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。     

Interferon-alpha-induced apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent and -independent manner

IFN-alpha regulates tumor cell growth at least through induction of apoptosis. We have recently demonstrated that IFN-alpha causes apoptosis through upregulation of TNF-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma and U266 myeloma cells. However, other cell lines such as Ramos and RPMI 8226 underwent apoptosis without any apparent involvement of TRAIL following IFN-alpha stimulation. In this study, we examined whether the IFN-alpha-induced upregulation of TRAIL is essential for the induction of apoptosis. IFN-alpha-induced early phase (48 h) of loss of DeltaPsim was substantially prevented in Daudi B lymphoma cells overexpressing the dominant-negative form of Fas-associated death domain (dnFADD) compared with vector control, whereas a late phase (72 h) of DeltaPsim was comparable to the control. The IFN-alpha-induced early phase of apoptosis was also reduced in the dnFADD-expressing cells, while the late phase of apoptosis was unaffected. IFN-alpha-induced upregulation of TRAIL protein in the dnFADD-expressing Daudi or U266 cells was comparable to their control cells, suggesting that FADD is not involved in the IFN-alpha-induced upregulation of TRAIL. Moreover, the early phase of mitochondrial depolarization was severely prevented by the presence of fusion protein of TRAIL receptor 1 and Fc portion of immunoglobulin (TRAIL-R1:Fc) and TRAIL-R2:Fc. Together, IFN-alpha induces apoptosis in a TRAIL-dependent or -independent manner, depending on the course of the apoptotic process.     

Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors

Patients with malignant gliomas have a very poor prognosis. To explore a novel and more effective approach for the treatment of malignant gliomas, a strategy that combined tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and radiation treatment (RT) was designed in this study. Plasmid pE4-GFP was constructed by including the radioinducible early growth response gene 1 (Egr-1) promoter, and it yielded the best response with fractionated RT. Plasmid pE4-TRAIL was constructed by including the Egr-1 promoter and evaluated using U251 and U87 glioma cells. In the assay of apoptosis and killing activities, pE4-TRAIL exhibited radioresponse. pE4-TRAIL combined with RT is capable of inducing cell death synergistically. The expression of TRAIL death receptors was evaluated; which may be influenced by RT. Glioma cells with wild-type p53 showed upregulated expression of death receptors, and more synergistic effects on killing activities are expected. pE4-TRAIL was transfected into the subcutaneous U251 glioma cells in nude mice by the in vivo electroporation method. In the mice treated with pE4-TRAIL and RT, apoptotic cells were detected in pathological sections, and a significant difference of tumor volumes was observed when compared with the other groups (P<0.001). Our results indicate that radioresponsive gene therapy may have great potential as a novel therapy because this therapeutic system can be spatially or temporally controlled by exogenous RT and provides specificity and safety.     

Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene and transferred them into malignant cells in vitro and tumors in vivo. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of activation of pro-caspase-8 and cleavage of poly(ADP-ribose) polymerase. The intratumoral delivery elicited tumor cell apoptosis and suppressed tumor growth. In comparison with Bax gene treatment, which is toxic to normal cells, TRAIL gene treatment caused no detectable toxicity in cultured normal fibroblasts nor in mouse hepatocytes after systemic gene delivery. Furthermore, coculture of cancer cells expressing TRAIL with those expressing green fluorescent protein (GFP) resulted in apoptosis of both cells, whereas coculture of Bax-expressing cells with GFP-expressing cells resulted in the cell death of the Bax-expressing cells only, which suggested that the transfer of the TRAIL gene resulted in bystander effects. Moreover, culture of cells with medium from TRAIL-expressing cells showed the proapoptotic activity and bystander effect of the TRAIL gene to be not transferable with medium. To further demonstrate the bystander effect of the TRAIL gene, we constructed plasmid vectors encoding GFP-TRAIL or GFP-Bik chimeric proteins. Transfection of the GFP-TRAIL gene into cancer cells resulted in the death of GFP-positive cells and their neighbors, whereas transfection of the GFP-Bik gene killed GFP-positive cells only. Finally, GFP-TRAIL genes, transfected into normal human fibroblasts or bronchial epithelial cells, did not kill such cells, whereas transfected GFP-Bik genes did. Thus, the direct transfer of the TRAIL gene led to selective killing of malignant cells with bystander effect, which suggests that the TRAIL gene could be valuable for treatment for cancers. Together, these results suggest that delivering the TRAIL gene to cancerous cells may be an alternative approach to cancer treatment.     

Sensitization of human glioblastomas to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by NF-kappaB inhibitors

Glioblastoma is the most malignant form of primary brain tumor in adults, with no effective therapy and a low survival rate. TRAIL is a member of the TNF family, which selectively induces apoptosis in certain neoplastic cells, but not normal cells. In this study, we investigated the sensitivity of 7 human glioblastoma cell lines to TRAIL and the expression in them of TRAIL receptors. TRAIL exhibited significant cytotoxicity in 5 of 7 glioma cell lines. These glioblastoma cell lines expressed TRAIL-R2, but not TRAIL-R1, R3, or R4. However, no correlation was observed between the TRAIL sensitivity and the TRAIL-R2 expression level, suggesting that there is an additional determinant of TRAIL sensitivity. Treatments with NF-kappaB inhibitors, such as LLnL, MG132, and SN50, significantly increased the sensitivity of glioma cells to TRAIL. These results suggested that activation of NF-kappaB is a protective mechanism against TRAIL-induced cell death in some glioma cells, and thus NF-kappaB inhibitors may be useful to improve the clinical treatment of glioblastoma with TRAIL.     

Role of antiapoptotic proteins in tumor necrosis factor-related apoptosis-inducing ligand and cisplatin-augmented apoptosis.

PURPOSE AND EXPERIMENTAL DESIGN: The purpose of this study was to examine the effect of combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin in human head and neck squamous cell carcinoma. HNSCC-6 cells were treated with 0.1-1 micro g/ml TRAIL and/or 1-10 micro g/ml cisplatin for 24 h. RESULTS: TRAIL alone or cisplatin alone caused minimal cytotoxicity. The combination of TRAIL and cisplatin synergistically enhanced apoptotic death, caspase-8 and caspase-3 activation, as well as poly(ADP-ribose) polymerase cleavage. However, the total cellular levels and the surface expression of TRAIL receptor proteins, such as death receptors 4 and 5 and decoy receptors 2 and 1, were not significantly changed by treatment with TRAIL and cisplatin. Interestingly, the level of the short form of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP(S)) but not the long form of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein was reduced through cleavage. Benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone a caspase-3 inhibitor, blocked the cleavage of FLIP(S) and caspase-3 activation. Overexpression of FLIP(S) protected cells from apoptotic death and FLIP(S) cleavage during treatment with TRAIL in combination with cisplatin. CONCLUSIONS: These results suggest that caspase-3 is responsible for FLIP(S) cleavage, and the cleavage of FLIP(S) is one of facilitating factors for TRAIL-induced apoptotic death.     

Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cell lines but not against normal cells. It has been hypothesized that this difference in TRAIL sensitivity between normal and transformed cells might be due to the expression of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by competition for limited amounts of TRAIL. To assess the regulation of resistance versus sensitivity to TRAIL in primary as well as transformed keratinocytes, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. Although TRAIL induced apoptosis in primary as well as transformed keratinocytes, a marked difference in sensitivity could be observed with primary keratinocytes (PK) being 5-fold less sensitive to TRAIL than transformed keratinocytes (TK). Yet both cell types exhibited similar TRAIL receptor surface expression, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity to TRAIL. Biochemical analysis of the signaling events induced by TRAIL revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly resulted in processing of caspase-8, which did not occur in TRAIL-resistant PK. These data indicate that an early block of TRAIL-induced apoptosis was present in PK compared with TK or PK treated with CHX. Interestingly, cellular FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and several other squamous cell carcinoma cell lines, decreased rapidly after treatment of PK with CHX, correlating with the increase in TRAIL sensitivity and caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFLIP(L)) in TK by transfection with a cFLIP(L) expression vector resulted in resistance to TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily regulated at the intracellular level rather than at the receptor level.     

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.     

Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through CHOP-independent DR5 upregulation

Death receptor DR5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, is a potent enhancer of TRAIL-induced apoptosis through upregulation of DR5 expression. Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNA (siRNA) attenuated curcumin plus TRAIL-induced apoptosis, showing that the critical role of DR5 in this cell death. Curcumin also induced the expression of a potential pro-apoptotic gene, C/EBP homologous protein (CHOP), both at its mRNA and protein levels. However, suppression of CHOP expression by small interfering RNA did not abrogate the curcumin-mediated induction of DR5 and the cell death induced by curcumin plus TRAIL, demonstrating that CHOP is not involved in curcumin-induced DR5 upregulation. Taken together, the present study demonstrates that curcumin enhances TRAIL-induced apoptosis by CHOP-independent upregulation of DR5.     

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression

Background  

TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression.     

Osteoprotegerin (OPG) binds with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): suppression of TRAIL-induced apoptosis in ameloblastomas

Osteoprotegerin (OPG) is a useful receptor in inhibiting Receptor Activator of NFkappaB Ligand (RANKL) in inducing osteoclastogenesis. Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) is a potent apoptosis-inducing ligand in ameloblastomas. Since OPG has been reported to bind to TRAIL as well, the effect of OPG in TRAIL's function in inducing apoptosis should also be investigated. To investigate on the expression of OPG in ameloblastomas, immuhistochemistry, immunofluorescence and Western blot were performed. From the immunohistochemistry results, we found that OPG was expressed in ameloblastoma tissues. Expression of OPG was clearly seen in AM-1 cells by immunofluorescence as well. Additionally, Western blot analysis confirmed OPG expression in ameloblastoma tissues and AM-1 cells. To investigate on the potential of TNFalpha, TRAIL and RANKL in inducing apoptosis, we performed an apoptosis assay. From the apoptosis assay, we found that TRAIL had the highest potential in inducing apoptosis compared to TNFalpha and RANKL. To investigate the binding of OPG with RANKL and TRAIL, we performed a binding assay. We noticed that OPG preferably bind with RANKL than TRAIL. However, at low levels of RANKL, marked binding of OPG with TRAIL was seen. As we suspected that the binding of OPG and TRAIL might cause the effect of TRAIL in inducing apoptosis in ameloblastomas, we combined the treatment of OPG and TRAIL in AM-1 cells. From the apoptosis assay, we found that under treatment of OPG, TRAIL's function in inducing apoptosis was suppressed. These data suggest that by binding with TRAIL, OPG suppressed TRAIL's function in inducing apoptosis in ameloblastomas.     

Role of tumor necrosis factor-related apoptosis-inducing ligand in interferon-induced apoptosis in human bladder cancer cells

Immunomodulators such as Bacillus Calmette-Guerin and interferon are clinically active in transitional cell carcinoma of the bladder, but their mechanisms of action remain unclear. Here we investigated the effects of IFNalpha on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and apoptosis in a panel of 20 human bladder cancer cell lines. Six (30%) displayed significant DNA fragmentation in response to increasing concentrations of IFNalpha (10-100,000 units/mL). In these lines IFNalpha induced early activation of caspase-8, and DNA fragmentation was blocked by a caspase-8-selective inhibitor (IETDfmk), consistent with the involvement of death receptor(s) in cell death. IFNalpha stimulated marked increases in TRAIL mRNA and protein in the majority of IFN-sensitive and IFN-resistant cell lines. A blocking anti-TRAIL antibody significantly inhibited IFN-induced DNA fragmentation in four of six IFN-sensitive cell lines, confirming that TRAIL played a direct role in cell death. Bortezomib (PS-341, Velcade), a potent TRAIL-sensitizing agent, increased sensitivity to IFNalpha in two of the IFN-resistant cell lines that produced large amounts of TRAIL in response to IFN treatment. Our data show that IFN-induced apoptosis in bladder cancer cells frequently involves autocrine TRAIL production. Combination therapy strategies aimed at overcoming TRAIL resistance may be very effective in restoring IFN sensitivity in a subset of human bladder tumors.     

Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in normal prostate epithelial cells

TRAIL is a pro-apoptotic cytokine believed to selectively kill cancer cells without harming normal ones. However, we found that in normal human prostate epithelial cells (PrEC) TRAIL is capable of inducing apoptosis as efficiently as in some tumor cell lines. At the same time, TRAIL did not cause apoptosis in several other human primary cell lines: aorta smooth muscle cells, foreskin fibroblasts, and umbilical vein endothelial cells. Compared to these primary cells, PrEC were found to contain significantly fewer TRAIL receptors DcR1 and DcR2 which are not capable of conducting the apoptotic signal. This result suggests that the unusual sensitivity of PrEC to TRAIL may result from their deficiency in anti-apoptotic decoy receptors. The protein synthesis inhibitor cycloheximide significantly enhanced TRAIL toxicity toward PrEC as measured by tetrazolium conversion but had little or no effect on other TRAIL-induced apoptotic responses. Although cycloheximide did not further accelerate the processing of caspases 3 and 8, it significantly enhanced cleavage of the caspase 3 substrate gelsolin, indicating that in PrEC a protein(s) with a short half-life may inhibit the activity of the executioner caspases toward specific substrates. As the majority of prostate cancers are derived from epithelial cells, our data suggest the possibility that TRAIL could be a useful treatment for the early stages of prostate cancer.     

Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand

Current therapies for gliomas fail to address their highly infiltrative nature. Standard treatments often leave behind microscopic neoplastic reservoirs, resulting in eventual tumor recurrence. Neural stem cells (NSCs) are capable of tracking disseminating glioma cells. To exploit this tropism to develop a therapeutic strategy that targeted tumor satellites, we inoculated human glioblastoma xenografts with tumor necrosis factor-related apoptosis-inducing ligand-secreting NSCs. This resulted in the dramatic induction of apoptosis in treated tumors and tumor satellites and was associated with significant inhibition of tumor growth. These results add credence to the potential of NSCs as therapeutically effective delivery vehicles for the treatment of intracranial glioma.