蛋白激酶C对Bcl-2、Bax调控兔脊髓空洞前状态神经元凋亡的影响

目的 探讨蛋白激酶C(PKC)对Bcl-2、Bax调控兔脊髓空洞前状态神经元凋亡的影响.方法 用成年新西兰白兔制作模型,术后1、3、7、14、21 d,用底物磷酸化法测定胞膜、胞质PKC活性;用TUNEL法和免疫组化检测脊髓神经元凋亡和Bcl-2、Bax表达.结果 Kaolin组动物胞膜PKC活性术后1d出现增加[(5.67±0.26) pmol·mg-1.min-1],7～14d达到最高水平[(13.27±3.15) pmol·mg-1·min -1],21 d开始回落[(8.85±1.56) pmol·mg -1·min -1],胞质的PKC活性则呈相反趋势;同时受试动物术后各个时点上颈髓均有神经元凋亡发生,以7～14 d最为多见[(37.75±4.36)％];Bcl-2和Bax表达均于术后1d开始增加[(7.50±1.15)％;(18.27±2.55)％],到术后7d达高峰[(17.64±4.52)％;(40.29±3.68)％],持续至14 d后下降.但后者明显强于前者.结论 脊髓空洞前状态发展过程中,出现了PKC的转位激活,其通过直接或间接途径上调Bcl-2和Bax表达,主要上调Bax,诱导神经元凋亡,参与了神经功能损害.     

Bcl-2核酶对SMMC7721细胞的促凋亡机制

目的 观察Bel-2核酶对SMMC-7721细胞的作用,探讨bcl-2抑制细胞凋亡的机制。方法 经脂质体介导的方法将PMTr-neo(正向Bcl-2核酶真核表达载体)导入SMMC 7721细胞中．细胞克隆转移扩大培养后,采用TUNEL,TRAP结合ELISA流式细胞仪,免疫组化技术检测SMMC 7721／PMTr-neo细胞增殖及细胞凋亡。结果 较对照组SMMC 7721／PMTr-neo细胞bcl-2表达水平显著下降,伴有显著细胞凋亡现象及端粒酶活性下降,结论 Bcl-2核酶可促进SMMC 7721细胞发生凋亡,并降低细胞端粒酶活性,为反义技术在肝癌治疗中应用提供理论依据。     

亚砷酸对肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响

目的探讨亚砷酸（AA）对人肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响。方法采用MTT比色法检测从作用后的BEL-7402细胞增殖抑制率,流式细胞术检测BEL-7402细胞周期及凋亡细胞,HE染色法观察凋亡细胞的形态,RT-PCR检测BEL．7402细胞的Bcl-2 mRNA,免疫组化法检测细胞的Bcl-2蛋白。结果1．0—8．0μmol／L的AA可使BEL-7402细胞增殖抑制率上升,能诱导BEL-7402细胞凋亡并阻滞细胞周期于S、G2／M期,呈剂量依赖性;8．0μmol／L的AA作用BEL-7402细胞48h后,细胞呈现明显的凋亡形态改变,其Bcl-2 mRNA及蛋白表达明显减弱。结论AA体外有抑制BEL-7402细胞增殖及诱导凋亡的作用,且呈时间、剂量依赖性,其作用机制可能与降低其Bcl-2表达有关。     

Retinoid-induced apoptosis in B-cell chronic lymphocytic leukaemia cells is mediated through caspase-3 activation and is independent of p53, the retinoic acid receptor,and differentiation

The aim of this study was to investigate the effects of all-trans retinoic acid (ATRA) on apoptosis induction, Bcl-2 family protein expression, and differentiation in B-cell chronic lymphocytic leukaemia (B-CLL) cells. ATRA induced apoptosis in all the B-CLL samples tested, and this was accompanied by a specific reduction in Bcl-2 and Mcl-1 protein expression in the apoptotic cells. In contrast, Bax, p21, and p53 expression was not altered in either the viable or apoptotic B-CLL cells, inferring that ATRA utilises a p53-independent cell death pathway. Caspase-3 activation was shown to be a prerequisite for ATRA-induced apoptosis, which was inhibited by the pan-caspase inhibitor Z-VAD-FMK and the caspase-9 inhibitor Z-LEHD-FMK. In addition, the retinoic acid receptor (RAR) antagonist AGN194310 failed to abrogate the apoptotic effects of ATRA, indicating that RAR binding was not necessary for ATRA-induced apoptosis. Furthermore, there was no evidence of ATRA-induced differentiation of the B-CLL cells in this study either in terms of altered morphology or immunophenotype. In summary these data indicate that ATRA induces apoptosis via the intrinsic apoptotic pathway, and this is independent of RAR binding, p53 activation, and cellular differentiation in B-CLL cells.     

缬沙坦对血管衰老中凋亡调控基因Bcl-2、Bax表达的影响

目的探讨缬沙坦对血管衰老中凋亡调控基因Bcl-2、Bax表达的影响。方法健康Wistar大鼠分为青年组、衰老组及缬沙坦组，测定血浆丙二醛（MDA）、超氧化物歧化酶（SOD）水平，同时采用恒速注人流体方法测定各组大鼠颈动脉血管的顺应性，利用免疫组织化学染色法、RT—PCR法和Western印迹法分析各组大鼠凋亡调控基因Bcl-2、Bax的mRNA及蛋白表达水平。结果与衰老组相比，缬沙坦组MDA浓度显著降低（P〈0．05），SOD浓度显著升高（P〈0．05），颈动脉血管的顺应性增高，其中弹性面积有显著性差异（P〈0．05），Bcl-2的mRNA及蛋白表达水平明显增高（P〈0．05），Bax的mRNA及蛋白表达水平降低（P〈0．05）。结论血管衰老有其特征性生理改变，Bcl-2、Bax的mRNA及蛋白表达的失衡可能是血管衰老的重要分子机制之一，缬沙坦有一定的逆转血管衰老的作用。     

Cyclin E but not bcl-2, bax or mcl-1 is differentially expressed in ZAP 70-positive and ZAP 70-negative B-CLL cells

The clinical course of chronic lymphocytic leukemia is variable. While some patients have indolent disease, others require aggressive treatment within a short time after diagnosis. Differences in the expression of proteins regulating cell cycle and apoptosis may be responsible for the heterogeneous course of the disease. Recently, protein ZAP 70 [zeta-chain (T-cell receptor) associated protein kinase 70 kDa] has been found to be differentially expressed within two biologic subgroups, characterized by the presence or absence of somatic mutations in specific immunoglobulin heavy-chain variable region genes. In the present work, we analyzed highly purified B-CLL cells from 60 patients for ZAP 70 expression and the expression of cyclin E, bcl-2, bax, and mcl-1 as well as the ratios of bcl-2/bax and mcl-1/bax. The results indicate that cyclin E is expressed significantly higher in ZAP 70-positive as in ZAP 70-negative samples. We did not observe significant differences within the expression of Bcl-2 family member proteins. We conclude that higher cyclin E expression in samples of ZAP 70-positive patients may reflect a larger proliferating compartment in vivo compared to ZAP 70-negative patients and that cyclin E may add prognostic information in this context for patients with B-CLL.     

Etodolac inhibits EBER expression and induces Bcl-2-regulated apoptosis in Burkitt's lymphoma cells

Cyclooxygenase-2 (COX-2) is reported to be an important cellular target for therapy in malignancies. The growth inhibitory effects of COX-2 inhibitors on malignancies have been demonstrated to be through not only COX-2 dependent, but also independent mechanisms. In this study, we showed that etodolac, COX-2 inhibitor, induced apoptosis via COX-2 independent pathway, and investigated the molecular details of etodolac-induced apoptosis in Burkitt's lymphoma cells. In Daudi and Raji Burkitt's lymphoma cell lines, which expressed no COX-2 enzyme, etodolac more strongly induced apoptosis compared to meloxicam. Moreover, etodolac did not induce apoptosis to normal B-lymphocytes. For the pathway of etodolac-induced apoptosis, reduction of anti-apoptotic bcl-2 mRNA and Bcl-2 protein, activation of Caspase-9 and -3, down-regulation of caspase inhibitors, c-IAP-1 and Survivin were involved. Moreover, EBER-1 and -2 expression in Epstein-Barr virus positive Daudi and Raji cells were reduced to result in down-regulation of Bcl-2 by treatment with etodolac. It has been reported that etodolac has stereoisomers, R- and S-etodolac. We found that racemate of etodolac more strongly induced apoptosis in Daudi and Raji cells compared to R- or S-etodolac. In conclusion, our findings indicated etodolac inhibited EBERs expression and induced apoptosis via a Bcl-2-regulated pathway. Moreover, racemate of etodolac more effectively induced apoptosis than R- and/or S-etodolac. Therefore, these activities of etodolac potentially extend to the treatment of patients with Burkitt's lymphoma resistant to chemotherapy.     

细胞凋亡在结肠腺癌组织中的变化及其相关蛋白的表达

目的:观察结肠腺癌组织细胞凋亡的变化,探讨抑制凋亡蛋白Bcl-2、Bcl-xL及Bcl-w在结肠腺癌中的作用.方法:收集14例我院手术切除结肠腺癌标本,肠镜取正常结肠黏膜组织27例,所有标本均经病理明确诊断.用TUNEL法检测其组织细胞凋亡水平,并用免疫组织化学PV法检测蛋白Bcl-2、Bcl-xL及Bcl-w的表达水平.结果:正常结肠黏膜组织的细胞凋亡指数明显高于结肠腺癌,有统计学显著性差异(t=3.35,P=0.002);结肠腺癌中Bcl-xL与Bcl-w的表达均高于正常结肠黏膜组织,有统计学差异(92.86% vs 11.11%;85.71% vs 0%,P<0.01或P<0.05);Bcl-2的表达与正常结肠黏膜组织没有统计学差异(P>0.05).结论:结肠腺癌的组织细胞凋亡明显低于正常结肠组织:结肠腺癌中抑制凋亡蛋白Bcl-xL与Bcl-w的高表达可能发挥了重要的作用.     

MicroRNA-15b 下调Bcl-2促进耐药肺癌细胞凋亡

目的 探讨微小RNA-15b （microRNA-15b,miR-15b）对人肺癌耐药细胞A549/DDP凋亡的影响.方法 采用体外转染法将MicroRNA-15b瞬时转染到A549/DDP细胞后,应用Real-time PCR检测A549/DDP细胞中MicroRNA-15b的表达情况;流式细胞仪（FCM）检测细胞凋亡变化;并用Western印迹法（Western blot）检测细胞中Bcl-2表达.结果 转染后miR-15b组的miR-15b表达水平显著增加（P〈0.05）;miR-15b组细胞凋亡率为：32.4%±5.1%,与Mock组（5.73%±1.2%）和miR-15b-Cont（6.24%±2.4%）比较,差异具有统计学意义（P〈0.05）;miR-15b组的Bcl-2表达量较对照组明显增加.结论 miR-15b可能通过下调Bcl-2的表达从而诱导A549/DDP细胞的凋亡,这可能为肺癌耐药的治疗提供新靶点.     

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.The sphingolipid ceramide has been widely shown to be an essential component of the mitochondrial phase of apoptosis progression. De novo synthesis of ceramides is catalyzed by six different ceramide synthases (CerS1–6), also referred to as “sphinganine N-acyl-transferases,” which generate ceramides with distinct fatty-acid chain lengths. Upon apoptosis induction, CerS activity is stimulated at mitochondria and mitochondria-associated membranes (MAMs), a distinct membrane compartment that links the endoplasmic reticulum to mitochondria (1). Ceramide production is necessary for Bcl2-associated X protein (Bax) to insert into the mitochondrial membranes, oligomerize, and subsequently form a pore resulting in mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release (2, 3).Reflecting the important role of ceramide in regulating apoptosis and therapy susceptibility, a role for CerSs in the pathobiology of cancer is beginning to emerge. In breast cancer, CerS2 acts as a proapoptotic protein that increases chemosensitivity and inhibits tumor growth. Consequently, reduced expression of CerS2 has been shown to be a negative prognostic indicator in breast cancer patients (4). Similarly, CerS6 promotes therapy-induced apoptosis in colon cancer cells (5, 6), head and neck squamous cell carcinoma, and lung carcinomas (7, 8), and CerS1 acts as a proapoptotic factor in multiple cancer cell lines (9, 10).Although it has been well established that CerS-mediated ceramide synthesis is an integral part of mitochondria-controlled intrinsic apoptosis signaling and is an important factor regulating tumorigenesis, specific mechanisms of CerS regulation during apoptosis are not well understood. Here, we have identified and characterized the atypical Bcl-2 family protein Bcl2-like 13 (Bcl2L13) as a CerS inhibitor with elevated expression in glioblastoma (GBM) and other solid and systemic human cancers, and potent tumorigenicity in an orthotopic GBM tumor model. A series of yeast two-hybrid (Y2H), immunoprecipitation, and molecular analyses of intrinsic apoptosis signaling revealed that Bcl2L13 blocks apoptosis in response to conventional and targeted therapy upstream of Bax activation and MOMP, at least in part by inhibiting CerS2 and CerS6 activity. Thus, our genetic and functional studies revealed, for the first time to our knowledge, that CerS activity is under the control of Bcl-2 family proteins and that the Bcl2L13–CerS2/6 signaling axis may represent a novel target to sensitize cancer cells toward extant therapies.     

Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.     

Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2

Abstract:  We have recently shown that melatonin antagonizes damage-induced apoptosis by interaction with the MT-1/MT-2 plasma membrane receptors. Here, we show that melatonin interferes with the intrinsic pathway of apoptosis at the mitochondrial level. In response to an apoptogenic stimulus, melatonin allows mitochondrial translocation of the pro-apoptotic protein Bax, but it impairs its activation/dimerization The downstream apoptotic events, i.e. cytochrome c release, caspase 9 and 3 activation and nuclear vesiculation are equally impaired, indicating that melatonin interferes with Bax activation within mitochondria. Interestingly, we found that melatonin induces a strong re-localization of Bcl-2, the main Bax antagonist to mitochondria, suggesting that Bax activation may in fact be antagonized by Bcl-2 at the mitochondrial level. Indeed, we inhibit the melatonin anti-apoptotic effect (i) by silencing Bcl-2 with small interfering RNAs, or with small-molecular inhibitors targeted at the BH3 binding pocket in Bcl-2 (i.e. the one interacting with Bax); and (ii) by inhibiting melatonin-induced Bcl-2 mitochondrial re-localization with the MT1/MT2 receptor antagonist luzindole. This evidence provides a mechanism that may explain how melatonin through interaction with the MT1/MT2 receptors, elicits a pathway that interferes with the Bcl-2 family, thus modulating the cell life/death balance.     

Silencing of Bcl-2 gene expression by siRNA transfection inhibits the protective effect of fluvastatin against cell apoptosis in human aortic endothelial cells

Objective To study the protective effect of fluvastatin,one of the HMG-CoA reductase inhibitors (statins),against oxygen radical-induced oxidative damages in human aortic endothelial cell,and the role of Bcl-2 in this protection.Methods Human aortic endothelial cells with or without Bcl-2 siRNA transfection were subjected to 1-100 nM of fluvastatin and 100 la hydrogen peroxide for 24 hours.Bcl-2 mRNA and protein expression were measured by Taqman quantitative PCR and Western blotting.Cell apoptosis was measured by normal and fluorescent microscopy and Cell Death Detection ELISA.Results In the Bcl-2-expressed cells,fluvastatin significantly reversed hydrogen peroxide-induced microscopic apoptosis and apoptotic DNA fragmentation,which were accompanied by a markedly upregulation of Bcl-2 expression by fluvastatin.However,the endothelial protection by fluvastatin was completely lost in Bcl-2 siRNA transfected cells.Conclusion Fluvastatin protects human endothelial cells against oxygen radical-induced cell apoptosis in vitro,and this protection seemed to be mediated in a Bcl-2 dependent pathway.(J Geriatr Cardil 12008;5:33-38)     

Melatonin suppresses NO-induced apoptosis via induction of Bcl-2 expression in PGT-beta immortalized pineal cells

In the present study, we investigated whether melatonin would prevent nitric oxide (NO)-induced apoptotic death of PGT-beta immortalized pineal cells. To examine the protective effect of melatonin, cytotoxicity assay, DNA fragmentation analysis, caspase-3 activity assay, and Western blotting for caspase-3 and poly(ADP-ribose) polymerase (PARP) were performed. Treatment of cells with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, was shown to induce apoptotic cell death in a dose-dependent manner, and pretreatment with melatonin (0.1 mm) attenuated the occurrence of NO-induced apoptotic cell death. DNA fragmentation in response to NO was also arrested by melatonin. Caspase-3 activity induced by NO was decreased with melatonin treatment. Furthermore, the active fragments of caspase-3 and PARP were almost completely absent following exposure to melatonin. To elucidate the protective mechanisms of action of melatonin, Western blot analyses for Bcl-2 expression and cytochrome c release were carried out. Pretreatment with melatonin (0.1 mm) induced the expression of Bcl-2 and suppressed the release of cytochrome c into the cytosol, thereby arresting NO-induced apoptotic cell death. These results suggest that the antiapoptotic effect of melatonin is associated with induction of Bcl-2 expression in PGT-beta cells, which in turn blocks caspase-3 activation and inhibits cytochrome c release into the cytosol.     

Bcl-2/bax、Bak介导的肝细胞凋亡在丁型肝炎病情重型化机制中的作用

目的　探讨Bcl 2 /bax、Bak及其介导的肝细胞凋亡在病情重型化机制中的作用。方法　采用刊TUNEL技术和免疫组化单、双标记染色 ,检测 77例丁肝病人肝组织中HDAg、bcl 2、bax和Bak表达及肝细胞凋亡情况。以HDV阴性的 67例乙型肝炎作对照。结果　Bcl 2、bax和Bak均以肝细胞浆表达为主。HDAg以肝细胞核表达为主。HDAg、bax和Bak与肝细胞凋亡表达及分布有相关性 ,四成分在各型肝炎中的表达强度有显著性差别意义 (P <0 .0 5 )。结论　HDAg、bax、Bak和肝细胞凋亡表达强度和阳性细胞分布均与肝组织炎症活动及病理损害程度相关 ,HDV感染可诱导肝细胞表达bax和Bak ,增强肝细胞凋亡 ,这一机制在丁型肝炎发病机理中可能有一定重要作用     

凋亡相关蛋白Bcl-2、Bax在消炎痛所致胃黏膜细胞凋亡中的作用

目的研究在体情况下消炎痛（Indomethacin,IND）所致胃黏膜细胞凋亡过程中Bcl-2、Bax蛋白表达的变化,以探讨其黏膜损伤机制。方法 SD大鼠胃内灌注不同剂量IND,灌胃后3h处死,采用TUNEL标记技术检测黏膜细胞凋亡;应用免疫组化方法检测Bcl-2、Bax蛋白表达的变化。结果①TUNEL标记显示对照组大鼠胃黏膜仅见少量凋亡细胞,IND使凋亡细胞数明显增加,计算机图像分析显示单位面积内阳性细胞平均像素点30mg/kg组为对照组的6.3倍（P〈0.01）,60～120mg/kg组分别为对照组的8.0、12.6和17.1倍,明显高于对照组（P〈0.01）;②免疫组化染色显示对照组大鼠Bcl-2在胃腺体部呈中等至强阳性表达,平均灰度值为113.8±9.6;而Bax蛋白仅在胃腺体及基底部呈弱阳性表达,平均灰度值为128.5±6.1。30mg/kgIND使Bcl-2表达明显减弱（P〈0.05vs对照组）,灰度值为124.8±6.3,60～90mg/kg组呈中等至弱阳性表达,灰度值分别为153.1±10.1、174.1±8.6,120mg/kg组仅见散在弱阳性细胞分布于胃腺体部,灰度值为222.3±14.6,表达均明显低于对照组（P〈0.01）,表达水平变化同细胞凋亡显著负相关（r=-0.9771,P〈0.01）;Bax表达在30～90mg/kg组呈中等阳性,明显高于对照组（P〈0.05）,灰度值分别为107.4±4.2、90.1±5.6、72.8±6.3,120mg/kg组在胃腺体及基底部见大量强阳性染色细胞分布,灰度值为69.8±6.2,表达明显高于对照组（P〈0.01）,其变化与细胞凋亡明显正相关（r=0.9725,P〈0.01）。结论 IND使凋亡抑制蛋白Bcl-2表达减弱和促凋亡蛋白Bax表达增强,从而降低了Bcl-2/Bax,导致胃黏膜细胞凋亡。     

Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis

Platelets are physiologically anucleated cells, derived from megakaryocytes, that undergo vesiculation and transformation into small particles when they are stimulated in vitro by ionomycin and other agents. Electron microscopy images suggest a similarity to apoptosis in cells with nuclei, which ends with cell disintegration and formation of apoptotic bodies. By PCR, we have demonstrated mRNA expression of bcl-2, bax_α and p53 in highly purified non-stimulated platelets. A side-scatter shift and a decrease in the Bcl-2/Bax protein ratio were observed by flow cytometry analysis after stimulation with ionomycin. The ionomycin-induced modifications were inhibited by the calpain I inhibitor calpeptin and, less effectively, by VAD-cmk, a broad-spectrum caspase inhibitor. However, caspase 3-like activity was very low, with only a twofold increase after ionomycin stimulation, as measured by the cleavage of the fluorogenic peptide substrate DEVD-AMC. Our data indicate that platelets may constitute a natural model for the analysis of cytoplasmic events in apoptosis.     

A green tea polyphenol, epigalocatechin-3-gallate, induces apoptosis of human hepatocellular carcinoma, possibly through inhibition of Bcl-2 family proteins

BACKGROUND/AIMS: A major polyphenol of green tea, epigallocatechin-3-gallate (EGCG), has previously been shown to induce cell-cycle arrest and apoptosis in various cancers. However, little is known about its effects on hepatocellular carcinomas (HCCs). METHODS: Four HCC cell lines, HLE, HepG2, HuH-7 and PLC/PRF/5, were treated with EGCG or vehicle. Cell viability was assessed by trypan blue staining and WST-8 assay. Cell-cycle, apoptosis and apoptosis-related proteins in HLE cells were evaluated by flow cytometry and Western blotting. The effect of EGCG was also studied in vivo using a xenograft model. The effect of co-treatment with EGCG and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was also assessed. RESULTS: EGCG inhibited the growth of all HCC cell lines at concentrations of 50-100 microg/ml. In HLE cells, EGCG induced apoptosis but not cell-cycle arrest and appears to have down-regulated Bcl-2alpha and Bcl-xl by inactivation of NF-kappaB. Oral administration of EGCG showed similar effects in HLE xenograft tumors. Co-treatment with EGCG and TRAIL synergistically induced apoptosis in HLE cells. CONCLUSIONS: EGCG induced apoptosis in HLE cells, both in vitro and in vivo. Moreover, it enhanced TRAIL-induced apoptosis. Therefore, EGCG treatment may be useful for improving the prognosis of HCCs.     

Chlorambucil resistance in B-cell chronic lymphocytic leukaemia is mediated through failed Bax induction and selection of high Bcl-2-expressing subclones

Our previous data have shown that high Bct-2/ Bax ratios in chronic lymphocytic leukaemia (B-CLL) correlate with in vitro apoptosis and clinical resistance. We have now monitored the in vitro viability of B-CLL cells in relation to Bcl-2 and Bax expression over a 48 h time course following exposure to chlorambucil. The results showed that Bax up-regulation was essential for chlorambucil-induced apoptosis in B-CLL cells and a 3-fold increase in expression within 4 h of exposure to drug was typically observed in sensitive cells; resistant cells failed to up-regulate Bax at all. In contrast, the constitutively high levels of Bcl-2 found in B-CLL cells were found to be down-regulated in apoptotic cells but the mean Bcl-2 expression in viable cells was increased, probably as a result of the loss of lower Bcl-2-expressing cells into the apoptotic compartment. Taken together, these data add further weight to the suggestion that Bcl-2/Bax ratios may be pivotal in determining the fate of B-CLL cells. Furthermore, the Bcl-2/Bax ratios found in apoptotic B lymphocytes were remarkably similar in the treated, untreated and normal control cells, which suggests that there is a universal Bcl-2/Bax ratio threshold for cell survival and cell death.