Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM_2.5) and cardiovascular disorders, but how PM_2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen‐activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM_2.5. H9c2 cells were incubated with PM_2.5 at 100–800 µg ml^–1 to evaluate the effects of PM_2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis‐related proteins as well as activation of MAPKs. PM_2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration‐dependent manner. PM_2.5 decreased the Bcl‐2/Bax ratio and increased cleaved caspase‐3 levels. A Western blots study showed up‐regulation of phosphorylated MAPKs including extracellular signal‐regulated protein kinases (ERKs), c‐Jun NH₂‐terminal kinases (JNKs) and p38 MAPK in the PM_2.5‐treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM_2.5 on apoptosis and the expression of related proteins. In conclusion, PM_2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM_2.5‐induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Thymoquinone suppresses the proliferation of renal cell carcinoma cells via reactive oxygen species‐induced apoptosis and reduces cell stemness

Thymoquinone is a phytochemical compound isolated from Nigella sativa and has various biological effects, including anti‐inflammation, antioxidation, and anticancer. Here, we further investigated the anticancer effects and associated molecular mechanism of 2‐methyl‐5‐isopropyl‐1,4‐benzoquinone (thymoquinone) on human renal carcinoma cell lines 786‐O and 786‐O‐SI3 and transitional carcinoma cell line BFTC‐909. Results showed that thymoquinone significantly reduced cell viability, inhibited the colony formation of renal cancer cells, and induced cell apoptosis and mitochondrial membrane potential change in both cancer cells. In addition, thymoquinone also triggered the production of reactive oxygen species (ROS) and superoxide and the activation of apoptotic and autophagic cascade. ROS inhibition suppressed the caspase‐3 activation and restored the decreased cell viability of 786‐O‐SI3 in response to thymoquinone. Autophagy inhibition did not restore the cell viability of 786‐O‐SI3 suppressed by thymoquinone. Moreover, thymoquinone suppressed the cell sphere formation and the expression of aldehyde dehydrogenase, Nanog, Nestin, CD44, and Oct‐4 in 786‐O‐SI3 cells. The tumor‐bearing model showed that thymoquinone in vivo inhibited the growth of implanted 786‐O‐SI3 cell. All these findings indicate that thymoquinone inhibits the proliferation of 786‐O‐SI3 and BFTC‐909 cell possibly due to the induction of ROS/superoxide and the consequent apoptosis, suggesting that thymoquinone may be a potential anticancer supplement for genitourinary cancer.     

二烯丙基二硫通过活性氧介导的JNK信号通路诱导人白血病K562细胞凋亡

目的研究二烯丙基二硫(DADS)诱导人白血病K562细胞凋亡的分子机制。方法应用MTT法检测细胞的活性;用流式细胞术检测细胞凋亡以及细胞内的活性氧(reac-tive oxygen species,ROS)水平;Western blot检测JNK以及磷酸化JNK的活化。结果 DADS能明显抑制K562细胞的增殖,呈时间和剂量依赖性;5.0 mg.L-1DADS处理K562细胞,细胞内ROS水平在1 h后明显增加,8 h达到高峰,随后又开始下降。随着DADS剂量的增加,JNK的活性明显增强,在DADS处理K562细胞8 h后,磷酸化的JNK达到最高值,而在随后的4 h又明显降低。Sp600125和NAC能明显减少磷酸化JNK的表达和抑制DADS诱导的细胞凋亡。结论 ROS是DADS诱导K562细胞凋亡过程中JNK活化的有效调节剂,DADS通过ROS介导的JNK活化诱导人白血病K562细胞凋亡。     

Chrysophanol‐induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential

Chrysophanol (1,8‐dihydroxy‐3‐methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol‐induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose‐ and time‐dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondria membrane potential (ΔΨ_m) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis‐associated protein levels including caspase‐3, caspase‐8, Apaf‐1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740–749, 2014.     

Perfluorooctane sulfonate induces apoptosis in lung cancer A549 cells through reactive oxygen species‐mediated mitochondrion‐dependent pathway

Perfluorooctane sulfonate (PFOS) is a widespread environmental contaminant that is detected in the lung of mammals. The mechanisms underlying PFOS‐induced lung cytotoxicity remain unclear. The main purpose of this study was to evaluate the cytotoxic effects of PFOS on human lung cancer A549 cells and its possible molecular mechanism. A549 cells were treated with PFOS (0, 25, 50, 100 and 200 μm ) and the cellular apoptosis, mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In this study, PFOS induced a dose‐dependent increase in A549 cell toxicity via an apoptosis pathway as characterized by increased percentage of sub‐G1, activation of caspase‐3 and ?9, and increased ratio of Bax/bcl‐2 mRNA expression. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N‐Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, can effectively block PFOS‐induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicate that PFOS induces apoptosis in A549 cells through a reactive oxygen species‐mediated mitochondrial dysfunction pathway mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

Apoptosis induced by para‐phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells

para‐phenylenediamine (p‐PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p‐PD‐induced cell death in normal Chang liver cells was investigated. The results demonstrated that p‐PD decreased cell viability in a dose‐dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub‐G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen‐activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p‐PD activated not only stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinases (JNK) and p38 MAPK but also extracellular signal‐regulated kinase (ERK). Cytotoxicity and apoptosis induced by p‐PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38‐specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p‐PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p‐PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p‐PD‐induced apoptosis. Western blot analysis revealed that p‐PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p‐PD and that a decreased ERK signal contributes to growth inhibition or apoptosis. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 981–990, 2014.     

D-氨基葡萄糖衍生物对Eca-109细胞内活性氧、线粒体膜电位的影响

目的：探讨2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖（2-[（3-carboxy-1-oxoprogy1）amino]-2-deoxy-D-Glucose,COPADG）诱导Eca-109细胞凋亡的机制。方法：不同浓度COPADG作用于人食管癌Eca-109细胞24h,检测Eca-109细胞的抑制率、凋亡率、细胞内活性氧（ROS）、线粒体跨膜电位。结果：Eca-109细胞凋亡率与COPADG浓度呈正相关（rs=1.0,P〈0.01）;Eca-109细胞线粒体膜电位水平与Eca-109细胞凋亡率相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞凋亡率呈正相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞线粒体膜电位水平呈负相关（rs=1.0,P〈0.01）。结论：COPADG可促进Eca-109细胞凋亡,提高Eca-109细胞内ROS水平,并降低线粒体膜电位水平。实验结果提示COPADG提高ROS水平,降低Eca-109细胞线粒体膜电位水平,启动细胞凋亡通路,促使Eca-109细胞凋亡,并且线粒体膜电位的下降是通过提高ROS实现的。     

Microcystin‐LR‐Caused ROS generation involved in p38 activation and tau hyperphosphorylation in neuroendocrine (PC12) cells

Microcystin‐LR (MC‐LR), a potent specific hepatotoxin produced by cyanobacteria, has recently been reported to show neurotoxicity. Our previous study demonstrated that MC‐LR caused the reorganization of cytoskeleton architectures and hyperphosphorylation of the cytoskeletal‐associated proteins tau and HSP27 in neuroendocrine PC12 cell line by direct PP2A inhibition and indirect p38 mitogen‐activated protein kinase (MAPK) activation. It has been shown that oxidative stress is extensively associated with MC‐LR toxicity, mainly resulting from an excessive production of reactive oxygen species (ROS). However, the mechanisms by which ROS mediates the cytotoxic action of MC‐LR are unclear. In the present study, we investigated whether ROS might play a critical role in MC‐LR‐induced hyperphosphorylation of microtubule‐associated protein tau and the activation of the MAPKs in PC12 cell line. The results showed that MC‐LR had time‐ and concentration‐dependent effects on ROS generation, p38‐MAPK activation and tau phosphorylation. The time‐course studies indicated similar biphasic changes in ROS generation and tau hyperphosphorylation, which started to increase within 1 h and reached the maximum level at 3 h followed by a decrease after prolonged treatment. Furthermore, pretreatment with the antioxidants, N‐acetylcysteine and vitamin C, significantly decreased MC‐LR‐induced ROS generation and effectively attenuated p38‐MAPK activation as well as tau hyperphosphorylation. Taken together, these findings suggest that ROS generation triggered by MC‐LR is a key intracellular event that contributes to an induction of p38‐MAPK activation and tau phosphorylation, and that blockade of this ROS‐mediated redox‐sensitive signal cascades may attenuate the toxic effects of MC‐LR. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 366–374, 2015.     

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U‐2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell‐cycle distribution, apoptotic cells in sub‐G1 phase, reactive oxygen species (ROS), Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). Comet assay and 4′‐6‐diamidino‐2‐phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis‐associated protein levels in U‐2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U‐2 OS cells. CECF‐stimulated activities of caspase‐8, caspase‐9, and caspase‐3, ROS, and Ca²⁺ production, decreased ΔΨ_m levels of in U‐2 OS cells. CECF increased protein levels of caspase‐3, caspase‐9, Bax, cytochrome c, GRP78, AIF, ATF‐6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell‐cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U‐2 OS cells via ROS‐modulated caspase‐dependent and ‐independent pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1020–1031, 2014.     

Reactive oxygen species mediate Terbufos‐induced apoptosis in mouse testicular cell lines via the modulation of cell cycle and pro‐apoptotic proteins

Terbufos (S‐t‐butylthiomethyl‐O,O‐diethyl phosphorodithioate) is a highly toxic organophosphate which is extensively used as an insecticide and nematicide. Chronic exposure to terbufos causes neuronal injury and predisposes to neurodegenerative diseases. Accumulating evidence has shown that the exposure to terbufos, as an occupational risk factor, may also cause reproductive disorders. However, the exact mechanisms of reproductive toxicity remain unclear. The present study aimed to investigate the toxic effect of terbufos on testicular cells and to explore the mechanism of toxicity on a cellular level. The cytotoxic effects of terbufos on mouse immortalized spermatogonia (GC‐1), spermatocytes (GC‐2), Leydig (TM3), and Sertoli (TM4) cell lines were assessed by MTT assays, caspase activation, flow cytometry, TUNEL assay, Western blot, and cell cycle analysis. The exposure to different concentrations of terbufos ranging from 50 to 800 μM for 6 h caused significant death in all the used testicular cell lines. Terbufos increased reactive oxygen species (ROS) production, reduced mitochondrial membrane potential, and initiated apoptosis, which was confirmed by a dose‐dependent increase in the number of TUNEL‐positive apoptotic cells. Blocking ROS production by N‐acetyl cysteine (NAC) protected GC‐1 cells from terbufos‐induced cell death. The results demonstrated that terbufos induces ROS, apoptosis, and DNA damage in testicular cell lines and it should be considered potentially hazardous to testis. Together, this study provided potential molecular mechanisms of terbufos‐induced toxicity in testicular cells and suggests a possible protective measure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1888–1898, 2016.     

Propyl gallate decreases the proliferation of Calu-6 and A549 lung cancer cells via affecting reactive oxygen species and glutathione levels

Propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester, PG) has an anti-proliferative effect in various cells. In this study, Calu-6 and A549 lung cancer cells were used to examine the anti-proliferative effect of PG in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG (100–1,600 μM) dose-dependently inhibited the proliferation of Calu-6 and A549 cells at 24 h, and PG at 800–1,600 μM strongly induced cell death in both cell lines. PG (800–1,600 μM) increased cellular metabolism in Calu-6 but not A549 cells at 4 h. PG either increased or decreased ROS levels, including O₂˙⁻ and ˙OH, depending on the incubation doses and times of 1 or 24 h. Even these effects differed between Calu-6 and A549 cell types. PG reduced the activity of superoxide dismutase (SOD) in Calu-6 cells, and it augmented the activity of catalase in A549 cells. PG dose-dependently increased the number of GSH depleted cells in both Calu-6 and A549 cells at 24 h. In addition, PG decreased GSH levels in both lung cancer cells at 1 h. Furthermore, diethyldithiocarbamate (DDC; an inhibitor of SOD) and 3-amino-1,2,4-triazole (AT; an inhibitor of catalase) differently affected cellular metabolism, ROS and GSH levels in PG-treated and PG-untreated Calu-6 and A549 cells at 1 h. In conclusion, PG dose-dependently decreased the proliferation of Calu-6 and A549 lung cancer cells, which was related to changes in ROS levels and the depletion of GSH.     

Multiwall carbon nano‐onions induce DNA damage and apoptosis in human umbilical vein endothelial cells

Growing evidence has indicated the potential adverse effects on cardiovascular system of some nanomaterials, including fullerenes. In this study, we have evaluated the biological effects of multiwall carbon nano‐onions (MWCNOs) (average size of 31.2 nm, ζ potential of 1.6 mV) on human umbilical vein endothelial cells (HUVECs). It was found that MWCNOs exhibited a dose‐dependent inhibitory effect on cell growth; EC₅₀ was 44.12 μg/mL. Thus, three concentrations were chosen (0.2, 1, and 5 μg/mL) for further experiments. Flow cytometry analysis revealed that 1 and 5 μg/mL MWCNOs could induce apoptosis in HUVECs, the apoptotic rates were 12% and 24% at 24 h after exposure. On the other hand, MWCNOs did not affect the cell cycle distribution during 24 h period. Using γH2AX foci formation as an indicator for DNA damage, it was shown that 5 μg/mL MWCNOs can induce γH2AX foci formation in HUVECs at 6, 12, and 24 h after treatment, whereas 0.2 μg/mL MWCNOs induced γH2AX foci formation only at 6 h after treatment. In addition, all three concentrations of MWCNOs induced the generation of reactive oxygen species (ROS), and inhibition of ROS generation can partially decrease the γH2AX foci formation induced by MWCNOs. Taken together, these data first suggested that MWCNOs can induce DNA damage and apoptosis in HUVECs, and that ROS might be involved in this process. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 442–450, 2013.     

Induction of apoptosis and reactive oxygen species production by N-nitrosopiperidine and N-nitrosodibutylamine in human leukemia cells

N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells.     

Identification and evaluation of 2 circulating microRNAs for non‐small cell lung cancer diagnosis

The circulating miRNAs are dysregulated in non‐small cell lung cancer (NSCLC) and have great promise for clinical diagnosis. The aim of this study was to screen and evaluate the reliability and diagnostic efficiency of candidate serum miRNAs for NSCLC diagnosis. We found that miR‐21 and let‐7a were significantly dysregulated in NSCLC patients (P < .001). When applied for NSCLC diagnosis, combined application of miR‐21 and let‐7a had a sensitivity of 0.790, specificity of 0.750, which is significantly higher than application alone of miR‐21(sensitivity 0.647; specificity 0.641), let‐7a (sensitivity 0.653; specificity 0.718) or computed tomography‐guided core‐needle biopsy (CTCB) (sensitivity 0.725; specificity 0.609) (P < .01). In conclusion, our study indicates that combined application of circulating miR‐21 and let‐7a has advantages over traditional CTCB technique.