Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.     

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.     

p21 promotes ceramide-induced apoptosis and antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.     

双酚A对中国林蛙生精细胞凋亡和Bax、Bcl-2表达的影响

为探讨双酚A(BPA)对两栖动物生精细胞凋亡及相关蛋白Bax和Bcl-2表达的影响.将雄性中国林蛙(Rana chensinensis)分别暴露于10-7、10-6、10-5 mol/L BPA水体中持续3 d、5 d、7 d,取其精巢组织.用原位末端转移酶法(TUNEL)和甲基绿-派诺宁法(Methyl Green-Pyronine)检测生精细胞凋亡,用免疫组织化学方法检测生精细胞的Bax和Bcl-2表达.结果显示,各BPA处理组中国林蛙生精细胞凋亡指数(Apoptotic index,AI)均显著高于对照组,10-6 mol/L与10-7 mol/L BPA处理组生精细胞的AI差异不显著,10-5 mol/L BPA处理组生精细胞的AI与前两组相比显著增高;在同一BPA浓度处理组,生精细胞AI随处理时间的延长而增高.与对照组相比,各BPA处理组Bax表达上调,Bcl-2表达下调,差异均显著;生精细胞AI与Bax/Bcl-2表达呈正相关.这些结果提示,BPA可导致中国林蛙的生精细胞凋亡,而生精细胞凋亡的发生与Bax/Bcl-2表达比值的变化密切相关.     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) adenocarcinoma cells. The IC₅₀ values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respectively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduction in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

鱼藤酮致帕金森大鼠的脑黑质中凋亡相关蛋白Bcl-2、Bax表达改变

目的研究鱼藤酮致帕金森病（PD）大鼠中脑黑质凋亡相关蛋白Bcl-2、Bax表达的改变。方法Wistar大鼠每日颈背部皮下注射鱼藤酮2mg（kg·d）（3～6周）造模,依据所建立的评分体系记录动物行为变化,在行为学有记分并停止给鱼藤酮4、10d时,中脑黑质病理切片免疫组化染色比较黑质区域Bcl-2、Bax的表达。结果在有行为学记分4d时,记4分和8分的大鼠中脑黑质Bcl-2表达均显著减少;所有PD大鼠中脑黑质Bax表达均显著增加;Bcl-2/Bax比率均显著减少;有记分4d时,行为学记分与Bcl-2/Bax比值成负相关性。结论细胞凋亡参与了鱼藤酮帕金森模型大鼠黑质多巴胺神经细胞的损伤。     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carboni-trile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) ade-nocarcinoma cells. The IC50 values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respec-tively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduc-tion in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

脂可平对实验性早期动脉粥样硬化内皮细胞凋亡及Bax、Bcl-2表达的影响

研究脂可平对大鼠血脂、早期动脉粥样硬化血管内皮细胞凋亡及Bax、Bcl-2表达的影响。应用高脂饲料复制SD大鼠早期动脉粥样硬化模型,分别给以脂可平、辛伐他汀片混悬液灌胃,实验10周后,全自动生化分析仪、流式细胞仪定量分别检测各组血脂、主动脉内皮细胞凋亡率及Bax、Bcl-2蛋白的表达;苏木素伊红(HE)染色观察主动脉组织形态学变化。两治疗组均可降低血脂,不同程度的改善了主动脉组织病理损伤,降低内皮细胞凋亡率,调控Bax、Bcl-2蛋白表达。本实验说明脂可平可降低血脂,干预动脉粥样硬化的始动环节及其发生并从蛋白水平调控早期动脉粥样硬化内皮细胞凋亡。     

Bcl-2和Bax在吗啡依赖雄性大鼠生殖细胞中的表达

目的：探讨B细胞淋巴瘤/白血病-2和人Bcl-2相关x蛋白（Bcl-2、Bax）在吗啡依赖大鼠睾丸生殖细胞中的表达及细胞凋亡可能机制,为治疗阿片类毒品造成的男性性功能减退提供理论依据。方法：以递增法每日给予雄性大鼠皮下注射盐酸吗啡针剂,建立吗啡依赖组。空白对照组注射等量生理盐水。实验成功后将两组大鼠睾丸组织作常规HE染色和免疫组化染色。结果：吗啡依赖组大鼠生精管壁细胞明显地出现上皮层次减少,仅有2~3层,细胞排列疏松,界限模糊,精子细胞和精子数目减少,并发现曲细精管腔内有脱落的生精细胞;免疫组化结果：吗啡依赖组大鼠生殖细胞中bcl-2的阳性表达率明显低于对照组（P〈0.01）,而生殖细胞中bax蛋白的阳性表达率明显高于对照组（P〈0.01）。结论：吗啡依赖可造成雄性大鼠生殖细胞凋亡数量显著增加,其机制可能是通过下调抑凋亡因子Bcl-2,上调促凋亡因子Bax,促进生殖细胞凋亡来实现的。     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

Resveratrol disrupts peroxynitrite-triggered mitochondrial apoptotic pathway: a role for Bcl-2

Resveratrol (3,4′,5-trihydroxystilbene) is a phytochemical believed to be partly responsible for the cardioprotective effects of red wine due to its numerous biological activities. Here, we studied biochemical pathways underlying peroxynitrite-mediated apoptosis in endothelial cells and potential mechanisms responsible for resveratrol cytoprotective action. Peroxynitrite triggered endothelial cell apoptosis through caspases-8, -9 and -3 activation implying both mitochondrial and death receptor apoptotic pathways. Resveratrol was able to prevent peroxynitrite-induced caspases-3 and -9 activation, but not caspase-8 activation. Additionally, peroxynitrite increased intracellular levels of Bax without affecting those of Bcl-2, increasing consequently the Bax/Bcl-2 ratio. This ratio decreased when cells where pre-incubated with 10 and 50 μM resveratrol, mainly due to resveratrol ability per se to increase Bcl-2 intracellular levels without affecting Bax intracellular levels. These results propose an additional mechanism whereby resveratrol may exert its cardioprotective effects and suggest a key role for Bcl-2 in the resveratrol anti-apoptotic action, especially in disrupting peroxynitrite-triggered mitochondrial pathway.     

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.     

Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells

Many laboratory studies and epidemiological observations confirm that nematodes prevent some immune-mediated diseases. The development of immunologically well-defined laboratory models of intestinal nematode infection has allowed significant advances to be made in understanding the immunological basis of effector mechanisms operating during infection under controlled laboratory conditions. The Heligmosomoides polygyrus- mouse system is used for studies of parasite immunomodulation. H. polygyrus causes a chronic, asymptomatic intestinal infection and effectively maintains both local and systemic tolerance to reduce allergic and autoimmune inflammation. However, exposure of mice to H. polygyrus antigen reduced spontaneous and glucocorticoid-induced apoptosis of CD4- positive T cells in mesenteric lymph node (MLN). In this study we evaluate the proliferation, cytokine secretion, cell cycle progression and expression of apoptosis related genes in MLN CD4 T cells of uninfected and H. polygyrus infected mice ex vivo and in vitro after restimulation with parasite excretory secretory antigen (ESAg), somatic antigen (SAg) and fraction 9 (F9Ag) of somatic antigen. For the first time we explain the influence of H. polygyrus antigens on the intrinsic pathway of apoptosis. We found that the proliferation provoked by fraction 9 and inhibition of apoptosis was dependent on a low Bax/Bcl-2 ratio, dramatical upregulation of survivin, D1 cyclin, P-glycoprotein, and loss of p27Kip1 protein with inhibition of active caspase-3 but not caspase- 8.     

Small hairpin RNA targeting at Bcl-2 increases cytarabine-induced apoptosis in Raji cells

Bcl-2, a prominent member of the Bcl-2 family proteins, is responsible for the dysregulation of apoptosis and resistance to chemotherapy. In this study, we inves-tigated whether small hairpin RNA （shRNA） targeting at Bcl-2 mRNA could enhance cytarabine （Ara-C）- induced apoptosis in Raji cells. Bcl-2 shRNA was trans-fected into Raji cells and the expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR and immunofluorescence. Cell proliferation was determined by MTT assay. Apoptosis was determined by morphological observation and flow cytometric analysis. Our results show that expression levels of Bcl-2 mRNA and protein from Raji cells transfected with Bcl-2 shRNA decreased, compared with either negative control shRNA group or untransfected cells group （P 〈 0.05）. Viability of cells transfected with Bcl-2 shRNA was less than the cells transfected with control shRNA and untransfected Raji cells, respectively （P〈 0.05）. Bcl-2 shRNA combined with Ara-C significantly inhibited the growth of cells （P 〈 0.05）. There was no difference in cell survival between control shRNA/Ara-C combination and cells treated with Ara-C alone. Using Giemsa staining, cells treated with Bcl-2 shRNA plus Ara-C at 48h displayed changes of apoptosis. Apoptotic rates of Raji cells treated with Bcl-2 shRNA combined with Ara-C significantly increased （P 〈 0.05）, compared with either control shRNA/Ara-C combi- nation or Ara-C-treated cells alone. Our results suggest that the shRNA against Bcl-2 mRNA could increase Ara-C-induced apoptosis in Raji cells.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

CDC20 regulates the cell proliferation and radiosensitivity of P53 mutant HCC cells through the Bcl-2/Bax pathway

Purpose: The incidence of hepatocellular carcinoma (HCC) is extremely high, and China accounts for approximately 50% of global liver cancer cases. Previous studies reported that CDC20 is involved in the occurrence and progression of a variety of malignant tumors. So, whether CDC20 will affect the development of HCC, we have conducted in-depth research on this.Methods: We selected Hep3B and HepG2 for cell culture, and performed siRNA transfection, lentiviral infection, western blot, MTS determination, cell cycle determination, apoptosis test, immunodeficiency test, clone survival test and subcutaneous parthenogenesis in nude mice.Results: Knockdown of CDC20 greatly enhanced the radiation efficacy on the growth retardation in HepG2, and protein level of CDC20 was decreased for the activation of P53 by radiation. Downregulation of CDC20 combined with radiation can inhibit proliferation, aggravate DNA damage, increase G2/M arrest, and promote apoptosis of HCC cells to a greater extent, and the relative survival fraction of HCC cells was gradually reduced with radiation dose increased in P53 mutated Hep3B cells. After knocking down CDC20 in HCC, Bcl-2 was down-regulated and Bax expression increased. Down-regulation of CDC20 can inhibit further invasion by promoting the radiosensitivity of HCC.Conclusion: In this study, we found that that CDC20 was highly expressed in HCC and participated in radio resistance of HCC cells with P53 mutation Bcl-2/Bax via signaling pathway. This study is the first to present evidence that CDC20 may play a role in improving the efficacy of radiotherapy in HCC.