Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.     

Phenotypic variations of TRAIL sensitivity in cloned populations of prostate cancer cells

Factors that regulate the induction of apoptosis of tumour cells are potential candidates for therapeutic intervention for the majority of cancers. Studying modifiers of apoptotic responses, such as members of the tumour necrosis factor receptor superfamily, may give clues as to how induction of apoptosis in tumours could be maximized to enhance the benefit of treatment regimes. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising anti‐tumour molecule since its activity is specific for tumour cell populations. TRAIL binds to death receptors, inducing apoptosis in susceptible cells. The mechanisms which determine whether tumour cells are susceptible to TRAIL are unclear, and several mechanisms have been proposed, including expression of osteoprotegerin (OPG), decoy receptors, and factors that affect intracellular signalling of pro‐apoptotic molecules, such as c‐FLIP. Here we show that experiments to modulate the activity of one of these factors, OPG, by over‐expression and also by stable knockdown of OPG expression, alters the TRAIL sensitivity of PC3 prostate cancer cells. However we show that some observed effects, which appear to support the hypothesis that OPG prevents TRAIL‐induced apoptosis of tumour cells, may be due to variation of the TRAIL response of sub‐clones of tumour cells, even within a cloned population. These results highlight potential limitations of experiments designed to test contribution of factors affecting intrinsic apoptosis susceptibility using cloned tumour cell populations. J. Cell. Biochem. 104: 1452–1464, 2008. © 2008 Wiley‐Liss, Inc.     

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

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

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D) 相似文献   

Osteoprotegerin Prevents Development of Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture and subsequent high mortality. Establishment of medical therapies for the prevention of AAAs requires further understanding of the molecular pathogenesis of this condition. This report details the possible involvement of Osteoprotegerin (OPG) in the prevention of AAAs through inhibition of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In CaCl₂-induced AAA models, both internal and external diameters were significantly increased with destruction of elastic fibers in the media in Opg knockout (KO) mice, as compared to wild-type mice. Moreover, up-regulation of TRAIL expression was observed in the media by immunohistochemical analyses. Using a culture system, both the TRAIL-induced expression of matrix metalloproteinase-9 in smooth muscle cells (SMCs) and the chemoattractive effect of TRAIL on SMCs were inhibited by OPG. These data suggest that Opg may play a preventive role in the development of AAA through its antagonistic effect on Trail.     

The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors     

Medication persistence over 2 years of follow-up in a cohort of early rheumatoid arthritis patients: associated factors and relationship with disease activity and with disability

Introduction

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.

Methods

Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.

Results

Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.

Conclusions

This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.     

TRAIL‐induced apoptosis of FHIT‐negative lung cancer cells is inhibited by FHIT re‐expression

Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)‐related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT‐negative non‐small lung cancer cell line Calu‐1. We generated four FHIT‐inducible Calu‐1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL‐induced apoptosis, without affecting TRAIL‐receptors surface expression. FHIT‐specific small interference RNA transfection of SV40‐immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL‐induced apoptosis. Of note, suramin‐mediated inhibition of FHIT enzymatic activity also enhanced TRAIL‐induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL‐induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL‐receptors and likely requires its dinucleoside‐triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands‐based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug‐induced epigenetic modulations aimed to restoring FHIT gene expression levels in non‐small cells lung tumors. J. Cell. Physiol. 220: 492–498, 2009. © 2009 Wiley‐Liss, Inc.     

Trail down-regulates the release of osteoprotegerin (OPG) by primary stromal cells

The soluble member of the TNF-R superfamily osteoprotegerin (OPG) is abundantly released under basal conditions by both mesenchymal stem cells (MSC) and fibroblasts and by endothelial cells upon stimulation with inflammatory cytokines. Since MSC, fibroblasts and endothelial cells represent key elements of the normal and tumor microenvironment and express detectable levels of surface TRAIL receptors, we investigated the effect of TRAIL on OPG release. Unexpectedly, recombinant TRAIL decreased the spontaneous OPG release in all cell types examined. Moreover, TRAIL decreased OPG release also in stromal cells co-cultured with lymphoma cells and counteracted the OPG induction by TN-alpha in HUVEC and MSC. Such down-regulation was not due to a masking effect in the ELISA quantification of the OPG released in the culture supernatants due to binding of OPG to its ligands (TRAIL and RANKL), as demonstrated by competition experiments with recombinant TRAIL and by the lack of RANKL release/induction. In addition, OPG down-regulation was not due to induction of cytotoxic effects by TRAIL, since the degree of apoptosis in response to TRAIL was negligible in all primary cell types. With regards to the possible molecular mechanism accounting for the down-regulation of OPG release by TRAIL, we found that treatment of MSC with TRAIL significantly decreased the phosphorylation levels of p38/MAPK. There is a suggestion that this pathway is involved in the stabilization of OPG mRNA. In this respect, the ability of TRAIL to decrease the release of OPG, in the absence of cell cytotoxicity, was mimicked by the p38/MAPK inhibitor SB203580.     

多囊卵巢综合征模型鼠颗粒细胞凋亡及TRAIL蛋白的表达

目的通过观察卵巢颗粒细胞凋亡及TRAIL(肿瘤坏死因子相关凋亡诱导配体)蛋白的表达情况,探讨颗粒细胞凋亡与PCOS发病的相关性及凋亡调控蛋白TRAIL在PCOS颗粒细胞凋亡中的作用。方法采用硫酸普拉睾酮钠诱导大鼠PCOS模型,3’-末端原位标记法(TUNEL)检测大鼠卵巢颗粒细胞凋亡情况,免疫组化染色及RT-PCR分析检测TRAIL蛋白及TRAIL mRNA在颗粒细胞的表达。结果PCOS组大鼠卵巢窦状卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达较对照组明显增强(P<0.01,P<0.05),窦前卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未发现凋亡征象及TRAIL蛋白表达。PCOS组大鼠卵巢颗粒细胞TRAIL mRNA的表达较对照组明显增强(P<0.01)。结论PCOS大鼠卵巢窦状卵泡颗粒细胞凋亡明显增强,TRAIL在PCOS大鼠卵巢颗粒细胞凋亡调控中发挥了作用。     

Modulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors in a human osteoclast model in vitro

TRAIL (TNF-related apoptosis-inducing ligand) has been shown to induce apoptosis by binding to TRAIL-R1 and -R2 death receptors, but not to TRAIL-R3 or -R4, its decoy receptors that lack the internal death domain. Osteoclasts (Ocs) are sensitive to TRAIL-induced apoptosis, and modulation of these receptors may change Oc sensitivity to TRAIL. Using human Oc cultures, we first investigated the gene expression profile of these receptors (TNFRSF10 -A, -B, -C, -D encoding TRAIL-Rs 1–4) by real time PCR after adding osteotropic factors during the last week of Oc cultures. We observed a significant decrease in the expression of TNFRSF10-A after the addition of TGFβ, and an increase in that of TNFRSF10-A and -B post-PTH stimulation. Protein expression of TRAIL-R1 and -R3 was upregulated in the presence of MIP-1α, but down-regulated in the presence of TGFβ (R1), TRAIL (R2) or OPG (R3). The percentage of Ocs expressing the TRAIL-R1 and/or -R2 at their surface was increased by MIP-1α and TRAIL, increased (R2) or decreased (R1) by TGFβ, and the percentage expressing TRAIL-R3 was increased by MIP-1α, TRAIL and RANKL. Although significant, the magnitude of all these changes was of about 10–15%. While a direct correlation between these changes and TRAIL-induced Oc apoptosis was less clear, a protective effect was observed in Ocs that had been treated with OPG, and an additive effect in Ocs pre-treated with TRAIL or TGFβ increased TRAIL sensitivity.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill transformed cells. We have studied the expression and functionality of the TRAIL apoptotic pathway in Ewing's sarcoma. We demonstrate that tumors from patients with Ewing's sarcoma express receptors TRAIL-R1 and -R2. Using a panel of nine Ewing's sarcoma cell lines TRAIL could induce apoptosis in seven cell lines. Preincubation with interferon-gamma rendered the two resistant cell lines sensitive. TRAIL was the most potent inducer of apoptosis when compared to Fas ligand or TNF. TRAIL-mediated apoptosis could be inhibited by various caspase-inhibitors. No difference in the surface expression of TRAIL-receptors was observed between sensitive and resistant cell lines. Also, all cell lines had similar levels of expression of Flice-like inhibitory protein (FLIP) on immunoblot. However, the two resistant cell lines had only very low level expression of caspase 8 on RNA and protein level. In summary, we show that Ewing's sarcoma expresses receptors for TRAIL, and that cells are exquisitely sensitive to TRAIL-mediated apoptosis. These results may warrant clinical trials with TRAIL in Ewing's sarcoma once the safety of TRAIL for humans has been established.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

TNF-related apoptosis-inducing ligand and its decoy receptor osteoprotegerin in nonischemic dilated cardiomyopathy

Apoptosis has been attributed an essential role in dilated cardiomyopathy (DCM) recently. We assessed expression of TNF-related apoptosis-inducing ligand (TRAIL) and its decoy receptor osteoprotegerin (OPG) in men with nonischemic DCM, who underwent coronary angiography and endomyocardial biopsy (EMB) after exclusion of coronary artery disease compared to control patients. TRAIL plasma concentrations were elevated in DCM (p=0.02 vs. controls), and were positively correlated with left ventricular enddiastolic diameter (r=0.15, p=0.04), whereas OPG plasma levels did not differ between both groups (p=0.96). In EMB of DCM patients, TRAIL and OPG protein were detected by immunohistochemistry but not in controls. Furthermore, gene expression in EMB or peripheral blood leukocytes (PBL) of DCM patients assessed by real-time PCR showed an increase of TRAIL mRNA in PBL (p=0.01 vs. controls), whereas OPG mRNA was upregulated in endomyocardial specimens (p<0.001 vs. controls). In conclusion, myocardial overexpression of antiapoptotic OPG in DCM patients may represent a compensatory mechanism to limit systemic activation of TRAIL in patients with congestive heart disease.     

Hypermethylation of the death-associated protein kinase promoter attenuates the sensitivity to TRAIL-induced apoptosis in human non-small cell lung cancer cells

Death-associated protein (DAP) kinase plays an important role in IFN-gamma, tumor necrosis factor (TNF)-alpha, or Fas-ligand induced apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family and can induce caspase-dependent apoptosis in cancer cells while sparing most of the normal cells. However, some of the cancer cell lines are insensitive to TRAIL, and such resistance cannot be explained by the dysfunction of TRAIL receptors or their known downstream targets. We reported previously that DAP kinase promoter is frequently methylated in non-small cell lung cancer (NSCLC), and such methylation is associated with a poor clinical outcome. To determine whether DAP kinase promoter methylation contributes to TRAIL resistance in NSCLC cells, we measured DAP kinase promoter methylation and its gene expression status in 11 NSCLC cell lines and correlated the methylation/expression status with the sensitivity of cells to TRAIL. Of the 11 cell lines, 1 had a completely methylated DAP kinase promoter and no detectable DAP kinase expression, 4 exhibited partial promoter methylation and substantially decreased gene expression, and the other 6 cell lines showed no methylation in the promoter and normal DAP kinase expression. Therefore, the amount of DAP kinase expression amount was negatively correlated to its promoter methylation (r = -0.77; P = 0.003). Interestingly, the cell lines without the DAP kinase promoter methylation underwent substantial apoptosis even in the low doses of TRAIL, whereas those with DAP kinase promoter methylation were resistant to the treatment. The resistance to TRAIL was reciprocally correlated to DAP kinase expression in 10 of the 11 cell lines at 10 ng/mL concentration (r = 0.91; P = 0.001). We treated cells resistant to TRAIL with 5-aza-2'-deoxycytidine, a demethylating reagent, and found that these cells expressed DAP kinase and became sensitive to TRAIL. These results suggest that DAP kinase is involved in TRAIL-mediated cell apoptosis and that a demethylating agent may have a role in enhancing TRAIL-mediated apoptosis in some NSCLC cells by reactivation of DAP kinase.     

Decreased Affinity of Recombinant Human Tumor Necrosis Factor-related Apoptosis-inducing Ligand (rhTRAIL) D269H/E195R to Osteoprotegerin (OPG) Overcomes TRAIL Resistance Mediated by the Bone Microenvironment

The bone marrow microenvironment provides important signals for the survival and proliferation of hematopoietic and malignant cells. In multiple myeloma, plasma cells are surrounded by stromal cells including osteoblasts. These stromal cells protect multiple myeloma cells from apoptosis induced by chemotherapeutic agents. Osteoprotegerin (OPG), a soluble receptor of the cytokine TNF-related apoptosis-inducing ligand (TRAIL), is secreted by osteoblasts and has been implicated in the prevention of cell death induced by TRAIL in malignant cells. Previously, we have designed death receptor-specific TRAIL variants that induce apoptosis exclusively via one of its death receptors. Here, we have studied in detail the interaction between recombinant human (rhTRAIL) variants and OPG. We show that a DR5-specific variant (rhTRAIL D269H/E195R) displays a significantly decreased affinity to OPG. Furthermore, this rhTRAIL variant shows a much higher activity when compared with rhTRAIL WT and retains its effectiveness in inducing cell death in multiple myeloma cell lines, in the presence of OPG secreted by stromal cells. We also demonstrate that stromal cells are largely insensitive to high concentrations of this rhTRAIL variant. In conclusion, rhTRAIL D269H/E195R is a potential therapy for multiple myeloma due to its high effectiveness and diminished binding to OPG.     

Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways

TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκβ and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.