Natural Diterpenoid Compound Elevates Expression of Bim Protein, Which Interacts with Antiapoptotic Protein Bcl-2, Converting It to Proapoptotic Bax-like Molecule

Lixia Zhao,Feng He,Haiyang Liu,Yushan Zhu,Weili Tian,Ping Gao,Hongping He,Wen Yue,Xiaobo Lei,Biyun Ni,Xiaohui Wang,Haijing Jin,Xiaojiang Hao,Jialing Lin,Quan Chen

doi:10.1074/jbc.m111.264481

Abstract

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.

Highlights

Bcl-2-related proteins regulate mitochondrial membrane permeabilization during apoptosis
Permeabilization of mitochondrial outer membrane (MOM) by Bax or Bak is required for the release of proapoptotic molecules, including cytochrome c, from mitochondria, a concept that was supported by the fact that bax/bak double knock-out
To further characterize the death process induced by S-3 in baxϪ/Ϫ/bakϪ/Ϫ cells, we used Annexin V (AV) staining to measure the exposure of phosphatidylserine on the cell surface, a hallmark of apoptosis

Summary

Background

Bcl-2-related proteins regulate mitochondrial membrane permeabilization during apoptosis. The diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer mem-. Brane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics. Apoptotic cytochrome c release from mitochondria is largely governed by interactions among proteins in the Bcl-25 family, which is divided into three functionally distinct groups [1, 2]. Binding to the multi-BH family members may induce an ␣-helical conformation in the BH3 region of Bim. Permeabilization of mitochondrial outer membrane (MOM) by Bax or Bak is required for the release of proapoptotic molecules, including cytochrome c, from mitochondria, a concept that was supported by the fact that bax/bak double knock-out

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION