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,1
,1
* 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;.
|| College of Life Sciences, 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
ABSTRACT
Cells without Bak and Bax are largely resistant to apoptosis (1;2), despite the presence of other key components of the apoptotic machinery. We screened 7,800 natural compounds and found several that could specifically induce caspase activation and the release of cytochrome c (cyto c) in the bak–/–/bax–/– cells. One of these was gossypol, a polyphenolic compound naturally found in cottonseed that has been used in antifertility trials. We found that gossypol, but not other Bcl-2-interacting molecules, induced cyto c release and loss of mitochondrial membrane potential (
m) independently of mPTP and Bak/Bax activation. Furthermore, we found that gossypol induced an allosteric change in Bcl-2 in both bak–/–/bax–/– cells and Bcl-2 overexpressing cells. This change in Bcl-2 conformation led to the release of cyto c in the presence of Bcl-2 and Bcl-xL in reconstituted proteoliposomes. We also observed that gossypol substantially reduced the growth of tumor xenografts from Bcl-2 overexpressing cells in nude mice. We conclude that gossypol converts the antiapoptotic molecule Bcl-2 into a proapoptotic molecule that can mediate the release of cyto c and induce apoptosis—Lei, X., Chen, Y., Du, G., Yu, W., Wang, X., Qu, H., Xia, B., He, H., Mao, J., Zong, W., Liao, X., L., Mehrpour, M., Hao, X., Chen, Q. Gossypol induces Bax/Bak-independent activation of apoptosis and cytochrome c release via a conformational change in Bcl-2.
Key Words: mitochondria • Bcl-2 inhibitors • drug screening
STUDIES HAVE SUGGESTED that, when activated, Bak and Bax, proapoptotic molecules of the Bcl-2 family of proteins, create discontinuity or pores in the outer mitochondrial membrane to mediate cyto c release (3
4
5)
. More specifically, Bax translocates to mitochondria where oligomerized Bak and Bax form a mega-channel or membrane pore (6, 7). Reports have shown that the mitochondrial apoptotic pathway is not activated in the absence of Bak/Bax-activating signals. Furthermore, cells lacking both Bak and Bax do not undergo apoptosis in response to death stimuli, such as DNA damaging agents, signal transduction through death receptors, growth factor deprivation, and ER stress (2, 8, 9), although they may undergo caspase-independent type II cell death (autophagic cell death) or programmed necrosis in response to high doses of DNA-damaging agents (8)
. Although bak–/–/bax–/– mice show certain developmental abnormalities, programmed cell death appears to proceed normally in vivo and in vitro under certain experimental conditions (10)
. Better understanding of the mechanisms of Bak/Bax-independent cell death is important because cancer cells lacking Bak or Bax, or harboring mutations of these proteins, fail to respond to chemotherapeutic drugs and death ligands (11)
.
Agents that overcome drug resistance in this type of cancer are of special interest in drug development and cancer therapy. It is desirable to search for small natural compounds from a library that comprises greater structural diversity, since natural compounds have favorable pharmacological properties with minimal side effects. Given that other key components of the apoptotic machinery remain intact in these cells, we screened a library of natural compounds for small molecules that specifically induced apoptosis in bak–/–/bax–/– cells. We identified a number of compounds that induced apoptosis, including gossypol, a polyphenolic compound found in cottonseed. We demonstrated that gossypol potently induced caspase-dependent apoptosis in the absence of Bak and Bax by converting Bcl-2 from an inhibitor to an activator of apoptosis.
MATERIALS AND METHODS
Cell lines and reagents
Simian virus 40 (SV40) transformed embryonic fibroblasts from bak–/–/bax–/– mice were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, USA) and penicillin/streptomycin at 37°C in a 5% CO2 humidified atmosphere. IM-9/Bcl-2 cells were maintained as described previously (12)
.
Gossypol [racemic mixture of two enantiomers (–)gossypol and (+)gossypol, purity 
95% (HPLC)], acridine orange, 3-MA, DPQ, and anti-Bax 6A7 monoclonal antibody (mAb) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3,3'-dihexyloxacarbocyanineiodide [DIOC6 (3)
] and anticyto c oxidase mAb were purchased from Molecular Probes (Eugene, OR, USA). Purified anticyto c was purchased from BD Transduction Labs (Lexington, KY, USA). Anti-Bcl-2 antibodies were purchased from BD Transduction, Abgent, and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies and enhanced chemiluminescence (ECL) reagents were purchased from Pharmingen (San Diego, CA, USA) and Pierce (Rockford, IL, USA), respectively. All other chemicals were purchased from Sigma unless otherwise specified.
Detection of cell death by Hoechst 33342 and Annexin-V-FITC
For Hoechst 33342 staining, cells were plated at a density of 5 x 104 cells/ml on glass coverslides in a six-well plate and treated with 20 µM gossypol. After 24 h, cells were stained with Hoechst 33342 in PBS for 15 min at room temperature in the dark. Cells were then washed three times with PBS and analyzed using a fluorescence microscope. At least 200 cells were counted. For quantitative analysis, apoptosis was evaluated by flow cytometry by Annexin V-FITC and propidium iodide (PI) staining following a standard protocol as described previously (13)
.
Cell staining with acridine orange
To detect whether gossypol induces the formation of accumulation of acidic vesicle organelles, which is characteristic of autophagy, cell staining was performed as described previously (14)
. After treatment with gossypol, acridine orange was added to a final concentration of 1 µg/ml for 15 min and cells were examined by fluorescence microscopy. For quantitative analysis using flow cytometry, cells were collected in PBS and stained with acridine orange for 15 min. Red fluorescence emission from 104 cells was measured with a FACScan using Cell Quest software.
Measurement of mitochondrial membrane potential
This assay was performed as described previously (12, 13). Briefly, after being treated with gossypol, cells were collected and 
m indicator DiOC6 (3)
[2 µl of 2 µM stock solution in dimethyl sulfoxide (DMSO)] was added to 0.4 ml cell suspension (4x105 cells/ml) in PBS (pH 7.2) and incubated at 37°C for 5 min. PI (5 µl of 500 µg/ml stock) was added before analysis. Analysis of m was performed by flow cytometry with excitation at 488 nm. Data were obtained and analyzed with Cell Quest software from the PI-negative cell population on a BD FACScan.
Cell fractionation assay
Cells treated with gossypol were fractionated by differential centrifugation as described previously (12, 15). Briefly, cells were homogenized with a Dounce homogenizer and the homogenate was centrifuged at 800 g for 5 min to remove unbroken cells and nuclei. The cytosolic fractions were obtained by further centrifugation at 100,000 g for 30 min.
Immunofluorescence microscopy
For cyto c subcellular localization, cells were grown on glass coverslips, washed with PBS, and fixed in 3.7% formaldehyde-PBS (+) solution. Cells were incubated in 0.1% Triton-X100-PBS (+). Primary antibody (Ab) (mouse anticyto c mAb) was diluted 1:200 in 2% BSA-PBS (+) and incubated with the cells at 4°C for 12 h. The FITC-conjugated secondary Ab was used at a 1:50 dilution in 2% BSA-PBS (+) solution and incubated at room temperature for 2 h.
Detection of activation of casapse in situ
Cells were collected and washed with PBS. CaspACETM FITC-VAD-FMK in situ marker was added to the cells to a final concentration of 10 µM and incubated for 20 min in the dark. Cells were then washed three times with PBS and resuspended in 400 µl PBS and analyzed with a FACScan.
Analysis for protein expression
Western blotting was performed as described previously (16)
. Briefly, cells were washed and lysed in buffer containing 150 mM NaCl; 25 mM HEPES, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 1 mM EGTA; 1 mM DTT; 50 µg/ml trypsin inhibitor; 1 mM PMSF; and 10 µg/mL aprotinin, leupeptin, and pepstatin. Proteins from total cell lysates were resolved on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 for 2 h at room temperature and then probed with indicated antibodies for incubation at 4°C overnight. Immune complexes were detected with HRP-conjugated secondary Ab and were visualized by ECL (Pierce).
Isolation of mouse mitochondria and measurement of mitochondrial function
Mitochondria were isolated from the liver of Balb/c mice as described previously (17, 18). Briefly, liver was minced on ice, transferred to isolated buffer, and homogenized with a glass-TeflonTM Potter homogenizer. Mitochondria were isolated by differential centrifugation in PT-1 buffer containing 250 mM sucrose; 2 mM HEPES, pH 7.4; 0.1 mM EDTA; and 0.1% fatty acid-free BSA. Mitochondria were washed twice and then resuspended in the same medium. All steps were performed on ice. Protein content of mitochondria was determined by the microbiuret method using BSA as a standard. Isolated mouse liver mitochondria were used for the following experiments: i) Western blotting for cyto c release. Isolated mitochondria (1 mg protein/ml) were incubated in a total vol of 50 µl PT-2 buffer (250 mM sucrose, 2 mM HEPES, 0.5 mM KH2PO4, and 4.2 mM potassium succinate, pH 7.4) in the presence or absence of gossypol for the indicated time at 25°C, followed by centrifugal separation of mitochondria (12,000 g, 10 min at 4°C). Aliquots of the supernatant (20 µl) were subjected to Western blotting. Cyto c was detected by anticyto c mAb. Equal protein loading was confirmed by immunodetection of cyto c oxidase subunit IV (COX-IV). ii) Determination of mitochondrial membrane potential (m). As described previously, m was measured. Briefly, isolated mitochondria (0.1 mg protein/ml) were loaded with 30 nM Rhodamine 123 and incubated at 25°C in the PT-2 medium. By measuring the m-dependent release of Rhodamine 123 from mitochondria using a spectrofluorimeter (Jobin-Yvon FluoroMax-2, excitation=505 nm and emission=534 nm), m was assessed. iii) Measurement of mitochondrial swelling. Mitochondrial swelling was monitored by the decrease of 90° light scatter at 520 nm in the PT-2 medium at 25°C using Jobin-Yvon FluoroMax-2 spectrofluorimeter as described (17, 19).
Immunoprecipitations for detecting Bax and Bcl-2 conformational change
Cells were lysed with 1% Chaps lysis buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 1% Chaps] containing protease inhibitors as described (7, 20). Total protein (500 µg) was incubated with 2 µg of anti-Bax 6A7 mAb in 500 µl of Chaps lysis buffer at 4°C overnight on a rotator. Immunoprecipitates were collected by incubating with 20 µl protein G agarose for 2 h at 4°C, followed by centrifugation for 1 min. The pellets were washed three times with Chaps lysis buffer, and beads were boiled in loading buffer and analyzed by Western blotting using the anti-Bax polyclonal antibody (pAb). For detection of Bcl-2 conformational change, cells were lysed in 1% Chaps buffer (1% Chaps; 14.5 mM KCl; 5 mM MgCl2; 1 mM EGTA; 1 mM EDTA; 20 mM Tris, pH 7.5) (21)
. Total protein (500 µg) was incubated with appropriate Ab and 20 µl protein G agarose beads overnight at 4°C. Beads were washed three times with 1% Chaps buffer before Western blotting.
Measurement of cyto c release in Bcl-xL and Bcl-2 liposomes
Liposomes were prepared by a standard method as described previously (22, 23). Briefly, 500 mg L-
-phosphatidyl choline was dissolved in 5 ml chloroform, and the solvent was then evaporated under nitrogen. A phospholipid mixture was reconstructed in 10 ml liposome buffer containing 50 mM KCl, 20 mM KH2PO4, 20 mM HEPES (pH 7.0), and 1 mM EDTA. After sonication, purified Bcl-xL or Bcl-2 (0.1 mg/ml, final concentration) was then mixed with liposomes and incubated for 20 min. FITC-conjugated cyto c was loaded into the proteoliposomes by three freeze-thaw cycles, and then the proteoliposomes were washed three times with the liposome buffer. Aliquots of the three types of liposomes were mixed with 50 µM gossypol and incubated for 1 h, and the reactions were terminated by centrifugation. The cyto c released in the supernatant was detected by immunoblotting assay.
Effect of gossypol on tumor growth
IM-9/Bcl-2 cells were harvested by centrifugation and suspended in HBSS. A tumor cell suspension (5x106 cells in 50 µl of HBSS) was injected into the back region of 6–8 wk old Balb/c mice using a 27-gauge needle. Two groups of animals (10 animals each group) were used. Animals were treated with 30 mg/kg/day of gossypol two days after injection. The mice were sacrificed 33 d after cell injection. Tumor size was monitored by measurement of the length (a) and width (b) of the tumor using a slide gauge. Tumor volumes (V) were calculated according to the formula: V = 1/6
[(a+b)/2]3.
Statistical analysis
Statistical analysis was performed using Student’s t test analysis, with P values < 0.05 considered significant.
RESULTS
Identification of gossypol inducing typical apoptosis in bak–/–/bax–/– cells
Using simian virus 40 (SV40) transformed embryonic fibroblasts from Bak-Bax double knockout mice (bak–/–/bax–/–) (1, 8), we first screened 7800 natural compounds for their effects on cell viability by using the MTT assay. Twenty-three compounds resulted in the reduction of MTT. We then stained the cells with acridine orange and examined them using a fluorescent microscope to identify nuclear condensation (morphological hallmark of apoptosis) or accumulation of acidic vesicles in the cytoplasm (marker of autophagic cell death) of bak–/–/bax–/– cells. One of these compounds, gossypol, was found to induce nuclear fragmentation in bak–/–/bax–/– cells (Fig. 1
A). Gossypol was originally identified as a male contraceptive drug (24, 25). Although it is not fully effective as a contraceptive, there has been renewed interest in the compound for the treatment of cancer (26, 27).
|
To further characterize the death process, we measured phosphatidylserine exposure, a defining morphological characteristic of apoptotic cells. Gossypol induced a significant increase in the size of the Annexin V positive population (apoptotic cells) in a time-dependent manner as compared to an untreated control (Fig. 1B
). The effect was maximal for cells exposed to 20 µM of Gossypol for 48 h (50% apoptotic cells). We next used CaspACETM FITC-VAD-FMK in situ Marker to detect the intracellular caspase activity in the individual cells. As shown in Fig. 1C
, gossypol induced caspase activation in a time-dependent manner. However, we did not detect caspase activity when bak–/–/bax–/– cells were treated with VP-16, cisplatin, or other related agents. We next measured the activation of effector caspases by determining the cleavage of the chromogenic caspase tetrapeptide substrate Ac-DEVD-pNA. The DEVD cleavage activity appeared 12 h after gossypol treatment and was further elevated in a time-dependent manner (data not shown). Moreover, z-VAD-fmk (100 µM), a pan-caspase inhibitor, could potently block gossypol-induced cell death (Fig. 1D
).
The autophagic inhibitor 3-Methylademine (3-MA) did not block cell death induced by gossypol (Fig. 1D
). The accumulation of acidic vesicle organelles was not observed after treatment with gossypol, while VP16, a positive control of autophagic cell death, induced the significant accumulation of acidic vesicle organelles in bak–/–/bax–/– cells as examined using fluorescent microscopy (Fig. 1E
). These results suggest that gossypol induces morphological and biochemical changes typical of apoptosis but not caspases-independent autophagic cell death.
Gossypol induced cyto c release in a mPTP-independent manner
To investigate the underlying mechanism of caspase activation in bak–/–/bax–/– cells, we analyzed cyto c release in bak–/–/bax–/– cells treated by gossypol. We found that gossypol induced cyto c release in a time-dependent manner. Cyto c appeared in the cytosol within 12 h of exposure to gossypol with an accompanying decrease of cyto c in the mitochondria (Fig. 2
A, top). The release of cyto c from mitochondria was concomitant with the appearance of caspase activity within cells (Fig. 1C
). Immunostaining of cyto c revealed that, in contrast to the punctate staining in untreated cells, cyto c staining became diffuse 24 h after treatment (Fig. 2A
, bottom), indicating the loss of cyto c from mitochondria and its translocation to the cytosol. To exclude the possibility that gossypol may activate intracellular pathways that induce cyto c release, we isolated mitochondria from bak–/–/bax–/– cells and found that exposure to gossypol directly induced the release of cyto c from mitochondria in a dose-dependent manner (Fig. 2B
) in the absence of Bak and Bax. These data clearly demonstrate that gossypol induces cyto c release from mitochondria to activate caspase and to induce apoptosis in the absence of Bak and Bax.
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Currently, there are two possible pathways that may mediate the release of cyto c from mitochondria (28)
. One involves Bax activation, its oligomerization, and its interaction with other mitochondrial membrane proteins (7)
. Alternatively, the opening of mitochondrial permeability transition pores (mPTP) results in the rupture of the outer mitochondrial membrane and, thereby, the nonspecific release of cyto c. To determine whether gossypol induces cyto c release from mitochondria via the opening of mPTP, we used isolated mitochondria from mouse liver and found that gossypol did not cause mitochondrial swelling, which is indicative of the opening of mPTP (Fig. 2C
). Nonetheless, gossypol could induce cyto c release in a time- (Fig. 2D
) and dose-dependent manner (data not show). The release of cyto c from mitochondria started at 10 min after treatment with 20 µM gossypol (Fig. 2D
), whereas the mPTP opening didn’t occur even 30 min after treatment with 20 µM gossypol (Fig. 2C
). These data further support the notion that gossypol-induced cyto c release from isolated mitochondria is not the result of mPTP opening. Due to its mild uncoupling effect, gossypol could reduce mitochondrial membrane potential (m) to a certain extent (Fig. 2E
). Cyclosporin (CsA) and Bonkrekic acid (BA), commonly used inhibitors of mPTP opening, could not inhibit gossypol-induced cyto c release in bak–/–/bax–/– cells (data not shown) or isolated mitochondria from mouse liver (Fig. 2F
). These results strongly suggest that gossypol-induced cyto c release is independent of mPTP opening.
Unlike gossypol, other Bcl-2 inhibitors cannot induce Bax/Bak and mPTP opening independent cyto c release
Other compounds such as HA14-1 (reported to be a Bcl-2 inhibitor) and chelerythrine (reported to be a Bcl-xL inhibitor) induced mitochondrial swelling (Fig. 3
A, B) and cyto c release (Fig. 3C
) from isolated mitochondria from mouse liver in a CsA-sensitive manner, whereas CsA itself had no effect on cyto c release (Fig. 3C
). Computer modeling suggested that gossypol, HA14–1, and chelerythrine could interact with Bcl-xL and Bcl-2 (29)
; we thus checked the effects of other Bcl-2 inhibitors on bak–/–/bax–/– cells. Interestingly, HA14–1 and chelerythrine could induce apoptosis in Bcl-2 overexpressing cells (see below), although they did not induce apoptosis (data not show) and cyto c release (Fig. 3D
) in bak–/–/bax–/– cells. These results suggest that gossypol has the distinct property of inducing the opening of mPTP and cyto c release in bak–/–/bax–/– cells and mouse liver.
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Gossypol induces allosteric Bcl-2 conformational change
In an attempt to identify the potential target(s) responsible for its apoptosis-inducing effects in the absence of Bak-Bax and opening of mPTP, we asked whether gossypol directly interacts with Bcl-2 to induce a change in Bcl-2 conformation. It has been suggested that conformational changes in Bcl-2/Bcl-xL convert them from antiapoptotic to proapoptotic (21)
. Using NMR chemical shift perturbation analysis, we found that gossypol, but not chelerythrine or antimycin A, interacted with Bcl-xL protein at the pocket region among its BH1, BH2, and BH3 domains (Fig. 4
A, B). Most of the residues involved in gossypol binding are located at the Bak BH3 peptide binding site (30)
. Careful analysis showed that this interaction also resulted in significant changes in the chemical environments of a number of amino acids outside the region. Therefore, similar to the binding of Bak and BH3I-1 (31)
, the binding of gossypol likely induces a conformational change in Bcl-xL to open its hydrophobic binding surface. To determine the direct effect of gossypol on Bcl-2 conformation, we used an Ab (Bcl-2/BH3-domain pAb) that recognizes the exposed epitope of BH3 in Bcl-2 molecule. Immunostaining detected by flow cytometric assay revealed that gossypol could significantly increase the fluorescent intensity of Bcl-2/BH3 domain pAb, but not mouse Ab, against the whole Bcl-2 protein (Fig. 4C
). Immunoprecipitation analysis with a specific Ab showed that gossypol induced a conformational change in Bcl-2 in bak–/–/bax–/– cells, but it did not induce a change in Bcl-2 expression levels (Fig. 4D
). To determine whether gossypol directly induces a change in Bcl-2 conformation, we treated mitochondria isolated from bak–/–/bax–/– cells with gossypol. The data depicted in Fig. 4E
show that the exposure of gossypol directly induces a change in Bcl-2 conformation in the absence of cytosol in a dose-dependent manner. In contrast, HA14–1 and chelerythrine did not induce the Bcl-2 conformational change in bak–/–/bax–/– cells (Fig. 4F
).
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To examine whether the interaction of gossypol with Bcl-2 or Bcl-xL has functional consequence for cyto c release, we reconstituted the proteoliposomes encapsulated with FITC-cyto c in the presence or absence of Bcl-2 and Bcl-xL as previously reported (22)
. Following treatment with gossypol, cyto c was released to supernatant in the presence of Bcl-2 and Bcl-xL (Fig. 4G
), but there was no cyto c release in the absence of Bcl-2 or Bcl-xL or in the untreated control. This was in sharp contrast to our previous observation that Bcl-xL could prevent VDAC-mediated cyto c release in the same reconstituted proteoliposome system (22)
.
Gossypol induces typical apoptosis in Bcl-2 overexpressing cells in a Bcl-2 conformational change-dependent manner without activation of Bax
We next asked whether gossypol would induce Bcl-2 conformational change in other systems. We found that gossypol induced apoptosis in IM-9 Bcl-2 overexpressing cells in a time- (Fig. 5
A) and dose-dependent manner (Fig. 5B
). Similar results were obtained with Bcl-xL stably overexpressed cells (data not shown). Gossypol induced the reduction of m, cyto c release (Fig. 5C
), and processing of caspase and PARP in a time-dependent manner (Fig. 5D
). Interestingly, other Bcl-2 inhibitors such as HA14–1, antimycin A, and chelerythrine induced apoptosis in Bcl-2 overexpressing cells (Fig. 5E
), although these inhibitors had no apoptotic effect on bak–/–/bax–/– cells (Fig. 1C
). Gossypol did not induce Bax conformational change (Fig. 5F
) or Bax homodimerization or oligomerization (Fig. 5G
), but it was found to induce Bax activation in the parental cells (unpublished data). Bcl-2 conformational changes were detected 4 h after treatment in IM-9/Bcl-2 cells, concomitant with the appearance of apoptosis and cyto c release, although treatment did not induce changes in Bcl-2 expression levels (Fig. 5H
). The compound did not kill white blood cells from healthy donors, although it induced apoptosis in IM-9 cells by activating Bax conformational change under identical experimental conditions (data not shown).
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We next tested the effects of gossypol on the growth of IM-9/Bcl-2 cells after implantation in nude mice. Two days after implantation of the IM-9/Bcl-2 cells (5x106) onto the backs of the mice, we began treating them daily with gossypol or a control physiological saline solution. As shown in Fig. 5I
, the tumor vol of the treated group [0.44±0.24 cm3] was significantly reduced compared to the control group [1.79+0.44 cm3]. As shown in Figure 5J
, gossypol inhibited tumor growth in the mice without adverse effects on body wt and activity. These results indicate that gossypol effectively inhibits the growth of IM-9/Bcl-2 cells in vivo without apparent adverse side effects.
DISCUSSION
In this paper, we first used bak–/–/bax–/– cells, which are resistant to most death stimuli (2, 8), to screen for small natural molecules that can induce caspase-dependent apoptosis. Our results show that gossypol can induce cyto c release from mitochondria to activate caspase-dependent apoptosis in the absence of both Bak and Bax. We further found that gossypol induced Bcl-2 conformational change, which may convert the protective molecule of Bcl-2/Bcl-xL into a killer molecule. Recent reports show that Bcl-2 conformational change occurs during the onset of apoptosis, although it has been suggested that this conformational change may be integral to its antiapoptotic function (32)
. Interaction of Bcl-2 with a nuclear orphan receptor may convert Bcl-2 from a protector to a killer molecule via conformational change (21)
. Photodynamic therapy and ursodeoxycholi acid also cause a conformational change in Bcl-2 and promotes HA14–1 to bind to Bcl-2 (33)
. To the best of our knowledge, our study is the first to identify a small molecule that induces Bcl-2 conformational change and to link this type of change with cyto c release. Our results suggest that, in addition to the rheostat balancing of protective Bcl-2 protein to Bak and Bax, the conformational status of the protective molecule is also important in determining the fate of a cell. This conversion could be reminiscent of Ced-9 in C. Elegans, whose genome contains no Bax homologue. It has been suggested that Ced-9 performs the functions of both Bax and Bcl-2 concomitant with its conformational change (34)
. It is possible that the exposure of Bcl-2’s BH3 domain and its insertion into the mitochondrial membrane may change the binding properties of Bcl-2 to other apoptosis-related proteins, or to lipids in the outer mitochondrial membrane, to create discontinuity or pores. Our results do not exclude other mechanisms such as hexokinase dissociation from mitochondria (35)
or the conformational changes of other antiapoptotic molecules such as Bcl-xL and Mcl-1.
A wide array of Bcl-2 inhibitors are reported to interact with Bcl-2 and induce tumor cell apoptosis or regression of tumors with single-agent treatment (36)
. These inhibitors bind to the same hydrophobic groove, leading to the disruption of Bcl-2’s heterodimerization with proapoptotic partners and inhibition of its pore forming activity. These inhibitors target mitochondria to induce cyto c release via a mechanism of CsA-inhibitable opening of PTP and in Bcl-2 overexpressing cells, but they had no killing effect on bak–/–/bax–/– cells in our experiments. Our results suggest that gossypol is distinct from these Bcl-2 inhibitors. The discovery that gossypol changes Bcl-2 from a protector to a killer will help further elucidate Bcl-2 function and its mechanism both in vitro and in vivo. This discovery also holds promise for cancer therapy, since over-expression of Bcl-2 or Bcl-xL has been observed in 80% of B-cell lymphomas, 90% of colorectal adenocarcinomas, and many other forms of cancer (37)
. Recently, gossypol was shown to inhibit the growth of several tumor cell lines in vitro and has been suggested to be a potential antitumor drug (27, 38, 39). Given its tolerable toxicity and ability to convert Bcl-2 to a proapoptotic molecule, gossypol holds promise as a more potent and specific agent targeting Bcl-2-regulated apoptosis, both alone or in combination with other anticancer agents. Further work is underway to screen and examine novel compounds that induce Bcl-2 conformational change to change the molecule from anti to proapoptotic.
ACKNOWLEDGMENTS
We gratefully acknowledge Professor Alastair Watson (Liverpool University, UK) and Dr. Dean Tang (M.D. Anderson Cancer Center, Austin, TX) for their thoughtful comments. We thank Dr. Honggang Wang for Bcl-2 plasmid, and Mrs. Jing Wang and Haijing Jin for their technical assistance. We would also like to thank Miss Lei Du and other lab members for their suggestions and comments, and Dr. Gerhard Wagner and Dr. Alexey Lugovskoy from Harvard for kindly providing the NMR signal assignments of Bcl-xL, as well as some important suggestions. We are also grateful to Dr Stephen W. Fesik for giving us chemical shift lists of Bcl-xL/bad and so on.
This work was supported by grants from the National Proprietary Basic Research Program (973 program project, No. 2002CB513100, 2004CB72000), a National Outstanding Young Investigator Fellowship (No. 30325013) from NSFC, and the "Knowledge Innovation Key Project" (Kscx2-sw-2010) from the Chinese Academy of Sciences awarded to Q.C. Grant 30125009 from the National Science Foundation of China to B.X.
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication January 24, 2006. Accepted for publication May 22, 2006.
REFERENCES
This article has been cited by other articles:
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  K. Balakrishnan, W. G. Wierda, M. J. Keating, and V. Gandhi Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells Blood, September 1, 2008; 112(5): 1971 - 1980. [Abstract] [Full Text] [PDF]
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 K. F. Wan, S.-L. Chan, S. K. Sukumaran, M.-C. Lee, and V. C. Yu Chelerythrine Induces Apoptosis through a Bax/Bak-independent Mitochondrial Mechanism J. Biol. Chem., March 28, 2008; 283(13): 8423 - 8433. [Abstract] [Full Text] [PDF]
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