肿瘤对肿瘤坏死因子相关凋亡诱导配体的耐受机制及增敏实验的研究

肿瘤坏死因子相关凋亡诱导配体(TRAIL)是近年来发现的凋亡分子,能选择性诱导转化细胞、肿瘤细胞和病毒感染细胞发生凋亡,而对大多数正常细胞几乎没有毒性。但如果TRAIL在肿瘤中介导的凋亡信号传递受阻或者出现其他信号传导通路的激活,就可引起肿瘤对TRAIL的耐受。如果TRAIL蛋白与化疗药物或其他方法联合应用,就可以上调DR4、DR5、caspase3、caspase8或Bax等促凋亡因子的表达,或下调Bcl-XL、cFLIP等凋亡抑制因子的表达,从而克服肿瘤细胞对TRAIL的耐受或提高对TRAIL的敏感性。本文主要概述肿瘤细胞对TRAIL的耐受机制及克服耐受、增强肿瘤细胞凋亡敏感性的实验研究进展。     

肿瘤坏死因子相关凋亡诱导配体与肿瘤免疫

TRAIL是肿瘤坏死因子超家族成员,广泛表达于多种组织细胞。TRAIL与其受体结合后,可启动信号转导,选择性地诱导肿瘤细胞凋亡,而对正常细胞毒性小或无损伤。一些药物和细胞因子可协同TRAIL诱导肿瘤细胞凋亡。但肿瘤细胞也可通过表达TRAIL,产生免疫逃逸或免疫赦免,且TRAIL的作用还受到NFκB或靶细胞表达TRAIL受体类型和数量的影响。     

地榆皂苷Ⅱ抑制肿瘤细胞增殖和诱导其凋亡的作用

目的 检测地榆皂苷Ⅱ对多种肿瘤细胞的体外增殖抑制作用,研究地榆皂苷Ⅱ诱导肿瘤细胞凋亡的分子机制.方法 采用SRB法检测地榆皂苷Ⅱ对肿瘤细胞的体外增殖抑制,通过Western blot来研究地榆皂苷Ⅱ对三大凋亡通路的影响.结果 地榆皂苷Ⅱ可上调MDA-MB-231细胞中活性氧的水平,上调CHOP的表达,激活内质网应激途径;下调Bcl-2的表达,上调Bax的表达,激活线粒体凋亡途径;上调DR4、DR5的表达,激活TRAIL外源凋亡通路.结论 地榆皂苷Ⅱ通过激活三大细胞凋亡通路来抑制肿瘤细胞的增殖,促进肿瘤细胞的凋亡.     

氧化应激在TRAIL诱导Hela细胞凋亡中的作用

目的 探讨氧化应激在肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)诱导人宫颈癌Hela细胞凋亡中的作用.方法 40μg·L-1 TRAIL处理细胞后,用MTT法检测TRAIL对细胞增殖的抑制作用;用分光光度法检测细胞内还原型谷胱甘肽(GSH)、丙二醛(MDA)含量;流式细胞仪检测胞浆内的活性氧(ROS)水平;激光共聚焦测定细胞线粒体膜电位(△Ψm);蛋白质印迹法检测Bcl-2、Cyt C、DR 4、DR 5蛋白量.结果 40 μg·L-1 TRAIL对Hela细胞的生长具有显著的抑制作用;使抗氧化因子GSH含量明显下降,氧化应激产物ROS、MDA含量明显增多;到6h时△Ψm不断降低,能明显下调抗凋亡蛋白Bcl-2的表达,引起线粒体Cyt C向细胞质的释放;同时上调死亡受体DR 5的表达.而抗氧化剂N-乙酰半胱氨酸(NAC)能抑制TRAIL引起的这些变化.结论 氧化应激在TRAIL诱导的Hela细胞凋亡中起着重要作用,可能与ROS激活线粒体途径和上调DR 5的表达密切相关.     

肿瘤坏死因子相关的凋亡诱导配体（TRAIL）可溶性胞外区在大肠杆菌中的高效表达

肿瘤坏死相关凋亡诱导配体（TNF－related apoptosis-inducing ligand,TRAIL),也称为凋亡素2配体（Apoptosisin 2 Ligand,Apo2L),是肿瘤坏死因子超家族成员,能够激发肿瘤细胞选择性地发生凋亡,并对正常组织细胞几乎无毒性。TRAIL通过细胞表达受体,即死亡受体4和5（Death Receptor 4,DR4 and Death Receptor5)发挥作用,而大多数肿瘤细胞表面表达这2种受体。为了深入研究TRAIL在肿瘤治疗中的价值,我们采用RT－PCR方法克隆了TRAIL基因,并对其细胞外可溶性区域（114－281氨基酸残基）在大肠杆菌中进行了表达。重组的TRAIL可溶性蛋白占细菌总蛋白的45％以上,初步分析具有TRAIL的生物学活性。     

肿瘤坏死因子相关凋亡诱导配体在消化系统肿瘤治疗中的研究进展

肿瘤坏死因子相关凋亡诱导配体（TRAIL）可通过特异性识别肿瘤细胞死亡受体而诱导并启动多种凋亡机制,发挥抑制肿瘤的增殖、浸润、转移的作用,但TRAIL对食管癌、胃癌、大肠癌等消化系统恶性肿瘤治疗并不敏感。TRAIL可通过联合使用某些小分子化合物,或通过调节凋亡相关蛋白的表达等来提高其抑癌的能力,然而其具体作用机制尚不清楚。就肿瘤坏死因子相关凋亡诱导配体在消化系统肿瘤治疗中的研究进展进行综述。     

线粒体通路和死亡受体通路在中华眼镜蛇毒组分诱导KG1a细胞凋亡中的作用

目的研究线粒体通路和死亡受体通路在中华眼镜蛇毒组分(naja naja actra venom component,NNAVC)诱导KG1a细胞凋亡过程中的作用。方法以人急性髓系白血病细胞株KG1a细胞为研究对象,采用Annexin V-FITC/PI荧光染色法进行凋亡细胞的形态学观察,流式细胞仪检测细胞凋亡率,分光光度法检测Caspase-9、Caspase-8和Caspase-3的活性,ELISA法检测胞质内细胞色素C的表达水平,流式细胞仪分析TRAIL死亡受体(DR4、DR5)和诱骗受体(DcR1、DcR2)的改变。结果 NNAVC作用后,荧光显微镜下可见明显的凋亡细胞,细胞凋亡率随药物作用浓度的增加而升高;Caspase-9、Caspase-8和Caspase-3蛋白被激活,胞质内细胞色素C的表达明显增加;流式细胞仪检测结果显示,NNAVC具有降低死亡受体DR4和诱骗受体DcR1的表达率,而提高DR5和DcR2的表达水平的作用。结论线粒体凋亡通路和死亡受体通路均参与NNAVC诱导KG1a细胞凋亡的过程,这可能是NNAVC发挥抗白血病作用的机制之一。     

三-(2,3-二溴丙基)异氰脲酸酯通过死亡受体信号通路诱导HepG2细胞凋亡

目的探讨环境外源性污染物三-(2,3-二溴丙基)异氰脲酸酯(TBC)诱导人肝癌细胞株HepG2凋亡的死亡受体途径。方法采用MTT法、流式细胞术确定TBC对HepG2细胞的毒性作用、细胞凋亡率,Western blot法对死亡受体信号通路关键蛋白Caspase-8、FADD、CD95/FAS、DR4、DR5、PARP和TRAIL的蛋白水平表达进行检测。结果 TBC处理浓度增加(0、12.5、25和50)μg/ml对HepG2细胞的增殖抑制作用增强,凋亡峰越明显,表明DNA合成受到抑制。且细胞内PARP蛋白表达量逐渐降低,细胞内的Caspase-8、FADD、CD95/FAS、DR4、DR5和TRAIL蛋白表达量逐渐升高,呈现剂量-效应关系。结论环境外源性污染物TBC具有细胞毒性,且可以通过死亡受体途径进行诱导细胞凋亡,为其他外源性化合物的毒理学研究和早期干预提供了理论基础。     

大蒜素对TRAIL诱导SKOV-3细胞凋亡影响的作用研究

目的研究大蒜素对肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导人卵巢癌细胞SKOV-3凋亡的作用机制。方法体外培养的人卵巢癌细胞SKOV-3,以大蒜素和重组人TRAIL蛋白单独及联合作用后MTT法检测细胞的生长抑制率;流式细胞仪检测大蒜素作用后细胞死亡受体DR4、DR5表达变化;Caspase活性检测试剂盒检测大蒜素作用后细胞Caspase-3、8活性变化。结果 （1）单独应用12.5、25、50、75mg/L的大蒜素、单独应用25、50、100、200ng/ml的TRAIL蛋白以及50mg/L的大蒜素联合100ng/ml的TRAIL蛋白处理SKOV-3细胞24、48、72h后,随着药物浓度和作用时间的增加,抑制率增高。各组抑制率与对照组比较,差异有统计学意义（P〈0.05）。（2）大蒜素作用48h后SKOV-3细胞TRAIL死亡受体DR4、DR5表达上调,DR4、DR5的FI值分别由用药前的1.78、1.94升高到2.27、2.58。用药前后DR4、DR5表达变化比较有统计学意义（P〈0.05）。（3）大蒜素作用48h后,SKOV-3细胞Caspase-3、8活性明显上调,Caspase-3、8活性的OD试验组/OD空白组的比值分别是对照组的2.49、2.08倍,用药前后Caspase-3、8活性变化比较差异有统计学意义（P〈0.05）。结论 （1）SKOV-3细胞是大蒜素和TRAIL敏感细胞株。（2）大蒜素可以增强TRAIL诱导细胞凋亡作用。（3）大蒜素上调死亡受体4、5的表达,提高Caspase-3、8活性是其增强TRAIL诱导肿瘤细胞凋亡的重要机制。     

凋亡相关蛋白Caspase研究进展

细胞凋亡,又称细胞程序性死亡,是指细胞在一定的生理或病理条件下,遵循自身的程序,自己结束其生命的过程。细胞凋亡是一个主动的、信号依赖的过程,可以由许多因素所诱导,如放射线照射、毒素、药物、缺血缺氧、病毒感染等。和细胞增殖一样,细胞凋亡也是受基因调控的精确过程,其途径主要有两条,一条是通过细胞膜上的死亡受体激活cas-pase,另一条是通过胞质内的线粒体途径释放细胞凋亡因子激活caspase。     

LBY135, a novel anti‐DR5 agonistic antibody induces tumor cell–specific cytotoxic activity in human colon tumor cell lines and xenografts

TRAIL (TNF‐related apoptosis‐inducing ligand) induces apoptosis on binding to DR4 and DR5 receptors on the surface of tumor cells. These receptors are of particular interest in the development of cancer therapeutics as they preferentially mediate tumor cell apoptosis. We have generated a chimeric anti‐DR5 agonistic antibody, LBY135, from its murine parental antibody, LCR211, identified using hybridoma technology. Both LCR211 and LBY135 specifically bind to DR5 with nanomolar affinity, mimic TRAIL to induce cell death in tumor cells, and have little effect on non‐transformed cells in vitro. The anti‐DR5 antibody reduced viability in 45% of a panel of 40 human colon cancer cell lines with IC₅₀ values of 20 nM or less. In vivo, using human colorectal tumor xenograft mouse models, LCR211 induced tumor regression and showed enhanced efficacy when combined with 5‐FU. Both in vitro evaluation of ADCC (antibody‐dependent cell‐mediated cytotoxicity) and CDC (complement‐dependent cytotoxicity), and in vivo studies using a non‐functional DR5 specific antibody or SCID‐Beige mice, suggested ADCC and CDC are unlikely to be the mechanism to ablate tumors in vivo. LBY135 and LCR211 appear to mediate cell death and tumor regression mainly through apoptosis, as demonstrated by the activation of caspase 3, caspase 8, M30, and TUNEL assay. In addition, the discovery of synergy between cross‐linked LBY135 and TRAIL not only revealed the unique epitope of LBY135, but also demonstrated an additional mechanism of action for LBY135 in vivo. LBY135 demonstrates promise as a novel therapeutic for cancer treatment and is currently in Phase I clinical trials. Drug Dev Res 69: 69–82, 2008. © 2008 Wiley‐Liss, Inc.     

J7, a methyl jasmonate derivative, enhances TRAIL-mediated apoptosis through up-regulation of reactive oxygen species generation in human hepatoma HepG2 cells

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L), a member of the TNF gene superfamily, induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, some human hepatoma cells are particularly resistant to the effects of TRAIL. In this study, we show that J7, a novel methyl jasmonate analogue, sensitizes TRAIL-resistant HepG2 human hepatocarcinoma cells to TRAIL-mediated apoptosis. Our results indicate that J7 substantially enhances TRAIL-induced apoptosis, compared with treatment with either agent alone. Combined treatment with J7 and TRAIL effectively induced Bid cleavage, down-regulation of XIAP, cIAP-1 and Bcl-xL, activation of caspases, and cleavage of poly(ADP-ribose) polymerase and phopholipase γ-1. In addition, generation of reactive oxygen species (ROS) showed a significant increase in cells following exposure to J7 in a time-dependent manner. However, the cytotoxic effects induced by co-treatment with J7 and TRAIL were markedly attenuated by caspase inhibitors, indicating an important role for caspases. Administration of N-acetyl cysteine, a scavenger of ROS, also resulted in significant inhibition of apoptosis induced by combinatory treatment with J7 and TRAIL. These results support a mechanism whereby J7 plus TRAIL induces apoptosis of HepG2 human hepatoma cells through a signaling cascade involving a ROS-mediated caspase pathway.     

Down-Regulation of Survivin by Nemadipine-A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.     

Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Intrinsic, as well as acquired, resistance to chemotherapy remains a major problem in the treatment of this disease. It is, therefore, of great importance to develop new, patient-tailored, treatment strategies for colorectal cancer patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts through the pro-apoptotic DR4 and DR5 receptors in tumor cells without harming normal cells and will soon be tested in clinical trials as a novel anti-cancer agent. However, not all human colon cancer cell lines are sensitive to TRAIL due to intrinsic or acquired TRAIL-resistance. This review discusses the mechanisms and modulation of TRAIL-resistance in colon cancer cells. Cell sensitivity to TRAIL can be affected by TRAIL-receptor expression at the cell membrane, DR4/DR5 ratio and functionality of TRAIL-receptors. Additional intracellular factors leading to TRAIL-resistance affect the caspase 8/c-FLIP ratio, such as loss of caspase 8 and caspase 10 due to mutations or gene methylation, CARP-dependent degradation of active caspase 8 and changes in caspase 8 or c-FLIP expression levels. Further downstream in the TRAIL apoptotic pathway, Bax mutations, or increased expression of IAP family members, in particularly XIAP and survivin, also cause resistance. Chemotherapeutic drugs, NSAIDs, interferon-gamma and proteasome inhibitors can overcome TRAIL-resistance by acting on TRAIL-receptor expression or changing the expression of pro- or anti-apoptotic proteins.     

Diarylheptanoid hirsutenone enhances apoptotic effect of TRAIL on epithelial ovarian carcinoma cell lines via activation of death receptor and mitochondrial pathway

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) induces apoptosis in various cancer cells. Diarylheptanoids such as hirsutenone and oregonin have been shown to have anti-inflammatory and anti-tumor effects. However, it is still unknown by which mechanism diarylheptanoids induce cell death. In addition, the effect of hirsutenone on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines is unknown. To assess the apoptosis promoting effect of hirsutenone, we investigated the effect of hirsutenone on the apoptotic effect of TRAIL using the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced nuclear damage, decrease in Bid, Bcl-2 and Bcl-xL protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (8, 9 and 3) and increase in tumor suppressor p53 levels. Hirsutenone enhanced the TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. The results suggest that hirsutenone may enhance the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Hirsutenone may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

肿瘤坏死因子诱导凋亡配体（TRAIL）抗肿瘤治疗研究进展

癌症是全球死亡率最高的疾病.严重影响了癌症患者的生活质量.细胞凋亡又称程序化细胞死亡,是多细胞有机体为调控机体发育、维护内环境稳定、由基因控制的细胞主动死亡过程.许多研究表明,肿瘤坏死因子诱导凋亡配体(TRAIL)能通过与死亡受体结合选择性引起肿瘤细胞凋亡而对正常细胞无明显影响,有望成为一种新的抗肿瘤制剂.本文就近几年TRAIL抗肿瘤研究的进展作一综述.     

Concepts in the use of TRAIL/Apo2L: an emerging biotherapy for myeloma and other neoplasias

TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) is a member of the TNF superfamily of death ligands that selectively induces apoptosis in tumour cells of diverse origins. In this report, we have reviewed recent studies examining TRAIL/Apo2L-induced apoptosis in multiple myeloma (MM), a B-cell malignancy which, in spite of its initial sensitivity to steroids, cytotoxic and high-dose chemotherapy, remains incurable. Recently, we demonstrated that TRAIL/Apo2L induces apoptosis of steroid- and chemotherapy-sensitive and resistant MM cell lines. Moreover, TRAIL/Apo2L selectively induced apoptosis of patient MM tumour cells while sparing non-malignant bone marrow and peripheral blood mononuclear cells. In addition, TRAIL/Apo2L inhibited the growth of human plasmacytomas xenografted into mice. Importantly, TRAIL/Apo2L-induced apoptosis was unaffected by IL-6, a potent growth and survival factor for MM cells which, as we and others have previously shown, blocks various pro-apoptotic signals including Fas ligand, which like TRAIL/Apo2L is also a member of the TNF family of ligands. In view of the potential clinical application of TRAIL/Apo2L to the treatment of MM, we have attempted to discern intracellular mechanisms of action and resistance for TRAIL/Apo2L in MM, along with strategies to increase sensitivity and overcome resistance of MM cells to TRAIL/Apo2L. These studies demonstrated that doxorubicin, an agent which is commonly used to treat MM patients, upregulated the expression of the DR5 death-signalling TRAIL receptor and synergistically enhanced the pro-apoptotic effect of TRAIL on MM cells. Moreover, NF-κB inhibitors such as SN50 (a cell permeable inhibitor of NF-κB nuclear translocation) as well as the proteasome inhibitor PS-341, which is currently in Phase II clinical trials, also enhanced the pro-apoptotic activity of TRAIL/Apo2L in MM cells. Lastly, TRAIL/Apo2L-induced apoptosis in MM cells was dependent on caspase-8 activation and inhibited by the caspase regulatory proteins FLIP and cIAP2. These studies provide a framework for the use of TRAIL/Apo2L as a single agent or as part of combination therapy for the treatment of MM.     

Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: involvement of Ca(2+) influx

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation.