斑蝥素诱导人胰腺癌细胞凋亡的实验研究

目的 探讨斑蝥素（Cantharidin）对人胰腺癌细胞凋亡的影响及其作用机制。方法采用MTT法观察斑蝥素对人胰腺癌细胞株SW1990细胞增殖的抑制作用。采用Hoechst33258染色、TUNNEL染色、流式细胞术检测细胞凋亡改变，并以RT—PCR和Westernblot检测凋亡调节基因p53和Bcl-2、Bax的表达。结果 5mol／L斑蝥素能明显抑制人胰腺癌SW1990细胞的生长，呈现凋亡特征。RT—PCR和Westernblot检测可见Bax、p53基因表达显著增加，而Bcl—2基因表达减少。结论 斑蝥素能诱导人胰腺癌细胞凋亡，其作用可能与上调p53、Bax基因和下调Bcl-2基因有关。     

斑蝥素抑制裸鼠胰腺癌移植瘤生长研究

目的 探讨斑蝥素对荷瘤裸鼠胰腺癌移植瘤生长的影响.方法 在培养人胰腺癌SW1990细胞的基础上建立荷瘤裸鼠胰腺癌模型,瘤内注射斑蝥素1 mg/kg体重作为斑蝥素组,瘤内注射等量生理盐水作为对照组,观测移植瘤大小改变及p53、Bcl-2和Bax蛋白表达的变化.结果 两组裸鼠移植瘤的体积随着时间的延长而明显增加,但斑蝥素组的增长幅度明显小于对照组(P＜0.05),移植后27 d瘤体缩小40%;斑蝥素组移植瘤Bcl-2无强阳性(+ +～+ + +)表达,p53强阳性表达6只(85.7%),Bax强阳性表达3只(42.9%),对照组强阳性表达分别为2只(33.3%)、3只(50%)和2只(33.3%).Bcl-2、p53表达两组差异显著(P＜0.05),而Bax表达无显著差异.结论 斑蝥素可明显抑制裸鼠胰腺癌移植瘤的生长,其机制可能与斑蝥素下调Bcl-2蛋白表达,上调p53蛋白表达有关.     

去甲斑蝥素诱导人肝癌细胞凋亡的研究

目的探讨去甲斑蝥素（NCTD）对人肝癌细胞SMMC-7721凋亡的影响。方法用MTT方法检测人肝癌细胞SMMC-7721的生长抑制率。应用流式细胞仪检测细胞周期和凋亡。结果 NCTD对人肝癌细胞SMMC-7721有生长抑制作用,随浓度升高、时间延长作用增强,呈剂量和时间效应关系。流式细胞仪示SMMC-7721细胞的S期细胞明显增多,G0/G1期细胞减少,凋亡率上升（P〈0.05或〈0.01）。结论 NCTD能显著抑制人肝癌SMMC-7721细胞的生长,其可能与抑制SMMC-7721细胞增殖,诱导细胞凋亡有关。     

斑蝥素对胰腺癌细胞系PANC1、CFPAC-1的凋亡诱导作用及机制研究

目的 研究斑蝥素对胰腺癌细胞系PANC1、CFPAC-1的凋亡诱导作用及机制。方法 用斑蝥素处理PANC1、CFPAC-1细胞。四甲基偶氮唑蓝(MTT)法检测细胞增殖;流式细胞术检测细胞凋亡;酶化学法检测caspase活性;RT-PCR及蛋白质印迹法检测凋亡相关基因的表达。结果 斑蝥素呈剂量依赖性明显抑制胰腺癌细胞系PANC1、CFPAC-1增殖及诱导细胞凋亡。10μmol/L斑蝥素处理细胞72 h,PANC1、CFPAC-1细胞的增殖抑制率最高,分别达(52.95 +6.34)％和(71.21 +6.30)％。处理24h,PANC1细胞的早期和晚期凋亡细胞分别从7.35％增加到24.89％、6.36％增加到17.73％;CFPAC-1细胞从6.39％增加到24.70％、9.21％增加到12.58％(P值均＜0.01)。PANC1细胞的caspase 8和caspase9活性分别为对照组的(155.8±11.5)％和(194.6±14.7)％;CFPAC-1细胞分别为对照组的(182.5±24.3)％和(215.8±12.2)％(P值均＜0.01)。促凋亡基因TNF-α、TRAILR1、TRAILR2、Bad、Bak和Bid表达增加,抗凋亡基因Bcl-2表达减少。结论 斑蝥素通过激活Caspase、上调促凋亡基因及下调抗凋亡基因的表达从而诱导胰腺癌细胞凋亡。     

氧化砷诱导胰腺癌细胞凋亡的实验研究

目的观察As2O3对胰腺癌细胞株体外生长和裸鼠腹腔种植腹水生成的影响及其作用机制.方法O.125～2 μmol/L的As2O3与胰腺癌细胞株SW-8902共同孵育,观察不同浓度、不同作用时间对胰腺癌细胞生长的抑制作用、作用后胰腺癌细胞的凋亡特征及Fas Fas L表达的变化.80只BALB/C-nu/nu裸鼠腹腔内接种胰腺癌细胞株SW-8902,并随机分为4组,然后分别腹腔内注射生理盐水及不同剂量的As2O3,观察各组裸鼠的生存时间.结果1～2 μmol/L的As2O3作用后,胰腺癌细胞呈典型的凋亡特征性改变,流式细胞仪检测在G1期前出现亚二倍体凋亡峰,DNA电泳呈现特征性Ladder,细胞核内可见染色质浓缩、碎裂和边聚.As2O3也可显著抑制荷胰腺癌裸鼠腹水的生成,延长生存期(P＜O.01),Fas、Fas-L表达在As2O3作用后2 d上升,3 d达最高,以后表达量下降.结论As2O3诱导胰腺癌细胞凋亡,抑制裸鼠胰腺癌腹腔种植及腹水的生成,延长生存期.Fas、Fas-L表达上调是As2O3诱导肿瘤细胞凋亡的途径之一.     

胰腺癌Bcl-2,P53蛋白表达和细胞凋亡

目的　探讨ｂｃｌ２ ,ｐ５３ 基因和细胞凋亡在胰腺癌发病机制中的作用以及它们之间相互关系．方法　应用ＡＢＣ 免疫组化技术检测５０ 例胰腺癌中Ｂｃｌ２ 和Ｐ５３ 蛋白表达,运用原位末端标记法观察肿瘤中细胞凋亡数量．结果　Ｐ５３ 蛋白表达阳性率为５４ ％ ,临床Ⅰ期阳性率（２６－７ ％ ）却显著低于Ⅱ期（６１－１ ％ ） 和Ⅲ＋ Ⅳ期（７０－６ ％ ,Ｐ＜ ０－０５） ;Ｂｃｌ２蛋白表达阳性率为６４ ％ ,临床Ⅰ期阳性率（９３－３ ％ ） ,显著高于Ⅱ期（５５－６ ％ ） 和Ⅲ＋ Ⅳ期（４７－１ ％ ,Ｐ＜ ０－０５） ;组织学Ⅲ级癌细胞中凋亡指数明显高于Ⅰ,Ⅱ级（ Ｐ＜ ０－０５） ,Ｂｃｌ２ 蛋白阴性病例中凋亡指数明显高于Ｂｃｌ２ 阳性者（ Ｐ＜ ０－０１） ．结论　Ｂｃｌ２ 是通过抑制细胞凋亡参与肿瘤的生长过程,Ｂｃｌ２和Ｐ５３ 蛋白表达之间 存在密切负相关（τ＝ － ０－１７４７ ,Ｐ＜ ０－０５） ．     

全反式维甲酸诱导胰腺癌细胞凋亡机制研究

目的 维甲酸类药物调节肿瘤细胞生长、分化和凋亡是其防治肿瘤的基础。本研究观察全反式维甲酸(ATRA)诱导体外胰腺癌细胞凋亡的可能机制。方法 胰腺癌细胞Patu8988与ATRA共同孵育。通过DNA断裂分析、TUNEL标记证实凋亡存在，并用流式细胞仪检测凋亡细胞比例。用RT-PCR和Western blot方法检测凋亡诱导过程中p53、bcl-2和bax基因的水平及其表达变化。结果 ATRA处理组细胞凋亡比例明显增加。TUNEL标记和DNA断裂分析发现典型凋亡特征。ATRA处理组bax和phospho-p53(Ser 46)表达上调，但bcl-2表达下调。结论 ATRA能诱导胰腺癌细胞凋亡，其分子机制可能与bcl-2/bax和p53的表达相关。     

XAF1基因诱导胰腺癌细胞株BxPC3凋亡的实验研究

目的 探讨Ad5/F35腺病毒介导的XAF1基因诱导胰腺癌细胞BxPC3凋亡及其可能的作用机制.方法 将前期构建的重组腺病毒Ad5/F35-XAF1感染胰腺癌细胞BxPc3.采用半定量RTPCR、蛋白质印迹检测法检测感染前后BxPC3细胞XAF1 mRNA和蛋白表达的变化;运用Annexin-V/PI和TUNEL法检测细胞的凋亡率;免疫蛋白印迹法检测细胞Caspase-3、PARP、Caspase-8和Bcl-2蛋白的表达.结果 Ad5/F35-XAF1感染后,BxPC3细胞的XAF1 mRNA和蛋白表达明显增高,与空白组和Ad5/F35-Null对照组比较,差异显著(P＜0.05);两种方法 检测的细胞凋亡率分别为(19.90±3.09)%、(9.29±2.13)%,较Ad5/F35-Null组的(6.72±0.76)%、(2.73±0.51)%和空白组的(7.22±1.53)%、(1.56±0.47)%均有显著差异(P＜0.01);Caspase-3、PARP、Caspase-8蛋白表达显著增强,Bcl-2表达降低.结论 Ad5F35-XAF1重组腺病毒感染胰腺癌细胞BxPC-3能明显诱导细胞的凋亡,其机制可能是激活死亡受体途径和线粒体途径.     

PUMA在吉西他滨诱导胰腺癌细胞凋亡过程中的作用

目的 研究吉西他滨体外对人胰腺癌AsPC-1细胞生长的作用机制.方法 用脂质体转染法将p53正向凋亡调控因子(PUMA)反义核酸(反义PUMA cNA)的真核表达载体pcDNA3.1-PUMAAS和空载体pcDNA3.1-导入AsPC-1细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆.将转染载体的AsPC-1阳性克隆细胞和未转染载体的AsPC-1细胞分别暴露于浓度1、5、10和15 μmol/ml的吉西他滨中作用72 h.RT-PCR和Western blotting检测不同组细胞经吉西他滨作用72 h后的PUMA表达;MTT检测细胞生长抑制,流式细胞仪(FCM)、Hoechst 33258荧光染色法和TUNEL法检测细胞凋亡.结果 吉西他滨促进AsPC-1细胞凋亡,抑制细胞生长,并有明显的剂量依赖性,在细胞凋亡的同时伴有PUMA表达的上调;当细胞转染PUMA反义核酸抑制PUMA表达后,受吉西他滨作用的细胞出现PUMA蛋白表达明显降低,同时伴有细胞凋亡的抑制及细胞的明显增殖.结论 吉西他滨促进体外AsPC-1细胞凋亡,并抑制其生长,其诱导凋亡与上调PUMA有关.     

Metformin induces apoptosis of pancreatic cancer cells

AIM：To assess the role and mechanism of metformin in inducing apoptosis of pancreatic cancer cells.METHODS：The human pancreatic cancer cell lines ASPC-1,BxPc-3,PANC-1 and SW1990 were exposed to metformin.The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of metformin was tested.RESULTS：In each pancreatic cancer cell line tested,metformin inhibited cell proliferation in a dose dependent manner in MTS（3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium assays）.Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction.Enzymelinked immunosorbent assay（ELISA） showed that metformin induced apoptosis in all pancreatic cancer cell lines.In Western blot studies,metformin induced poly-ADP-ribose polymerase（PARP） cleavage（an indicator of caspase activation） in all pancreatic cancer cell lines.The general caspase inhibitor（VAD-fmk） completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1,the caspase-8 specific inhibitor（IETD-fmk） and the caspase-9 specific inhibitor（LEHD-fmk） only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells.We also observed that metformin treatment dramatically reduced epidermal growth factor receptor（EGFR） and phosphorylated mitogen activated protein kinase（P-MAPK） in both a time-and dose-dependent manner in all cell lines tested.CONCLUSION：Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines.And the metformin-induced apoptosis is associated with PARP cleavage,activation of caspase-3,-8,and-9 in a time-and dose-dependent manner.Hence,both caspase-8 and-9-initiated apoptotic signaling pathways contribute to metformin-induced apoptosis in pancreatic cell lines.     

Melatonin induces apoptosis in human neuroblastoma cancer cells

Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.     

Arsenic trioxide induces apoptosis of human gastrointestinal cancer cells

AIM:To investigate the changes in apoptosis in gastrointestinal cancer cells from patients with gastrointestinal cancers treated with arsenic trioxide(As2O3);and to study the possible molecular mechanisms of such changes by detecting the expression levels of p53and Bcl-2.METHODS:Twenty patients with gastrointestinal adenocarcinoma based on endoscopic and biopsy findings(ten patients with gastric cancer and ten patients with colorectal cancer)who received treatment in our hospital between August 2007 and December 2008were included in this study.None of the patients had received anti-tumour agents prior to As2O3 treatment.As2O3 was administered intravenously at a dose of 0.01g/d diluted with 5%glucose in normal saline for 2-3h for 3 consecutive days before surgery.Morphological changes associated with apoptosis of gastrointestinal cancer cells were observed by light microscopy.Changes in the apoptotic index induced by As2O3 were investigated using the terminal deoxynucleotidyl transferase dUTP nick end labelling method.Expression levels of p53 and Bcl-2 proteins in gastrointestinal cancer tissues were determined by immunohistochemistry.RESULTS:The apoptotic index of human gastrointestinal cancer cells was higher in cells from patients treated with As2O3 than in those not treated(P<0.05).p53 protein expression in gastrointestinal tissues was unchanged by As2O3(P>0.05).However,Bcl-2 protein expression in gastrointestinal tissues was downregulated by As2O3(P<0.01).CONCLUSION:These results demonstrate that As2O3treatment in patients with gastrointestinal cancers can induce apoptosis in gastrointestinal cancer cells and down-regulate Bcl-2 protein expression.     

蟾毒灵介导JNK信号通路诱导人胰腺癌细胞的凋亡

目的:研究蟾毒灵诱导人胰腺癌细胞凋亡以及凋亡相关基因表达的JNK信号转导通路,揭示其抗胰腺癌的部分机制.方法:MTT法观察蟾毒灵对人胰腺癌BxPC-3细胞的生长抑制作用;0.16、0.32、0.64mg/L蟾毒灵分别作用人胰腺癌BxPC-3细胞48h后,流式细胞仪(flow cytometry,FCM)检测细胞周期和细胞凋亡;Western印迹法检测蟾毒灵作用BxPC-3细胞后SAPK/JNK信号通路的激活情况,荧光定量PCR检测Survivin基因mRNA的表达水平;并比较阻断JNK信号通路后丹参酮ⅡA对胰腺癌细胞凋亡Survivin基因mRNA的表达.结果:MTT法测得蟾毒灵对人胰腺癌BxPC-3细胞具有显著的抑制作用,其作用效果与剂量和作用时间成正相关;0.16、0.32、0.64mg/L浓度蟾毒灵作用人胰腺癌细胞后的细胞凋亡率分别为19.36%±0.39%、40.69%±0.44%、59.63%±1.14%,与对照组2.24%±0.37%比较均有显著性差异(P<0.01);蟾毒灵作用人胰腺癌细胞1h后JNK信号通路被激活,2h达峰值;阻断JNK信号通路后,凋亡率明显降低(P<0.01);0.32mg/L蟾毒灵作用人胰腺癌细胞48h后Survivin mRNA的表达明显下降;阻断JNK信号通路后,蟾毒灵作用人胰腺癌细胞的Survivin mRNA的表达明显上升.结论:蟾毒灵通过JNK信号转导通路下调人胰腺癌BxPC-3细胞Survivin mRNA的表达,可能是其诱导胰腺癌细胞凋亡的机制.     

靶向5-LOX的siRNA诱导胰腺癌细胞的凋亡

目的:采用RNA干扰技术(siRNA)阻断5-LOX基因的表达,观察其抑制人胰腺癌细胞增殖及诱导细胞凋亡的作用.方法:构建靶向5-LOx的siRNA质粒表达载体,采用Lipofectamine-2000转染人胰腺癌细胞株SW1990,采用RT-PCR检测RNA干扰后5-LOX mRNA表达,MTT法检测细胞的增殖抑制率,流式细胞仪检测细胞凋亡率.结果:靶向5-LOX序列特异性的三条siRNA(组1、组2、组3)可以有效地抑制SW1990细胞5-LOX基因表达,其表达抑制率分别为19.6%±1.9%、55.4%±2.6%和55.2%±2.7%.转染靶向5-LOX siRNA的三个质粒表达载体可以显著抑制SW1990细胞的增殖, 细胞接种24 h后,三组增殖抑制率分别为5.37%±1.19%、11.63%±1.25%和13.67%±1.04%:48 h后其增殖抑制率分别为16.13%±1.5%、26.63%±1.22%和25.47%±1.67%, 各siRNA组增殖抑制率高于空白组和阴性对照组(均P<0.05),转染后24 h和48 h三组细胞凋亡率分别为5.56%±1.05%、11.45%±1.44%、12.13%±1.36%和7.37%±1.23%、18.75%±1.5%和22.02%±1.45%,均高于空白对照组和阴性对照组(P<0.05).结论:所构建的靶向5-LOX的siRNA质粒表达载体可以有效阻断SW1990细胞5-LOX基因表达,显著地抑制SW1990细胞增殖,并在一定程度上诱导其凋亡.     

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 μM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.