Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11

Because apoptosis is deregulated in most cancers, apoptosis-modulating approaches offer an attractive opportunity for clinical therapy of many tumors, including that of the prostate. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to treatment with Apo2 ligand (Apo2L) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), when using a nontagged, Zn-bound recombinant trimeric version that is devoid of any exogeneous sequences and therefore least likely to be immunogenic in human patients and that has been optimized for maximum efficacy and minimum toxicity. When combined with the topoisomerase I inhibitor CPT-11 (irinotecan), Apo2L/TRAIL exhibits enhanced apoptotic activity in C4-2 cells cultured in vitro as well as xenografted as tumors in vivo. Apoptosis both in vitro and in vivo was characterized by two major molecular events. First, apoptosis induction was accompanied by changes in expression levels of the Bcl-2 family genes and their products. However, whereas combination treatment applied to in vitro cell culture was characterized by a significant up-regulation and activation of Bax and down-regulation of Bcl-xL, the treatment applied to tumors induced Bak and Bcl-xS, whereas Bcl-omega and Bcl-xL were down-regulated. Because there are multiple members of the Bcl-2 family (24 members to date), these data indicate that, under different biological conditions, different proteins may be responsible for activating apoptosis and provide evidence for a differential regulation of the multidomain Bcl-2 protein-encoding genes, bax and bak. Increased Bax expression led to its activation, translocation to the mitochondria, and release of cytochrome c. In addition, this combination treatment induced apoptosis through potent activation of caspase-8 and the proapoptotic protein Bid, resulting in activation of effector caspase-3 and cleavage of its cellular target protein, poly(ADP-ribose) polymerase (PARP), events blocked by the pan-caspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk). Activation of multiple caspases and PARP cleavage were also observed in the C4-2 tumors treated with doses resulting in effective tumor control at 42 days after Apo2L/TRAIL plus CPT-11 treatment. Down-regulation of Bax by small interference (RNA) (siRNA) in C4-2 cells significantly prevented PARP cleavage and apoptosis. Strikingly, similar experiments in cells stably expressing a dominant-negative death receptor DR5 led to complete ablation of PARP cleavage and apoptosis, indicating the essential role of both mitochondrial and receptor-mediated apoptotic pathways. Our data indicate that the combined treatment of Apo2L/TRAIL and CPT-11 achieves tumor control in prostate cancer tumors through regulation of Bcl-2 family proteins and potent activation of caspases.     

Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells

Myeloma cells grow only in the bone marrow closely associated with bone,suggesting that this microenvironment provides critical signals for their growth and survival. Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor family, which binds to the ligand for receptor activator of nuclear factor kappa B and inhibits bone resorption. However, it is unclear whether OPG can also bind to other TNF family members, such as TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L), and, by inhibiting their activity, function as a survival factor for myeloma cells. In the present study MG63 osteoblast-like cells and primary bone marrow stromal cells were both shown to produce OPG, whereas human myeloma cells did not produce OPG but down-regulated release of OPG from MG63 cells. TRAIL/Apo2L induced apoptosis in myeloma cells, and this could be prevented with the addition of recombinant OPG. Medium conditioned by MG63 cells was also shown to inhibit TRAIL/Apo2L-induced apoptosis, an effect that was reversed by the addition of soluble receptor activator of nuclear factor kappa B ligand. Medium conditioned by cocultures of MG63 cells with myeloma cells had a reduced effect on TRAIL/Apo2L-induced apoptosis, reflecting the decreased concentrations of OPG in cocultures of myeloma cells with bone cells. These observations suggest that OPG may function as a paracrine survival factor in the bone marrow microenvironment in multiple myeloma.     

Cysteine 230 modulates tumor necrosis factor-related apoptosis-inducing ligand activity

Biologically active tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein is known to form a homotrimer in solution. Unexpectedly, the recombinant active human TRAIL protein purified from bacteria produced two bands (a Mr 21,000 monomer derived from the disruption of the trimer in SDS gels and a Mr 42,000 dimer) on nonreducing SDS gels. The treatment of this TRAIL protein with DTT, a reducing agent, abolished formation of the Mr 42,000 band, suggesting that the Mr 42,000 band was the result of intermolecular disulfide bridge formation. Inspection of the amino acid sequence of human TRAIL protein identified a unique cysteine residue at position 230, and subsequent site-directed mutagenesis revealed that this amino acid residue is responsible for the appearance of the Mr 42,000 dimer. The binding analysis using the TRAIL protein and a TRAIL receptor (death receptor 5) revealed that both the dimer and the trimer bind to death receptor 5 with similar affinity. Interestingly, mutation of cysteine 230 to glycine completely abolished the apoptotic activity of TRAIL protein. The disruption of the dimer in the mixture of TRAIL dimer and trimer increased the apoptotic activity slightly, suggesting that the dimer has less apoptotic activity than the trimer. Therefore, our data indicate that cysteine 230 is not only required for TRAIL function but also modulates the apoptotic activity of TRAIL by forming an intermolecular disulfide bridge.     

Apo2l/Tumor necrosis factor-related apoptosis-inducing ligand prevents breast cancer-induced bone destruction in a mouse model

Breast cancer is the most common carcinoma that metastasizes to bone. To examine the efficacy of recombinant soluble Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against breast cancer growth in bone, we established a mouse model in which MDA-MB-231 human breast cancer cells were transplanted directly into the marrow cavity of the tibiae of athymic nude mice producing osteolytic lesions in the area of injection. All vehicle-treated control animals developed large lesions that established in the marrow cavity, eroded the cortical bone, and invaded the surrounding soft tissue, as assessed by radiography, micro-computed tomography, and histology. In contrast, animals treated with recombinant soluble Apo2L/TRAIL showed significant conservation of the tibiae, with 85% reduction in osteolysis, 90% reduction in tumor burden, and no detectable soft tissue invasion. Tumor cells explanted from Apo2L/TRAIL-treated animals were significantly more resistant to the effects of Apo2L/TRAIL when compared with the cells explanted from the vehicle-treated control animals, suggesting that prolonged treatment with Apo2/TRAIL in vivo selects for a resistant phenotype. However, such resistance was readily reversed when Apo2L/TRAIL was used in combination with clinically relevant chemotherapeutic drugs, including taxol, etoposide, doxorubicin, cisplatin, or the histone deacetylase inhibitor suberoylanilide hydroxamic acid. These studies show for the first time that Apo2L/TRAIL can prevent breast cancer-induced bone destruction and highlight the potential of this ligand for the treatment of metastatic breast cancer in bone.     

Direct stimulation of apoptotic signaling by soluble Apo2l/tumor necrosis factor-related apoptosis-inducing ligand leads to selective killing of glioma cells.

Apo2 ligand tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor family that interacts with cell surface "death receptors" (DR4 and DR5) to initiate programmed cell death. Apo2L/TRAIL also binds to "decoy" receptors (DcR1 and DcR2) that can antagonize its interaction with DR4 and DR5. In recent studies, Apo2L/TRAIL has been noted to produce selective toxicity toward certain neoplastic cells versus normal cells. The decoy receptors may in part contribute to this selectivity, because they are expressed in various normal tissues but are present at low or undetectable levels in certain types of neoplastic cells. In the current study, we examined the potential therapeutic applicability of recombinant soluble Apo2L/TRAIL by investigating its effects in vitro and in vivo against a series of cell lines derived from malignant gliomas, which are often resistant to conventional treatment modalities. In cell proliferation assays, Apo2L/TRAIL produced a striking decrease in cell numbers, with a median inhibitory concentration of 30-100 ng/ml, in the TP53 wild-type high-grade glioma cell lines U87 and A172, the TP53-mutated T98G, and the TP53-deleted LN-Z308. In contrast, no significant effects were observed in non-neoplastic astrocytes at concentrations up to 3000 ng/ml. Clonogenic assays showed that exposure to Apo2L produced a time-dependent decrease in the viability of glioma-derived cell lines. This correlated with the induction of apoptosis as assessed by a terminal transferase-catalyzed in situ end-labeling assay. Pretreatment of the cells with the caspase inhibitors Acetyl-Asp-Glu-Val-L-aspartic acid aldehyde or Acetyl-Tyr-Val-Ala-Asp-chlormethylketone (200 microM) largely eliminated the effects of Apo2L/TRAIL. Administration of Apo2L/TRAIL (0.3, 1, 3, 10, and 30 mg/kg/day for 7 days via i.p. infusion) to nude mice harboring established intracranial U87 xenografts produced a significant, dose-dependent prolongation of survival versus control animals. Survival in the control group was 27 +/- 1.7 days, compared with more than 50 days in each of the treatment groups (P < 0.001). At the 30 mg/kg dose level, 100% of animals survived for 120 days without evidence of tumor, a substantial improvement in comparison with lower dose levels (P < 0.01). No overt toxicity was apparent even at the highest Apo2L dose. We conclude that soluble Apo2L/TRAIL is effective in inducing apoptosis in high-grade glioma cells in vitro. Because this ligand appears to exhibit selective cytotoxicity for glioma cells versus non-neoplastic cells in vitro and demonstrates significant activity in vivo when administered systemically in an otherwise uniformly fatal central nervous system glioma model system, Apo2L may constitute a useful therapeutic agent for these challenging tumors.     

High expression of tumor necrosis factor-related apoptosis-inducing ligand is associated with favorable ovarian cancer survival.

PURPOSE: The molecular determinants of survival in ovarian cancer are poorly understood. Using expression microarrays, we recently found that high expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene is associated with prolonged survival in advanced ovarian cancer. TRAIL has also been shown to synergize with chemotherapeutic agents to induce apoptosis in ovarian cancer cell lines. We therefore sought to confirm the association between TRAIL expression and survival in a larger group of women with ovarian cancer. EXPERIMENTAL DESIGN: TRAIL expression was measured using quantitative real-time PCR in 120 epithelial ovarian cancers (11 stage I/II, 109 stage III/IV) and 8 normal ovarian surface epithelial samples. RESULTS: Ovarian cancers demonstrated 10-fold higher mean TRAIL expression than normal ovarian epithelial samples (P < 0.001). Among ovarian cancers, high TRAIL expression was associated with prolonged survival and was 2.2-fold higher in cancers from patients who lived more than 5 years compared with patients who died within 1 year (P = 0.03). CONCLUSIONS: TRAIL expression is higher in ovarian cancers relative to normal ovarian epithelium. High TRAIL expression is associated with favorable ovarian cancer survival, which may be attributable to increased chemosensitivity of cancers that express the most TRAIL. The use of TRAIL to enhance sensitivity of ovarian cancers to therapy represents an appealing molecular therapeutic strategy worthy of further investigation.     

Targeting the tumor necrosis factor-related apoptosis-inducing ligand path in neuroblastoma

The identification of the tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) a few years ago generated considerable enthusiasm for it as a potential cancer therapeutic agent. This is because TRAIL shows potent apoptosis inducing activity in a wide spectrum of transformed cell lines but not in cell lines derived from normal tissue origin. As the details in the signal transduction pathway of TRAIL-induced apoptosis are clarified, various defects of TRAIL pathway have been identified in TRAIL resistant cancer cells. Neuroblastoma is the most common extracranial solid tumor in children and those with a poor prognosis require more sensitive therapies. Unlike other cancer cells, most neuroblastoma cell lines are resistant to TRAIL induced apoptosis and the resistance correlates with caspase 8 deficiency, which is attributed to the methylation of the gene. Interferon (IFN)-gamma induces caspase 8 expression in most neuroblastoma cell lines regardless of the methylation status but fails to sensitize most NB to TRAIL. Further analysis indicates a TRAIL receptor deficiency contributes to TRAIL resistance in NB. Multiple lesions suggest that this path may play an important role in tumorigenesis and/ or evasion from therapies. Furthermore it indicates that the clinical application of TRAIL in NB will require a multi-modality approach. Important questions remain unanswered: How does IFN-gamma induce caspase 8 and why is the induction heterogeneous? How to stimulate the caspase 8 induction in cells that fail to respond to IFN-gamma? How to target other TRAIL pathway lesions with the clinically feasible approaches?     

肿瘤坏死因子相关凋亡诱导配体对肿瘤细胞PRDXs表达影响的观察

目的:探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肿瘤细胞中过氧化氧化还原蛋白(PRDXs)表达的影响及其机制。方法:选取肿瘤细胞,设立对照组和TRAIL处理组;用实时定量PCR法和蛋白质印迹法检测PRDXs在各种肿瘤细胞中的表达;用PCR法克隆PRDX4启动子,用萤光素酶含量测定其含量。结果:与对照组相比,TRAIL处理组中PRDX1、PRDX2、PRDX3、PRDX5和PRDX6mRNA及蛋白表达差异无统计学意义,P>0.05;PRDX4mRNA和蛋白的表达显著降低,P<0.01;放线菌素D处理8h时,TRAIL处理组与对照组中PRDX4mRNA降解近50%,差异无统计学意义,P>0.05;TRAIL显著降低pPRDX4/-979的活性呈剂量依赖方式。结论:TRAIL在转录水平上下调肿瘤细胞中PRDX4基因的表达,对PRDX4mRNA的稳定性没有影响。     

Simultaneously targeting bcl-2 and Akt pathways sensitizes nasopharyngeal carcinoma to tumor necrosis factor-related apoptosis-inducing ligand

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing despite the much improved technologies in its treatment. Thus, finding more effective treatment for NPC has become an urgent priority. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Bcl-2 and Akt contribute to TRAIL resistance in some cancer cells. In this study, CNE-2 was found to be the most resistant NPC cell line to TRAIL, and whether Bcl-2 small-interfering RNA (siRNA) and phosphatidylinositol 3-kinase (PI3-K) inhibitors (LY294002 and Wortmannin) could prevent TRAIL resistance in CNE-2 was also investigated. Results showed that both Bcl-2 siRNA and PI3-K inhibitors could prevent TRAIL resistance in CNE-2. Bcl-2 siRNA sensitized CNE-2 by activating the intrinsic apoptotic pathway and PI3-K inhibitors sensitized CNE-2 by activating both intrinsic and extrinsic pathways. Further, simultaneously targeting Bcl-2 and Akt was found to be a more efficient approach to prevent TRAIL resistance in CNE-2 and this synergistic effect happened at the level of Bid downstream. At last, the combinative treatments did not enhance toxicity of TRAIL in MRC5, a human benign fibroblast cell line. This study suggests that simultaneously targeting Bcl-2 and Akt pathway might be effective in preventing TRAIL resistance of NPC cells.     

肿瘤坏死因子相关凋亡诱导配体及其受体与肺癌治疗

肿瘤坏死因子相关凋亡诱导配体(TRAIL)是近年新发现的一种凋亡诱导配体,可以激活多信号传导途径诱导肿瘤细胞凋亡,而对正常细胞没有杀伤作用.TRAIL能诱导肺肿瘤细胞发生凋亡而对正常细胞没有毒性,这为肺癌的治疗提供了新的途径.     

Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo.

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analogue of vitamin E, is a strong inducer of apoptosis, whereas alpha-tocopherol (alpha-TOH) lacks apoptogenic activity (J. Neuzil et al., FASEB J., 15: 403-415, 2001). Here we investigated the possible antineoplastic activities of alpha-TOH and alpha-TOS and further explored the potential of alpha-TOS as an antitumor agent. Using nude mice with colon cancer xenografts, we found that alpha-TOH exerted modest antitumor activity and acted by inhibiting tumor cell proliferation. In contrast, alpha-TOS showed a more profound antitumor effect, at both the level of inhibition of proliferation and induction of tumor cell apoptosis. alpha-TOS was nontoxic to normal cells and tissues, triggered apoptosis in p53(-/-) and p21(Waf1/Cip1(-/-)) cancer cells, and exerted a cooperative proapoptotic activity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) due to differences in proapoptotic signaling. Finally, alpha-TOS cooperated with tumor necrosis factor-related apoptosis-inducing ligand in suppression of tumor growth in vivo. Vitamin E succinate is thus a potent and highly specific anticancer agent and/or adjuvant of considerable therapeutic potential.     

Transduction of tumor necrosis factor-related apoptosis-inducing ligand into hematopoietic cells leads to inhibition of syngeneic tumor growth in vivo

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell death in many types of tumor and transformed cells but not in normal cells. This tumor-selective property has made TRAIL a promising candidate for the development of cancer therapy. However, safety issues are a concern because certain preparations of recombinant TRAIL protein were reported to induce toxicity in normal human hepatocytes in culture. In addition, previous studies on tumor selectivity of exogenous TRAIL protein were carried out in xenograft models, which do not directly address the tumor selectivity issue. It was not known whether exogenous or overexpression of TRAIL in a syngeneic system could induce tumor cell death while leaving normal tissue cells unharmed. Thus, the tumor selectivity of TRAIL-induced apoptosis remains to be further characterized. In our study, we established mice that overexpress TRAIL by retroviral-mediated gene transfer in bone marrow cells followed by bone marrow transplantation. Our results show that TRAIL overexpression is not toxic to normal tissues, as analyzed by hematologic and histologic analyses of tissue samples from TRAIL-transduced mice. We show for the first time that TRAIL overexpression in hematopoietic cells leads to significant inhibition of syngeneic tumor growth in certain tumor lines. This approach may be used further to identify important molecules that regulate the sensitivity of tumor cells to TRAIL-induced cell death in vivo.     

Tumor necrosis factor-related apoptosis-inducing ligand cooperates with anticancer drugs to overcome chemoresistance in antiapoptotic Bcl-2 family members expressing jurkat cells.

PURPOSE: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-x(L) overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model EXPERIMENTAL DESIGN: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-x(L) overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC(6) and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. RESULTS: Bcl-2 and Bcl-x(L) overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-x(L) Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation CONCLUSIONS: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.     

Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma

The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.     

肿瘤坏死因子相关的凋亡诱导配体对胰腺癌细胞抗肿瘤作用的研究

Objective To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL).Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry.Procaspase-8 and procaspase-3 were determined by Western blot. Results The stable overexpression of TRAIL was detected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore,co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P ＜ 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and themechanisms involved in this effect may be proapoptosis and bystander effect.     

Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and has recently been shown to exert tumoricidal activity in vivo in the absence of any observable toxicity. The signaling pathways triggered by TRAIL stimulation and the mechanisms involved in resistance against TRAIL-mediated apoptosis are still poorly defined. We show here that TRAIL-induced apoptosis involves late dissipation of mitochondrial membrane potential (delta psi(m)) and cytochrome c release. These events follow activation of caspase-8 and caspase-3 and induction of DNA fragmentation. In addition, caspase-8-deficient cells are resistant against TRAIL-induced apoptosis, and inhibition of caspase-8 but not caspase-9 prevents mitochondrial permeability transition and apoptosis. In contrast, various Bcl-2- or Bcl-xL-overexpressing tumor cell lines are sensitive to TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced mitochondrial permeability transition compared with control transfectants. This indicates that TRAIL-induced apoptosis depends on caspase-8 activation rather than on the disruption of mitochondrial integrity. Because most chemotherapeutic drugs used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery, we tested whether drug-resistant tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells overexpressing Bcl-2 or Bcl-xL become resistant to apoptosis induced by the chemotherapeutic drug etoposide. However, these cells are not protected or are only marginally protected against TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have acquired resistance to chemotherapeutic drugs by overexpression of Bcl-2 or Bcl-xL. These data will influence future treatment strategies involving TRAIL.     

Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.     

Enhanced apoptosis and tumor regression induced by a direct agonist antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2.

PURPOSE: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy.EXPERIMENTAL DESIGN: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor-expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model.RESULTS: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor-expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor.CONCLUSION: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.