Expression and function of TNF-family proteins and receptors in human osteoblasts

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFalpha were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFalpha receptors and identified subpopulations with high surface-bound TNFalpha. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFalpha except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFalpha, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.     

Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis

Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.     

TRAIL受体在膀胱癌中的表达及意义

目的：了解肿瘤坏死因子相关诱导凋亡配体（TRAIL）受体在膀胱癌组织中的表达及意义。方法：采用RT－PCR及Northern blot方法检测TRAIL受体在膀胱癌组织及正常膀胱粘膜中的表达。结果：死亡受体DR4、DR5在膀胱癌组织及正常膀胱粘膜中呈强表达,候受体DcR-1在正常膀胱粘膜呈强表达,假受体DcR-2未见表达。结论：TRAIL基因在膀胱移行上皮细胞癌凋亡机制中可能发挥重要作用。     

Sensitivity of prostate cells to TRAIL-induced apoptosis increases with tumor progression: DR5 and caspase 8 are key players

BACKGROUND: As advanced prostate cancers are resistant to currently available chemotherapies, we evaluated the cytotoxic effect of TNF-related apoptosis-inducing ligand (TRAIL) and characterized the involvement of its five receptors DR4, DR5, DcR1, DcR2, and osteoprotegerin (OPG) and of the death-inducing signaling complex (DISC)-forming proteins caspase 8 and c-FLIP in prostate cell lines. METHODS: We used six prostate cell lines, each corresponding to a particular stage in prostate tumorigenesis, and analyzed TRAIL sensitivity in relation to TRAIL receptors' expression. RESULTS: TRAIL sensitivity was correlated with tumor progression and DR5 expression levels and apoptosis was exclusively mediated by DR5. DcR2 was significantly more abundant in tumor cells than in non-neoplastic ones and may contribute to partial resistance to TRAIL in some prostate tumor cells. Conversely, non-tumoral cells secreted high levels of OPG, which can protect them from apoptosis. Finally, caspase 8 expression levels were as DR5 directly correlated to TRAIL sensitivity in prostate tumor cells. CONCLUSION: TRAIL-induced apoptosis is closely related to the balanced expression of its different receptors in prostate cancer cells and their modulation could be of potential clinical value for advanced tumor treatment.     

Thapsigargin potentiates TRAIL-induced apoptosis in giant cell tumor of bone

TNF-related apoptosis-inducing ligand (TRAIL) is capable of causing apoptosis in tumor cells but not in normal cells; however, it has been shown that certain types of tumor cells are resistant to TRAIL-induced apoptosis. In this study, we examined the potentiation of TRAIL-induced apoptosis in the stromal-like tumor cells of giant cell tumor of bone (GCT). We show that both mRNA and protein of TRAIL receptors—death receptors (DR4, DR5) and decoy receptors (DcR1, DcR2) are present in GCT stromal tumor cells. However, the expression profiles in all GCT clones tested do not readily correlate with their differential sensitivity to TRAIL. To this end, we selected thapsigargin (TG), an agent known to cause perturbations in intracellular Ca²⁺ homeostasis to enhance the apoptotic action of TRAIL. When added alone, neither TRAIL nor TG induces a therapeutically important magnitude of cell death in GCT tumor cells. Interdependently, scheduled treatment of the cultures with TG followed by subsequent addition of TRAIL resulted in a significant synergistic apoptotic activity, while in contrast, no obvious augmentation was seen when TRAIL was added before TG. This effect was in accord with our observation that TG predominantly up-regulated both mRNA and protein expression of DR5, as well as DR4 mRNA while down-regulating DcR1 protein in GCT stromal-like tumor cells. Taken together, our findings suggest that TG is able to sensitize tumor cells of GCT to TRAIL-induced cell death, perhaps in part through up-regulating the death receptor DR5 and down-regulating the decoy receptor DcR1. These findings provide an additional insight into the design of new treatment modalities for patients suffering from GCT.     

TRAIL及其受体在肾脏疾病的研究进展

肿瘤坏死因子(TNF)相关的凋亡诱导配体(TRAIL)是肿瘤坏死因子超家族中重要的促凋亡细胞因子。TRAIL通过与死亡受体(DR)结合发挥促凋亡效应,同时该效应受诱骗受体(DcR)的调节,进而发挥抗凋亡、促增殖等作用。TRAIL同各类受体结合所致的病理损伤效应参与糖尿病肾病(DN)、狼疮肾炎(LN)、移植性肾病等肾脏疾病进展,并与其预后密切相关。本文将简要介绍TRAIL、TRAIL受体的结构与功能及其在肾脏疾病中的作用。     

肿瘤坏死因子相关凋亡诱导配体受体单克隆抗体对人肾癌细胞的毒性作用

目的　探讨肿瘤坏死因子相关凋亡诱导配体受体 (TRAIL R)的单克隆抗体对肾癌细胞的毒性作用和机制。方法　在人肾癌细胞系ACHN和Caki 1中 ,应用四甲基偶氮唑盐 (MTT)方法分析细胞毒效应 ,应用流式细胞术检测相关凋亡诱导配体受体 (TRAIL R)的表达 ,应用荧光染色和AnnexinV标记检测细胞凋亡的发生 ,应用比色法分析多种Caspase活性在凋亡过程中的变化。结果　抗TRAIL R2单克隆抗体 (ETR2 )对人肾癌细胞系ACHN和Caki 1都具有明显的细胞毒性 (P <0 .0 1) ,而抗TRAIL R1单克隆抗体 (ETR1)的细胞毒性不显著 (P >0 .0 5 )。TRAIL R2在两种细胞表面均高表达 (ACHN :97.9% ,Caki 1:98.7% ) ,TRAIL R1表达微弱 (ACHN :7.6% ,Caki 1:7.5 4% )。ETR2通过诱导细胞凋亡发挥细胞毒作用 ,Caspase 8, 9, 6和 3活性在凋亡过程中明显增高 (P <0 .0 1)。结论　TRAIL R2的单克隆抗体在体外可诱导肾癌细胞发生凋亡 ,其细胞毒性与TRAIL R2的表达有关 ,在凋亡过程中内源性与外源性凋亡通路均被激活。     

Causative agents of osteomyelitis induce death domain-containing TNF-related apoptosis-inducing ligand receptor expression on osteoblasts

Bacteria and their products are potent inducers of bone destruction. While inflammatory damage during conditions such as osteomyelitis is associated with increased formation and activity of bone-resorbing osteoclasts, it is likely that bone loss also results from the elimination of the cells responsible for matrix deposition. Consistent with this notion, we have previously demonstrated that bone-forming osteoblasts undergo apoptosis following bacterial challenge and that this cell death is due, at least in part, to the actions of TNF-related apoptosis-inducing ligand (TRAIL). In the present study, we demonstrate that primary osteoblasts constitutively express death domain containing TRAIL receptors. Importantly, we show that cell surface expression of the death-inducing receptors DR4 and DR5 on murine and human osteoblasts is restricted to cells infected with the principle causative agents of osteomyelitis, Staphylococcus aureus and Salmonella. In addition, we show that the robust constitutive production by osteoblasts of the decoy TRAIL receptor, OPG, is inhibited following bacterial infection. Finally, we report that while exogenous administration of TRAIL fails to activate apoptosis signaling pathways in uninfected osteoblasts, acute bacterial exposure sensitizes these cells to this ligand. Based upon these findings we suggest a model in which bacterially challenged osteoblasts express TRAIL while concomitantly decreasing the production of the decoy receptor OPG and upregulating cell surface death receptor expression. Such an increase in TRAIL bioavailability and induced sensitivity of infected osteoblasts to this ligand would result in apoptotic cell death of this bone-forming population, providing an additional mechanism underlying inflammatory bone loss during diseases such as osteomyelitis.     

Enhanced sensitivity of bladder cancer cells to tumor necrosis factor related apoptosis inducing ligand mediated apoptosis by cisplatin and carboplatin

PURPOSE: The development and acquisition of multiple drug resistance in cancer cells are a consequence of cancer chemotherapy and remain a major obstacle in treatment. Therefore, there is an obvious need for alternative approaches, such as immunotherapy and gene therapy. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of the tumor necrosis factor ligand families and it selectively induces apoptosis against cancer cells. Several cytotoxic anticancer drugs also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. We reasoned that combination treatment of cancer cells with TRAIL and drugs may overcome this resistance. We evaluated whether bladder cancer cells are sensitive to TRAIL mediated cytotoxicity and whether TRAIL may synergize with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells. MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. RESULTS: Human T24 bladder cancer line was relatively resistant to TRAIL and TRAIL was not cytotoxic against normal bladder cells. Treatment of T24 cells with TRAIL in combination with 5-fluorouracil or mitomycin C did not overcome resistance to these agents. However, treatment of T24 cells with a combination of TRAIL and cisplatin resulted in a synergistic cytotoxic effect. Synergy was also achieved in the cisplatin resistant T24 line (T24/CDDP), 2 other bladder cancer lines and 3 freshly derived bladder cancer cells. The combination of TRAIL and carboplatin resulted in a synergistic cytotoxic effect on T24 cells. However, the combination of TRAIL and trans-diamminedichloroplatinum (II) resulted in an antagonistic cytotoxic effect. The synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Treating T24 cells with cisplatin enhanced the expression of bax but not bcl-2. Incubation of T24 cells with TRAIL increased the intracellular accumulation of cisplatin. CONCLUSIONS: This study demonstrates that combination treatment of bladder cancer cells with TRAIL and cisplatin overcomes their resistance. The sensitization obtained with established cisplatin resistant and freshly isolated bladder cancer cells required low subtoxic concentrations of cisplatin, supporting the in vivo potential application of a combination of TRAIL and cisplatin for treating TRAIL resistant and cisplatin resistant bladder cancer.     

Regulation of the resistance to TRAIL-induced apoptosis as a new strategy for pancreatic cancer

BACKGROUND: Tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in a wide variety of tumor cells, but it does not cause toxicity in the majority of normal cells. Therefore, TRAIL could become a suitable agent for anticancer therapies. However, a number of tumor cell lines are known to be resistant to TRAIL-induced apoptosis. The purpose of this study was to determine the mechanisms of resistance to TRAIL in pancreatic cancer cells. METHODS: In human pancreatic cancer cell lines, the sensitivity to TRAIL-induced apoptosis was tested. The expression of TRAIL receptors (DR4, DR5, DcR1, and DcR2) and the expression of death signal-transducing proteins were investigated. In the TRAIL-resistant pancreatic cancer cells, effects of cycloheximide, a protein synthesis inhibitor, on death signal-transducing proteins were tested. Finally, the effects of the combined treatment with cycloheximide and TRAIL on the induction of apoptosis and on the expression of death signal-transducing proteins were examined. RESULTS: Pancreatic cancer cells responded to TRAIL in a different way. Resistant cell lines, AsPC-1, Suit-2, and CFPAC-1, expressed higher levels of FLIP-S protein, one of the splice variants of FLIP. Cycloheximide reduced the expression of FLIP in the resistant cells. Combined treatment with cycloheximide and TRAIL induced cleaved forms of caspases and simultaneously restored the sensitivity to TRAIL-induced apoptosis in the resistant cells. CONCLUSIONS: Pancreatic cancer cells are resistant to TRAIL-induced apoptosis via strong expression of the anti-apoptotic protein FLIP-S. Suppression of FLIP-S by cycloheximide restored sensitivity to TRAIL-induced apoptosis in resistant cancer cells. These findings may provide useful information for the development of TRAIL-based therapeutic strategies aimed at restoring the functionality of apoptotic pathways in pancreatic cancer cells.     

A tripartite anoikis-like mechanism causes early isolated islet apoptosis

BACKGROUND: This study examines the mechanisms of early isolated islet apoptosis (II-APO) and loss of functional islet mass. METHODS: Rhesus islets were isolated for transplantation, and an aliquot was used for in vitro molecular studies of II-APO. These studies used Western blotting to examine caspase activation and perinuclear envelope protein cleavage that are associated with II-APO and used immunofluorescence analysis of Annexin V and mitochondrial permeability index to examine spontaneous and tripartite anoikis-like (TRAIL) mechanism--induced II-APO. RESULTS: Caspase 6 was prominently activated in association with spontaneous II-APO, which occurred after overnight culture. In contrast, caspase 7, 8, and 9 were not activated. Cleavage of focal adhesion kinase and Lamin, substrates of caspase 6, was also evident in spontaneous II-APO. II-APO was exaggerated by the addition of the TRAIL mechanism. The TRAIL mechanism--induced II-APO was blocked by the caspase 6 inhibitor, VEID, and by the soluble fusion proteins, DR4 or DR5, which act as decoy receptors. In vivo studies in diabetic severe combined immunodeficiency disease mice showed that rhesus islets were cytoprotected by either ex vivo gene transfer of Bcl-2 or treatment of the isolated islet with VEID. CONCLUSIONS: These studies suggest 3 major mechanisms involved in II-APO: caspase 6 activation, a TRAIL-induced apoptosis pathway, and the mitochondrial-associated apoptosis pathway. Inhibition of these II-APO pathways may improve isolated islet survival and reduce functional islet mass loss, which compromises the stable reversal of diabetes.     

肿瘤坏死因子相关凋亡诱导配体受体在人肝细胞肝癌的原发灶及其门脉癌栓中的表达及意义

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体DR4、DR5、DcR1、DcR2在人肝细胞肝癌的原发灶及其门脉癌栓中的表达及意义.方法 采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)法,检测20例人肝细胞肝癌的原发灶及其门脉癌栓和20例未发生转移的原发性肝癌组织中TRAIL受体mRNA的表达水平.结果 死亡受体DR4、DB5在无转移的肝癌组织分别为(3.59±0.87)、(1.98±0.54),伴有门脉癌栓的肝癌原发灶组织(分别设定为1)及其门脉癌栓组织中的表达量分别为(0.62±0.28)、(0.31±0.12),呈递减趋势(P<0.05).诱捕受体DcR1、DcR2在伴有门脉癌栓的肝癌组织中的表达量分别为(0.29±0.04)、(0.54±0.08),显著低于无转移的肝癌组织(分别设定为1)(P<0.05).而在伴发PVTT的HCC中,门脉癌栓组织与其肝癌原发灶组织中DcR的表达量差异无统计学意义(P>0.05).DR的表达水平与肿瘤的分化程度(r=0.461,P<0.05)及门静脉浸润情况(r=0.587,P<0.05)呈显著正相关.DR的表达水平与肿瘤的大小及血清甲胎蛋白(AFP)浓度无明显相关(P>0.05).结论 TRAIL死亡受体DR的表达下调可能与肝癌的恶性进展密切相关.TRAIL 途径诱导凋亡在肝癌转移过程中可能起到重要的作用.     

白细胞介素12通过抑制survivin表达加强肿瘤坏死因子相关凋亡诱导配体诱导的肝癌细胞凋亡

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肝细胞癌(HCC)的治疗作用,以及TRAIL耐药的可能机制和逆转耐药方案。方法 采用原位杂交方法观察HCC与正常肝组织中TRAIL的表达差异。采用不同浓度的sTRAIL(可溶性)处理HCC细胞株及真核表达质粒pIRES-EGFP-sTRAIL转染HCC细胞株,观察sTRAIL的抑癌疗效。建立裸鼠肝癌模型,观察sTRAIL的体内抑癌作用。进一步,检测HCC中survivin的表达并采用反义寡核苷酸封闭治疗。最后,观测sTRAIL和IL-12联合抗癌效果。结果 肝癌组织DR表达量显著强于正常肝组织DR表达量。60例肝癌组织中54例不表达诱捕受体DcRl,25例不表达DcR2,而20例正常肝组织均表达DcR。两种．HCC细胞株中DcR1表达缺失。经sTRAIL(100ng/ml)处理24h,HCC细胞凋亡发生率约10％,而Jurkat细胞凋亡率达70％以上。体外pIRES-EGFP-sTRAIL转染对肝癌细胞杀伤作用不敏感。体内直接瘤体注射pIRES-EGFP-sTRAIL可对裸鼠肝癌无明显抑制作用。HCC高表达survivin,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12使survivin表达明显下调,显著加强TRAIL对HCC细胞的杀伤作用。结论 HCC对TRAIL诱导的凋亡有耐药现象。survivin参与HCC对TRAIL的耐药机制,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12可通过抑制siurvivin表达增强TRAIL对HCC杀癌作用。联合基因治疗(如TRAIL和IL-12基因)可能成为一种有前途HCC治疗方案。     

TRAIL受体在人骨肉瘤组织中的表达

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体在骨肉瘤细胞中的表达情况。方法 应用逆转录—聚合酶链反应(RT—PCR)对22例骨肉瘤组织标本、MG—63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞TRAILR1—R4 mRNA表达进行检测。结果 22例骨肉瘤组织标本中15例同时表达TRAILR1、-R2和-R3，4例同时表达TRAIL-R1和-R2，只表达TRAIL-R1或-R2者3例，所有标本中均未检测到TRAIL，-R4表达；MG-63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞则均检测到TRAILR1、-R2和-R3的联合表达。结论 死亡受体TRAIL-R1，R2在骨肉瘤中的普遍表达，是TRAIL诱导骨肉瘤细胞凋亡的分子基础；死亡受体和诱骗受体的差异性分布，并非TRAIL对选择性杀伤肿瘤细胞的关键性因素，可能还受其它因子调控。     

Activin A Causes Cancer Cell Aggressiveness in Esophageal Squamous Cell Carcinoma Cells

BACKGROUND: Expression of activin A is associated with lymph node metastasis and clinical stage in esophageal cancer. METHODS: To clarify the aggressive behavior of tumors with high activin A expression, we used the beta subunit of activin A to establish stable activin betaA (Act-betaA)-transfected carcinoma cells in two human esophageal carcinoma cell lines, KYSE110 and KYSE140. The biological behavior of these cells was compared with that in mock-transfected cells from the same cell lines. We focused our attention on cell growth and tumorigenesis, and proliferation and apoptosis. RESULTS: Both Act-betaA-transfected carcinoma cell lines showed a higher growth rate than the mock-transfected carcinoma cells. In an in vitro invasion assay and a xenograft analysis, the Act-betaA-transfected carcinoma cells showed far higher proliferation in vitro and a higher potency for tumorigenesis in vivo, respectively. Moreover, in an analysis of apoptosis via Fas stimulation, the Act-betaA-transfected carcinoma cells showed a higher tolerance to apoptosis compared with the mock-transfected carcinoma cells. Moreover, anti-activin-neutralizing antibody-treated squamous cell cancer cell lines inhibited their migration. CONCLUSIONS: Collectively, these data indicate that continuous high expression of activin A in esophageal carcinoma cells is not related to tumor suppression, but rather to tumor progression in vitro and in vivo. The inhibition of activin might be one of the methods to attenuate tumor aggressiveness.     

Enhanced proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand on temozolomide-resistant glioma cells

OBJECT: Death receptor targeting is an attractive approach in experimental treatment for tumors such as malignant gliomas, which are resistant to radiation and chemotherapy. Among the family of cytokines referred to as death li gands, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted clinical interest. The aim of this study was to assess whether TRAIL can be used as an adjuvant to temozolomide (TMZ) for apoptosis induction in malignant glioma cell lines. METHODS: Six human malignant glioma cell lines (A172, U87, U251, T98, U343, and U373) were exposed to human (h)TRAIL, TMZ, or an hTRAIL/TMZ combined treatment. Cell viability was assayed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and phase-contrast microscopy. Cell apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and quantified using flow cytometric analysis. The apoptosis signaling cascade was studied with Western blotting. The additive effects of hTRAIL and TMZ resulted in a significant decrease in cell viability and an increased apoptotic rate. Expression of the death receptors DR5 and DR4 in two cell lines (A172 and U251) upregulated significantly when they were used in combination hTRAIL/TMZ treatment (p < 0.05 compared with baseline control), leading to activation of caspase-8 and caspase-3 (p < 0.05 compared with baseline control) and confirming an extrinsic apoptotic pathway. A cell intrinsic pathway through mitochondrial cytochrome c was not activated. CONCLUSIONS: Based on this work, one may infer that hTRAIL should be considered as an adjuvant treatment for TMZ-resistant human malignant gliomas.     

Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo

BACKGROUND: Tumor necrosis factor related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo-2L) is a novel anticancer agent, capable of inducing apoptosis preferentially in tumor and transformed cells. TRAIL-R1/death receptor (DR)4 and TRAIL-R2/DR5 are members of the tumor necrosis factor (TNF) receptor family, and can be activated by the TRAIL. We examined the clinical potential of chemotherapeutic drugs and TRAIL for the treatment of prostate cancer. METHODS: Prostate and bladder cancer cells were exposed to chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) and TRAIL. Cell viability was measured by sodium 3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) assay; expressions of death receptors and Bcl-2 family members were measured by Western blotting, ELISA and ribonuclease protection assay. PC-3 tumor cells xenografted athymic nude mice were exposed to chemotherapeutic drugs and TRAIL, either alone or in combination, to measure tumor growth and survival of mice. Apoptosis was measured by annexin V-FITC/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. Caspase-3 activity was measured by the Western blotting and immunohistochemistry. RESULTS: TRAIL induced apoptosis with varying sensitivity. Chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) significantly augmented TRAIL-induced apoptosis in cancer cells through up-regulation of DR4, DR5, Bax, and Bak, and induction of caspase activation. Mitochondrial pathway enhanced the synergistic interactions between drugs and TRAIL. The sequential treatment of mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity, and apoptosis, inhibited angiogenesis, completely eradicated the established tumors, and enhanced survival of mice. CONCLUSIONS: Chemotherapeutic drugs can be used to enhance the therapeutic potential of TRAIL in prostate cancer.     

Ionizing radiation enhances the therapeutic potential of TRAIL in prostate cancer in vitro and in vivo: Intracellular mechanisms

BACKGROUND: We assessed the influence of sequential treatment of ionizing radiation followed by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on intracellular mechanisms of apoptosis of prostate tumor cells in vitro and in vivo. METHODS: Prostate normal and cancer cells were exposed to irradiation and TRAIL. Four- to 6-week-old athymic nude mice were injected s.c. with PC-3 tumor cells. Tumor bearing mice were exposed to irradiation and TRAIL, either alone or in combination (TRAIL after 24 hr of irradiation), and tumor growth, apoptosis, and survival of mice were examined. Expressions of death receptors, Bcl-2 family members, and caspase were measured by Western blotting, ELISA, and ribonuclease protection assay; tumor cellularity was assessed by H&E staining; inhibition of p53 was performed by RNA interference (RNAi) technology, and apoptosis was measured by annexin V/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. RESULTS: Irradiation significantly augmented TRAIL-induced apoptosis in prostate cancer cells through upregulation of DR5, Bax, and Bak, and induction of caspase activation. Dominant negative FADD and p53 siRNA inhibited the synergistic interaction between irradiation and TRAIL. The pretreatment of cells with irradiation followed by TRAIL significantly enhanced more apoptosis than single agent alone or concurrent treatment. Furthermore, irradiation sensitized TRAIL-resistant LNCaP cells to undergo apoptosis. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through activation of caspase-3, induction of Bax and Bak, and inhibition of Bcl-2, and completely eradicated the established tumors with enhanced survival of nude mice. CONCLUSION: The sequential treatment with irradiation followed by TRAIL can be used as a viable option to enhance the therapeutic potential of TRAIL in prostate cancer.