死亡受体Fas在TRAIL 联合三氧化二砷诱导人胃癌细胞凋亡中的作用

目的： 探讨Fas在肿瘤坏死因子相关凋亡诱导配体 (tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)与三氧化二砷 (arsenic trioxide,As2O3)联合诱导人胃癌细胞凋亡过程中的作用。方法：采用体外培养人低分化胃腺癌细胞SGC-7901,分别以Fas正义、反义寡核苷酸转染SGC-7901细胞获得Sense、Antisense细胞。以0.5 μg/ml As2O3+0.2 μg/ml TRAIL分别处理未转染细胞、Sense组细胞以及Antisense组细胞,采用MTT法检测24、48、72 h后细胞增殖情况;倒置显微镜观察联合作用48 h后细胞集落形态;DAPI染色法、流式细胞仪技术检测48 h时细胞凋亡率;Western blot法检测48 h细胞中Fas、FADD以及Parp表达情况。结果：与未转染组和Sense组细胞比较,转染Fas反义寡核苷酸后,降低了TRAIL与As2O3联合抑制细胞增殖、促进凋亡的作用 (P<0.05),48 h时效果最为明显;荧光显微镜下可见死亡细胞明显减少;Western blot结果显示细胞中Fas、FADD表达降低,Parp活化程度降低 (均P<0.05)。结论：Fas在TRAIL与As2O3联合抑制人胃癌细胞增殖、促进细胞凋亡过程中起重要作用。     

Failure of Bcl-2 to block mitochondrial dysfunction during TRAIL-induced apoptosis. Tumor necrosis-related apoptosis-inducing ligand

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis in a wide variety of tumor cells but not in normal cells. Oncogene Bcl-2 can protect cells from apoptosis induced by various stress stimuli. However, it is not clear whether Bcl-2 can regulate TRAIL-induced apoptosis. The objective of this study was to investigate whether Bcl-2 can regulate apoptosis induced by TRAIL. TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Delta psi(m)), and the redistribution of mitochondrial cytochrome c. TRAIL has no effect on Delta psi(m) and apoptosis in Jurkat cells deficient in either FADD or caspase-8, suggesting both FADD and caspase-8 are required for TRAIL signaling. Overexpression of Bcl-2 delays, but does not inhibit, TRAIL-induced Delta psi(m), cytochrome c release from mitochondria and apoptosis, whereas etoposide-induced apoptosis is blocked by Bcl-2. XIAP, cowpox virus CrmA and baculovirus p35 inhibits TRAIL-induced apoptosis. These data suggest that TRAIL can be used to kill Bcl-2 positive cells that can not be killed by other class of chemotherapeutic drugs.     

Adenovirus E1A reverses the resistance of normal primary human lung fibroblast cells to TRAIL through DR5 upregulation and caspase 8-dependent pathway

Expression of the adenovirus serotype 5 (Ad5) E1A enhances tumor cells to apoptosis by TNF-alpha, Fas-ligand and TNF-related apoptosis-inducing ligand (TRAIL). In this study, we found that E1A expression reversed the resistance of normal primary human lung fibroblast cells (P-HLF) to TRAIL-induced apoptosis. Furthermore, TRAIL dramatically induced apoptosis of P-HLF cells that expressed E1A following either infection with Ad-E1A or transfection with pcDNA3-E1A. Further results demonstrated that E1A specifically upregulated DR5 levels but had nearly no effect on the levels of DR4. E1A dramatically upregulated the exogenous TRAIL, and then increased a substantial amount of TRAIL on the surface of P-HLF cells treated with the expression vectors, both Ad-TRAIL and pIRES-EGFP-TRAIL. The dominant negative FADD mutation (FADD-DN) results revealed that the apoptosis in Ad-E1A and Ad-TRAIL coinfected P-HLF cells was completely blocked following inhibition of the death receptors-associated apoptosis-inducing molecules FADD. Moreover, the caspase 8 inhibitor (Z-IETD-FMK) could efficiently block caspase 8 activation and resulted in inhibition of caspase 3 activation and cleavage. However, The caspase 9 specific inhibitor (Z-LEHD-FMK) could not counteract the synergistic effect of TRAIL-induced apoptosis in combination with E1A, and caspase 3 activation and cleavage were not inhibited by Z-LEHD-FMK. Thus, our results suggest that adenovirus E1A sensitizes P-HLF cells to TRAIL-induced apoptosis involving DR5 upregulation and the caspase 8-dependent pathway. These findings provide the first direct evidence for molecular mechanisms of adenovirus E1A gene products to sensitize normal cells to TRAIL-mediated apoptosis.     

miR-139过表达降低急性髓系白血病TRAIL耐受性

目的:探讨miR-139过表达对急性髓系白血病肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)耐受性的影响。方法:采用浓度为100、200、300和400 ng/ml TRAIL处理人急性髓系白血病K562、HL-60细胞及相对应的多药耐药K562/A02和HL-60/ADM细胞,采用CCK8法计算IC50值,以IC50法评价急性髓系白血病细胞对TRAIL的敏感性。RT-PCR检测miR-139在 TRAIL敏感细胞和耐药细胞中的表达。采用脂质体2000将miR-139 mimics转染至TRAIL耐药细胞株,RT-PCR检测转染效果,CCK8法检测细胞活力及IC50值,流式细胞仪检测细胞凋亡率。结果:在K562、K562/A02、HL-60和HL-60/ADM细胞中,HL-60/ADM细胞对TRAIL最为敏感,而K562细胞对TRAIL的耐药性最强。与敏感细胞株HL-60/ADM相比,miR-139在耐药细胞株K562中表达显著下降。转染miR-139 mimics后,K562细胞中miR-139表达和细胞凋亡率明显升高,而细胞存活率及IC50值明显降低。结论:miR-139在TRAIL耐药细胞株K562中低表达,过表达miR-139可能通过促进TRAIL诱导的细胞凋亡降低K562细胞对TRAIL的耐受性。     

TRAIL和ACNU对实验性大鼠脑胶质瘤的治疗作用

[目的]观察TRAIL和嘧啶亚硝脲（ACNU）对实验性大鼠脑胶质瘤的治疗作用,并探讨TRAIL和ACNU联合在诱导细胞凋亡中的作用机制。[方法]制备Wistar大鼠C6细胞胶质瘤动物模型,并随机分成4组：TRAIL＋ACNU治疗组（A组）、ACNU治疗组（B组）、TRAIL治疗组（C组）、生理盐水对照组（D组）。观察动物的生存状况及肿瘤大体情况,测量肿瘤的体积、计算肿瘤的抑制率;观察TRAILR2/DR5免疫组织化学表达变化情况;电镜观察肿瘤细胞变化;流式细胞仪检测肿瘤细胞凋亡情况。[结果]A组与B组的大鼠生存较好,而C组与D组的大鼠生存差。肿瘤体积分别为A组（19.00±2.59）mm3、B组（68.76±5.56）mm3、C组（230.31±13.94）mm3、D组（238.84±10.64）mm3。除C组和D组相比无显著差异外,4组间两两比较均有显著差异（P〈0.05）。电子显微镜观察,A组和B组可见明显凋亡小体,C组与D组少见。肿瘤细胞凋亡率A组（20.38%±1.62%）大于B组（14.85%±2.41%）,差异有显著性（P〈0.05）;C组（0.44%±0.21%）与D组（0.35%±0.24%）相比较差异无显著性（P〉0.05）。[结论]ACNU引起肿瘤细胞凋亡,并可诱导大鼠脑胶质瘤细胞表达DR5,使其对TRAIL诱导的凋亡敏感,TRAIL和ACNU体内联合应用具有协同作用。     

Cytotoxic drug-induced, p53-mediated upregulation of caspase-8 in tumor cells

Apoptosis resistance is crucially involved in cancer development and progression, represents the leading cause for failure of anticancer therapy and is caused, for example, by downregulation of proapoptotic intracellular signaling molecules such as caspase-8. We found that the cytotoxic drugs methotrexate (MTX) and 5-fluorouracil (5-FU) were both able to sensitize resistant tumor cells for induction of apoptosis by p53-mediated upregulation of caspase-8. Increase in caspase-8 messenger RNA and protein expression disabled tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced proliferation and restored sensitivity toward TRAIL-induced apoptosis which was inhibited by transfection of p53 decoy oligonucleotides, p53 shRNA and caspase-8 shRNA. Upregulation of caspase-8 and sensitization toward TRAIL-induced apoptosis was found both in a broad panel of tumor cell lines with downregulated caspase-8 and in TRAIL-resistant primary tumor cells of children with acute leukemia. Taken together, we have identified caspase-8 as an important p53 target gene regulated by cytotoxic drugs. These findings highlight a new drug-induced modulation of physiological apoptosis pathways, which may be involved in successful anticancer therapy using MTX and 5-FU in leukemia and solid tumors over decades.     

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.     

AAF-cmk sensitizes tumor cells to trail-mediated apoptosis

TRAIL is a member of the tumor necrosis factor (TNF) superfamily. This cytokine is cytotoxic for a high proportion of tumor cells, but could be also toxic for normal cells. There is a need to find other agents able to potentiate the antitumor effects of this cytokine. In our study, we found that Ala-Ala-Phe-chloromethylketone (AAF-cmk) augmented cytotoxic activity of TRAIL or TNF against human leukemic cells. Flow cytometry studies and electron microscopy revealed that apoptosis was primarily responsible for this potentiation. Altogether, our studies indicate that AAF-cmk might effectively sensitize human leukemia cells to apoptosis induced by TRAIL and TNF.     

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIP_L, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIP_L expression. Downregulation of c-FLIP_L expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIP_L conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIP_L and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas.     

转染TRAIL的内皮祖细胞对人卵巢癌裸鼠皮下移植瘤的抑制作用

背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactor-relatedapoptosisinducingligand,TRAIL)有广谱的抗瘤作用,且对正常组织细胞无毒性,因此有望应用于肿瘤基因治疗。内皮祖细胞(endothelialprogenitorcells,EPCs)在体内能定向归巢于肿瘤,参与肿瘤新生血管的建立。本研究以EPC为载体,观察TRAIL转染EPCs对人卵巢上皮癌裸鼠皮下移植瘤的治疗作用。方法:用磁珠分离法从脐血中分离EPCs,并进行体外培养扩增。用脂质体将带有GFP-TRAIL基因的质粒转入EPCs(TRAIL-EPCs)。将转染后的EPC经尾静脉注入3AO卵巢癌裸鼠皮下移植瘤模型。流式细胞仪检测各组中绿色荧光蛋白(Green-Fluoroprotein,GFP)表达情况,观察各组移植瘤体积的变化,计算抑瘤率。结果:静脉注射转染TRAIL后的EPC,对卵巢上皮癌裸鼠皮下移植瘤生长具有明显抑制作用,对照组裸鼠的瘤重(0.226±0.209)g,而TRAIL细胞因子治疗组、GFP-TRAIL转染组裸小鼠的瘤重分别为(0.118±0.164)g、(0.075±0.084)g;TRAIL细胞因子组的抑瘤率为48.1%,TRAIL转染组抑瘤率为66.9%。肿瘤组织石蜡切片HE染色检查显示TRAIL细胞因子组,TRAIL转染组转对照组有更多的出血坏死区。TRAIL细胞因子组,TRAIL转染组均无明显毒副作用的表现。结论:TRAIL细胞因子和TRAIL-EPCs对人卵巢癌裸鼠皮下移植瘤均有明显的抑制作用,EPC在裸鼠皮下移植瘤模型体内有一定的导向作用,有希望成为基因治疗的载体。     

腺相关病毒携带hTERT启动子调控的TRAIL基因特异性杀伤肿瘤细胞

背景与目的:腺相关病毒(adeno-associated virus)作为载体已被广泛用于肿瘤的基因治疗研究.肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factorrelated apoptosis.inducing ligand,TRAIL)基因可迅速诱导多种肿瘤细胞的凋亡.是一个安全有效的肿瘤杀伤基因.本研究旨在构建能在肿瘤细胞内特异性表达TRAIL基因的靶向腺相关病毒,并探讨其体外抗肿瘤效应的可能机制.方法:利用肿瘤特异性启动子端粒酶逆转录酶(human telomerase reverse transcfiptase,hTERT)构建特异性杀伤肿瘤细胞的腺相关病毒载体pAAV-hTERT-TRAIL.通过与pAAV-Rc、pHelper共转染HEK293细胞包装出病毒AAV-hTERT-TRAIL.将该病毒体外转染人结肠癌SW620细胞、人肝癌HepG2细胞、人肺癌A549细胞和正常细胞NHLF、MRC5后,检测TRAIL基因的肿瘤特异性表达.MTT法检测其对细胞增殖的影响,ELISA、Western blot法以及流式细胞仪检测细胞的凋亡,并分析其体外抗肿瘤效应的可能机制.结果:成功包装出病毒AAVhTERT-TRAIL.RT-PCR、Western blot和免疫组化法均证实AAV-hTERT-TRAIL能介导TRAIL基因在肿瘤细胞内特异性表达,但在正常细胞内不表达.以100 MOI AAV-IlTERTTRAIL感染细胞96 h后,SW620、A549和HepG2细胞的增殖率分别是41.55%、44.29%、49.95%,NHLF和MRC5细胞的增殖率分别是84.59%和87.22%.Western blot检测发现AAV-hTERT-TRAIL可激活Caspase通路.流式细胞仪和ELISA方法检测证实AAV-hTERT-TRAIL可诱导细胞凋亡.结论:hTERT的存在增强了腺相关病毒所携带TRAIL基因表达的肿瘤靶向性和对正常细胞的安全性.由它调控的杀伤基因可介导肿瘤细胞特异性的细胞毒效应.     

磁性纳米颗粒介导基因治疗乳腺癌实验研究

 目的 探讨新型基因载体PEI包裹的磁性纳米颗粒polyMAG-1000介导TRAIL基因治疗乳腺癌的可行性，观察TRAIL基因转染乳腺癌细胞后的治疗作用。方法 以polyMAG-1000为基因载体，联结TRAIL质粒DNA后转染人乳腺癌细胞株MCF-7细胞，用Tunel法检测细胞的凋亡，用流式细胞仪检测细胞的凋亡率，以空载体作为阴性对照；脂质体转染TRAIL基因作阳性对照。结果 Tunel法可见MCF-7细胞经polyMAG1000和脂质体转染TRAIL基因后均可见发生凋亡的细胞，流式细胞仪检测polyMAG-1000转染的细胞凋亡率为25．11％±2．85％，脂质体转染的细胞凋亡率为18．31％±2．44％（P〈0．05）。结论 TRAlL对乳腺癌细胞MCF-7具有凋亡效应，PEI包裹的磁性纳米颗粒介导的TRAIL基因治疗在肿瘤的基因治疗中具有应用前景。     

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.     

Caspase-mediated p65 cleavage promotes TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is cytotoxic to a wide variety of transformed cells, but not to most normal cells, implying potential therapeutic value against advanced cancer. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood compared with other TNF family members. Specifically, it is not yet understood how TRAIL controls nuclear factor kappaB (NF-kappaB) activation and overcomes its anti-apoptotic effect. We explored the regulation of NF-kappaB activity by TRAIL and its role in apoptosis. TRAIL combined with IkappaBalpha-"superrepressor" induced potent apoptosis of SK-Hep1 hepatoma cells at low concentrations of TRAIL that do not independently induce apoptosis. Apoptosis by high concentrations of TRAIL was not affected by IkappaBalpha-superrepressor. Although TRAIL alone did not induce NF-kappaB activity, TRAIL combined with z-VAD significantly increased NF-kappaB activation. Analysis of the NF-kappaB activation pathway indicated that TRAIL unexpectedly induced cleavage of p65 at Asp97, which was blocked by z-VAD, accounting for all of these findings. p65 expression abrogated apoptosis and increased NF-kappaB activity in TRAIL-treated cells. Cleavage-resistant p65D97A further increased NF-kappaB activity in TRAIL-treated cells, whereas the COOH-terminal p65 fragment acted as a dominant-negative inhibitor. XIAP levels were increased by TRAIL in combination with z-VAD, whereas XIAP levels were decreased by TRAIL alone. Cleavage of p65 was also detected after FRO thyroid cancer cells were treated with TRAIL. These results suggest that TRAIL induces NF-kappaB activation, but simultaneously abrogates NF-kappaB activation by cleaving p65, and thereby inhibits the induction of anti-apoptotic proteins such as XIAP, which contributes to the strong apoptotic activity of TRAIL compared with other TNF family members.     

顺铂增加人脑胶质瘤细胞U251对TRAIL敏感性的实验研究

目的 研究顺铂在增加人脑胶质瘤细胞U251对肿瘤坏死因子相关凋亡诱导配体(TRAIL)敏感性的作用,并探讨其分子机制.方法 通过倒置荧光显微镜下观察重组腺病毒载体携带的绿色荧光蛋白的表达确定最适感染复数MOI;运用MTT法检测细胞增殖活性;倒置荧光显微镜下观察Hoechst33342染色细胞凋亡形态;PI染色后通过流式细胞术检测诱导凋亡作用,RT-PCR法检测凋亡相关基因.结果 感染后TRAIL基因的表达明显上调.顺铂增敏TRAIL组与单独组相比,有显著抑制细胞增殖作用(P<0.05),Hoechst33342染色观察有明显的核固缩,核碎裂.流式细胞术检测细胞有凋亡峰的出现,增敏组与单独组有显著性差异(P<0.05).RT-PCR检测到TRAIL、DB5、caspase-3基因上调,survivin基因下调,DB4表达无明显变化.结论 顺铂可以增加人脑胶质瘤细胞U251对TRAIL的敏感性,其分子机制可能与DR5、caspase-3基因上调,survivin基因下调有关.     

TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cells. Examining primary cells of children with untreated acute leukemia, TRAIL induced apoptosis in 50% of cells, but to our surprise attenuated spontaneous apoptosis in the remaining samples or, most importantly, even mediated proliferation. We therefore examined tumor cell lines of leukemic and nonleukemic origin with apoptosis resistance towards TRAIL because of absent Caspase-8 or dysfunctional FADD. In all cell lines tested, TRAIL treatment increased cell numbers in average to 163% within 4 days and accelerated doubling time from 24 to 19 h. TRAIL-mediated proliferation was completely abrogated by blockade of NF-kappaB activation using proteasome inhibitors or in RIP-negative, IKKgamma-negative cells or in cells overexpressing dominant-negative IkappaBalpha. Our data describe the biological significance of TRAIL-mediated activation of NF-kappaB in cancer cells resistant to TRAIL-mediated apoptosis: TRAIL leads to an increase in tumor cell count by a prosurvival and possibly mitogenic function. Given the promising therapeutic potential of TRAIL as a novel anticancer drug, TRAIL-mediated survival or proliferation of target cells may restrict its use to apoptosis-sensitive tumors.     

IG20 (MADD splice variant-5), a proapoptotic protein, interacts with DR4/DR5 and enhances TRAIL-induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC

Recently, we identified Insulinoma-Glucagonoma clone 20 (IG20) that can render cells more susceptible to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. In addition, it can slow cell proliferation, and enhance drug- and radiation-induced cell death. TNF-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in some cancer cells and render others susceptible to cotreatment with drugs and irradiation, with little or no effect on most normal cells. In this study, we investigated the potential of IG20 to enhance TRAIL-induced apoptosis and found that it can render cells more susceptible to TRAIL treatment through enhanced activation of caspases. Further, we showed that this effect can be suppressed by caspase inhibitors, p35 and CrmA, and a dominant-negative Fas-associated death domain-containing protein (DN-FADD). Results from colocalization and immunoprecipitation studies showed that IG20 can interact with TRAIL death receptors (DR), DR4 and DR5 and increase recruitment of FADD and caspase-8 into the TRAIL death-inducing signaling complex (DISC). These results indicate that IG20 is a novel protein that can enhance TRAIL-induced apoptosis by facilitating DISC formation.     

The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation

Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.     

DJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8 recruitment to FADD

DJ-1 was initially identified as an oncogene product involved in human tumorigenesis in cooperation with Ras. Increased DJ-1 expression is associated with tumorigenesis in many cancers, whereas the loss of DJ-1 function is linked to an autosomal recessive form of Parkinson's disease (PD). It has been reported that DJ-1 protects cells from TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, the mechanism by which DJ-1 is involved is still largely unknown. Here we show that DJ-1 inhibits TRAIL-induced apoptosis by blocking Fas-associated protein death domain (FADD)-mediated pro-caspase-8 activation. Wild-type DJ-1, but not the PD-associated mutant L166P, binds to FADD to inhibit the formation of the death-inducing signaling complex (DISC). DJ-1 competes with pro-caspase-8 to bind to FADD at the death effector domain, thereby repressing the recruitment and activation of pro-caspase-8 to the active form of caspase-8. Thus, our study suggests that DJ-1 protects against TRAIL-induced apoptosis through the regulation of DISC formation.