To TNF-related Apoptosis-inducing Ligand Research Articles

Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20.

Hepatitis B virus (HBV) infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx) is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD), and the formation of death-inducing signaling complex (DISC), thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.

Elevated Pressure Enhanced Trail-Induced Apoptosis in Hepatocellular Carcinoma Cells Via Erk1/2-Inactivation.

The high frequency of intrinsic resistance to TNF-related apoptosisinducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.

Abstract 2271: Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists

Abstract Metastatic renal cell carcinoma (RCC) is incurable. IFNα or IL-2 immunotherapy has been used for RCC for more than 20 years, but response rates remain low and these cytokines can generate toxic. More recently, administration of multikinase inhibitors or anti-VEGF receptor mAb has become popular, but these agents are largely palliative and tumor regression is common. The shortcomings in current therapeutic options for patients with metastatic RCC provide the rationale for the development of novel treatment protocols. Triptolide, a diterpene triepoxide from the medicinal herb Tripterygium wilfordii, can suppress cell growth and induce apoptosis in a range of human tumor cells. Further, triptolide also sensitizes tumor cells to TNF-related apoptosis-inducing ligand (TRAIL). The objective of this study was to determine the effectiveness of combining triptolide with TRAIL receptor agonists against RCC. In vitro, the combination of triptolide and recombinant human TRAIL protein decreased cell viability in the human RCC cell line ACHN cells compared with either treatment alone. This decrease in viability correlated with increased induction of apoptosis (positive Annexin V staining) and caspase activation. The triptolide-induced sensitization was accompanied by decreased HSP70, HSP27, and HSF1 expression, as well as increased surface expression of TRAIL-R2. Similar in vitro treatment of the mouse renal cell adenocarcinoma cell line, Renca, demonstrated that triptolide enhanced the killing of these cells by membrane-bound TRAIL or agonistic anti-DR5 mAb. In vivo treatment of mice bearing orthotopic and metastatic Renca tumors showed that combination therapy consisting of minnelide (a water soluble version of triptolide) and agonistic anti-DR5 mAb resulted in significantly decreased tumor burden and increased animal survival compared to either therapy alone. Collectively, our data suggest that triptolide sensitizes RCC cells to TRAIL-induced apoptosis, which is accompanied by decreased heat shock protein expression and altered TRAIL receptor expression. These data also suggest that the combination of TRAIL receptor agonists and triptolide may prove to be an effective treatment for metastatic RCC, especially at doses that show suboptimal tumoricidal activity when used individually. Citation Format: Thomas S. Griffith, Erik L. Brincks, Tamara A. Kucaba, Britnie R. James, Veena Sangwan, Sulagna Banerjee, Ashok Saluja. Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2014-2271

Osteoprotegerin expression in triple-negative breast cancer cells promotes metastasis.

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells.

Inhibition of CK2: An Attractive Therapeutic Target for Cancer Treatment.

Summary: The invention in this patent application relates to pyrrolo[1,5-A]pyrimidine derivatives represented generally by formula (I). These compounds are inhibitors of CK2 and may potentially be used in the prevention and treatment of CK2-related conditions and diseases including cancer. The casein kinase 2 (CK2) is a serine/threonine-selective protein kinase that exists as a tetramer of two catalytic (α and/or α 0 ) subunitsand two regulatory(β) subunits.The level ofCK2 innormalcells is tightlyregulatedto performa variety ofessential roles suchascellcyclecontrol,cellulardifferentiationandproliferation,andpromotingcellsurvivalviamodulationofapoptoticpathways. Studies have shown that elevated levels of CK2 are associated with cancer, while targeted overexpression of CK2 in transgenic animalmodelsresultsinneoplasticgrowth.CK2phosphorylatesandregulatestheactivityandstabilityofmultipletumorsuppressor proteins such as PML, p53, and PTEN, as well as oncogenes and transcriptional activatorssuch as c-Myc, c-Myb, c-Jun, NFkB, and β-catenin. Recent studies provided evidence that CK2 can act as suppressor of apoptosis via phosphorylation of pro-apoptotic proteins, which protect them from caspase mediated cleavage. Studies also showed that inhibition of CK2 sensitizes cells to TNFrelated apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis, ionizing radiation and chemotherapeutic agents. Thus,inhibitionofCK2maybeaneffectivetherapeutictargetfortreatmentofcancer.Theinventorsstatedthatthecurrentlyavailable small molecule inhibitors of CK2 lack the necessary balance of potency, selectivity, and drug-like properties for consideration as therapeutic agents. The CK2 inhibitors described in this patent application may possess improved properties that would provide a potentially useful treatment for cancer.

MiR-34a/c-Dependent PDGFR-α/β Downregulation Inhibits Tumorigenesis and Enhances TRAIL-Induced Apoptosis in Lung Cancer

Lung cancer is the leading cause of cancer mortality in the world today. Although some advances in lung cancer therapy have been made, patient survival is still poor. MicroRNAs (miRNAs) can act as oncogenes or tumor-suppressor genes in human malignancy. The miR-34 family consists of tumor-suppressive miRNAs, and its reduced expression has been reported in various cancers, including non-small cell lung cancer (NSCLC). In this study, we found that miR-34a and miR-34c target platelet-derived growth factor receptor alpha and beta (PDGFR-α and PDGFR-β), cell surface tyrosine kinase receptors that induce proliferation, migration and invasion in cancer. MiR-34a and miR-34c were downregulated in lung tumors compared to normal tissues. Moreover, we identified an inverse correlation between PDGFR-α/β and miR-34a/c expression in lung tumor samples. Finally, miR-34a/c overexpression or downregulation of PDGFR-α/β by siRNAs, strongly augmented the response to TNF-related apoptosis inducing ligand (TRAIL) while reducing migratory and invasive capacity of NSCLC cells.

Su1730 Activation of Notch Pathway Plays a Role in Pancreatic Cancer Treatment Failure

BACKGROUND: Recurrence of colorectal cancers after surgery and chemotherapy may be partly explained by the presence of chemoresistant cancer stem cells (CSC). Given the highly heterogeneous nature of CSCs it is possible that further characterization might identify a subset of CSC that eventually develop micro-metastases. Galectin-3 (Gal3) has been implicated in tumor progression, metastasis, and drug-resistance. We have previously reported that cell surface Gal3 renders colon cancer cells resistant to TNF-related Apoptosis-Inducing Ligand (TRAIL). Here, our aim was to determine how Gal3 is related to expression of stem cell markers and chemoresistance in different colon cancer cell populations. METHODS: We used the highly metastatic colon cancer cell line LiM6 and its TRAIL-resistant derivative LIM6-TR as cellular model. CSC populations were identified and isolated based on the expression of 5 established CSC-Markers CD24, CD44, CD166, EpCAM, and ALDH. Surface Gal3 in these CSCs was monitored by flow cytometry. Self-renewal, a functional CSC hallmark of CSC, was measured in sphere-formation assays. Response to TRAIL wasmeasured by APO-BRDU TUNEL assays. RESULTS: Only 15% of LIM6 and LIM6-TR cells were CSC as defined by the 5 markers (CD24+, CD44+, CD166+, EpCAM+, ALDH+). Surface Gal3 analysis identified two subpopulations within the CSC pool of LiM6 (54% Gal3+, 46% Gal3 negative). In contrast, CSC from LIM6-TR were uniformly Gal3+. Of note, not all Gal3+ colorectal cells can be attributed with CSC properties, since only 25-30% of Gal3+ CRCs express the remaining CSC markers simultaneously. Sphere assays demonstrated that the parental LIM6-TR had a 2-fold higher sphere forming ability than LIM6. The isolated Gal3+ CSCs formed 3.3-fold more spheres than parental Lim6 cells, while a 90% decrease in sphere formation was observed in the GAL3 negative CSC subpopulation. Depletion of Gal3 in LiM6-TR cells resulted in the loss of the Gal3+ CSC subpopulation, diminished sphere formation, and resensitization to TRAIL. Sphere formation of GAL+ CSCs in the presence of the Gal3 inhibitor LacNAc [5mM] led to a 60% reduction in sphere-formation. CONCLUSIONS: Our findings indicate that surface Gal3 is responsible for high sphere formation ability and TRAIL-resistance in a subpopulation of CSC. Further experiments will determine whether surface Gal3 in combination with other CSC markers can help to identify metastatic and chemoresistant CSCs in the circulation of CRC patients.

Su1729 Targeting Both Active and Quiescent Cancer (stem) Cells Using Combined Therapy

BACKGROUND: Recurrence of colorectal cancers after surgery and chemotherapy may be partly explained by the presence of chemoresistant cancer stem cells (CSC). Given the highly heterogeneous nature of CSCs it is possible that further characterization might identify a subset of CSC that eventually develop micro-metastases. Galectin-3 (Gal3) has been implicated in tumor progression, metastasis, and drug-resistance. We have previously reported that cell surface Gal3 renders colon cancer cells resistant to TNF-related Apoptosis-Inducing Ligand (TRAIL). Here, our aim was to determine how Gal3 is related to expression of stem cell markers and chemoresistance in different colon cancer cell populations. METHODS: We used the highly metastatic colon cancer cell line LiM6 and its TRAIL-resistant derivative LIM6-TR as cellular model. CSC populations were identified and isolated based on the expression of 5 established CSC-Markers CD24, CD44, CD166, EpCAM, and ALDH. Surface Gal3 in these CSCs was monitored by flow cytometry. Self-renewal, a functional CSC hallmark of CSC, was measured in sphere-formation assays. Response to TRAIL wasmeasured by APO-BRDU TUNEL assays. RESULTS: Only 15% of LIM6 and LIM6-TR cells were CSC as defined by the 5 markers (CD24+, CD44+, CD166+, EpCAM+, ALDH+). Surface Gal3 analysis identified two subpopulations within the CSC pool of LiM6 (54% Gal3+, 46% Gal3 negative). In contrast, CSC from LIM6-TR were uniformly Gal3+. Of note, not all Gal3+ colorectal cells can be attributed with CSC properties, since only 25-30% of Gal3+ CRCs express the remaining CSC markers simultaneously. Sphere assays demonstrated that the parental LIM6-TR had a 2-fold higher sphere forming ability than LIM6. The isolated Gal3+ CSCs formed 3.3-fold more spheres than parental Lim6 cells, while a 90% decrease in sphere formation was observed in the GAL3 negative CSC subpopulation. Depletion of Gal3 in LiM6-TR cells resulted in the loss of the Gal3+ CSC subpopulation, diminished sphere formation, and resensitization to TRAIL. Sphere formation of GAL+ CSCs in the presence of the Gal3 inhibitor LacNAc [5mM] led to a 60% reduction in sphere-formation. CONCLUSIONS: Our findings indicate that surface Gal3 is responsible for high sphere formation ability and TRAIL-resistance in a subpopulation of CSC. Further experiments will determine whether surface Gal3 in combination with other CSC markers can help to identify metastatic and chemoresistant CSCs in the circulation of CRC patients.

Abstract LB-244: MiR-34a/c-dependent PDGFR-α/β reduction inhibits tumorigenesis and sensitizes TRAIL-induced apoptosis in lung cancer.

Abstract Lung cancer is the most common cause of cancer death worldwide. Despite years of research, the prognosis for patients with lung cancer remains dismal. The most frequent type, non-small cell lung cancer (NSCLC) (85%), shows an overall five year survival of 15%. Isoforms of platelet-derived growth factor receptor (PDGFR) and its ligand, PDGF, constitute a family of receptors and ligands involved in proliferative and prosurvival signaling within cells. MicroRNAs (miRNAs), a class of ~ 22 nt endogenous RNAs, are important regulators of gene expression and have been implicated in the regulation of critical processes that are deregulated in cancer cells, as proliferation differentiation and apoptosis. Alterations in miRNA expression in cancer have been documented in numerous studies and suggest that miRNAs critically contribute to the cancer cell phenotype. In mammalians, the miR-34 family comprises three processed miRNAs that are encoded by two different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript. In this study we found that miR-34a/c and not miR-34b, suppress the metastatic capacity of NSCLC by targeting platelet-derived growth factor receptor alpha and beta (PDGFR-α and PDGFR-β). We also observed an inverse relation between PDGFR-α/β mRNAs and miR-34a/c expression in lung tumor compared to normal lung samples. Finally, miR34-a/c-dependent PDGFR-α/β suppression or knocking-down of PDGFR-α/β by siRNAs sensitized NSCLC cells to TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. Taken together our findings could be helpful for future anti-tumor therapy of lung cancer. Citation Format: Young-Jun Jeon, Gerard J. Nuovo, Justin Midddleton, Pooja Joshi, Natalya Nazaryan, Gianpiero Di Leva, Giulia Romano, Melissa Crawford, Patrick Nana-Sinkam, Michela Garofalo, Carlo M. Croce. MiR-34a/c-dependent PDGFR-α/β reduction inhibits tumorigenesis and sensitizes TRAIL-induced apoptosis in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-244. doi:10.1158/1538-7445.AM2013-LB-244

Hepatitis C Virus Sensitizes Host Cells to TRAIL-Induced Apoptosis by Up-Regulating DR4 and DR5 via a MEK1-Dependent Pathway

BackgroundHepatitis C virus (HCV) is the leading cause of liver fibrosis, cirrhosis and hepatocellular carcinoma. It is believed that continuous liver cell apoptosis contributes to HCV pathogenesis. Recent studies have shown that HCV infection can sensitize host cells to TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis, but the mechanism by which HCV regulates the TRAIL pathway remains unclear.Methods and ResultsUsing a sub-genomic replicon and full length virus, JFH-1, we demonstrate that HCV can sensitize host cells to TRAIL-induced apoptosis by up-regulating two TRAIL receptors, death receptor 4 (DR4) and death receptor 5 (DR5). Furthermore, the HCV replicon enhanced transcription of DR5 via Sp1, and the HCV-mediated up-regulation of DR4 and DR5 required MEK1 activity. HCV infection also stimulated the activity of MEK1, and the inhibition of MEK1 activity or the knockdown of MEK1 increased the replication of HCV.ConclusionsOur studies demonstrate that HCV replication sensitizes host cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 via a MEK1 dependent pathway. These findings may help to further understand the pathogenesis of HCV infection and provide a therapeutic target.

MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.

Multi-level disruption of the extrinsic apoptotic pathway mediates resistance of leukemia cells to TNF-related apoptosis-inducing ligand (TRAIL)

Disruption of apoptotic pathways belongs to commonly reported molecular mechanisms that underlie cancer drug resistance. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo2L) is a cytokine of the TNF family with selective anti-tumor activity and minimal toxicity toward healthy tissues. Primary leukemia cells are, however, largely intrinsically resistant to TRAIL-induced apoptosis. In this study we analyzed molecular differences between TRAIL-resistant K562 cell line and TRAIL-sensitive K562 clones. We demonstrate that TRAIL-sensitive K562 cells differ from the TRAIL-resistant cell line by cell surface downregulation of TRAIL decoy receptor 1, upregulation of both TRAIL death receptors, enhanced assembly and improved functioning of the death-inducing signaling complex, and increased cytoplasmic protein expression of CASP8 and key proapoptotic BCL2 members BID, BIM, BAD and BAK. The molecular basis of the intrinsic leukemia cell TRAIL resistance thus appears a consequence of the multi-level disruption of the extrinsic apoptotic pathway. The results of this study also suggest that the leukemia TRAIL-resistance is functional, leaving a possibility of overcoming the resistance by preexposure of the leukemia cells to potent TRAIL sensitizers, e.g. BH3-mimetics.

EAG1: A Potassium Channel Involved in Cancer

There is increasing evidence for the causal involvement of ion channels in cancer. Being extracellularly accessible, they constitute a novel and promising targets to treat cancer. Kv10.1 is among the best-characterized ion channels implicated in cancer. Kv10.1 is practically not detected in normal tissues outside the central nervous system, but is aberrantly expressed with very high frequency (>70%) in tumor cells from diverse origin. Its potential to favor tumor progression is maintained to a large extent even if it is unable to permeate ions. We have therefore searched for other mechanisms that mediate and/or favor its malignancy and found that: a) siRNA against Kv10.1, inhibition of channel gating by astemizol and imipramine (both unspecific Kv10.1 blockers) or by a monoclonal blocking antibody are all able to reduce proliferation rates in cell lines expressing the channel. b) Kv10.1 has a high turnover rate at the membrane and is rapidly ubiquitinated and degraded. c) it's expression greatly increases the secretion of VEGF under hypoxia, favoring angiogenesis in a manner similar to the Hypoxia Inducible Factor (HIF). d) Kv10.1 can be detected in the inner nuclear membrane and even potassium currents can be recorded from these membranes, indicating that it is functional. e) miRNA 34 regulates Kv10.1 and p53 in a reciprocal manner.In retrospective studies it could be shown that Kv10.1 expression in hemaotopetic cells correlates with poor prognosis in a specific leukemia (AML M4).Linking a Fab-fragment specific against Kv10.1 to TNF-related apoptosis inducing ligand (TRAIL), it was possible to selectively induce apoptosis in cancer cells expressing Kv10.1 by an autocrine, and adjacent non-Kv10.1 bearing cells by a paracrine mechanism.

Oligomycin A enhances apoptotic effect of TRAIL through CHOP‐mediated death receptor 5 expression

Development of resistance to TNF-related apoptosis-inducing ligand (TRAIL) in tumor cells is one of the important problems in cancer treatment. Despite the previous report demonstrating that oligomycin suppressed TNF-induced apoptosis, in our screening of small molecules enhancing cancer cell death to TRAIL, oligomycin A (OMA) was found to enhance TRAIL-induced apoptosis in HeLa cells. CCAAT/enhancer-binding protein homologous protein (CHOP) was found to directly bind to death receptor 5 (DR5) promoter through endoplasmic reticulum stress (ER-stress) signaling and sensitize the cells to TRAIL. Among ER-stress associated proteins, OMA triggered the inositol-requiring enzyme 1 (IRE1) signaling pathway, leading to X-binding protein 1 (XBP1) splicing, CHOP expression and DR5 upregulation. In contrast, small-interfering RNA (siRNA) of CHOP reduced the number of apoptotic cells in response to the co-treatment of TRAIL and OMA. Collectively, our data suggest that OMA enhances apoptotic death of cervical cancer cells to TRAIL through upregulation of CHOP-mediated DR5 expression following ER-stress.

Smac Mimetic Compounds Potentiate Interleukin-1β-mediated Cell Death

Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1β. Here, we investigated the potential impact of IL-1β on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1β treatment, which required IL-1β-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1β-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1β treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1β treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.

Targeted inhibition of XIAP expression reverses resistance of colon cancer cells to TNF-related apoptosis-inducing ligand

Objective To study the resistance changes of colon cancer cells SW1116 to TNF-related apoptosis-inducing ligand ( TRAIL) after targeted inhibition of X-linked inhibitor of apoplosis protein (XIAP) expression by small hoop RNA plasmid. Methods Colon cancer cells were transfected by XIAP short hairpin RNA (shRNA) plasmid with liposorae. Inhibition rate and cell viability were examined 48 h post-transfection. SW1116 cells were exposed to TRAIL at concentrations of 25, 50, 100, 200 μLg/L, and their sensitivity to TRAIL was evaluated after inhibition of the XIAP expression. Caspase-3 activity in tumor cells was also evaluated by Western blotting. Results XIAP expression was decreased significantly after XIAP shRNA transfection (inhibition rate 60% ) , cell growing rate was inhibited (inhibition rate 24% ) , resistance to TRAIL was reversed, sensitivity to TRAIL was increased, cell growing inhibition rate reached 64% in 200 μg/L of TRAIL, and Caspase-3 activity was increased in tumor cells. Conclusion Targeted inhibition of XIAP expression restored and increased sensitivity of colon cancer cells to TRAIL. Key words: Colon carcinoma; RNAi; XIAP

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

BackgroundWhile deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.Principal FindingsIn mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC50 (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC50 (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.Conclusions/SignificanceTogether, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.

The contact allergen nickel sensitizes primary human endothelial cells and keratinocytes to TRAIL‐mediated apoptosis

Primary endothelial cells are fully resistant to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Here, we demonstrate that certain environmental conditions, such as exposure to the widespread allergen nickel, can dramatically increase the susceptibility of naturally resistant primary endothelial cells or keratinocytes to TRAIL-induced apoptosis. While nickel treatment increased surface expression of the apoptosis-inducing TRAIL receptors TRAIL-R1 and TRAIL-R2, it also up-regulated the apoptosis-deficient TRAIL-R4, suggesting that modulation of TRAIL receptor expression alone is unlikely to fully account for the dramatic sensitization effect of nickel. Further analysis of candidate mediators revealed that nickel strongly repressed c-FLIP at mRNA and protein levels. Accordingly, increased activation of Caspase-8 and Caspase-3 following nickel treatment was observed. Importantly, depletion of c-FLIP by RNA interference could largely recapitulate the effect of nickel and sensitize endothelial cells to TRAIL-dependent apoptosis in the absence of nickel pre-treatment. Conversely, ectopic expression of c-FLIPL largely protected nickel-treated cells from TRAIL-mediated apoptosis. Our data demonstrate that one key mechanism of sensitization of primary human endothelial cells or keratinocytes is transcriptional down-regulation of c-FLIP. We hypothesize that environmental factors, exemplified by the contact allergen nickel, strongly modulate death ligand sensitivity of endothelial cells and keratinocytes thus influencing vascular and epidermal function and integrity under physiological and pathophysiological conditions.

Quercetin 에 의한 사람백혈병 세포의 TRAIL 에 대한 감수성 증가: DNA-PK/Akt 신호전달경로의 관여

Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, some cancer cells show either partial or complete resistance to TRAIL. Human leukemic K562 and CEM cells also show resistance to TRAIL-induced apoptosis. Novel molecular target and treatment strategies are required to overcome TRAIL resistance of human leukemia cells. Therefore, the purpose of this study was to target key anti-apoptotic molecules deciding TRAIL resistance for sensitization of TRAIL-resistant K562 and CEM cells, and to evaluate the effect of quercetin as a TRAIL sensitizer on these TRAIL-resistant cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in K562 cells by inhibition of the DNA-PK/Akt signaling pathway, which leads to enhancement of TRAIL-mediated activation of caspases and concurrent cleavage of PARP and up-regulation of Bax. The findings suggest that the DNA-PK/Akt signaling pathway plays an essential role in regulating cells to escape from TRAIL-induced apoptosis, and quercetin could act in synergy with TRAIL to increase apoptosis by inhibition of the DNA-PK/Akt signaling pathway, which overcomes TRAIL-resistance of K562 and CEM cells. This study suggests that DNA-PK might interfere with TRAIL-induced apoptosis in human leukemic cells through activation of the Akt signaling pathway.