mda-7/IL-24对裸鼠肝癌移植瘤的生长抑制和促凋亡作用

目的: 探讨mda-7/IL-24对裸鼠肝癌细胞移植瘤的生长抑制和促凋亡作用及其相关机制.方法:构建携带mda-7基因的重组腺病毒载体Ad.mda-7.以HepG2细胞皮下接种建立裸鼠肝癌移植瘤模型,采用瘤内单点注射的方法分别给予Ad.GFP、Ad.mda-7和ALLN(毒胡萝卜素,N-Ac-L-L-norleucinal)+Ad.mda-7,观察瘤质量和瘤体积的变化,通过免疫组化和TUNEL法检测肿瘤组织内凋亡相关蛋白caspase-3活化、细胞增殖相关抗原ki-67和微血管密度、细胞凋亡率,并通过Western blotting检测caspase-12、caspase-3和Bax的表达.结果:Ad.mda-7治疗组和Ad.GFP对照组肿瘤体积分别为(312.6±30.2)mm3和(520.6±30.0)mm3(P<0.01),两组的肿瘤质量分别为(0.321±0.031)g和(0.534±0.030)g(P<0.01);Ad.mda-7治疗后瘤细胞的凋亡率显著高于对照组(P<0.01);Ad.mda-7可抑制肝癌组织ki-67表达、微血管密度和促进caspase-3的表达. 经 ALLN 处理的裸鼠,明显抑制Ad.mda-7对肝癌细胞的致凋亡作用(P<0.05),并且下调Ad.mda-7诱导的caspase-12、caspase-3和Bax的表达.结论:Ad.mda-7可显著抑制裸鼠肝癌移植瘤的生长和新生血管的形成,并通过内质网应激通路显著诱导肿瘤细胞的凋亡.     

腺病毒介导mda-7/IL-24基因感染对卵巢癌耐药细胞凋亡的影响

背景与目的:Mda-7/IL-24基因是一个具有选择性的诱导肿瘤细胞凋亡和细胞因子免疫调节功能双重作用的基因,在抗肿瘤基因治疗方面有很好的应用前景,本研究利用构建的mda-7/IL-24重组腺病毒感染卵巢癌耐药细胞株,了解其对卵巢癌耐药细胞的抗肿瘤作用.方法:利用腺病毒AdEasy 1载体构建含目的基因的Ad.mda-7/IL-24,感染两种卵巢癌耐药细胞株OVCAR-3和OVCAR-8/TR,Western blot法检测感染后MDA-7/IL-24蛋白表达,用Hoechst33258凋亡染色和流式细胞仪检测感染后细胞的凋亡和细胞周期改变.结果:Ad.mda-7/IL-24经测序、酶切电泳、感染后Western blot检测MDA-7蛋白表达,表明构建成功;感染Ad.mda-7后72 h内OVCAR-3细胞最高凋亡率为14.1%,OVCAR-8/TR细胞为32.4%,明显高于空载体组和未感染组.结论:成功构建了Ad.mda-7/IL-24重组腺病毒,用其感染卵巢癌耐药细胞可诱导细胞凋亡.     

δ-生育三烯酚诱导人肝癌HepG2细胞凋亡的机制研究

目的:探讨δ-生育三烯酚诱导人肝癌HepG2细胞凋亡的作用机制.方法:应用MTT法检测δ-生育三烯酚对人肝癌HepG2细胞增殖的影响,应用高内涵活细胞成像系统检测δ-生育三烯酚对人肝癌HepG2细胞凋亡率、细胞周期以及线粒体膜电位的影响,Western印迹法检测δ-生育三烯酚对人肝癌HepG2细胞凋亡相关蛋白(如caspase-3、caspase-8、caspase-9、Bcl-2、Bax、tBid和cytochrome C)表达的影响.结果:δ-生育三烯酚呈浓度依赖性地抑制肝癌HepG2细胞生长并诱导其凋亡,其机制为δ-生育三烯酚降低线粒体膜电位,并诱导cytochrome C从线粒体释放到细胞质中,调控Bcl-2家族蛋白表达(如上调Bax及tBid蛋白的表达,下调Bcl-2蛋白的表达),继而引起caspase-3、caspase-8和caspase-9的活化,最终导致肝癌 HepG2细胞凋亡.结论:δ-生育三烯酚可能通过线粒体途径及膜死亡受体途径共同诱导人肝癌细胞 HepG2凋亡.     

pIRES-IL-24对Bel-7402肝癌细胞抑制的实验研究

目的:观察白细胞介素-24(IL24)基因对肝癌细胞系Bel-7402生长的抑制作用,为肝癌的基因治疗提供理论基础.方法:将真核分泌表达载体pIRES-IL-24转染肝癌细胞系Bel-7402.RT-PCR检测IL-24基因的表达,蛋白质印迹法及ELlSA检测IL-24蛋白的表达,MTT法检测IL-24对肝癌细胞的生长抑制和杀伤作用,流式细胞仪检测细胞的凋亡和细胞周期.结果:pIRES-IL-24能够在Bel-7402中高效表达.细胞培养上清液中IL-24蛋白表达浓度为124.1 ng/mL.IL-24能明显抑制Bel-7402肝癌细胞的生长,转染后第4天抑制率为46.3%,与对照组比较,P＜0.05.IL-24促进肝癌细胞的凋亡,凋亡率41.0%,与对照组比较,P＜0.05.细胞周期分析显示,IL-24阻滞肝癌细胞在G2/M期.结论:重组表达载体pIRES-IL-24介导IL-24基因在人肝癌细胞中高效表达,可杀伤肝癌细胞Bel-7402,促进细胞增殖阻滞及诱导肿瘤细胞凋亡.     

Smac对肝癌细胞HepG2的体内外抗肿瘤效应

目的研究腺病毒Ad.smac体内外肝癌细胞HepG2的抗肿瘤效应。方法通过表达Smac的腺病毒Ad.smac和对照Ad.GFP作用于肝癌细胞HepG2,评价Ad.smac对HepG2细胞的体外杀伤作用;利用平板克隆形成实验和肿瘤细胞体内致瘤性实验,探讨Smac对HepG2细胞致瘤性的影响;通过对荷瘤裸鼠的抑瘤性实验研究Ad.smac的体内抗肿瘤作用。结果Ad.smac对肝癌细胞HepG2体外有很强的杀伤作用,50MOIAd.smac作用于Smac细胞HepG248h,细胞死亡率达58%,对照组无明显死亡细胞;感染Ad.smacHepG2细胞形成的肿瘤明显小于对照组,P=0.000;Ad.smac治疗荷HepG2细胞瘤的裸鼠,生存率由33%提高到100%。结论Ad.smac有应用于抗肿瘤治疗的前景。     

Smac/DIABLO和Survivin shRNA联合转染对大肠癌Lovo细胞凋亡的影响

背景与目的:Smac/DIABLO是一种新发现的促凋亡蛋白,通过阻滞凋亡抑制蛋白(包括Survivin),激活caspase-9和caspase-3而发挥作用.本实验研究线粒体促凋亡蛋白Smac/DIABLO和沉默凋亡抑制基因Survivin联合应用对大肠癌细胞凋亡的影响及其促凋亡机制.方法:构建Survivin的shRNA-EGFP质粒和Smac-pcDNA3.1质粒,将其用脂质体单、共转染至大肠癌Lovo细胞;Western blot检测survivin基因和Smac基因的蛋白表达;Ho-chest 33258染色观察凋亡核形态学变化,并粗略计数凋亡率;流式细胞仪PI单染测凋亡周期;caspase-3活性检测试剂盒检测caspase-3活性.结果:转染48 h后单、共转染组的Smac蛋白水平增加而Survivin蛋白水平下降;单、共转染组Hochest 33258染色可见明显的凋亡核形态学变化,凋亡率高于各对照组,且共转染组凋亡率(18.5±1.7)%高于单转染组(9.6±1.8)%、(15.0±0.3)%;Smac组G0/G1期增多为(51.0±6.2)%,而Survivin shRNA组S期细胞增多为(53.3±1.3)%(P＜0.05);caspase-3活性比Smac和Survivin shRNA共转组为(169±3)%,高于Smac组(143±5)%、Survivin shRNA组(152±6)%,且都高于各对照组(P＜0.05).结论:Smac/DIABLO和Sur-vivin shRNA协同作用激活caspase-3途径,抑制大肠癌Lovo细胞生长而促进细胞凋亡,Smac阻滞细胞周期于G0/G1期而RNA干扰Survivin后细胞周期阻滞于S期.     

线粒体促凋亡蛋白Smac/DIABLO及其与肿瘤的关系

Smac/DIABLO即第2个线粒体衍生的半胱天冬蛋白酶激活剂/低pI的IAP直接结合蛋白,是近年发现的一种线粒体促凋亡蛋白,存在于线粒体并通过拮抗凋亡抑制蛋白(IAPs)的作用激活caspase-9和caspase-3而诱导细胞凋亡,并可促进肿瘤坏死因子相关的凋亡促使配体(TRAIL)诱导的凋亡。Smac/DIABLO还可以通过其N-或C-末端促进肿瘤细胞凋亡、增强肿瘤免疫及影响肿瘤细胞周期等机制抗肿瘤,并且增加肿瘤对放化疗的敏感性。本文主要就Smac/DIABLO的结构功能、作用机制及与肿瘤发生和治疗的关系作一综述。     

人mda-7/IL-24对淋巴瘤细胞Namalwa的抑制作用

目的:研究黑素瘤分化相关基因-7(melanoma differentiation associated gene-7,mda-7,又称IL-24)对淋巴瘤细胞系Namalwa细胞的抑制作用。方法:用半定量RT-PCR方法检测10个造血系统恶性肿瘤细胞系(Namalwa、Raji、K562、NB4、U937、Ramous、CEM、KG1a、HL60、J6-1)中mda-7/IL-24的表达情况。用RT-PCR方法从活化的人外周血单个核细胞(PBMC)中克隆mda-7/IL-24编码区,构建真核表达载体pTarget-IL-24。经测序鉴定后,用脂质体法转染Namalwa细胞,筛选稳定表达细胞株。转染细胞经RT-PCR和Western blotting证实mda-7/IL-24的表达。通过MTF法、集落形成试验、流式细胞术、裸鼠体内成瘤实验来评价mda-7/IL-24对肿瘤细胞增殖、生长特性、集落形成、凋亡情况、体内致瘤能力的作用。结果:10个造血系统恶性肿瘤细胞系中未检测到mda-7/IL-24的表达;转染重组质粒pTarget-IL-24的Namalwa细胞在mRNA与蛋白水平都有mda-7/IL-24的表达;稳定表达mda-7/IL-24的Namalwa细胞增殖活力以及集落形成能力与转染空载体的对照组相比明显下降(P<0.05);但是两者的凋亡率比较无统计学意义。裸鼠体内移植瘤实验结果显示稳定表达mda-7/IL-24的Namalwa细胞株的致瘤性明显低于转染空载体的对照组(P<0.05)。结论:mda-7/IL-24对来源于Burkitt淋巴瘤的Namalwa细胞系具有明显的增殖抑制作用,为Burkitt淋巴瘤的基因治疗提供了新的思路。     

蛋白激酶B抑制线粒体Smac释放与卵巢癌顺铂耐药的关系

目的 探讨蛋白激酶B(Akt)和线粒体促凋亡蛋白(Smac)在顺铂诱导的卵巢癌细胞凋亡中的关系及Akt在卵巢癌顺铂耐药中的分子机制.方法 应用Western blot检测顺铂作用前后卵巢癌顺铂敏感细胞OV2008、A2780s和顺铂耐药细胞C13*、A2780cp中Smac含量.将Smac siRNA和Smac N7多肽分别导人0V2008和C13*细胞中,应用流式细胞仪测定细胞的凋亡率,观察稳定转染Akt2的A2780s(A2780s-AAkt2)细胞和转染Akt1/2siRNA的C13*细胞对顺铂耐药性的改变.结果顺铂能导致OV2008、A2780s细胞线粒体释放Smac,并引起细胞凋亡(P＜0.05);但在C13*和A2780cp细胞中无此反应(P＞0.05).转染Smac siRNA后的0V2008细胞,其Smac表达降低,对顺铂的耐药性增加;转染Smac N7多肽能增加C13*细胞对顺铂的敏感性;Akt2过度表达可抑制A2780s细胞Smac释放,并对顺铂产生了耐药性;应用Akt1/2 siRNA后可下调C13*细胞中Akt1/2的表达,使C13*细胞对顺铂的敏感性增加.结论 顺铂对卵巢癌细胞的杀伤在一定程度上是通过线粒体释放Smac所致;Akt抑制了线粒体Smac释放与卵巢癌化疗耐药部分相关.     

IL-24对C6细胞JNK及caspase-3表达的影响

目的:白细胞介素24(interleukin 24,IL-24)是由黑素瘤分化相关基因-7(melanoma differentiation -associated gene-7,mda-7)编码的一种分泌蛋白.以转入外源性IL-24基因的C6/IL-24细胞、空载体C6/pLXSN细胞以及亲代C6细胞为研究对象,探讨外源性IL-24体外抑制胶质瘤细胞增殖的相关机制.方法:应用MTT法体外观察IL-24对胶质瘤细胞增长的抑制作用.Western blot方法检测c-Jun氨基末端蛋白激酶(c-Jun NH2-terminal kinases,JNK)和半胱氨酸蛋白酶-3(caspase-3)蛋白的表达.探讨外源性IL-24体外抑制胶质瘤细胞增殖的相关机制.结果:C6/IL-24细胞与C6/pLXSN和亲代C6细胞相比,其吸光值降低,细胞生长曲线显示其体外增殖能力降低.与C6/pLXSN及C6细胞比较,C6/IL-24细胞的JNK和caspase-3蛋白表达增高(P＜0.05).C6/pLXSN与C6细胞比较无显著性差异(P＞0.05).结论:外源性IL-24基因可抑制C6胶质瘤细胞增殖.外源性IL-24基因对胶质瘤细胞诱导凋亡作用,与其上调并激活JNK及caspase-3表达有关.     

mda-7/IL-24 induces apoptosis in human HepG2 hepatoma cells by endoplasmic reticulum stress

mda-7/IL-24 shows tumor-suppressor activity in a broad spectrum of human cancer cells. However, the molecular mechanism by which mda-7/IL-24 induces apoptosis is not well understood and most likely involves different pathways depending on the tumor. We examined the apoptotic effect of the adenovirus-mediated mda-7/IL-24 (Ad.mda-7) on human HepG2 hepatoma cells. We found that blocking the endoplasmic reticulum (ER) stress inhibited apoptosis induced by Ad.mda-7 and down-regulated the expression of caspase-12, Bax and caspase-3. The treatment of subcutaneous tumor xenografts of HepG2 cells with Ad.mda-7 inhibited tumor growth and angiogenesis. As in the in vitro studies, we found that blocking ER stress prevented Ad.mda-7 from inducing apoptosis in liver cancer cells in vivo. Our studies suggest that Ad.mda-7 induces apoptosis of HepG2 cells mainly through activation of the ER stress pathway.     

Melanoma differentiation associated gene-7, mda-7/interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species

Mda-7/IL-24 (Ad.mda-7) is a novel cytokine gene belonging to the interleukin (IL) 10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 causes growth suppression and apoptosis in a wide spectrum of cancer cells, including prostate, without harming normal cells. We now demonstrate that Ad.mda-7 selectively induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and reactive oxygen species (ROS) production. Antioxidants (N-acetyl-L-cysteine and Tiron) and inhibitors of mitochondrial permeability transition (cyclosporine A and bongkrekic acid) inhibit Ad.mda-7-induced mitochondrial dysfunction and apoptosis. Conversely, agents augmenting ROS production (arsenic trioxide, NSC656240, and PK11195) facilitate Ad.mda-7-induced apoptosis. Ectopic expression of Bcl-2 and Bcl-x(L) inhibits mitochondrial changes, ROS production, and apoptosis providing additional support for an association between mitochondrial dysfunction and Ad.mda-7 action. These studies present definitive evidence that changes in mitochondrial function and ROS production are key components associated with selective killing of prostate cancer cells by mda-7/IL-24.     

Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human colon cancer cells.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. We determined the effect of ectopic Bcl-2 expression on TRAIL-induced apoptosis and whether the small molecule Bcl-2 inhibitor, HA14-1, could increase TRAIL sensitivity. EXPERIMENTAL DESIGN: SW480 human colon cancer cells were stably transfected with the PC3-Bcl-2 plasmid or vector alone. Cells were incubated with recombinant human TRAIL +/- HA14-1 or caspase-9 inhibitor (Z-LEHD-FMK). Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate labeling and DNA fragmentation factor 45 (DFF45) cleavage. Clonigenic survival was also studied. Caspase activation was determined by immunoblotting or colorimetric assay. The cytosolic expression of Bid, Bax, and XIAP and release of cytochrome c and Smac/DIABLO were determined by immunoblotting. RESULTS: Bcl-2 overexpression partially protected SW480 cells from a dose-dependent induction of apoptosis by TRAIL, as did a caspase-9 inhibitor, and increased their clonogenic survival. Bcl-2 overexpression attenuated TRAIL-induced cleavage of caspase-8, indicating its activation upstream and downstream of mitochondria, as well as cleavage of Bid and caspase-3. Bcl-2 inhibited TRAIL-induced Bax translocation, cytosolic release of cytochrome c and Smac/DIABLO, and the downstream cleavage of XIAP and DFF45. Coadministration of HA14-1 and TRAIL increased apoptosis in SW480/Bcl-2 cells by restoring Bax redistribution and cytochrome c release. CONCLUSIONS: Bcl-2 confers apoptosis resistance to TRAIL by inhibiting a mitochondrial amplification step and by inactivating downstream XIAP in SW480 cells. HA14-1 reversed Bcl-2-mediated TRAIL resistance, suggesting a novel strategy for increasing TRAIL sensitivity in Bcl-2-overexpressing colon cancers.     

Eradication of therapy-resistant human prostate tumors using a cancer terminator virus

Terminal prostate cancer is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or Bcl-x(L). Adenovirus-mediated delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a secreted cytokine having cancer-selective apoptosis-inducing properties, profoundly inhibits prostate cancer cell growth. However, forced overexpression of Bcl-2 or Bcl-x(L) renders prostate cancer cells resistant to Ad.mda-7. We constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV generates large quantities of MDA-7/IL-24 as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal prostate epithelial cells and parental and Bcl-2- or Bcl-x(L)-overexpressing prostate cancer cells confirmed cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 (CTV) into xenografts derived from DU-145-Bcl-x(L) cells in athymic nude mice completely eradicated not only primary tumors but also distant tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for advanced prostate cancer patients with metastatic disease.     

Ad.mda-7 (IL-24) selectively induces apoptosis in hepatocellular carcinoma cell lines, suppresses metastasis, and enhances the effect of doxorubicin on xenograft tumors

Overexpression of the melanoma differentiation associated gene-7 (MDA-7)/IL-24 in vitro generally results in the growth suppression and induction of apoptosis of diverse human tumor cells. In this study, we investigated the effects of overexpression of the MDA-7/IL-24 gene in human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Adenovirus-mediated overexpression of MDA-7 facilitated the MDA-7/IL-24-induced apoptosis and G2/M arrest in HCC cells, but not in the normal liver cell line L02, and the effect was independent of the p53 status. Inhibition of metastasis and angiogenesis was correlated with decreasing expression of STAT3, P-STAT3, MMP-2, VEGF, and TGF-beta genes, regulated by STAT3 in MHCCLM6 cells. We also showed that Ad.mda-7 combined with doxorubicin (ADM) had significantly enhanced antitumor and antimetastatic effects in vivo, accompanied by the downregulation of VEGF, MMP-2, and TGF-beta genes and the upregulation of E-cadherin genes. These data suggested that MDA-7/IL-24 induces its selective antitumor properties in HCC cells by promoting apoptosis independent of p53 status, inhibiting subcutaneous tumor growth and metastasis, and increasing the effect of chemotherapeutic agents. MDA-7/IL-24 represents a new class of cancer suppressor genes that may be useful in the targeted therapy of HCC.     

Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G(2)/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-x(L) or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.     

Regulation of the mitochondrial checkpoint in p53-mediated apoptosis confers resistance to cell death

The p53 tumor suppressor protein inhibits tumor formation, in part by inducing apoptosis, which is inhibited by anti-apoptotic Bcl-2 family members Bcl-2 and adenovirus E1B 19K. We have identified p53-apoptotic signaling events which are targeted for inhibition by E1B 19K. Apoptotic signaling by p53 induced a Bid-independent conformational change in Bax, a Bax-Bak interaction, release of cytochrome c and Smac/DIABLO from mitochondria, caspase-9 and -3 activation, cleavage of known caspase substrates, and apoptosis. When p53-dependent apoptosis was blocked by E1B 19K expression, E1B 19K bound Bak, and the Bax-Bak interaction was inhibited. Cytochrome c and Smac/DIABLO release from mitochondria was also inhibited in E1B 19K expressing cells and cells remained viable. After a prolonged p53 death stimulus, the inhibition of the mitochondrial death checkpoint by E1B 19K failed, and cytochrome c and Smac/DIABLO were released from mitochondria, and became degraded. Despite this eventual failure to inhibit the mitochondrial checkpoint, caspase-9 and -3 were not activated, and cells remained viable even upon treatment with an exogenous death stimulus. Thus, p53 induces apoptosis in part through Bax and Bak, and even an incomplete inhibition of this mitochondrial checkpoint may be sufficient to confer resistance to cell death.     

Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria

We have previously shown that Smac/DIABLO release from mitochondria appears to be the principal pathway by which TRAIL induces apoptosis of human melanoma. We report that TRAIL-induced release of Smac/DIABLO appears to be downregulated by concomitant signaling through the MEK Erk1/2 kinase pathway and that this inhibits TRAIL-induced apoptosis. Inhibition of Erk1/2 signaling by either the MEK inhibitor U0126 or a dominant-negative mutant of MKK1 markedly sensitized melanoma cells to TRAIL-induced apoptosis. The site in the apoptotic pathway acted on by U0126 appeared to be downstream of caspase-8 and Bid but upstream of caspase-3 in that the levels of proteolytic cleavage of caspase-8 and Bid by TRAIL were similar in cells with or without exposure to U0126. Caspase-3 activation and cleavage of its substrates, PARP, ICAD and XIAP, were however increased by cotreatment with U0126. This was associated with a rapid reduction in mitochondrial transmembrane potential (MMP) and increased release of Smac/DIABLO into the cytosol. Exploration of events leading to the changes in MMP revealed an increased translocation of Bax from the cytosol to mitochondria in the presence of U0126. There was also a delayed decrease in the levels of expression of Mcl-1. Bcl-2 and Bcl-X(L). Over expression of Bcl-2 blocked TRAIL-induced apoptosis in the presence of U0126. Cytochrome c appeared not to play a major role in sensitization of melanoma to TRAIL in that caspase-9 activation was not detected in most of the cell lines. These results suggest that Erk1/2 signaling may protect melanoma cells against TRAIL-induced apoptosis by inhibiting the relocation of Bax from the cytosol to mitochondria and that this may reduce TRAIL-mediated release of Smac/DIABLO and induction of apoptosis.     

Role of Smac in human leukaemic cell apoptosis and proliferation

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.