Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.     

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy

Apo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24 h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL. RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.     

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.     

The histone deacetylase inhibitor, suberoylanilide hydroxamic acid, overcomes resistance of human breast cancer cells to Apo2L/TRAIL

While the apoptosis-inducing ligand Apo2L/TRAIL is a promising new agent for the treatment of cancer, the sensitivity of cancer cells for induction of apoptosis by Apo2L/TRAIL varies considerably. Identification of agents that can be used in combination with Apo2L/TRAIL to enhance apoptosis in breast cancer cells would increase the potential utility of this agent as a breast cancer therapeutic. Here, we show that the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize Apo2L/TRAIL-resistant breast cancer cells to Apo2L/TRAIL-induced apoptosis. Importantly, neither Apo2L/TRAIL alone, nor in combination with SAHA, affected the viability of normal human cells in culture. Apo2L/TRAIL-resistant MDA-MB-231 breast cancer cells, generated by long-term culture in the continuous presence of Apo2L/TRAIL, were resensitized to Apo2L/TRAIL-induced apoptosis by SAHA. The sensitization of these cells by SAHA was accompanied by activation of caspase 8, caspase 9 and caspase 3 and was concomitant with Bid and PARP cleavage. The expression of the proapoptotic protein, Bax, increased significantly with SAHA treatment and high levels of Bax were maintained in the combined treatment with Apo2L/TRAIL. Treatment with SAHA increased cell surface expression of DR5 but not DR4. Interestingly, SAHA treatment also resulted in a significant increase in cell surface expression of DcR1. Taken together, our findings indicate that the use of these 2 agents in combination may be effective for the treatment of breast cancer.     

Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells. The objectives of this study are to investigate the intracellular mechanisms by which TRAIL induces apoptosis. The death receptor Fas, upon ligand binding, trimerizes and recruits the adaptor protein FADD through the cytoplasmic death domain of Fas. FADD then binds and activates procaspase-8. It is unclear whether FADD is required for TRAIL-induced apoptosis. Here we show that the signaling complex of DR4/DR5 is assembled in response to TRAIL binding. FADD and caspase-8, but not caspase-10, are recruited to the receptor, and cells deficient in either FADD or caspase-8 blocked TRAIL-induced apoptosis. In addition, TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Deltapsi(m)), the cleavage of BID, and the redistribution of mitochondrial cytochrome c. Treatment of Jurkat cells with cyclosporin A delayed TRAIL-induced Deltapsi(m), caspase-3 activation and apoptosis. Similarly, Overexpression of Bcl-2 or Bcl-X(L) delayed, but did not inhibit, TRAIL-induced Deltapsi(m) and apoptosis. In contrast, XIAP, cowpox virus CrmA and baculovirus p35 inhibited TRAIL-induced apoptosis. These data suggest that death receptors (DR4 and DR5) and Fas receptors induced apoptosis through identical signaling pathway, and TRAIL-induced apoptosis via both mitochondrial-dependent and -independent pathways.     

Enhanced tumor killing by Apo2L/TRAIL and CPT-11 co-treatment is associated with p21 cleavage and differential regulation of Apo2L/TRAIL ligand and its receptors

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G(2)M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G(2)-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.     

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand cooperates with chemotherapy to inhibit orthotopic lung tumor growth and improve survival

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a tumor necrosis factor superfamily member that induces apoptosis through the death receptors DR4 and/or DR5 in various cancer cell types but not in most normal cells. Several lung cancer cell lines express DR4 and DR5 and undergo apoptosis in vitro in response to Apo2L/TRAIL. We investigated the efficacy of recombinant soluble human Apo2L/TRAIL and its interaction with chemotherapy in xenograft models based on human NCI-H460 non-small cell lung carcinoma cells. In vitro, Taxol enhanced caspase activation and apoptosis induction by Apo2L/TRAIL. In vivo, Apo2L/TRAIL or Taxol plus carboplatin chemotherapy partially delayed progression of established subcutaneous tumor xenografts, whereas combined treatment caused tumor regression and a substantially longer growth delay. Apo2L/TRAIL, chemotherapy, or the combination of both inhibited growth of preformed orthotopic lung parenchymal tumors versus control by 60%, 57%, or 97%, respectively (all P < 0.01; n = 8-10). Furthermore, combination treatment improved day-90 survival relative to control (7 of 15 versus 1 of 15; P = 0.0003 by Mantel-Cox) as well as to Apo2L/TRAIL (3 of 14; P = 0.031) or chemotherapy (3 of 15; P = 0.035). These studies provide evidence for in vivo activity of Apo2L/TRAIL against lung tumor xenografts and underscore the potential of this ligand for advancing current lung cancer treatment strategies.     

Cotreatment with STI-571 enhances tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL or apo-2L)-induced apoptosis of Bcr-Abl-positive human acute leukemia cells.

Bcr-Abl tyrosine kinase inhibitor STI-571 induces differentiation and apoptosis of HL-60/Bcr-Abl (with ectopic expression of p190 Bcr-Abl) and K562 (with endogenous expression of p210 Bcr-Abl) cells (Blood, 96: 2246-2253, 2000). Cotreatment with STI-571 partially overcomes the resistance to antileukemic drug-induced apoptosis of HL-60/Bcr-Abl and K562 cells. Tumor necrosis factor (TNF) alpha-related apoptosis-inducing ligand (Apo-2L/TRAIL), after binding with its signaling death receptors (DR4 and DR5), triggers the intrinsic "mitochondrial" pathway of apoptosis more efficiently in the cancer than do normal cells. In the present studies, we compared the apoptotic effects of Apo-2L/TRAIL, with or without cotreatment with STI-571, in HL-60/neo, HL-60/Bcr-Abl, and K562 cells. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells are relatively resistant to Apo-2L/TRAIL-induced apoptosis. In HL-60/Bcr-Abl and K562 versus HL-60/neo cells, Apo-2L/TRAIL caused less cytosolic accumulation of cytochrome c and the processing of caspase-9 and -3. This was also associated with decreased processing of caspase-8, c-FLIP(L) and Bid. Reduced effects of Apo-2L/TRAIL in Bcr-Abl-positive leukemic cells were not attributable to diminished expression of DR4 and DR5, or higher expressions of the decoy receptors DcR1 and -2 or c-FLIP(L). Cotreatment with STI-571 significantly enhanced Apo-2L/TRAIL-induced apoptosis (P < 0.01) as well as increased the processing of caspase-9 and -3 and XIAP, without affecting the levels of DR4, DR5, decoy receptors, or c-FLIP(L). Cotreatment with STI-571 did not enhance Apo-2L/TRAIL-induced apoptosis of HL-60/neo cells. These studies suggest that a combined treatment with STI-571 may be an effective strategy to selectively sensitize Bcr-Abl-positive leukemic blasts to Apo-2L/TRAIL-induced apoptosis.     

Sensitization of tumor cells to Apo2 ligand/TRAIL-induced apoptosis by inhibition of casein kinase II

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.     

Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.     

ROS and CHOP are critical for dibenzylideneacetone to sensitize tumor cells to TRAIL through induction of death receptors and downregulation of cell survival proteins

Because tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells, it is being tested in cancer patients. Unfortunately, patients develop resistance to the cytokine, therefore, agents that can sensitize cells to TRAIL are urgently needed. In this study, we investigated whether dibenzylideneacetone (DBA) can sensitize cancer cells to TRAIL and potentiates TRAIL-induced apoptosis. As indicated by accumulation of the membrane phospholipid phosphatidylserine, DNA breaks, intracellular esterase activity, and activation of caspase-8, -9, and -3, we concluded that DBA potentiated TRAIL-induced apoptosis in colon cancer cells. DBA also converted TRAIL resistant-cells to TRAIL-sensitive. When examined for the mechanism, we found that DBA decreased the expression of antiapoptotic proteins and decoy receptor-2 and increased proapoptotic proteins. DBA also induced both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 by small interfering RNA (SiRNA) reduced the sensitizing effect of DBA on TRAIL-induced apoptosis. In addition, DBA increased the expression of CHOP proteins. Knockdown of CHOP by siRNA decreased the induction of DBA-induced DR5 expression and apoptosis. Induction of receptors by DBA, however, was p53-independent, as deletion of p53 had no effect on receptor induction. We observed that DBA-induced induction of DR5 and DR4 was mediated through generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of death receptors and suppression of cell survival proteins by DBA. Overall, our results show that DBA potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of ROS and CHOP mediated pathways.     

Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo

Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL/Apo-2 ligand) induces apoptosis in the majority of cancer cells without appreciable effect in normal cells. Here, we report the effects of TRAIL on apoptosis in several human breast cancer cell lines, primary memory epithelial cells, and immortalized nontransformed cell lines, and we examine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in breast cancer cells in vitro and in vivo. TRAIL induced apoptosis with different sensitivities, and the majority of cancer cell lines were resistant to TRAIL. The chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, camptothecin, and Adriamycin) induced death receptors (DRs) TRAIL receptor 1/DR4 and TRAIL receptor 2/DR5, and successive treatment with TRAIL resulted in apoptosis of both TRAIL-sensitive and -resistant cells. Actinomycin D sensitized TRAIL-resistant cells through up-regulation of caspases (caspase-3, -9, and -8). TRAIL induces apoptosis in Adriamycin-resistant MCF7 cells already expressing high levels of death receptors DR4 and DR5. The pretreatment of breast cancer cells with chemotherapeutic drugs followed by TRAIL reversed their resistance by triggering caspase-3, -9, and -8 activation. The sequential treatment of nude mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity and apoptosis in xenografted tumors. Complete eradication of established tumors and survival of mice were achieved without detectable toxicity. Thus, the sequential administration of chemotherapeutic drugs followed by TRAIL may be used as a new therapeutic approach for cancer therapy.     

TRAIL and apoptosis induction by TNF-family death receptors

Tumor necrosis factor-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) family of ligands capable of initiating apoptosis through engagement of its death receptors. TRAIL selectively induces apoptosis of a variety of tumor cells and transformed cells, but not most normal cells, and therefore has garnered intense interest as a promising agent for cancer therapy. TRAIL is expressed on different cells of the immune system and plays a role in both T-cell- and natural killer cell-mediated tumor surveillance and suppression of suppressing tumor metastasis. Some mismatch-repair-deficient tumors evade TRAIL-induced apoptosis and acquire TRAIL resistance through different mechanisms. Death receptors, members of the TNF receptor family, signal apoptosis independently of the p53 tumor-suppressor gene. TRAIL treatment in combination with chemo- or radiotherapy enhances TRAIL sensitivity or reverses TRAIL resistance by regulating the downstream effectors. Efforts to identify agents that activate death receptors or block specific effectors may improve therapeutic design. In this review, we summarize recent insights into the apoptosis-signaling pathways stimulated by TRAIL, present our current understanding of the physiological role of this ligand and the potential of its application for cancer therapy and prevention.     

Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of DR5

Sulforaphane is a chemopreventive agent present in various cruciferous vegetables, including broccoli. Here, we show that treatment with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in combination with subtoxic doses of sulforaphane significantly induces rapid apoptosis in TRAIL-resistant hepatoma cells. Neither TNF-alpha- nor Fas-mediated apoptosis was sensitized in hepatoma cells by cotreatment with sulforaphane, suggesting that sulforaphane can selectively sensitize cells to TRAIL-induced apoptosis but not to apoptosis mediated by other death receptors. We found that sulforaphane treatment significantly up-regulated mRNA and protein levels of DR5, a death receptor of TRAIL. This was accompanied by an increase in the generation of reactive oxygen species (ROS). Pretreatment with N-acetyl-l-cysteine and overexpression of catalase inhibited sulforaphane-induced up-regulation of DR5 and almost completely blocked the cotreatment-induced apoptosis. Furthermore, the sulforaphane-mediated sensitization to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs for DR5. These results collectively indicate that sulforaphane-induced generation of ROS and the subsequent up-regulation of DR5 are critical for triggering and amplifying TRAIL-induced apoptotic signaling. We also found that sulforaphane can sensitize both Bcl-xL- and Bcl-2-overexpressing hepatoma cells to TRAIL-induced apoptosis, indicating that treatment with a combination of TRAIL and sulforaphane may be a safe strategy for treating resistant hepatomas.     

Targeting death-inducing receptors in cancer therapy

Deregulated cell death pathways may lead to the development of cancer, and induction of tumor cell apoptosis is the basis of many cancer therapies. Knowledge accumulated concerning the molecular mechanisms of apoptotic cell death has aided the development of new therapeutic strategies to treat cancer. Signals through death receptors of the tumor necrosis factor (TNF) superfamily have been well elucidated, and death receptors are now one of the most attractive therapeutic targets in cancer. In particular, DR5 and DR4, death receptors of TNF-related apoptosis-inducing ligand (TRAIL or Apo2L), are interesting targets of antibody-based therapy, since TRAIL may also bind decoy receptors that may prevent TRAIL-mediated apoptosis, whereas TRAIL ligand itself selectively induces apoptosis in cancer cells. Here, we review the potential therapeutic utility of agonistic antibodies against DR5 and DR4 and discuss the possible extension of this single-antibody-based strategy when combined with additional modalities that either synergizes to cause enhanced apoptosis or further engage the cellular immune response. Rational design of antibody-based therapies combining the induction of tumor cell apoptosis and activation of tumor-specific adaptive immunity enables promotion of distinct steps of the antitumor immune response, thereby enhancing tumor-specific lymphocytes that can eradicate TRAIL/DR5-resistant mutating, large established and heterogeneous tumors in a manner that does not require the definition of individual tumor-specific antigens.     

Enhancement of TRAIL/Apo2L-mediated apoptosis by adriamycin through inducing DR4 and DR5 in renal cell carcinoma cells

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies in humans. We show that adriamycin (ADR) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L have a synergistic cytotoxic effect against RCC cells. This synergistic cytotoxicity was obtained in ACHN, A704, Caki-1 and Caki-2 human RCC cell lines and freshly derived RCC cells from 6 patients. This synergistic effect, however, was not achieved in 5 samples of freshly isolated normal kidney cells. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of TRAIL/Apo2L and ADR was realized by inducing apoptosis. Sequential treatment with ADR followed by TRAIL/Apo2L induced significantly more cytotoxicity than the reverse treatment. ADR increased the expression of DR4 and DR5 in RCC cells, but not in the normal kidney cells. Furthermore, the synergistic cytotoxicity was significantly inhibited by DR4:Fc and DR5:Fc fusion proteins, which inhibit TRAIL/Apo2L-mediated apoptosis. In addition, caspase activity assays and treatment of caspase inhibitors demonstrated that the combination treatment with ADR and TRAIL/Apo2L activated caspase cascade, including caspase-9, -8, -6 and -3, which were the downstream molecules of death receptors. These findings indicate that ADR sensitizes RCC cells to TRAIL/Apo2L-mediated apoptosis through induction of DR4 and DR5, suggesting that the combination therapy of TRAIL/Apo2L and ADR might be effective for RCC therapy.     

PG490-mediated sensitization of lung cancer cells to Apo2L/TRAIL-induced apoptosis requires activation of ERK2

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the family of programmed cell death-inducing cytokines. Apo2L/TRAIL induces apoptosis in a wide variety of tumor cells. Tumor cells that are resistant to Apo2L/TRAIL-induced apoptosis can be sensitized by chemotherapeutic drugs and other agents via an unknown mechanism. Here we report that PG490 (triptolide), a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii and used in traditional Chinese medicine, sensitizes lung cancer but not normal human bronchial epithelial cells to Apo2L/TRAIL-induced apoptosis. Sensitization was accompanied by caspase-3 and caspase-8 activation, whereas no cleavage of caspase-9 was observed. Determination of cell surface receptors by flow cytometry demonstrated no difference in Apo2L/TRAIL-R1 and -R2 expression, the two receptors with functional death domains, between resistant and sensitized cells. In cells treated with the combination of Apo2L/TRAIL and PG490, we observed activation of ERK2, a member of the mitogen-activated protein kinase family. Furthermore, sensitization could be blocked by the ERK inhibitor U0126 but not the p38 inhibitor SB203580, suggesting that activation of ERK2 is required for this effect. In addition, sensitization of lung cancer cells was also seen in ex vivo culture of lung cancer tissue from four patients who underwent surgery. Immunohistochemical staining showed a clear reduction in proliferation cell nuclear antigen (PCNA) in tissue treated with Apo2L/TRAIL and PG490. In conclusion, apoptosis induced by the combination of Apo2L/TRAIL and PG490 warrants further evaluation as a potential new strategy for the treatment of lung cancer.     

Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in susceptible cells by binding to death receptors 4 (DR4) and 5 (DR5). TRAIL preferentially induces apoptosis in transformed cells and the identification of mechanisms by which TRAIL-induced apoptosis can be enhanced may lead to novel cancer chemotherapeutic strategies. Here we show that reovirus infection induces apoptosis in cancer cell lines derived from human breast, lung and cervical cancers. Reovirus-induced apoptosis is mediated by TRAIL and is associated with the release of TRAIL from infected cells. Reovirus infection synergistically and specifically sensitizes cancer cell lines to killing by exogenous TRAIL. This sensitization both enhances the susceptibility of previously resistant cell lines to TRAIL-induced apoptosis and reduces the amount of TRAIL needed to kill already sensitive lines. Sensitization is not associated with a detectable change in the expression of TRAIL receptors in reovirus-infected cells. Sensitization is associated with an increase in the activity of the death receptor-associated initiator caspase, caspase 8, and is inhibited by the peptide IETD-fmk, suggesting that reovirus sensitizes cancer cells to TRAIL-induced apoptosis in a caspase 8-dependent manner. Reovirus-induced sensitization of cells to TRAIL is also associated with increased cleavage of PARP, a substrate of the effector caspases 3 and 7.     

Lipoxygenase inhibitors induce death receptor 5/TRAIL-R2 expression and sensitize malignant tumor cells to TRAIL-induced apoptosis

Lipoxygenases induce malignant tumor progression and lipoxygenase inhibitors have been considered as promising anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. Combined treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and TRAIL markedly induced apoptosis in Jurkat T-cell leukemia cells at suboptimal concentrations for each agent. The combined treatment efficiently activated caspase-3, -8 and -10, and Bid. The underling mechanism by which NDGA enhanced TRAIL-induced apoptosis was examined. NDGA did not change the expression levels of anti-apoptotic factors, Bcl-x(L), Bcl-2, cIAP-1, XIAP and survivin. The expression of death receptor-related genes was investigated and it was found that NDGA specifically up-regulated the expression of death receptor 5 (DR5) at mRNA and protein levels. Down-regulation of DR5 by small interfering RNA prevented the sensitizing effect of NDGA on TRAIL-induced apoptosis. Furthermore, NDGA sensitized prostate cancer and colorectal cancer cells to TRAIL-induced apoptosis. In contrast, NDGA neither enhanced TRAIL-induced apoptosis nor up-regulated DR5 expression in normal peripheral blood mononuclear cells. Another lipoxygenase inhibitor, AA861, also up-regulated DR5 and sensitized Jurkat and DU145 cells to TRAIL. These results indicate that lipoxygenase inhibitors augment the apoptotic efficiency of TRAIL through DR5 up-regulation in malignant tumor cells, and raise the possibility that the combination of lipoxygenase inhibitor and TRAIL is a promising strategy for malignant tumor treatment.