Sestrin2通过抑制砷化物诱导的氧化应激反应发挥拮抗细胞凋亡的保护性作用

目的:探讨Sestrin2在砷化物诱导细胞凋亡反应中的作用及机制。方法:体外培养人肝癌细胞HepG2,以砷化物为刺激源,用免疫印迹和RT-PCR方法检测Sestrin2在砷化物刺激HepG2细胞前后的表达水平差异;用流式细胞术检测敲低Sestrin2前后以及抑制氧化应激反应前后细胞凋亡水平变化情况;用活性氧(ROS)检测试剂盒分析敲低Sestrin2前后细胞在砷化物刺激后的氧化应激水平变化情况。结果:砷化物刺激HepG2细胞后Sestrin2表达水平显著上调;敲低HepG2细胞中Sestrin2表达水平后,细胞凋亡水平明显升高,说明Sestrin2的诱导表达是砷化物诱导细胞凋亡反应中的保护性事件;在敲低Sestrin2表达水平后,砷化物诱导的ROS产生效应和氧化应激反应程度明显加剧;抗氧化剂NAC能够显著逆转Sestrin2对细胞凋亡的保护性效应。结论:Sestrin2在砷化物诱导细胞凋亡反应中可通过抑制ROS产生而发挥拮抗细胞凋亡的保护性作用。     

1-磷酸鞘氨醇改善缺氧诱导的肺上皮细胞损伤

目的:如何减轻缺氧造成的肺损伤是平原人群进入高原环境时面临的难题。本研究旨在探索外源性1-磷酸鞘氨醇(S1P)对低氧暴露诱导肺上皮细胞损伤的改善作用。方法:对肺上皮细胞(BEAS 2B细胞)进行4 h不同浓度的S1P预处理,之后放入低氧培养箱(氧气浓度为1%)模拟24 h和48 h的低氧暴露,检测细胞的增殖活性、早期凋亡以及线粒体相关功能;通过实时荧光定量PCR检测受体基因(S1PR1-3)的表达水平。结果:外源性S1P预处理可在BEAS 2B细胞中显著提高S1PR3的表达水平;对于24 h-48 h的急性低氧暴露,给予1μM浓度的S1P预处理时对细胞具有显著的保护作用,主要表现在线粒体功能改善、细胞增殖活性提升及早期凋亡率下降,包括:线粒体膜电位(MMP)和三磷酸腺苷(ATP)水平显著升高(P<0.0005),线粒体活性氧(ROS)产生显著减少(P<0.0001),从而显著提高了细胞的增殖活性(P<0.005),并降低早期凋亡率。结论:外源性S1P预处理能通过改善低氧诱导的氧化应激损伤保护肺上皮细胞。S1P在预防急性高原病、改善高原反应方面具有潜在应用价值。     

敲低额外11-19白血病融合基因蛋白通过激活线粒体通路诱导非小细胞肺癌A549细胞凋亡

目的 探讨额外11-19白血病融合基因蛋白（extra eleven-nineteen leukemia fusion gene protein, EEN）对非小细胞肺癌（non-small cell lung cancer, NSCLC）的影响及其作用机制。方法 用免疫组织化学染色检测EEN在NSCLC组织和癌旁组织的表达水平,Western blot检测EEN在正常肺上皮细胞BEAS-2B细胞和NSCLC细胞A549细胞中的水平。在A549细胞中转染sh-EEN后,使用MTT测定细胞活性,Annexin V-FITC/PI流式细胞术和TUNEL染色分析细胞凋亡,使用荧光探针DCFH-DA测量细胞内ROS生成水平,使用JC-1染色测定线粒体膜电位的变化,通过Western blot分析凋亡相关蛋白水平。结果 与癌旁组织相比,EEN在NSCLC组织中表达上调;与BEAS-2B细胞相比,EEN在A549细胞中水平明显升高。沉默A549细胞EEN后,A549细胞生长受到抑制,细胞凋亡显著增加,ROS水平升高,线粒体膜电位丢失,Cyt C释放入细胞质,同时伴随着Bcl-2降低、Bax升高和C...     

ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究

该文研究了活性氧(reactive oxygen species, ROS)在镉(Cadmium, Cd)诱导HK-2细胞氧化损伤和凋亡中的作用。不同浓度CdCl2处理HK-2细胞不同时间后,通过MTT法、DCFH-DA标记、JC-1染色、彗星实验和流式细胞术分别检测细胞活性、ROS、线粒体膜电位Δψm、DNA损伤及细胞凋亡情况。结果显示, CdCl2处理引起HK-2细胞形态皱缩、变圆,活性下降,且呈时间和剂量依赖性; CdCl2处理导致ROS水平升高、线粒体膜电位Δψm下降、DNA损伤和caspase-3活化,最终导致细胞凋亡,且60μmol/L处理组及高浓度组与对照组相比差异极显著(P0.01)。采用ROS清除剂NAC与CdCl2共处理细胞24 h,发现细胞形态明显恢复、ROS水平显著降低、线粒体膜电位Δψm显著升高、彗星尾部长度和DNA百分比显著下降、凋亡细胞减少(P0.01)。综上所述, ROS介导了Cd诱导的HK-2细胞氧化损伤和凋亡。     

亚硒酸钠诱导SW480细胞凋亡过程中活性氧的作用

为探讨亚硒酸钠诱导人结肠癌SW480细胞凋亡的机理,将荧光探针2′,7′－二氯荧光黄乙二脂（2′,7′－DCFH－DA）、罗丹明123（rhodamine123）负载人结肠癌细胞,利用多光子成像系统测定胞内活性氧（ROS）、线粒体跨膜电位（△Ψm）的变化。结果发现（1）Na2SeO3作用SW480细胞,可导致细胞凋亡和胞内的ROS增加。SOD、过氧化氢酶可降低凋亡率并抑制ROS的增加。（2）线粒体电子传递链抑制剂鲁藤酮及氰化钠可抑制OS增加。（3）Na2SeO3可导致线粒体的跨膜电位的下降。表明Na2SeO3作用细胞可导致来源于线粒体的ROS增加,ROS介导亚硒酸钠诱导细胞凋亡。     

缺氧条件下GC-1细胞凋亡的作用机制研究

以小鼠精原细胞系GC-1细胞为研究对象,探讨缺氧条件下GC-1细胞凋亡潜在的分子机制。首先,采用CCK-8 (Cell Counting Kit-8)法检测不同缺氧时间处理下的细胞活力,以确定细胞缺氧损伤的时间;然后,通过化学荧光法检测GC-1细胞中活性氧(reactive oxygen species, ROS)的含量,采用JC-1法检测线粒体的膜电位,采用比色法检测ATP的含量,采用线粒体荧光探针法检测线粒体的数量与分布,并采用透射电镜观察细胞的超微结构;最后,利用实时荧光定量PCR检测线粒体信号通路相关基因胱天蛋白酶(caspase)-3、caspase-9、细胞色素c (cytochrome c, Cyt-c)、Bax和Bcl-2的m RNA表达水平。结果显示,缺氧36 h后,细胞活力为(60.36±5.40)%,符合后续实验需求;在缺氧条件下, GC-1细胞中的ROS含量显著增加, ATP含量显著下降,线粒体膜电位下降、数量减少,同时细胞中促凋亡相关基因的表达水平上调,抗凋亡因子Bcl-2的基因表达水平下调。实验结果初步表明,缺氧可导致GC-1细胞线粒体功能障碍, ROS/线...     

Genistein对卵巢癌铂类耐药细胞株CP70增殖凋亡的影响及与细胞内ROS的相关性

目的:探讨Genistein对卵巢癌铂类耐药细胞CP70增殖、凋亡的影响及与细胞内活性氧水平的关系。方法:采用MTT法检测Genistein对CP70细胞增殖的影响;流式细胞仪分析不同药物处理后对细胞凋亡的影响,线粒体膜电位及细胞内ROS水平的变化情况。结果:Genistein对CP70细胞增殖表现出剂量和时间依赖性的抑制作用,并能诱导其凋亡;Genistein作用于CP70细胞后,可使其线粒体膜电位降低,并引发了细胞内ROS水平的显著升高;ROS抑制剂NAC预处理CP70细胞后,有效抑制了ROS的产生,并降低了细胞凋亡率,与未加NAC组相比差异有显著性(P0.05)。结论:Genistein能抑制铂类耐药卵巢癌细胞CP70的增殖,并促进其凋亡,这与细胞内ROS水平的升高有关,可能是Genistein抗肿瘤诱导细胞凋亡的机制之一。     

IscU2对非小细胞肺癌增殖、迁移、侵袭能力的影响及其作用机制的研究

该文通过shRNA干扰技术敲低IscU2干扰细胞IscU2的表达,研究了干扰IscU2对非小细胞肺癌(NSCLC)细胞NCI-H520增殖、迁移及侵袭能力的影响。构建了稳定低表达IscU2的非小细胞肺癌细胞系NCI-H520;采用CCK-8和平板克隆实验检测细胞的增殖能力;流式细胞仪检测细胞周期、凋亡、ROS、线粒体膜电位变化情况;Transwell实验检测细胞迁移及侵袭能力;Western blot检测相关蛋白的表达。结果表明,干扰IscU2后,非小细胞肺癌细胞的增殖及克隆形成能力降低;细胞周期停滞在G1/G0期,同时伴随有p-AKT和Cyclin D1蛋白含量的下降;细胞晚期凋亡率明显增加,凋亡蛋白Cleaved-caspase3和Cleaved-PARP表达上调;细胞迁移和侵袭能力降低,上皮标志物E-Cadherin表达上调,间质标志物N-Cadherin和Snail表达下调;细胞ROS积累和线粒体膜电位下降。该研究结果表明,干扰IscU2显著抑制非小细胞肺癌的增殖、迁移、侵袭能力和上皮–间质转化,这为非小细胞肺癌的诊断和治疗提供了新的潜在靶点和视角。     

大蒜素诱导人食管癌EC-109细胞凋亡

为寻找毒副作用小并且治疗效果好的抗癌药物,研究大蒜素对人食管癌EC-109细胞凋亡的影响,同时探讨了大蒜素引发细胞凋亡的可能机制。通过激光共聚焦显微镜观察细胞形态变化,琼脂糖凝胶电泳检测DNA片段化情况,流式细胞术检测细胞凋亡率和线粒体膜电位变化,qRT-PCR和Western blotting检测细胞凋亡相关基因Bax、Bcl-2的mRNA和蛋白表达水平。结果显示,大蒜素作用人食管癌EC-109细胞48 h后,线粒体膜电位显著降低,并且早期凋亡细胞和晚期凋亡细胞所占百分比均显著增加。同时,与对照组相比,Bax mRNA和蛋白水平均显著升高(p<0.05),Bcl-2 mRNA和蛋白水平均显著降低(p<0.05)。据此,本研究得出大蒜素可诱导人食管癌EC-109细胞凋亡,并呈剂量依赖性,有潜在的药用价值。     

华蟾素对MDA - MB -231细胞线粒体膜电位及活性氧的影响

目的:研究华蟾素诱导乳腺癌MDA - MB - 231细胞凋亡过程中,细胞内活性氧(ROS)及线粒体膜电位(△Ψm)的变化,探讨华蟾素对乳腺癌细胞的作用机制.方法:用不同浓度的华蟾素作用于MDA - MB - 231细胞24h后,分别用荧光探针罗丹明123和荧光探针DCFH-DA进行荧光染色,用流式细胞仪检测细胞内线粒体膜电位和活性氧的变化.结果:不同浓度的华蟾素作用于MDA - MB - 231细胞后,随着药物浓度的增加(0、12.5、25、37.5、50μg/ml),细胞内的ROS水平显著升高,荧光强度从3 609±24上升为6 263±35;同时,线粒体膜电位(△Ψm)显著下降,荧光强度从242±6降低到173±4.结论:华蟾素作用细胞后,使得细胞内活性氧水平显著升高,同时,线粒体膜电位显著下降,推测华蟾素对MDA - MB - 231细胞通过线粒体途径诱发细胞凋亡.     

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.     

Silencing FUNDC1 alleviates chronic obstructive pulmonary disease by inhibiting mitochondrial autophagy and bronchial epithelium cell apoptosis under hypoxic environment

Chronic obstructive pulmonary disease (COPD) is a major global epidemic with increasing incidence worldwide. The pathogenesis of COPD is involved with mitochondrial autophagy. Recently, it has been reported that FUN14 domain containing 1 (FUNDC1) is a mediator of mitochondrial autophagy. Therefore, we hypothesized that FUNDC1 was involved in cigarette smoke (CS)-induced COPD progression by regulating mitochondrial autophagy. In vitro cigarette smoke extract (CSE)-treated human bronchial epithelial cell (hBEC) Beas-2B cell line and in vivo CS-induced COPD mouse models were developed, in which FUNDC1 expression was measured. Next, whether FUNDC1 interacted with dynamin-related protein 1 (DRP1) in COPD was investigated. The functional mechanism of FUNDC1 in COPD was evaluated through gain- or loss-of-function studies. Then, pulmonary function, mitochondrial transmembrane potential (MTP) and mucociliary clearance (MCC) were examined. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and expression of autophagy-specific markers (light chain 3 [LC3] II, LC3 I, and Tom20) were measured. Finally, apoptosis and mitochondrial autophagy were assessed. FUNDC1 was highly expressed in CSE-treated hBECs and COPD mice. Meanwhile, FUNDC1 was proved to interact with DRP1 in CSE-treated cells. Moreover, in CSE-treated hBECs, silencing FUNDC1 was observed to reduce levels of IL-6 and TNF-α, and MTP but increase MCC, and inhibit CSE-induced mitochondrial autophagy and Beas-2B cell apoptosis, which was consistent with the trend in COPD mouse models. In addition, pulmonary function of COPD mouse models was increased in response to FUNDC1 silencing. Finally, silencing of DRP1 also inhibited mitochondrial autophagy and Beas-2B cell apoptosis. Collectively, FUNDC1 silencing could suppress the progression of COPD by inhibiting mitochondrial autophagy and hBEC apoptosis through interaction with DRP1, highlighting a potential therapeutic target for COPD treatment.     

Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real‐time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45 min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage‐independent clathrin‐mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨ_m). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N‐acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.     

Honokiol ameliorates cigarette smoke-induced damage of airway epithelial cells via the SIRT3/SOD2 signalling pathway

Cigarette smoking can cause damage of airway epithelial cells and contribute to chronic obstructive pulmonary disease (COPD). Honokiol is originally isolated from Magnolia obovata with multiple biological activities. Here, we investigated the protective effects of honokiol on cigarette smoke extract (CSE)-induced injury of BEAS-2B cells. BEAS-2B cells were treated with 300 mg/L CSE to construct an in vitro cell injury model, and cells were further treated with 2, 5 and 10 μM honokiol, then cell viability and LDH leakage were analysed by CCK-8 and LDH assay kits, respectively. Apoptosis was detected by flow cytometry analysis. ELISA was used to measure the levels of tumour necrosis factor (TNF)-ɑ, IL-1β, IL-6, IL-8 and MCP-1. The results showed that honokiol (0.5–20 μM) showed non-toxic effects on BEAS-2B cells. Treatment with honokiol (2, 5 and 10 μM) reduced CSE (300 mg/L)-induced decrease in cell viability and apoptosis in BEAS-2B cells. Honokiol also decreased CSE-induced inflammation through inhibiting expression and secretion of inflammatory cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-8 and MCP-1. Moreover, honokiol repressed CSE-induced reactive oxygen species (ROS) production, decrease of ATP content and mitochondrial biogenesis, as well as mitochondrial membrane potential. Mechanistically, honokiol promoted the expression of SIRT3 and its downstream target genes, which are critical regulators of mitochondrial function and oxidative stress. Silencing of SIRT3 reversed the protective effects of honokiol on CSE-induced damage and mitochondrial dysfunction in BEAS-2B cells. These results indicated that honokiol attenuated CSE-induced damage of airway epithelial cells through regulating SIRT3/SOD2 signalling pathway.     

白藜芦醇对高糖条件下大鼠晶状体上皮细胞线粒体活性氧产生及内质网的影响

目的：探讨白藜芦醇（resveratrol,Res）对高糖条件下大鼠晶状体上皮细胞（LECs）凋亡、线粒体活性氧产生以及内质网表达的影响。方法：用含30 mmol·L-1葡萄糖浓度的培养基体外培养LECs,随后加入25 mg·L-1Res共培养48 h。流式细胞术检测LECs细胞凋亡情况和线粒体膜电位的变化。激光共聚焦显微镜观察线粒体活性氧变化情况,并用免疫组化法检测内质网表达。结果：在高糖培养条件下,与对照组相比,LECs死亡率明显增高,线粒体膜电位降低,活性氧增多。内质网阳性率明显下降。经Rev干预后,细胞凋亡率显著降低,线粒体膜电位和内质网阳性率均升高,活性氧产生明显减少（P〈0.05）。结论：白藜芦醇能在一定程度上减轻糖尿病性白内障大鼠晶状体凋亡的发生并维持正常细胞器功能,从而延缓白内障的发生和发展。     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.     

Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells

We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4-6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5-9% and 5-20% of complex-I activity and 8-75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ρ(0) cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells.