Adiponectin and colon cancer: evidence for inhibitory effects on viability and migration of human colorectal cell lines

Exogenous hydrogen peroxide (H₂O₂) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H₂O₂-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H₂O₂ inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H₂O₂-treated lung cancer cells. H₂O₂ increased intracellular ROS levels, including that of O ₂ ^·? , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H₂O₂ triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O ₂ ^·? levels in H₂O₂-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O ₂ ^·? , in H₂O₂-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H₂O₂-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H₂O₂ induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H₂O₂-induced oxidative stress and GSH depletion.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX–MAPK pathway

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP–human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal–regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.     

Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure

Cadmium (Cd) is one of the most toxic heavy metal pollutants in the aquatic environment and can induce the formation of reactive oxygen species (ROS) that cause oxidative stress. In present study, we cloned catalase (CAT) and glutathione peroxidase (GPX) cDNA, and investigated its time- and dose-related effects of three Cd concentrations (0.01, 0.05 or 0.1 ppm) on mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), CAT, GPX) in the gill and changes enzyme levels in the hemolymph of the Pacific oyster, Crassostrea gigas. The cDNA indentified encoded proteins of 516 and 244 amino acids corresponding to CAT and GPX, respectively. BLAST analysis from other species indicated that the residues essential to the enzymatic function of CAT and GPX proteins of C. gigas are highly conserved. Cd treatment significantly increased antioxidant enzyme mRNA expression in the gill in a time- and dose-dependent manner. The mRNA expression at 0.1 ppm Cd concentration increased up to 3 days (CAT, GPX) or 7 days (SOD) and then decreased by 7 days (CAT, GPX) or 11 days (SOD). Aspartate aminotransferase, alanine amintransferase and hydrogen peroxide (H(2)O(2)) concentrations levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results suggest that antioxidant enzymes play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.     

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.     

2,3,7,8-tetrachlorobenzo-p-dioxin inhibits proliferation of SK-N-SH human neuronal cells through decreased production of reactive oxygen species

Oxidative stress has been known to be involved in the mechanism of toxic effects of various agents on many cellular systems. In this study we investigated the role of reactive oxygen species (ROS) in 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced neuronal cell toxicity using SK-N-SH human neuroblastoma cells. TCDD inhibited proliferation of the cells in a dose-dependent manner, which was revealed by MTT staining, counting of cells stained with trypan blue and [ 3 H]thymidine uptake assay. TCDD also suppressed the basal generation of ROS in a time- and concentration-dependent manner assessed by 2',7'-dichlorofluorescein fluorescence. In addition, TCDD induced a dose-dependent inhibition of lipid peroxidation, a biomarker of oxidative stress, whereas it significantly increased the level of glutathione (GSH), an intracellular free radical scavenger in the cells. Moreover, TCDD altered the activities of major antioxidant enzymes; increase in superoxide dismutase (SOD) and catalase, but decrease in glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Red). Pretreatment with l -buthionine- S , R -sulfoximine (BSO, 50 μM), an inhibitor of GSH synthesis, significantly prevented the TCDD-induced reduction in lipid peroxidation and cell proliferation. Interestingly, exogenous application of an oxidant, H 2 O 2 (50 μM) markedly restored the inhibited cell proliferation induced by TCDD. Taken together, these results suggest that alteration of cellular redox balance may mediate the TCDD-induced inhibition of proliferation in human neuronal cells.     

Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells

Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H₂O₂-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H₂O₂ in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H₂O₂-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC_1,2 receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.     

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.     

The effects of MAPK inhibitors on antimycin A-treated Calu-6 lung cancer cells in relation to cell growth, reactive oxygen species, and glutathione

Antimycin A (AMA) inhibits succinate oxidase, NADH oxidase, and mitochondrial electron transport chain between cytochrome b and c. We recently demonstrated that AMA inhibited the growth of Calu-6 lung cancer cells through apoptosis. Here, we investigated the effects of AMA and/or MAPK inhibitors on Calu-6 lung cancer cells in relation to cell growth, cell death, reactive oxygen species (ROS), and GSH levels. Treatment with AMA inhibited the growth of Calu-6 cells at 72 h. AMA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). While ROS were decreased in AMA-treated Calu-6 cells, O ₂ ^?? among ROS was increased. AMA also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor intensified cell death, MMP (ΔΨ_m) loss, and GSH depletion in AMA-treated Calu-6 cells. JNK inhibitor also increased cell death, MMP (ΔΨ_m) loss, and ROS levels in these cells. Treatment with p38 inhibitor magnified cell growth inhibition by AMA and increased cell death, MMP (ΔΨ_m) loss, ROS level, and GSH depletion in AMA-treated cells. Conclusively, all the MAPK inhibitors slightly intensified cell death in AMA-treated Calu-6 cells. The changes of ROS and GSH by AMA and/or MAPK inhibitors were in part involved in cell growth and death in Calu-6 cells.     

CT2-3, a novel magnolol analogue suppresses NSCLC cells through triggering cell cycle arrest and apoptosis

Magnolol, a major bioactive component found in Magnolia officinalis with anti-inflammation and anti-oxidation activities as well as minimized cytotoxic effects. Although magnolol has a wide range of clinical applications, the anti-tumor activity of magnolol is not efficient. Herein, we reported the synthesis and anti-cancer activities of three novel magnolol analogues CT2-1, CT2-2, CT2-3, among which CT2-3 revealed more efficient anti-non-small cell lung cancer (NSCLC) activity than magnolol. Our data showed that CT2-3 could significantly inhibit the proliferation of human NSCLC cells in a dose-dependent manner. In addition, we revealed CT2-3 could induce cell cycle arrest through down-regulating mRNA expression of CDK4, CDK6 and cyclin D1. Moreover, we verified that CT2-3 could cause ROS generation, leading to apoptosis of human NSCLC cells. Further more, we also provided strong evidences that CT2-3 down-regulates the expression of c-Myc and topoisomerases, and contributes to the apoptosis of human NSCLC cells. Taken together, the current study is the first to report a promising new chemotherapeutic drug candidate CT2-3 that can efficiently eliminate human NSCLC cells through triggering cell cycle arrest as well as ROS-mediated and c-Myc/topoisomerases-mediated apoptosis.     

Retracted: Sapylin inhibits lung cancer cell proliferation and promotes apoptosis by attenuating PI3K/AKT signaling

Sapylin (OK-432) revealed biological properties in cancers. In this study, the effect of sapylin on lung cancer cell A549 was investigated. A549 cell lines were treated with sapylin (0.1, 0.5, and 1 KE/mL) for different time intervals. A549 cell proliferation and apoptosis was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide/Ki67 assay and flow cytometry, respectively. Western blot was used to determine the expressions of proteins involved in proliferation, apoptosis, and phosphoinositide 3-kinase/serine/threonine kinase (PI3K/AKT), Wnt3a/β-catenin signaling pathway. Level of intracellular reactive oxygen species (ROS) was insured by using the ROS kit. Sapylin inhibited A549 cell viability and the expressions of proliferation-related proteins (cyclin E1 and D1) in dose- and time-dependent manners. Sapylin promoted apoptosis in a dose- and time-dependent manners. Sapylin also promoted the expressions of apoptotic proteins (cleaved caspase-3 and 8) in dose- and time-dependent manners. Furthermore, sapylin increased the intracellular concentration of ROS in a dose-dependent manner. Besides, the high expression of ROS level might induce inhibition of cell viability and increase cell apoptosis. The mechanistic study revealed that sapylin inactivated the PI3K/AKT and Wnt3a/β-catenin signaling pathways. Our findings suggest that sapylin inhibits proliferation and promotes apoptosis in lung cancer cells, thus providing a new theoretical basis for the treatment of lung cancer.     

Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-alpha led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-alpha-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.     

2,3,7,8-Tetrachlorobenzo- p -dioxin Inhibits Proliferation of SK-N-SH Human Neuronal Cells Through Decreased Production of Reactive Oxygen Species

Oxidative stress has been known to be involved in the mechanism of toxic effects of various agents on many cellular systems. In this study we investigated the role of reactive oxygen species (ROS) in 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced neuronal cell toxicity using SK-N-SH human neuroblastoma cells. TCDD inhibited proliferation of the cells in a dose-dependent manner, which was revealed by MTT staining, counting of cells stained with trypan blue and [ 3 H]thymidine uptake assay. TCDD also suppressed the basal generation of ROS in a time- and concentration-dependent manner assessed by 2',7'-dichlorofluorescein fluorescence. In addition, TCDD induced a dose-dependent inhibition of lipid peroxidation, a biomarker of oxidative stress, whereas it significantly increased the level of glutathione (GSH), an intracellular free radical scavenger in the cells. Moreover, TCDD altered the activities of major antioxidant enzymes; increase in superoxide dismutase (SOD) and catalase, but decrease in glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Red). Pretreatment with l -buthionine- S , R -sulfoximine (BSO, 50 &#119 M), an inhibitor of GSH synthesis, significantly prevented the TCDD-induced reduction in lipid peroxidation and cell proliferation. Interestingly, exogenous application of an oxidant, H 2 O 2 (50 &#119 M) markedly restored the inhibited cell proliferation induced by TCDD. Taken together, these results suggest that alteration of cellular redox balance may mediate the TCDD-induced inhibition of proliferation in human neuronal cells.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1–SIRT2 protein. Purified PEP-1–SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H₂O₂)-induced cell death and cytotoxicity. Also, transduced PEP-1–SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1–SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1–SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1–SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1–SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.     

Apoptosis in arsenic trioxide-treated Calu-6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.     

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2′,7′-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 μM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.