Upregulation Of DR5 Research Articles

RETRACTED: Seleno-cyclodextrin sensitises human breast cancer cells to TRAIL-induced apoptosis through DR5 induction and NF-κB suppression

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/policies/article-withdrawal). This article has been retracted at the request of This article has been retracted at the request of the authors and the Editor-in-Chief. The authors contacted the Editors of the European Journal of Cancer regarding oversights or labelling errors introduced in Fig. 2 and Fig.7 during data processing. The authors provided the raw data, however the Editors conclude that the raw data management cannot be relied upon and the article needs to be retracted.

Abstract 3323: Neural stem cells expressing TRAIL as therapeutic modality for treatment of experimental malignant glioma

Abstract Introduction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent with apoptotic activity against cancer cells. However, many of human glioblastoma multiforme (GBM) have been shown to be resistant to treatment with soluble TRAIL. Neural stem cells (NSCs) possess an ability to migrate to the main tumor and its satellites. NSCs engineered to express membrane bound TRAIL (NSC-TRAIL) may help to overcome resistance of glioma cells to soluble TRAIL. Therefore, the aim of this study was to investigate if treatment with NSCs-TRAIL is a feasible therapeutic approach for experimental glioma alone or in combination with proteasome inhibitors. Methods. Full size TRAIL cDNA was cloned from mRNA obtained from Jurkat cells by PCR amplification and subsequently sub-cloned in lentiviral vector. NSCs were modified to express membrane bound TRAIL by infection with lentiviral particles encoding TRAIL. Membrane integrity assay was used to evaluate toxicity of soluble TRAIL and proteasome inhibitors in glioma cells. Immunocytochemistry and flow cytometry were used to analyze apoptosis in glioma cells. Intracranial glioma xenograft model in nude mice was utilized for evaluation of therapeutic properties of NSCs-TRAIL alone or in combination with proteasome inhibitors. Results. Glioma cell lines were resistant to treatment with soluble TRAIL and proteasome inhibitors alone. Sensitivity of glioma cells to TRAIL was significantly increased in combined treatment with proteasome inhibitors in vitro. Up-regulation of TRAIL receptor 2 (DR5) in glioma cells exposed to proteasome inhibitors was recorded in time dependent manner. Co-culture with NSCs-TRAIL alone were able to induce apoptosis in glioma cells. The apoptotic effect of NSCs-TRAIL was magnified by treatment with proteasome inhibitor, bortezomib. Animals, received intracranial injection of glioma cells with NSCs-TRAIL at 1:0.5 and 1:1 ratio demonstrated 20 and 80% survival over 3 month, respectively. The survival of animals was improved to 40% and 100% in control NSCs and NSCs-TRAIL experimental groups which received bortezomib therapy. No up-regulation of DR5 was observed in the tumor samples from animals treated with bortezomib. Mice with established intracranial glial xenografts treated with NSCs-TRAIL alone and in combination with bortezomib showed improvement in survival in comparison with animals bearing tumor alone or injected with control NSCs. Conclusion. This study demonstrates that NSCs expressing membrane bound TRAIL are able to trigger apoptosis in glioma cells in vitro and improve survival of animals in vivo. Treatment of glioma cells with proteasome inhibitors significantly increased their sensitivity to TRAIL in vitro. Proteasome inhibitor, bortezomib, improved survival of animals treated with NSCs but its effect was not specific to the NSCs-TRAIL treated mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2011-3323

Abstract 5482: c-Met and DR5 co-expression is an independent prognostic marker for better survival in colorectal carcinoma

Abstract Recently, dysregulated over-expression of hepatocyte growth factor (HGF) and its receptor, c-Met has been reported in variety of cancers. However, its role in colo-rectal carcinoma (CRC) has not been fully elucidated. Therefore, we investigated the role of c-Met in a large series of Middle Eastern CRC patient samples and CRC cell lines. c-Met was over-expressed in 86.8% of CRC and was strongly associated with expression of p-AKT (p<0.0001), DR5 (p=0.0084) and Ki67 (p=0.011) by immuno-histochemistry. Co-expression of c-Met and DR5 was associated with less aggressive phenotype, a better overall survival (p=0.0027) and was an independent prognostic marker. In vitro, PHA665752, a selective c-Met inhibitor, inhibited growth and induced apoptosis in CRC cells. Additionally, PHA665752 treatment caused dephosphorylation of c-Met, AKT and its downstream effector targets FOXO1, GSK-3 and p-Bad. Interestingly, PHA665752 treatment of CRC cell lines also caused up-regulation of DR5 and combination treatment with TRAIL and PHA665752 significantly synergized CRC cells to undergo apoptosis. In-vivo, co-treatment of CRC xenograft with PHA665752 and TRAIL significantly reduced tumor volume and weight. Protein analysis of in-vivo xenografts by immuno-blotting showed that co-treatment with TRAIL and PHA665752 inactivated met and AKT more effectively as compared to treatment with TRAIL or PHA665752 alone. Finally, activation and cleavage of caspase-3 was more pronounced in CRC xenografts treated with combination of TRAIL and PHA665752. Altogether, these data demonstrate a significant correlation between expression of c-Met and DR5 in CRC patients. Furthermore, inhibition of c-Met signaling by PHA665752 in combination with TRAIL significantly inhibited cell growth and induced apoptosis in CRC cell lines suggesting that this may have significant clinical implications as a therapeutic target in the treatment of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2011-5482

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.

Abstract 3499: Identification of a novel mechanism by which the BH3 mimetic obatoclax sensitizes non-Hodgkin's lymphoma tumor cells to TRAIL-mediated apoptosis

Abstract The BH3 mimetic chemical antagonist obatoclax (GX15-070, Gemin X Pharmaceuticals) has been used in vitro and nowadays also in clinical trials for its potential therapeutic effects in both solid and lymph node malignancies. The effects of obatoclax treatment alone or in combination with cytotoxic drugs results in the reversal of tumor cell resistance. Studies by us and others have reported that inhibition of over expressed anti-apoptotic gene products such as Bcl-2 and Bcl-XL can reverse resistance to TRAIL. Obatoclax was shown to sensitize TRAIL-resistant solid tumors to TRAIL-apoptosis, inhibiting diverse anti-apoptotic molecules like Bcl-2, Bcl-XL and Mcl-1 through direct binding. We hypothesized that treatment of B-NHL cell lines with Obatoclax will result in tumor cell sensitization to TRAIL apoptosis by a different mechanism. B-NHL Ramos cell line were treated with different concentrations of Obatoclax (7-28 nM) and TRAIL (2.5-20 ng/ml) resulting in significant potentiation of apoptosis and synergy. Western analysis revealed that treatment with obatoclax resulted in significant inhibition of several members of the Bcl-2 family such as inhibition of Mcl-1, Bcl-XL, XIAP, and cIAP 1/2. In addition, obatoclax dissociated Bak from Mcl-1 as determined by immunoprecipitation. We found that, obatoclax inhibited the expression/activity of several members of the NF-κB pathway including phospho-p65, IKK2, and phosphor-IκB-α. The NF-κB activity inhibition by obatoclax was assessed by EMSA. We examined whether obatoclax-induced sensitization to TRAIL resulted from both signaling of DR4 and DR5. Treatment of Ramos cells with DR4 siRNA or DR5 siRNA and then treated with obatoclax and TRAIL resulted in the sensitization of cells treated with DR4 siRNA but not DR5 siRNA. We have previously reported that DR5 is negatively regulated by the transcription repressor YY1, upstream of NF-κB. We found that obatoclax inhibited YY1 transcription and expression. The direct role of YY1 inhibition by obatoclax in both DR5 upregulation and sensitization to TRAIL was determined in transfected cells with YY1 siRNA. Such cells showed upregulation of DR5 and sensitivity to TRAIL apoptosis. The inhibitory effect of obatoclax on Mcl-1 activity and the Mcl-1 direct role in sensitization was examined by treatment of cells with Mcl-1 siRNA. Such cells reversed their sensitivity to TRAIL-mediated apoptosis. Overall, the present findings reveal a new mechanism of obatoclax-induced sensitization to TRAIL by inhibiting NF-κB and downstream Mcl-1 and YY1. Both Mcl-1 and YY1 play a direct role in tumor cells sensitization to TRAIL mediated apoptosis. The present findings identify several new therapeutic targets modified by obatoclax whose modifications can reverse tumor cell resistance to TRAIL mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3499. doi:10.1158/1538-7445.AM2011-3499

Abstract 4502: The small molecule TIC10 has potent anticancer efficacy mediated by induction of TRAIL production in normal and tumor cells

Abstract TRAIL is an attractive anti-neoplastic agent due to its minimal toxicity and its potent ability to induce apoptosis in cancer cells. Clinical trials with intravenous administration of recombinant human TRAIL in cancer patients have been limited by the short serum half-life of TRAIL and molecular resistance mechanisms including loss of cell surface death receptor expression and/or pro-survival expression changes in proteins that regulate apoptosis such as Mcl-1. Although TRAIL death receptor-targeted agonistic antibodies have an increased half-life, they act on one of the two pro-apoptotic TRAIL receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In order to pharmacologically increase tumor and host TRAIL production as a novel anti-neoplastic mechanism, to improve the pharmacokinetic properties of TRAIL by increasing its effective half-life, and perhaps its local concentration within the tumor microenvironment, we screened for small molecules capable of inducing the endogenous TRAIL gene. A cell-based bioluminescence reporter screen was conducted in TRAIL-resistant Bax-null human HCT116 colon cancer cells using the TRAIL gene promoter yielded TRAIL-inducing compound 10 (TIC10). TIC10 induces TRAIL at the message, membrane-bound, and soluble levels in a time- and dose-dependent manner in TRAIL-sensitive and TRAIL-resistant human cancer cell lines as well as normal human fibroblasts without any effect on cell cycle profile. TIC10 induces cell death in a number of cancer cell lines without evidence of cell cycle arrest, a characteristic of HDAC inhibitors that are the only other pharmacological agent currently shown to upregulate TRAIL. Lack of structural homology with HDAC inhibitors along with this observation suggest a novel mechanism of TRAIL-upregulation. Western blot analysis revealed that TIC10 sensitizes to TRAIL-mediated apoptosis by tumor cell-specific upregulation of DR5 and downregulation of Mcl-1. Human xenograft experiments demonstrate that TIC10 is highly effective as a multi- or single-dose agent with similar efficacy to TRAIL in TRAIL-sensitive xenografts. Some TRAIL-resistant xenografts such as RKO demonstrate TIC10 superiority to TRAIL with no incidence of toxicity. TIC10 causes highly increased protein levels of TRAIL and cleaved caspase-8, increased TUNEL staining indicating apoptosis, in addition to striking decrease in tumor cell density by histology in tumors of human xenografts in mice. Mice lacking tumors demonstrate an upregulation of TRAIL protein levels in serum. The effect of TIC10 on normal cell production of TRAIL provides a novel mechanism for killing tumor cells through a bystander effect in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2011-4502

Abstract 4778: Resveratrol suppresses constitutive activation of AKT via generation of reactive oxygen species and induces apoptosis in diffuse large B cell lymphoma cell lines

Abstract We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in the pathogenesis of diffuse large B cell lymphoma (DLBCL). In an attempt to identify newer agents that can be utilized to target these deregulated pathways, we found that Resveratrol (trans-3,4′, 5-trihydroxystilbene) is a naturally occurring polyphenolic compound that has an antioxidant and chemopreventive effects on various cancer cells. In the current investigation, we therefore investigated the role of resveratrol-mediated chemopreventive effect on various DLBCL cell lines. Resveratrol caused a dose dependent inhibition of cell viability in DBCL cell lines. To see whether this inhibition was due to cell cycle arrest or apoptosis, we found that resveratrol induced apoptosis in all DLBCL cell lines. We next investigated the action of resveratrol on PI3-kinase/AKT pathway and found that resveratrol treatment resulted in inhibition of constitutively activated AKT and its downstream targets, p-FOXO1, p-GSK3 and p-Bad via generation of reactive oxygen species (ROS). Inactivation of Bad led to conformational changes in Bax protein causing loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This led to activation of caspase-9, caspase-3, and poly-(ADP)-ribose polymerase cleavage leading to caspase-dependent apoptosis. In addition, resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; however, this up-regulation did not result in apoptosis. Finally, co-treatment of DLBCL with sub-toxic doses of TRAIL and resveratrol induced efficient apoptosis. Altogether, these data suggest a novel function for resveratrol, acting as a suppressor of AKT/PKB pathway via generation of ROS in DLBCL cells, and raise the possibility that this agent may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway either alone or in combination with other chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2011-4778

Abstract 4101: Off-target lapatinib activity up-regulates TRAIL death receptors in colon cancer cells

Abstract Lapatinib is a dual HER2/EGFR inhibitor that is FDA-approved for the treatment of HER2+ metastatic breast cancer when combined with capecitabine. Colon cancer cells express high levels of EGFR and to a lesser extent, HER2, and therefore, we set out to identify effective therapeutic combinations involving lapatinib in colon cancer. We screened for therapeutic partners of lapatinib using SW620 cells, a cell line that expresses mutant p53 and is resistant to various chemotherapies and apoptosis-inducing agents. We found that high concentrations of lapatinib (10 microM) failed to induce cell death as a single agent or in combination with chemotherapy. However, lapatinib significantly enhanced the pro-apoptotic effects of TRAIL, a finding that we confirmed in a panel of colon cancer cell lines. Lapatinib also sensitized colon cancer cells to the agonistic TRAIL receptor antibodies mapatumumab and lexatumumab. We then tested this novel combination in vivo, and found that tumors from mice treated with lapatinib/TRAIL combinations exhibited increased staining for cleaved caspase-8, a biomarker of death receptor activity, compared to lapatinib or TRAIL treatment alone. Furthermore, the combination therapy suppressed tumor growth, whereas single agent treatments had little effect. Mechanistically, we found that lapatinib up-regulates both pro-apoptotic TRAIL death receptors, DR4 and DR5. Lapatinib increased DR5 mRNA, but had no effects on DR5 protein stability, suggesting that lapatinib up-regulates death receptors through a pre-translational or translational mechanism. Interestingly, other EGFR and HER2 inhibitors, individually or in combination, failed to increase death receptor expression or TRAIL sensitivity, leading us to hypothesize that this activity of lapatinib was due to an off-target effect. In support of this idea, lapatinib induced death receptor up-regulation in SW620 cells, which lack EGFR expression and have undetectable levels of HER2 signaling. In addition, lower lapatinib concentrations, which completely inhibited EGFR and HER2, were failed to induce death receptor expression and increase TRAIL sensitivity, further demonstrating the off-target nature of this effect. High concentrations of lapatinib increased phosphorylation of c-Jun, extracellular signal related kinase (ERK) and c-Jun N-terminal kinase (JNK). RNAi-mediated knockdown experiments proved that c-Jun and JNK, but not ERK, were required for lapatinib-induced DR5 up-regulation. We conclude that high concentrations of lapatinib, through an EGFR/HER2-indpendent mechanism, up-regulate TRAIL death receptors and increase TRAIL sensitivity by a mechanism involving the JNK/c-Jun signaling pathway. From a clinical perspective, therapeutic benefit may be gained from this off-target activity by using high doses of lapatinib and introducing TRAIL receptor-activating agents into the treatment regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2011-4101

Amiodarone sensitizes human glioma cells but not astrocytes to TRAIL-induced apoptosis via CHOP-mediated DR5 upregulation

Amiodarone is a widely used anti-arrhythmic drug that inhibits diverse ion channels, including the Na(+)/Ca(2+) exchanger (NCX), L-type Ca(2+) channels, and Na(+) channels. Here, we report that subtoxic doses of amiodarone and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induced apoptosis of various glioma cells. Treatment of U251MG glioma cells with amiodarone increased intracellular Ca(2+) levels and enhanced the expression of the endoplasmic reticulum (ER) stress-inducible transcription factor C/EBP homologous protein (CHOP). This upregulation of CHOP was followed by marked upregulation of the TRAIL receptor, DR5. Suppression of DR5 expression by small interfering (si) RNAs almost completely blocked amiodarone/TRAIL-induced apoptosis in U251MG glioma cells, demonstrating that DR5 is critical to this cell death. siRNA-mediated CHOP suppression reduced amiodarone-induced DR5 upregulation and attenuated the cell death induced by amiodarone plus TRAIL. In addition, omitting Ca(2+) from the external medium using ethylene glycol tetraacetic acid markedly inhibited this cell death, reducing the protein levels of CHOP and DR5. These results suggest that amiodarone-induced influx of Ca(2+) plays an important role in sensitizing U251MG cells to TRAIL-mediated apoptosis through CHOP-mediated DR5 upregulation. Furthermore, subtoxic doses of bepridil and cibenzoline, two other anti-arrhythmic drugs with NCX-inhibitor activity, also sensitized glioma cells to TRAIL-mediated apoptosis, via the upregulation of both CHOP and DR5. Notably, amiodarone/TRAIL cotreatment did not induce cell death in astrocytes, nor did it affect the expression of CHOP or DR5 in these cells. These results collectively suggest that a combined regimen of amiodarone plus TRAIL may offer an effective therapeutic strategy for safely and selectively treating resistant gliomas.

Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma

We provide evidence that thymoquinone (TQ), a natural compound isolated from Nigella sativa, induces growth inhibition and apoptosis in several primary effusion lymphoma (PEL) cell lines. Our data demonstrate that TQ treatment results in down-regulation of constitutive activation of AKT via generation of reactive oxygen species (ROS) and it causes conformational changes in Bax protein, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This leads to activation of caspase-9, caspase-3, and polyadenosine 5′-diphosphate ribose polymerase cleavage, leading to caspase-dependent apoptosis. Pretreatment of PEL cells with N-acetylcysteine, a scavenger of ROS, prevented TQ-mediated effects. In addition, subtoxic doses of TQ sensitized PEL cells to TRAIL via up-regulation of DR5. Altogether, these findings demonstrate that TQ is a potent inducer of apoptosis in PEL cells via release of ROS. They also raise the possibility that incorporation of TQ in treatment regimens for primary effusion lymphomas may provide a novel approach to sensitizing malignant cells and provide a molecular basis for such future translational efforts.

High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde

BackgroundTumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.ResultsPC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.ConclusionThis study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells

To investigate the apoptotic activities of casticin in hepatocellular carcinoma (HCC) cells and its molecular mechanisms. PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide (MTT) assay. The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay (ELISA) detection kit, flow cytometry (FCM) after propidium iodide (PI) staining and DNA agarose gel electrophoresis. The caspase activities were measured using ELISA. Reactive oxygen species (ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCFH-DA) probe labeling. Intracellular glutathione (GSH) content was measured using a glutathione assay kit. The expression of death receptor (DR)4 and DR5 proteins was analyzed by Western blotting and FCM. Casticin significantly inhibited the growth of human HCC (PLC/PRF/5 and Hep G2) cells in a dose-dependent manner (P < 0.05). Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner. The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil (26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h. Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3, -8 and -9 in a concentration-dependent manner (P < 0.05). Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder. Casticin reduced the GSH content (P < 0.05), but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells. The thiol antioxidants, acetylcysteine (NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis. In contrast, the nonthiol antioxidants, butylated hydroxyanisole and mannitol failed to do so. In the HCC cells treated with casticin for 24 h, DR5 protein level was increased. The expression of DR5 protein induced by casticin was inhibited by NAC. Pretreatment with DR5/Fc chimera protein, a blocking antibody, effectively attenuated the induction of apoptosis by casticin. Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.

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.

Immunotherapy Targeting Colon Cancer Stem Cells

In the last 10 years, cancer stem cells have interested the scientific community because this small tumorigenic population is also associated with tumor progression in human patients and specific targeting of cancer stem cells could be a strategy to eradicate cancers currently resistant to conventional therapy. Clinical studies have recently demonstrated that adding immune therapy to chemotherapy has survival benefits in comparison with chemotherapy alone that can sensitize tumors to immune cell-mediated killing (e.g., increasing sensitivity of tumor cells to subsequent cytotoxicity by T cells via upregulation of death receptors DR5 and Fas). However, loss of MHC molecules is often observed in cancer cells, rendering tumor cells resistant to CD8 T-cell-mediated cytotoxicity. For this reason, we review the role of other T-cell subsets, such as γδ T and NK cells that are able to efficiently recognize and kill tumor cells and that could be used in passive or active immunotherapy in cancer stem cell eradication.

Efficacy of TRAIL treatment against HPV16 infected cervical cancer cells undergoing senescence following siRNA knockdown of E6/E7 genes

In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug ‘tumor necrosis factor-related apoptosis inducing ligand’ (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular β-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment.

Piceatannol enhances TRAIL-induced apoptosis in human leukemia THP-1 cells through Sp1- and ERK-dependent DR5 up-regulation

Although piceatannol (PIC) is known to mediate anti-cancer, anti-inflammatory, and anti-oxidant activities, little is known about the mechanism of PIC in terms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In this study, we examined whether combined treatment with PIC and TRAIL synergistically induces apoptosis in THP-1 leukemia cells. Results indicate that PIC substantially enhances TRAIL-induced cell death including DNA fragmentation and poly(ADP-ribose) polymerase cleavage. Consistent with TRAIL-induced apoptosis, PIC significantly increased the mRNA and protein expression levels of DR5, a death receptor of TRAIL. Further, PIC enhanced DR5 promoter activity via Sp1 activation. Interestingly, the DR5 chimera antibodies significantly suppressed PIC and TRAIL-mediated apoptosis. The inhibitor of ERK also decreased PIC and TRAIL-induced apoptosis by blocking DR5 expression. In conclusion, our results suggest that PIC sensitizes TRAIL-induced-apoptosis via Sp1- and ERK-dependent DR5 up-regulation.

Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

BackgroundResveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Methodology/Principal FindingsResveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin) and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM) induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by resveratrol.Conclusion/SignificanceThese data suggest that FOXO transcription factors mediate anti-proliferative and pro-apoptotic effects of resveratrol, in part due to activation of extrinsic apoptosis pathway.

Cisplatin sensitizes human hepatocellular carcinoma cells, but not hepatocytes and mesenchymal stem cells, to TRAIL within a therapeutic window partially depending on the upregulation of DR5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell apoptosis in many types of tumors, but not in normal cells. This tumor-selective property has made TRAIL a promising approach for the development of cancer therapy. However, hepatocellular carcinoma (HCC) cells display a striking resistance to TRAIL. Although some chemotherapeutic agents can overcome this resistance, safety issues remain a concern because the combination of these agents and TRAIL has been reported to induce toxicity in normal hepatocytes. In this study, we examined whether cisplatin could reverse TRAIL resistance in HCC cells with different p53 status and evaluated the toxicity of combination TRAIL and cisplatin to normal hepatocytes and mesenchymal stem cells (MSCs). We observed that cisplatin could efficiently sensitize HCC cells, but not hepatocytes and MSCs to TRAIL-induced apoptosis within a wide therapeutic window. The apoptosis of HCC cells only partially depended on the upregulation of DR5 and the status of p53. In addition, we provide favorable evidence supporting the feasibility of the combination of chemotherapy and MSCs transduced with TRAIL.

MS-275 Sensitizes TRAIL-Resistant Breast Cancer Cells, Inhibits Angiogenesis and Metastasis, and Reverses Epithelial-Mesenchymal Transition In vivo

Histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) show promise for the treatment of cancers. The purpose of this study was to examine the molecular mechanisms by which HDAC inhibitor MS-275 sensitizes TRAIL-resistant breast cancer cells in vivo, inhibits angiogenesis and metastasis, and reverses epithelial-mesenchymal transition (EMT). BALB/c nude mice were orthotopically implanted with TRAIL-resistant invasive breast cancer MDA-MB-468 cells and treated intravenously with MS-275, TRAIL, or MS-275 followed by TRAIL, 4 times during first 3 weeks. Treatment of mice with TRAIL alone had no effect on tumor growth, metastasis, angiogenesis, and EMT. In comparison, MS-275 sensitized TRAIL-resistant xenografts by inducing apoptosis, inhibiting tumor cell proliferation, angiogenesis, metastasis, and reversing EMT. Treatment of nude mice with MS-275 resulted in downregulation of NF-κB and its gene products (cyclin D1, Bcl-2, Bcl-X(L), VEGF, HIF-1α, IL-6, IL-8, MMP-2, and MMP-9) and upregulation of DR4, DR5, Bax, Bak, and p21(/CIP1) in tumor cells. Furthermore, MS-275-treated mice showed significantly reduced tumor growth and decreased circulating vascular VEGFR2-positive endothelial cells, CD31-positive or von Willebrand factor-positive blood vessels, and lung metastasis compared with control mice. Interestingly, MS-275 caused "cadherin switch" and reversed EMT as shown by the upregulation of E-cadherin and downregulation of N-cadherin and transcription factors Snail, Slug, and ZEB1. In conclusion, sequential treatments of mice with MS-275 followed by TRAIL may target multiple pathways to reverse EMT and inhibit tumor progression, angiogenesis, and metastasis and represent a novel therapeutic approach to treat cancer.

C-Jun NH2-terminal kinase-dependent upregulation of DR5 mediates cooperative induction of apoptosis by perifosine and TRAIL

BackgroundPerifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis.ResultsThe combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA) inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK) and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured.ConclusionsDR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species.