Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL*

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G1 cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G 1 cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bclx L , Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.
TRAIL (TNF-related apoptosis-inducing ligand), a member of the TNF superfamily, has strong antitumor activity in a wide range of tumor cell types and minimal cytotoxicity in most normal cells (1)(2)(3). TRAIL mediates its anticancer effects through two transmembrane agonistic receptors, TRAIL death receptor (DR) 3 -4 (4) and TRAIL DR5 (5), and three antagonistic receptors, transmembrane decoy receptor (DcR)-1, transmembrane DcR2 (6,7), and osteoprotegerin, a soluble receptor that lacks the transmembrane domain (8). The binding of TRAIL to DR4 and DR5 leads to caspase-8 and caspase-10 activation, which then activates downstream executioner caspase-3, -6, and -7 to mediate apoptosis (9). Because of the selectivity of TRAIL toward tumor cells, both TRAIL and agonistic antibodies against its receptor are currently being studied in clinical trials as a cancer treatment in combination with various chemotherapeutic agents (10). Although most cancer cells express TRAIL functional receptors DR4 and DR5, resistance to the cytotoxic effects of TRAIL is common. Many molecular mechanisms may account for the resistance of these cancer cells to TRAIL (11).
Several studies have shown that chemotherapy sensitizes cancer cells to TRAIL by recruiting the mitochondria-dependent caspase activation cascade (12)(13)(14). Effective TRAIL-based combination therapy can be achieved by up-regulating DR4 and DR5 expression and directly targeting tumor cell mitochondria to stimulate their apoptosis-inducing properties (15). The ability of mitochondria to mediate apoptosis is tightly regulated by members of the Bcl-2 superfamily (16). Molecules commonly referred to as BH3 mimetics bind to the BH3 domain-binding pockets of Bcl-2 and Bcl-x L to inhibit anti-apoptotic function (17). Suppressing anti-apoptotic protein expression with antisense RNA or siRNA has been shown to sensitize cancer cells to TRAIL (18). As a result, agents that down-regulate such anti-apoptotic proteins as survivin, cFLIP (cellular FLICE (FADD-like interleukin-1␤-converting enzyme) inhibitory protein), Bcl-2, Bcl-x L , and XIAP (X-linked inhibitor of apoptosis protein) can potentially sensitize cancer cells to the cytotoxic effects of TRAIL (11,18).
Among the candidate sensitizers is gossypol, a polyphenol derived from cottonseed oil that was once used as an anti-fertility agent and that has recently been shown to have anticancer properties (19). In part through their ability to bind to and inactivate BH3 domain-containing anti-apoptotic proteins (20), gossypol and its analogs inhibit the growth of a wide variety of human cancer cells in culture and xenograft models (21,22).
We investigated whether gossypol potentiates TRAIL-induced cancer cell apoptosis, and if so, how gossypol potentiates this effect. We found that gossypol potentiates TRAIL-induced apoptosis by up-regulating TRAIL receptor DR5 through the ROS (reactive oxygen species)-ERK-CHOP (CAAT/enhancer-binding protein homologous protein) pathway and by down-regulating the expression of cell survival proteins Bclx L , Bcl-2, XIAP, cFLIP, and survivin.
LIVE/DEAD Assay-To measure apoptosis, we used the LIVE/DEAD cell viability assay (Invitrogen), which determines intracellular esterase activity and plasma membrane integrity, to assess cell viability. Calcein-AM, a non-fluorescent polyanionic dye, is retained by live cells, in which it produces green fluorescence through enzymatic (esterase) conversion. In addition, the ethidium homodimer enters cells with damaged membranes and binds to nucleic acids, thereby producing a red fluorescence in dead cells. Briefly, treated and untreated cells were stained with 5 mol/liter ethidium homodimer and 5 mol/liter calcein-AM and incubated at 37°C for 30 min. The cells were then analyzed under a Labophot-2 fluorescence microscope (Nikon).
Analysis of Cell-surface Expression of DR4 and DR5-To analyze the cell-surface expression of DR4 and DR5, cells were treated with 5 M gossypol for 24 h, stained with phycoerythrin-conjugated mouse anti-human DR4 or DR5 monoclonal antibody (R&D Systems) for 45 min at 4°C according to the manufacturer's instructions, resuspended, and analyzed by flow cytometry with phycoerythrin-conjugated mouse IgG2B as an isotype control.
Propidium Iodide Staining for DNA Fragmentation-Cells were pretreated with gossypol for 8 h and then exposed to TRAIL for 24 h. Propidium iodide staining for DNA content analysis was performed as described previously (23). A total of 10,000 events were analyzed by flow cytometry using an excitation wavelength set at 488 nm and an emission wavelength set at 610 nm.
Measurement of Reactive Oxygen Species-To detect intracellular reactive oxygen species (ROS), we preincubated HCT116 cells with 20 M dichlorofluorescein diacetate for 15 min at 37°C before treating the cells with gossypol. After 30 min of incubation, the increase in fluorescence resulting from the oxidation of dichlorofluorescein diacetate to dichlorofluorescein was measured by flow cytometry as described previously (24). The mean fluorescence intensity at 530 nm was calculated for at least 10,000 cells at a flow rate of 250 -300 cells/s.
Transfection with siRNA-HCT116 cells were plated in each well of 6-well plates and allowed to adhere for 24 h. On the day of transfection, 12 l of HiPerFect transfection reagent (Qiagen) was added to 50 nmol/liter siRNA in a final volume of 100 l of culture medium. After 48 h of transfection, cells were treated with gossypol for 8 h and then exposed to TRAIL for 24 h.

RESULTS
The objective of this study was to determine whether gossypol, a polyphenol derived from cottonseed oil, can modulate TRAIL-induced apoptosis and, if so, to determine the mechanism by which gossypol modulates the effect of TRAIL. Most studies were carried out in human colorectal cancer cell line HCT116; however, we showed that the effect of gossypol was not restricted to these cells.
Gossypol Sensitizes Colon Cancer Cells to TRAIL-mediated Apoptosis-We used the LIVE/DEAD cell viability assay to investigate the effect of gossypol on TRAIL-induced apoptosis and found that a subtoxic dose of gossypol induced apoptosis in HCT116 cells in a dose-dependent manner. We also observed that TRAIL treatment alone induced apoptosis in 10% of the HCT116 cells. However, treating cells with TRAIL plus gossypol induced apoptosis in 51% of the HCT116 cells (Fig. 1A).
We also examined apoptosis by determining the size of the sub-G 1 cell pool. We found that apoptosis was induced at 12.1% by gossypol, 17.4% by TRAIL, and 47% by the combination of the two (Fig. 1B).
We examined the effect of gossypol on TRAIL-induced cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, which detects mitochondrial activity. Briefly, cells were pretreated with various concentrations of gossypol for 8 h and then exposed to TRAIL for 24 h. Cells were stained with propidium iodide, and the sub-G 1 fraction was analyzed by flow cytometry. C, cells were pretreated with various concentrations of gossypol (GP) for 8 h, the medium was removed, and the cells were exposed to TRAIL (25 ng/ml) for an additional 24 h. Cell viability was then analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. D, cells were pretreated with the indicated concentrations of gossypol for 8 h and then rinsed. The cells were then treated with TRAIL (25 ng/ml) for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting using antibodies against caspase-9, caspase-8, caspase-3, and PARP.
HCT116 cells were moderately sensitive to either gossypol or TRAIL alone. However, pretreatment with gossypol significantly enhanced TRAIL-induced cytotoxicity in dose-dependent manner (Fig. 1C).
We also investigated whether gossypol enhances TRAIL-induced activation of caspase-8, -9, and -3 and consequent PARP cleavage and found that gossypol enhanced TRAIL-induced activation of all three caspases, thus leading to increased PARP cleavage (Fig. 1D). These results suggest that gossypol enhances TRAIL-induced apoptosis.
Gossypol Down-regulates the Expression of Various Cell Survival Proteins-Because numerous anti-apoptotic proteins such as survivin, cFLIP, XIAP, Bcl-2, and Bcl-x L have been shown to regulate TRAIL-induced apoptosis (25,26), we investigated whether gossypol potentiates TRAIL-induced apoptosis by regulating these cell survival proteins. HCT116 cells were exposed to different concentrations of gossypol for 24 h and then examined for Bcl-x L , Bcl-2, survivin, XIAP, cFLIP, cIAP-1 (cellular inhibitor of apoptosis protein 1), and TRAF1 (TNF receptor-associated factor 1) expression. Gossypol inhibited Bcl-x L , Bcl-2, survivin, and XIAP expression ( Fig. 2A). The effect of gossypol on the expression of Bcl-x L and Bcl-2 was quite dramatic, whereas the down-regulation of cIAP-1, cFLIP, and TRAF1 was not very pronounced. These results suggest that the gossypol-induced down-regulation of certain cell survival proteins could be one mechanism by which TRAIL-induced apoptosis is potentiated.
Gossypol Up-regulates the Expression of Bax-We next examined whether gossypol could modulate the expression of pro-apoptotic proteins. We found that gossypol dramatically up-regulated the expression of Bax in a dose-dependent manner (Fig. 2B).
Gossypol Induces the Expression of TRAIL Receptor DR5-To determine whether gossypol can modulate DR4 and/or DR5 expression in HCT116 cells, we treated HCT116 cells with gossypol for 24 h and then used Western blot analysis to examine the cells for TRAIL receptor expression. Gossypol increased DR5 expression in a dose-dependent manner but had no effect on DR4 expression or DcR1 or DcR2 induction (Fig. 3A). Gossypol also induced DR5 expression in a time-dependent manner (Fig. 3B). These findings suggest that DR5 up-regulation could be a mechanism by which gossypol enhances the apoptotic effects of TRAIL in HCT116 cells.  To determine whether gossypol induces expression of the death receptor on the cell surface, we analyzed the cell-surface expression of DR5 and DR4 in cells using the flow cytometry method. After cell exposure to gossypol, the level of DR5 on the cell surface increased (Fig. 3C, left panel). However, the level of DR4 on the cell surface was not significant (Fig. 3C, right panel). Collectively, these results indicate that gossypol up-regulates the expression of DR5 on the cell surface.
Up-regulation of DR5 by Gossypol Is Not Cell Type-specific-We also investigated whether up-regulation of DR5 by gossypol is specific to HCT116 cells or also occurs in other cell types.  (Fig. 3D). Together, these findings suggest that the up-regulation of DR5 by gossypol is not specific to cell type.
DR5 Induction by Gossypol Is Needed for TRAIL-induced Apoptosis-To determine the role of DR5 in TRAIL-induced apoptosis, we used siRNA specific to DR5 to down-regulate the expression of this receptor. Transfection of cells with siRNA for DR5 but not with the control siRNA reduced gossypol-induced DR5 expression (Fig. 4A). However, DR5 siRNA had a minimal effect on the gossypol-induced up-regulation of DR4.
We next used an esterase staining assay (the LIVE/DEAD cell viability assay) to examine whether suppression of DR5 expres-sion by siRNA could abrogate the effect of gossypol on TRAILinduced apoptosis. Our results showed that the effect of gossypol on TRAIL-induced apoptosis was effectively abolished in cells transfected with DR5 siRNA (Fig. 4B, middle panel), whereas cells transfected with control siRNA were not affected (Fig. 4B, lower panel). Taken together, these results indicate that induction of TRAIL receptor DR5 is critical for sensitization of tumor cells to the effect of gossypol on TRAIL-induced apoptosis.
Gossypol-induced DR5 Up-regulation Is Mediated through Induction of CHOP-Several studies have shown that the upregulation of DR5 is mediated through p53, peroxisome prolif- HCT116 cells were cultured in 6-well plates and transfected the next day with DR5 siRNA or control siRNA (scrambled RNA (scRNA)). Twenty-four hours after transfection, cells were reseeded in 6-well plates or chamber slides and treated with gossypol. A, cells were incubated with 5 M gossypol for 24 h and subjected to preparation of whole-cell lysates and Western blot analysis. B, cells were exposed to 2 M gossypol for 8 h, washed with PBS to remove gossypol, and then treated with TRAIL for an additional 24 h. Cell death was determined by the LIVE/DEAD cell viability assay.

FIGURE 5. Up-regulation of CHOP is required for gossypol-induced DR5 expression.
A, HCT116 cells (1 ϫ 10 6 cells/well) were incubated with 5 M gossypol for the indicated times, and whole-cell lysates were subjected to Western blot analysis using relevant antibodies. PPAR␥, peroxisome proliferator-activated receptor-␥. B, HCT116 cells (3 ϫ 10 5 cells/well) were transfected with either CHOP siRNA or control siRNA (scrambled RNA (scRNA)). Twentyfour hours after transfection, cells were reseeded in 6-well plates or chamber slides. Cells were treated with 5 M gossypol for 24 h, and whole-cell lysates were analyzed by Western blotting. The same blots were stripped and reprobed with anti-␤-actin antibody to verify equal protein loading. C, cells were exposed to 2 M gossypol for 8 h, washed with PBS to remove gossypol, and then treated with 25 ng/ml TRAIL. Cell death was determined by the LIVE/ DEAD cell viability assay.

Gene Silencing of CHOP Abrogates Gossypol-induced Up-regulation of DR5 Expression and Its Effect on TRAIL-induced
Apoptosis-To elucidate the role of CHOP in gossypol-induced up-regulation of the death receptor, we examined gene silencing of CHOP by siRNA. Whereas gossypol up-regu-lated the expression of DR5 in nontransfected and control-transfected (scrambled RNA) cells, transfection with CHOP siRNA significantly suppressed gossypol-induced DR5 up-regulation (Fig. 5B, upper panel). CHOP siRNA had no effect on gossypol-induced DR4 expression (Fig.  5B, middle panel).
We next used an esterase staining assay to examine whether the suppression of CHOP by siRNA could abrogate the sensitizing effects of gossypol on TRAIL-induced apoptosis. Gossypol significantly reduced the effects (from 58 to 24%) on TRAIL-induced apoptosis (Fig.  5C, middle panel), whereas treatment with control siRNA had no effect (Fig. 5C, lower panel). These results indicate that CHOP plays a major role in DR5 up-regulation and contributes to the sensitizing effect of gossypol on TRAIL-induced apoptosis.

Gossypol-induced TRAIL Receptor Up-regulation Requires ERK but Not p38 MAPK and JNK-Because
various studies have suggested that MAPK activation plays a role in TRAIL receptor induction (30,31), we investigated whether the activation of ERK1/2, p38 MAPK, or JNK plays a role in gossypol-induced DR5 induction. We first investigated the gossypol-induced activation of these kinases. Gossypol activated JNK, ERK1/2, and p38 MAPK in a dose-dependent manner (Fig. 6A).
Gene Silencing of ERK Abolishes DR5 Up-regulation and TRAIL Sensitization-To determine whether silencing of ERK blocks DR5 induction and abrogates TRAIL-induced cell death, we transfected HCT116 cells with control siRNA and specific siRNA against ERK1. After 48 h, cells were exposed to gossypol To determine the involvement of ERK1/2, p38 MAPK, and JNK in the up-regulation of DR5, we treated HCT116 cells (1 ϫ 10 6 cells/well) with 5 M gossypol for various durations. Whole-cell extracts were prepared, and Western blotting was used to analyze the extracts for phosphorylated (p) JNK, ERK1/2, and p38 MAPK expression. A, the same blots were stripped and reprobed with anti-JNK1, anti-ERK1/2, and anti-p38 MAPK antibodies to verify equal protein loading. B, to confirm the role of MAPK, we performed inhibitor studies. We pretreated HCT116 cells (1 ϫ 10 6 cells/well) with p38 MAPK inhibitor (SB202190), JNK inhibitor (SP600125), and ERK1/2 inhibitor (PD98059) for 1 h and then treated the cells with 5 M gossypol for 24 h. Whole-cell extracts were then prepared, and Western blotting was used to analyze the extracts for DR5 and CHOP expression. The same blots were stripped and reprobed with antibodies for non-phosphorylated proteins to verify equal protein loading. C, blockade of ERK reversed the effect of gossypol on TRAIL-mediated cell death. Cells were transfected with either ERK1 siRNA or control siRNA (scrambled RNA (scRNA)). After 48 h, cells were treated with 5 M gossypol for 24 h, and whole-cell extracts were subjected to Western blotting (left panel). Cells and siRNA-transfected cells were pretreated with 2 M gossypol for 8 h, washed with PBS to remove gossypol, and then incubated with 25 ng/ml TRAIL for an additional 24 h (right panel). Cell death was determined by the LIVE/DEAD cell viability assay.
for 24 h and harvested, and lysates were subjected to Western blotting. The results indicated that the expression of ERK1 was significantly reduced in cells treated with specific siRNA compared with that in untreated cells or cells treated with control siRNA (Fig. 6C, left panel). The reduction in ERK1 expression by the siRNA correlated with suppression of gossypol-induced up-regulation of CHOP and DR5; however, the deletion of ERK1 had no effect on DR4 expression (Fig.  6C, left panel). Furthermore, siRNA suppression of ERK1 expression significantly reduced gossypol enhancement of TRAIL-induced apoptosis. However, control siRNA had no effect (Fig. 6C, right panel). These results suggest that CHOP-dependent DR5 up-regulation contributes to the sensitizing effect of gossypol on TRAIL-induced apoptosis.

Gossypol-induced TRAIL Receptor Up-regulation and Apoptosis
Potentiation Are ROS-dependent-Because numerous studies have implicated ROS in TRAIL receptor induction, we investigated whether gossypol mediates its effects through ROS. To investigate the ability of gossypol to induce ROS, we treated cells with gossypol and used dichlorofluorescein diacetate as a probe to measure the increase in ROS levels in cells. Gossypol induced ROS in a dose-dependent manner (Fig. 7A). We next examined whether ROS regulates gossypol-induced TRAIL receptors. We found that pretreating cells with N-acetylcysteine reduced the gossypol-induced upregulation of DR5 expression (Fig. 7B). Under these conditions, N-acetylcysteine also suppressed gossypol-induced ERK phosphorylation. Thus, these data suggest that gossypol-induced ROS plays a critical role in activation of ERK, which in turn leads to induction of the TRAIL receptor.
Next, we investigated whether ROS plays any role in gossypolinduced TRAIL potentiation. As shown in Fig. 7C, gossypol significantly enhanced TRAIL-induced apoptosis in HCT116 cells, and pretreatment of cells with N-acetylcysteine markedly reduced gossypolinduced enhancement from 51 to 19%. Then, we determined whether N-acetylcysteine could reverse gossypol-enhanced caspase activation. We found that N-acetylcysteine abolished the effect of gossypol on TRAILinduced procaspase and PARP cleavage (Fig. 7D), suggesting that ROS plays a critical role in mediating the effects of gossypol on TRAIL-induced apoptosis. Whole-cell extracts were prepared, and Western blotting was used to analyze the extracts for caspase and PARP expression. ␤-Actin was used as a loading control.

Gossypol Induces Endoplasmic Reticulum (ER) Stress-ER
stress is known to induce ER stress marker proteins (e.g. GRP78, ATF4, and phospho-eIF2␣), leading to expression of CHOP. Whether gossypol induces these ER stress marker proteins was examined. Our results showed that gossypol did indeed induce all of these markers of ER stress (Fig. 8A). Together, these results suggest that gossypol does induce ER stress.
Effect of Gossypol on TRAIL Receptors Is Independent of a BH3 Mimetic-Gossypol is a well known BH3 mimetic (20,32) and binds to the BH3 domain-binding pockets of Bcl-2 and Bcl-x L . Whether the effect of gossypol on TRAIL receptors is through BH3 mimetic action was examined. For this, we used Bcl-x L siRNA to down-regulate the expression of Bcl-x L in HCT116 cells and investigated the effect of gossypol on expression of death receptors. Down-regulation of Bcl-x L by specific siRNA had no effect on the up-regulation of DR5 or CHOP induced by gossypol (Fig. 8B). These results thus indicate that induction of death receptors by gossypol is not mediated through its role as a BH3 mimetic.

DISCUSSION
Although both TRAIL and agonistic antibodies to TRAIL receptors are currently in clinical trials for treatment of cancer patients, resistance of tumor cells to apoptosis is one of the major hurdles in the usefulness of this cytokine. Thus, in this study, we investigated the ability of gossypol, a polyphenol derived from cotton seed oil and once tested in men as an antifertility drug, to modulate TRAIL signaling in cancer cells, and our findings indicate that gossypol potentiates TRAIL-induced apoptosis in cancer cells by inducing DR5 expression and by down-regulating cell survival proteins linked to tumor cell resistance to TRAIL. Gossypol up-regulation of DR5 appears to be mediated through activation of the ROS-ERK-CHOP pathway (Fig. 8C).
We found that gossypol could enhance TRAIL-induced apoptosis in human colorectal cancer cells. These results are in agreement with those of Yeow et al. (33), who reported that gossypol sensitizes thoracic cancer cells to TRAIL but did not address the mechanism of sensitization. In this study, we identified several sensitization mechanisms. One of the mechanisms of sensitization involved is the regulation of anti-apoptotic proteins. Gossypol down-regulated the expression of Bcl-2, Bcl-x L , and survivin, all of which have been linked to tumor cell resistance to TRAIL (11,34,35). Indeed, down-regulation of XIAP, Bcl-2, and Bcl-x L expression has been shown to sensitize tumor cells to TRAIL (34,36,37). Song et al. (34) showed that the dissociation of Bad from Bcl-x L and an increase in the intracellular level of Bcl-x L are responsible for the acquisition of TRAIL resistance in tumor cells.
Our results are also in agreement with previous studies showing that survivin down-regulation enhances TRAIL-induced apoptosis; for instance, the flavonoid kaempferol has been shown to sensitize human glioma cells to TRAIL-mediated apoptosis by inducing the proteasomal degradation of survivin (38). Embelin, an XIAP inhibitor, has also been shown to enhance TRAIL-mediated apoptosis in malignant glioma cells (39).
We found that, in addition to down-regulating cell survival proteins, gossypol selectively induced DR5 expression. We have shown that DR5 up-regulation is critical for sensitization of cells to TRAIL, as gene silencing of the receptor (DR5) abolished TRAIL-induced apoptosis. Thus, DR5 up-regulation can sensitize cells to TRAIL-induced apoptosis (40). Numerous mechanisms have been described for induction of the death receptor, including ROS generation, p53 induction, and NF-B, DDIT3 (DNA damage-inducible transcript 3), peroxisome proliferator-activated receptor-␥, and MAPK activation (27-29, 40, 41). We found that gossypol-induced DR5 induction was independent of JNK and p38 MAPK activation but that ERK1/2 activation was required. Gossypol activated this kinase, but inhibition of ERK1/2 abolished DR5 induction. We found that the gene silencing of ERK1 led to suppression of gossypol-induced DR5 expression, which suppressed gossypol enhancement of TRAIL-induced cell death. These findings are similar FIGURE 8. Gossypol induces ER stress. A, HCT116 cells were treated with various concentrations of gossypol for 24 h. Whole-cell extracts were prepared, and Western blotting was used to analyze relevant antibodies. p, phosphorylated. B, the effect of gossypol on TRAIL-receptors was independent of a BH3 mimetic. Cells were transfected with either Bcl-x L siRNA or control siRNA (scrambled RNA (scRNA)). After 48 h, cells were treated with 5 M gossypol for 24 h, and whole-cell extracts were subjected to Western blotting. C, shown is the proposed mechanism by which gossypol potentiates the effect of TRAIL on apoptosis. NAC, N-acetylcysteine.
to those of a previous study showing that the induction of death receptors by LY303511 (a PI3K inhibitor) and zerumbone requires ERK1/2 activation (41). However, both of these studies showed that JNK is also required for death receptor induction.
In this study, DR5 up-regulation was also mediated through CHOP induction. We found that gossypol induced CHOP and that the gene silencing of CHOP by siRNA blocked the effect of gossypol on the induction of death receptors and on TRAILinduced apoptosis. Our findings are similar to those of other studies that indicated that CHOP binds to the DR5 promoter and up-regulates this receptor expression (29,42,43). Although p53 induction has also been linked to DR5 induction (27), we found that gossypol-induced up-regulation of TRAIL receptor DR5 was independent of p53.
In this study, we found that perhaps the most important upstream signal linked to gossypol modulation of TRAIL receptors is ROS. Our findings demonstrate that gossypol induces the production of ROS. Furthermore, the quenching of ROS by the antioxidant N-acetylcysteine abolishes the effect of gossypol on induction of CHOP and DR5. We found that quenching ROS also abolished gossypol potentiation of TRAIL-induced apoptosis. Our findings are in agreement with those reported in previous studies using sulforaphane, zerumbone, and celastrol for DR5 induction that indicated that ROS has a major role in modulation of TRAIL receptor DR5 (23,44,45).
Gossypol may also potentiate the effect of TRAIL by inhibiting NF-B activation. Studies have shown that TRAIL can activate NF-B (46), death receptor induction is linked with NF-B activation (40), NF-B activation can block TRAIL-induced apoptosis (6), and gossypol can suppress NF-B activation (47). The role of gossypol as a BH3 mimetic may also contribute to tumor cell sensitization to TRAIL. Many pro-apoptotic and anti-apoptotic proteins with the BH3 domain have been identified (48), and gossypol and its analogs bind to and interact with anti-apoptotic proteins Bcl-2 and Bcl-x L to induce apoptosis in cancer cells (20,49,50).
Various BH3 mimetics, including gossypol, are now in clinical trials for treatment of cancer (51). The results of this study indicate that gossypol contributes to the sensitivity of tumor cells to TRAIL through numerous mechanisms (Fig. 8C). Additional studies of gossypol in animals are needed to fully realize the potential of gossypol therapy for cancer patients.