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.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Death receptors, Fas and TRAIL receptors, are involved in human osteoclast apoptosis

Survival and apoptosis are crucial aspects of the osteoclast life cycle. Although osteoclast survival has been extensively studied, little is known about the mechanisms involved in human osteoclast apoptosis. In the present study, cord blood monocytes (CBMs) were used as the source of human osteoclast precursors. When cultured in the presence of M-CSF and RANKL, CBMs formed multinucleated cells that expressed RANK and calcitonin receptor, and were able to resorb bone. These cells expressed TRAIL receptors (R1-R4). Surprisingly, although TRAIL-receptor expression was not detectable in osteoclasts from normal bone, osteoclasts from myeloma specimens did express TRAIL receptors to a variable extent. Significantly, we have shown for the first time that this pathway is indeed functional in human osteoclasts, and that apoptosis occurred and was significantly greater in the presence of TRAIL. In addition, we have shown that a Fas-activating antibody is also able to induce osteoclast apoptosis, as did TGFbeta, whereas the survival factor M-CSF decreased apoptosis. Overall, these findings suggest that death receptors, TRAIL receptors and Fas, could be involved in osteoclast apoptosis in humans.     

Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells, but not in normal cells. However, more and more tumor cells remain resistant to TRAIL, which limited its application for cancer therapy. Expression of the adenovirus serotype 5 (Ad5) E1A sensitizes tumor cells to apoptosis by TNF-alpha, Fas-ligand, and TRAIL. Here we asked whether E1A overcomes this resistance and enhances TRAIL-induced apoptosis in the tumor cells. Our results revealed that the tumor cell lines, HeLa and HepG2, with infection by Ad-E1A, were highly sensitive to TRAIL-induced apoptosis. Importantly, we found that in normal primary human lung fibroblast cells (HLF) TRAIL is capable of inducing apoptosis in combination with E1A as efficiently as in some tumor cell lines. The adenovirus type 5 encoding proteins, E1B19K and E3 gene products, have been shown to inhibit E1A and TRAIL-induced apoptosis of HLF cells by using the recombinant adenovirus AdDeltaE1B55K, with mutation of E1B55K, containing E1B19K and complete E3 region. Further results demonstrated that the expression of DR5 and TRAIL was down-regulated in the AdDeltaE1B55K co-infected HLF cells. These findings suggest that TRAIL may play an important role in limiting virus infections and the ability of adenovirus to inhibit killing may prolong acute and persistent infections. The results from this study have also suggested the possibility that the combination of E1A with TRAIL could be used in the treatment of human malignancy, or in the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy.     

Sensitization of mesothelioma to TRAIL apoptosis by inhibition of histone deacetylase: role of Bcl-xL down-regulation

The TNF-related apoptosis-inducing ligand (TRAIL) is an immunological inducer of apoptosis selectively killing many, but not all, cancer cells. Malignant mesothelioma (MM) is fatal neoplasia with no current treatment, most likely due to high resistance of MM cells towards inducers of apoptosis, including TRAIL. We studied whether inhibition of histone deacetylase (HDAC), recently shown to sensitize malignant cells to a variety of apoptogenic substances, renders MM cells susceptible to TRAIL. Indeed, sub-apoptotic doses of the HDAC inhibitor suberohydroxamic acid (SBHA) sensitized MM cells to TRAIL apoptosis. Of the apoptotic mediators tested, the anti-apoptotic protein Bcl-x(L) was strongly down-regulated by combined treatment of the cells with SBHA and TRAIL but not by the HDAC inhibitor alone, while little or no change in the expression of other Bcl-2 family members highly expressed in MM cells, including Mcl-1 and Bax, was observed. Our data suggest a cross-talk between HDAC inhibition and TRAIL that results in modulation of expression of specific apoptotic mediators, and point to the potential of their combinatorial use in treatment of TRAIL-resistant neoplastic disease.     

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.     

Bim plays a crucial role in synergistic induction of apoptosis by the histone deacetylase inhibitor SBHA and TRAIL in melanoma cells

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor – induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X_L, Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim_EL. Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma. This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council, Australia. X.D. Zhang is a Cancer Institute NSW Fellow.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis

We evaluated the ability of alpha-tocopheryl succinate (alpha-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that alpha-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of alpha-TOS restored expression of DR4 and DR5. The ability of alpha-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli.     

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.