Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.     

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.     

Artesunate induces apoptosis via a Bak-mediated caspase-independent intrinsic pathway in human lung adenocarcinoma cells

This report is designed to explore the exact molecular mechanism by which artesunate (ART), a semisynthetic derivative of the herbal antimalaria drug artemisinin, induces apoptosis in human lung adenocarcinoma (ASTC‐a‐1 and A549) cell lines. ART treatment induced ROS‐mediated apoptosis in a concentration‐ and time‐dependent fashion accompanying the loss of mitochondrial potential and subsequent release of Smac and AIF indicative of intrinsic apoptosis pathway. Blockage of casapse‐8 and ‐9 did not show any inhibitory effect on the ART‐induced apoptosis, but which was remarkably prevented by silencing AIF. Of the utmost importance, ART treatment induced the activation of Bak but not Bax, and silencing Bak but not Bax remarkably inhibited ART‐induced apoptosis and AIF release. Furthermore, although ART treatment did not induced a significant down‐regulation of voltage‐dependent anion channel 2 (VDAC2) expression and up‐regulation of Bim expression, silencing VDAC2 potently enhanced the ART‐induced Bak activation and apoptosis which were significantly prevented by silencing Bim. Collectively, our data firstly demonstrate that ART induces Bak‐mediated caspase‐independent intrinsic apoptosis in which Bim and VDAC2 as well as AIF play important roles in both ASTC‐a‐1 and A549 cell lines, indicating a potential therapeutic effect of ART for lung cancer. J. Cell. Physiol. 227: 3778–3786, 2012. © 2012 Wiley Periodicals, Inc.     

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.     

Isoegomaketone induces apoptosis through caspase-dependent and caspase-independent pathways in human DLD1 cells

Isoegomaketone (IK) is an essential oil component of Perilla frutescens (L.), but the mechanism by which IK induces apoptosis has never been studied. The purpose of this study was to elucidate the IK-induced apoptotic pathway in DLD1 human colon cancer cells. We observed that IK treatment over 24 h significantly inhibited cell viability in a dose-dependent manner. We also found that IK triggered cleavage of PARP. Moreover, IK treatment resulted in cleavage of caspase-8, -9, and -3 in a dose- and time-dependent manner. IK treatment also resulted in cleavage of Bid and translocation of Bax, and triggered the release of cytochrome c from the mitochondria to the cytoplasm. Furthermore, it resulted in the translocation of apoptosis inducing factor (AIF), a caspase-independent mitochondrial apoptosis factor, from the mitochondria into the nucleus. Overall, these results suggest that IK induces apoptosis through caspase-dependent and capase-independent pathways in DLD1 cells.     

Cardiac volume overload rapidly induces oxidative stress-mediated myocyte apoptosis and hypertrophy

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated the role of oxidative stress in the initial phases of cardiac remodeling induced in an animal model by volume overload. As plausible candidates for a connection between oxidative stress and cardiomyocyte apoptosis or hypertrophy, we explored the behaviour of two MAPKs, specifically JNK and ERK. At 48 h of overload, the greatest increase in oxidative stress coincided with a peak of cardiomyocyte apoptosis. This was possibly induced through the mitochondrial metabolism, as evidenced by the release of cytochrome c and a significant increase in the active forms of caspase-9 and -3, but not caspase-8. Oxidative stress markers significantly decreased at 96 h of overload, combined with a marked attenuation of apoptosis and the appearance of hypertrophy. The highest levels of JNK and the lowest levels of ERK phosphorylation were observed at 48 h of overload. Conversely, a sharp increase in ERK phosphorylation was detected at 96 h of overload coinciding with the hypertrophic response. Together these results show that oxidative stress is an early and transient event in myocardial volume overload. They suggest that oxidative stress mediates amplitude dependent apoptotic and hypertrophic responses in cardiomyocytes through the selective activation of, respectively, JNK and ERK.     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Resveratrol induces mitochondria-mediated AIF and to a lesser extent caspase-9-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Resveratrol (RV), a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts, has an ability to inhibit various stages of carcinogenesis in vitro and in vivo. In this report, we explored the roles of intrinsic and extrinsic apoptotic pathways during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. After exposure of cells to different concentrations of RV, we found that RV induced concentration-dependent apoptosis. Fluorometric substrates assay and western blotting (WB) analysis showed that caspase-8 was not activated, which was further verified by monitoring the cleavage of Bid to tBid using fluorescence resonance energy transfer (FRET) microscopy imaging inside single living cells, indicating that extrinsic apoptotic pathway was not involved in RV-induced apoptosis. In addition, inhibition of caspases-3 or -9 but not caspase-8 using the specific inhibitors of caspases modestly but significantly attenuated RV-induced apoptosis. Moreover, flow cytometry (FCM) analysis showed that RV treatment induced time-dependent loss of mitochondrial membrane potential (?ψ(m)), in combination with the activation of caspases-3 and -9; we therefore concluded that RV-induced apoptosis involved the intrinsic apoptotic pathway. It is noteworthy that RV treatment induced translocation of AIF from mitochondria to nucleus in a time dependent manner, and that knockdown of AIF remarkably attenuated RV-induced apoptosis. Collectively, our findings demonstrate that RV induces caspase-8-independent apoptosis via AIF and to a lesser extent caspase-9-dependent mitochondrial pathway in ASTC-a-1 cells.     

Role of AIF in human coronary artery endothelial cell apoptosis

Apoptosis-inducing factor (AIF), which exerts its effect via a caspase-independent pathway, has been suggested to be a mediator of cell injury. We have recently identified the expression of AIF in human coronary artery endothelial cells (HCAECs). The present study was designed to determine the pathophysiological role of AIF in oxidized low-density lipoprotein (ox-LDL)-induced apoptosis of HCAECs. The cells were cultured and treated with ox-LDL (40 microg/ml) for 24 h. Ox-LDL increased AIF expression, caused apoptosis of HCAECs (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and large-scale DNA fragmentation), and induced translocation of AIF from the cytoplasm to the nucleus (fluorescence immunocytochemistry). Pretreatment of HCAECs with a caspase inhibitor (ZVAD-fmk) did not influence AIF-mediated apoptosis in response to ox-LDL. We developed a specific antisense oligonucleotide targeted to the 5'-TCG CCG AAA TGT TCC GGT GTG GA-3' portion of the human AIF mRNA sequence (AIF-AS) to bind a complementary sequence overlapping the translational start site. Pretreatment of cells with the AIF-AS for 24 h resulted in suppression of ox-LDL-upregulated AIF protein, as measured by immunoblot analysis. AIF-AS also reduced apoptosis and AIF translocation (P < 0.01 vs. ox-LDL alone). Next, we constructed a recombinant AIF plasmid by inserting whole-length AIF cDNA into the expression vector pcDNA3.1 with a cytomegalovirus promoter. HCAECs transfected with plasmid showed a two- to fourfold increase in AIF expression, extensive apoptosis, and translocation of AIF from the cytoplasm to the nucleus. These results from two approaches indicate that AIF plays an important role in ox-LDL-induced endothelial injury.     

The rhizome of Trillium tschonoskii Maxim. extract induces apoptosis in human lung cancer cells

Trillium tschonoskii Maxim. has been used to treat several diseases including cancers in folk medicine. However, the mechanisms responsible for T. tschonoskii extract-induced apoptosis are not clear. This study was mainly undertaken to identify the major biochemical changes in a lung cancer cell line upon treatment with an T. tschonoskii extract (TTME), and to investigate the functional relationship between these changes. The n-butanol extract was used to evaluate the mechanism of induction of apoptosis in A549 human lung cancer cells and its effects on mitochondrial function and production of reactive oxygen species (ROS). The n-butanol extract of T. tschonoskii has cytotoxic, antiproliferative, and morphological effects on the lung cancer cell line. T. tschonoskii mainly leads to apoptosis of cancer cells with a concomitant increase in the release of cytochrome c and a loss of mitochondrial membrane potential in a dose-dependent manner. A rapid increase in the level of intracellular ROS and an accumulation of cells in the G2/M and S phase of the cell cycle were also observed in treated cells. These observations suggest that the n-butanol extract of T. tschonoskii has promising anticancer activities, which could be useful in cancer treatment.     

Buforin IIb induces endoplasmic reticulum stress-mediated apoptosis in HeLa cells

Buforin IIb, a novel cell-penetrating anticancer peptide derived from histone H2A, has been reported to induce mitochondria-dependent apoptosis in tumor cells. However, increasing evidence suggests that endoplasmic reticulum and mitochondria cooperate to signal cell death. In this study, we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization. Two-dimensional PAGE coupled with MALDI-TOF and western blot analysis showed that buforin IIb treatment of HeLa cells resulted in upregulation of ER stress proteins. PBA (ER stress inhibitor) and BAPTA/AM (Ca²⁺ chelator) pretreatment rescued viability of buforin IIb-treated cells through abolishing phosphorylation of SAPK/JNK and p38 MAPK. SP600125 (SAPK/JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria. Taken together, our data suggest that the ER stress pathway has an important role in the buforin IIb-induced apoptosis in HeLa cells.     

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

Crystalline silica induces apoptosis in human endothelial cells in vitro

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.     

Increased oxidative stress induces apoptosis in human cystic fibrosis cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells.