青蒿琥酯通过增加活性氧簇生成诱导人肝癌HepG2细胞凋亡

目的:探讨青蒿琥酯诱导人肝癌Hep G2细胞凋亡的机制及活性氧簇(ROS)在青蒿琥酯诱导Hep G2细胞凋亡中的作用。方法:采用MTT法观查青蒿琥酯对人肝癌Hep G2细胞存活的影响,Hoechst 33258荧光染色法观察细胞凋亡形态的变化,流式细胞术检测Hep G2细胞的凋亡率,DCFH-DA检测细胞凋亡过程中ROS的变化。Western blot检测细胞内凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和细胞色素C(Cyt C)蛋白水平的变化。采用NADPH氧化酶抑制剂夹竹桃麻素(apocynin)预处理Hep G2细胞,Western blot检测NADPH氧化酶亚基p47~(phox)和p22~(phox)蛋白表达水平,流式细胞术检测ROS变化。结果:与对照组相比,青蒿琥酯作用于Hep G2细胞24 h后,细胞存活率明显减少(P0.05);细胞核呈致密浓染色,细胞凋亡比例升高(P0.05);ROS明显升高(P0.05);Western blot结果显示,青蒿琥酯作用后细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,cleaved caspase-3和Cyt C蛋白水平升高。Apocynin预处理能降低青蒿琥酯给药组细胞内p47~(phox)和p22~(phox)蛋白表达及ROS的生成。结论:青蒿琥酯能诱导Hep G2细胞凋亡,其凋亡过程可能与ROS的生成增加相关。     

Noxa在凋亡中的作用及机制

Noxa是内源性途径凋亡中Bc l-2家族促凋亡的BH3-on ly亚家族成员之一,在诱导细胞凋亡中起重要作用。它通过定位于线粒体,引起线粒体释放细胞色素C以形成凋亡体,启动caspase级联反应而诱导细胞发生凋亡。Noxa的表达主要受p53调控,但是多种刺激因子可以通过p53或者不依赖p53等多种途径诱导Noxa表达上调,最后由Noxa来诱导细胞发生凋亡。     

诱导细胞凋亡青蒿琥酯抗食管癌作用机制研究

 [摘要] 目的 研究青蒿琥酯诱导食管癌细胞凋亡作用及探讨青蒿琥酯抗食管癌作用机制。方法 不同浓度的青蒿琥酯(Artesunate, Art)(0、10、20、40μg/ml) 作用Eca109细胞24h,流式细胞术（Flow cytometry, FCM）方法检测细胞凋亡、周期及细胞中bcl-2和bax蛋白的表达量。结果 青蒿琥酯作用Eca109细胞24h后,与对照组相比,细胞凋亡率显著增高P<0.05,且具有剂量依赖性。青蒿琥酯组与对照组相比,Eca109细胞中bcl-2蛋白表达水平及细胞增殖指数显著降低P<0.05,而bax蛋白表达量显著增高P<0.05,且具有剂量依赖性。结论 青蒿琥酯可以通过调节Eca109细胞中bcl-2、bax蛋白表达水平和细胞增殖,从而诱导Eca109细胞产生凋亡,起到抗食管癌作用。     

蛇床子素通过抑制SIRT1的表达增强阿霉素对p53野生型前列腺癌细胞的凋亡诱导活性

目的:探讨中药活性成分蛇床子素对阿霉素抗前列腺癌作用的影响及机制。方法:MTT法检测前列腺癌细胞系LNCaP在阿霉素和蛇床子素处理下的细胞活力。Western blot实验检测阿霉素和蛇床子素对LNCaP细胞中沉默信息调节因子1(SIRT1)、p53、乙酰化p53和Puma的表达水平、细胞色素C的释放水平及caspase-9和caspase-3活化水平的影响。流式细胞术检测阿霉素和蛇床子素对LNCaP细胞凋亡的影响。结果:蛇床子素联合治疗能明显提高阿霉素对p53野生型前列腺癌细胞系LNCaP的杀伤力。蛇床子素处理能显著抑制LNCaP细胞中SIRT1的表达,转染SIRT1过表达质粒后,蛇床子素、阿霉素联合治疗对LNCaP细胞的杀伤力受到显著抑制(P0.05)。蛇床子素联合阿霉素显著升高LNCaP细胞p53蛋白的表达水平和乙酰化水平,转染p53 si RNA后,蛇床子素对阿霉素的协同作用明显减弱。蛇床子素联合阿霉素显著诱导LNCaP细胞细胞色素C从线粒体释放到细胞质中,增强细胞中的caspase-9及下游caspase-3的活性并诱导细胞发生凋亡。结论:蛇床子素通过下调前列腺癌LNCaP细胞中SIRT1的表达促进阿霉素诱导的p53依赖的细胞凋亡。     

hTERT干扰对HepG2肝癌细胞Smac表达的影响

目的检测RNAi靶向抑制hTERT基因表达促HepG2肝癌细胞凋亡与Smac的关系,探讨hTERT干扰促进细胞凋亡的可能机制。方法收集hTERT基因RNAi真核表达载体稳定转染第20代的HepG2肝癌转染细胞、对照细胞（转染空载体质粒）及未转染细胞,通过Westernblot检测胞内XIAP和caspase-3,以及线粒体和胞浆Smac的表达;采用共聚焦显微镜观察线粒体膜电位变化。结果与未转染细胞和对照细胞相比,转染细胞内XIAP表达明显减少（P〈0．05）,caspase-3表达明显增加（P〈0．05）,线粒体Smac表达明显减少（P〈0．05）,而胞浆Smac表达明显增加（P〈0．05）;转染细胞线粒体膜电位明显降低。结论RNAi靶向抑制hTERT基因表达可能通过降低HepG2肝癌细胞线粒体膜电位,引起Smac从线粒体释放入胞浆,进而抑制XIAP表达,促进caspase-3表达增加诱导细胞凋亡。     

野生型PTEN基因增强青蒿琥酯抑制K562细胞的作用

 目的: 探讨野生型PTEN转染人白血病K562细胞系对青蒿琥酯敏感性的影响及其分子作用机制。方法: 将野生型PTEN以腺病毒为载体转染(感染复数为200)人白血病K562细胞(Ad-WT-PTEN),同时以转染空载体腺病毒(Ad)及未转染细胞为对照组,与青蒿琥酯(ART)联合作用,观察野生型PTEN增强青蒿琥酯抑制K562细胞的作用。根据IC₅₀计算PTEN对青蒿琥酯的增敏倍数。以四甲基偶氮唑蓝(MTT)法检测细胞活力,流式细胞术检测细胞凋亡率,real-time PCR检测PTEN 的mRNA水平,Western blot检测PTEN、蛋白激酶B(Akt)及磷酸化Akt(p-Akt)的蛋白水平;caspase活性检测试剂盒检测caspase-3/7的活性。结果: Ad-WT-PTEN转染K562细胞后,对青蒿琥酯敏感性明显增加,依据IC₅₀计算增敏倍数为2.25倍。至第3天,Ad-WT-PTEN +ART组较Ad+ART组细胞活力下降、凋亡率升高。Ad-WT-PTEN转染K562细胞后PTEN 的mRNA及蛋白表达明显增加,p-Akt水平及caspase-3/7活性下调,以PTEN及青蒿琥酯联合作用组下调尤为明显。结论: 野生型PTEN可能通过降低K562细胞Akt磷酸化的水平,并增加caspase-3/7活性,增强细胞对青蒿琥酯的敏感性。     

青蒿琥酯通过调控PI3K/Akt信号通路抑制肝癌细胞增殖促进凋亡

目的 探讨青蒿琥酯对人肝癌细胞系HepG2的增殖和凋亡的影响及可能机制.方法 体外培养人肝癌细胞系HepG2,分别用浓度为12.5、25、50、100mg/L青蒿琥酯作用不同时间后,采用CCK-8实验检测细胞增殖情况;随后选择最佳浓度的青蒿琥酯作用HepG2细胞24h后,流式细胞术检测细胞凋亡,Western blot检测Bax、Bcl-2、PI3K、p-PI3K、Akt和p-Akt的水平.结果 不同浓度的青蒿琥酯处理细胞24h后,青蒿琥酯均能显著抑制肝癌细胞HepG2的增殖,且该作用呈现剂量依赖性.流式细胞仪检测发现青蒿琥酯能显著促进肝癌细胞HepG2的凋亡,同时Western blot也进一步证实,青蒿琥酯处理后HepG2细胞中的抑凋亡蛋白Bcl-2显著下调,促凋亡蛋白Bax表达显著上调;同时发现青蒿琥酯能够降低p-PI3K和p-Akt的水平,但未磷酸化的PI3K和未磷酸化的Akt含量变化无显著性差异.结论 青蒿琥酯可以抑制肝癌细胞HepG2的增殖,促进细胞凋亡,该作用与调控PI3K和Akt的磷酸化水平密切相关.     

幽门螺杆菌感染通过 MDM2 / P53 通路调控食管癌细胞凋亡的作用及机制

目的 研究幽门螺杆菌 (helicobacter pylori, Hp) 感染通过 MDM2 / P53 通路调控食管癌细胞凋 亡的作用及机制。 方法 收集手术切除的食管癌组织, 检测 Hp 感染情况及 MDM2、 P53 的表达水平。 培养 食管癌 TE-1 细胞株, 给予 Hp 感染、 生理盐水 (NS) 或泛素-蛋白酶体抑制剂 PS341 干预、 转染阴性对照 (NC) siRNA 或 MDM2 siRNA, 检测细胞活力、 凋亡率、 MDM2 及 P53 的表达水平、 P53 的泛素化水平。 结果 Hp 阳性的食管癌组织中 MDM2 的表达水平高于 Hp 阴性的食管癌组织、 P53 的表达水平低于 Hp 阴性 的食管癌组织 (P< 0. 05); Hp 组 TE-1 细胞的细胞活力、 MDM2 表达水平、 P53 泛素化水平高于对照组, 凋亡率、 P53 表达水平低于对照组; PS341 + Hp 组 TE-1 细胞的 P53 的表达水平高于 NS + Hp 组 (P< 0. 05); si-MDM2 + Hp 组 TE-1 细胞的细胞活力、 MDM2 的表达水平、 P53 的泛素化水平均低于 si-NC + Hp 组, 凋亡 率、 P53 的表达水平高于 si-NC + Hp 组 (P< 0. 05)。 结论 Hp 感染抑制食管癌细胞凋亡的作用与增加 MDM2 表达、 促进 P53 的泛素化降解有关。     

依托泊苷通过线粒体信号通路释放细胞色素C诱导Jurkat白血病细胞凋亡

目的：研究在人类Jurkat白血病细胞株中依托泊苷诱导凋亡的分子机制，揭示由依托泊苷启动的凋亡信号通路。 方法：分别用annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞仪测定annexin V阳性和出现亚二倍体DNA的凋亡细胞。以3,3＇-dihexyloxyacarbocyanine iodide ［DiOC6(3)］为染色剂，采用流式细胞术检测细胞线粒体膜电位的变化。采用离心技术分离细胞的胞浆与线粒体。细胞色素c从线粒体转入胞浆，caspase-3的激活，多聚二磷酸腺苷核糖聚合酶（PARP）的切割等蛋白质的表达由免疫印迹技术（Western blotting）检测。 结果：依托泊苷诱导Jurkat白血病细胞凋亡，细胞凋亡与依托泊苷的作用时间呈线性关系。广谱的caspase抑制剂zVAD.fmk可抑制依托泊苷诱导的DNA片段化和磷脂酰丝氨酸外翻。依托泊苷引起的线粒体膜电位下降早于DNA片段化和磷脂酰丝氨酸外翻，形成明显对照的是zVAD.fmk不能阻断依托泊苷诱导的线粒体膜电位的下降。依托泊苷介导细胞色素c从线粒体释放到胞浆，激活caspase-3，caspase-3的底物PARP被切割。 结论：依托泊苷诱导Jurkat白血病细胞株凋亡的机制是降低线粒体膜电位和释放细胞色素c到细胞浆启动线粒体信号转导通路, 最终激活caspase而导致细胞凋亡。     

氧化应激诱导HepG2肝癌细胞凋亡的研究(英)

目的：直接暴露细胞于活性氧能诱导发生凋亡，本文研究氧化应激诱导HepG2肝癌细胞的死亡及其机制。方法：暴露细胞于2 mmol/L过氧化氢产生氧化应激，用DNA凝胶电泳检测细胞凋亡，用荧光染色法检测细胞线粒体膜电位变化，Western blotting检测细胞浆中细胞色素c变化，fluorometric assay kit检测caspase活性变化。结果：氧化应激作用于HepG2细胞后12 h开始发生凋亡；氧化应激作用后4 h，细胞线粒体膜电位明显下降；胞浆中细胞色素c浓度呈时间依赖性增高；氧化应激作用8 h、12 h后细胞内caspase-3、caspase-9活性分别升高6.7及3.6倍，但caspase-8活性无变化。结论：氧化应激能诱导HepG2肝癌细胞发生凋亡，其途径与线粒体通路及caspase激活有关。     

二氢杨梅素联合顺铂显著诱导PC3细胞中Noxa和Bim过表达

目的:探讨二氢杨梅素对前列腺癌顺铂化疗的辅助作用并研究其机制。方法:MTT法检测前列腺癌细胞系LNCaP和PC3在不同浓度二氢杨梅素和顺铂处理下的细胞活力。Western blot实验检测顺铂和二氢杨梅素联用对PC3细胞FOXO1、Noxa和Bim表达水平、细胞色素C从线粒体中的释放及caspase-9和caspase-3活化水平的影响。免疫共沉淀实验检测PC3细胞中凋亡蛋白酶激活因子1(Apaf-1)和caspase-9的相互作用。流式细胞术检测PC3细胞的凋亡率。结果:二氢杨梅素辅助治疗可明显提高顺铂对前列腺癌的体外抗肿瘤活性(P0.05)。二氢杨梅素处理能显著促进PC3细胞FOXO1的表达,转染FOXO1小干扰RNA(siRNA)后,二氢杨梅素对顺铂的辅助治疗效果受到明显抑制。二氢杨梅素联合顺铂能显著诱导PC3细胞中Noxa和Bim的过表达,细胞色素C的释放,Apaf-1和caspase-9的相互作用,caspase-9和caspase-3的活化及凋亡的发生。转染FOXO1 siRNA后,二氢杨梅素联合顺铂对PC3细胞的凋亡诱导途径受到显著抑制。结论:二氢杨梅素可能通过FOXO1-Bim/Noxa途径增强顺铂对前列腺癌细胞的体外杀伤活性。     

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells

Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.     

Induction of apoptosis by S-allylmercapto-L-cysteine, a biotransformed garlic derivative, on a human gastric cancer cell line

Epidemiological and experimental carcinogenesis studies provide evidence that certain components of garlic have anti-cancer activity. Although the biotransformed garlic derivative S-allylmercapto-L-cysteine (SAMC) has been reported to show an inhibitory effect on tumorigenesis, the mechanisms are poorly understood. The present study investigated the effect of SAMC on the growth of human gastric cancer SNU-1 cells. Upon treatment with SAMC, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis, including DNA fragmentation and an increase in the sub-diploid population. The anti-proliferative and apoptotic effect of SAMC was associated with the induction of Bax, p53, and caspase-9, rather than the induction of Bcl-2 and p21. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of SAMC, which mediates cell death. These results suggest that the apoptotic effect of SAMC on gastric cancer SNU-1 cells may be connected with caspase-3 activation through the induction of Bax and p53, rather then Bcl-2 and p21.     

Involvement of the mitochondrial pathway in bruceine D-induced apoptosis in Capan-2 human pancreatic adenocarcinoma cells

The fruit of Brucea javanica L. is a common herb used in Chinese medicine for the treatment of a variety of cancers. Our research group has previously identified bruceine D (BD), a quassinoid found abundantly in B. javanica, to have potent cytotoxic effect on a number of pancreatic cancer cell lines, including Panc-1, SW1990 and Capan-1 cells. In the present study, we showed that BD was also able to inhibit the growth of the Capan-2 human pancreatic adenocarcinoma cell line, but it exerted only modest cytotoxicity on the WRL68 human hepatocyte cell line and a human pancreatic progenitor cell line. The antiproliferative effects of BD were comparable to those exhibited by camptothecin and gemcitabine in our culture system. We found a dose-dependent decrease of the mitochondrial membrane potential in BD-treated Capan-2 cells as measured by the JC-1 assay. BD exposure was able to attenuate the expression of Bcl-2 protein in Capan-2 cells as detected by western blot analysis. In addition, the expression of both caspase 9 and caspase 3 in BD-treated Capan-2 cells was significantly accentuated. Moreover, BD was capable of inducing the fragmentation of genomic DNA in Capan-2 cells as evidenced by Hoechst staining. Cell cycle analysis demonstrated that BD could increase the percentage of Capan-2 cells in the subG1 phase in a dose-related manner. An increase in the apoptosis of Capan-2 cells was also observed by Annexin V and PI staining. These results unequivocally indicate that BD induces cytotoxicity in Capan-2 cells via the induction of cellular apoptosis involving the mitochondrial pathway.     

The levels of MDM2 protein are decreased by a proteasome-mediated proteolysis prior to caspase-3-dependent pRb and PARP cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.     

Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3

Induction of cell death by p14^ARF is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21^CDKN1. Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21^CDKN1 or p53-reconstituted DU145 prostate cancer cells, we show that p14^ARF-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14^ARF in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14^ARF-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14^ARF-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21^CDKN1 strongly enhances p14^ARF-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14^ARF-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14^ARF-triggered apoptosis is enhanced by loss of p21^CDKN1-mediated cell cycle checkpoint control.     

Apoptosis-promoted tumorigenesis: γ-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death

Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in γ-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from γ-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.     

An ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line

Iris nertschinskia, an ornamental plant, is utilized in traditional East Asian medicine for the treatment of skin diseases. However, the biological activity underlying its therapeutic effects remains to be established. In this study, we investigated the anti-tumor effect of the plant extract on MCF7 human breast cancer cells. An ethanol extract of Iris nertschinskia triggered cell death in a dose-dependent manner. Moreover, treatment with the extract promoted p53 phosphorylation in MCF7 cells. Increased phosphorylation of p53, in turn, led to induction of Bax protein, a key regulator of p53-dependent apoptotic cell death, as well as of caspase-7 cleavage in MCF7 cells. Consistently, cells treated with p53-specific siRNA or the caspase inhibitor, Z-VAD, resisted apoptotic cell death induced by the Iris nertschinskia extract. Our results suggest that p53 sensitizes tumor cells to the ethanol extract of Iris nertschinskia by Bax protein induction and caspase-dependent apoptosis.