Upregulation Of Death Receptor Research Articles

Phenethyl isothiocyanate upregulates death receptors 4 and 5 and inhibits proliferation in human cancer stem-like cells.

BackgroundThe cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.MethodsCancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers’ analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.ResultsPEITC attenuated proliferation of CD44high/+/CD24low/–, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.ConclusionsThe anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells.

A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the −301 to −253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.

Cariporide sensitizes leukemic cells to tumor necrosis factor related apoptosis-inducing ligand by up-regulation of death receptor 5 via endoplasmic reticulum stress–CCAAT/enhancer binding protein homologous protein dependent mechanism

CCAAT/enhancer binding protein homologous protein (CHOP) expression increases when Na+–H+ exchanger 1 (NHE1) is inhibited. Endoplasmic reticulum (ER) stress has been shown to trigger tumor cell death through CHOP. We therefore hypothesized that NHE1 activity correlates with ER stress and confers pharmaceutical potential to NHE1 inhibitor as an anti-tumor agent. The present study showed that treatment with the NHE1 inhibitor cariporide led to ER stress-induced up-regulation of the death receptor 5 (DR5) which is mediated by CHOP at the transcriptional level. We also determined that ER stress-induced Janus kinase (JNK) activation was responsible for the modulation of CHOP. Combining cariporide with tumor necrosis factor related apoptosis-inducing ligand (TRAIL) led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of CHOP. This study provides a potential mechanistic rationale for the use of NHE1 inhibitor in combination with DR5 agonists to induce apoptosis in leukemia.

Role of Activating Transcription Factor 3 (ATF3) in Endoplasmic Reticulum (ER) Stress-induced Sensitization of p53-deficient Human Colon Cancer Cells to Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-mediated Apoptosis through Up-regulation of Death Receptor 5 (DR5) by Zerumbone and Celecoxib

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers cell death upon binding to its ligand, TNF-related apoptosis-inducing ligand (TRAIL), and a combination of TRAIL and agents that increase the expression of DR5 is expected to be a novel anticancer therapy. In this report, we demonstrate that the stress response gene ATF3 is required for endoplasmic reticulum stress-mediated DR5 induction upon zerumbone (ZER) and celecoxib (CCB) in human p53-deficient colorectal cancer cells. Both agents activated PERK-eIF2α kinases and induced the expression of activating transcription factor 4 (ATF4)-CCAAT enhancer-binding protein (C/EBP) homologous protein, which were remarkably suppressed by reactive oxygen species scavengers. In the absence of ATF3, the induction of DR5 mRNA and protein was abrogated significantly, and this was associated with reduced cell death by cotreatment of TRAIL with ZER or CCB. By contrast, exogenous expression of ATF3 caused a more rapid and elevated expression of DR5, resulting in enhanced sensitivity to apoptotic cell death by TRAIL/ZER or TRAIL/CCB. A reporter assay demonstrated that at least two ATF/cAMP response element motifs as well as C/EBP homologous protein motif at the proximal region of the human DR5 gene promoter were required for ZER-induced DR5 gene transcription. Taken together, our results provide novel insights into the role of ATF3 as an essential transcription factor for p53-independent DR5 induction upon both ZER and CCB treatment, and this may be a useful biomarker for TRAIL-based anticancer therapy.

Beta amyloid-induced upregulation of death receptor 6 accelerates the toxic effect of N-terminal fragment of amyloid precursor protein

Amyloid precursor protein (APP) plays essential roles in the development of the Alzheimer's disease. Although full-length APP has been thoroughly studied, the role of the cleavage fragments especially the N-terminal fragments (N-APPs) in Alzheimer's disease pathogenesis was still elusive. In this study, we demonstrated that application of recombinant APP18-286 could enhance beta amyloid (Aβ)-induced neuronal injuries which were related to the activation of apoptosis proteins. Aβ treatment could induce a slight increase of N-APPs release. In addition, expression of death receptor 6 (DR6) was increased in Aβ-treated neurons and APP transgenic mice. Moreover, the effect of APP18-286 on Aβ-induced injuries could be suppressed by the application of recombinant DR641-341 and DR6 antibody. Furthermore, pull-down assay revealed that APP18-286 could bind both exogenous and endogenous DR6. Aβ promoted APP18-286 targeting to neuron which was accompanied with the increase of DR6 expression, whereas downregulation of DR6 by interference RNA could alleviate the binding of N-APPs to neuron and also suppressed Aβ-dependent toxic effect with N-APPs. These results suggested that APP N-terminal fragments might play neurotoxic roles in Aβ-induced neuronal injuries through cell surface DR6.

Antitumor effects of a sirtuin inhibitor, tenovin-6, against gastric cancer cells via death receptor 5 up-regulation.

Up-regulated sirtuin 1 (SIRT1), an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53). Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5). In the KatoIII cell line (TP53-null), DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased susceptibility of wild-type p53 cancer cells to oxidative stress and therapeutic agents

Sirtuin 1 (Sirt1) plays an important role in cellular redox balance and resistance to oxidative stress. Sirt1 exhibits oncogenic properties in wild-type p53 cancer cells, whereas it acts as a tumor suppressor in p53-mutated cancer cells. Here, we investigated the effects of metformin on Sirt1 expression in several cancer cell lines. Using human cancer cell lines that exhibit differential expression of p53, we found that metformin reduced Sirt1 protein levels in cancer cells bearing wild-type p53, but did not affect Sirt1 protein levels in cancer cell lines harboring mutant forms of p53. Metformin-induced p53 protein levels in wild-type p53 cancer cells resulted in upregulation of microRNA (miR)-34a. The use of a miR-34a inhibitor confirmed that metformin-induced miR-34a was required for Sirt1 downregulation. Metformin suppressed peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (Pgc-1α) expression and its downstream target Nrf2 in MCF-7 cells. Genetic tools demonstrated that the reduction of Sirt1 and Pgc-1α by metformin caused Nrf2 downregulation via suppression of PPARγ transcriptional activity. Metformin reduced heme oxygenase-1 and superoxide dismutase 2 but upregulated catalase expression in MCF-7 cells. Metformin-treated MCF-7 cells had no increase in basal levels of reactive oxygen species but were more susceptible to oxidative stress. Furthermore, upregulation of death receptor 5 by metformin-mediated Sirt1 downregulation enhanced the sensitivity of wild-type p53 cancer cells to TRAIL-induced apoptosis. Our results demonstrated that metformin induces miR-34a to suppress the Sirt1/Pgc-1α/Nrf2 pathway and increases susceptibility of wild-type p53 cancer cells to oxidative stress and TRAIL-induced apoptosis.

Ilimaquinone induces death receptor expression and sensitizes human colon cancer cells to TRAIL-induced apoptosis through activation of ROS-ERK/p38 MAPK–CHOP signaling pathways

TRAIL induces apoptosis in a variety of tumor cells. However, development of resistance to TRAIL is a major obstacle to more effective cancer treatment. Therefore, novel pharmacological agents that enhance sensitivity to TRAIL are necessary. In the present study, we investigated the molecular mechanisms by which ilimaquinone isolated from a sea sponge sensitizes human colon cancer cells to TRAIL. Ilimaquinone pretreatment significantly enhanced TRAIL-induced apoptosis in HCT 116 cells and sensitized colon cancer cells to TRAIL-induced apoptosis through increased caspase-8, -3 activation, PARP cleavage, and DNA damage. Ilimaquinone also reduced the cell survival proteins Bcl2 and Bcl-xL, while strongly up-regulating death receptor (DR) 4 and DR5 expression. Induction of DR4 and DR5 by ilimaquinone was mediated through up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). The up-regulation of CHOP, DR4 and DR5 expression was mediated through activation of extracellular-signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Finally, the generation of ROS was required for CHOP and DR5 up-regulation by ilimaquinone. These results demonstrate that ilimaquinone enhanced the sensitivity of human colon cancer cells to TRAIL-induced apoptosis through ROS-ERK/p38 MAPK–CHOP-mediated up-regulation of DR4 and DR5 expression, suggesting that ilimaquinone could be developed into an adjuvant chemotherapeutic drug.

P0159 Bidens pilosa extract induces apoptosis through up-regulation of death receptor 5 in human colon carcinoma cell lines

Background Bidens pilosa Linn. var. radiata is a tropical weed widely present in tropical and subtropical regions, including Miyako Island, Okinawa, Japan. The whole plant or its leaves and stem are used in various folk medicines and as a popular ingredient in herbal tea for its anti-inflammatory, anti-septic, liver-protective, blood-pressure lowering, and hypoglycaemic effects. There are no reports of the effects of B. pilosa on human colorectal cancer cells. Methods We investigated the effects of B pilosa on the human carcinoma cell lines HCT116 and Caco-2. The former has wild-type p53, the latter has mutant p53. B pilosa was provided as an extract powder from Musashi Immune laboratory Co. (Miyako Island, Okinawa, Japan). Findings B pilosa (0.25, 0.5, and 1 mg/ml in medium) demonstrated dose-dependent growth inhibition on both cell lines, assessed by WST-8 assay (Nacalai Tesque, Kyoto, Japan). B pilosa induced apoptosis in both HCT116 and Caco-2 cell lines, assessed by DNA ladder. In addition, a cleaved caspase-8 was expressed in both cell lines, as assessed by western blot. In both cell lines, in spite of the p53-genetic status, B pilosa also increased the expression death receptor 5 (DR5), a TRAIL receptor that activates caspase-8, leading directly to the activation of caspase-3. Interpretation Our results suggest that B pilosa has the potential to prevent or treat for colon cancers through induction of apoptosis by a p53-independent pathway.

Indomethacin Sensitizes TRAIL-Resistant Melanoma Cells to TRAIL-Induced Apoptosis through ROS-Mediated Upregulation of Death Receptor 5 and Downregulation of Survivin

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention owing to its selective killing of tumor cells but not normal cells. Melanoma shows weak response to TRAIL because of its low level of TRAIL death receptors. Here, we investigated whether indomethacin, a nonsteroidal anti-inflammatory drug, can potentiate TRAIL-induced apoptosis in melanoma cells. We showed that indomethacin was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. Mechanistically, indomethacin induced cell surface expression of death receptor 5 (DR5) in melanoma cells and also in various types of cancer cells. DR5 knockdown abolished the enhancing effect of indomethacin on TRAIL responses. Induction of the DR5 by indomethacin was found to be p53 independent but dependent on the induction of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP abolished indomethacin-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, indomethacin-induced reactive oxygen species (ROS) production preceded upregulation of CHOP and DR5, and consequent sensitization of cells to TRAIL. We also found that indomethacin treatment downregulated survivin via ROS and the NF-κB-mediated signaling pathways. Interestingly, indomethacin also converted TRAIL-resistant melanoma MeWo and SK-MEL-5 cells into TRAIL-sensitive cells. Taken together, our results indicate that indomethacin can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of survivin.

Mechanistic elucidation of the antitumor properties of withaferin a in breast cancer.

Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels that are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK (ribosomal S6 kinase) in breast cancer cells. Pursuing this observation, we defined activation of extracellular signal-regulated kinase (ERK)-RSK and ETS-like transcription factor 1 (Elk1)-CHOP (C-EBP homologous protein) kinase pathways in upregulating transcription of the death receptor 5 (DR5). Through this route, WFA acted as an effective DR5 activator capable of potentiating the biologic effects of celecoxib, etoposide, and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and mouse mammary tumor virus (MMTV)-neu mouse models in a manner associated with activation of the ERK/RSK axis, DR5 upregulation, and elevated nuclear accumulation of Elk1 and CHOP. Together, our results offer mechanistic insight into how WFA inhibits breast tumor growth.

Digitoxin sensitizes glioma cells to TRAIL-mediated apoptosis by upregulation of death receptor 5 and downregulation of survivin

Glioblastoma multiforme is the most lethal and aggressive astrocytoma among primary brain tumors in adults. However, most glioblastoma cells have been reported to be resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we have shown that digitoxin (DT), a clinically approved cardiac glycoside for heart failure, can induce TRAIL-mediated apoptosis of glioblastoma cells. DT in noncytotoxic doses (20 nmol/l) can increase TRAIL-induced apoptosis in TRAIL-resistant U87MG glioblastoma cells. Treatment with DT led to apoptosis and a robust reduction in the levels of the antiapoptotic protein survivin by inducing its proteasomal degradation; however, it did not affect the levels of many other apoptosis regulators. Moreover, silencing survivin with small interfering RNAs sensitized glioma cells to TRAIL-induced apoptosis, underscoring the functional role of survivin depletion in the TRAIL-sensitizing actions of DT. We demonstrate that inactivation of survivin and death receptor 5 expression by DT is sufficient to restore TRAIL sensitivity in resistant glioma cells. Our results suggest that combining DT with TRAIL treatments may be useful in the treatment of TRAIL-resistant glioma cells.

Induction of apoptosis by a hexane extract of aged black garlic in the human leukemic U937 cells.

BACKGROUND/OBJECTIVESIn this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells.MATERIALS/METHODSCytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay.RESULTSExposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade.CONCLUSIONSBased on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.

Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL-mediated apoptosis.

Bats are a major reservoir of emerging infectious viruses. Many of these viruses are highly pathogenic to humans however bats remain asymptomatic. The mechanism by which bats control viral replication is unknown. Here we utilize an integrated approach of proteomics informed by transcriptomics to compare the response of immortalized bat and human cells following infection with the highly pathogenic bat-borne Hendra virus (HeV). The host response between the cell lines was significantly different at both the mRNA and protein levels. Human cells demonstrated minimal response eight hours post infection, followed by a global suppression of mRNA and protein abundance. Bat cells demonstrated a robust immune response eight hours post infection, which led to the up-regulation of apoptosis pathways, mediated through the tumor necrosis factor-related apoptosis inducing ligand (TRAIL). HeV sensitized bat cells to TRAIL-mediated apoptosis, by up-regulating death receptor transcripts. At 48 and 72 hours post infection, bat cells demonstrated a significant increase in apoptotic cell death. This is the first study to comprehensively compare the response of bat and human cells to a highly pathogenic zoonotic virus. An early induction of innate immune processes followed by apoptosis of virally infected bat cells highlights the possible involvement of programmed cell death in the host response. Our study shows for the first time a side-by-side high-throughput analysis of a dangerous zoonotic virus in cell lines derived from humans and the natural bat host. This enables a way to search for divergent mechanisms at a molecular level that may influence host pathogenesis.

Peroxisome proliferator-activated receptor γ ligand troglitazone and TRAIL synergistically induce apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to cause apoptosis in several types of malignant tumor cells through its interaction with the death domain-containing receptor, death receptor 5 (DR5). In the present study, we showed that co-treatment with troglitazone (TGZ), a synthetic ligand of peroxisome proliferator-activated receptor γ (PPARγ), and TRAIL synergistically induced apoptosis through DR5 upregulation in human colon cancer DLD-1 cells. TGZ elevated DR5 expression at the promoter level through the CCAAT/enhancer-binding protein homologous protein (CHOP) binding site. These results suggest that combined treatment with TGZ and TRAIL may be promising as a new therapy against malignant tumors.

Azadirone, a Limonoid Tetranortriterpene, Induces Death Receptors and Sensitizes Human Cancer Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) through a p53 Protein-independent Mechanism: EVIDENCE FOR THE ROLE OF THE ROS-ERK-CHOP-DEATH RECEPTOR PATHWAY

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. We investigated whether azadirone, a limonoidal tetranortriterpene, can sensitize human tumor cells to TRAIL. Results indicate that azadirone sensitized cancer cells to TRAIL. The limonoid induced expression of death receptor (DR) 5 and DR4 but did not affect expression of decoy receptors in cancer cells. The induction of DRs was mediated through activation of ERK and through up-regulation of a transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) as silencing of these signaling molecules abrogated the effect of azadirone. These effects of azadirone were cancer cell-specific. The CHOP binding site on the DR5 gene was required for induction of DR5 by azadirone. Up-regulation of DRs was mediated through the generation of reactive oxygen species (ROS) as ROS scavengers reduced the effect of azadirone on ERK activation, CHOP up-regulation, DR induction, and TRAIL sensitization. The induction of DRs by this limonoid was independent of p53, but sensitization to TRAIL was p53-dependent. The limonoid down-regulated the expression of cell survival proteins and up-regulated the proapoptotic proteins. The combination of azadirone with TRAIL was found to be additive at concentrations lower than IC50, whereas at higher concentrations, the combination was synergistic. Overall, this study indicates that azadirone can sensitize cancer cells to TRAIL through ROS-ERK-CHOP-mediated up-regulation of DR5 and DR4 signaling, down-regulation of cell survival proteins, and up-regulation of proapoptotic proteins.

Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis

Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.

Ibuprofen enhances TRAIL-induced apoptosis through DR5 upregulation

Numerous human chemoprevention studies have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) possess chemopreventive effects against a variety of malignant tumors. However, there have been many clinical studies on aspirin, but not ibuprofen, even though ibuprofen is one of the most clinically and safely used NSAIDs showing potent anti-inflammatory effects. Moreover, we reported that many chemopreventive agents enhance the apoptosis-inducing effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is known to be crucial for cancer prevention. We, therefore, investigated whether ibuprofen enhances the cytocidal effect of TRAIL and found that ibuprofen markedly stimulated the apoptosis-inducing efficacy of TRAIL against human colon cancer HCT116 cells. As detected by western blot analysis and real-time RT-PCR, ibuprofen upregulated the expression of death receptor 5 (DR5), a TRAIL receptor. TRAIL-induced apoptosis enhanced by ibuprofen was effectively decreased by a caspase inhibitor and dominant-negative DR5. Noteworthy, co-treatment of ibuprofen with TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMCs). These results demonstrated that ibuprofen and TRAIL synergistically induced apoptosis in human colon cancer HCT116 cells but not in normal PBMCs, raising the possibility that ibuprofen may be promising as a safe chemopreventive agent against colon cancer.

CAPE promotes TRAIL-induced apoptosis through the upregulation of TRAIL receptors via activation of p38 and suppression of JNK in SK-Hep1 hepatocellular carcinoma cells

Caffeic acid phenethyl ester (CAPE), a phenolic compound derived from honeybee propolis, has been reported to possess anticancer activities in several types of malignant cells. Here, we show that treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with CAPE significantly sensitized SK-Hep1 cells to TRAIL-induced apoptosis. The sensitization to TRAIL was accompanied by the activation of extrinsic and intrinsic apoptotic pathways, leading to the activation of caspases, mitochondrial disruption and PARP cleavage. Moreover, TRAIL receptors, such as DR4 and DR5 were significantly upregulated by CAPE treatment, and both DR4/Fc and DR5/Fc chimera markedly abrogated apoptosis induced by CAPE and TRAIL, demonstrating the critical role of these death receptors in combination-induced apoptosis. The effect of CAPE on mitogen-activated protein kinases (MAPKs) was further examined, where CAPE treatment resulted in the activation of p38 and the inhibition of JNK, without affecting levels of phospho-ERK. Our results showed that p38 and JNK exhibited the opposite role in SK-Hep1 cells. The inhibition of p38, using SB203580, blocked the CAPE-induced expression of death receptors and attenuated the combination‑induced apoptosis, suggesting the pro-apoptotic role of p38. In contrast, JNK-specific inhibition, by SP600125, triggered upregulation of DR4 and DR5, and sensitized SK-Hep1 cells to TRAIL, indicating that the CAPE-induced suppression of JNK may contribute to the sensitizing effect of CAPE through the upregulation of death receptors. Taken together, these results indicate that CAPE potentiated TRAIL-induced apoptosis in SK-Hep1 cells, through upregulation of TRAIL receptors via modulation of p38 and JNK signaling pathways.

Early Changes in mRNA and Protein Expression Related to Cancer Treatment by Modulated Electrohyperthermia

Modulated electrohyperthermia (mEHT), generated by capacitive coupled, modulated 13.56 MHz radiofrequency, is a noninvasive technique for targeted tumor treatment based on elevated ion concentration and electric admittance in malignant tumors. In this study, we tested early changes in protein expression related to tumor destruction upon a single shot of 30-minute mEHT treatment of xenografted human colorectal cell line (HT29) implanted into the femoral region of Balb/c nu/nu mice. Treatment-related mRNA expression profiling was done using the human genome U133 Plus 2.0 Arrays. Apoptosis protein arrays and immunohistochemistry were performed for validating changes at the protein level. The mEHT treatment resulted in major expression changes in 48 genes including several heat-shock proteins. Apoptosis protein arrays revealed the upregulation of death receptors, Bcl-2 superfamily mitochondrial apoptosis regulatory proteins, and heat-shock proteins, which were also confirmed in situ. Within 24-hour post-treatment, mEHT resulted in the upregulation apoptosis induction and heat-shock-related gene and protein expression in HT29 colorectal cancer xenografts contributing to tumor destruction.