凋亡抑制基因XIAP在前列腺癌组织中的表达及临床意义

目的探讨前列腺组织中凋亡抑制基因XIAP的表达及其与临床病理特征的关系。方法应用RT-PCR和免疫组化(SP)法,检测XIAP在62例前列腺癌组织中的表达情况。结果62例前列腺癌组织中XIAP基因高表达,正常的前列腺组织中XIAP均无明显表达。62例前列腺癌组织中XIAP蛋白阳性表达共有43例(69．4％),低分化组XIAP蛋白阳性表达率(94．4％)明显高于中分化(70．0％)和高分化组(35．7％),其差异有统计学意义(P<0．05);XIAP蛋白阳性表达与前列腺癌的临床分期比较亦有统计学意义,临床分期愈晚,XIAP蛋白阳性表达率愈高(P<0．05)。结论XIAP与前列腺癌的发生发展有关,检测前列腺癌组织中XIAP表达可能对判断前列腺癌预后有一定意义。     

氧化砷诱导胰腺癌细胞凋亡的实验研究

目的: 观察不同浓度As2O3对胰腺癌细胞生长的影响. 方法: 0.1~2μmol/LAs2O3与胰腺癌细胞株SW-1990、SW-8902共同孵育,观察不同浓度As2O3作用、不同时间对胰腺癌细胞的生长抑制情况. 结果: 经1~2μmol/LAs2O3处理的胰腺癌细胞呈典型的凋亡特征性改变:用HE染色光镜观察可见凋亡小体形成,流式细胞仪检测在G1期前出现亚二倍体凋亡峰,细胞DNA抽提电泳后发现凋亡特征性Ladder. 结论: As2O3可诱导胰腺癌细胞凋亡.     

XIAP siRNA、Embelin对TRAIL诱导肝癌细胞生长抑制和细胞凋亡的影响

目的 研究X-连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis,XIAP) siRNA或XIAP拮抗剂Embelin对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)所致肝癌细胞HepG2生长抑制和细胞凋亡的影响.方法 HepG2细胞转染XIAP siRNA或者阴性对照,然后给予TRAIL处理.此外,HepG2细胞给予TRAIL、Embelin或者联合处理.XIAP表达水平变化、生长抑制、caspase-3活性分别用Western blot、MTT测定、caspase-3荧光法检测.切割PARP的表达水平用Western blot测定.结果 XIAP蛋白表达水平在转染XIAP siRNA后显著下调.与阴性对照相比,XIAP siRNA显著增强TRAIL在100 ng/ml(6.8％±1.2％比11.8％±4.0％,P＜0.05)和1000 ng/ml(18.9％±2.0％比26.6％±1.5％,P＜0.01)的生长抑制作用.在转染XIAP siRNA后,TRAIL诱导caspase-3的活性和PARP的切割显著增强.此外,Embelin显著增强TRAIL对HepG2细胞的生长抑制(P＜0.01)、caspase-3活化(P＜0.01)和PAPR切割.结论 XIAP siRNA或Embelin在肝细胞癌的临床治疗方面具有潜在应用前景.     

Caspase-3 drives apoptosis in pancreatic cancer cells after treatment with gemcitabine

Pancreatic cancer remains a highly chemoresistant malignancy. Gemcitabine, the most effective firstline agent available, acts by disrupting cellular replication. Caspases belong to a family of proteases that function as key components of the apoptotic death machinery. We investigated the mechanisms by which gemcitabine blocks proliferation and whether it can induce apoptosis in pancreatic cancer cells. Quiescent pancreatic cancer cells (BxPC-3) were stimulated to proliferate (10% fetal calf serum) with or without gemcitabine, PS-341 (26S proteasome inhibitor), or both. Proliferation was measured by MTT assay and apoptosis by propidium iodine staining. To determine activation of the apoptotic regulatory cell proteins, caspase-3 and cleavage of poly(ADP-ribose)polymerase (PARP) into its 85-kDa fragment were assessed by Western blotting. Gemcitabine at even low doses (10 μmol/L) significantly inhibited cellular proliferation, whereas PS-341 (10 nmol/L) had no effect. With combined treatment, PS-341 potentiated the antiproliferative effects of gemcitabine (P = 0.001). At 48 hours, the apoptotic fraction was greatly enhanced by the presence of PS-341 compared with gemcitabine alone. Caspase-3 accumulated as early as 30 minutes and was associated with cleavage of PARP to its apoptotic fragment. Gemcitabine, a nucleoside analogue, may in part exert its antiproliferative effects by directing pancreatic cancer cells to a default pathway of apoptosis. 26S proteasome inhibition potentiates this effect, suggesting its potential clinical value against chemoresistance in pancreatic cancer. Presented at the Fourth Biennial Congress of the American Hepato-Pancreato-Biliary Association (AHPBA), Miami Beach, Florida, February Presented at the Fourth Biennial Congress of the American Hepato-Pancreato-Biliary Association (AHPBA), Miami Beach, Florida, February Supported by grants from the American Hepato-Pancreato-Biliary Association, Ethicon Research Fellowship, and National Pancreas Foundation.     

人工合成Smac拟肽促进前列腺癌细胞凋亡的实验研究

目的探讨人工合成的小分子Smac拟肽对前列腺癌细胞增殖的抑制作用和诱导细胞调亡作用。方法应用固相多肽合成技术人工合成Smac融合多肽,以激光共聚焦显微镜观察肽段的细胞定位;以不同浓度处理前列腺癌细胞,应用MTT比色法、Hoechst染色、流式细胞术检测细胞增殖和凋亡情况。结果激光共聚焦检测显示小分子肽段能进入细胞内,没有表现出某一特定细胞器的聚集性;MTT结果显示Smac拟肽对前列腺癌LNCap和PC-3细胞的增殖抑制具有浓度依赖性和时间依赖性（高浓度Smac拟肽）;Hoechst染色发现LNCap和PC-3细胞经高浓度（200μg/mL）作用48 h时细胞凋亡明显;同样浓度Smac拟肽作用LNCap和PC-3细胞48 h后用流式细胞仪分析,结果同样显示细胞明显凋亡。结论小分子Smac拟肽能有效地诱导前列腺癌细胞的凋亡,为前列腺癌的生物治疗提供了新的思路。     

Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells

BACKGROUND: Cardiac glycosides may induce oncolytic effects in cancers. This study was to evaluate bufalin and cinobufagin effects on the proliferation of prostate cancer cell lines named LNCaP, DU145, and PC3. METHODS: Cell proliferation was measured by MTT assay. The cytotoxic effects were determined by lactate dehydrogenase measurements. The intracellular calcium concentration ([Ca(2+)](i)) was measured by a dual-wavelength spectrometer system. TUNEL assay and flow cytometry were performed to measure percentage of apoptotic cells. A colorimetric assay was to measure caspases activities. RESULTS: Bufalin and cinobufagin inhibited proliferation of cancer cells at doses of 0.1, 1, or 10 microM after 2-4 days of culture. Cytotoxicity of bufalin and cinobufagin on the DU145 and LNCaP cells was dose-dependent. Bufalin or cinobufagin increased [Ca(2+)](i) and apoptosis in cancer cells after a 24-hr culture as well as caspase 3 activities in DU145 and PC3 cells and caspase 9 activities in LNCaP cells. CONCLUSIONS: Bufalin and cinobufagin may inhibit the proliferation of prostate cancer cell lines associated with sustained elevation of the [Ca(2+)](i) and that of apoptosis.     

The equilibrium of XIAP and Smac/DIABLO expression is gradually deranged during the development and progression of testicular germ cell tumours

Overexpression of the inhibitor of apoptosis protein (IAP) XIAP (BIRC4) and downregulation of its antagonist Smac/DIABLO (DIABLO) are associated with the onset and progression of various malignancies. In this study, real-time RT-PCR was used to quantify the mRNA expression of XIAP and Smac/DIABLO in normal testicular tissue (n = 19), testicular carcinoma in situ (CIS; n = 4), testicular seminomas (n = 64) and non-seminomatous germ cell tumours (NSGCT; n = 35). XIAP and Smac/DIABLO were commonly expressed in normal and malignant testicular tissue with no apparent differences in XIAP mRNA levels among the histologic subgroups. Smac/DIABLO levels, on the other hand, gradually decreased from normal testicular tissue to CIS and seminomas and finally to NSGCT (p < 0.001). An inverse trend was observed when calculating the XIAP-to-Smac/DIABLO ratio (p < 0.001). This ratio differed when comparing normal testicular tissue with CIS (p = 0.014), seminomas (p < 0.001) and NSGCT (p < 0.001) and when comparing seminomas with NSGCT (p = 0.002), whereas no such difference was observed between CIS and seminomas (p = 0.302). TGCT patients dichotomized by the overall median XIAP-to-Smac/DIABLO ratio were more likely to present with a high ratio in clinical stage (CS) III than in CS I or II (p = 0.034). These data indicate that the balance of mRNA expression between XIAP and Smac/DIABLO is altered in favour of antiapoptotic XIAP during the development and progression of TGCT. Thus the expression of proapoptotic Smac/DIABLO is lowest in NSGCT and stage III tumours.     

转染XIAP基因对丝裂霉素诱导膀胱癌细胞凋亡的影响

目的：探讨XIAP基因对低剂量丝裂霉素C(MMC)诱导膀胱癌细胞凋亡的影响。方法：脂质体介导XIAP基因转染膀胱癌T24细胞3天后．用逆转录聚合酶链反应(RT-PCR)检测XIAP基因的表达．用低剂量MMC诱导细胞凋亡启动,用MTT比色法分析检测癌细胞生长活性．用3末端脱氧核苷酰转移酶介导生物素标记法检测细胞凋亡率。结果：各组癌细胞在低剂量MMC的诱导下发生凋亡、与单用MMC组比较．转染XIAP基因的癌细胞组的凋亡率显著降低。结论：XIAP基因在癌细胞中的过度表达可以明显降低化疗药物诱导的膀胱癌细胞凋亡,其可能在膀胱癌多药耐药中起一定作用。     

靶向抑制XIAP基因后结肠癌细胞对凋亡诱导配体耐药性的变化

目的 观察应用短发夹RNA(shRNA)干扰质粒靶向抑制X连锁凋亡抑制蛋白(XIAP)基因后,结肠癌细胞SW1116对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)耐药性的变化.方法 采用脂质体包裹方法,将干扰质粒转染结肠癌细胞SW1116,48 h后检测转染前后XIAP蛋白表达和结肠癌细胞生长活性的变化;应用TRAIL药物25、50、100、200 μg/L梯度浓度作用于结肠癌细胞,24 h后噻唑蓝(MTT)比色法检测抑制XIAP表达后结肠癌细胞对TRAIL敏感性的变化,并利用蛋白印迹方法检测细胞内凋亡相关因子Caspase-3活性的改变.结果 转染干扰质粒后XIAP的表达能够得到有效抑制(抑制率60%),细胞的生长活性得到抑制(抑制率24%),结肠癌细胞对TRAIL的耐药得到逆转,对TRAIL的敏感性显著提高,在200μg/L浓度下细胞生长抑制率可达64%,肿瘤细胞内的Caspase-3的表达活性得到提高.结论 靶向抑制XIAP基因的表达能够恢复和增强结肠癌细胞对TRAIL的敏感性.     

肾细胞癌中X染色体相关的凋亡抑制蛋白的表达与细胞凋亡的关系

目的 :探讨X染色体相关的凋亡抑制蛋白 (XIAP)在肾细胞癌中的表达及其与细胞凋亡的关系。方法 :应用免疫组织化学技术和DNA原位末端标记技术 (TUNEL)检测 2 8例肾细胞癌中XIAP的表达与细胞凋亡。结果 :2 8例肾细胞癌XIAP阳性率为 5 7.1% ,9例正常肾组织XIAP阳性率为 11.1% ,差异有统计学意义 (P<0 .0 1)。XIAP表达与肾细胞癌病理类型、病理分期和病理分级无明显相关 (P >0 .0 5 )。XIAP表达与AI呈负相关 (P <0 .0 1)。结论 :XIAP在肾细胞癌中表达对判断肾细胞癌的生物学特性无明显价值 ,但却为靶向XIAP的抗肾细胞癌治疗提供了理论依据。     

胰腺癌中XIAP和Survivin的表达及其相关性

目的:研究X染色体连锁的凋亡抑制因子(XIAP)和SurvMn在胰腺癌中的表达并探讨二者之间的关系.方法:采用免疫组织化学法对26例胰腺癌组织中XIAP和Survivin的表达进行检测并积分;采用RT-PCT检测胰腺癌细胞Panc-1、AsPC-1中XIAP和Survivin基因的表达.结果:26例胰腺癌组织中XIAP和Survivin的阳性率分别为88.5%(23/26)和92.3%(24/26),其表达积分分别为7.40 4±4.12和8.53±3.83,二者共同阳性率为84.6%(22/26);表达强弱与肿瘤大小均无明显关系;分化越差的肿瘤,二者的表达越高(P<0.05).2株胰腺癌细胞中均有XIAP和Survivin的基因表达.XIAP和Survivin在胰腺癌组织和细胞中均存在相关性(P<0.05).结论:XIAP和Survivin的表达水平均与胰腺癌的分化程度有关,二者在胰腺癌组织和细胞中均存在相关性,可能在胰腺癌的转化以及耐药方面发挥重要作用.     

Synergy is achieved by complementation with Apo2L/TRAIL and actinomycin D in Apo2L/TRAIL-mediated apoptosis of prostate cancer cells: role of XIAP in resistance

BACKGROUND: Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis. METHODS: We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot. RESULTS: Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D. CONCLUSION: This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.     

Smac/Diablo与X-连锁凋亡抑制蛋白在ATP耗竭再恢复诱导肾小管上皮细胞凋亡中的作用

目的 研究线粒体蛋白Smac/Diablo和细胞X-连锁凋亡抑制蛋白(XIAP)在ATP耗竭及再恢复导致肾小管上皮细胞凋亡中的作用和机制。 方法 应用代谢抑制剂暂时性阻断人肾小管上皮细胞(HK-2细胞)内ATP的生成,再换用含糖的培养液使细胞内ATP再恢复,诱导肾小管上皮细胞凋亡。应用Hoechst33342检测肾小管上皮细胞凋亡的发生。用间接免疫荧光检测Smac/Diablo在细胞内的分布。分别提取胞质蛋白和细胞总蛋白,以Western印迹检测胞质中Smac/Diablo、XIAP和活化半胱氨酸天冬氨酸蛋白酶3前体(pro-caspase-3)的蛋白水平。 结果 肾小管上皮细胞内ATP耗竭及再恢复时,Hoechst33342染色可见HK-2细胞核固缩和凋亡小体的形成;间接免疫荧光可见Smac/Diablo由线粒体释放至胞质;Western印迹可见胞质内Smac/Diablo的含量增多( P < 0.01);XIAP和pro-caspase 3的蛋白水平降低(P < 0.05)。 结论 肾小管上皮细胞内ATP耗竭及再恢复时,Smac/Diablo释放至胞质,XIAP蛋白水平降低,进而激活caspase 3,介导肾小管上皮细胞凋亡。     

Effects of the proteasome inhibitor bortezomib on gene expression profiles of pancreatic cancer cells

BACKGROUND: Pancreatic cancer remains a highly chemoresistant malignancy. Gemcitabine is a widely used clinical chemotherapeutic agent against locally advanced and metastatic pancreatic cancer. Proteasome inhibitor bortezomib has been shown to result in enhanced cytotoxicity and apoptosis when used alone or in combination with gemcitabine in pancreatic cancer cell lines. MATERIALS AND METHODS: To determine the effect of bortezomib on gene expression profile of pancreatic adenocarcinoma cells with different sensitivity to gemcitabine, we used Affymetrix HG U133A 2.0 GeneChip (Santa Clara, CA) and measured changes induced by bortezomib in pancreatic cancer cell lines with high (BxPC-3) and low (PANC-1) sensitivity to gemcitabine, at time points 24 h. Selected genes were subsequently validated by quantitative real-time polymerase chain reaction. RESULTS: Forty-four common genes in both PANC-1 and BxPC-3 cells were identified as up-regulated (>3-fold) induced by bortezomib analyzed by microarray, which are associated with multiple cytotoxic and cytoprotective effects. Bcl-2 was repressed by bortezomib in both PANC-1 and BxPC-3 cells, while no changes induced in either cell by bortezomib were disclosed in all five members of nuclear factor-kappa B family. Other interesting genes related to apoptosis or drug metabolism, such as TP53 and ABCB1 (mdr1), were not found differentially expressed in common. CONCLUSIONS: Bortezomib exhibits antitumor effects toward pancreatic cancer in vitro and in vivo. Genes with divergent apoptotic effects are induced by bortezomib, which may become promising targets for pancreatic cancer treatment.     

龙葵碱对胰腺癌细胞裸鼠移植瘤的抑制作用及机制研究

目的 探讨龙葵碱对胰腺癌细胞SW1990裸鼠移植瘤的抑制作用并初步研究其作用机制。方法 建立胰腺癌细胞SW1990的裸鼠荷瘤模型,随机分为对照组和实验组,每组10只。肿瘤接种成功第2周起对照组和实验组分别腹腔注射溶媒（含1‰二甲亚砜）1 mL、龙葵碱10 mg/kg,隔天给药1次,共给药2周。每次给药结束用游标卡尺记录肿瘤大小。给药结束后颈椎脱臼处死裸鼠,测量瘤体重量并计算抑瘤率,留取新鲜肿瘤组织用RT-PCR和免疫组化检测Bcl-2、Bax和Caspase-3基因mRNA和蛋白表达情况。结果 与对照组相比,实验组裸鼠肿瘤生长受到显著抑制（P＜0.05）,实验组的肿瘤组织Bcl-2表达阳性率明显降低（实验组90% vs 对照组46%,P＜0.01）,Bax（实验组38% vs 对照组70%,P＜0.01）和Caspase-3（实验组 22% vs 对照组74%,P＜0.01）的表达阳性率明显升高。结论 龙葵碱在裸鼠体内能显著抑制人胰腺癌细 胞SW1990的增殖,此抑制作用呈明显的时间依赖性。龙葵碱发挥上述作用的机制可能在于诱导胰腺癌细胞凋亡。     

Cisplatin inhibits the expression of X-linked inhibitor of apoptosis protein in human LNCaP cells

The purpose of the present study is to investigate the role of X-linked inhibitor of apoptosis protein (XIAP) in the regulation of apoptosis induced by cisplatin in human prostate cancer cell line (LNCaP). We examined the effects of cisplatin on cell growth and apoptosis in LNCaP by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), flow cytometric analyses, and caspase-3 activity assay. In addition, to clarify the roles of the XIAP, we established clonal cell lines that overexpressed XIAP. The effects of cisplatin on the XIAP expression in the induction of apoptosis in LNCaP were examined by RT-PCR and immunoblot analyses. Although the growth rates were reduced in a dose- and time-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in XIAP stably overexpressing cell lines. In addition, we found that cisplatin-induced apoptosis following activation of caspase-3, and that the overexpression of XIAP inhibited apoptosis by attenuating caspase-3 activity. Interestingly, treatment of LNCaP cells with 10 and 100 μM cisplatin for 48 h significantly decreased the expression of XIAP at both the protein and mRNA levels in a dose-dependent manner. Furthermore, 10-μM cisplatin treatment of LNCaP decreased XIAP mRNA and protein in a time-dependent manner. These results suggest that cisplatin induces apoptosis by the inhibition of XIAP expression, and that XIAP plays an important role in the regulation of cisplatin-induced apoptosis in LNCaP cells. The ability of cisplatin to down-regulate XIAP may be an important mechanism in chemosensitivity.     

Smac基因诱导前列腺癌细胞凋亡的实验研究

目的：观察Smac基因对前列腺癌细胞凋亡的影响。方法：前列腺癌细胞株（PC-3）在含小牛血清的DMEM-F12培养基中培养、传代。通过脂质体介导将Smac基因导入前列腺癌细胞株，通过RT-PCR法检测SmacmRNA的表达，同时用SABC免疫组化法分析Smac的表达。采用细胞计数法检测细胞生长抑制率，流式细胞仪观察细胞凋亡情况。结果：转染Smac基因后，RT-PCR可检测出Smac的特异条带，SABC结果转染组Smae蛋白表达显著性增强，前列腺癌细胞的生长明显受抑制（P〈0．05）。细胞周期分析可见凋亡峰，凋亡率35．2％。结论：转入Smac基因可显著促进前列腺癌细胞细胞的凋亡。     

曲古抑菌素A诱导膀胱癌细胞p27kip1、XIAP表达的实验研究

目的 观察组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)对体外培养膀胱癌细胞生长情况及相关基因(p27kip1、XIAP)表达的影响,并探讨其可能的作用机制.方法 采用MTT法检测不同浓度TSA(100、200、400、800nmol/L)对人膀胱癌BIU-87细胞生长的影响;流式细胞术(FCM)检测TSA处理后膀胱癌细胞周期分布的变化及对膀胱癌细胞凋亡的影响;Western blot检测TSA作用BIU-87细胞后p27kip1、XIAP蛋白表达的变化.结果 TSA体外能明显抑制BIU-87细胞的生长,且抑制作用呈明显的剂量、时间依赖性.FCM检测示处理后膀胱癌细胞阻滞于G0-G1期,Western blot结果示TSA处理能显著诱导p27kip1蛋白的表达和显著抑制XIAP蛋白的表达.结论 TSA可通过诱导细胞凋亡,使细胞阻滞于G1期而发挥体外抗膀胱癌作用,其作用机制可能涉及相关基因(p27kip1、XIAP)表达的调控.     

南瓜蛋白诱导人胰腺癌细胞凋亡的线粒体机制

目的 探讨南瓜蛋白(CUS)诱导人胰腺癌细胞(PANC)-1凋亡的线粒体机制.方法 0、2.5、10.0、40.0 mg/L的CUS处理胰腺癌PANC-1 72 h,荧光显微镜和流式细胞仪观察线粒体膜电位(△Ψm)的变化,Western blot检测胞质细胞色素C(Cyt-C)的含量变化和细胞半胱氨酰天冬氨酸特异性蛋白酶-9(Caspase-9)和Caspase-3、B细胞淋巴瘤/白血病-2(bcl-2)和B细胞淋巴瘤/白血病-2相关X蛋白(bax)蛋白的表达水平.结果 0、2.5、10.0、40.0 mg/L CUS处理PANC-1 72 h后,△Ψm逐渐下降,分别为167.23±8.27、123.56±7.26、83.25±5.36和40.45±5.87,差异有统计学意义(P＜0.05);Western blot结果显示线粒体Cyt-C释放到细胞质的量逐渐增加,灰度值分别为0.29±0.05、1.69±0.35、1.87±0.40、2.47±0.32(P＜0.05);Caspase-9、Caspase-3和bax蛋白的表达逐渐增强,且其作用呈剂量依赖性(Caspase-9灰度值分别为0.15±0.03、0.19±0.05、0.49±0.09、0.89±0.08,P＜0.05;Caspase-3灰度值分别为0.88±0.08、1.81±0.19、2.36±0.38、2.92±0.24,P ＜0.05;bax灰度值分别为0.79±0.18、1.66±0.31、2.61±0.41、3.67±0.24,P＜0.05);而bcl-2蛋白表达无明显变化(bcl-2灰度值分别为0.88±0.08、0.82±0.18、0.84±0.04、0.82±0.13,P＞0.05).结论 线粒体途径在CUS诱导胰腺癌细胞凋亡中起重要作用,其机制可能是CUS通过激活促凋亡蛋白bax表达增加,降低△Ψm,促进线粒体Cyt-C的释放,从而激活Caspase系列酶,诱导胰腺癌细胞凋亡.