytochrome C (Cyt. C) release from intact cells and isolated mitochondria is not affected by the absence of Bax and Bak. (A) Bax and Bak were dispensable for chelerythrine-induced Cyt. C release in intact cells. WT and bax and bak double-knockout (DKO) murine embryonic fibroblasts (MEFs) were treated with chelerythrine at the indicated concentrations for 16 h (left panel) or 10 µM of chelerythrine for the indicated duration (right panel) and harvested for cytosolic and pellet fractions. Proteins in the preparations were subjected to SDS-PAGE, and immunoblotted with Cyt. C, actin (loading control for cytosolic fraction) and HSP60 antibodies (loading control for pellet fraction). (B) Chelerythrine induced rapid release of Cyt. C that preceded Bax translocation and integration in WT MEFs. MEFs were treated with 10 µM chelerythrine for the indicated durations and harvested for cytosolic and pellet fractions as described in (A). For Bax and VDAC integration analysis, mitochondria isolated from chelerythrine-treated (10 µM) WT and DKO MEFs for the indicated durations were resuspended in 0.1 M NaCO3 (pH 10.5), and incubated on ice for 20 min followed by sonication for 5 min. Mitochondria were repelleted by centrifugation (100,000 rpm, 20 min) and immunobloted for the indicated proteins. (C) Chelerythrine, but not Bim BH3 peptides, was efficacious in inducing Cyt. C release from mitochondria isolated from DKO MEFs. Isolated mitochondria were incubated at room temperature with the indicated concentrations of either Bim BH3 peptide (left panel) or chelerythrine (right panel). Supernatant (S/N) and pellet fractions containing mitochondria were then subjected to SDS-PAGE, and immunoblotted with Cyt. C and HSP60 antibodies (loading control for pellet fraction). Data are representative of at least 3 experiments.

Published in The Journal of Biological Chemistry (2008) Vol. 283:13, 8423–8433

For more information of Victor C. YU's Lab, Please Click here.