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Gossypol induces Bax/Bak-independent activation of apoptosis and cytochrome c release via a conformational change in Bcl-2

Xiaobo Lei^*,,1, Yingyu Chen^*,,1, Guanhua Du, Wenyu Yu^||, Xiaohui Wang^*, Hong Qu^||, Bin Xia^||, Hongping He^¶, Jianhua Mao^*, Weixing Zong^#, Xudong Liao^*, Maryam Mehrpour^*,**, Xiaojiang Hao^¶ and Quan Chen^*,2

^* The Laboratory of Apoptosis and Cancer Biology, The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, P.R. China;

Graduate School of the Chinese Academy of Sciences, Beijing, P.R. China;

Center for Human Disease Genomics, Peking University, Beijing, P.R. China;

Institute of Materia Medica, Academy Medical Science and Peking Union Medical College, Beijing, P.R. China;

^|| Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing, P.R. China;

^¶ The State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Beijing, P.R. China;

^# Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA;

^** INSERM U-487, Laboratoire de Cytokines et Immunologie des tumeurs Humaines, Institut Gustave Roussy PR1 and IFR 54, Villejuif, France

²Correspondence: The Laboratory of Apoptosis and Cancer Biology, The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Bei Si Huan Xi Road 25, Haidian District, Beijing 100080, P.R. China. E-mail: chenq{at}ioz.ac.cn

SPECIFIC AIMS

Cells lacking both Bak and Bax are largely resistant to apoptosis induced by a number of death stimuli, including DNA-damaging agents, signal transduction through death receptors, growth factor deprivation, and ER stress. These cells may undergo caspase-independent type II cell death (autophagic cell death) or programmed necrosis in response to high doses of DNA-damaging agents. Given that the other key components of the apoptotic machinery at both pre- and postmitochondrial levels are intact in those bak^–/–/bax^–/– cells, we undertook the large-scale screening of a natural compounds library for small molecules that could induce apoptosis in bak^–/–/bax^–/– cells.

PRINCIPAL FINDINGS

1. We screened out compounds that can activate caspase-dependent apoptosis in Bak/Bax double knockout (bak^–/–/bax^–/–) cells
Using simian virus 40 (SV40) transformed bak^–/–/bax^–/– embryonic fibroblasts, we first tested the inhibitory activity of a library of 7800 natural compounds on cell viability using the MTT assay. 23 compounds that resulted in a reduction of MTT staining, and in cells treated with those compounds, we applied acridine orange staining, Annexin V staining and an assay for intracellular caspase activity. One of these compounds, gossypol, a polyphenolic compound naturally occurring in cottonseed that has been clinically used for male contraceptive, was found to induce nuclear fragmentation and apoptosis in bak^–/–/bax^–/– cells (Fig. 1 A, B). Gossypol-induced apoptosis is dependent on caspase activation and z-VAD-fmk (100 µM), a pan-caspase inhibitor that could potently block gossypol-induced cell death (Fig. 1C, D ). However, other related agents did not induce caspase activation in bak^–/–/bax^–/– cells (Fig. 1C ).

View larger version (28K): [in this window] [in a new window]   Figure 1. Apoptosis in bak–/–/bax–/– cells induced by gossypol. A) bak–/–/bax–/– cells were treated with 20 µM gossypol for 24 h, followed by fixation and stained with Hoechst33342, and visualized by fluorescent microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. Bar diagram shows the ratio of Hoechst positive cells. Data were the mean value of three independent experiments with each count of no less than 200 cells. *P < 0.05. B) Flow cytometric analysis of apoptosis in bak–/–/bax–/– cells following treatment with 20 µM gossypol for the indicated times. The percentage of apoptotic cells was determined by Annexin V/PI staining. C) bak–/–/bax–/– cells were treated with 20 µM gossypol for the indicated time or with indicated compound for 48 h, and the percentage of cells with activated caspases were characterized as those that stained with the FITC in situ marker. Data represent the mean values of three independent experiments. *P < 0.05. D) bak–/–/bax–/– cells were exposed to 20 µM gossypol in the presence of 100 µM zVAD-fmk or 5 mM autophagic inhibitor 3-MA for 24 h. The percentage of cell death was determined by Annexin V/PI staining followed by flow cytometric analysis. Results are expressed as the mean (±SD) for three independent experiments. *P < 0.05.

2. We found that gossypol induced cyto c release in a mitochondrial permeability transition pore independent manner in bak^–/–/bax^–/– cells
We found that gossypol induced cyto c release from mitochondria to the cytosol in bak^–/–/bax^–/– cells. Therefore, we aimed to understand the mechanism of gossypol-induced apoptosis and cyto c release in the absence of Bak and Bax. Since mPTP opening is one of the pathways for cyto c release, we used isolated mitochondria from mouse liver to determine whether gossypol induces cyto c release from mitochondria via the pathway, and found that gossypol did not induce the opening of mPTP. Cyclosporin (CsA), a commonly used inhibitor of mPTP, could not inhibit gossypol-induced cyto c release. These data suggest that gossypol-induced cyto c release is independent of mPTP opening.

3. We compared the apoptotic effects of gossypol with other Bcl-2 inhibitors
Gossypol was found to interact with Bcl-xL and Bcl-2. Gossypol is different from other known Bcl-2 inhibitors, such as HA14–1 and chelerythrine, which did not induce apoptosis and cyto c release in bak^–/–/bax^–/– cells but did induce apoptosis in Bcl-2 overexpressing IM-9 cells.

4. We found that gossypol induced Bcl-2 conformational change and present data suggest that altered Bcl-2 is responsible for cyto c release in the absence of Bak and Bax
Using NMR chemical shift perturbation analysis (Fig. 2 A) and a specific antibody (Ab) that recognizes the exposed epitope of BH3 in the altered Bcl-2 molecule (Fig. 2 ), we showed that gossypol induced conformational change in Bcl-2/Bcl-xL, thus converting their antiapoptotic function into a proapoptotic one. Using reconstituted proteoliposomes encapsulated with FITC-cyto c, in the presence or absence of Bcl-2 and Bcl-xL, we showed that the interaction of gossypol with Bcl-2 or Bcl-xL has functional consequence for cyto c release.

View larger version (35K): [in this window] [in a new window]   Figure 2. Gossypol induces allosteric Bcl-2 conformational change, which may be responsible for cyto c release. A) Superimposition of 1H-15N HSQC spectra recorded at different Bcl-xL/gossypol ratios: without gossypol (black peaks), 2:1(green peaks), and 1:1(magenta peaks). The residues for which the NH signals showed chemical shift or intensity changes on binding of gossypol are labeled by residue type and number. B) Interaction region between gossypol and Bcl-xL. The dotted blue part is the Bak peptide. All the residues whose NH chemical shifts affected by gossypol titration are indicated in pink. These residues are mostly located at BH1, BH2, and BH3 (also labeled in figure), which form the hydrophobic pocket. C) bak–/–/bax–/– cells were treated with or without gossypol for 24 h, and then immunostained with indicated Bcl-2 Ab, followed by FITC-conjugated secondary Ab, and identified by flow cytometry. Control (purple) and gossypol treated (green). D) bak–/–/bax–/– cells were exposed to 20 µM gossypol for indicated time. Following treatment, cells were lysed in Chaps lysis buffer and immunoprecipitated with the indicated anti-Bcl-2 Ab. Immunoprecipitates were subjected to immunoblotting using anti-Bcl-2 Ab. E) Isolated mitochondria from bak–/–/bax–/– cells were exposed to different concentrations of gossypol as indicated at 25°C for 60 min. After centrifugation, mitochondria were lysed in Chaps buffer and immunoprecipitation was performed as described above.

5. Gossypol could be useful for fighting cancers with dysregulated Bcl-2 family proteins
We found that gossypol overcame Bcl-2-conferred drug resistance in lymphoblast cells by inducing a conformational change in Bcl-2 but not activating Bax. In vivo study showed that gossypol inhibited tumor growth in nude mice without adverse effects on body wt and activity.

CONCLUSIONS AND SIGNIFICANCE

In this study, we used bak^–/–/bax^–/– cells, which are resistant to most death stimuli, to specifically screen for small natural molecules that can induce caspase-dependent apoptosis. Our results reveal that gossypol can induce cyto c release from mitochondria to activate caspase-dependent apoptosis in the absence of both Bak and Bax. We found that gossypol induced Bcl-2 conformational change, which may convert it from a protective to a killer molecule. To our best knowledge, this is the first study describing a small molecule that can induce Bcl-2 conformational change and links this change to cyto c release. Our results suggest that, in addition of the rheostat balancing of protective Bcl-2 protein to proapoptotic Bcl-2 family proteins, the conformational status of Bcl-2 is also important in determining the fate of the cell. This conversion could be reminiscent of Ced-9 in C. Elegans, in which the genome contains no Bax homologue and Ced-9 may perform functions similar to that of both Bax and Bcl-2 by conformational change. The discovery that gossypol can change Bcl-2 from a protector to a killer will be help further elucidate Bcl-2 function. This discovery also holds promise for cancer therapy, as overexpression of Bcl-2 or Bcl-xL has been observed in 80% B-cell lymphoma, 90% of colorectal adenocarcinomas, and many other forms of cancer. Given its tolerable toxicity, gossypol represents a promising lead for the development of more potent and specific agents targeting Bcl-2-regulated apoptosis both alone or in combination with other anticancer agents.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram of apoptotic mechanism induced by gossypol. We found that gossypol induces Bax/Bak independent apoptosis. It can directly impact on mitochondria to induce the reduction of m and cytochrome c release without affecting mPTP opening. Our results showed that the interaction between gossypol with Bcl-2 and the subsequent Bcl-2 conformational change play critical roles for the apoptotic effects.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5665fje

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