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-x_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.     

Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.     

The Herbal Compound Cryptotanshinone Restores Sensitivity in Cancer Cells That Are Resistant to the Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells but not normal cells. However, TRAIL resistance due to low level of TRAIL receptor expression is widely found in cancer cells and hampers its development for cancer treatment. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are urgently needed. We investigated whether tanshinones, the major bioactive compounds of Salvia miltiorrhiza (danshen), can up-regulate TRAIL receptor expression. Among the major tanshinones being tested, cryptotanshinone (CT) showed the best ability to induce TRAIL receptor 2 (DR5) expression. We further showed that CT was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. CT-induced DR5 induction was not cell type-specific, as DR5 induction was observed in other cancer cell types. DR5 knockdown abolished the enhancing effect of CT on TRAIL responses. Mechanistically, induction of the DR5 by CT was found to be p53-independent but dependent on the induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). Knockdown of CHOP abolished CT-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, CT-induced ROS production preceded up-regulation of CHOP and DR5 and consequent sensitization of cells to TRAIL. Interestingly, CT also converted TRAIL-resistant lung A549 cancer cells into TRAIL-sensitive cells. Taken together, our results indicate that CT can potentiate TRAIL-induced apoptosis through up-regulation of DR5.     

Acrolein sensitizes human renal cancer Caki cells to TRAIL-induced apoptosis via ROS-mediated up-regulation of death receptor-5 (DR5) and down-regulation of Bcl-2

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,β-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5.     

Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.     

Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53-independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK

Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53(-/-) cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins.     

Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53?/?) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Casticin Potentiates TRAIL-Induced Apoptosis of Gastric Cancer Cells through Endoplasmic Reticulum Stress

Background

Casticin is one of the main active components obtained from Fructus Viticis and has been reported to exert anti-carcinogenic activity on a variety of cancer cells but the precise mechanism underlying this activity remains unclear.

Materials and Methods

Apoptotic activities of casticin (1.0 µmol/l) and TRAIL (25, 50 ng/ml) alone or in combination in the gastric cancer cell lines BGC-823, SGC-7901 and MGC-803 were detected by the use of a cell apoptosis ELISA detection kit, flow cytometry (FCM) with propidium iodide (PI) staining and activities of caspase-3, -8 and -9 by ELISA and cleavage of polyADP-ribose polymerase (PARP) protein using western blot analysis. Death receptors (DR) expression levels were evaluated using FCM analysis and western blotting. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to measure the increase in reactive oxygen species (ROS) levels in cells. Multiple interventions, such as siRNA transfection and pharmacological inhibitors were used to explore the mechanisms of these actions.

Results

Subtoxic concentrations of casticin significantly potentiated TRAIL-induced cytotoxicity and apoptosis in BGC-823, SGC-7901 and MGC-803 cells. Casticin dramatically upregulated DR5 receptor expression but had no effects on DR4 or decoy receptors. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by the co-application of TRAIL and casticin. Gene silencing of the CCAAT/enhancer binding protein homologous protein (CHOP) and pretreatment with salubrinal, an endoplasmic reticulum (ER) stress inhibitor, attenuated casticin-induced DR5 receptor expression, and apoptosis and ROS production. Casticin downregulated the expression levels of the cell survival proteins cFLIP, Bcl-2, XIAP, and survivin. In addition, casticin also induced the expressions of DR5 protein in other gastric cancer cells (SGC-7901 and MGC-803).

Conclusion/Significance

Casticin enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

Rosiglitazone promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we show that rosiglitazone sensitizes human renal cancer cells to TRAIL-mediated apoptosis, but not normal human mesangial cells. Furthermore, because rosiglitazone-enhanced TRAIL-mediated apoptosis is induced in various types of cancer cells but is not interrupted by Bcl-2 overexpression, this combinatory treatment may provide an attractive strategy for cancer treatment. We found that treatment with rosiglitazone significantly induces DR5 expression at both its mRNA and its protein levels, accompanying the generation of reactive oxygen species (ROS). Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNAs attenuated rosiglitazone plus TRAIL-induced apoptosis, showing the critical role of DR5 in this cell death. Pretreatment with GSH significantly inhibited rosiglitazone-induced DR5 up-regulation and the cell death induced by the combined treatment with rosiglitazone and TRAIL, suggesting that ROS mediate rosiglitazone-induced DR5 up-regulation, contributing to TRAIL-mediated apoptosis. However, both DR5 up-regulation and sensitization of TRAIL-mediated apoptosis induced by rosiglitazone are likely PPARgamma-independent, because a dominant-negative mutant of PPARgamma and a potent PPARgamma inhibitor, GW9662, failed to block DR5 induction and apoptosis. Interestingly, we also found that rosiglitazone treatment induced down-regulation of cellular FLICE-inhibitory protein (c-FLIPs), and ectopic expression of c-FLIPs attenuated rosiglitazone plus TRAIL-mediated apoptosis, demonstrating the involvement of c-FLIPs in this apoptosis. Taken together, the results of this study demonstrate that rosiglitazone enhances TRAIL-induced apoptosis in various cancer cells by ROS-mediated DR5 up-regulation and down-regulation of c-FLIPs.     

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrated that human prostate cancer cells, but not normal prostate cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by quercetin. Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. We have shown that quercetin can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 and LNCaP cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or quercetin (10-200 microM) for 4 h. Quercetin, which caused no cytotoxicity by itself, promoted TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly(ADP-ribose) polymerase) cleavage were both enhanced by quercetin. Western blot analysis showed that combined treatment with TRAIL and quercetin did not change the levels of TRAIL receptors (death receptors DR4 and DR5, and DcR2 (decoy receptor 2)) or anti-apoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, quercetin promoted the dephosphorylation of Akt. Quercetin-induced potent inhibition of Akt phosphorylation. Taken together, the present studies suggest that quercetin enhances TRAIL-induced cytotoxicity by activating caspases and inhibiting phosphorylation of Akt.     

Death receptor 5 promoter-enhancing compounds isolated from Catimbium speciosum and their enhancement effect on TRAIL-induced apoptosis

The TRAIL/death-receptor signaling pathway has been considered a promising target for selective cancer therapy, although some malignant tumors exhibit TRAIL resistance. We previously found that isoflavonoid enhanced TRAIL-induced apoptosis in TRAIL-resistant cells, which is achieved through up-regulation of death receptor 5 (DR5). In our screening program targeting DR5 promoter enhancement activity, activity-guided fractionations of the extract of Catimbium speciosum led to the isolation of six compounds. Of the isolates, cardamomin (6), the most potent compound, enhanced the expressions of DR5 and DR4 and decreased the Bcl-xL level in TRAIL-resistant DLD1 cells. The combination of 6 and TRAIL synergistically enhanced TRAIL-induced apoptosis against TRAIL-resistant cells upon the activation of caspase-8, 9, and 3. In addition, enhancement of apoptosis by 6 was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, TRAIL-neutralizing fusion proteins, indicating that 6 sensitize TRAIL-resistant cells to TRAIL through the induction of DR5 and DR4. Also, up-regulation of DR5 by 6 paralleled that of CCAAT/enhancer-binding protein-homologous protein (CHOP).     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Down-regulation of DcR2 sensitizes androgen-dependent prostate cancer LNCaP cells to TRAIL-induced apoptosis

Background

Dysregulation of many apoptotic related genes and androgens are critical in the development, progression, and treatment of prostate cancer. The differential sensitivity of tumour cells to TRAIL-induced apoptosis can be mediated by the modulation of surface TRAIL receptor expression related to androgen concentration. Our previous results led to the hypothesis that downregulation of TRAIL-decoy receptor DcR2 expression following androgen deprivation would leave hormone sensitive normal prostate cells vulnerable to the cell death signal generated by TRAIL via its pro-apoptotic receptors. We tested this hypothesis under pathological conditions by exploring the regulation of TRAIL-induced apoptosis related to their death and decoy receptor expression, as also to hormonal concentrations in androgen-sensitive human prostate cancer, LNCaP, cells.

Results

In contrast to androgen-insensitive PC3 cells, decoy (DcR2) and death (DR5) receptor protein expression was correlated with hormone concentrations and TRAIL-induced apoptosis in LNCaP cells. Silencing of androgen-sensitive DcR2 protein expression by siRNA led to a significant increase in TRAIL-mediated apoptosis related to androgen concentration in LNCaP cells.

Conclusions

The data support the hypothesis that hormone modulation of DcR2 expression regulates TRAIL-induced apoptosis in LNCaP cells, giving insight into cell death induction in apoptosis-resistant hormone-sensitive tumour cells from prostate cancer. TRAIL action and DcR2 expression modulation are potentially of clinical value in advanced tumour treatment.