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Sample records for mitochondrial apoptotic pathways

  1. Carbon disulfide induces rat testicular injury via mitochondrial apoptotic pathway.

    PubMed

    Guo, Yinsheng; Wang, Wei; Dong, Yu; Zhang, Zhen; Zhou, Yijun; Chen, Guoyuan

    2014-08-01

    Carbon disulfide (CS2), one of the most important volatile organic chemicals, was shown to have serious impairment to male reproductive system. But the underline mechanism is still unclear. In the present study, we aim to investigate the male germ cell apoptosis induced by CS2 exposure alone and by co-administration with cyclosporin A (CsA), which is the inhibitor of membrane permeability transition pore (MPTP). It was shown that CS2 exposure impaired ultrastructure of germ cells, increased the numbers of apoptotic germ cells, accumulated intracellular level of calcium, elevated ROS level, and increased activities of complexes of respiratory chain. Meanwhile, exposure to CS2 dramatically decreased the mitochondrial transmembrane potential (ΔΨm) and levels of ATP and MPTP opening. Exposure to CS2 can also cause a significantly dose-dependent increase in the expression levels of Bax, Cytc, Caspase-9, and Caspase-3, but decreased the expression level of Bcl-2. Moreover, co-administration of CsA with CS2 can reverse or alleviate the above apoptotic damage effects of CS2 on testicular germ cells. Taken together, our findings suggested that CS2 can cause damage to testicular germ cells via mitochondrial apoptotic pathway, and MPTP play a crucial role in this process. PMID:24582363

  2. The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage

    PubMed Central

    Varanita, Tatiana; Soriano, Maria Eugenia; Romanello, Vanina; Zaglia, Tania; Quintana-Cabrera, Rubén; Semenzato, Martina; Menabò, Roberta; Costa, Veronica; Civiletto, Gabriele; Pesce, Paola; Viscomi, Carlo; Zeviani, Massimo; Di Lisa, Fabio; Mongillo, Marco; Sandri, Marco; Scorrano, Luca

    2015-01-01

    Summary Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo. PMID:26039448

  3. BCDO2 acts as a carotenoid scavenger and gatekeeper for the mitochondrial apoptotic pathway

    PubMed Central

    Lobo, Glenn P.; Isken, Andrea; Hoff, Sylvia; Babino, Darwin; von Lintig, Johannes

    2012-01-01

    Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as β,β-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway. PMID:22764054

  4. Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells.

    PubMed

    Predescu, Sanda A; Predescu, Dan N; Knezevic, Ivana; Klein, Irene K; Malik, Asrar B

    2007-06-01

    Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway. PMID:17405881

  5. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    NASA Astrophysics Data System (ADS)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  6. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease

    PubMed Central

    Wang, Yan; Cai, Biao; Shao, Jing; Wang, Ting-ting; Cai, Run-ze; Ma, Chang-ju; Han, Tao; Du, Jun

    2016-01-01

    Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer’s disease. A rat model of Alzheimer’s disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25–35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer’s disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genistein in Alzheimer’s disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  7. DIEPOXYBUTANE ACTIVATES THE MITOCHONDRIAL APOPTOTIC PATHWAY AND MEDIATES APOPTOSIS IN HUMAN LYMPHOBLASTS THROUGH OXIDATIVE STRESS

    PubMed Central

    Yadavilli, Sridevi; Martinez-Ceballos, Eduardo; Snowden-Aikens, Janana; Hurst, Angela; Joseph, Tranole; Albrecht, Thomas; Muganda, Perpetua M.

    2007-01-01

    Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1, 3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multi-systems organ toxicity. We recently reported that DEB-induced cell death in TK6 lymphoblasts was due to the occurrence of apoptosis, and not necrosis. In this study, we investigated the molecular mechanisms responsible for DEB-induced apoptosis in these cells. Bax and Bak were found to be over-expressed and activated, and the mitochondrial trans-membrane potential was attenuated in cells undergoing DEB-induced apoptosis. Cytochrome c was depleted from the mitochondria of TK6 cells undergoing apoptosis, and was released into the cytosol in Jurkat-T lymphoblasts exposed to the same concentrations of DEB. Executioner caspase 3 was deduced to be activated by initiator caspase 9. DEB induced reactive oxygen species (ROS) formation, and the ROS scavenger N-acetyl-L-cysteine effectively blocked DEB-induced apoptosis in TK6 cells. Collectively, these results demonstrate that the mitochondrial apoptotic pathway is activated to mediate DEB-induced apoptosis in human TK6 lymphoblasts. These results further demonstrate that DEB-induced apoptosis is also mediated by the DEB-induced generation of ROS. This is the first report to examine the mechanism of DEB-induced apoptosis in human lymphoblasts. PMID:17693053

  8. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation

    PubMed Central

    Moncunill-Massaguer, Cristina; Saura-Esteller, José; Pérez-Perarnau, Alba; Palmeri, Claudia Mariela; Núñez-Vázquez, Sonia; Cosialls, Ana M.; González-Gironès, Diana M.; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-01-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa−/−/Bim−/− MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway. PMID:26497683

  9. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation.

    PubMed

    Moncunill-Massaguer, Cristina; Saura-Esteller, José; Pérez-Perarnau, Alba; Palmeri, Claudia Mariela; Núñez-Vázquez, Sonia; Cosialls, Ana M; González-Gironès, Diana M; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-12-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway. PMID:26497683

  10. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Hsu, Shu-Shong; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Tseng, Hui-Wen; Kuo, Chun-Chi; Jan, Chung-Ren

    2015-01-01

    Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca(2+) levels ([Ca(2+)]i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca(2+)]i rises which were reduced by removing extracellular Ca(2+). Eugenol-induced [Ca(2+)]i rises were not altered by store-operated Ca(2+) channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca(2+)]i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca(2+)]i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca(2+)]i rises by inducing PLC-dependent release of Ca(2+) from the endoplasmic reticulum and caused Ca(2+) influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway. PMID:25455450

  11. Sequence and expression variations in 23 genes involved in mitochondrial and non-mitochondrial apoptotic pathways and risk of oral leukoplakia and cancer.

    PubMed

    Datta, Sayantan; Ray, Anindita; Singh, Richa; Mondal, Pinaki; Basu, Analabha; De Sarkar, Navonil; Majumder, Mousumi; Maiti, Guruparasad; Baral, Aradhita; Jha, Ganga Nath; Mukhopadhyay, Indranil; Panda, Chinmay; Chowdhury, Shantanu; Ghosh, Saurabh; Roychoudhury, Susanta; Roy, Bidyut

    2015-11-01

    Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer. PMID:26403071

  12. HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway

    PubMed Central

    Tian, Xin; Zhao, Lei; Song, Xianjing; Yan, Youyou; Liu, Ning; Li, Tianyi; Yan, Bingdi

    2016-01-01

    Objectives. Elevated plasma homocysteine (Hcy) could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27), a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs) and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO) level, increase of endothelin-1 (ET-1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway. PMID:27190988

  13. Activation of the Mitochondrial Apoptotic Pathway Produces Reactive Oxygen Species and Oxidative Damage in Hepatocytes That Contribute to Liver Tumorigenesis.

    PubMed

    Hikita, Hayato; Kodama, Takahiro; Tanaka, Satoshi; Saito, Yoshinobu; Nozaki, Yasutoshi; Nakabori, Tasuku; Shimizu, Satoshi; Hayashi, Yoshito; Li, Wei; Shigekawa, Minoru; Sakamori, Ryotaro; Miyagi, Takuya; Hiramatsu, Naoki; Tatsumi, Tomohide; Takehara, Tetsuo

    2015-08-01

    Chronic hepatitis, including viral hepatitis and steatihepatitis, is a well-known high-risk condition for hepatocellular carcinoma. We previously reported that continuous hepatocyte apoptosis drives liver tumors in hepatocyte-specific Bcl-xL or Mcl-1 knockout mice. In this study, we further examine the underlying cellular mechanisms of generating tumors in apoptosis-prone liver. In cultured hepatocytes, the administration of ABT-737, a Bcl-xL/-2/-w inhibitor, led to production of reactive oxygen species (ROS) as well as activation of caspases. Mitochondria isolated from murine liver, upon administration of truncated-Bid, a proapoptotic Bcl-2 family protein, released cytochrome c and produced ROS, which was dependent on mitochondrial respiration. Hepatic apoptosis, regeneration, accumulation of oxidative damages, and tumorigenesis observed in hepatocyte-specific Mcl-1 knockout mice were substantially attenuated by further deficiency of Bax or Bid, suggesting that a balance of mitochondrial Bcl-2 family proteins governs generation of oxidative stress and other pathologies. Whole-exome sequencing clarified that C>A/G>T transversion, which is often caused by oxidative DNA damage in proliferating cells, was a frequently observed mutation pattern in liver tumors of Mcl-1 knockout mice. The administration of antioxidant L-N-acetylcysteine did not affect apoptosis, compensatory regeneration, or fibrotic responses but significantly reduced oxidative DNA damage and incidence and multiplicity of live tumors in Mcl-1 knockout mice. In conclusion, activation of the mitochondrial apoptotic pathway in hepatocytes accumulates intracellular oxidative damages, leading to liver tumorigenesis, independently of liver regeneration or fibrosis. This study supports a concept that antioxidant therapy may be useful for suppressing liver carcinogenesis in patients with chronic liver disease. PMID:26038117

  14. Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells.

    PubMed

    Grebenová, Dana; Kuzelová, Katerina; Smetana, Karel; Pluskalová, Michaela; Cajthamlová, Hana; Marinov, Iuri; Fuchs, Ota; Soucek, Josef; Jarolím, Petr; Hrkal, Zbynek

    2003-02-01

    We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway. PMID:12633980

  15. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    PubMed Central

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  16. Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.

    PubMed

    Shan, Ming; Fan, Ting-Jun

    2016-09-01

    Carteolol is a frequently used nonselective β-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and -3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway. PMID:27216471

  17. A Small Molecule That Protects the Integrity of the Electron Transfer Chain Blocks the Mitochondrial Apoptotic Pathway.

    PubMed

    Jiang, Xian; Li, Li; Ying, Zhengxin; Pan, Chenjie; Huang, Shaoqiang; Li, Lin; Dai, Miaomiao; Yan, Bo; Li, Ming; Jiang, Hui; Chen, She; Zhang, Zhiyuan; Wang, Xiaodong

    2016-07-21

    In response to apoptotic stimuli, mitochondria in mammalian cells release cytochrome c and other apoptogenic proteins, leading to the subsequent activation of caspases and apoptotic cell death. This process is promoted by the pro-apoptotic members of the Bcl-2 family of proteins, such as Bim and Bax, which, respectively, initiate and execute cytochrome c release from the mitochondria. Here we report the discovery of a small molecule that efficiently blocks Bim-induced apoptosis after Bax is activated on the mitochondria. The cellular target of this small molecule was identified to be the succinate dehydrogenase subunit B (SDHB) protein of complex II of the mitochondrial electron transfer chain (ETC). The molecule protects the integrity of the ETC and allows treated cells to continue to proliferate after apoptosis induction. Moreover, this molecule blocked dopaminergic neuron death and reversed Parkinson-like behavior in a rat model of Parkinson's disease. PMID:27447985

  18. A methoxy derivative of resveratrol analogue selectively induced activation of the mitochondrial apoptotic pathway in transformed fibroblasts

    PubMed Central

    Gosslau, A; Chen, M; Ho, Ci-T; Chen, K Y

    2005-01-01

    Resveratrol (R-3), a trihydroxy trans-stilbene from grape, inhibits multistage carcinogenesis in animal models. A resveratrol derivative 3,4,5,4′-tetrahydroxystilbene (R-4) exhibits potent growth inhibitory effect against transformed human cells. Here we report that 3,4,5,4′-tetramethoxystilbene (MR-4), converted from R-4, was more potent against cancer cell lines (WI38VA, IMR-90SV, HeLa, LNCaP, HT-29, and HepG2), but had almost no inhibitory effect on the growth of normal cells (WI38, IMR-90, BJ-T) at the concentrations tested. The IC50 value of MR-4 on the growth inhibition of transformed WI38VA human cells was 0.5 μM, as compared to the value of greater than 50 μM for the normal WI38 cells. Resveratrol, however, did not exhibit such clear differential effect and the IC50 value of R-3 for WI38VA cells was about 50 μM. The growth inhibitory effect of MR-4 correlated with the induction of apoptosis in the transformed cells. When normal WI38 cells and transformed WI38VA cells were compared, MR-4 induced increases of the Bax/Bcl-2 mRNA ratio, p53 and Bax protein level, activation of caspases, and DNA fragmentation in transformed, but not in normal cells. Further analysis revealed that MR-4 caused a rapid appearance of perinuclear aggregation of mitochondria in WI38VA but not in WI38 cells, suggesting that the mitochondria could serve as an early target of MR-4. R-3 also induced apoptosis and mitochondrial clustering but only at a much higher concentration, close to 500 μM. Taken together, the specific activation of the mitochondria-mediated apoptotic pathway could be a major reason for the striking differential growth inhibitory effect of MR-4. PMID:15668717

  19. Mitochondrial Apoptotic Pathway Is Activated by H2O2-Mediated Oxidative Stress in BmN-SWU1 Cells from Bombyx mori Ovary

    PubMed Central

    Chen, Peng; Hu, Yan-Fen; Wang, La; Xiao, Wen-Fu; Bao, Xi-Yan; Pan, Chun; Yi, Hua-Shan; Chen, Xiang-Yun; Pan, Min-Hui; Lu, Cheng

    2015-01-01

    Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 μM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 μM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects. PMID:26225758

  20. Mitochondrial Apoptotic Pathway Is Activated by H2O2-Mediated Oxidative Stress in BmN-SWU1 Cells from Bombyx mori Ovary.

    PubMed

    Chen, Peng; Hu, Yan-Fen; Wang, La; Xiao, Wen-Fu; Bao, Xi-Yan; Pan, Chun; Yi, Hua-Shan; Chen, Xiang-Yun; Pan, Min-Hui; Lu, Cheng

    2015-01-01

    Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 μM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 μM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects. PMID:26225758

  1. Sinulariolide induced hepatocellular carcinoma apoptosis through activation of mitochondrial-related apoptotic and PERK/eIF2α/ATF4/CHOP pathway.

    PubMed

    Chen, Yi-Jen; Su, Jui-Hsin; Tsao, Chia-Yu; Hung, Chun-Tzu; Chao, Hsiang-Hao; Lin, Jen-Jie; Liao, Ming-Hui; Yang, Zih-Yan; Huang, Han Hisang; Tsai, Feng-Jen; Weng, Shun-Hsiang; Wu, Yu-Jen

    2013-01-01

    Sinulariolide, an active compound isolated from the cultured soft coral Sinularia flexibilis, has potent anti-microbial and anti-tumorigenesis effects towards melanoma and bladder cancer cells. In this study, we investigated the effects of sinulariolide on hepatocellular carcinoma (HCC) cell growth and protein expression. Sinulariolide suppressed the proliferation and colony formation of HCC HA22T cells in a dose-dependent manner and induced both early and late apoptosis according to flow cytometry, Annexin V/PI stain and TUNEL/DAPI stain analyses. A mechanistic analysis demonstrated that sinulariolide-induced apoptosis was activated through a mitochondria-related pathway, showing up-regulation of Bax, Bad and AIF, and down- regulation of Bcl-2, Bcl-xL, MCl-1 and p-Bad. Sinulariolide treatment led to loss of the mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and caspase-3. Sinulariolide-induced apoptosis was significantly blocked by the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. The increased expression of cleaved PARP also suggested that caspase-independent apoptotic pathway was involved. In the western blotting; the elevation of ER chaperones GRP78; GRP94; and CALR; as well as up-regulations of PERK/eIF2α/ATF4/CHOP; and diminished cell death with pre-treatment of eIF2α phosphatase inhibitor; salubrinal; implicated the involvement of ER stress-mediated PERK/eIF2α/ATF4/CHOP apoptotic pathway following sinulariolide treatment in hepatoma cells. The current study suggested sinulariolide-induced hepatoma cell cytotoxicity involved multiple apoptotic signal pathways. This may implicate that sinulariolide is a potential compound for the treatment of hepatocellular carcinoma. PMID:23973991

  2. RhoBTB2 (DBC2) functions as a multifunctional tumor suppressor in thyroid cancer cells via mitochondrial apoptotic pathway

    PubMed Central

    Wang, Chuan-Jiang; Yang, Dong; Luo, Ying-Wei

    2015-01-01

    Thyroid cancer is the most common endocrine malignancy worldwide. Tumor suppressor gene RhoBTB2 (also known as Deleted in Breast Cancer 2, DBC2) was observed in various carcinomas, however, no reports showed the effects of RhoBTB2 on thyroid cancer. In our study, we found that RhoBTB2 decreases proliferation, increases apoptosis, inhibits mobility, and induces mitochondria damage in SW579 cells through increased Bax and decreased Bcl-2 and Bcl-xL protein expression. The effects of RhoBTB2 on SW579 cells were inversed by using butin (an inhibitor of the mitochondrial apoptosis pathway). Our results suggest that RhoBTB2 suppresses the growth of SW579 cells through a mitochondrial apoptosis pathway. PMID:26131191

  3. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways

    PubMed Central

    YUAN, HONGYAN; WANG, JUAN; WANG, FENGLI; ZHANG, NANNAN; LI, QIONGSHU; XIE, FEI; CHEN, TANXIU; ZHAI, RUIPING; WANG, FANG; GUO, YINGYING; NI, WEIHUA; TAI, GUIXIANG

    2015-01-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  4. Zinc deficiency mediates alcohol-induced apoptotic cell death in the liver of rats through activating ER and mitochondrial cell death pathways

    PubMed Central

    Sun, Qian; Zhong, Wei; Zhang, Wenliang; Li, Qiong; Sun, Xiuhua; Tan, Xiaobing; Sun, Xinguo; Dong, Daoyin

    2015-01-01

    Hepatic zinc deficiency has been well documented in alcoholic patients, but the mechanisms by which zinc deficiency mediates cell death have not been well defined. The objectives of this study were to determine whether alcohol perturbs subcellular zinc homeostasis and how organelle zinc depletion may link with cell death pathways. Wistar rats were pair-fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure significantly reduced zinc level in isolated hepatic endoplasmic reticulum (ER) and mitochondria. Among the detected zinc transporters, ER Zrt/Irt-like protein (ZIP)13 and mitochondrial ZIP8, which transport zinc from ER and mitochondria to cytosol, were significantly increased. Mitochondrial zinc transporter (ZnT) 4, which transports zinc from cytosol to mitochondria, was also increased. ER phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4, and C/EBP homologous protein were significantly upregulated, and mitochondrial cytochrome c release and Bax insertion were detected in association with caspase-3 activation and apoptotic cell death. To define the role of zinc deficiency in ER and mitochondrial stress, H4IIEC3 cells were treated with 3 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine for 6 h with or without supplementation with zinc or N-acetylcysteine (NAC). The results demonstrated that zinc deprivation induced caspase-3 activation and apoptosis in association with ER and mitochondria dysfunction, which were inhibited by zinc as low as 10 μM but not by 2 mM NAC. These results suggest that chronic ethanol exposure induced in ER and mitochondrial zinc deficiency might activate intrinsic cell death signaling pathway, which could not be effectively rescued by antioxidant treatment. PMID:25767260

  5. Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK.

    PubMed

    Dalal, Suman; Zha, Qinqin; Singh, Mahipal; Singh, Krishna

    2016-07-01

    Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK. PMID:27262843

  6. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    SciTech Connect

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-09-15

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

  7. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  8. Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

    SciTech Connect

    Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H.-H.; Chang, Chia-Che; Lee, Tsung-Han

    2014-09-15

    Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.

  9. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    PubMed Central

    2011-01-01

    Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-)C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. Conclusions The

  10. Synthesis of Apoptotic New Quinazolinone-Based Compound and Identification of its Underlying Mitochondrial Signalling Pathway in Breast Cancer Cells.

    PubMed

    Zahedifard, Maryam; Faraj, Fadhil Lafta; Paydar, Mohammadjavad; Looi, Chung Yeng; Hasandarvish, Pouya; Hajrezaie, Maryam; Kamalidehghan, Behnam; Majid, Nazia Abdul; Khalifa, Shaden A M; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen; El-Seedi, Hesham R

    2015-01-01

    The anti-carcinogenic effect of the new quinazolinone compound, named MMD, was tested on MCF-7 human breast cancer cell line. The synthesis of quinazolinone-based compounds attracted strong attention over the past few decades as an alternative mean to produce analogues of natural products. Quinazolinone compounds sharing the main principal core structures are currently introduced in the clinical trials and pharmaceutical markets as anti-cancer agents. Thus, it is of high clinical interest to identify a new drug that could be used to control the growth and expansion of cancer cells. Quinazolinone is a metabolite derivative resulting from the conjugation of 2-aminobenzoyhydrazide and 5-methoxy-2- hydroxybenzaldehyde based on condensation reactions. In the present study, we analysed the influence of MMD on breast cancer adenoma cell morphology, cell cycle arrest, DNA fragmentation, cytochrome c release and caspases activity. MCF-7 is a type of cell line representing the breast cancer adenoma cells that can be expanded and differentiated in culture. Using different in vitro strategies and specific antibodies, we demonstrate a novel role for MMD in the inhibition of cell proliferation and initiation of the programmed cell death. MMD was found to increase cytochrome c release from the mitochondria to the cytosol and this effect was enhanced over time with effective IC50 value of 5.85 ± 0.71 μg/mL detected in a 72-hours treatment. Additionally, MMD induced cell cycle arrest at G0/G1 phase and caused DNA fragmentation with obvious activation of caspase-9 and caspases-3/7. Our results demonstrate a novel role of MMD as an anti-proliferative agent and imply the involvement of mitochondrial intrinsic pathway in the observed apoptosis. PMID:25808938

  11. Puerarin attenuates glucocorticoid-induced apoptosis of hFOB1.19 cells through the JNK- and Akt-mediated mitochondrial apoptotic pathways.

    PubMed

    Yu, Dongdong; Mu, Shuai; Zhao, Danyang; Wang, Guangbin; Chen, Zhiguang; Ren, Hongfei; Fu, Qin

    2015-08-01

    Puerarin is an active component of Pueraria lobata, which is a commonly used Chinese herbal medicine for the treatment of osteoporosis. The present study aimed to evaluate the osteoprotective effect of puerarin on glucocorticoid (GC)-induced apoptosis of osteoblasts in vitro. The effects of puerarin on dexamethasone (DEX)-induced cell apoptosis were assessed using enzyme-linked immunosorbent assay and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and found that the viability of hFOB1.19 cells was significantly increased following exposure to between 10(-6) and 10(-10) M puerarin, with a maximal anti-apoptotic effect at a concentration of 10(-8) M. In addition, compared with the control group, puerarin upregulated the transcription and protein levels of B-cell lymphoma-2 and downregulated B-cell-associated X protein in the hFOB1.19 cells. Puerarin attenuated the DEX-induced release of cytochrome c and cleavage of caspase-3, and treatment with puerarin inhibited the c-Jun N-terminal kinase (JNK) pathway and activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in the hFOB1.19 cells. Furthermore, the Akt inhibitor, LY294002, partly eliminated the protective effect of puerarin on DEX-induced apoptosis, and puerarin combined with the JNK inhibitor, SP600125, suppressed DEX-induced apoptosis to a lesser extent than in the cells treated with SP600125 alone. These results suggested that the JNK and PI3K/Akt signaling pathways mediate the inhibitory effects of puerarin on apoptosis in the hFOB1.19 cells. In conclusion, puerarin prevented DEX-induced apoptosis of hFOB1.19 cells via inhibition of the JNK pathway and activation of the PI3K/Akt signaling pathway in the cells, dependent on the mitochondrial apoptotic pathway. These results support puerarin as a promising target in the treatment of GC-induced osteoporosis. PMID:26101183

  12. HIF-1α inhibition by 2-methoxyestradiol induces cell death via activation of the mitochondrial apoptotic pathway in acute myeloid leukemia.

    PubMed

    Zhe, Nana; Chen, Shuya; Zhou, Zhen; Liu, Ping; Lin, Xiaojing; Yu, Meisheng; Cheng, Bingqing; Zhang, Yaming; Wang, Jishi

    2016-06-01

    The bone marrow microenvironment plays an important role in the development and progression of AML. Leukemia stem cells are in a hypoxic condition, which induces the expression of HIF-1α. Aberrant activation of HIF-1α is implicated in the poor prognosis of patients with acute myeloid leukemia (AML). Herein, we investigated the expression of HIF-1α in AML and tested 2-methoxyestradiol (2ME2) as a candidate HIF-1α inhibitor for the treatment of AML. We found that HIF-1α was overexpressed in AML. HIF-1α suppression by 2ME2 significantly induced apoptosis of AML cells, and it outperformed traditional chemotherapy drugs such as cytarabine. At the same time, 2ME2 downregulated the transcriptional levels of VEGF, GLUT1 and HO-1 in cellular assays. Additionally, 2ME2 displayed antileukemia activity in bone marrow blasts from AML patients, but showed little effect on normal cells. 2ME2-induced activation of mitochondrial apoptotic pathway is mediated by reactive oxygen species (ROS), which decreased the slight effect of drug on normal cells. Our data show that supression of HIF-1α expression significantly reduced the survival of AML cell lines, suggesting that 2ME2 may represent a powerful therapeutic approach for patients with AML. PMID:27082496

  13. Involvement of the mitochondrial apoptotic pathway and nitric oxide synthase in dopaminergic neuronal death induced by 6-hydroxydopamine and lipopolysaccharide.

    PubMed

    Singh, Sarika; Kumar, Sachin; Dikshit, Madhu

    2010-01-01

    The primary pathology in Parkinson's disease patients is significant loss of dopaminergic neurons in the substantia nigra through multiple mechanisms. We previously have demonstrated the involvement of nitric oxide (NO) in the dopaminergic neurodegeneration induced by 6-hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) in rats. The present study was undertaken to investigate further the role of NO in the mitochondria-mediated apoptosis of dopaminergic neurons during the early time period after administration of 6-OHDA and LPS. Measurement of dopamine and its metabolites, TH immunolabeling, cytochrome-c release, mitochondrial complex-I and caspase-3 activity assessment was performed in both the 6-OHDA- and LPS-induced experimental models of Parkinson's disease. Significant decreases in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), tyrosine hydroxylase (TH) immunolabeling and mitochondrial complex-I activity were observed, with increase in cytochrome-c release and caspase-3 activation. Dopmaine and its metabolite levels, mitochondrial complex-I activity and caspase-3 activity were significantly reversed with treatment of the NOS inhibitor, L-NAME. The reduction in the extent of cytochrome-c release responded variably to NOS inhibition in both the models. The results obtained suggest that NO contributes to mitochondria-mediated neuronal apoptosis in the dopaminergic neurodegeneration induced by 6-OHDA and LPS in rats. PMID:20594414

  14. Ethanol extract of Forsythia suspensa root induces apoptosis of esophageal carcinoma cells via the mitochondrial apoptotic pathway

    PubMed Central

    ZHAO, LIANMEI; YAN, XI; SHI, JUAN; REN, FENGZHI; LIU, LIHUA; SUN, SHIPING; SHAN, BAOEN

    2015-01-01

    Forsythia suspensa root is used in the treatment of fever and jaundice in Traditional Chinese Medicine. In the present study, the anti-tumor activity of the ethanolic extract of Forsythia suspensa root (FSREE) against esophageal carcinoma cells was investigated in vitro and in vivo and its anti-cancer mechanism was examined. The results revealed that FSREE, rather than Forsythia suspensa ethanolic extracts from the leaf (FSLEE) and fruit (FSFEE) exhibited marked anti-tumor activity towards human esophageal cancer cells. FSREE induced cancer cell apoptosis and growth arrest by downregulating B-cell lymphoma (Bcl)-2, Bcl-extra large and myeloid cell leukemia 1, while upregulating Bcl-2-associated X protein, Bcl-2 antagonist of cell death and phorbol-12-myristate-13-acetate-induced protein 1. This led to the activation of poly(ADP ribose) polymerase, caspase-3 and caspase-9, but not caspase-8. Furthermore, the anti-cancer activity of FSREE was associated with a decreased level of phosphorylated Janus kinase/signal transducer and activator of transcription 3 and extracellular-signal-regulated kinase signaling activity. It was also observed that the levels of cytochrome c were elevated in the cytoplasm, accounting for the loss of mitochondrial membrane potential in the TE-13 cells upon treatment with FSEER. In addition, FSEER inhibited the growth of esophageal cancer cells in xenograft models and no detectable toxicity was present in the lung or liver tissues. These observations provided further evidence of the anti-tumor effect of FSEER and may be of importance to further examine the potential role of Forsythia suspensa root as a therapeutic agent in esophageal carcinoma therapy. PMID:25373392

  15. Organization of the Mitochondrial Apoptotic BAK Pore

    PubMed Central

    Aluvila, Sreevidya; Mandal, Tirtha; Hustedt, Eric; Fajer, Peter; Choe, Jun Yong; Oh, Kyoung Joon

    2014-01-01

    The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.” PMID:24337568

  16. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells

    SciTech Connect

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ahmad, Javed; Siddiqui, Maqsood A.; Dwivedi, Sourabh; Khan, Shams T.; Musarrat, Javed

    2013-12-01

    localization of NPs. • ZnFe{sub 2}O{sub 4}-NPs induce DNA damage and mitochondrial dysfunction in WISH cells. • ZnFe{sub 2}O{sub 4}-NPs activate inflammatory and oxidative stress signaling in WISH cells. • Elevation of p53, CASP 3, bax and bcl 2 genes affirms intrinsic apoptotic pathway.

  17. UVA1 radiation triggers two different final apoptotic pathways.

    PubMed

    Godar, D E

    1999-01-01

    Because ultraviolet-A1 (UVA1; 340-400 nm) radiation is used therapeutically, this in vitro study addressed the question "how does it work?" To begin addressing this question, UVA1 radiation was first established to reduce the survival of transformed T and B lymphocytes in a linear dose-dependent manner using clonogenic reproductive assays, and that cell death occurs by apoptosis using transmission electron microscopy, Annexin V, and flow cytometry. The primary mechanism was determined to be immediate pre-programmed cell death, an apoptotic mechanism that does not require protein synthesis post-insult, by quantifying the apoptotic cells over time in the absence or presence of a translation inhibitor. To explore how UVA1 radiation induces immediate pre-programmed cell death apoptosis, reactive oxygen species and mitochondrial activity were altered during exposure using a variety of agents, while a specific fluorescent probe, 5,5',6,6'tetrachloro- 1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, was used to examine mitochondrial transmembrane depolarization. To show that UVA1 mediates singlet-oxygen damage to the mitochondrial membranes, X-rays, UVB (290-320 nm), 8-methoxypsoralen and UVA, vitamin K3, anti-Fas antibody, and blocking antibody were the negative controls, while rose bengal or protoporphyrin IX with visible light were the positive controls. Cyclosporine A, which inhibits the mitochondrial megapore from opening, was used with singlet-oxygen and superoxide-anion generators to distinguish between the two final apoptotic pathways. The collective results show that UVA1 radiation primarily mediates singlet-oxygen damage triggering immediate pre-programmed cell death apoptosis (T < 20 min) by immediately opening the cyclosporine A-sensitive ("S" site) mitochondrial megapore, while superoxide anions initiate another cyclosporine A-insensitive ("P" site) final apoptotic pathway. PMID:9886256

  18. Endoplasmic reticulum stress-induced apoptotic pathway and mitochondrial dysregulation in HeLa cells treated with dichloromethane extract of Dillenia suffruticosa

    PubMed Central

    Wan Nor Hafiza, Wan Abd Ghani; Yazan, Latifah Saiful; Tor, Yin Sim; Foo, Jhi Biau; Armania, Nurdin; Rahman, Heshu Sulaiman

    2016-01-01

    Ethyl acetate and dichloromethane extract of Dillenia suffruticosa (EADS and DCMDS, respectively) can be a potential anticancer agent. The effects of EADS and DCMDS on the growth of HeLa cervical cancer cells and the expression of apoptotic-related proteins had been investigated in vitro. Cytotoxicity of the extracts toward the cells was determined by 5-diphenyltetrazolium bromide assay, the effects on cell cycle progression and the mode of cell death were analyzed by flow cytometry technique, while the effects on apoptotic-related genes and proteins were evaluated by quantitative real-time polymerase chain reaction, and Western blot and enzyme-linked immunosorbent assay, respectively. Treatment with DCMDS inhibited (P < 0.05) proliferation and induced apoptosis in HeLa cells. The expression of cyclin B1 was downregulated that led to G2/M arrest in the cells after treatment with DCMDA. In summary, DCMDS induced apoptosis in HeLa cells via endoplasmic reticulum stress-induced apoptotic pathway and dysregulation of mitochondria. The data suggest the potential application of DCMDS in the treatment of cervical cancer. PMID:27041866

  19. Endoplasmic reticulum stress-induced apoptotic pathway and mitochondrial dysregulation in HeLa cells treated with dichloromethane extract of Dillenia suffruticosa.

    PubMed

    Wan Nor Hafiza, Wan Abd Ghani; Yazan, Latifah Saiful; Tor, Yin Sim; Foo, Jhi Biau; Armania, Nurdin; Rahman, Heshu Sulaiman

    2016-01-01

    Ethyl acetate and dichloromethane extract of Dillenia suffruticosa (EADS and DCMDS, respectively) can be a potential anticancer agent. The effects of EADS and DCMDS on the growth of HeLa cervical cancer cells and the expression of apoptotic-related proteins had been investigated in vitro. Cytotoxicity of the extracts toward the cells was determined by 5-diphenyltetrazolium bromide assay, the effects on cell cycle progression and the mode of cell death were analyzed by flow cytometry technique, while the effects on apoptotic-related genes and proteins were evaluated by quantitative real-time polymerase chain reaction, and Western blot and enzyme-linked immunosorbent assay, respectively. Treatment with DCMDS inhibited (P < 0.05) proliferation and induced apoptosis in HeLa cells. The expression of cyclin B1 was downregulated that led to G2/M arrest in the cells after treatment with DCMDA. In summary, DCMDS induced apoptosis in HeLa cells via endoplasmic reticulum stress-induced apoptotic pathway and dysregulation of mitochondria. The data suggest the potential application of DCMDS in the treatment of cervical cancer. PMID:27041866

  20. p66Shc mediates high-glucose and angiotensin II-induced oxidative stress renal tubular injury via mitochondrial-dependent apoptotic pathway

    PubMed Central

    Xiao, Li; Nie, Jing; Liu, Fu-you; Ling, Guang-hui; Zhu, Xue-jing; Tang, Wen-bin; Chen, Wen-cui; Xia, Yun-cheng; Zhan, Ming; Ma, Ming-ming; Peng, You-ming; Liu, Hong; Liu, Ying-hong; Kanwar, Yashpal S.

    2010-01-01

    p66Shc, a promoter of apoptosis, modulates oxidative stress response and cellular survival, but its role in the progression of diabetic nephropathy is relatively unknown. In this study, mechanisms by which p66Shc modulates high-glucose (HG)- or angiotensin (ANG) II-induced mitochondrial dysfunction were investigated in renal proximal tubular cells (HK-2 cells). Expression of p66Shc and its phosphorylated form (p-p66Shc, serine residue 36) and apoptosis were notably increased in renal tubules of diabetic mice, suggesting an increased reactive oxygen species production. In vitro, HG and ANG II led to an increased expression of total and p-p66Shc in HK-2 cells. These changes were accompanied with increased production of mitochondrial H2O2, reduced mitochondrial membrane potential, increased translocation of mitochondrial cytochrome c from mitochondria into cytosol, upregulation of the expression of caspase-9, and ultimately reduced cell survival. Overexpression of a dominant-negative Ser36 mutant p66Shc (p66ShcS36A) or treatment of p66Shc- or PKC-β-short interfering RNAs partially reversed these changes. Treatment of HK-2 cells with HG and ANG II also increased the protein-protein association between p-p66Shc and Pin1, an isomerase, in the cytosol, and with cytochrome c in the mitochondria. These interactions were partially disrupted with the treatment of PKC-β inhibitor or Pin1-short interfering RNA. These data suggest that p66Shc mediates HG- and ANG II-induced mitochondrial dysfunctions via PKC-β and Pin1-dependent pathways in renal tubular cells. PMID:20739391

  1. ER-Dependent Ca++-mediated Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic and ATM-JNK Signal Pathways in Gallic Acid-treated Human Oral Cancer Cells.

    PubMed

    Lu, Yao-Cheng; Lin, Meng-Liang; Su, Hong-Lin; Chen, Shih-Shun

    2016-02-01

    Release of calcium (Ca(++)) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using inhibitor of ER Ca(++) release dantrolene and inhibitor of mitochondrial Ca(++) uptake Ru-360, we demonstrated that Ca(++) release from the ER was associated with generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Small interfering RNA-mediated suppression of protein kinase RNA-like endoplasmic reticulum kinase inhibited tunicamycin-induced induction of 78 kDa glucose-regulated protein, C/EBP homologous protein, pro-caspase-12 cleavage, cytosolic Ca(++) increase and apoptosis, but did not attenuate the increase in cytosolic Ca(++) level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)-mediated c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis by GA was blocked by dantrolene. The specificity of ROS-mediated ATM-JNK activation was confirmed by treatment with N-acetylcysteine, a ROS scavenger. Blockade of ATM activation by specific inhibitor KU55933, short hairpin RNA, or kinase-dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro-caspase-3 cleavage, and apoptosis induced by GA. Taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic and ATM-JNK signal pathways, likely through ER Ca(++)-mediated ROS production. PMID:26851027

  2. Apoptotic effect of novel Schiff Based CdCl2(C14H21N3O2) complex is mediated via activation of the mitochondrial pathway in colon cancer cells

    PubMed Central

    Hajrezaie, Maryam; Paydar, Mohammadjavad; Looi, Chung Yeng; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Salga, Muhammad Saleh; Karimian, Hamed; Shams, Keivan; Zahedifard, Maryam; Majid, Nazia Abdul; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen

    2015-01-01

    The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies. PMID:25764970

  3. Glucose regulated protein 78 (GRP78) inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via AKT/mitochondrial apoptotic pathway.

    PubMed

    Xie, Jie; Tao, Zhong-Hua; Zhao, Jiang; Li, Ting; Wu, Zheng-Hua; Zhang, Jin-Feng; Zhang, Jian; Hu, Xi-Chun

    2016-06-01

    The underlying mechanism of gemcitabine resistance during breast cancer treatment remains unclear. Glucose regulated protein 78 (GRP78) frequently triggered by anticancer agents, was substantially elevated in gemcitabine resistant sublines. Ectopic expression of GRP78 changes gemcitabine chemosensitivity and apoptosis levels in breast cancer cells. Further experiments showed an involvement of caspase 9, not caspase 8, in gemcitabine resistance and GRP78-mediated chemosensitivity, suggesting that mitochondria apoptotic pathway was activated by GRP78. This finding was further supported by the observations of AKT activation, Bcl-2 increase, Bax and Bim decrease. Conclusively, GRP78 plays a vital role in gemcitabine resistance and clinical strategy to improve gemcitabine efficacy in breast cancer by manipulating GRP78 should be explored. PMID:27012209

  4. Mangiferin Attenuates Diabetic Nephropathy by Inhibiting Oxidative Stress Mediated Signaling Cascade, TNFα Related and Mitochondrial Dependent Apoptotic Pathways in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2014-01-01

    Oxidative stress plays a crucial role in the progression of diabetic nephropathy in hyperglycemic conditions. It has already been reported that mangiferin, a natural C-glucosyl xanthone and polyhydroxy polyphenol compound protects kidneys from diabetic nephropathy. However, little is known about the mechanism of its beneficial action in this pathophysiology. The present study, therefore, examines the detailed mechanism of the beneficial action of mangiferin on STZ-induced diabetic nephropathy in Wister rats as the working model. A significant increase in plasma glucose level, kidney to body weight ratio, glomerular hypertrophy and hydropic changes as well as enhanced nephrotoxicity related markers (BUN, plasma creatinine, uric acid and urinary albumin) were observed in the experimental animals. Furthermore, increased oxidative stress related parameters, increased ROS production and decreased the intracellular antioxidant defenses were detected in the kidney. Studies on the oxidative stress mediated signaling cascades in diabetic nephropathy demonstrated that PKC isoforms (PKCα, PKCβ and PKCε), MAPKs (p38, JNK and ERK1/2), transcription factor (NF-κB) and TGF-β1 pathways were involved in this pathophysiology. Besides, TNFα was released in this hyperglycemic condition, which in turn activated caspase 8, cleaved Bid to tBid and finally the mitochorndia-dependent apoptotic pathway. In addition, oxidative stress also disturbed the proapoptotic-antiapoptotic (Bax and Bcl-2) balance and activated mitochorndia-dependent apoptosis via caspase 9, caspase 3 and PARP cleavage. Mangiferin treatment, post to hyperglycemia, successfully inhibited all of these changes and protected the cells from apoptotic death. PMID:25233093

  5. Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells.

    PubMed

    Chang, Hong-Tai; Chou, Chiang-Ting; Lin, You-Sheng; Shieh, Pochuen; Kuo, Daih-Huang; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-04-01

    Esculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca(2+) homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca(2+) signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca(2+) concentration ([Ca(2+)]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by the store-operated Ca(2+) channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca(2+)-free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca(2+)]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca(2+)]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through 2-APB-sensitive store-operated Ca(2+) entry. Furthermore, esculetin activated Ca(2+)-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstract The summary of esculetin

  6. Gallic acid induces apoptosis and enhances the anticancer effects of cisplatin in human small cell lung cancer H446 cell line via the ROS-dependent mitochondrial apoptotic pathway.

    PubMed

    Wang, Ruixuan; Ma, Lijie; Weng, Dan; Yao, Jiahui; Liu, Xueying; Jin, Faguang

    2016-05-01

    via the ROS-dependent mitochondrial apoptotic pathway. PMID:26987028

  7. Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

    PubMed

    Afnan, Quadri; Kaiser, Peerzada J; Rafiq, Rather A; Nazir, Lone A; Bhushan, Shashi; Bhardwaj, Subhash C; Sandhir, Rajat; Tasduq, Sheikh A

    2016-06-01

    Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1β and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage. PMID:26836460

  8. Sulfiredoxin-1 protects against simulated ischaemia/reperfusion injury in cardiomyocyte by inhibiting PI3K/AKT-regulated mitochondrial apoptotic pathways

    PubMed Central

    Zhang, Jiankai; He, Zhangyou; Guo, Jinhua; Li, Zhe; Wang, Xiaohong; Yang, Chun; Cui, Xiaojun

    2016-01-01

    Reactive oxygen species (ROS)-triggered cardiac cell injury is recognized as the major contributor for the pathogenesis progression of ischaemic cardiovascular diseases. Sulfiredoxin-1 (Srx-1) is an endogenous antioxidant and exerts the crucial neuroprotective effects in cerebral ischaemia. However, its function and the underlying mechanism in ischaemic heart diseases remain poorly defined. Here, a dramatical decrease in Srx-1 was validated in H9c2 cardiomyocytes upon simulated ischaemia–reperfusion (SI/R) injury. Moreover, Srx-1 protected H9c2 cells from SI/R-injured injury as the evidences that Srx-1 up-regulation attenuated the inhibitory effects on cell viability, lactate dehydrogenase (LDH) and cell apoptosis upon SI/R treatment. Knockdown of Srx-1 accelerated cell injury upon SI/R. Mechanism assay corroborated that SI/R treatment noticeably aggravated the loss of mitochondrial membrane potential (Δψm), which was remarkably abated in Ad-Srx-1 groups. Importantly, Srx-1 elevation substantially reduced cytochrome c release, the activity of caspase-9 and caspase-3, accompany with the subsequent decrease in the cleavage of poly (ADP ribose) polymerase (PARP). Concomitantly, overexpression of Srx-1 also decreased the expression of pro-apoptosis protein Bax and increased anti-apoptotic Bcl-2 expression. Further analysis substantiated that Srx-1 treatment remarkably induced the activation of PI3K/AKT signalling. Preconditioning with LY294002 dramatically decreased Srx-1-enhanced cell resistance to SI/R injury. Importantly, LY294002 mitigated the inhibitory effects of Srx-1 on Δψm loss, cytochrome c release, caspase-9/3 activity, and the expression of Bcl-2 family. Together, these results suggested that Srx-1 might protect cardiomyocyte injury upon SI/R by suppressing PI3K/AKT-mediated mitochondria dependent apoptosis, revealing a promising therapeutic agent against ischaemic cardiovascular diseases. PMID:26992405

  9. Apoptotic pathways in pancreatic ductal adenocarcinoma

    PubMed Central

    Hamacher, Rainer; Schmid, Roland M; Saur, Dieter; Schneider, Günter

    2008-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer related death. Despite the advances in understanding of the molecular pathogenesis, pancreatic cancer remains a major unsolved health problem. Overall, the 5-year survival rate is less than 5% demonstrating the insufficiency of current therapies. Most cytotoxic therapies induce apoptosis and PDAC cells have evolved a plethora of molecular mechanisms to assure survival. We will present anti-apoptotic strategies working at the level of the death receptors, the mitochondria or involving the caspase inhibitors of the IAP family. Furthermore, the survival function of the phosphotidylinositol-3' kinase (PI3K)/AKT- and NF-kappaB-pathways are illustrated. A detailed molecular knowledge of the anti-apoptotic mechanisms of PDAC cells will help to improve therapies for this dismal disease and therapeutic strategies targeting the programmed cell death machinery are in early preclinical and clinical development. PMID:18652674

  10. Costunolide induces G1/S phase arrest and activates mitochondrial-mediated apoptotic pathways in SK-MES 1 human lung squamous carcinoma cells

    PubMed Central

    HUA, PEIYAN; ZHANG, GUANGXIN; ZHANG, YIFAN; SUN, MEI; CUI, RANJI; LI, XIN; LI, BINGJIN; ZHANG, XINGYI

    2016-01-01

    Despite the availability of several therapeutic options, a safer and more effective modality strategy is required for the treatment of lung cancer. Costunolide, a sesquiterpene lactone which isolated from the Saussurea lappa, has potent anticancer properties. In the present study, the effects of costunolide on cell viability, the cell cycle and apoptosis in SK-MES-1 human lung squamous carcinoma cells were investigated. Costunolide induced morphological changes and inhibited growth of SK-MES-1 cells growth. Flow cytometric analysis data demonstrated that costunolide significantly induced apoptosis of SK-MES-1 cells and induced cell cycle arrest at G1/S phase in a dose-dependent manner. Through upregulation in the expression of p53 and Bax, and downregulation in the expression of Bcl-2 and activation of caspase-3, costunolide-induced apoptosis was confirmed by western blot analysis. In addition, the significant loss of mitochondrial membrane potential indicated that costunolide may induce apoptosis via the mitochondria-dependent pathway in SK-MES-1 cells. These results highlight the potential effects of costunolide as an anti-cancer agent in a human lung squamous carcinoma cell line. PMID:27073552

  11. [Sphingolipid-mediated apoptotic signaling pathways].

    PubMed

    Cuvillier, Olivier; Andrieu-Abadie, Nathalie; Ségui, Bruno; Malagarie-Cazenave, Sophie; Tardy, Claudine; Bonhoure, Elisabeth; Levade, Thierry

    2003-01-01

    Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate. PMID:14708343

  12. The Modulation of Apoptotic Pathways by Gammaherpesviruses

    PubMed Central

    Banerjee, Shuvomoy; Uppal, Timsy; Strahan, Roxanne; Dabral, Prerna; Verma, Subhash C.

    2016-01-01

    Apoptosis or programmed cell death is a tightly regulated process fundamental for cellular development and elimination of damaged or infected cells during the maintenance of cellular homeostasis. It is also an important cellular defense mechanism against viral invasion. In many instances, abnormal regulation of apoptosis has been associated with a number of diseases, including cancer development. Following infection of host cells, persistent and oncogenic viruses such as the members of the Gammaherpesvirus family employ a number of different mechanisms to avoid the host cell’s “burglar” alarm and to alter the extrinsic and intrinsic apoptotic pathways by either deregulating the expressions of cellular signaling genes or by encoding the viral homologs of cellular genes. In this review, we summarize the recent findings on how gammaherpesviruses inhibit cellular apoptosis via virus-encoded proteins by mediating modification of numerous signal transduction pathways. We also list the key viral anti-apoptotic proteins that could be exploited as effective targets for novel antiviral therapies in order to stimulate apoptosis in different types of cancer cells. PMID:27199919

  13. Strong anticancer potential of nano-triterpenoid from Phytolacca decandra against A549 adenocarcinoma via a Ca(2+)-dependent mitochondrial apoptotic pathway.

    PubMed

    Das, Jayeeta; Das, Sreemanti; Paul, Avijit; Samadder, Asmita; Khuda-Bukhsh, Anisur Rahman

    2014-06-01

    We isolated a triterpenoid from an ethanolic extract of Phytolacca decandra and nanoencapsulated it with biodegradable nontoxic polymers of poly(lactide-co-glycolide) to examine if the nanoform of this hitherto unexplored betulinic-acid derivative (NdBA) could produce a stronger anticancer effect by rendering better drug bioavailability and targeted delivery than the nonencapsulated betulinic-acid derivative (dBA). The nanoparticles were characterized with the help of physicochemical and morphological studies involving dynamic light scattering and atomic force microscopy. A549 cancer cells were exposed to NdBA and dBA at the IC50 doses of 50 μg/mL and 100 μg/mL, respectively. Mitochondrial dysfunction-mediated apoptosis was determined by examining the changes in the intracellular calcium content, the reactive oxygen species accumulation, the cytochrome c release, the upregulation of Bcl-2-associated-X protein (Bax) and caspase 3, the downregulation of B cell lymphoma 2, and the mitochondrial membrane potential (ΔΨm) depolarization. Apoptosis was also verified by acridine orange staining observed under fluorescence microscopy and annexin V-fluorescein isothiocyanate/propidium iodide staining through flow cytometric studies. The levels of intracellular adenosine triphosphate/adenosine diphosphate ratio decreased, and the ATPase activity increased more strikingly in A549 cells exposed to NdBA than in A549 cells exposed to dBA. Overall results showed that both drugs directly target the mitochondrial oxidative phosphorylation system, with NdBA having a stronger effect, indicating NdBA to be a better candidate for the development of an anticancer drug for use against lung adenocarcinomas. PMID:24929458

  14. Evidence for a Novel, Caspase-8-Independent, Fas Death Domain-Mediated Apoptotic Pathway

    PubMed Central

    Katsanis, Emmanuel

    2004-01-01

    Certain caspase-8 null cell lines demonstrate resistance to Fas-induced apoptosis, indicating that the Fas/FasL apoptotic pathway may be caspase-8-dependent. Some reports, however, have shown that Fas induces cell death independent of caspase-8. Here we provide evidence for an alternative, caspase-8-independent, Fas death domain-mediated apoptotic pathway. Murine 12B1-D1 cells express procaspase-3, -8, and -9, which were activated upon the dimerization of Fas death domain. Bid was cleaved and mitochondrial transmembrane potential was disrupted in this apoptotic process. All apoptotic events were completely blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK, but not by other peptide caspase inhibitors. Cyclosporin A (CsA), which inhibits mitochondrial transition pore permeability, blocked neither pore permeability disruption nor caspase activation. However, CsA plus caspase-8 inhibitor blocked all apoptotic events of 12B1-D1 induced by Fas death domain dimerization. Our data therefore suggest that there is a novel, caspase-8-independent, Z-VAD-FMK-inhibitable, apoptotic pathway in 12B1-D1 cells that targets mitochondria directly. PMID:15123887

  15. Activation of mitochondrial apoptotic pathway in mantle cell lymphoma: high sensitivity to mitoxantrone in cases with functional DNA-damage response genes.

    PubMed

    Ferrer, Ana; Marcé, Silvia; Bellosillo, Beatriz; Villamor, Neus; Bosch, Francesc; López-Guillermo, Armando; Espinet, Blanca; Solé, Francesc; Montserrat, Emili; Campo, Elias; Colomer, Dolors

    2004-11-25

    Mantle cell lymphoma (MCL) is a mature B-cell proliferation characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course, and poor response to chemotherapy. The majority of drugs currently used in the treatment of lymphoproliferative disorders induce cell death by triggering apoptosis, but few data concerning drug-induced apoptosis in MCL have been reported. We have analysed the mechanisms of drug-induced cell death in four cell lines with the t(11;14) and in primary cells from 10 patients with MCL. Mitoxantrone, a topoisomerase II inhibitor, induced a strong cytotoxic effect in three cell lines (JVM-2, REC-1, and Granta 519), and in primary MCL cells. This cytotoxic effect due to apoptosis induction was observed despite the presence of either p53 or ATM abnormalities. However, no cytotoxic effect was detected after incubation with DNA-damaging agents in the NCEB-1 cell line, carrying p53 and ATM alterations, despite the presence of functional mitochondrial machinery. These results support that mitoxantrone can be effective in the treatment of MCL but that this activity requires the integrity of functional DNA-damage response genes. PMID:15480431

  16. HDAC inhibitor misprocesses bantam oncomiRNA, but stimulates hid induced apoptotic pathway

    PubMed Central

    Bhadra, Utpal; Mondal, Tanmoy; Bag, Indira; Mukhopadhyay, Debasmita; Das, Paromita; Parida, Bibhuti B.; Mainkar, Prathama S.; Reddy, Chada Raji; Bhadra, Manika Pal

    2015-01-01

    Apoptosis or programmed cell death is critical for embryogenesis and tissue homeostasis. Uncontrolled apoptosis leads to different human disorders including immunodeficiency, autoimmune disorder and cancer. Several small molecules that control apoptosis have been identified. Here, we have shown the functional role of triazole derivative (DCPTN-PT) that acts as a potent HDAC inhibitor and mis-express proto onco microRNA (miRNA) bantam. To further understanding the mechanism of action of the molecule in apoptotic pathway, a series of experiments were also performed in Drosophila, a well known model organism in which the nature of human apoptosis is very analogous. DCPTN-PT mis processes bantam microRNA and alters its down regulatory target hid function and cleavage of Caspase-3 which in turn influence components of the mitochondrial apoptotic pathway in Drosophila. However regulatory microRNAs in other pro-apoptotic genes are not altered. Simultaneously, treatment of same molecule also affects the mitochondrial regulatory pathway in human tumour cell lines suggesting its conservative nature between fly and human. It is reasonable to propose that triazole derivative (DCPTN-PT) controls bantam oncomiRNA and increases hid induced apoptosis and is also able to influence mitochondrial apoptotic pathway. PMID:26442596

  17. Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

    PubMed

    Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

    2013-10-25

    Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. PMID:24021799

  18. IF1 limits the apoptotic-signalling cascade by preventing mitochondrial remodelling

    PubMed Central

    Faccenda, D; Tan, C H; Seraphim, A; Duchen, M R; Campanella, M

    2013-01-01

    Mitochondrial structure has a central role both in energy conversion and in the regulation of cell death. We have previously shown that IF1 protects cells from necrotic cell death and supports cristae structure by promoting the oligomerisation of the F1Fo-ATPsynthase. As IF1 is upregulated in a large proportion of human cancers, we have here explored its contribution to the progression of apoptosis and report that an increased expression of IF1, relative to the F1Fo-ATPsynthase, protects cells from apoptotic death. We show that IF1 expression serves as a checkpoint for the release of Cytochrome c (Cyt c) and hence the completion of the apoptotic program. We show that the progression of apoptosis engages an amplification pathway mediated by: (i) Cyt c-dependent release of ER Ca2+, (ii) Ca2+-dependent recruitment of the GTPase Dynamin-related protein 1 (Drp1), (iii) Bax insertion into the outer mitochondrial membrane and (iv) further release of Cyt c. This pathway is accelerated by suppression of IF1 and delayed by its overexpression. IF1 overexpression is associated with the preservation of mitochondrial morphology and ultrastructure, consistent with a central role for IF1 as a determinant of the inner membrane architecture and with the role of mitochondrial ultrastructure in the regulation of Cyt c release. These data suggest that IF1 is an antiapoptotic and potentially tumorigenic factor and may be a valuable predictor of responsiveness to chemotherapy. PMID:23348567

  19. Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection

    PubMed Central

    Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.

    2015-01-01

    Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined. The application of pifithrin μ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis. PMID:26703711

  20. Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection.

    PubMed

    Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K; Liebert, Uwe G

    2015-12-01

    Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined. The application of pifithrin μ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis. PMID:26703711

  1. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    PubMed

    Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. PMID:23707954

  2. Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells.

    PubMed

    Tian, K Y; Liu, X J; Xu, J D; Deng, L J; Wang, G

    2015-05-01

    Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway. PMID:25760023

  3. Apoptotic pathways as a therapeutic target for colorectal cancer treatment

    PubMed Central

    Abraha, Aman M; Ketema, Ezra B

    2016-01-01

    Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment. PMID:27574550

  4. Apoptotic pathways as a therapeutic target for colorectal cancer treatment.

    PubMed

    Abraha, Aman M; Ketema, Ezra B

    2016-08-15

    Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment. PMID:27574550

  5. Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal?

    PubMed Central

    Lokhmatikov, Alexey V.; Voskoboynikova, Natalia; Cherepanov, Dmitry A.; Skulachev, Maxim V.; Steinhoff, Heinz-Jürgen; Skulachev, Vladimir P.; Mulkidjanian, Armen Y.

    2016-01-01

    Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane—liposomes of pure bovine heart CL—we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature. PMID:27313834

  6. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death.

    PubMed

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-05-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  7. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death

    PubMed Central

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-01-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  8. Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

    SciTech Connect

    Grondin, Melanie; Marion, Michel; Denizeau, Francine; Averill-Bates, Diana A. . E-mail: averill.diana@uqam.ca

    2007-07-01

    Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl{sup -}/HCO{sub 3} {sup -} exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.

  9. Investigation of the apoptotic pathway induced by benzimidazole-oxindole conjugates against human breast cancer cells MCF-7.

    PubMed

    Lakshma Nayak, Vadithe; Nagaseshadri, Bobburi; Vishnuvardhan, M V P S; Kamal, Ahmed

    2016-07-15

    In our previous studies, benzimidazole-oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP. PMID:27262596

  10. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA

    PubMed Central

    Rongvaux, Anthony; Jackson, Ruaidhrí; Harman, Christian C.D.; Li, Tuo; West, A. Phillip; de Zoete, Marcel R.; Wu, Youtong; Yordy, Brian; Lakhani, Saquib A.; Kuan, Chia-Yi; Taniguchi, Tadatsugu; Shadel, Gerald S.; Chen, Zhijian J.; Iwasaki, Akiko; Flavell, Richard A.

    2014-01-01

    The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify of a mechanism by which mitochondria and downstream pro-apoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response, and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a pro-inflammatory type of cell death. PMID:25525875

  11. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia.

    PubMed

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD(+) biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD(+) synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  12. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia

    PubMed Central

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  13. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    SciTech Connect

    Nicolaou, Katerina A.; Liapis, Vasilis; Evdokiou, Andreas; Constantinou, Constantina; Magiatis, Prokopios; Skaltsounis, Alex L.; Koumas, Laura; Costeas, Paul A.; Constantinou, Andreas I.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer The effects of 6BIO and 7BIO are evaluated against five breast cancer cell lines. Black-Right-Pointing-Pointer 6BIO induces a caspase dependent apoptotic effect via the intrinsic pathway. Black-Right-Pointing-Pointer 7BIO promotes G{sub 2}/M cells cycle arrest. Black-Right-Pointing-Pointer 7BIO triggers a caspase-8 mediated apoptotic pathway. Black-Right-Pointing-Pointer 7BIO triggers and a caspase independent pathway. -- Abstract: Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3 Prime -oxime (6BIO) and 7-bromoindirubin-3 Prime -oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G{sub 2}/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.

  14. Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.

    PubMed

    Micali, Nicola; Ferrai, Carmelo; Fernandez-Diaz, Luis C; Blasi, Francesco; Crippa, Massimo P

    2009-03-01

    The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression. PMID:19103748

  15. Cedrol induces autophagy and apoptotic cell death in A549 non-small cell lung carcinoma cells through the P13K/Akt signaling pathway, the loss of mitochondrial transmembrane potential and the generation of ROS.

    PubMed

    Zhang, Shi-Yi; Li, Xue-Bo; Hou, Sheng-Guang; Sun, Yao; Shi, Yi-Ran; Lin, Song-Sen

    2016-07-01

    The objective of the present study was to determine the anticancer effects of cedrol in A549 human non-small cell lung cancer cells by examining the effects of cedrol on apoptosis induction, the phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway, autophagy, reactive oxygen species (ROS) generation and mitochondrial transmembrane potential (MTP). The anticancer effects of cedrol were examined using A549 human lung carcinoma cells as an in vitro model. Cell viability was determined using MTT and lactate dehydrogenase (LDH) assays, and an inverted phase contrast microscope was used to examine the morphological changes in these cells. Cedrol‑triggered autophagy was confirmed by transmission electron microscopy (TEM) analysis of the cells, as well as by western blot analysis of microtubule-associated protein light-chain 3 (LC3)B expression. Intracellular ROS generation was measured by flow cytometry using 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CM-DCFH2-DA) staining and MTP was measured using flow cytometry. The results demonstrated that cedrol reduced cell viability and induced cell apoptosis in a dose-dependent manner. Mechanistic evaluations indicated that cedrol induced apoptosis by reducing the MTP and by decreasing the levels of phosphorylated (p-)PI3K and p-Akt. Cedrol induced autophagy, which was confirmed by TEM analysis, by increasing intracellular ROS formation in a concentration-dependent manner, which was almost completely reversed by N-acetyl-L-cysteine (NAC) and tocopherol. Taken together, these findings reveal that cedrol inhibits cell proliferation and induces apoptosis in A549 cells through mitochondrial and PI3K/Akt signaling pathways. Our findings also reveal that cedrol induced pro-death autophagy by increasing intracellular ROS production. PMID:27177023

  16. The mitochondrial p53 pathway

    PubMed Central

    Vaseva, Angelina V.; Moll, Ute M.

    2010-01-01

    p53 is one of the most mutated tumor suppressors in human cancers and as such has been intensively studied for a long time. p53 is a major orchestrator of the cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, senescence, DNA repair and genetic stability. For a long time it was thought that these functions of p53 solely rely on its function as a transcription factor, and numerous p53 target genes have been identified [1]. In the last 8 years however, a novel transcription-independent proapoptotic function mediated by the cytoplasmic pool of p53 has been revealed. p53 participates directly in the intrinsic apoptosis pathway by interacting with the multidomain members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization. Our review will discuss these studies, focusing on recent advances in the field. PMID:19007744

  17. CGP57380 enhances efficacy of RAD001 in non-small cell lung cancer through abrogating mTOR inhibition-induced phosphorylation of eIF4E and activating mitochondrial apoptotic pathway.

    PubMed

    Wen, Qiuyuan; Wang, Weiyuan; Luo, Jiadi; Chu, Shuzhou; Chen, Lingjiao; Xu, Lina; Zang, Hongjing; Alnemah, Mohannad Ma; Ma, Jian; Fan, Songqing

    2016-05-10

    The mammalian target of rapamycin (mTOR) is a potentially important therapeutic target in a broad range of cancer types. mTOR inhibitors such as rapamycin and its analogs (rapalogs) have been proven effective as anticancer agents in non-small cell lung cancer (NSCLC), whereas they strongly enhance phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and activation of Akt, which cause resistance to mTOR-targeted therapy after an initial response. Rapamycin induces eIF4E phosphorylation by activating MAPK-interacting kinases (Mnks), and therefore targeting Mnk/eIF4E pathway represents a potential therapeutic strategy for the treatment of NSCLC. Here, our results showed that over-expression of p-Mnk1 and p-eIF4E was significantly associated with poor overall survival of NSCLC patients and high expression of p-Mnk1 might act as an independent prognostic biomarker for these patients. Meanwhile, inhibiting Mnk1 expression by Mnk inhibitor (CGP57380) could abrogate rapalogs (RAD001)-induced eIF4E phosphorylation and Akt activation. Furthermore, combination of CGP57380 and RAD001 could induce NSCLC cells apoptosis via activating intrinsic mitochondrial pathway, and exert synergistic antitumor efficacy both in vitro and in vivo. In conclusion, combination of targeting both mTOR and Mnk/eIF4E signaling pathways to enhance effectiveness of mTOR-targeted cancer therapy might be significant innovation for the personalized treatment of NSCLC. PMID:27050281

  18. Apoptosis Induction by 13-Acetoxyrolandrolide through the Mitochondrial Intrinsic Pathway

    PubMed Central

    Acuña, Ulyana Muñoz; Matthew, Susan; Pan, Li; Kinghorn, A. Douglas; Swanson, Steven M.; Carcache de Blanco, Esperanza J.

    2014-01-01

    The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13-acetoxyrolandrolide, an NF-κB inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with inhibition of the NFκB pathway included dose-dependent inhibition of the NF-κB subunit p65 (RelA) and inhibition of upstream mediators IKKβ and oncogenic K-Ras. The inhibitory concentration (IC50) of 13-acetoxyrolandrolide on K-Ras was 7.7 μM. The downstream effects of the inhibition of NF-κB activation were also investigated in vitro. After 24 h of treatment with 13-acetoxyrolandrolide, the mitochondrial transmembrane potential (ΔΨm) was depolarized in human colon cancer (HT-29) cells. The mitochondrial oxidative phosphorylation was also negatively affected and reduced levels of NAD(P)H were detected after 2 h of 13-acetoxyrolandrolide exposure. Furthermore, the expression of the pro-apoptotic protein caspase-3 increased in a concentration-dependent manner. Cell flow cytometry showed that 13-acetoxyrolandrolide induced cell cycle arrest at G1, suggesting that treated cells had undergone caspase-3-mediated apoptosis, suggesting negative effects on cancer cell proliferation. These results suggest that 13-acetoxyrolandrolide inhibits NF-κB and K-Ras as well as promotes cell death mediated through the mitochondrial apoptotic pathway. PMID:24338805

  19. Infrared radiation affects the mitochondrial pathway of apoptosis in human fibroblasts.

    PubMed

    Frank, Sandra; Oliver, Lisa; Lebreton-De Coster, Corinne; Moreau, Carole; Lecabellec, Marie-Thérèse; Michel, Laurence; Vallette, François M; Dubertret, Louis; Coulomb, Bernard

    2004-11-01

    We have previously observed that near-infrared (IR) pre-irradiation protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. Here, we show that IR pre-irradiation of human fibroblasts inhibited UVB activation of caspase-9 and -3, leading us to study early events in the mitochondrial apoptotic pathway after IR irradiation. IR irradiation led to a partial release of cytochrome c and Smac/Diablo but not apoptosis-inducing factor (AIF). This was accompanied by a slight but transient decrease in the mitochondrial membrane potential (Deltapsim) and by the insertion of Bax into mitochondrial membrane. Early apoptotic events in the mitochondrial pathway thus occurred after IR irradiation despite a lack of caspase-9 and -3 activation. This could be explained by the induction by IR of the expression of heat shock protein Hsp27, which is known to prevent apoptosome assembly. Furthermore, the balance between pro-apoptotic (i.e., Bax) and anti-apoptotic (i.e., Bcl-2 or Bcl-xL) proteins, which was rather pro-apoptotic after IR exposure, became anti-apoptotic 24 h later, suggesting a protective effect. Together, these actions could also contribute to prepare the cell to resist UVB-triggered apoptosis. Finally, isolated rat liver mitochondria-released cytochrome c in response to IR, demonstrating that mitochondria were a primary target of IR radiation. PMID:15482467

  20. Regulators in the apoptotic pathway during spermatogenesis: Killers or guards?

    PubMed

    Xu, Ya-Ru; Dong, Hong-Shan; Yang, Wan-Xi

    2016-05-15

    Apoptosis occurs at any time in the ontogeny of the testis, especially during the first wave of spermatogenesis. However, the exact mechanisms by which homeostasis of apoptosis and survival in GCs and mature sperm are orchestrated remain unclear. Three pathways during the process of apoptosis in mammals are discussed extensively. The three pathways are extrinsic pathway, mitochondrial pathway and endoplasmic reticulum pathway. Based on that, many factors, such as growth factors (SCF, FGF, TGF), hormones (FSH, LH, E2, MIS), partial oxygen pressure, and testis specific genes are involved in apoptosis and survival process. The pathways of apoptosis adopted by the GCs and sperm depend on the types of stimuli they receive. Diverse pathways are initiated in heat-stress induced apoptosis of GCs and the destiny of GCs suppressed by hyperglycemia is mainly regulated by a rheostat of total oxidants and anti-oxidants which leading to intrinsic pathway. In this review, we provide an overview of three classic pathways and important factors involved in the process of germ cell apoptosis and survival, and discuss the recent advances made in understanding of the molecular mechanisms of spermatogenic cells and sperm response to stress-inducers, such as heat stress and hyperglycemia. All the findings may provide clues to the control of male fertility or treating germ cell tumors and other testis associated pathological conditions, at the same time, a novel idea may result in devising much safer contraception with high efficiency. PMID:26861610

  1. Mevalonate Pathway Blockade, Mitochondrial Dysfunction and Autophagy: A Possible Link

    PubMed Central

    Tricarico, Paola Maura; Crovella, Sergio; Celsi, Fulvio

    2015-01-01

    The mevalonate pathway, crucial for cholesterol synthesis, plays a key role in multiple cellular processes. Deregulation of this pathway is also correlated with diminished protein prenylation, an important post-translational modification necessary to localize certain proteins, such as small GTPases, to membranes. Mevalonate pathway blockade has been linked to mitochondrial dysfunction: especially involving lower mitochondrial membrane potential and increased release of pro-apoptotic factors in cytosol. Furthermore a severe reduction of protein prenylation has also been associated with defective autophagy, possibly causing inflammasome activation and subsequent cell death. So, it is tempting to hypothesize a mechanism in which defective autophagy fails to remove damaged mitochondria, resulting in increased cell death. This mechanism could play a significant role in Mevalonate Kinase Deficiency, an autoinflammatory disease characterized by a defect in Mevalonate Kinase, a key enzyme of the mevalonate pathway. Patients carrying mutations in the MVK gene, encoding this enzyme, show increased inflammation and lower protein prenylation levels. This review aims at analysing the correlation between mevalonate pathway defects, mitochondrial dysfunction and defective autophagy, as well as inflammation, using Mevalonate Kinase Deficiency as a model to clarify the current pathogenetic hypothesis as the basis of the disease. PMID:26184189

  2. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    PubMed Central

    Venkatarame Gowda Saralamma, Venu; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-01-01

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer. PMID:26393583

  3. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells].

    PubMed

    Qiu, Qiang; Wang, Zhen; Jiang, Jian-ming; Li, Dian-dong

    2007-02-01

    Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities. PMID:17518039

  4. Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway.

    PubMed

    Yang, Joo-Sung; Ramanathan, Mathura P; Muthumani, Karuppiah; Choo, Andrew Y; Jin, Sung-Ha; Yu, Qian-Chun; Hwang, Daniel S; Choo, Daniel K; Lee, Mark D; Dang, Kesen; Tang, Waixing; Kim, J Joseph; Weiner, David B

    2002-12-01

    West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade. PMID:12498651

  5. Mitochondrial Retrograde Signaling: Triggers, Pathways, and Outcomes

    PubMed Central

    da Cunha, Fernanda Marques; Torelli, Nicole Quesada; Kowaltowski, Alicia J.

    2015-01-01

    Mitochondria are essential organelles for eukaryotic homeostasis. Although these organelles possess their own DNA, the vast majority (>99%) of mitochondrial proteins are encoded in the nucleus. This situation makes systems that allow the communication between mitochondria and the nucleus a requirement not only to coordinate mitochondrial protein synthesis during biogenesis but also to communicate eventual mitochondrial malfunctions, triggering compensatory responses in the nucleus. Mitochondria-to-nucleus retrograde signaling has been described in various organisms, albeit with differences in effector pathways, molecules, and outcomes, as discussed in this review. PMID:26583058

  6. Overexpression of Amyloid- β Protein Precursor Induces Mitochondrial Oxidative Stress and Activates the Intrinsic Apoptotic Cascade

    PubMed Central

    Bartley, Matthew G.; Marquardt, Kristin; Kirchhof, Danielle; Wilkins, Heather M.; Patterson, David; Linseman, Daniel A.

    2015-01-01

    Alzheimer's disease (AD) is a debilitating cognitive disorder which is characterized pathologically by amyloid-β plaques and neurofibrillary tangles. Aberrant processing of amyloid beta protein precursor (AβPP) into amyloid-β fragments underlies the formation of senile plaques. Moreover, amyloid-β fragments, particularly Aβ42, exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down Syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces the activation of caspase-3 and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and its overexpression causes glutathione (GSH)-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP were significantly inhibited by the co-expression of Bcl-2 or treatment with either GSH or Boc, a pan-caspase inhibitor. Furthermore, the mPTP inhibitor, cyclosporin A, also significantly protected CHO cells from apoptosis induced by AβPP overexpression. Finally, co-treatment with a β-secretase inhibitor did not significantly protect CHO cells from AβPP overexpression and Aβ42 levels were undetectable in transfected CHO cells. Therefore, the mechanism of AβPP induced MOS and apoptosis is independent of the production of Aβ42. However, a γ-secretase inhibitor showed significant protection of the CHO cells against AβPP overexpression. Thus, suggesting a possible role of the AβPP intracellular domain in this apoptotic

  7. Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

    PubMed Central

    Ponomarev, Eugeny D.; Tsytsykova, Alla; Armant, Myriam; Vorobjev, Ivan A.

    2014-01-01

    High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE+ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations. PMID:24394470

  8. Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease

    PubMed Central

    JIA, DONG-PEI; WANG, SONG; ZHANG, BAO-CHAO; FANG, FANG

    2015-01-01

    In previous years, increasing evidence has indicated that paraptosis and mitochondrial-mediated apoptosis may be associated with Alzheimer's disease (AD). However, the association between paraptosis and mitochondrial-mediated apoptosis, and the pathological processes underlying AD, remain elusive. In the present study, the β-amyloid precursor protein gene, and the gene mutations PS1M146L and L286V, were transfected to an SH-SY5Y cell line to establish an AD cell model. Subsequently, an MTT assay was used to examine the cell viability of the AD cell model, while a TUNEL assay was employed to observe the number of positively stained apoptotic cells. Cytoplasmic vacuolization was examined using light microscopy and images were photographed. Furthermore, western blot analysis was utilized to detect the expression of golden biomarkers of the mitochondrial pathway, including Bcl-2 and Bax. The paraptosis inhibitor, cycloheximide, was selected to treat the AD model cells in order to observe the association between paraptosis and mitochondrial-mediated apoptosis. The results indicated that the decrease in the cell viability of the AD cells was initiated at 24 h, as compared with the normal cells (P<0.05). TUNEL-positive stained cells were observed at 48 h, which was later compared with the cell death initiation. In addition, examination of cytoplasmic vacuolization using microscopy indicated that there were a small number of paraptosis cells present at 24 h. The expression levels of Bcl-2 was significantly decreased, while Bax was significantly increased at 48 h. Furthermore, cycloheximide treatment was demonstrated to significantly increase Bcl-2 expression, while decreasing Bax expression (P>0.05). In conclusion, the occurrence of paraptosis was demonstrated in the early pathological stages of AD, which may subsequently damage the mitochondria and trigger mitochondrial pathway-mediated apoptosis. Thus, paraptosis may trigger programmed cell death directly, or indirectly

  9. Protective effect of FGF21 on type 1 diabetes-induced testicular apoptotic cell death probably via both mitochondrial- and endoplasmic reticulum stress-dependent pathways in the mouse model.

    PubMed

    Jiang, Xin; Zhang, Chi; Xin, Ying; Huang, Zhifeng; Tan, Yi; Huang, Yadong; Wang, Yonggang; Feng, Wenke; Li, Xiaokun; Li, Wei; Qu, Yaqin; Cai, Lu

    2013-05-10

    Fibroblast growth factor 21 (FGF21) is a novel member identified and was reported to express predominantly in pancreas, liver and adipose tissue, and relatively less in other organs, such as the testis. However, the role of FGF21 in the testis has never been addressed. The present study examined FGF21 expression at mRNA level by real-time RT-PCR assay in the testis of fasting and non-fasting mice or mice with type 1 diabetes that was induced with streptozotocin. We also examined the effect of Fgf21 gene deletion or supplementation of the exogenous FGF21 on the testicular apoptotic cell death spontaneously or induced by type 1 diabetes in FGF21 knockout (FGF21-KO) mice. Deletion of Fgf21 gene does not affect testicular cell proliferation, but significantly increases the spontaneous incidence of testicular TUNEL positive cells with increases in the Bax/Bcl2 expression ratio and apoptosis-inducing factor (AIF) expression. Diabetes induced significant increases in testicular TUNEL positive cells, Bax/Bcl2 expression ratio, AIF expression, CHOP and cleaved caspase-12 expression, and oxidative damage, but did not change the expression of cleaved caspase-3 and caspase-8. Deletion of Fgf21 gene also significantly enhances diabetes-induced TUNEL positive cells along with the increased expression of Bax/Bcl2 ratio, AIF, CHOP, cleaved caspase-12, and oxidative damage, which was significantly prevented by the supplementation of exogenous FGF21. These results suggest that Fgf21 gene may involve in maintaining normal spermatogenesis and also protect the germ cells from diabetes-induced apoptotic cell death probably via the prevention of diabetes-induced oxidative damage. PMID:23499715

  10. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

    PubMed

    Thangam, Ramar; Sathuvan, Malairaj; Poongodi, Arasu; Suresh, Veeraperumal; Pazhanichamy, Kalailingam; Sivasubramanian, Srinivasan; Kanipandian, Nagarajan; Ganesan, Nalini; Rengasamy, Ramasamy; Thirumurugan, Ramasamy; Kannan, Soundarapandian

    2014-07-17

    Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release. PMID:24702929

  11. Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation.

    PubMed

    Zeng, Changjun; Tang, Keyi; He, Lian; Peng, Wenpei; Ding, Li; Fang, Donghui; Zhang, Yan

    2014-06-01

    Artificial insemination (AI) with post-thawed boar spermatozoa results in low farrowing rates and reduced litter sizes mainly due to cryoinjury or damages to spermatozoa during cryopreservation. Low viability and motility of post-thawed boar spermatozoa are highly associated with apoptosis during cryopreservation. Although glycerol is widely used a cryoprotectant (CPA) for boar spermatozoa cryopreservation, the mechanism and relationship between glycerol and apoptosis-related gene expression needs to be clarified. In this study, we treated boar spermatozoa with different concentrations of glycerol in lactose egg yolk (LEY) extender to evaluate the apoptosis-related gene expression and protease activities of caspases. These results show that: (1) low concentrations of glycerol (2% and 3%) were more suitable for boar spermatozoa cryopreservation; (2) apoptosis-related genes involved in intrinsic mitochondrial and extrinsic death receptor apoptotic signaling pathways were widely expressed in different concentrations of glycerol treated boar spermatozoa; (3) there was a significant positive correlation (r=0.840, P=0.037) between the percentage of Annexin V(+)/PI(+) staining spermatozoa and caspase-6/9 protease activity. In conclusion, 2% and 3% glycerol have the best anti-apoptotic effects, and the expression of Fas/FasL and Bcl-2/Bax have a strong correlation with spermatozoa parameters. PMID:24680861

  12. Cytotoxic activity of the novel heterocyclic compound G-11 is primarily mediated through intrinsic apoptotic pathway.

    PubMed

    Saleh, Ayman M; Aziz, Mohammad A; Abdou, Ibrahim M; Taha, Mutasem O; Al-Qudah, Mahmoud A; Abadleh, Mohammed M; Aljada, Ahmad; Rizvi, Syed A

    2016-07-01

    Natural and chemically synthesized heterocyclic compounds have been explored for their potential use as anticancer agents. We had synthesized non-natural heterocyclic analogs and evaluated their anti-tumor activity by measuring effect on cell proliferation and induction of apoptosis in different cell lines. Previously, we identified a pyrazole-containing compound (G-11) showing cytotoxic effect towards leukemia and lymphoma cell lines. In this study, we further investigated the mechanistic aspects of anticancer properties of G-11 in HL-60 cell line. We demonstrated that cytotoxic effect of G-11 is mediated by caspase-dependent apoptosis. However, the involvement of mitochondrial dysfunction induced by G-11 was independent of caspases. G-11 triggered generation of ROS, caused disruption of mitochondrial transmembrane potential, increased release of cytochrome c to the cytosol, and altered the expression of Bcl-2 and Bax proteins. These results suggest significant involvement of intrinsic apoptotic pathway. This study comprehensively details the possible mechanisms of action of a novel heterocyclic compound which could find its potential use as an anticancer agent. PMID:27154302

  13. Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways

    PubMed Central

    Timmins, Jenelle M.; Ozcan, Lale; Seimon, Tracie A.; Li, Gang; Malagelada, Cristina; Backs, Johannes; Backs, Thea; Bassel-Duby, Rhonda; Olson, Eric N.; Anderson, Mark E.; Tabas, Ira

    2009-01-01

    ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis. PMID:19741297

  14. Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway-Effect of SH reagents.

    PubMed

    Lofrumento, Dario Domenico; La Piana, Gianluigi; Palmitessa, Valeria; Abbrescia, Daniela Isabel; Lofrumento, Nicola Elio

    2016-07-01

    Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours. PMID:27129925

  15. Mcl-1 involvement in mitochondrial dynamics is associated with apoptotic cell death

    PubMed Central

    Morciano, Giampaolo; Giorgi, Carlotta; Balestra, Dario; Marchi, Saverio; Perrone, Daniela; Pinotti, Mirko; Pinton, Paolo

    2016-01-01

    The B-cell lymphoma-2 (Bcl-2) family proteins are critical regulators of apoptosis and consist of both proapoptotic and antiapoptotic factors. Within this family, the myeloid cell leukemia factor 1 (Mcl-1) protein exists in two forms as the result of alternative splicing. The long variant (Mcl-1L) acts as an antiapoptotic factor, whereas the short isoform (Mcl-1S) displays proapoptotic activity. In this study, using splice-switching antisense oligonucleotides (ASOs), we increased the synthesis of Mcl-1S, which induced a concurrent reduction of Mcl-1L, resulting in increased sensitivity of cancer cells to apoptotic stimuli. The Mcl-1 ASOs also induced mitochondrial hyperpolarization and a consequent increase in mitochondrial calcium (Ca2+) accumulation. The high Mcl-1S/L ratio correlated with significant hyperfusion of the entire mitochondrial network, which occurred in a dynamin-related protein (Drp1)–dependent manner. Our data indicate that the balance between the long and short variants of the Mcl-1 gene represents a key aspect of the regulation of mitochondrial physiology. We propose that the Mcl-1L/S balance is a novel regulatory factor controlling the mitochondrial fusion and fission machinery. PMID:26538029

  16. PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

    PubMed Central

    Arena, G; Gelmetti, V; Torosantucci, L; Vignone, D; Lamorte, G; De Rosa, P; Cilia, E; Jonas, E A; Valente, E M

    2013-01-01

    Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1–Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer. PMID:23519076

  17. Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

    SciTech Connect

    Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2011-11-15

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles ({approx} 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25-400 {mu}g/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase C{delta} (PKC{delta}), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: Black-Right-Pointing-Pointer Mn nanoparticles

  18. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis.

    PubMed

    Shiraishi, J; Tatsumi, T; Keira, N; Akashi, K; Mano, A; Yamanaka, S; Matoba, S; Asayama, J; Yaoi, T; Fushiki, S; Fliss, H; Nakagawa, M

    2001-10-01

    Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation. PMID:11557554

  19. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1.

    PubMed

    Fond, Aaron M; Lee, Chang Sup; Schulman, Ira G; Kiss, Robert S; Ravichandran, Kodi S

    2015-07-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  20. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1

    PubMed Central

    Fond, Aaron M.; Lee, Chang Sup; Schulman, Ira G.; Kiss, Robert S.; Ravichandran, Kodi S.

    2015-01-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane–initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell–initiated pathway functioned independently of the liver X receptor (LXR) sterol–sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  1. Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers.

    PubMed

    Marques-Aleixo, I; Santos-Alves, E; Balça, M M; Rizo-Roca, D; Moreira, P I; Oliveira, P J; Magalhães, J; Ascensão, A

    2015-08-20

    We here investigate the effects of two exercise modalities (endurance treadmill training-TM and voluntary free-wheel activity-FW) on the brain cortex and cerebellum mitochondrial bioenergetics, permeability transition pore (mPTP), oxidative stress, as well as on proteins involved in mitochondrial biogenesis, apoptosis, and quality control. Eighteen male rats were assigned to sedentary-SED, TM and FW groups. Behavioral alterations and ex vivo brain mitochondrial function endpoints were assessed. Proteins involved in oxidative phosphorylation (OXPHOS, including the adenine nucleotide translocator), oxidative stress markers and regulatory proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, -SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α, TFAM) were evaluated. Apoptotic signaling was measured through quantifying caspase 3, 8 and 9-like activities, Bax, Bcl2, CypD, and cofilin expression. Mitochondrial dynamics (Mfn1/2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin, p62)-related proteins were also measured by Western blotting. Only the TM exercise group showed increased spontaneous alternation and exploratory activity. Both exercise regimens improved mitochondrial respiratory activity, increased OXPHOS complexes I, III and V subunits in both brain subareas and decreased oxidative stress markers. Increased resistance to mPTP and decreased apoptotic signaling were observed in the brain cortex from TM and in the cerebellum from TM and FW groups. Also, exercise increased the expression of proteins involved in mitochondrial biogenesis, autophagy and fusion, simultaneous with decreased expression of mitochondrial fission-related protein DRP1. In conclusion, physical exercise improves brain cortex and cerebellum mitochondrial function, decreasing oxidative stress and apoptotic related markers. It is also possible that favorable alterations in mitochondrial biogenesis, dynamics and autophagy signaling induced by exercise

  2. Mitochondrial dysfunction: a crucial event in okadaic acid (ICV) induced memory impairment and apoptotic cell death in rat brain.

    PubMed

    Kamat, Pradeep K; Tota, Santoshkumar; Shukla, Rakesh; Ali, Shakir; Najmi, Abul Kalam; Nath, Chandishwar

    2011-12-01

    Mitochondrial abnormalities have been identified in a large proportion of neurodegenerative diseases. Recently we have reported that intracerebroventricular (ICV) administration of okadaic acid (OKA) causes memory impairment in rat. However involvement of mitochondrial function in OKA induced memory impairment and neuronal damage has not been determined. OKA (200 ng) was administered by ICV route. After 13th day of OKA administration memory function was evaluated by Morris Water Maze test. Following completion of behavioral studies on 16th day, mitochondrial membrane potential, Ca(2+) and reactive oxygen species were evaluated in mitochondrial preparation of cortex, hippocampus, striatum and cerebellum of rat brain. While ATP, mitochondrial activity, lipid peroxidation and nitrite were investigated in synaptosomal preparation of rat brain areas. The activities and mRNA expression of apoptotic factors, caspase-3 and caspase-9, were studied in rat brain regions. The neuronal damage was also confirmed by histopathological study. OKA treated rats showed memory impairment including increased Ca(2+) and reactive oxygen species and decreased mitochondrial membrane potential, ATP and mitochondrial activity in mitochondrial preparation. There was a significant increase in lipid peroxidation and nitrite in synaptosomal preparations. Preventive treatment daily for 13 days with antidementic drugs, donepezil (5 mg/kg, p.o) and memantine (10 mg/kg, p.o), significantly attenuated OKA induced mitochondrial dysfunction, apoptotic cell death, memory impairment and histological changes. Mitochondrial dysfunction appeared as a key factor in OKA induced memory impairment and apoptotic cell death. This study indicates that clinically used antidementic drugs are effective against OKA induced adverse changes at behavioral, cellular, and histological levels and mitochondrial dysfunction. PMID:21893081

  3. B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

    PubMed

    Besnault, L; Schrantz, N; Auffredou, M T; Leca, G; Bourgeade, M F; Vazquez, A

    2001-07-15

    We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein. PMID:11441077

  4. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.

    PubMed

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H; Hustedt, Eric J; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the 'α3/α5' interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known 'α6:α6' interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  5. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore

    PubMed Central

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H.; Hustedt, Eric J.; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  6. Enhanced apoptotic cancer cell killing after Foscan photodynamic therapy combined with fenretinide via de novo sphingolipid biosynthesis pathway.

    PubMed

    Boppana, Nithin B; DeLor, Jeremy S; Van Buren, Eric; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Korbelik, Mladen; Separovic, Duska

    2016-06-01

    We and others have shown that stresses, including photodynamic therapy (PDT), can disrupt the de novo sphingolipid biosynthesis pathway, leading to changes in the levels of sphingolipids, and subsequently, modulation of cell death. The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent reaction, giving rise to dihydroceramide, which is then converted in a desaturase-dependent reaction to ceramide. In this study we tested the hypothesis that combining Foscan-mediated PDT with desaturase inhibitor fenretinide (HPR) enhances cancer cell killing. We discovered that by subjecting SCC19 cells, a human head and neck squamous cell carcinoma cell line, to PDT+HPR resulted in enhanced accumulation of C16-dihydroceramide, not ceramide. Concomitantly, mitochondrial depolarization was enhanced by the combined treatment. Enhanced activation of caspase-3 after PDT+HPR was inhibited by FB. Enhanced clonogenic cell death after the combination was sensitive to FB, as well as Bcl2- and caspase inhibitors. Treatment of mouse SCCVII squamous cell carcinoma tumors with PDT+HPR resulted in improved long-term tumor cures. Overall, our data showed that combining PDT with HPR enhanced apoptotic cancer cell killing and antitumor efficacy of PDT. The data suggest the involvement of the de novo sphingolipid biosynthesis pathway in enhanced apoptotic cell killing after PDT+HPR, and identify the combination as a novel more effective anticancer treatment than either treatment alone. PMID:27085050

  7. Mitochondrial Cell Death Pathways in Caenorhabiditis elegans.

    PubMed

    Seervi, Mahendra; Xue, Ding

    2015-01-01

    Programmed cell death is an evolutionarily conserved process essential for animal development and tissue homeostasis. Mitochondria have been demonstrated to play a central role in regulating both the activation and the execution of apoptosis. In particular, mitochondria release multiple proapoptotic factors from its intermembrane space, leading to both caspase-dependent and -independent cell death. Despite the pivotal roles of invertebrate animal models, Caenorhabiditis elegans and Drosophila melanogaster, in deciphering conserved pathways and mechanisms of programmed cell death, the importance of mitochondria to apoptosis of invertebrates remains elusive and largely unexplored. Recent studies have corroborated significant association between mitochondria and apoptosis in C. elegans, making it a thrust area of investigations. In this review, we detail the roles of mitochondrial proteins in mediating execution of cell death in C. elegans, including chromosome fragmentation, phosphatidylserine externalization, and elimination of mitochondria, and discuss the potential roles of mitochondria in the activation of C. elegans cell death. The combination of traditional powerful genetic tools and the emergence of the multiple new reverse genetic techniques, including the highly efficient CRISPR/Cas9 gene-editing method, should make C. elegans an ideal animal model for analyzing mitochondrial cell death pathways and associated regulatory mechanisms. PMID:26431563

  8. Crosstalk between tumor suppressors p53 and PKCδ: Execution of the intrinsic apoptotic pathways.

    PubMed

    Dashzeveg, Nurmaa; Yoshida, Kiyotsugu

    2016-07-28

    p53 and PKCδ are tumor suppressors that execute apoptotic mechanisms in response to various cellular stresses. p53 is a transcription factor that is frequently mutated in human cancers; it regulates apoptosis in transcription-dependent and -independent ways in response to genotoxic stresses. PKCδ is a serine/threonine protein kinase and mutated in human cancers. Available evidence shows that PKCδ activates p53 by direct and/or indirect mechanisms. Moreover, PKCδ is also implicated in the transcriptional regulation of p53 in response to DNA damage. Recent findings demonstrated that p53, in turn, binds onto the PKCδ promoter and induces its expression upon DNA damage to facilitate apoptosis. Both p53 and PKCδ are associated with the apoptotic mechanisms in the mitochondria by regulating Bcl-2 family proteins to provide mitochondrial outer membrane permeabilization. This review discusses the crosstalk between p53 and PKCδ in the context of apoptotic cell death and cancer therapy. PMID:27130668

  9. Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes.

    PubMed

    Gulbins, Erich; Sassi, Nicola; Grassmè, Heike; Zoratti, Mario; Szabò, Ildikò

    2010-01-01

    Mitochondria have been shown to play a pivotal role in apoptotic signalling in various cell types. We have recently reported that in lymphocytes the voltage-gated potassium channel Kv1.3, known to reside in the plasma membrane, is active also in the inner mitochondrial membrane. Upon induction of apoptosis, outer-membrane inserted Bax binds to and inhibits Kv1.3 resulting in hyperpolarization, an increase in reactive oxygen species production and cytochrome c release. In cells lacking Kv1.3 these events do not take place. Here, we present new data which further corroborates an important role of this channel in the sequence of events leading to Bax-induced cytochrome c release. Recombinant Kv1.3, when pre-incubated with Bax, prevents the actions of Bax at the level of mitochondria. Furthermore, we report the presence of Kv1.3 protein in mitochondria from PC3 and MCF-7 cancer cells, suggesting that this channel might play a role in the apoptotic signalling not only in lymphocytes but also in other cells. PMID:20114030

  10. Plasma-activated medium induces A549 cell injury via a spiral apoptotic cascade involving the mitochondrial-nuclear network.

    PubMed

    Adachi, Tetsuo; Tanaka, Hiromasa; Nonomura, Saho; Hara, Hirokazu; Kondo, Shin-ichi; Hori, Masaru

    2015-02-01

    Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Nonthermal atmospheric pressure plasma can be applied to living cells and tissues and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only directly, but also by indirect treatment with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the inhibitory effects of PAM on A549 cell survival and elucidate the signaling mechanisms responsible for cell death. PAM maintained its ability to suppress cell viability for at least 1 week when stored at -80°C. The severity of PAM-triggered cell injury depended on the kind of culture medium used to prepare the PAM, especially that with or without pyruvate. Hydrogen peroxide (H2O2) and/or its derived or cooperating reactive oxygen species reduced the mitochondrial membrane potential, downregulated the expression of the antiapoptotic protein Bcl2, activated poly(ADP-ribose) polymerase-1, and released apoptosis-inducing factor from mitochondria with endoplasmic reticulum stress. However, the activation of caspase 3/7 and attenuation of cell viability by the addition of caspase inhibitor were not observed. The accumulation of adenine 5'-diphosphoribose as a product of the above reactions activated transient receptor potential melastatin 2, which elevated intracellular Ca(2+) levels and subsequently led to cell death. These results demonstrated that H2O2 and/or other reactive species in PAM disturbed the mitochondrial-nuclear network in cancer cells through a caspase-independent apoptotic pathway. Moreover, damage to the plasma membrane by H2O2-cooperating charged species not only induced apoptosis, but also increased its permeability to extracellular reactive species. These phenomena were also detected in PAM-treated HepG2 and MCF-7 cells. PMID:25433364

  11. Stevioside induced ROS-mediated apoptosis through mitochondrial pathway in human breast cancer cell line MCF-7.

    PubMed

    Paul, S; Sengupta, S; Bandyopadhyay, T K; Bhattacharyya, A

    2012-01-01

    Stevioside is a diterpene glycoside found in the leaf of Stevia rebaudiana, a traditional oriental medicinal herb, which has been shown to have various biological and ethno-medicinal activities including antitumor activity. In this study, we investigated the effects of stevioside on the cytotoxicity, induction of apoptosis, and the putative pathways of its action in human breast cancer cells (MCF-7). For the analysis of apoptotic pathway, measurement of reactive oxygen species (ROS) and assessment of mitochondrial transmembrane potential (MTP) were achieved. We showed that stevioside was a potent inducer of apoptosis and it conveyed the apoptotic signal via intracellular ROS generation; thereby inducing change in MTP and induction of mitochondrial mediated apoptotic pathway. Taken together, our data indicated that stevioside induces the ROS-mediated mitochondrial permeability transition and results in the increased expression of apoptotic proteins such as Bax, Bcl-2 and Caspase-9. Effect of stevioside on stress-related transcription factors like NF-E2-related factor-2 opens up a new vista for further studies. This is the first report on the mechanism of the antibreast cancer (in vitro) activity of stevioside. PMID:23061910

  12. Ethanol promotes T cell apoptosis through the mitochondrial pathway

    PubMed Central

    Kapasi, Aditi A; Patel, Geeta; Goenka, Anuj; Nahar, Nilay; Modi, Neeraj; Bhaskaran, Madhu; Reddy, Krishna; Franki, Nicholas; Patel, Jaimita; Singhal, Pravin C

    2003-01-01

    Clinical reports suggest that acute ethanol intoxication is often associated with lymphopenia. Previously, ethanol was reported to invoke thymocyte apoptosis. We studied the effect of ethanol on T cell apoptosis. In addition, we evaluated the molecular mechanism of ethanol-induced T cell apoptosis. Human T cells harvested from healthy subjects after an alcohol drinking binge showed enhanced T cell apoptosis (before, 0·4 ± 0·2% versus after, 19·6 ± 2·5% apoptotic lymphocytes/field; P < 0·001). In in vitro studies, ethanol in a concentration of 50 mm and higher enhanced the apoptosis of Jurkat cells. DNA isolated from ethanol-treated Jurkat cells displayed integer multiples of 180 base pairs. Ethanol decreased Jurkat cell expression of Bcl-2, whereas ethanol increased Jurkat cell expression of Bax. Jurkat cells treated with ethanol also showed translocation of cytochrome C into cytosol. Moreover, a caspase-9 inhibitor partially inhibited ethanol-induced Jurkat cell apoptosis. In in vivo studies, after binge drinking, T cell expression of Bcl-2 also decreased. In addition, binge drinking induced the cleavage of caspase-3, suggesting activation of caspase-3 in T cells. These results suggest that ethanol promotes T cell apoptosis through the activation of intrinsic or mitochondrial pathway. PMID:12603597

  13. Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-L-carnitine to mouse fibroblasts in culture

    SciTech Connect

    Pillich, Rudolf Tito; Scarsella, Gianfranco; Risuleo, Gianfranco . E-mail: gianfranco.risuleo@uniroma1.it

    2005-05-15

    It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl-L-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl-L-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptotic death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl-L-carnitine stimulates cell proliferation.

  14. Apoptosis and apoptotic pathway in actinic prurigo by immunohistochemistry

    PubMed Central

    Cuevas-González, Juan-Carlos; García-Vázquez, Francisco-Javier; Rodríguez-Lobato, Erika; Farfán-Morales, José-Eduardo

    2016-01-01

    Background Actinic prurigo (AP) is an idiopathic photodermatosis, this entity requires exposure to UV-B and -A to develop lesions. Apoptosis is a physiological death program that can be initiated by a permanently active mechanism (extrinsic pathway) or irreparable damage (intrinsic pathway). Material and Methods Descriptive study, the sample size comprised 64 paraffin blocks of tissue with a diagnosis of AP. In H&E-stained slides, the diagnosis of AP was corroborated, and 1-µm-thick sections were processed for immunohistochemistry (IHC). A database was constructed with SPSS version 20, Inc., Chicago, IL, USA, and descriptive statistics were analyzed by X2 test and comparison of means. Results A total of 64 cases were processed, of which 40 (62.5%) were cheilitis AP and 24 (37.5%) were AP in the skin. Of the 40 cheilitis samples, 27 were positive for Bcl-2 and caspase 3 (67.5%), p53 was expressed in 30 (75%). Of the skin lesions,p53 and caspase 3 were expressed in 18 of 24 cases (75%), and 13 were positive for Bcl-2 (54%). Conclusions We propose that apoptosis is the last step in the type IV subtype a-b hypersensitivity response-activation of the intrinsic pathway indicates that external factors, such as UV-A and -B are the trigger. Key words:Apoptosis, actinic prurigo, cheilitis actinic prurigo. PMID:26615506

  15. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

    PubMed Central

    Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2014-01-01

    Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998

  16. Proteasome Impairment Induces Recovery of Mitochondrial Membrane Potential and an Alternative Pathway of Mitochondrial Fusion

    PubMed Central

    Shirozu, Ryohei; Yashiroda, Hideki

    2015-01-01

    Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of the short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to the long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment. PMID:26552703

  17. Antitumor effects of traditional Chinese medicine targeting the cellular apoptotic pathway

    PubMed Central

    Xu, Huanli; Zhao, Xin; Liu, Xiaohui; Xu, Pingxiang; Zhang, Keming; Lin, Xiukun

    2015-01-01

    Defects in apoptosis are common phenomena in many types of cancer and are also a critical step in tumorigenesis. Targeting the apoptotic pathway has been considered an intriguing strategy for cancer therapy. Traditional Chinese medicine (TCM) has been used in the People’s Republic of China for thousands of years, and many of the medicines have been confirmed to be effective in the treatment of a number of tumors. With increasing cancer rates worldwide, the antitumor effects of TCMs have attracted more and more attention globally. Many of the TCMs have been shown to have antitumor activity through multiple targets, and apoptosis pathway-related targets have been extensively studied and defined to be promising. This review focuses on several antitumor TCMs, especially those with clinical efficacy, based on their effects on the apoptotic signaling pathway. The problems with and prospects of development of TCMs as anticancer agents are also presented. PMID:26056434

  18. Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs, mitochondria, calpain and caspase12 dependent pathways.

    PubMed

    Pal, Sankhadeep; Ghosh, Manoranjan; Ghosh, Shatadal; Bhattacharyya, Sudip; Sil, Parames C

    2015-09-01

    Atorvastatin (ATO), a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, is used widely for the treatment of hypercholesterolemia and hypertriglyceridemia. Application of this drug has now been made somehow limited because of ATO associated several acute and chronic side effects. The present study has been carried out to investigate the dose-dependent hepatic tissue toxicity in ATO induced oxidative impairment and cell death in mice. Administration of ATO enhanced ALT, ALP level, increased reactive oxygen species (ROS) production and altered the pro oxidant-antioxidant status of liver by reducing intracellular GSH level, anti-oxidant enzymes activities and increasing intracellular lipid peroxidation. Our experimental evidence suggests that ATO markedly decreased mitochondrial membrane potential, disturbed the Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased the levels of Apaf1, caspase-9, -3, cleaved PARP protein and ultimately led to apoptotic cell death. Besides, ATO distinctly increased the phosphorylation of p38, JNK, and ERK MAPKs, enhanced Caspase12 and calpain level. Histological studies also support the dose-dependent toxic effect of ATO in these organs pathophysiology. These results reveal that ATO induces hepatic tissue toxicity via MAPKs, mitochondria and ER dependent signaling pathway, in which calcium ions and ROS act as the pivotal mediators of the apoptotic signaling. PMID:26051349

  19. BAD-mediated apoptotic pathway is associated with human cancer development

    PubMed Central

    STICKLES, XIAOMANG B; MARCHION, DOUGLAS C; BICAKU, ELONA; SAWAH, ENTIDHAR AL; ABBASI, FOROUGH; XIONG, YIN; ZGHEIB, NADIM BOU; BOAC, BERNADETTE M; ORR, BRIAN C; JUDSON, PATRICIA L; BERRY, AMY; HAKAM, ARDESHIR; WENHAM, ROBERT M; APTE, SACHIN M; BERGLUND, ANDERS E; LANCASTER, JOHNATHAN M

    2015-01-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway

  20. The JAK2/STAT3 and mitochondrial pathways are essential for quercetin nanoliposome-induced C6 glioma cell death

    PubMed Central

    Wang, G; Wang, J J; Chen, X L; Du, S M; Li, D S; Pei, Z J; Lan, H; Wu, L B

    2013-01-01

    The formulation of quercetin nanoliposomes (QUE-NLs) has been shown to enhance QUE antitumor activity in C6 glioma cells. At high concentrations, QUE-NLs induce necrotic cell death. In this study, we probed the molecular mechanisms of QUE-NL-induced C6 glioma cell death and examined whether QUE-NL-induced programmed cell death involved Bcl-2 family and mitochondrial pathway through STAT3 signal transduction pathway. Downregulation of Bcl-2 and the overexpression of Bax by QUE-NL supported the involvement of Bcl-2 family proteins upstream of C6 glioma cell death. In addition, the activation of JAK2 and STAT3 were altered following exposure to QUE-NLs in C6 glioma cells, suggesting that QUE-NLs downregulated Bcl-2 mRNAs expression and enhanced the expression of mitochondrial mRNAs through STAT3-mediated signaling pathways either via direct or indirect mechanisms. There are several components such as ROS, mitochondrial, and Bcl-2 family shared by the necrotic and apoptotic pathways. Our studies indicate that the signaling cross point of the mitochondrial pathway and the JAK2/STAT3 signaling pathway in C6 glioma cell death is modulated by QUE-NLs. In conclusion, regulation of JAK2/STAT3 and ROS-mediated mitochondrial pathway agonists alone or in combination with treatment by QUE-NLs could be a more effective method of treating chemical-resistant glioma. PMID:23907460

  1. Modulation of Apoptotic Pathways of Macrophages by Surface-Functionalized Multi-Walled Carbon Nanotubes

    PubMed Central

    Jiang, Yuanqin; Zhang, Honggang; Wang, Yange; Chen, Min; Ye, Shefang; Hou, Zhenqing; Ren, Lei

    2013-01-01

    Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47phox and p67phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity. PMID:23755279

  2. Multiple Pathways Influence Mitochondrial Inheritance in Budding Yeast

    PubMed Central

    Frederick, Rebecca L.; Okamoto, Koji; Shaw, Janet M.

    2008-01-01

    Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Δ and deletions of genes that affect mitochondrial function or inheritance, including mmr1Δ. Synthetic sickness of gem1Δ mmr1Δ double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution. PMID:18245340

  3. Chronic Cigarette Smoke Extract Treatment Selects For Apoptotic Dysfunction and Mitochondrial mutations in Minimally Transformed Oral Keratinocytes

    PubMed Central

    Chang, Steven S.; Jiang, Wei Wen; Smith, Ian; Glazer, Chad; Sun, Wen-Yue; Mithani, Suhail; Califano, Joseph A.

    2009-01-01

    Cigarette smoke demonstrates a carcinogenic effect through chronic exposure, not acute exposures. However, current cell line models study only the acute effects of cigarette smoke. Using a cell line model, we compared the effects of acute versus chronic cigarette-smoke-extract (CSE) on mitochondria in minimally-transformed oral keratinocytes (OKF6). OKF6 cells were treated with varying concentrations of CSE for 6-months. Cells were analyzed monthly by flow cytometry for mitochondrial-membrane-potential (MMP), cytochrome-c release, caspase-3 activation and viability after CSE-exposure. At each time point the same assays were performed after 24hrs of valinomycin (MMP depolarizing agent) treatment. The mitochondrial-DNA of chronically CSE-treated cells was sequenced. After 6-months of CSE-treatment, the cells were increasingly resistant to CSE-mediated and valinomycin induced cell death. In addition, chronic CSE-treatment caused chronic depolarization of MMP, cytochrome c release, and caspase activation. Cells grown in the presence of only CSE vapor also exhibited the same resistance and chronic baseline apoptotic activation. Mitochondrial DNA sequencing found that chronic CSE treated cells had more amino acid changing mitochondrial mutations than acutely treated cells. CSE treatment of normal cells select for apoptotic dysfunction as well as mitochondrial mutations. These findings suggest that chronic tobacco exposure induce carcinogenesis via selection of apoptosis resistance and mitochondrial mutation in addition to previously known genotoxic effects that were found by acute treatments. Chronic models of tobacco exposure on upper aerodigestive epithelia may be more insightful than models of acute exposure in studying head and neck carcinogenesis PMID:19634139

  4. Rilmenidine suppresses proliferation and promotes apoptosis via the mitochondrial pathway in human leukemic K562 cells.

    PubMed

    Srdic-Rajic, Tatjana; Nikolic, Katarina; Cavic, Milena; Djokic, Ivana; Gemovic, Branislava; Perovic, Vladimir; Veljkovic, Nevena

    2016-01-01

    Imidazoline I1 receptor signaling is associated with pathways that regulate cell viability leading to varied cell-type specific phenotypes. We demonstrated that the antihypertensive drug rilmenidine, a selective imidazoline I1 receptor agonist, modulates proliferation and stimulates the proapoptotic protein Bax thus inducing the perturbation of the mitochondrial pathway and apoptosis in human leukemic K562 cells. Rilmenidine acts through a mechanism which involves deactivation of Ras/MAP kinases ERK, p38 and JNK. Moreover, rilmenidine renders K562 cells, which are particularly resistant to chemotherapeutic agents, susceptible to the DNA damaging drug doxorubicin. The rilmenidine co-treatment with doxorubicin reverses G2/M arrest and triggers apoptotic response to DNA damage. Our data offer new insights into the pathways associated with imidazoline I1 receptor activation in K562 cells suggesting rilmenidine as a valuable tool to deepen our understanding of imidazoline I1 receptor signaling in hematologic malignancies and to search for medicinally active agents. PMID:26598394

  5. Attenuation of Magnesium Sulfate on CoCl₂-Induced Cell Death by Activating ERK1/2/MAPK and Inhibiting HIF-1α via Mitochondrial Apoptotic Signaling Suppression in a Neuronal Cell Line.

    PubMed

    Huang, Chih-Yang; Hsieh, You-Liang; Ju, Da-Tong; Lin, Chien-Chung; Kuo, Chia-Hua; Liou, Yi-Fan; Ho, Tsung-Jung; Tsai, Chang-Hai; Tsai, Fuu-Jen; Lin, Jing-Ying

    2015-08-31

    Magnesium sulfate (MgSO₄) ameliorates hypoxia/ischemia-induced neuronal apoptosis in a rat model. This study aimed to investigate the mechanisms governing the anti-apoptotic effect of MgSO₄ on cobalt chloride (CoCl₂)-exposed NB41A3 mouse neuroblastoma cells. MgSO₄ increased the viability of NB41A3 cells treated with CoCl₂ in a dose-dependent manner. MgSO₄ treatment was shown to lead to an increase in the anti-apoptotic Bcl-2 family proteins, with a concomitant decrease in the pro-apoptotic proteins. MgSO₄ also attenuated the CoCl₂-induced disruption of mitochondrial membrane potential (ΔΨ(m)) and reduced the release of cytochrome c form the mitochondria to the cytosol. Furthermore, exposure to CoCl₂ caused activation of the hypoxia-inducible factor 1α (HIF-1α). On the other hand, MgSO₄ markedly reduced CoCl₂-induced HIF-1α activation and suppressed HIF-1α downstream protein BNIP3. MgSO₄ treatment induced ERK1/2 activation and attenuated CoCl₂-induced activation of p38 and JNK. Addition of the ERK1/2 inhibitor U0126 significantly reduced the ability of MgSO₄ to protect neurons from CoCl₂-induced mitochondrial apoptotic events. However, incubation of cultures with the p38 and JNK inhibitors did not significantly affect MgSO₄-mediated neuroprotection. MgSO₄ appears to suppress CoCl₂-induced NB41A3 cell death by activating ERK1/2/ MAPK pathways, which further modulates the role of Bcl-2 family proteins and mitochondria in NB41A3 cells. Our data suggest that MgSO₄ may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways. PMID:26211648

  6. Molecular adaptations of apoptotic pathways and signaling partners in the cerebral cortex of human cocaine addicts and cocaine-treated rats.

    PubMed

    Alvaro-Bartolomé, M; La Harpe, R; Callado, L F; Meana, J J; García-Sevilla, J A

    2011-11-24

    Cocaine induces apoptotic effects in cultured cells and in the developing brain, but the aberrant activation of cell death in the adult brain remains inconclusive, especially in humans. This postmortem human brain study examined the status of canonical apoptotic pathways, signaling partners, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, in prefrontal cortex (PFC) of a small but well-characterized cohort of cocaine abusers (n=10). For comparison, the chosen targets were also quantified in the cerebral cortex of cocaine-treated rats. In the PFC of cocaine abusers, FS7-associated cell surface antigen (Fas) receptor aggregates and Fas-associated death domain (FADD) adaptor were reduced (-26% and -66%, respectively) as well as the content of mitochondrial cytochrome c (-61%). In the same brain samples of cocaine abusers, the proteolytic cleavage of PARP-1 was increased (+39%). Nuclear PARP-1 degradation, possibly a consequence of increased mitochondrial oxidative stress, involved the activation of apoptosis-inducing factor (AIF) and not that of caspase-3. In the PFC of cocaine abusers, several signaling molecules associated with cocaine/dopamine and/or apoptotic pathways were not significantly altered, with the exception of anti-apoptotic truncated DARPP-32 (t-DARPP), a truncated isoform of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), whose content was decreased (-28%). Chronic exposure to cocaine in rats, including withdrawal for 3 days, did not alter Fas-FADD receptor complex, cytochrome c, caspase-3/fragments, AIF, PARP-1 cleavage, and associated signaling in the cerebral cortex. Chronic cocaine and abstinence, however, increased the content of t-DARPP (+39% and +47%) in rat brain cortex. The major findings indicate that cocaine addiction in humans is not associated with abnormal activation of extrinsic and intrinsic apoptotic pathways in PFC. The downregulation of Fas-FADD receptor complex and cytochrome c

  7. Fermented Brown Rice Extract Causes Apoptotic Death of Human Acute Lymphoblastic Leukemia Cells via Death Receptor Pathway.

    PubMed

    Horie, Yukiko; Nemoto, Hideyuki; Itoh, Mari; Kosaka, Hiroaki; Morita, Kyoji

    2016-04-01

    Mixture of brown rice and rice bran fermented with Aspergillus oryzae, designated as FBRA, has been reported to reveal anti-carcinogenic and anti-inflammatory effects in rodents. Then, to test its potential anti-cancer activity, the aqueous extract was prepared from FBRA powder, and the effect of this extract on human acute lymphoblastic leukemia Jurkat cells was directly examined. The exposure to FBRA extract reduced the cell viability in a concentration- and time-dependent manner. The reduction of the cell viability was accompanied by the DNA fragmentation, and partially restored by treatment with pan-caspase inhibitor. Further studies showed that FBRA extract induced the cleavage of caspase-8, -9, and -3, and decreased Bcl-2 protein expression. Moreover, the expression of tBid, DR5, and Fas proteins was enhanced by FBRA extract, and the pretreatment with caspase-8 inhibitor, but not caspase-9 inhibitor, restored the reduction of the cell viability induced by FBRA extract. These findings suggested that FBRA extract could induce the apoptotic death of human acute lymphoblastic leukemia cells probably through mainly the death receptor-mediated pathway and supplementarily through the tBid-mediated mitochondrial pathway, proposing the possibility that FBRA was a potential functional food beneficial to patients with hematological cancer. PMID:26769704

  8. The cytotoxic and pro-apoptotic effects of phenylephrine on corneal stromal cells via a mitochondrion-dependent pathway both in vitro and in vivo.

    PubMed

    Zhao, Jun; Qiu, Yue; Tian, Cheng-Lei; Fan, Ting-Jun

    2016-08-01

    Phenylephrine (PHE), a selective α1-adrenergic receptor agonist, is often used as a decongestant for mydriasis prior to cataract surgery, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of PHE to the cornea and its cellular and molecular mechanisms remain unknown. To provide references for secure medication and prospective therapeutic interventions of PHE, we investigated the cytotoxicity of PHE to corneal stroma and its possible mechanisms using an in vitro model of human corneal stromal (HCS) cells and an in vivo model of cat keratocytes. We found that PHE, above the concentration of 0.0781125% (1/128 of its clinical therapeutic dosage), had a dose- and time-dependent cytotoxicity to HCS cells by inducing morphological abnormality and viability decline, as well as S phase arrest. Moreover, PHE induced apoptosis of HCS cells by inducing plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation. Furthermore, PHE could induce activations of caspase-3 and -9, disruption of mitochondrial transmembrane potential, downregulation of anti-apoptotic Bcl-xL, upregulation of pro-apoptotic Bax, along with upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. The cytotoxic and pro-apoptotic effects of PHE were also proven by the induced apoptotic-like ultrastructural alterations of keratocytes in vivo. Taken together, our results suggest that PHE has a significant cytotoxicity to corneal stroma cells both in vitro and in vivo by inducing cell apoptosis, and the pro-apoptotic effect of PHE is achieved via a Bcl-2 family proteins-mediated mitochondrion-dependent pathway. PMID:27344612

  9. The BARD1 BRCT domain contributes to p53 binding, cytoplasmic and mitochondrial localization, and apoptotic function.

    PubMed

    Tembe, Varsha; Martino-Echarri, Estefania; Marzec, Kamila A; Mok, Myth T S; Brodie, Kirsty M; Mills, Kate; Lei, Ying; DeFazio, Anna; Rizos, Helen; Kettle, Emma; Boadle, Ross; Henderson, Beric R

    2015-09-01

    BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function. PMID:26022179

  10. Oridonin induces apoptosis through the mitochondrial pathway in human gastric cancer SGC-7901 cells.

    PubMed

    Gao, Shiyong; Tan, Huixin; Zhu, Nan; Gao, Haiyu; Lv, Chunyu; Gang, Jian; Ji, Yubin

    2016-06-01

    Oridonin is one of the most important antitumor active ingredients of Rabdosia rubescens. Recently published studies from our laboratory have demonstrated that oridonin was able to arrest human gastric cancer SGC-7901 cells at G2/M phase. However, little is known about inducing apoptosis in gastric cancer. The aim of this study was to investigate the effect of oridonin on antineoplastic capability of SGC-7901 cells and the detailed molecular mechanism of oridonin-mediated intrinsic pathway of apoptosis. Cell proliferation was assessed by MTT assay while apoptosis induced by oridonin was determined by Hoechst 33342 staining assay and Annexin V/PI double staining assay. Early apoptotic rate was stained by Annexin V/PI and detected by flow cytometry. Apoptosis pathway was analyzed by western blot analysis of Bcl-2, Bax, cytochrome c and caspase-3 expression. The results showed that oridonin was able to inhibit the SGC-7901 cell proliferation, the 50% growth inhibition (IC50) was 22.74 µM. Oridonin could induce cell apoptosis of SGC-7901 cells and the early apoptotic rates induced by 0, 20, 40, 80 µmol/l oridonin were 1.53±0.67, 3.33±0.29, 84.80±0.82 and 96.43±0.51%, respectively. Western blot analysis revealed that oridonin downregulated Bcl-2 protein (the anti-apoptotic factor) and upregulated Bax protein (pro-apoptotic factor), eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. Furthermore, oridonin induced the release of cytochrome c from the mitochondria to the cytosol and the activation of caspase-3. Taken together, the current study suggested that oridonin induced apoptosis in SGC-7901 cells via the mitochondrial signal pathway, which may represent one of the major mechanisms of oridonin-mediated apoptosis in SGC-7901 cells. PMID:27082253

  11. Simian virus 40 infection triggers a balanced network that includes apoptotic, survival, and stress pathways.

    PubMed

    Butin-Israeli, Veronika; Drayman, Nir; Oppenheim, Ariella

    2010-04-01

    The infection process by simian virus 40 (SV40) and entry of its genome into nondividing cells are only partly understood. Infection begins by binding to GM1 receptors at the cell surface, cellular entry via caveolar invaginations, and trafficking to the endoplasmic reticulum, where the virus disassembles. To gain a deeper insight into the contribution of host functions to this process, we studied cellular signaling elicited by the infecting virus. Signaling proteins were detected by Western blotting and immunofluorescence staining. The study was assisted by a preliminary proteomic screen. The contribution of signaling proteins to the infection process was evaluated using specific inhibitors. We found that CV-1 cells respond to SV40 infection by activating poly(ADP-ribose) polymerase 1 (PARP-1)-mediated apoptotic signaling, which is arrested by the Akt-1 survival pathway and stress response. A single key regulator orchestrating the three pathways is phospholipase C-gamma (PLCgamma). The counteracting apoptotic and survival pathways are robustly balanced as the infected cells neither undergo apoptosis nor proliferate. Surprisingly, we have found that the apoptotic pathway, including activation of PARP-1 and caspases, is absolutely required for the infection to proceed. Thus, SV40 hijacks the host defense to promote its infection. Activities of PLCgamma and Akt-1 are also required, and their inhibition abrogates the infection. Notably, this signaling network is activated hours before T antigen is expressed. Experiments with recombinant empty capsids, devoid of DNA, indicated that the major capsid protein VP1 alone triggers this early signaling network. The emerging robust signaling network reflects a delicate evolutionary balance between attack and defense in the host-virus relationship. PMID:20089643

  12. Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway

    PubMed Central

    Wang, Xiaoliang; Lau, Waynebond; Wang, Yajing; Xing, Yuan; Zhang, Xing; Ma, Xinliang; Gao, Feng

    2013-01-01

    Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway. PMID:23976968

  13. Targeting the apoptotic pathway with BCL-2 inhibitors sensitizes primary chronic lymphocytic leukemia cells to vesicular stomatitis virus-induced oncolysis.

    PubMed

    Tumilasci, Vanessa Fonseca; Olière, Stephanie; Nguyên, Thi Lien-Ahn; Shamy, April; Bell, John; Hiscott, John

    2008-09-01

    Chronic lymphocytic leukemia (CLL) is characterized by clonal accumulation of CD5(+) CD19(+) B lymphocytes that are arrested in the G(0)/G(1) phase of the cell cycle and fail to undergo apoptosis because of overexpression of the antiapoptotic B-cell CLL/lymphoma 2 (BCL-2) protein. Oncolytic viruses, such as vesicular stomatitis virus (VSV), have emerged as potential anticancer agents that selectively target and kill malignant cells via the intrinsic mitochondrial pathway. Although primary CLL cells are largely resistant to VSV oncolysis, we postulated that targeting the apoptotic pathway via inhibition of BCL-2 may sensitize CLL cells to VSV oncolysis. In the present study, we examined the capacity of EM20-25--a small-molecule antagonist of the BCL-2 protein--to overcome CLL resistance to VSV oncolysis. We demonstrate a synergistic effect of the two agents in primary ex vivo CLL cells (combination index of 0.5; P < 0.0001). In a direct comparison of peripheral blood mononuclear cells from healthy volunteers with primary CLL, the two agents combined showed a therapeutic index of 19-fold; furthermore, the combination of VSV and EM20-25 increased apoptotic cell death in Karpas-422 and Granta-519 B-lymphoma cell lines (P < 0.005) via the intrinsic mitochondrial pathway. Mechanistically, EM20-25 blocked the ability of the BCL-2 protein to dimerize with proapoptotic BAX protein, thus sensitizing CLL to VSV oncolytic stress. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in treatment-resistant hematological malignancies, such as CLL, with characterized defects in the apoptotic response. PMID:18579592

  14. Tat-NOL3 protects against hippocampal neuronal cell death induced by oxidative stress through the regulation of apoptotic pathways.

    PubMed

    Sohn, Eun Jeong; Shin, Min Jea; Eum, Won Sik; Kim, Dae Won; Yong, Ji In; Ryu, Eun Ji; Park, Jung Hwan; Cho, Su Bin; Cha, Hyun Ju; Kim, Sang Jin; Yeo, Hyeon Ji; Yeo, Eun Ji; Choi, Yeon Joo; Im, Seung Kwon; Kweon, Hae Young; Kim, Duk-Soo; Yu, Yeon Hee; Cho, Sung-Woo; Park, Meeyoung; Park, Jinseu; Cho, Yong-Jun; Choi, Soo Young

    2016-07-01

    Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein 3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide (H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein (Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species (ROS), DNA fragmentation and reduced mitochondrial membrane potential (ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3 and -8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury. PMID:27221790

  15. Arsenic trioxide induces oxidative stress, DNA damage, and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells

    PubMed Central

    2014-01-01

    Background Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells. Methods In the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization. Results ATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells. Conclusion Taken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death. PMID:24887205

  16. Gemfibrozil pretreatment resulted in a sexually dimorphic outcome in the rat models of global cerebral ischemia-reperfusion via modulation of mitochondrial pro-survival and apoptotic cell death factors as well as MAPKs.

    PubMed

    Mohagheghi, Fatemeh; Ahmadiani, Abolhassan; Rahmani, Behrouz; Moradi, Fatemeh; Romond, Nathalie; Khalaj, Leila

    2013-07-01

    Inducers of mitochondrial biogenesis are widely under investigation for use in a novel therapeutic approach in neurodegenerative disorders. The ability of Gemfibrozil, a fibrate, is investigated for the first time to modulate mitochondrial pro-survival factors involved in the mitochondrial biogenesis signaling pathway, including peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A (TFAM) in the brain. Gemfibozil is clinically administered to control hyperlipidemia. It secondarily prevents cardiovascular events such as cardiac arrest in susceptible patients. In this study, pretreatment of animals with gemfibrozil prior to ischemia-reperfusion (I/R) resulted in a sexually dimorphic outcome. While the expression of NRF-1 and TFAM were induced in gemfibrozil-pretreated met-estrous females, they were suppressed in males. Gemfibrozil also proved to be neuroprotective in met-estrous females, as it inhibited caspase-dependent apoptosis while in males it led to hippocampal neurodegeneration via activation of both the caspase-dependent and caspase-independent apoptosis. In the mitogen-activated protein kinase (MAPKs) pathway, gemfibrozil pretreatment induced the expression of extracellular signal-regulated kinases (ERK1/2) in met-estrous females and reduced it in males. These findings correlatively point to the sexual-dimorphic effects of gemfibrozil in global cerebral I/R context by affecting important factors involved in the mitochondrial biogenesis, MAPKs, and apoptotic cell death pathways. PMID:23288702

  17. Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

    PubMed Central

    Santambrogio, Paolo; Erba, Benedetta Gaia; Campanella, Alessandro; Cozzi, Anna; Causarano, Vincenza; Cremonesi, Laura; Gallì, Anna; Della Porta, Matteo Giovanni; Invernizzi, Rosangela; Levi, Sonia

    2011-01-01

    Background Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. Design and Methods We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Results Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Conclusions Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation. PMID:21712541

  18. Mitochondrial Respiratory Pathways Inhibition in Rhizopus oryzae Potentiates Activity of Posaconazole and Itraconazole via Apoptosis

    PubMed Central

    Shirazi, Fazal; Kontoyiannis, Dimitrios P.

    2013-01-01

    The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents. PMID:23696824

  19. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

    SciTech Connect

    Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke . E-mail: ryosuket@kuhp.kyoto-u.ac.jp; Miura, Masayuki . E-mail: miura@mol.f.u-tokyo.ac.jp

    2007-05-18

    Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.

  20. Mitochondrial respiratory pathways inhibition in Rhizopus oryzae potentiates activity of posaconazole and itraconazole via apoptosis.

    PubMed

    Shirazi, Fazal; Kontoyiannis, Dimitrios P

    2013-01-01

    The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents. PMID:23696824

  1. Mitochondrial translocation of APE1 relies on the MIA pathway.

    PubMed

    Barchiesi, Arianna; Wasilewski, Michal; Chacinska, Agnieszka; Tell, Gianluca; Vascotto, Carlo

    2015-06-23

    APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival. PMID:25956655

  2. Pomegranate peel extract polyphenols induced apoptosis in human hepatoma cells by mitochondrial pathway.

    PubMed

    Song, Bingbing; Li, Jia; Li, Jianke

    2016-07-01

    This study was aimed to investigate the influence of pomegranate peel polyphenols (PPPs) on the proliferation and apoptosis of HepG2 cells (a kind of human hepatoma cells) and the related mechanism. The inverted fluorescence microscope and the flow cytometer (FCM) were used to test the changes of the cellular morphology, cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm). The kit was used to measure the activities of caspase-3/9, and Western Blot was used to detect the expressions of apoptosis-associated proteins including p53, Bcl-2/Bax, Cyt-c and PARP. The results showed that the cells cycle of HepG2 arrested at the S-phase by PPPs and the amount of the early apoptotic cells and ROS level were increased obviously, the level of Cyt-c and the activity of Caspase-3/9 markedly were also increased by PPPs, as well as the ratio of Bax/Bcl-2 and the protein expressions of P53. It was concluded that PPPs could inhibit the growth of HepG2 cells by blocking the cell cycle and inducing the mitochondrial apoptotic pathway in a dose-dependent manner. PMID:27120393

  3. Genetic modulation of apoptotic pathways fails to alter disease course in tripeptidyl-peptidase 1 deficient mice.

    PubMed

    Kim, Kwi-Hye; Sleat, David E; Bernard, Ora; Lobel, Peter

    2009-03-27

    Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, incurable neurodegenerative disease of children caused by the loss of the lysosomal protein tripeptidyl-peptidase 1 (TPP1). Previous studies have suggested that Bcl-2-dependent apoptotic pathways are involved in neuronal cell death in LINCL patients and, as a result, anti-apoptotic treatments that increase Bcl-2 activity have been proposed as a potential therapeutic approach. In this study, we have directly investigated whether targeting anti-apoptotic pathways may be of value in LINCL in a mouse model of this disease that lacks TPP1 and which recapitulates many aspect of the human disease, including a greatly shortened life-span. Our approach was to genetically modify apoptotic pathways and determine the effects of these changes on the severe neurodegenerative phenotype of the LINCL mouse. LINCL mice were generated that either lacked the pro-apoptotic p53 or had increased levels of anti-apoptotic Bcl-2, changes that would exacerbate or ameliorate neuronal death, respectively, should pathways involving these proteins be important. Neither modification affected the shortened life-span of the LINCL mouse. These results suggest that either neuronal death in LINCL does not occur via apoptosis or that it occurs via apoptotic pathways not involving p53 or Bcl-2. Alternatively, pathways involving p53 and/or Bcl-2 may be involved in neuronal death under normal circumstances but may not be the only routes to this end. Importantly, our findings suggest that targeting pathways of cell death involving p53 or Bcl-2 do not represent useful directions for developing effective treatment. PMID:19429009

  4. Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand?

    PubMed Central

    Baig, S; Seevasant, I; Mohamad, J; Mukheem, A; Huri, H Z; Kamarul, T

    2016-01-01

    Underneath the intricacy of every cancer lies mysterious events that impel the tumour cell and its posterity into abnormal growth and tissue invasion. Oncogenic mutations disturb the regulatory circuits responsible for the governance of versatile cellular functions, permitting tumour cells to endure deregulated proliferation, resist to proapoptotic insults, invade and erode normal tissues and above all escape apoptosis. This disruption of apoptosis has been highly implicated in various malignancies and has been exploited as an anticancer strategy. Owing to the fact that apoptosis causes minimal inflammation and damage to the tissue, apoptotic cell death-based therapy has been the centre of attraction for the development of anticancer drugs. Increased understanding of the molecular pathways underlying apoptosis has enabled scientists to establish unique approaches targeting apoptosis pathways in cancer therapeutics. In this review, we reconnoitre the two major pathways (intrinsic and extrinsic) targeted cancer therapeutics, steering toward chief modulators of these pathways, such as B-cell lymphoma 2 protein family members (pro- and antiapoptotic), inhibitor of apoptosis proteins, and the foremost thespian of extrinsic pathway regulator, tumour necrosis factor-related apoptosis-inducing agent. Together, we also will have a look from clinical perspective to address the agents (drugs) and therapeutic strategies adopted to target these specific proteins/pathways that have entered clinical trials. PMID:26775709

  5. A novel pathway for phagosome maturation during engulfment of apoptotic cells

    PubMed Central

    Kinchen, Jason M.; Doukoumetzidis, Kimon; Almendinger, Johann; Stergiou, Lilli; Tosello-Trampont, Annie; Sifri, Costi D.; Hengartner, Michael O.; Ravichandran, Kodi S.

    2010-01-01

    The efficient removal of apoptotic cells is critical for the physiological well-being of the organism1â4; defects in corpse removal have been linked to autoimmune disease4, 5. While several players regulating the early steps of corpse recognition and internalization have been characterized6, the molecules and mechanisms relevant to the subsequent processing of the internalized corpses are poorly understood. Here, we identify a novel pathway for the processing of internalized apoptotic cells in C. elegans and in mammals. First, we show that RAB-5 and RAB-7 are sequentially recruited to phagosomes containing apoptotic corpses as they mature within phagocytes, and that both proteins are required for efficient corpse clearance. We then used targeted genetic screens to identify players regulating the recruitment and/or retention of Rab5 and Rab7 to phagosomes. Seven members of the HOPS complex (a Rab7 activator/effector complex) were required for Rab7 localization or retention on phagosomes. In an effort to identify factors that regulate Rab5 recruitment, we undertook an unbiased reverse genetic screen and identified 61 genes potentially required for corpse removal. In-depth analysis of two candidate genes, vps-34 and dyn-1/dynamin, showed accumulation of internalized, but undegraded corpses within abnormal phagosomes that are defective in RAB-5 recruitment. Using a series of genetic and biochemical experiments in worms and mammalian cells, we ordered these proteins in a pathway, with DYN-1 functioning upstream of VPS-34, in the recruitment/retention of Rab5 to the nascent phagosome. Further, we identified a novel biochemical complex containing Vps34, dynamin and Rab5GDP, providing a mechanism for Rab5 recruitment to the nascent phagosome. PMID:18425118

  6. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells.

    PubMed

    Fu, Meili; Wan, Fuqiang; Li, Zhengling; Zhang, Fenghua

    2016-03-01

    The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation-inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D, a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. PMID:26773495

  7. Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP Pathway Activators of the Unfolded Protein Response

    PubMed Central

    2015-01-01

    Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50 = 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel. PMID:25530830

  8. Mitochondrial pathway of apoptosis is ancestral in metazoans

    PubMed Central

    Bender, Cheryl E.; Fitzgerald, Patrick; Tait, Stephen W. G.; Llambi, Fabien; McStay, Gavin P.; Tupper, Douglas O.; Pellettieri, Jason; Alvarado, Alejandro Sánchez; Salvesen, Guy S.; Green, Douglas R.

    2012-01-01

    The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in vertebrates. In this pathway, proapoptotic members of the Bcl-2 family cause mitochondrial outer membrane permeabilization (MOMP), allowing the release of cytochrome c, which interacts with Apaf-1 to trigger caspase activation and apoptosis. Despite conservation of Bcl-2, Apaf-1, and caspases in invertebrate phyla, the existence of the mitochondrial pathway in any invertebrate is, at best, controversial. Here we show that apoptosis in a lophotrochozoan, planaria (phylum Platyhelminthes), is associated with MOMP and that cytochrome c triggers caspase activation in cytosolic extracts from these animals. Further, planarian Bcl-2 family proteins can induce and/or regulate cell death in yeast and can replace Bcl-2 proteins in mammalian cells to regulate MOMP. These results suggest that the mitochondrial pathway of apoptosis in animals predates the emergence of the vertebrates but was lost in some lineages (e.g., nematodes). In further support of this hypothesis, we surveyed the ability of cytochrome c to trigger caspase activation in cytosolic extracts from a variety of organisms and found this effect in cytosolic extracts from invertebrate deuterostomes (phylum Echinodermata). PMID:22416118

  9. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    SciTech Connect

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  10. mitochondrial pathway for biosynthesis of lipid mediators

    PubMed Central

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-01-01

    The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury. PMID:24848241

  11. Upregulation of extrinsic apoptotic pathway in curcumin-mediated antiproliferative effect on human pancreatic carcinogenesis.

    PubMed

    Youns, Mahmoud; Fathy, Gihan Mahmoud

    2013-12-01

    Pancreatic cancer is one of the most lethal human cancers, with almost identical incidence and mortality rates. Curcumin, derived from the rhizome of Curcuma longa, has a long history of use as coloring agent and for a wide variety of disorders. Here, the antiproliferative activity of curcumin and its modulatory effect on gene expression of pancreatic cancer cell lines were investigated. The effect of curcumin on cellular proliferation and viability was monitored by sulphurhodamine B assay. Apoptotic effect was evaluated by flow cytometry and further confirmed by measuring amount of cytoplasmic histone-associated DNA fragments. Analysis of gene expression was performed with and without curcumin treatment using microarray expression profiling techniques. Array results were confirmed by real-time PCR. ingenuity pathway analysis (IPA) has been used to classify the list of differentially expressed genes and to indentify common biomarkergenes modulating the chemopreventive effect of curcumin. Results showed that curcumin induces growth arrest and apoptosis in pancreatic cancer cell lines. Its effect was more obvious on the highly COX-2 expressing cell line. Additionally, the expression of 366 and 356 cancer-related genes, involved in regulation of apoptosis, cell cycle, metastasis, was significantly altered after curcumin treatment in BxPC-3 and MiaPaCa-2 cells, respectively. Our results suggested that up-regulation of the extrinsic apoptotic pathway was among signaling pathways modulating the growth inhibitory effects of curcumin on pancreatic cancer cells. Curcumin effect was mediated through activation of TNFR, CASP 8, CASP3, BID, BAX, and down-regulation of NFκB, NDRG 1, and BCL2L10 genes. PMID:23794119

  12. The study of the Oxytropis kansuensis-induced apoptotic pathway in the cerebrum of SD rats

    PubMed Central

    2013-01-01

    Background Locoweeds cause significant livestock poisoning and economic loss all over the world. Animals can develop locoism, a chronic neurological disease, after grazing on locoweeds. Oxytropis kansuensis is a variety of locoweed that contains swainsonine as its main toxic ingredient. The purpose of this study was to investigate the apoptotic pathway induced in the cerebrum by swainsonine. Results Twenty-four Sprague-Dawley rats were randomly divided into four groups (experimental groups I, II, III and a control group) and 6 SD rats of each group were feed in 3 cages separately. Rats were penned as groups and fed with feeds containing 15% (SW content 0.03‰), 30% (SW content 0.06‰), or 45% (SW content 0.09‰) O. kansuensis for experimental groups I, II, and III, respectively, or complete feed in the case of the control group. One hundred and nineteen days after poisoning, and all rats showed neurological disorders at different degrees, which were considered to be successful established a chronic poisoning model of O. kansuensis. rats were sacrificed and the expression of Fas, FasL, Bcl-2, Bax as well as cleaved caspase-3, -8 and -9 proteins in brain tissues were detected by Western blot. The results showed that SW treatment up-regulated Fas and Fas ligand (FasL) (P < 0.05), and that there was an increase in Bax and a decrease in Bcl-2 protein (P < 0.01). Moreover, SW treatment significantly increases the activation of caspase-3, 8 and -9, the key effectors in apoptosis pathway (P < 0.01). Conclusion Our data suggest that SW induces apoptosis in cells of the brain through death receptor and mitochondria-mediated, caspase-dependent apoptotic pathways in the brain tissue of SD rats. PMID:24148892

  13. Methane attenuates retinal ischemia/reperfusion injury via anti-oxidative and anti-apoptotic pathways.

    PubMed

    Liu, Lin; Sun, Qinglei; Wang, Ruobing; Chen, Zeli; Wu, Jiangchun; Xia, Fangzhou; Fan, Xian-Qun

    2016-09-01

    Retinal ischemia/reperfusion injury (IRI) may cause incurable visual impairment due to neural regeneration limits. Methane was shown to exert a protective effect against IRI in many organs. This study aims to explore the possible protective effects of methane-rich saline against retinal IRI in rat. Retinal IRI was performed on the right eyes of male Sprague-Dawley rats, which were immediately injected intraperitoneally with methane-saturated saline (25ml/kg). At one week after surgery, the number of retinal ganglion cells (RGCs), total retinal thickness, visual function were measured by hematoxylin and eosin staining, FluoroGold anterograde labeling and flash visual evoked potentials. The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-Hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), caspase-3, caspase-9, B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in retinas were assessed by immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. As expected, methane treatment significantly improved the retinal IRI-induced RGC loss, total retinal layer thinning and visual dysfunction. Moreover, methane treatment significantly reduced the levels of oxidative stress biomarkers (8-OHdG, 4-HNE, MDA) and increased the antioxidant enzyme activities (SOD, CAT, GPx) in the retinas with IRI. Meanwhile, methane treatment significantly increased the anti-apoptotic gene (Bcl-2) expression and decreased the pro-apoptotic gene (Bax) expression, accompanied by the suppression of caspase-3 and caspase-9 activity. Thus, these data demonstrated that methane can exert a neuroprotective role against retinal IRI through anti-oxidative and anti-apoptotic pathways. PMID:27208496

  14. Anti-apoptotic role of the sonic hedgehog signaling pathway in the proliferation of ameloblastoma

    PubMed Central

    KANDA, SHIORI; MITSUYASU, TAKESHI; NAKAO, YU; KAWANO, SHINTARO; GOTO, YUICHI; MATSUBARA, RYOTA; NAKAMURA, SEIJI

    2013-01-01

    Sonic hedgehog (SHH) signaling pathway is crucial to growth and patterning during organogenesis. Aberrant activation of the SHH signaling pathway can result in tumor formation. We examined the expression of SHH signaling molecules and investigated the involvement of the SHH pathway in the proliferation of ameloblastoma, the most common benign tumor of the jaws. We used immunohistochemistry on ameloblastoma specimens and immunocytochemistry and reverse transcription-PCR on the ameloblastoma cell line AM-1. We also used the inhibitors of SHH signaling, SHH neutralizing antibody and cyclopamine, to assess the effects of SHH on the proliferation of AM-1 cells. We detected expression of SHH, patched, GLI1, GLI2 and GLI3 in the ameloblastoma specimens and AM-1 cells. The proliferation of these cells was significantly inhibited in the presence of SHH neutralizing antibody or cyclopamine; this was confirmed by BrdU incorporation assays. Furthermore, in the presence of SHH neutralizing antibody, nuclear translocation of GLI1 and GLI2 was abolished, apoptosis was induced, BCL-2 expression decreased and BAX expression increased. Our results suggest that the SHH signaling pathway is constitutively active in ameloblastoma and plays an anti-apoptotic role in the proliferation of ameloblastoma cells through autocrine loop stimulation. PMID:23835807

  15. Cd-Induced Apoptosis through the Mitochondrial Pathway in the Hepatopancreas of the Freshwater Crab Sinopotamon henanense

    PubMed Central

    Liu, Dongmei; Yang, Jian; Li, Yingjun; Zhang, Meng; Wang, Lan

    2013-01-01

    Cd is one of the most common pollutants in the environment that also induces the apoptosis. To explore the mechanism of apoptosis in the hepatopancreas, freshwater crab S. henanense were treated with 0, 3.56, 7.12, 14.25, 28.49 and 56.98 mg/L Cd for 72 h. Apoptosis was noticeable in every treatment group and necrosis was observed clearly in the high concentration Cd groups. Classical apoptotic bodies were found by transmission electronic microscopy, which revealed chromatin condensation under nuclear membrane and mitochondrial membrane rupture. An increasing number of autolysosomes, damaged rough endoplamic reticulum and Golgi complex were observed as the Cd concentration increase. Brown colored apoptotic cells were detected by the TUNEL test in all Cd-treatment groups. The apoptosis index increased following the elevation of Cd concentration and got 32.9% in the highest Cd group. Caspase-9 and caspase-3 activities increased in the lower Cd treatment groups but no changes in the higher Cd concentration groups (comparing to the control group). The activity of caspase-8 did not change significantly. No significant change in the content of mitochondrial cytochrome c (cyt c) in Cd exposed groups except the decrease in the 56.98 mg/L group. In crabs treated with 3.56, 7.12 and 14.25 mg/L Cd, hyperpolarization of mitochondrial membrane potential (Δψm) significantly increased. These results implied that apoptosis in the hepatopancreas induced by Cd occurrs through the mitochondrial caspase-dependent pathway. However, whether there are other apoptotic pathways needs to be studied further. PMID:23894343

  16. Effects of 10-hydroxycamptothecin on intrinsic mitochondrial pathway in silkworm BmN-SWU1 cells.

    PubMed

    Pan, Chun; Hu, Yan-Fen; Song, Juan; Yi, Hua-Shan; Wang, La; Yang, Yi-Ying; Wang, Yong-Ping; Zhang, Man; Pan, Min-Hui; Lu, Cheng

    2016-02-01

    10-Hydroxycamptothecin (HCPT), a plant alkaloid isolated from Camptotheca acuminate, is known as a planted-derived insecticide, however, the specific mechanism in insect cells is still unclear. In this study, we treated the ovarian cell line of the silkworm, BmN-SWU1, with different HCPT doses for durations ranging from 0 to 72h. The apoptosis morphology was evident after 72h of incubation and included cell protuberance, concentrated cytoplasm and apoptotic bodies. We observed DNA fragmentation and cell apoptosis after HCPT treatment. The disruption of mitochondrial distribution, activation of the intracellular mitochondrial permeability transition pore, and release of cytochrome c during HCPT-induced apoptosis in dose and time-dependent manner indicate the involvement of mitochondria in BmN-SWU1 cells. Caspase-9 and -3 activities increased gradually with the duration of incubation time. In conclusion, HCPT has a significant effect to initiate the intrinsic mitochondrial pathway in silkworm cells, providing a theoretical basis for better application of plant-derived insecticide in pest control. PMID:26821653

  17. Towards the small and the beautiful: a small dibromotyrosine derivative from Pseudoceratina sp. sponge exhibits potent apoptotic effect through targeting IKK/NFκB signaling pathway.

    PubMed

    Su, Jui-Hsin; Chen, Yu-Cheng; El-Shazly, Mohamed; Du, Ying-Chi; Su, Chiang-Wen; Tsao, Chia-Wei; Liu, Li-Lian; Chou, Yalan; Chang, Wen-Been; Su, Yin-Di; Chiang, Michael Y; Yeh, Yao-Tsung; Lu, Mei-Chin

    2013-09-01

    A dibromotyrosine derivative, (1'R,5'S,6'S)-2-(3',5'-dibromo-1',6'-dihydroxy-4'-oxocyclohex-2'-enyl) acetonitrile (DT), was isolated from the sponge Pseudoceratina sp., and was found to exhibit a significant cytotoxic activity against leukemia K562 cells. Despite the large number of the isolated bromotyrosine derivatives, studies focusing on their biological mechanism of action are scarce. In the current study we designed a set of experiments to reveal the underlying mechanism of DT cytotoxic activity against K562 cells. First, the results of MTT cytotoxic and the annexin V-FITC/PI apoptotic assays, indicated that the DT cytotoxic activity is mediated through induction of apoptosis. This effect was also supported by caspases-3 and -9 activation as well as PARP cleavage. DT induced generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) as indicated by flow cytometric assay. The involvement of ROS generation in the apoptotic activity of DT was further corroborated by the pretreatment of K562 cells with N-acetyl-cysteine (NAC), a ROS scavenger, which prevented apoptosis and the disruption of MMP induced by DT. Results of cell-free system assay suggested that DT can act as a topoisomerase II catalytic inhibitor, unlike the clinical anticancer drug, etoposide, which acts as a topoisomerase poison. Additionally, we found that DT treatment can block IKK/NFκB pathway and activate PI3K/Akt pathway. These findings suggest that the cytotoxic effect of DT is associated with mitochondrial dysfunction-dependent apoptosis which is mediated through oxidative stress. Therefore, DT represents an interesting reference point for the development of new cytotoxic agent targeting IKK/NFκB pathway. PMID:24065159

  18. Towards the Small and the Beautiful: A Small Dibromotyrosine Derivative from Pseudoceratina sp. Sponge Exhibits Potent Apoptotic Effect through Targeting IKK/NFκB Signaling Pathway

    PubMed Central

    Su, Jui-Hsin; Chen, Yu-Cheng; El-Shazly, Mohamed; Du, Ying-Chi; Su, Chiang-Wen; Tsao, Chia-Wei; Liu, Li-Lian; Chou, Yalan; Chang, Wen-Been; Su, Yin-Di; Chiang, Michael Y.; Yeh, Yao-Tsung; Lu, Mei-Chin

    2013-01-01

    A dibromotyrosine derivative, (1′R,5′S,6′S)-2-(3′,5′-dibromo-1′,6′-dihydroxy-4′-oxocyclohex-2′-enyl) acetonitrile (DT), was isolated from the sponge Pseudoceratina sp., and was found to exhibit a significant cytotoxic activity against leukemia K562 cells. Despite the large number of the isolated bromotyrosine derivatives, studies focusing on their biological mechanism of action are scarce. In the current study we designed a set of experiments to reveal the underlying mechanism of DT cytotoxic activity against K562 cells. First, the results of MTT cytotoxic and the annexin V-FITC/PI apoptotic assays, indicated that the DT cytotoxic activity is mediated through induction of apoptosis. This effect was also supported by caspases-3 and -9 activation as well as PARP cleavage. DT induced generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) as indicated by flow cytometric assay. The involvement of ROS generation in the apoptotic activity of DT was further corroborated by the pretreatment of K562 cells with N-acetyl-cysteine (NAC), a ROS scavenger, which prevented apoptosis and the disruption of MMP induced by DT. Results of cell-free system assay suggested that DT can act as a topoisomerase II catalytic inhibitor, unlike the clinical anticancer drug, etoposide, which acts as a topoisomerase poison. Additionally, we found that DT treatment can block IKK/NFκB pathway and activate PI3K/Akt pathway. These findings suggest that the cytotoxic effect of DT is associated with mitochondrial dysfunction-dependent apoptosis which is mediated through oxidative stress. Therefore, DT represents an interesting reference point for the development of new cytotoxic agent targeting IKK/NFκB pathway. PMID:24065159

  19. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles

    PubMed Central

    Sugiura, Ayumu; McLelland, Gian-Luca; Fon, Edward A; McBride, Heidi M

    2014-01-01

    The last decade has been marked by tremendous progress in our understanding of the cell biology of mitochondria, with the identification of molecules and mechanisms that regulate their fusion, fission, motility, and the architectural transitions within the inner membrane. More importantly, the manipulation of these machineries in tissues has provided links between mitochondrial dynamics and physiology. Indeed, just as the proteins required for fusion and fission were identified, they were quickly linked to both rare and common human diseases. This highlighted the critical importance of this emerging field to medicine, with new hopes of finding drugable targets for numerous pathologies, from neurodegenerative diseases to inflammation and cancer. In the midst of these exciting new discoveries, an unexpected new aspect of mitochondrial cell biology has been uncovered; the generation of small vesicular carriers that transport mitochondrial proteins and lipids to other intracellular organelles. These mitochondrial-derived vesicles (MDVs) were first found to transport a mitochondrial outer membrane protein MAPL to a subpopulation of peroxisomes. However, other MDVs did not target peroxisomes and instead fused with the late endosome, or multivesicular body. The Parkinson's disease-associated proteins Vps35, Parkin, and PINK1 are involved in the biogenesis of a subset of these MDVs, linking this novel trafficking pathway to human disease. In this review, we outline what has been learned about the mechanisms and functional importance of MDV transport and speculate on the greater impact of these pathways in cellular physiology. PMID:25107473

  20. Phloroglucinol induces apoptosis via apoptotic signaling pathways in HT-29 colon cancer cells

    PubMed Central

    KANG, MI-HYE; KIM, IN-HYE; NAM, TAEK-JEO NG

    2014-01-01

    Phloroglucinol is a polyphenolic compound that is used to treat and prevent several human diseases, as it exerts beneficial biological activities, including anti-oxidant, anti-inflammatory and anticancer properties. The aim of the present study was to investigate the effects of phloroglucinol on apoptotic signaling pathways in HT-29 colon cancer cells. The results indicated that phloroglucinol suppressed cell viability and induced apoptosis in HT-29 cells in a concentration-dependent manner. Phloroglucinol treatment of HT-29 cells resulted in characteristic apoptosis-related changes: altered Bcl-2 family proteins, cytochrome c release, and activation of caspase-3 and caspase-8. This study also showed that proteins involved in apoptosis were stimulated by treatment with phloroglucinol. These findings demonstrated that phloroglucinol exerts anticancer activity in HT-29 colon cancer cells through induction of apoptosis. PMID:25070748

  1. Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

    PubMed

    Esmekaya, Meric Arda; Seyhan, Nesrin; Kayhan, Handan; Tuysuz, Mehmet Zahid; Kurşun, Ayşe Canseven; Yağcı, Münci

    2013-01-01

    In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway. PMID:23723005

  2. MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

    PubMed Central

    Li, Senlin; Tang, Yijun; Wei, Bo; Yu, Huansha; Wang, Chen

    2014-01-01

    Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2−/− cells, but not Jnk1−/− cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7−/−) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2−/− mice, but not Jnk1−/− mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses. PMID:24651600

  3. In vitro analysis of the role of the mitochondrial apoptosis pathway in CSBE therapy against human gastric cancer

    PubMed Central

    JI, YU-BIN; YU, LEI

    2015-01-01

    The caper plant (Capparis spinosa L.) was a common Uyghur folk medicine, and is a member of the Capparidaceae family. In a previous study, the n-butanol extract of C. spinosa L. (CSBE) was demonstrated to exert anti-tumor activity; however, the underlying mechanism is currently not understood. The present study aimed to elucidate the mechanism underlying the CSBE-induced mitochondrial apoptotic pathway, in order to investigate the anti-tumor effects of this plant extract. CSBE-induced apoptosis of the SGC-7901 human gastric cancer cell line was observed, and alterations in the expression levels and localization of initiators, markers, and executors of the mitochondrial apoptosis pathway were analyzed. Following treatment of SGC-7901 cells with CBSE, proliferation was inhibited and apoptosis was induced; and these effects were associated with mitochondrial membrane potential disruption, cytochrome c release into the cytoplasm, and caspase-9 and caspase-3 activation. CSBE may have induced SGC-7901 cell apoptosis by upregulating the expression of B-cell lymphoma-2 (BCL-2)-associated X protein, and downregulating the expression of BCL-2. The results of the present study suggested that CSBE may induce SGC-7901 cell apoptosis via activation of the mitochondrial apoptosis pathway. PMID:26668648

  4. The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine*

    PubMed Central

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-01-01

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal. PMID:25713076

  5. The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-04-17

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal. PMID:25713076

  6. Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells

    PubMed Central

    Huo, Haizhong; Zhou, Zhiyuan; Qin, Jian; Liu, Wenyong; Wang, Bing; Gu, Yan

    2016-01-01

    We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent. PMID:27171435

  7. 2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

    PubMed

    Schweikl, Helmut; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Buchalla, Wolfgang; Krifka, Stephanie

    2014-03-01

    Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which

  8. Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-{kappa}B, p38 and JNK MAPK pathway

    SciTech Connect

    Ghosh, Jyotirmoy; Das, Joydeep; Manna, Prasenjit

    2009-10-01

    Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-{kappa}B (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-{kappa}B and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-{kappa}B activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-{kappa}B activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection

  9. Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

    PubMed

    Wu, Yuqiu; Kazumura, Kimiko; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2015-10-01

    Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed. PMID:25863936

  10. A polysaccharide from pumpkin induces apoptosis of HepG2 cells by activation of mitochondrial pathway.

    PubMed

    Shen, Weixi; Guan, Yuanyuan; Wang, Jingfang; Hu, Yu; Tan, Qian; Song, Xiaowei; Jin, Yinghua; Liu, Ying; Zhang, Yanqiao

    2016-04-01

    Purified white polysaccharide (PPW) is a homogenous polysaccharide isolated from pumpkin, with an average molecular weight of 34 kDa. In this study, we aimed at examining the anti-proliferative activity of PPW against hepatocellular carcinoma (HCC) HepG2 cells and the underlying mechanisms. We found that PPW-induced inhibition of cell proliferation in HepG2 cells was associated with the induction of apoptosis. Exposure of HepG2 cells to PPW (100, 200, and 400 μg/mL) resulted in a loss of mitochondrial membrane potential (Δψm) and the release of cytochrome c from the mitochondria to the cytosol. Also, Western blot analysis revealed dose-dependent increase of pro-apoptotic Bax protein and decrease of anti-apoptotic Bcl-2 protein in PPW-treated cells. Besides, caspase-9 and caspase-3 activities were also enhanced in HepG2 cells followed by PPW treatment. Additionally, the cleavage of poly (ADP-ribose) polymerase (PARP) was observed in PPW-treated HepG2 cells, which altogether account for apoptotic cell death. These results suggested that PPW-induced apoptosis involved a caspase-3-mediated mitochondrial pathway and may have potential as a cancer chemopreventive and therapeutic agent for the prevention and treatment of HCC. PMID:26555544

  11. Huperzine A attenuates hepatic ischemia reperfusion injury via anti-oxidative and anti-apoptotic pathways.

    PubMed

    Xu, Zhe; Wang, Yang

    2014-08-01

    Hepatic ischemia reperfusion (HI/R) injury may occur during liver transplantation and remains a serious concern in clinical practice. Huperzine A (HupA), an alkaloid isolated from the Chinese traditional medicine Huperzia serrata, has been demonstrated to possess anti‑oxidative and anti‑apoptotic properties. In the present study, a rat model of HI/R was established by clamping the hepatic artery, the hepatoportal vein and the bile duct with a vascular clamp for 30 min followed by reperfusion for 6 h under anesthesia. HupA was injected into the tail vein 5 min prior to the induction of HI/R at doses of 167 and 500 µg/kg. The histopathological assessment of the liver was performed using hematoxylin and eosin staining. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed in the serum samples. The tissue levels of superoxide dismutase (SOD), catalase (CAT), malondiadehyde (MDA) and glutathione (GSH) were also measured spectrophotometrically. Furthermore, the protein expression of caspase‑3, Bcl‑2 and Bax in hepatic tissues was detected via western blot analysis. Treatment of Wistar rats with HupA at doses of 167 and 500 µg/kg markedly attenuated HI/R injury as observed histologically. In addition, the significant reductions of serum ALT and AST were observed in HupA‑treated ischemic rats. Furthermore, HupA treatment enhanced the activity of hepatic tissue SOD, CAT and GSH, but decreased the MDA tissue content. Western blot analysis revealed elevated levels of Bcl‑2 expression but decreased Bax and caspase‑3 tissue expression at the protein level in the HupA‑treated group. The present data suggest that HupA attenuates the HI/R injury of rats through its anti‑oxidative and anti‑apoptotic signaling pathways. PMID:24888717

  12. Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers.

    PubMed

    Thomas, Rusha; Kim, Myoung H

    2007-02-01

    Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors. PMID:16924414

  13. Mitochondrial function in ageing: coordination with signalling and transcriptional pathways.

    PubMed

    Yin, Fei; Sancheti, Harsh; Liu, Zhigang; Cadenas, Enrique

    2016-04-15

    Mitochondrial dysfunction entailing decreased energy-transducing capacity and perturbed redox homeostasis is an early and sometimes initiating event in ageing and age-related disorders involving tissues with high metabolic rate such as brain, liver and heart. In the central nervous system (CNS), recent findings from our and other groups suggest that the mitochondrion-centred hypometabolism is a key feature of ageing brains and Alzheimer's disease. This hypometabolic state is manifested by lowered neuronal glucose uptake, metabolic shift in the astrocytes, and alternations in mitochondrial tricarboxylic acid cycle function. Similarly, in liver and adipose tissue, mitochondrial capacity around glucose and fatty acid metabolism and thermogenesis is found to decline with age and is implicated in age-related metabolic disorders such as obesity and type 2 diabetes mellitus. These mitochondrion-related disorders in peripheral tissues can impact on brain functions through metabolic, hormonal and inflammatory signals. At the cellular level, studies in CNS and non-CNS tissues support the notion that instead of being viewed as autonomous organelles, mitochondria are part of a dynamic network with close interactions with other cellular components through energy- or redox-sensitive cytosolic kinase signalling and transcriptional pathways. Hence, it would be critical to further understand the molecular mechanisms involved in the communication between mitochondria and the rest of the cell. Therapeutic strategies that effectively preserves or improve mitochondrial function by targeting key component of these signalling cascades could represent a novel direction for numerous mitochondrion-implicated, age-related disorders. PMID:26293414

  14. Grape seed proanthocyanidins induce apoptosis through the mitochondrial pathway in nasopharyngeal carcinoma CNE-2 cells.

    PubMed

    Yao, Kai; Shao, Jingjing; Zhou, Keyuan; Qiu, Haitao; Cao, Fengxiang; Li, Caihong; Dai, De

    2016-08-01

    Although modern radiotherapy offers excellent local control in the treatment of nasopharyngeal carcinoma (NPC), current therapeutic decisions remain burdensome due to the frequency of local recurrence and treatment failure at distant sites. One potential and promising strategy for the prevention or treatment of cancers is the use of bioactive components of plant origin, including dietary plant products. Herein, we studied one class of these bioactive compounds, grape seed proanthocyanidins (GSPs), and explored their effect on NPC CNE-2 cells, as well as the primary mechanism underlying this effect. Our results revealed that treatment of human NPC CNE-2 cells with GSPs reduced cell viability in a dose- and time-dependent manner, and moreover, markedly induced cell cycle arrest at the G2/M phase, leading to induction of apoptosis. In addition, we found that the underlying mechanism was associated with increased expression of the pro-apoptotic protein Bax, decreased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PRAP) protein, and the loss of mitochondrial membrane potential (MMP) (Δψm). Furthermore, GSPs upregulated the Bcl-2 homology 3 (BH3)-only proteins, Bim and Bad, in a concentration-dependent manner. Taken together, these data supported our hypothesis that, in human NPC CNE-2 cells, GSPs could induce apoptosis through the mitochondrial pathway and ultimately reduce cell viability. Collectively, the results discussed above provide substantive evidence for the potential of GSPs as an effective bioactive phytochemical for the treatment of NPC. PMID:27277418

  15. Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic pathway in leukemic cells.

    PubMed

    Chiruvella, Kishore K; Kari, Vijayalakshmi; Choudhary, Bibha; Nambiar, Mridula; Ghanta, Rama Gopal; Raghavan, Sathees C

    2008-12-10

    Methyl angolensate (MA), a natural tetranortriterpenoid, purified from Soymida febrifuga is examined for the first time for its anticancer properties. We find that MA inhibits growth of T-cell leukemia and chronic myelogenous leukemia cells in a time- and dose-dependent manner. Accumulation of cells in the subG1 peak, annexin V binding and DNA fragmentation suggested induction of apoptosis. Besides, upregulation of BAD (proapoptotic) and downregulation of BCL2 (antiapoptotic) gene products further supported induction of apoptosis. Loss of mitochondrial membrane potential, activation of caspase 9, caspase 3, cleavage of PARP, downregulation of Ku70/80 and phosphorylation of MAP kinases suggested that MA could induce intrinsic pathway of apoptosis in leukemic cells. PMID:19022252

  16. Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

    PubMed

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

    2013-03-01

    Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. PMID:23415873

  17. Potent antitumor efficacy of ST13 for colorectal cancer mediated by oncolytic adenovirus via mitochondrial apoptotic cell death.

    PubMed

    Yang, Min; Cao, Xin; Yu, Ming Can; Gu, Jin Fa; Shen, Zong Hou; Ding, Miao; Yu, De Bing; Zheng, Shu; Liu, Xin yuan

    2008-04-01

    ST13 is a cofactor of heat shock protein 70 (Hsp70). To date, all data since the discovery of ST13 in 1993 until more recent studies in 2007 have proved that ST13 is downregulated in tumors and it was proposed to be a tumor suppressor gene, but no work reported its antitumor effect and apoptotic mechanism. In the work described in this paper, ST13 was inserted into ZD55, an oncolytic adenovirus with the E1B 55-kDa gene deleted, to form ZD55-ST13, which exerts an excellent antitumor effect in vitro and in an animal model of colorectal carcinoma SW620 xenograft. ZD55-ST13 inhibited tumor cells 100-fold more than Ad-ST13 and ZD55-EGFP in vitro. However, ZD55-ST13 showed no damage of normal fibroblast MRC5 cells. In exploring the mechanism of ZD55-ST13 in tumor cell killing, we found that ZD55-ST13-infected SW620 cells formed apoptotic bodies and presented obvious apoptosis phenomena. ZD55-ST13 induced the upregulation of Hsp70, the downregulation of antiapoptotic gene Bcl-2, and the release of cytochrome c. Cytochrome c triggered apoptosis by activating caspase-9 and caspase-3, which cleave the enzyme poly(ADP-ribose) polymerase in ZD55-ST13-infected SW620 cells. In summary, overexpressed ST13 as mediated by oncolytic adenovirus could exert potent antitumor activity via the intrinsic apoptotic pathway and has the potential to become a novel therapeutic for colorectal cancer gene therapy. PMID:18355116

  18. Memantine blocks mitochondrial OPA1 and cytochrome c release, and subsequent apoptotic cell death in glaucomatous retina

    PubMed Central

    Ju, Won-Kyu; Kim, Keun-Young; Angert, Mila; Duong-Polk, Karen X.; Lindsey, James D.; Ellisman, Mark H.; Weinreb, Robert N.

    2009-01-01

    Purpose To determine whether intraocular pressure (IOP) elevation alters OPA1 expression and triggers OPA1 release, as well as whether the uncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist memantine blocks OPA1 release and subsequent apoptotic cell death in glaucomatous DBA/2J mouse retina. Methods Preglaucomatous DBA/2J mice received memantine (5 mg/kg, i.p. injection, twice a day for 3 months) and IOP in the eyes was measured monthly. RGC loss was counted following Fluoro-Gold labeling. OPA1, Dnm1, Bcl-2 and Bax mRNA were measured by Taqman qPCR. OPA1 protein was assessed by immunohistochemistry and Western blot. Apoptotic cell death was assessed by TUNEL staining. Results Memantine treatment significantly increased RGC survival in glaucomatous DBA/2J mice. Memantine treatment increased the 75 kDa OPA1 isoform but did not alter the 80 and 90 kDa isoforms. The isoforms of OPA1 were significantly increased in the cytosol of the vehicle-treated glaucomatous retinas but were significantly decreased in memantine-treated glaucomatous retinas. OPA1 immunoreactivity was decreased in the photoreceptors of both vehicle- and memantine-treated glaucomatous retinas but was increased in the outer plexiform layer of only the memantine-treated glaucomatous retinas. Memantine blocked apoptotic cell death in the GCL, increased Bcl-2 gene expression, and decreased Bax gene expression. Conclusions OPA1 release from mitochondria in glaucomatous mouse retina is inhibited by blockade of glutamate receptor activation. Because this OPA1 effect was accompanied by increased Bcl-2 expression, decreased Bax expression and apoptosis blockade, glutamate receptor activation in the glaucomatous retina may involve a distinct mitochondria-mediated cell death pathway. PMID:18936150

  19. Manumycin A induces apoptosis in malignant pleural mesothelioma through regulation of Sp1 and activation of the mitochondria-related apoptotic pathway.

    PubMed

    Kim, Ka Hwi; Chae, Jung-Il; Oh, Hana; Cho, Jin Hyoung; Lee, Ra-Ham; Yoon, Goo; Cho, Seung-Sik; Cho, Young-Sik; Lee, Mee-Hyun; Liu, Kangdong; Lee, Hyun-Jeong; Shim, Jung-Hyun

    2016-07-01

    Manumycin A (Manu A) is a natural product isolated from Streptomyces parvulus and has been reported to have anti-carcinogenic and anti-biotic properties. However, neither its molecular mechanism nor its molecular targets are well understood. Thus, the aim of the present study was to explore the possibility that Manu A has cancer preventive and chemotherapeutic effects on malignant pleural mesothelioma (MPM) through regulation of Sp1 and induction of mitochondrial cell death pathway. Manu A inhibited the cell viability of MSTO-211H and H28 cells in a concentration‑dependent manner as determined by MTS assay. IC50 values were calculated as 8.3 and 4.3 µM in the MSTO-311H and H28 cells following 48 h incubation, respectively. Manu A induced a significant increase in apoptotic indices as shown by DAPI staining, Annexin V assay, multi-caspase activity and mitochondrial membrane potential assay. The downregulation of Sp1 mRNA and protein expression by Manu A led to apoptosis by suppressing Sp1-regulated proteins (cyclin D1, Mcl-1 and survivin). Manu A decreased the protein levels of BID, Bcl-xL and PARP while it increased Bax levels. Manu A caused depolarization of the mitochondrial membrane with induction of CHOP, DR4 and DR5. Our results demonstrated that Manu A exerted anticancer effects by inducing apoptosis via inhibition of the Sp1-related signaling pathway in human MPM. PMID:27176604

  20. BL-038, a Benzofuran Derivative, Induces Cell Apoptosis in Human Chondrosarcoma Cells through Reactive Oxygen Species/Mitochondrial Dysfunction and the Caspases Dependent Pathway.

    PubMed

    Liu, Ju-Fang; Chen, Chien-Yu; Chen, Hsien-Te; Chang, Chih-Shiang; Tang, Chih-Hsin

    2016-01-01

    Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-xL) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells. PMID:27618007

  1. Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization.

    PubMed

    Polonio-Vallon, Tilman; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Hofmann, Thomas G

    2014-01-01

    Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm. PMID:24196445

  2. Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization

    PubMed Central

    Polonio-Vallon, Tilman; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Hofmann, Thomas G

    2014-01-01

    Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm. PMID:24196445

  3. Mitochondrial pathways in sarcopenia of aging and disuse muscle atrophy

    PubMed Central

    Calvani, Riccardo; Joseph, Anna-Maria; Adhihetty, Peter J.; Miccheli, Alfredo; Bossola, Maurizio; Leeuwenburgh, Christiaan; Bernabei, Roberto; Marzetti, Emanuele

    2014-01-01

    Muscle loss during aging and disuse is a highly prevalent and disabling condition, but knowledge about cellular pathways mediating muscle atrophy is still limited. Given the postmitotic nature of skeletal myocytes, the maintenance of cellular homeostasis relies on the efficiency of cellular quality control mechanisms. In this scenario, alterations in mitochondrial function are considered a major factor underlying sarcopenia and muscle atrophy. Damaged mitochondria are not only less bioenergetically efficient, but also generate increased amounts of reactive oxygen species, interfere with cellular quality control mechanisms, and display a greater propensity to trigger apoptosis. Thus, mitochondria stand at the crossroad of signaling pathways that regulate skeletal myocyte function and viability. Studies on these pathways have sometimes provided unexpected and counterintuitive results, which suggests that they are organized into a complex, heterarchical network that is currently insufficiently understood. Untangling the complexity of such a network will likely provide clinicians with novel and highly effective therapeutics to counter the muscle loss associated with aging and disuse. In this review, we summarize the current knowledge on the mechanisms whereby mitochondrial dysfunction intervenes in the pathogenesis of sarcopenia and disuse atrophy, and highlight the prospect of targeting specific processes to treat these conditions. PMID:23154422

  4. Metabolomics for undergraduates: Identification and pathway assignment of mitochondrial metabolites.

    PubMed

    Marques, Ana Patrícia; Serralheiro, Maria Luisa; Ferreira, António E N; Freire, Ana Ponces; Cordeiro, Carlos; Silva, Marta Sousa

    2016-01-01

    Metabolomics is a key discipline in systems biology, together with genomics, transcriptomics, and proteomics. In this omics cascade, the metabolome represents the biochemical products that arise from cellular processes and is often regarded as the final response of a biological system to environmental or genetic changes. The overall screening approach to identify all the metabolites in a given biological system is called metabolic fingerprinting. Using high-resolution and high-mass accuracy mass spectrometry, large metabolome coverage, sensitivity, and specificity can be attained. Although the theoretical concepts of this methodology are usually provided in life-science programs, hands-on laboratory experiments are not usually accessible to undergraduate students. Even if the instruments are available, there are not simple laboratory protocols created specifically for teaching metabolomics. We designed a straightforward hands-on laboratory experiment to introduce students to this methodology, relating it to biochemical knowledge through metabolic pathway mapping of the identified metabolites. This study focuses on mitochondrial metabolomics since mitochondria have a well-known, medium-sized cellular sub-metabolome. These features facilitate both data processing and pathway mapping. In this experiment, students isolate mitochondria from potatoes, extract the metabolites, and analyze them by high-resolution mass spectrometry (using an FT-ICR mass spectrometer). The resulting mass list is submitted to an online program for metabolite identification, and compounds associated with mitochondrial pathways can be highlighted in a metabolic network map. PMID:26537432

  5. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

    PubMed

    Syed, Deeba N; Lall, Rahul K; Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-12-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  6. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma

    PubMed Central

    Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-01-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up- regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16 days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMPK-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  7. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway.

    PubMed

    Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

    2015-01-01

    Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation. PMID:26526304

  8. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway

    PubMed Central

    Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

    2015-01-01

    Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation. PMID:26526304

  9. Polysaccharide from Pleurotus nebrodensis induces apoptosis via a mitochondrial pathway in HepG2 cells.

    PubMed

    Cui, Haiyan; Wu, Shufen; Sun, Yanping; Wang, Tiantian; Li, Zhenjing; Chen, Mianhua; Wang, Changlu

    2016-01-01

    A novel alkali extractable polysaccharide (designated as PNA-2) was purified from Pleurotus nebrodensis and the effects of purified PNA-2 on the proliferation and apoptosis of human hepatic cancer cells (HepG2) were investigated in this study. The results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that PNA-2 inhibited the proliferation of HepG2 cells by apoptosis induction, which was also characterized using scanning electron microscopy (SEM). Moreover, the expression of apoptosis-associated mRNA, proteins and the cell-cycle arrest at the G0/G1 phase was determined using RT-qPCR, Western blot and flow cytometry, respectively. A notable inhibition of the migration rate of PNA-2-treated HepG2 cells was observed using a cell scratch assay. DNA damage was observed using a comet assay and AO/EB staining in HepG2 cells, which were exposed to PNA-2. Induction of the mitochondria-mediated intrinsic apoptotic pathway by PNA-2 was indicated by the loss of mitochondrial membrane potential (ΔΨm), Bcl-2 dysregulation and cytochrome c release. All the results suggested that the mitochondria-mediated intrinsic apoptotic pathway could be involved in PNA-2-mediated apoptosis of human liver carcinoma cells HepG2. Finally, the results indicated that PNA-2 significantly suppressed tumor growth in HepG2 tumor-bearing mice, indicating that PNA-2 may be developed as a candidate drug or functional food factor to prevent or treat liver cancer. PMID:26506946

  10. AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation.

    PubMed

    Morita, Akinori; Ariyasu, Shinya; Wang, Bing; Asanuma, Tetsuo; Onoda, Takayoshi; Sawa, Akiko; Tanaka, Kaoru; Takahashi, Ippei; Togami, Shotaro; Nenoi, Mitsuru; Inaba, Toshiya; Aoki, Shin

    2014-08-01

    In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo. PMID:25026551

  11. Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells

    PubMed Central

    CHENG, CHUN-HSIANG; CHENG, YEN-PO; CHANG, ING-LIN; CHEN, HSIN-YAO; WU, CHIA-CHIEH; HSIEH, CHEN-PU

    2016-01-01

    Dodecyl gallate (DG) is a gallic acid ester that has been shown to inhibit tumor growth. The aim of this study was to investigate the mechanism by which DG induces antiproliferative and apoptotic effects in MG-63 human osteosarcoma cells. Dose- and time-dependent cytotoxic effects of DG were determined using an MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) of DG in MG-63 cells was 31.15 µM at 24 h, 10.66 µM at 48 h, and 9.06 µM at 72 h. Flow cytometric analysis demonstrated that exposure to 20 and 40 µM DG resulted in an increase in the sub-G1 phase population and in S-phase cell cycle arrest. Furthermore, western blot analysis of apoptosis-related protein expression revealed an increase in the activation of caspases 8 and 3, cleavage of poly (ADPribose) polymerase (PARP), and disruption of mitochondrial membrane permeability was measured by flow cytometry. An increase in the Bax/Bcl-2 ratio and a decrease in the expression of inhibitor of apoptosis protein (IAP) family members, namely X-linked inhibitor of apoptosis protein and survivin, were also observed following DG treatment. These data provide insight into the molecular mechanisms governing the ability of DG to induce apoptosis in human osteosarcoma cells in vitro. PMID:26707422

  12. Secondary Metabolites from Endophytic Fungus Penicillium pinophilum Induce ROS-Mediated Apoptosis through Mitochondrial Pathway in Pancreatic Cancer Cells.

    PubMed

    Koul, Mytre; Meena, Samdarshi; Kumar, Ashok; Sharma, Parduman Raj; Singamaneni, Venugopal; Riyaz-Ul-Hassan, Syed; Hamid, Abid; Chaubey, Asha; Prabhakar, Anil; Gupta, Prasoon; Singh, Shashank

    2016-03-01

    The endophytic fungus strain MRCJ-326, isolated from Allium schoenoprasum, which is also known as Snow Mountain Garlic or Kashmiri garlic, was identified as Penicillium pinophilum on the basis of morphological characteristics and internal transcribed spacer region nucleotide sequence analysis. The endophytic fungus extract was subjected to 2D-SEPBOX bioactivity-guided fractionation and purification. The anthraquinone class of the bioactive secondary metabolites were isolated and characterized as oxyskyrin (1), skyrin (2), dicatenarin (3), and 1,6,8-trihydroxy-3-hydroxy methylanthraquinone (4) by spectral analysis. Dicatenarin and skyrin showed marked growth inhibition against the NCI60/ATCC panel of human cancer cell lines with least IC50 values of 12 µg/mL and 27 µg/mL, respectively, against the human pancreatic cancer (MIA PaCa-2) cell line. The phenolic hydroxyl group in anthraquinones plays a crucial role in the oxidative process and bioactivity. Mechanistically, these compounds, i.e., dicatenarin and skyrin, significantly induce apoptosis and transmit the apoptotic signal via intracellular reactive oxygen species generation, thereby inducing a change in the mitochondrial transmembrane potential and induction of the mitochondrial-mediated apoptotic pathway. Our data indicated that dicatenarin and skyrin induce reactive oxygen species-mediated mitochondrial permeability transition and resulted in an increased induction of caspase-3 apoptotic proteins in human pancreatic cancer (MIA PaCa-2) cells. Dicatenarin showed a more pronounced cytotoxic/proapopotic effect than skyrin due to the presence of an additional phenolic hydroxyl group at C-4, which increases oxidative reactive oxygen species generation. This is the first report from P. pinophilum secreating these cytotoxic/proapoptotic secondary metabolites. PMID:26848704

  13. Lithospermic acid B protects beta-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1

    SciTech Connect

    Lee, Byung-Wan; Chun, Sung Wan; Kim, Soo Hyun; Lee, Yongho; Kang, Eun Seok; Cha, Bong-Soo; Lee, Hyun Chul

    2011-04-01

    Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo. Pretreatment with 50 {mu}M LAB significantly reduced the 1000 U/mL INF-{gamma} and 100 U/mL IL-1{beta}-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against the cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxigenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo. Our results suggest that the cytoprotective effects of LAB on pancreatic {beta}-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.

  14. MiADMSA reverses impaired mitochondrial energy metabolism and neuronal apoptotic cell death after arsenic exposure in rats

    SciTech Connect

    Dwivedi, Nidhi; Mehta, Ashish; Yadav, Abhishek; Binukumar, B.K.; Gill, Kiran Dip; Flora, Swaran J.S.

    2011-11-15

    Arsenicosis, due to contaminated drinking water, is a serious health hazard in terms of morbidity and mortality. Arsenic induced free radicals generated are known to cause cellular apoptosis through mitochondrial driven pathway. In the present study, we investigated the effect of arsenic interactions with various complexes of the electron transport chain and attempted to evaluate if there was any complex preference of arsenic that could trigger apoptosis. We also evaluated if chelation with monoisoamyl dimercaptosuccinic acid (MiADMSA) could reverse these detrimental effects. Our results indicate that arsenic exposure induced free radical generation in rat neuronal cells, which diminished mitochondrial potential and enzyme activities of all the complexes of the electron transport chain. Moreover, these complexes showed differential responses towards arsenic. These early events along with diminished ATP levels could be co-related with the later events of cytosolic migration of cytochrome c, altered bax/bcl{sub 2} ratio, and increased caspase 3 activity. Although MiADMSA could reverse most of these arsenic-induced altered variables to various extents, DNA damage remained unaffected. Our study for the first time demonstrates the differential effect of arsenic on the complexes leading to deficits in bioenergetics leading to apoptosis in rat brain. However, more in depth studies are warranted for better understanding of arsenic interactions with the mitochondria. -- Research highlights: Black-Right-Pointing-Pointer Arsenic impairs mitochondrial energy metabolism leading to neuronal apoptosis. Black-Right-Pointing-Pointer Arsenic differentially affects mitochondrial complexes, I - III and IV being more sensitive than complex II. Black-Right-Pointing-Pointer Arsenic-induced apoptosis initiates through ROS generation or impaired [Ca{sup 2+}]i homeostasis. Black-Right-Pointing-Pointer MiADMSA reverses arsenic toxicity via intracellular arsenic- chelation, antioxidant

  15. Levosimendan suppresses oxidative injury, apoptotic signaling and mitochondrial degeneration in streptozotocin-induced diabetic cardiomyopathy.

    PubMed

    Akhtar, Md Sayeed; Pillai, Krishna Kolappa; Hassan, Quamrul; Ansari, Shahid Husain; Ali, Javed; Akhtar, Mohammed; Najmi, Abul Kalam

    2016-01-01

    Diabetic cardiomyopathy plays a major role in morbidity and mortality among cardiovascular disorder-related complications. This study was designed to explore long-term benefits of Levosimendan (LEVO) along with Ramipril and Insulin. Diabetic cardiomyopathy was induced using streptozotocin (STZ) at the dose of 25 mg/kg/body weight/day for three consecutive days in Wistar rats. Rats were randomly divided into 10 groups and treatments were started after 2 weeks of STZ administration. A gradual but severe hyperglycemia ((§§§)p < 0.001) was observed in all STZ-treated groups except those received insulin (2  U/day). LEVO alone and in combination with Ramipril and Insulin normalized (**p < 0.01) mean arterial pressure and heart rate, restored catalase, superoxide dismutase, malondialdehyde, glutathione level and also attenuated (***p < 0.001) the raised serum levels of creatine kinase-heart type, lactate dehydrogenase, tumor necrosis factor-alpha, C-reactive protein, and caspase-3 level in heart tissue altered after STZ treatment. Myofibril degeneration, mitochondrial fibrosis and vacuolization occurred after STZ treatment, were also reversed by LEVO in combination with Ramipril and Insulin. The combination of LEVO with Ramipril and Insulin improved hemodynamic functions, maintained cardiac enzymes and ameliorated myofibril damage in diabetic cardiomyopathy. PMID:26207881

  16. Polygala tenuifolia polysaccharide PTP induced apoptosis in ovarian cancer cells via a mitochondrial pathway.

    PubMed

    Zhang, Fubin; Song, Xiaowei; Li, Li; Wang, Jingfang; Lin, Leyuan; Li, Cong; Li, Hongtao; Lv, Yanju; Jin, Yinghua; Liu, Ying; Hu, Yu; Xin, Tao

    2015-04-01

    One purified polysaccharide protein tyrosine phosphatase (PTP) was isolated from the roots of Polygala tenuifolia. The aim of the present study is to investigate the antitumor effect of PTP on human ovarian cancer OVCAR-3 cells and explore the molecular mechanism of the action involved. The results of MTT assay and apoptosis detection assay showed that PTP inhibited cellular proliferation of OVCAR-3 cells and induced apoptotic cellular death via arresting cell circle at the G0/G1 phase. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis identified that bcl-2 gradually decreased at both transcription and protein levels after PTP treatment for 48 h in OVCAR-3 cells, while those of bax, cytochrome c, caspase-3, and caspase-9 increased. In addition, the low expression of NF-κB in PTP-treated OVCAR-3 cells would trigger the extrinsic pathway of programmed cell death signaling in tumor cells. These results together suggest that PTP may induce apoptosis of OVCAR-3 cells through a mitochondrial pathway. PMID:25501282

  17. Enhanced Hsp70 expression protects against acute lung injury by modulating apoptotic pathways.

    PubMed

    Aschkenasy, Gabriella; Bromberg, Zohar; Raj, Nichelle; Deutschman, Clifford S; Weiss, Yoram G

    2011-01-01

    The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury. Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFα. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co-immunoprecipitation and a caspase-3 activity assay. TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes. We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery. PMID:22132083

  18. Overexpression of long non-coding RNA PVT1 in ovarian cancer cells promotes cisplatin resistance by regulating apoptotic pathways.

    PubMed

    Liu, Enling; Liu, Zheng; Zhou, Yuxiu; Mi, Ruoran; Wang, Dehua

    2015-01-01

    Ovarian cancer is the most lethal gynecologic malignancy. Cisplatin is a very effective cancer chemotherapy drug, but cisplatin resistance is a crucial problem of therapy failure. Overexpression of PVT1 has been demonstrated in ovarian cancer. The mRNA level of PVT1 in ovarian cancer tissues of cisplatin-resistant patients and cisplatin-sensitive patients, cisplatin-resistant cells SKOV-3/DDP and A2780/DDP, cisplatin-sensitive cells SKOV-3 and A2780 were determined by qRT-PCR. The influence of the knockdown or overexpression of PVT1 on cisplatin resistance was measured by measuring the cytotoxicity of cisplatin and the apoptotic rate of ovarian cancer cells was detected by CCK-8 assay and flow cytometry, respectively. The mRNA levels and protein expression of TGF-β1, Smad4, p-Smad4 and Caspase-3 in apoptotic pathways were determined. The mRNA level of PVT1 was significantly higher in ovarian cancer tissues of cisplatin-resistant patients and cisplatin-resistant cells. SKOV-3/DDP and A2780/DDP cell viability and the percentage of apoptotic cells after transfection with PVT-1 siRNA and treated with cisplatin was markedly lower and higher than the control, respectively. Moreover, the overexpression of PVT1 exhibited the anti-apoptotic property in SKOV-3 and A2780 cells after transfection with LV-PVT1-GFP and treated with cisplatin. The mRNA levels and protein expression of TGF-β1, p-Smad4 and Caspase-3 were much higher in cisplatin-resistant cells transfected with siPVT1. Overexpression of LncRNA PVT1 in ovarian cancer promotes cisplatin resistance by regulating apoptotic pathways. PMID:26884974

  19. Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways

    SciTech Connect

    Chen Yawen; Huang Chunfa; Yang Chingyao; Yen Chengchieh; Tsai Kehsung; Liu Shinghwa

    2010-03-15

    Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl{sub 2}) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl{sub 2} significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl{sub 2}-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl{sub 2} increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl{sub 2} possessed ability in apoptosis induction. HgCl{sub 2} also displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl{sub 2} could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl{sub 2} could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl{sub 2}-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl{sub 2}-treated HIT-T15 cells. Taken together, these results suggest that HgCl{sub 2}-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.

  20. Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways.

    PubMed

    Ravichandran, Kodi S

    2011-10-28

    Prompt and efficient clearance of apoptotic cells is necessary to prevent secondary necrosis of dying cells and to avoid immune responses to autoantigens. Recent studies have shed light on how apoptotic cells through soluble "find-me" signals advertise their presence to phagocytes at the earliest stages of cell death. Phagocytes sense the find-me signal gradient, and in turn the presence of dying cells, and migrate to their vicinity. The apoptotic cells also expose specific "eat-me" signals on their surface that are recognized by phagocytes through specific engulfment receptors. This review covers the recent progress in the areas of find-me and eat-me signals and how these relate to prompt and immunologically silent clearance of apoptotic cells. PMID:22035837

  1. Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways

    PubMed Central

    Ravichandran, Kodi S.

    2011-01-01

    Prompt and efficient clearance of apoptotic cells is necessary to prevent secondary necrosis of dying cells, and to avoid immune responses to autoantigens. Recent studies have shed light on how apoptotic cells through soluble ‘find-me signals’ advertise their presence to phagocytes at the earliest stages of cell death. Phagocytes sense the find-me signal gradient, and in turn the presence of dying cells, and migrate to their vicinity. The apoptotic cells also expose specific eat-me signals on their surface that are recognized by phagocytes through specific engulfment receptors. This review covers the recent progress in the areas of find-me and eat-me signals, and how these relate to prompt and immunologically silent clearance of apoptotic cells. PMID:22035837

  2. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

    PubMed Central

    Chen, Ying-Hui; Wang, Jo-Yu; Pan, Bo-Syong; Mu, Yi-Fen; Lai, Meng-Shao; So, Edmund Cheung; Wong, Thian-Sze; Huang, Bu-Miin

    2013-01-01

    Purpose The present study aims to investigate whether the combination treatment of cordycepin (an extracted pure compound from Cordyceps sinensis) and cisplatin (a platinum-based chemotherapy drug) has better apoptotic effect in head and neck squamous cell carcinoma (HNSCC). Methods The apoptotic influences of cordycepin and/or cisplatin treatments to human OC3, OEC-M1, and FaDu HNSCC cells were investigated by morphological observations, viability assay, flow cytometry assay, and Western blotting methods. Results Data showed that the cell death phenomenon increased as the dosage of cordycepin or cisplatin increased, and it appeared more in cordycepin plus cisplatin cotreatment among three cell lines. Cell survival rates significantly decreased as the dosage of cordycepin or cisplatin increased, and the better apoptotic effects were observed in cotreatment. Cell cycle analysis further demonstrated that percentages of subG1 cells in cordycepin or cisplatin treatments significantly increased, suggesting that cells underwent apoptosis, and cordycepin plus cisplatin induced many more subG1 cells. Furthermore, cordycepin or cisplatin induced caspase-8, caspase-9, caspase-3, and poly adenosine diphosphate-ribose polymerase protein cleavages, and stimulated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 protein phosphorylations. Moreover, cordycepin plus cisplatin cotreatment significantly activated those proteins with much better effects among three cell lines. Conclusion Cordycepin plus cisplatin have better apoptotic effect by activating caspase activation with possible MAPK pathway involvement in HNSCC cells. PMID:23926438

  3. Mechanisms of andrographolide-induced platelet apoptosis in human platelets: regulatory roles of the extrinsic apoptotic pathway.

    PubMed

    Lien, Li-Ming; Su, Cheng-Chen; Hsu, Wen-Hsien; Lu, Wan-Jung; Chung, Chi-Li; Yen, Ting-Lin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

    2013-11-01

    Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by inhibition of the p38 MAPK/(●) HO-NF-κB-ERK2 cascade. Although platelets are anucleated cells, apoptotic machinery apparatus recently has been found to regulate platelet activation and limit platelet lifespan. Therefore, we further investigated the regulatory effects of andrographolide on platelet apoptotic events. In this study, apoptotic signaling events for caspase-3, -8, and Bid were time (10-60 min)- and dose (25-100 μΜ)-dependently activated by andrographolide in human platelets. Andrographolide could also disrupt mitrochondrial membrane potential. In addition, caspase-8 inhibitor (z-IETD-fmk, 50 μΜ) was found to reverse andrographolide-induced caspase-8 activation, whereas the antagonistic anti-Fas receptor (ZB4, 500 ng/mL) and anti-tumor necrosis factor-R1 (H398, 10 µg/mL) monoclonal antibodies did not. In conclusion, this study for the first time demonstrated that andrographolide might limit platelet lifespan by initiating the caspase-8-dependent extrinsic apoptotic pathway, in spite of no direct evidence that death receptors are involved in this process proved. Overall, the various medicinal properties of andrographolide suggest its potential value in treating patients with thromboembolic disorders. PMID:23292890

  4. A new view of the lethal apoptotic pore.

    PubMed

    Basañez, Gorka; Soane, Lucian; Hardwick, J Marie

    2012-01-01

    Cell death by apoptosis is indispensable for proper development and tissue homeostasis in all multicellular organisms, and its deregulation plays a key role in cancer and many other diseases. A crucial event in apoptosis is the formation of protein-permeable pores in the outer mitochondrial membrane that release cytochrome c and other apoptosis-promoting factors into the cytosol. Research efforts over the past two decades have established that apoptotic pores require BCL-2 family proteins, with the proapoptotic BAX-type proteins being direct effectors of pore formation. Accumulating evidence indicates that other cellular components also cooperate with BCL-2 family members to regulate the apoptotic pore. Despite this knowledge, the molecular pathway leading to apoptotic pore formation at the outer mitochondrial membrane and the precise nature of this outer membrane pore remain enigmatic. In this issue of PLOS Biology, Kushnareva and colleagues describe a novel kinetic analysis of the dynamics of BAX-dependent apoptotic pore formation recapitulated in native mitochondrial outer membranes. Their study reveals the existence of a hitherto unknown outer mitochondrial membrane factor that is critical for BAX-mediated apoptotic pore formation, and challenges the currently popular view that the apoptotic pore is a purely proteinaceous multimeric assembly of BAX proteins. It also supports the notion that membrane remodeling events are implicated in the formation of a lipid-containing apoptotic pore. PMID:23049484

  5. Induction of apoptosis in HL-60 cells through the ROS-mediated mitochondrial pathway by ramentaceone from Drosera aliciae.

    PubMed

    Kawiak, Anna; Zawacka-Pankau, Joanna; Wasilewska, Aleksandra; Stasilojc, Grzegorz; Bigda, Jacek; Lojkowska, Ewa

    2012-01-27

    Ramentaceone (1) is a naphthoquinone constituent of Drosera aliciae that exhibits potent cytotoxic activity against various tumor cell lines. However, its molecular mechanism of cell death induction has still not been determined. The present study demonstrates that 1 induces apoptosis in human leukemia HL-60 cells. Typical morphological and biochemical features of apoptosis were observed in 1-treated cells. Compound 1 induced a concentration-dependent increase in the sub-G1 fraction of the cell cycle. A decrease in the mitochondrial transmembrane potential (ΔΨm) was also observed. Furthermore, 1 reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and Bak, induced cytochrome c release, and increased the activity of caspase 3. The generation of reactive oxygen species (ROS) was detected in 1-treated HL-60 cells, which was attenuated by the pretreatment of cells with a free radical scavenger, N-acetylcysteine (NAC). NAC also prevented the increase of the sub-G1 fraction induced by 1. These results indicate that ramentaceone induces cell death through the ROS-mediated mitochondrial pathway. PMID:22250825

  6. Apoptosis induced by farrerol in human gastric cancer SGC-7901 cells through the mitochondrial-mediated pathway.

    PubMed

    Liu, Enli; Liang, Taigang; Wang, Xiaojian; Ban, Shurong; Han, Lingge; Li, Qingshan

    2015-09-01

    Farrerol, a typical flavanone isolated from the Chinese medicinal plant Rhododendron dauricum L., has been found to show various biological activities. However, to the best of our knowledge, its inhibitory actions against cancer cells have not been reported as yet. Therefore, the present study aimed to investigate the cytotoxic and apoptotic effects of farrerol on human gastric cancer SGC-7901 cells. Farrerol showed a 50% inhibition of SGC-7901 cell growth at a concentration of 40.4 μmol/l for 24 h according to MTT assays. The cell morphology results indicated that SGC-7901 cells treated with farrerol showed several features of apoptotic cell death, which was also confirmed by the Annexin-V FITC/PI double-staining assay. Further studies showed that farrerol treatment induced the attenuation of mitochondrial membrane potential, accompanied by the release of Cyt-c and the activation of caspase-9 and caspase-3. Furthermore, farrerol decreased the gene expression of Bcl-2, whereas the gene expression level of Bax was found to increase after farrerol treatment. These combined results indicated that farrerol can induce apoptosis through a mitochondrial-mediated pathway. PMID:26061993

  7. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes

    PubMed Central

    2013-01-01

    Background The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. Methods THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. Results P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. Conclusions P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species

  8. Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

    PubMed Central

    Chelh, Ilham; Meunier, Bruno; Picard, Brigitte; Reecy, Mark James; Chevalier, Catherine; Hocquette, Jean-François; Cassar-Malek, Isabelle

    2009-01-01

    Background Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. Results Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. Conclusion All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3ε protein, TCTP/GSK-3β). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo. PMID:19397818

  9. Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes.

    PubMed

    Kumar, Anoop; Sasmal, D; Bhaskar, Amand; Mukhopadhyay, Kunal; Thakur, Aman; Sharma, Neelima

    2016-07-01

    Deltamethrin (DLM) is a well-known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration-dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration-dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N-acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase-dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 808-819, 2016. PMID:25534813

  10. AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

    SciTech Connect

    Morita, Akinori; Ariyasu, Shinya; Wang, Bing; Asanuma, Tetsuo; Onoda, Takayoshi; Sawa, Akiko; Tanaka, Kaoru; Takahashi, Ippei; Togami, Shotaro; Nenoi, Mitsuru; Inaba, Toshiya; Aoki, Shin

    2014-08-08

    Highlights: • A bidentate HQ derivative, AS-2, suppresses p53-dependent apoptosis by DNA damage. • AS-2 does not significantly affect nuclear p53 response. • UV-excited blue emission of AS-2 clearly showed its extranuclear localization. • AS-2 prevents mitochondrial dysfunction despite the increase of mitochondrial p53. • AS-2 protects mice from a radiation dose that causes lethal hematopoietic syndrome. - Abstract: In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also

  11. MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway

    PubMed Central

    Tan, Guosheng; Wu, Linwei; Tan, Jinfu; Zhang, Bing; Tai, William Chi-shing; Xiong, Shiqiu; Chen, Wei; Yang, Jianyong; Li, Heping

    2016-01-01

    Apoptosis resistance in human hepatocellular carcinoma (HCC) is a significant factor in carcinogenesis. Therefore, understanding the molecular mechanisms involved in apoptosis resistance is crucial for developing anticancer therapies. Importantly, small non-coding microRNAs (miRNAs) have been reported as key biomarkers for detecting tumour onset and progression. In the present study, we demonstrate that miR-1180 is upregulated in HCC. Ectopic expression of miR-1180 has an anti-apoptotic effect in HCC, while miR-1180 inhibition increases cell apoptosis, both in vitro and in vivo. Moreover, our results show that miR-1180 directly targets key inhibitors of the nuclear factor (NF)-κB signaling pathway (i.e., OTUD7B and TNIP2) and the pro-apoptotic Bcl-2 associated death promoter (BAD) protein by post-transcriptional downregulation. Therefore, the anti-apoptotic function of miR-1180 in HCC may occur through NF-κB pathway activation via downregulation of its negative regulators. In conclusion, our study reveals the critical role of miR-1180 during apoptosis resistance in HCC. PMID:26928365

  12. Phosphorylation of ASPP2 by RAS/MAPK Pathway Is Critical for Its Full Pro-Apoptotic Function

    PubMed Central

    Slee, Elizabeth; Lu, Xin

    2013-01-01

    We reported recently that apoptosis-stimulating protein of p53 (ASPP) 2, an activator of p53, co-operates with oncogenic RAS to enhance the transcription and apoptotic function of p53. However, the detailed mechanism remains unknown. Here we show that ASPP2 is a novel substrate of mitogen-activated protein kinase (MAPK). Phosphorylation of ASPP2 by MAPK is required for RAS-induced increased binding to p53 and increased transactivation of pro-apoptotic genes. In contrast, an ASPP2 phosphorylation mutant exhibits reduced p53 binding and fails to enhance transactivation and apoptosis. Thus phosphorylation of ASPP2 by RAS/MAPK pathway provides a novel link between RAS and p53 in regulating apoptosis. PMID:24312625

  13. Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway.

    PubMed

    Ducker, Gregory S; Chen, Li; Morscher, Raphael J; Ghergurovich, Jonathan M; Esposito, Mark; Teng, Xin; Kang, Yibin; Rabinowitz, Joshua D

    2016-06-14

    One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions. PMID:27211901

  14. Dietary anthocyanins protect endothelial cells against peroxynitrite-induced mitochondrial apoptosis pathway and Bax nuclear translocation: an in vitro approach.

    PubMed

    Paixão, Joana; Dinis, Teresa C P; Almeida, Leonor M

    2011-10-01

    Anthocyanins have received increasing attention because of their relatively high intake in humans and wide range of potential health-promoting effects, including anti-atherogenic properties. Evidences support their vascular protective effects but the involved molecular mechanisms have not been well clarified. The endothelium seems to have a central role in atherogenesis and apoptosis is emerging as a crucial event in this disease progression. Following our previous work on the biochemical pathways underlying peroxynitrite-triggered apoptosis in endothelial cells, here we investigated potential mechanisms responsible for the cytoprotective actions of three common anthocyanins, namely cyanidin- delphinidin- and pelargonidin-3-glucoside, against this process. Beyond their antioxidant properties, all these flavonoids, possessing either catecholic or monophenolic structures, were able to counteract peroxynitrite-induced apoptotic effects in endothelial cells through the inhibition of several crucial signaling cascades. Actually, pre-incubation of cells with 25 μM anthocyanins prevented them from peroxynitrite-mediated apoptosis, which was evaluated by the loss of mitochondrial membrane potential, caspases-9 and-3 activation, the increase in cytoplasmatic Bax levels and the inactivation of the PI3 K/Akt pathway. Moreover, they counteracted the translocation of Bax into the nucleus, as observed by immunocytochemistry and immunoblot, an event shown for the first time in endothelial cells apoptotic process. Such cellular actions could not be inferred from their in vitro antioxidant properties. These results suggest a potential role of dietary anthocyanins in the modulation of several apoptotic signaling pathways triggered by peroxynitrite in endothelial cells, supporting mechanistically their health benefits in the context of prevention of endothelial dysfunction and, ultimately, of atherosclerosis. PMID:21785847

  15. Parthenolide induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits FAK-mediated cell invasion.

    PubMed

    Kwak, Sang Won; Park, Eon Sub; Lee, Chung Soo

    2014-01-01

    The natural product parthenolide induces apoptosis in cancer cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to parthenolide is not clear. In addition, it is unclear whether parthenolide-induced apoptosis is mediated by the formation of reactive oxygen species and the depletion of GSH contents, and the effect of parthenolide on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of parthenolide exposure on apoptosis, cell adhesion, and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that parthenolide may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of parthenolide appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Parthenolide inhibited fetal bovine serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase-dependent activation of cytoskeletal-associated components. Therefore, parthenolide might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy. PMID:24065392

  16. Activated Protein C Protects Myocardium Via Activation of Anti-apoptotic Pathways of Survival in Ischemia-reperfused Rat Heart

    PubMed Central

    Ding, Jia-Wang; Yang, Jun; Liu, Zhao-Qi; Zhang, Yan; Yang, Jian; Li, Song; Li, Li

    2010-01-01

    Activated protein C (APC) is known to be beneficial on ischemia reperfusion injury in myocardium. However, the protection mechanism of APC is not fully understood. The purpose of this study was to investigate the effects and possible mechanisms of APC on myocardial ischemic damage. Artificially ventilated anaesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 hr of reperfusion. Rats were randomly divided into four groups; Sham, I/R, APC preconditioning and postconditioning group. Myocardial infarct size, apoptosis index, the phosphorylation of ERK1/2, Bcl-2, Bax and cytochrome c genes and proteins were assessed. In APC-administrated rat hearts, regardless of the timing of administration, infarct size was consistently reduced compared to ischemia/reperfusion (I/R) rats. APC improved the expression of ERK1/2 and anti-apoptotic protein Bcl-2 which were significantly reduced in the I/R rats. APC reduced the expression of pro-apoptotic genes, Bax and cytochrome c. These findings suggest that APC produces cardioprotective effect by preserving the expression of proteins and genes involved in anti-apoptotic pathways, regardless of the timing of administration. PMID:21060750

  17. The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila

    PubMed Central

    Xu, D; Wang, Y; Willecke, R; Chen, Z; Ding, T; Bergmann, A

    2008-01-01

    Caspases are essential components of the apoptotic machinery in both vertebrates and invertebrates. Here, we report the isolation of a mutant allele of the Drosophila effector caspase drICE as a strong suppressor of hid- (head involution defective-) induced apoptosis. This mutant was used to determine the apoptotic role of drICE. Our data are consistent with an important function of drICE for developmental and irradiation-induced cell death. Epistatic analysis suggests that drICE acts genetically downstream of Drosophila inhibitor of apoptosis protein 1 (Diap1). However, although cell death is significantly reduced in drICE mutants in all assays, it is not completely blocked. A double-mutant analysis between drICE and death caspase-1 (dcp-1), another effector caspase, reveals that some cells (type I) strictly require drICE for apoptosis, whereas other cells (type II) require either drICE or dcp-1. Thus, these data demonstrate a barely appreciated complexity in the apoptotic pathway, and are consistent with current models about effector caspase regulation in both vertebrates and invertebrates. PMID:16645642

  18. The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila.

    PubMed

    Xu, D; Wang, Y; Willecke, R; Chen, Z; Ding, T; Bergmann, A

    2006-10-01

    Caspases are essential components of the apoptotic machinery in both vertebrates and invertebrates. Here, we report the isolation of a mutant allele of the Drosophila effector caspase drICE as a strong suppressor of hid- (head involution defective-) induced apoptosis. This mutant was used to determine the apoptotic role of drICE. Our data are consistent with an important function of drICE for developmental and irradiation-induced cell death. Epistatic analysis suggests that drICE acts genetically downstream of Drosophila inhibitor of apoptosis protein 1 (Diap1). However, although cell death is significantly reduced in drICE mutants in all assays, it is not completely blocked. A double-mutant analysis between drICE and death caspase-1 (dcp-1), another effector caspase, reveals that some cells (type I) strictly require drICE for apoptosis, whereas other cells (type II) require either drICE or dcp-1. Thus, these data demonstrate a barely appreciated complexity in the apoptotic pathway, and are consistent with current models about effector caspase regulation in both vertebrates and invertebrates. PMID:16645642

  19. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway

    SciTech Connect

    Chen, T.-G.; Chen, T.-L.; Chang, H.-C.; Tai, Y.-T.; Cherng, Y.-G.; Chang, Y.-T.; Chen, R.-M. . E-mail: rmchen@tmu.edu.tw

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  20. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway.

    PubMed

    Chen, Tyng-Guey; Chen, Ta-Liang; Chang, Huai-Chia; Tai, Yu-Ting; Cherng, Yih-Giun; Chang, Ya-Ting; Chen, Ruei-Ming

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  1. Fucoidan Derived from Undaria pinnatifida Induces Apoptosis in Human Hepatocellular Carcinoma SMMC-7721 Cells via the ROS-Mediated Mitochondrial Pathway

    PubMed Central

    Yang, Lili; Wang, Peisheng; Wang, Huaxin; Li, Qiaomei; Teng, Hongming; Liu, Zhichao; Yang, Wenbo; Hou, Lin; Zou, Xiangyang

    2013-01-01

    Fucoidans, fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia and breast cancer and in lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan extracted from the brown seaweed Undaria pinnatifida was investigated in human hepatocellular carcinoma SMMC-7721 cells, and the underlying mechanisms of action were investigated. SMMC-7721 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as chromatin condensation and marginalization, a decrease in the number of mitochondria, and in mitochondrial swelling and vacuolation. Fucoidan-induced cell death was associated with depletion of reduced glutathione (GSH), accumulation of high intracellular levels of reactive oxygen species (ROS), and accompanied by damage to the mitochondrial ultrastructure, depolarization of the mitochondrial membrane potential (MMP, Δψm) and caspase activation. Moreover, fucoidan led to altered expression of factors related to apoptosis, including downregulating Livin and XIAP mRNA, which are members of the inhibitor of apoptotic protein (IAP) family, and increased the Bax-to-Bcl-2 ratio. These findings suggest that fucoidan isolated from U. pinnatifida induced apoptosis in SMMC-7721 cells via the ROS-mediated mitochondrial pathway. PMID:23752353

  2. 13-acetoxysarcocrassolide induces apoptosis on human gastric carcinoma cells through mitochondria-related apoptotic pathways: p38/JNK activation and PI3K/AKT suppression.

    PubMed

    Su, Ching-Chyuan; Chen, Jeff Yi-Fu; Din, Zhong-Hao; Su, Jui-Hsin; Yang, Zih-Yan; Chen, Yi-Jen; Wang, Robert Y L; Wu, Yu-Jen

    2014-10-01

    13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ΔΨm, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways. PMID:25342459

  3. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    SciTech Connect

    Wu, Cheng Tien; Weng, Te I.; Chen, Li Ping; Chiang, Chih Kang; Liu, Shing Hwa

    2013-01-01

    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  4. Mitochondrial Gene Expression Profiles and Metabolic Pathways in the Amygdala Associated with Exaggerated Fear in an Animal Model of PTSD

    PubMed Central

    Li, He; Li, Xin; Smerin, Stanley E.; Zhang, Lei; Jia, Min; Xing, Guoqiang; Su, Yan A.; Wen, Jillian; Benedek, David; Ursano, Robert

    2014-01-01

    The metabolic mechanisms underlying the development of exaggerated fear in post-traumatic stress disorder (PTSD) are not well defined. In the present study, alteration in the expression of genes associated with mitochondrial function in the amygdala of an animal model of PTSD was determined. Amygdala tissue samples were excised from 10 non-stressed control rats and 10 stressed rats, 14 days post-stress treatment. Total RNA was isolated, cDNA was synthesized, and gene expression levels were determined using a cDNA microarray. During the development of the exaggerated fear associated with PTSD, 48 genes were found to be significantly upregulated and 37 were significantly downregulated in the amygdala complex based on stringent criteria (p < 0.01). Ingenuity pathway analysis revealed up- or downregulation in the amygdala complex of four signaling networks – one associated with inflammatory and apoptotic pathways, one with immune mediators and metabolism, one with transcriptional factors, and one with chromatin remodeling. Thus, informatics of a neuronal gene array allowed us to determine the expression profile of mitochondrial genes in the amygdala complex of an animal model of PTSD. The result is a further understanding of the metabolic and neuronal signaling mechanisms associated with delayed and exaggerated fear. PMID:25295026

  5. Rapamycin, an mTOR inhibitor, induced apoptosis via independent mitochondrial and death receptor pathway in retinoblastoma Y79 cell

    PubMed Central

    Wang, Yan-Dong; Su, Yong-Jing; Li, Jian-Ying; Yao, Xiang-Chao; Liang, Guang-Jiang

    2015-01-01

    Rapamycin is helpful in the treatment of certain cancers by inhibiting mTOR (mammalian target of rapamycin) pathway. Here, rapamycin mediated apoptosis were investigated in human retinoblastoma Y79 cells. The MTT assay showed that the IC50 value of rapamycin against Y79 cells was 0.136 ± 0.032 μmol/L. Flow cytometry analysis indicated that the percentage of apoptotic cells was increased from 2.16 ± 0.41% to 12.24 ± 3.10%, 20.16 ± 4.22%, and 31.32 ± 5.78% after 0.1, 0.2, and 0.4 μmol/L rapamycin or without rapamycin treatment for 48 hours. Flow cytometry analysis showed that rapamycin induced mitochondrial membrane potential (∆Ψm) collapse in Y79 cells in a concentration-dependent manner. Western blot assay showed that rapamycin led to release of cytochrome c from mitochondrial membranes to cytosol. Further Western blot assays showed that rapamycin induced activation of caspase-9 and caspase-8 and the cleavage of caspase-3. Rapamycin induced cleavages of caspase-3 and apoptosis was inhibited by both Z-LETD-FMK and Z-IETD-FMK treatment. Together, all these results illustrated that rapamycin induced apoptosis in human retinoblastoma Y79 cells involvement of both intrinsic and extrinsic pathways. PMID:26379864

  6. Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells.

    PubMed

    Hamzeloo-Moghadam, Maryam; Aghaei, Mahmoud; Fallahian, Faranak; Jafari, Seyyed Mehdi; Dolati, Masoumeh; Abdolmohammadi, Mohammad Hossein; Hajiahmadi, Sima; Esmaeili, Somayeh

    2015-02-01

    Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy. PMID:25342596

  7. The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

    SciTech Connect

    Park, Jeehye; Lee, Gina; Chung, Jongkyeong

    2009-01-16

    The two Parkinson's disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.

  8. Targeting the mitochondrial apoptotic pathway: a preferred approach in hematologic malignancies?

    PubMed Central

    Brinkmann, K; Kashkar, H

    2014-01-01

    Acquired resistance toward apoptosis represents one of the hallmarks of human cancer and a major cause of the inefficacy of most anticancer treatment regimens. Based on its ability to inhibit apoptosis, the B-cell lymphoma/leukemia 2 (Bcl-2) protein family has garnered the most attention as a promising therapeutic target in cancer. Accordingly, efforts have lately been focused on the development of drugs targeting Bcl-2 proteins with considerable therapeutic success, particularly in hematologic malignancies. Here, we review the previous studies and highlight the pivotal role of the Bcl-2 protein family in the homeostasis of hematologic tissue compartment. This knowledge provides more insight into why some cancers are more sensitive to Bcl-2 targeting than others and will foster the clinical evaluation of Bcl-2-targeting strategies in cancer by avoiding severe on-target side effects in the development of healthy tissues. PMID:24603326

  9. Withania somnifera alleviates parkinsonian phenotypes by inhibiting apoptotic pathways in dopaminergic neurons.

    PubMed

    Prakash, Jay; Chouhan, Shikha; Yadav, Satyndra Kumar; Westfall, Susan; Rai, Sachchida Nand; Singh, Surya Pratap

    2014-12-01

    Maneb (MB) and paraquat (PQ) are environmental toxins that have been experimentally used to induce selective damage of dopaminergic neurons leading to the development of Parkinson's disease (PD). Although the mechanism of this selective neuronal toxicity in not fully understood, oxidative stress has been linked to the pathogenesis of PD. The present study investigates the mechanisms of neuroprotection elicited by Withania somnifera (Ws), a herb traditionally recognized by the Indian system of medicine, Ayurveda. An ethanolic root extract of Ws was co-treated with the MB-PQ induced mouse model of PD and was shown to significantly rescue canonical indicators of PD including compromised locomotor activity, reduced dopamine in the substantia nigra and various aspects of oxidative damage. In particular, Ws reduced the expression of iNOS, a measure of oxidative stress. Ws also significantly improved the MB + PQ mediated induction of a pro-apoptotic state by reducing Bax and inducing Bcl-2 protein expression, respectively. Finally, Ws reduced expression of the pro-inflammatory marker of astrocyte activation, GFAP. Altogether, the present study suggests that Ws treatment provides nigrostriatal dopaminergic neuroprotection against MB-PQ induced Parkinsonism by the modulation of oxidative stress and apoptotic machinery possibly accounting for the behavioural effects. PMID:25403619

  10. Myristicin from nutmeg induces apoptosis via the mitochondrial pathway and down regulates genes of the DNA damage response pathways in human leukaemia K562 cells.

    PubMed

    Martins, Célia; Doran, Carolina; Silva, Inês C; Miranda, Claudia; Rueff, José; Rodrigues, António S

    2014-07-25

    Myristicin, an allylbenzene, is a major active component of various spices, such as nutmeg and cinnamon, plants from the Umbelliferae family or in some essential oils, such as oils of clove or marjoram. Human exposure to myristicin is low but widespread due to consumption of these spices and essential oils, added to food (e.g. cola drinks) or in traditional medicine. Occasionally high dose exposure occurs, leading to various clinical symptoms, however the molecular mechanisms underlying them are unknown. Our previous studies revealed that myristicin is not genotoxic and yet presented apoptotic activity. Therefore, in this work we assessed the apoptotic mechanisms induced by myristicin in human leukaemia cells. In order to gain further insight on the potential of myristicin to modulate gene expression we also analysed alterations in expression of 84 genes associated with the DNA damage response pathway. The results obtained show that myristicin can induce apoptosis as characterised by alterations in the mitochondrial membrane potential, cytochrome c release, caspase-3 activation, PARP-cleavage and DNA fragmentation. The gene expression profile revealed an overall down regulation of DNA damage response genes after exposure to myristicin, with significant under-expression of genes associated with nucleotide excision repair (ERCC1), double strand break repair (RAD50, RAD51) and DNA damage signalling (ATM) and stress response (GADD45A, GADD45G). On the whole, we demonstrate that myristicin can alter mitochondrial membrane function, induce apoptosis and modulate gene expression in human leukaemia K562 cells. This study provides further detail on the molecular mechanisms underlying the biological activity of myristicin. PMID:24792648

  11. Corosolic acid analogue, a natural triterpenoid saponin, induces apoptosis on human hepatocarcinoma cells through mitochondrial pathway in vitro.

    PubMed

    Qu, Liping; Zhang, Huiqing; Yang, Yanlong; Yang, Geliang; Xin, Hailiang; Ling, Changquan

    2016-08-01

    Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine. Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines. Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40 μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4'-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays. Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24 h, the IC50 values were 34.6, 30.8 and 30.5 μg/mL, respectively. TEO (40 μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio. Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway. PMID:26810384

  12. METHOTREXATE AND MYOTREXATE INDUCE APOPTOSIS IN HUMAN MYOMA FIBROBLASTS (T hES CELL LINE) VIA MITOCHONDRIAL PATHWAY.

    PubMed

    Kastratović, Tatjana; Arsenijević, Slobodan; Matović, Zoran; Mitrović, Marina; Nikolić, Ivana; Milosavljević, Zoran; Protrka, Zoran; Šorak, Marija; Đurić, Janko

    2015-01-01

    Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. Although the local application of low doses of methotrexate (MTX) is used as an effective treatment of the myomas, myotrexate could be a promising new drug. This study investigated the cytotoxic and apoptotic effects of both MTX and myotrexate in human fibroblasts derived from the uterine fibroids (T hES cell line). The myotrexate adduct is an aqueous solution of MTX and L-arginine. Cells were treated with a graded concentrations of both MTX and myothrexate (0.1-16 µM) for 24 h. The cytotoxicity was assayed by MTT test, apoptosis was evaluated by Annexin V-FITC assay and their possible role in apoptosis was determined by immnu- flourescence. Both MTX and myotrexate induced apoptosis in T hES cells in a dose dependent manner (p < 0.001). Myotrexate significantly increased the percentage of AnnexinV positive cells, BAX/Bcl-2 ratio and subsequent caspase-3 activation compared to the MTX treated cells (p < 0.05). Both MTX or myotrexate treatment showed a diffuse staining of cytochrome c indicating its release from mitochondria to the cytosol, suggesting that their mechanisms of action most likely involves the mitochondrial apoptotic pathway. PMID:26642654

  13. E. adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat

    PubMed Central

    Hu, Yanchun; Luo, Biao; Wu, Lei; Qiao, Yan; Mo, Quan; Xu, Ruiguang; Zhou, Yancheng; Ren, Zhihua; Zuo, Zhicai; Deng, Junliang; Peng, Guangneng; He, Wei; Wei, Yahui

    2015-01-01

    The cytotoxicity effects of E. adenophorum on cell cycle and apoptosis of renal cells in Saanen goat was evaluated by TUNEL, DAPI, AO/EB staining, DNA fragmentation assay, Caspase activity, Western-blot, qRT-PCR and flow cytometry analysis. 16 saanen goats randomly divided into four groups were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The Results showed that E. adenophorum induced typical apoptotic features of renal cells. E. adenophorum significantly suppressed renal cells viability, caused cell cycle activity arrest and induced typical apoptotic features in a dose-dependent manner. However, the protein levels of Fas/FasL, Bid and caspase-8 did not appear significant changes in the process of E. adenophorum-induced apoptosis. Moreover, E. adenophorum administration slightly decreased Bcl-2 expression, promoted Bax translocation to mitochondria, triggered the release of Cyt c from mitochondria into cytosol and activated caspase-9, -3, and cleaved PARP. The mitochondrial p53 translocation was significantly activated, accompanied by a significant increase in the loss of ΔΨm, Cyt c release and caspase-9 activation. Above all, these data suggest that E. adenophorum induces renal cells apoptosis via the activation of mitochondria-mediated apoptosis pathway in renal cells. These findings may provide new insights to understand the mechanisms involved in E. adenophorum-caused cytotoxicity of renal cells. PMID:26382060

  14. Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

    PubMed

    Dai, BaoDi; Wang, Yan; Li, DeDong; Xu, Yi; Liang, RongMei; Zhao, LanXue; Cao, YongBing; Jia, JianHui; Jiang, YuanYing

    2012-01-01

    Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway. PMID:23028789

  15. Highly efficient synthetic iron-dependent nucleases activate both intrinsic and extrinsic apoptotic death pathways in leukemia cancer cells.

    PubMed

    Horn, Adolfo; Fernandes, Christiane; Parrilha, Gabrieli L; Kanashiro, Milton M; Borges, Franz V; de Melo, Edésio J T; Schenk, Gerhard; Terenzi, Hernán; Pich, Claus T

    2013-11-01

    The nuclease activity and the cytotoxicity toward human leukemia cancer cells of iron complexes, [Fe(HPClNOL)Cl2]NO3 (1), [Cl(HPClNOL)Fe(μ-O)Fe(HPClNOL)Cl]Cl2·2H2O (2), and [(SO4)(HPClNOL)Fe(μ-O)Fe(HPClNOL)(SO4)]·6H2O (3) (HPClNOL=1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), were investigated. Each complex was able to promote plasmid DNA cleavage and change the supercoiled form of the plasmid to circular and linear ones. Kinetic data revealed that (1), (2) and (3) increase the rate of DNA hydrolysis about 278, 192 and 339 million-fold, respectively. The activity of the complexes was inhibited by distamycin, indicating that they interact with the minor groove of the DNA. The cytotoxic activity of the complexes toward U937, HL-60, Jukart and THP-1 leukemia cancer cells was studied employing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence and electronic transmission microscopies, flow cytometry and a cytochrome C release assay. Compound (2) has the highest activity toward cancer cells and is the least toxic for normal ones (i.e. peripheral blood mononuclear cells (PBMCs)). In contrast, compound (1) is the least active toward cancer cells but displays the highest toxicity toward normal cells. Transmission electronic microscopy indicates that cell death shows features typical of apoptotic cells, which was confirmed using the annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) assay. Furthermore, our data demonstrate that at an early stage during the treatment with complex (2) mitochondria lose their transmembrane potential, resulting in cytochrome C release. A quantification of caspases 3, 9 (intrinsic apoptosis pathway) and caspase 8 (extrinsic apoptosis pathway) indicated that both the intrinsic (via mitochondria) and extrinsic (via death receptors) pathways are involved in the apoptotic stimuli. PMID:23933562

  16. Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS-dependent mitochondrial signalling pathway

    PubMed Central

    Song, Xiaodong; Wang, Bingsi; Lin, Shengcui; Jing, Lili; Mao, Cuiping; Xu, Pan; Lv, Changjun; Liu, Wen; Zuo, Ji

    2014-01-01

    Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion-mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H2O2- or bleomycin (BLM)-induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs-II) in vivo and in vitro. Our data show that AST blocks H2O2- or BLM-induced ROS generation and dose-dependent apoptosis in AECs-II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase-9, caspase-3, Nrf-2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs-II cells through the ROS-dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment. PMID:25215580

  17. Hydrogen peroxide induces apoptosis via a mitochondrial pathway in chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhuang, Cai-ping; Liang, Qian; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H2O2), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H2O2 on cell viability. H2O2 treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H2O2 for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H2O2 treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H2O2 induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

  18. Global Proteomics and Pathway Analysis of Pressure-overload Induced Heart Failure and Its Attenuation by Mitochondrial Targeted Peptides

    PubMed Central

    Dai, Dao-Fu; Hsieh, Edward J.; Chen, Tony; Menendez, Lorena G.; Basisty, Nathan B.; Tsai, Lauren; Beyer, Richard P.; Crispin, David A.; Shulman, Nicholas J.; Szeto, Hazel H.; Tian, Rong; MacCoss, Michael J.; Rabinovitch, Peter S.

    2013-01-01

    Background We investigated the protective effects of mitochondrial-targeted antioxidant and protective peptides, SS31 and SS20, on cardiac function, proteomic remodeling and signaling pathways. Methods and Results We applied an improved label-free shotgun proteomics approach to evaluate the global proteomics changes in transverse aortic constriction (TAC) induced heart failure, and the associated signaling pathway changes using Ingenuity Pathway Analysis (IPA). We found 538 proteins significantly changed after TAC, which mapped to 53 pathways. The top pathways were in the categories of actin cytoskeleton, mitochondrial function, intermediate metabolism, glycolysis / gluconeogenesis and citrate cycle. Concomitant treatment with SS31 ameliorated the congestive heart failure phenotypes and mitochondrial damage induced by TAC, in parallel with global attenuation of mitochondrial proteome changes, with an average of 84% protection of mitochondrial and 69% of non-mitochondrial protein changes. This included significant amelioration of All the IPA pathways noted above. SS20 had only modest effects on heart failure and this tracked with only partial attenuation of global proteomics changes; furthermore, while actin cytoskeleton pathways were significantly protected in SS20, mitochondrial and metabolic pathways essentially were not. Conclusions This study elucidates the signaling pathways significantly changed in pressure-overload induced heart failure. The global attenuation of TAC-induced proteomic alterations by the mitochondrial targeted peptide SS-31 suggests that perturbed mitochondrial function may be an upstream signal to many of pathway alterations in TAC and supports the potential clinical application of mitochondrial-targeted peptide drugs for the treatment heart failure. PMID:23935006

  19. Apoptosis of human cholangiocarcinoma cell lines induced by β-escin through mitochondrial caspase-dependent pathway.

    PubMed

    Shen, Dong-Yan; Kang, Jin-He; Song, Wei; Zhang, Wen-Qing; Li, Wen-Gang; Zhao, Yan; Chen, Qing-Xi

    2011-10-01

    The study aimed to evaluate the effects of β-escin on human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and to explore its mechanisms. Cell growth, cell cycle and apoptosis were investigated, respectively, by MTT assay, single PI and FITC/PI double-staining flow cytometry, and fluorescence microscopy. The protein expression was determined by western blotting. The study revealed that β-escin inhibited cholangiocarcinoma cell growth in a dose- and time-dependent manner, and the cell cycle of QBC939 and Sk-ChA-1 cells was arrested in the G2/M phase, and MZ-ChA-1 cells in G1 phase. Apoptosis of the three cholangiocarcinoma cell lines induced by β-escin was associated with the collapse of the mitochondrial membrane potential and the activation of caspase-3. The apoptotic effect of β-escin was suppressed by pancaspase inhibitor z-VAD-fmk. Molecular dissection revealed that the antiapoptotic protein bcl-2 was down-regulated after cholangiocarcinoma cell lines were treated with β-escin, while the protein levels of bax and p53 were unchanged. Apoptosis was accompanied by an increase in reactive oxygen species (ROS). These results suggest that β-escin induces apoptosis of cholangiocarcinoma cells through an intrinsic mitochondrial caspase-dependent pathway, and the increase in the bax/bcl-2 ratio and ROS may play important roles in β-escin-induced apoptosis of cholangiocarcinoma cells. PMID:21394804

  20. Allicin sensitizes hepatocellular cancer cells to anti-tumor activity of 5-fluorouracil through ROS-mediated mitochondrial pathway.

    PubMed

    Zou, Xuejing; Liang, Jiyun; Sun, Jingyuan; Hu, Xiaoyun; Lei, Ling; Wu, Dehua; Liu, Li

    2016-08-01

    Drug resistance and hepatic dysfunction are the two major factors that limit the application of chemotherapy for hepatocellular carcinoma (HCC). It has been reported that allicin has the hepatic protective effect and antitumor activity. Hence allicin may be an ideal enhancer to chemotherapy regimen of HCC. In the present study, we demonstrated that allicin enhanced 5-fluorouracil (5-FU) inducing cytotoxicity in HCC cells. In vivo experiment, combined treatment group with allicin (5 mg/kg/d; every two days for 3 weeks) and 5-FU (20 mg/kg/d; 5 consecutive days) showed a dramatic inhibitory effect on the growth of HCC xenograft tumors in nude mice. The co-treatment group showed highly apoptotic level compared with 5-FU treated alone. Cells combined treatment with allicin and 5-FU increased intracellular reactive oxygen species (ROS) level, reduced mitochondrial membrane potential (ΔΨm), activated caspase-3 and PARP, and down-regulated Bcl-2 compared with DMSO, allicin and 5-FU treated alone. Moreover, the increase of activated caspase-3 and PARP was blocked by the ROS inhibitor antioxidant N-acetyl cysteine (NAC). In conclusion, this is the first study to demonstrate that allicin sensitized HCC cells to 5-FU induced apoptosis through ROS-mediated mitochondrial pathway. These results provided evidences for the combination used of allicin and 5-FU as a novel chemotherapy regimen in HCC. PMID:27177453

  1. Radiation Sensitivity in a Preclinical Mouse Model of Medulloblastoma Relies on the Function of the Intrinsic Apoptotic Pathway.

    PubMed

    Crowther, Andrew J; Ocasio, Jennifer K; Fang, Fang; Meidinger, Jessica; Wu, Jaclyn; Deal, Allison M; Chang, Sha X; Yuan, Hong; Schmid, Ralf; Davis, Ian; Gershon, Timothy R

    2016-06-01

    While treatments that induce DNA damage are commonly used as anticancer therapies, the mechanisms through which DNA damage produces a therapeutic response are incompletely understood. Here we have tested whether medulloblastomas must be competent for apoptosis to be sensitive to radiotherapy. Whether apoptosis is required for radiation sensitivity has been controversial. Medulloblastoma, the most common malignant brain tumor in children, is a biologically heterogeneous set of tumors typically sensitive to radiation and chemotherapy; 80% of medulloblastoma patients survive long-term after treatment. We used functional genetic studies to determine whether the intrinsic apoptotic pathway is required for radiation to produce a therapeutic response in mice with primary, Shh-driven medulloblastoma. We found that cranial radiation extended the survival of medulloblastoma-bearing mice and induced widespread apoptosis. Expression analysis and conditional deletion studies showed that Trp53 (p53) was the predominant transcriptional regulator activated by radiation and was strictly required for treatment response. Deletion of Bax, which blocked apoptosis downstream of p53, was sufficient to render tumors radiation resistant. In apoptosis-incompetent, Bax-deleted tumors, radiation activated p53-dependent transcription without provoking cell death and caused two discrete populations to emerge. Most radiated tumor cells underwent terminal differentiation. Perivascular cells, however, quickly resumed proliferation despite p53 activation, behaved as stem cells, and rapidly drove recurrence. These data show that radiation must induce apoptosis in tumor stem cells to be effective. Mutations that disable the intrinsic apoptotic pathways are sufficient to impart radiation resistance. We suggest that medulloblastomas are typically sensitive to DNA-damaging therapies, because they retain apoptosis competence. Cancer Res; 76(11); 3211-23. ©2016 AACR. PMID:27197166

  2. Induction of apoptosis by the tropical seaweed Pylaiella littoralis in HT-29 cells via the mitochondrial and MAPK pathways

    NASA Astrophysics Data System (ADS)

    Ye, Bo-Ram; Kim, Junseong; Kim, Min-Sun; Jang, Jiyi; Oh, Chulhong; Kang, Do-Hyung; Qian, Zhong-Ji; Jung, Won-Kyo; Choi, Il-Whan; Heo, Soo-Jin

    2013-12-01

    We demonstrated that an extract from Pylaiella littoralis, collected from the Federate States of Micronesia (FSM), could inhibit the proliferation of tumor cells. P. littoralis extract (PLE) showed anti-proliferative activities in the tumorigenic cells tested, ranging from 20.2% to 67.9%. The highest inhibitory activity, in HT-29 cells, was selected for further experiments. PLE showed no cytotoxic effect in normal cells and inhibited the growth of HT-29 cells depending on concentration and incubation time. PLE-treated HT-29 cells showed the typical morphological characteristics of apoptosis, such as apoptotic body formation and DNA fragmentation. PLE also induced mitochondrial membrane potential depolarization and resulted in increased mitochondrial membrane permeability, compared with untreated cells. PLE decreased Bcl-2 protein and increased Bax protein expression, activating caspase-3 and poly (ADP-ribose) polymerase (PARP) expression via the caspase pathway. PLE also increased the phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), and it reduced cell viability in treatment cells with specific inhibitors such as PD98059 (a specific inhibitor of ERK), SP600125 (a specific inbibitor of JNK), and SB 203580 (a specific inbibitor of p38 MAPK). via the the mitogen-activated protein kinases (MAPKs) pathway. These results suggest that PLE inhibits the proliferation of HT-29 cells by affecting the caspase and MAPK pathways involved in the induction of apoptosis. Thus, we suggest that P. littoralis extract might be potential candidate agents for the treatment of human colorectal cancer.

  3. T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity

    PubMed Central

    Burger, Megan L; Leung, Kenneth K; Bennett, Margaux J; Winoto, Astar

    2014-01-01

    T cell self-tolerance is thought to involve peripheral tolerance and negative selection, involving apoptosis of autoreactive thymocytes. However, evidence supporting an essential role for negative selection is limited. Loss of Bim, a Bcl-2 BH3-only protein essential for thymocyte apoptosis, rarely results in autoimmunity on the C57BL/6 background. Mice with T cell-specific over-expression of Bcl-2, that blocks multiple BH3-only proteins, are also largely normal. The nuclear receptor Nur77, also implicated in negative selection, might function redundantly to promote apoptosis by associating with Bcl-2 and exposing its potentially pro-apoptotic BH3 domain. Here, we report that T cell-specific expression of a Bcl2 BH3 mutant transgene results in enhanced rescue of thymocytes from negative selection. Concomitantly, Treg development is increased. However, aged BH3 mutant mice progressively accumulate activated, autoreactive T cells, culminating in development of multi-organ autoimmunity and lethality. These data provide strong evidence that negative selection is crucial for establishing T cell tolerance. DOI: http://dx.doi.org/10.7554/eLife.03468.001 PMID:25182415

  4. Cardiac arrest triggers hippocampal neuronal death through autophagic and apoptotic pathways.

    PubMed

    Cui, Derong; Shang, Hanbing; Zhang, Xiaoli; Jiang, Wei; Jia, Xiaofeng

    2016-01-01

    The mechanism of neuronal death induced by ischemic injury remains unknown. We investigated whether autophagy and p53 signaling played a role in the apoptosis of hippocampal neurons following global cerebral ischemia-reperfusion (I/R) injury, in a rat model of 8-min asphyxial cardiac arrest (CA) and resuscitation. Increased autophagosome numbers, expression of lysosomal cathepsin B, cathepsin D, Beclin-1, and microtubule-associated protein light chain 3 (LC3) suggested autophagy in hippocampal cells. The expression of tumor suppressor protein 53 (p53) and its target genes: Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) were upregulated following CA. The p53-specific inhibitor pifithrin-α (PFT-α) significantly reduced the expression of pro-apoptotic proteins (Bax and PUMA) and autophagic proteins (LC3-II and DRAM) that generally increase following CA. PFT-α also reduced hippocampal neuronal damage following CA. Similarly, 3-methyladenine (3-MA), which inhibits autophagy and bafilomycin A1 (BFA), which inhibits lysosomes, significantly inhibited hippocampal neuronal damage after CA. These results indicate that CA affects both autophagy and apoptosis, partially mediated by p53. Autophagy plays a significant role in hippocampal neuronal death induced by cerebral I/R following asphyxial-CA. PMID:27273382

  5. Cardiac arrest triggers hippocampal neuronal death through autophagic and apoptotic pathways

    PubMed Central

    Cui, Derong; Shang, Hanbing; Zhang, Xiaoli; Jiang, Wei; Jia, Xiaofeng

    2016-01-01

    The mechanism of neuronal death induced by ischemic injury remains unknown. We investigated whether autophagy and p53 signaling played a role in the apoptosis of hippocampal neurons following global cerebral ischemia-reperfusion (I/R) injury, in a rat model of 8-min asphyxial cardiac arrest (CA) and resuscitation. Increased autophagosome numbers, expression of lysosomal cathepsin B, cathepsin D, Beclin-1, and microtubule-associated protein light chain 3 (LC3) suggested autophagy in hippocampal cells. The expression of tumor suppressor protein 53 (p53) and its target genes: Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) were upregulated following CA. The p53-specific inhibitor pifithrin-α (PFT-α) significantly reduced the expression of pro-apoptotic proteins (Bax and PUMA) and autophagic proteins (LC3-II and DRAM) that generally increase following CA. PFT-α also reduced hippocampal neuronal damage following CA. Similarly, 3-methyladenine (3-MA), which inhibits autophagy and bafilomycin A1 (BFA), which inhibits lysosomes, significantly inhibited hippocampal neuronal damage after CA. These results indicate that CA affects both autophagy and apoptosis, partially mediated by p53. Autophagy plays a significant role in hippocampal neuronal death induced by cerebral I/R following asphyxial-CA. PMID:27273382

  6. Droxinostat, a Histone Deacetylase Inhibitor, Induces Apoptosis in Hepatocellular Carcinoma Cell Lines via Activation of the Mitochondrial Pathway and Downregulation of FLIP1

    PubMed Central

    Liu, Jing; Li, Guangming; Wang, Xiang; Wang, Liang; Zhao, Rui; Wang, Juanxia; Kong, Yin; Ding, Jie; Li, Juan; Zhang, Lingyi

    2016-01-01

    Background: The current chemotherapeutic outcomes for hepatocellular carcinoma (HCC) are not encouraging, and long-term survival of this patient group remains poor. Recent studies have demonstrated the utility of histone deacetylase inhibitors that can disrupt cell proliferation and survival in HCC management. However, the effects of droxinostat, a type of histone deacetylase inhibitor, on HCC remain to be established. Methods: The effects of droxinostat on HCC cell lines SMMC-7721 and HepG2 were investigated. Histone acetylation and apoptosis-modulating proteins were assessed via Western blot. Proliferation was examined with 3-(4, 5 dimetyl-2-thiazolyl)-2, 5-diphenyl 2H-tetrazolium bromide, cell proliferation, and real-time cell viability assays, and apoptosis with flow cytometry. Results: Droxinostat inhibited proliferation and colony formation of the HCC cell lines examined. Hepatoma cell death was induced through activation of the mitochondrial apoptotic pathway and downregulation of FLIP expression. Droxinostat suppressed histone deacetylase (HDAC) 3 expression and promoted acetylation of histones H3 and H4. Knockdown of HDAC3 induced hepatoma cell apoptosis and histone H3 and H4 acetylation. Conclusions: Droxinostat suppresses HDAC3 expression and induces histone acetylation and HCC cell death through activation of the mitochondrial apoptotic pathway and downregulation of FLIP, supporting its potential application in the treatment of HCC. PMID:26947884

  7. Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis.

    PubMed

    Fuenzalida, Karen; Quintanilla, Rodrigo; Ramos, Patricio; Piderit, Daniela; Fuentealba, Rodrigo A; Martinez, Gabriela; Inestrosa, Nibaldo C; Bronfman, Miguel

    2007-12-21

    Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2. PMID:17965419

  8. Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

    PubMed Central

    Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

    2013-01-01

    Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

  9. Constitutive p53 heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors of BCL-xL.

    PubMed

    Le Pen, J; Laurent, M; Sarosiek, K; Vuillier, C; Gautier, F; Montessuit, S; Martinou, J C; Letaï, A; Braun, F; Juin, P P

    2016-01-01

    Proapoptotic molecules directly targeting the BCL-2 family network are promising anticancer therapeutics, but an understanding of the cellular stress signals that render them effective is still elusive. We show here that the tumor suppressor p53, at least in part by transcription independent mechanisms, contributes to cell death induction and full activation of BAX by BH3 mimetic inhibitors of BCL-xL. In addition to mildly facilitating the ability of compounds to derepress BAX from BCL-xL, p53 also provides a death signal downstream of anti-apoptotic proteins inhibition. This death signal cooperates with BH3-induced activation of BAX and it is independent from PUMA, as enhanced p53 can substitute for PUMA to promote BAX activation in response to BH3 mimetics. The acute sensitivity of mitochondrial priming to p53 revealed here is likely to be critical for the clinical use of BH3 mimetics. PMID:26844698

  10. Ochratoxin a inhibits mouse embryonic development by activating a mitochondrion-dependent apoptotic signaling pathway.

    PubMed

    Hsuuw, Yan-Der; Chan, Wen-Hsiung; Yu, Jau-Song

    2013-01-01

    Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development. PMID:23296271

  11. Yeast as a tool to study signaling pathways in mitochondrial stress response and cytoprotection.

    PubMed

    Zdralević, Maša; Guaragnella, Nicoletta; Antonacci, Lucia; Marra, Ersilia; Giannattasio, Sergio

    2012-01-01

    Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, in addition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role of mitochondria in cell death is well recognized. Mitochondrial dysfunction has been associated with the death process and the onset of numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interest exists in pharmacological manipulation of mitochondrial metabolism and signaling. The yeast Saccharomyces cerevisiae has proven a valuable model organism in which several intracellular processes have been characterized in great detail, including the retrograde response to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidences of mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in the cross-talk between prosurvival and prodeath pathways. PMID:22454613

  12. Yeast as a Tool to Study Signaling Pathways in Mitochondrial Stress Response and Cytoprotection

    PubMed Central

    Ždralević, Maša; Guaragnella, Nicoletta; Antonacci, Lucia; Marra, Ersilia; Giannattasio, Sergio

    2012-01-01

    Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, in addition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role of mitochondria in cell death is well recognized. Mitochondrial dysfunction has been associated with the death process and the onset of numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interest exists in pharmacological manipulation of mitochondrial metabolism and signaling. The yeast Saccharomyces cerevisiae has proven a valuable model organism in which several intracellular processes have been characterized in great detail, including the retrograde response to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidences of mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in the cross-talk between prosurvival and prodeath pathways. PMID:22454613

  13. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zhu, Yue-Yong; Huang, Hong-Yan; Wu, Yin-Lian

    2015-10-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‑123 DNA‑binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‑dependent, as well as time‑dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‑G1 (apoptotic) phase of the cell cycle, in a dose‑dependent manner. Staining with Annexin V‑fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  14. Hippocampal Proteomic and Metabonomic Abnormalities in Neurotransmission, Oxidative Stress, and Apoptotic Pathways in a Chronic Phencyclidine Rat Model.

    PubMed

    Wesseling, Hendrik; Want, Elizabeth J; Guest, Paul C; Rahmoune, Hassan; Holmes, Elaine; Bahn, Sabine

    2015-08-01

    Schizophrenia is a neuropsychiatric disorder affecting 1% of the world's population. Due to both a broad range of symptoms and disease heterogeneity, current therapeutic approaches to treat schizophrenia fail to address all symptomatic manifestations of the disease. Therefore, disease models that reproduce core pathological features of schizophrenia are needed for the elucidation of pathological disease mechanisms. Here, we employ a comprehensive global label-free liquid chromatography-mass spectrometry proteomic (LC-MS(E)) and metabonomic (LC-MS) profiling analysis combined with the targeted proteomics (selected reaction monitoring and multiplex immunoassay) of serum and brain tissues to investigate a chronic phencyclidine (PCP) rat model in which glutamatergic hypofunction is induced through noncompetitive NMDAR-receptor antagonism. Using a multiplex immunoassay, we identified alterations in the levels of several cytokines (IL-5, IL-2, and IL-1β) and fibroblast growth factor-2. Extensive proteomic and metabonomic brain tissue profiling revealed a more prominent effect of chronic PCP treatment on both the hippocampal proteome and metabonome compared to the effect on the frontal cortex. Bioinformatic pathway analysis confirmed prominent abnormalities in NMDA-receptor-associated pathways in both brain regions, as well as alterations in other neurotransmitter systems such as kainate, AMPA, and GABAergic signaling in the hippocampus and in proteins associated with neurodegeneration. We further identified abundance changes in the level of the superoxide dismutase enzyme (SODC) in both the frontal cortex and hippocampus, which indicates alterations in oxidative stress and substantiates the apoptotic pathway alterations. The present study could lead to an increased understanding of how perturbed glutamate receptor signaling affects other relevant biological pathways in schizophrenia and, therefore, support drug discovery efforts for the improved treatment of patients

  15. Selenium (sodium selenite) causes cytotoxicity and apoptotic mediated cell death in PLHC-1 fish cell line through DNA and mitochondrial membrane potential damage.

    PubMed

    Selvaraj, Vellaisamy; Tomblin, Justin; Yeager Armistead, Mindy; Murray, Elizabeth

    2013-01-01

    Elevated concentration of selenium poses a toxic threat to organisms inhabiting aquatic ecosystems influenced by excessive inputs from anthropogenic sources. Selenium is also an essential micronutrient in living things, particularly in fish, and provides antioxidant properties to tissues. Whole fish and hepatocytes in primary culture show selenite toxicity above threshold levels. The present study was designed to investigate the process by which selenite exposure causes cellular toxicity and apoptotic and necrotic cell death in fish hepatoma cell line PLHC-1. PLHC-1 cells were exposed to various selenite concentrations (1, 10, 50 and 100 μM) for 10, 20 and 40 h intervals. The 24h inhibitory concentration 50 (IC₅₀) of selenite in PLHC-1 cell line was found to be 237 μM. Flow cytometery data showed that selenite exposed cells promote apoptotic and necrotic mediated cell death when selenite concentrations were ≥10 μM compared to control. Selenite exposure was associated with a significant increase of caspase-3 activities suggesting the induction of apoptosis. Selenite exposure at high levels (≥10 μM) and longer exposure times (≥20 h) induces mitochondrial membrane potential damage (ΔΨ(m)), DNA damage and elevated production of ROS which could be associated with cell death. PMID:23158585

  16. The Amaryllidaceae Isocarbostyril Narciclasine Induces Apoptosis By Activation of the Death Receptor and/or Mitochondrial Pathways in Cancer Cells But Not in Normal Fibroblasts1

    PubMed Central

    Dumont, Patrick; Ingrassia, Laurent; Rouzeau, Sébastien; Ribaucour, Fabrice; Thomas, Stéphanie; Roland, Isabelle; Darro, Francis; Lefranc, Florence; Kiss, Robert

    2007-01-01

    Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation. PMID:17898872

  17. Exploration of Bcl-2 family and caspases-dependent apoptotic signaling pathway in Zearalenone-treated mouse endometrial stromal cells.

    PubMed

    Hu, Jin; Xu, Minglong; Dai, Yujian; Ding, Xiaolin; Xiao, Cheng; Ji, Hongjun; Xu, Yinxue

    2016-08-01

    Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. Although the toxicity of ZEA have been widely studied in a number of cell types, the mechanistic role of ZEA on apoptosis of endometrial stromal cells (ESCs) remains poorly understood. The objective of this study was to determine the effects of ZEA on apoptosis of mouse ESCs and explore the signaling pathway underlying the cytotoxicity of ZEA. The results showed that ZEA treatment caused obvious apoptosis in ESCs as determined by the flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) revealed that ZEA treatment increased the ratio of Bax/Bcl-2. The enzymatic activity assays revealed that caspases-3 and caspase-9 were activated by ZEA treatment in a dose-dependent manner. In addition, flow cytometry show that the apoptotic percentages of cells pretreated with Z-VAD-FMK and Z-LEHD-FMK were markedly reduced compared to the ZEA-treated cells. Overall, the results suggested that ZEA induced obvious apoptosis in ESCs via a Bcl-2 family and caspases-dependent signaling pathway. PMID:27286704

  18. Osthole prevents anti-Fas antibody-induced hepatitis in mice by affecting the caspase-3-mediated apoptotic pathway.

    PubMed

    Okamoto, Toshihiro; Kawasaki, Toru; Hino, Okio

    2003-02-15

    Fas (Apo-1/CD95) ligand, which is a type II membrane protein, is a major inducer of apoptosis. Osthole is a coumarin derivative present in medicinal plants. The effect of osthole on hepatitis induced by anti-Fas antibody in mice was studied. Pretreatment of mice with osthole (10, 50, and 100 mg/kg, i.p.) prevented the elevation of plasma alanine aminotransferase (ALT) caused by anti-Fas antibody (175 microg/kg, i.v.). Administration of osthole to mice even at a dose of 10 mg/kg significantly inhibited of anti-Fas antibody-induced elevation of plasma ALT. Capase-3 is a cysteine protease, and treatment of mice with anti-Fas antibody caused an elevation of caspase-3 activity at 3.5 and 6 hr. Pretreatment of mice with osthole (100 mg/kg, i.p.) inhibited the elevation of caspase-3 activity caused by anti-Fas antibody. However, the addition of osthole (up to 10(-4)M) to a liver cytosol fraction isolated from mice treated with anti-Fas antibody did not inhibit caspase-3 activity in vitro. Thus, treatment of mice with osthole inhibited caspase-3 activity by an effect upstream of caspase-3 activation. The livers of mice treated with anti-Fas antibody contained apoptotic and dead cells; osthole attenuated the development of this apoptosis and cell death. The present results show that osthole prevented anti-Fas antibody-induced hepatitis by inhibiting the Fas-mediated apoptotic pathway. PMID:12566097

  19. Distinct muscle apoptotic pathways are activated in muscles with different fiber types a rat model of critical illness myopathy

    PubMed Central

    Barnes, Benjamin T.; Confides, Amy L.; Rich, Mark M.; Dupont-Versteegden, Esther E.

    2015-01-01

    Critical illness myopathy (CIM) is associated with severe muscle atrophy and fatigue in affected patients. Apoptotic signaling is involved in atrophy and is elevated in muscles from patients with CIM. In this study we investigated underlying mechanisms of apoptosis-related pathways in muscles with different fiber type composition in a rat model of CIM using denervation and glucocorticoid administration (denervation and steroid-induced myopathy, DSIM). Soleus and tibialis anterior (TA) muscles showed severe muscle atrophy (40–60% of control muscle weight) and significant apoptosis in interstitial as well as myofiber nuclei that was similar between the two muscles with DSIM. Caspase-3 and −8 activities, but not caspase-9 and −12, were elevated in TA and not in soleus muscle, while the caspase-independent proteins endonuclease G (EndoG) and apoptosis inducing factor (AIF) were not changed in abundance nor differentially localized in either muscle. Anti-apoptotic proteins HSP70, −27, and apoptosis repressor with a caspase recruitment domain (ARC) were elevated in soleus compared to TA muscle and ARC was significantly decreased with induction of DSIM in soleus. Results indicate that apoptosis is a significant process associated with DSIM in both soleus and TA muscles, and that apoptosis-associated processes are differentially regulated in muscles of different function and fiber type undergoing atrophy due to DSIM. We conclude that interventions combating apoptosis with CIM may need to be directed towards inhibiting caspase-dependent as well as -independent mechanisms to be able to affect muscles of all fiber types. PMID:25740800

  20. Harmine induces cell cycle arrest and mitochondrial pathway-mediated cellular apoptosis in SW620 cells via inhibition of the Akt and ERK signaling pathways.

    PubMed

    Liu, Jiming; Li, Qiang; Liu, Zhilong; Lin, Liuming; Zhang, Xiangqiang; Cao, Mingrong; Jiang, Jianwei

    2016-06-01

    Harmine, a β-carboline alkaloid isolated from the seeds of Peganum harmala, possesses both antitumor and anti‑nociceptive effects and inhibits human DNA topoisomerase. However, no detailed data are available concerning the mechanisms of harmine in human colorectal carcinoma SW620 cells. In the present study, we demonstrated that harmine inhibited the proliferation of SW620 cells in a dose-dependent manner using MTT and clone formation assays, and the IC50 value of harmine on the growth inhibition of SW620 cells for 48 h was 5.13 µg/ml. PI staining showed that harmine altered the cell cycle distribution by decreasing the proportion of cells in the G0-G1 phase and increasing the proportion in the S and G2-M phase. The expression level of cyclin D1 was decreased, while the expression of cyclin A, E2 and B1, CDK1/cdc2, Myt-1 and p-cdc2 (Tyr15) were increased, which was in accordance with the S and G2/M phase arrest. Hoechst 33258 staining revealed nuclear fragmentation, chromosomal condensation and cell shrinkage in the SW620 cells treated with harmine. Flow cytometry revealed that the percentage of apoptotic sub-G1 cells increased from 7.19 to 26.58%, while in the control group, sub-G1 cells only increased from 1.53 to 1.60%. Furthermore, early and late apoptotic cells were increased from 11.96 to 26.38% when incubated with the indicated concentration of harmine for 48 h, while in the control group, <8% of cells underwent apoptosis. JC-1 staining revealed that harmine decreased mitochondrial transmembrane potential (ΔΨm). The apoptosis of SW620 cells was also detected by western blot analysis, showing caspase-3 and -9, and PARP activation; the downregulation of Bcl-2, Mcl-1, Bcl-xL; and the upregulation of Bax. The expression of p-ERK, p-Akt (Ser473) and p-Akt (Thr308) was inhibited, and phosphorylation of downstream targets of Akt, such as p-FoxO3a and p-GSK‑3β were also attenuated. In conclusion, harmine induces cell cycle arrest and

  1. Adiponectin ameliorates the apoptotic effects of paraquat on alveolar type II cells via improvements in mitochondrial function

    PubMed Central

    HE, YARONG; ZOU, LIQUN; ZHOU, YAXIONG; HU, HAI; YAO, RONG; JIANG, YAOWEN; LAU, WAYNE BOND; YUAN, TUN; HUANG, WEN; ZENG, ZHI; CAO, YU

    2016-01-01

    Previous studies have demonstrated that excessive reactive oxygen/nitrogen species (ROS/RNS)-induced apoptosis is an important feature of the injury to the lung epithelium in paraquat (PQ) poisoning. However the precise mechanisms of PQ-induced dysfunction of the mitochondria, where ROS/RNS are predominantly produced, remain to be fully elucidated. Whether globular adiponectin (gAd), a potent molecule protective to mitochondria, regulates the mitochondrial function of alveolar type II cells to reduce PQ-induced ROS/RNS production remains to be investigated. The current study aimed to investigate the precise mechanisms of PQ poisoning in the mitochondria of alveolar type II cells, and to elucidate the role of gAd in protecting against PQ-induced lung epithelium injury. Therefore, lung epithelial injury was induced by PQ co-culture of alveolar type II A549 cells for 24 h. gAd was administrated to and removed from the injured cells in after 24 h. PQ was observed to reduce cell viability and increase apoptosis by ~1.5 fold in A549 cells. The oxidative/nitrative stress, resulting from ROS/RNS and disordered mitochondrial function were evidenced by increased O2−., NO production and reduced mitochondrial membrane potential (ΔΨ), adenosine 5′-triphosphate (ATP) content in PQ-poisoned A549 cells. gAd treatment significantly reversed the PQ-induced cell injury and mitochondrial dysfunction in A549 cells. The protective effects of gAd were partly abrogated by an adenosine 5′-monophosphate-activated protein kinase (AMPK) inhibitor, compound C. The results suggest that reduced ΔΨ and ATP content may result in PQ-induced mitochondrial dysfunction of the lung epithelium, which constitutes a novel mechanism for gAd exerting pulmonary protection against PQ poisoning via AMPK activation. PMID:27220901

  2. Mitochondrial Regulatory Pathways in the Pathogenesis of Alzheimer's Disease.

    PubMed

    Adiele, Reginald C; Adiele, Chiedukam A

    2016-07-01

    Alzheimer's disease (AD) is an age-associated neurodegenerative brain disorder with progressive cognitive decline that leads to terminal dementia and death. For decades, amyloid-beta (Aβ) and neurofibrillary tangle (NFT) aggregation hypotheses have dominated studies on the pathogenesis and identification of potential therapeutic targets in AD. Little attention has been paid to the mitochondrial molecular/biochemical pathways leading to AD. Mitochondria play a critical role in cell viability and death including neurons and neuroglia, not only because they regulate energy and oxygen metabolism but also because they regulate cell death pathways. Mitochondrial impairment and oxidative stress are implicated in the pathogenesis of AD. Interestingly, current therapeutics provide symptomatic benefits to AD patients resulting in the use of preventive trials on presymptomatic subjects. This review article elucidates the pathophysiology of AD and emphasizes the need to explore the mitochondrial pathways to provide solutions to unanswered questions in the prevention and treatment of AD. PMID:27392851

  3. Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice.

    PubMed

    Niessen, Markus; Krause, Katrin; Horst, Ina; Staebler, Norma; Klaus, Stephanie; Gaertner, Stefanie; Kebeish, Rashad; Araujo, Wagner L; Fernie, Alisdair R; Peterhansel, Christoph

    2012-04-01

    The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants. PMID:22268146

  4. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

    PubMed

    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. PMID:25875815

  5. The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

    PubMed Central

    Frank, Daniel O.; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. PMID:25875815

  6. Review of the apoptosis pathways in pancreatic cancer and the anti-apoptotic effects of the novel sea cucumber compound, Frondoside A.

    PubMed

    Li, X; Roginsky, A B; Ding, X-Z; Woodward, C; Collin, P; Newman, R A; Bell, R H; Adrian, T E

    2008-09-01

    Pancreatic cancer cells are resistant to the growth-inhibitory and apoptosis-inducing effects of conventional chemotherapeutic agents. There are multiple genetic and epigenetic events during the process of carcinogenesis that enable the cancer cells to avoid normal growth constraints and apoptosis. Investigation of the mechanisms involved has led to multiple strategies that encourage cell death and apoptosis to occur. The pathways involved are summarized in this review, together with some recently developed strategies to promote cell death in this cancer and with a particular focus on the frondoside A, a novel triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A inhibited proliferation of AsPC-1 human pancreatic cancer cells in a concentration- and time-dependent manner, as measured by (3)H-thymidine incorporation and cell counting. In concert with inhibition of cell growth, frondoside A induced significant morphological changes consistent with apoptosis. Propidium iodide DNA staining showed an increase of sub-G0/G1 cell population of apoptotic cells induced by frondoside A. Frondoside A-induced apoptosis was confirmed by annexin V binding and TUNEL assay. Furthermore, western blotting showed a decrease in expression of Bcl-2 and Mcl-1, an increase in Bax expression, activation of caspases 3, 7, and 9, and an increase in the expression of the cyclin-dependent kinase inhibitor, p21. These findings show that frondoside A induced apoptosis in human pancreatic cancer cells through the mitochondrial pathway and activation of the caspase cascade. Finally, a very low concentration of frondoside A (10 mug/kg/day) inhibited growth of AsPC-1 xenografts in athymic mice. In conclusion, new chemotherapeutic agents are desperately needed for pancreatic cancer because of the poor responsiveness to currently available treatment options. Frondoside A has potent growth inhibitory effects on human pancreatic cancer cells, and the inhibition

  7. A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

    PubMed Central

    Büttner, Sabrina; Ruli, Doris; Vögtle, F-Nora; Galluzzi, Lorenzo; Moitzi, Barbara; Eisenberg, Tobias; Kepp, Oliver; Habernig, Lukas; Carmona-Gutierrez, Didac; Rockenfeller, Patrick; Laun, Peter; Breitenbach, Michael; Khoury, Chamel; Fröhlich, Kai-Uwe; Rechberger, Gerald; Meisinger, Chris; Kroemer, Guido; Madeo, Frank

    2011-01-01

    Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-XL and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms. PMID:21673659

  8. Melittin induces human gastric cancer cell apoptosis via activation of mitochondrial pathway

    PubMed Central

    Kong, Gui-Mei; Tao, Wen-Hua; Diao, Ya-Li; Fang, Peng-Hua; Wang, Ji-Jun; Bo, Ping; Qian, Feng

    2016-01-01

    AIM: To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro. METHODS: SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential (MMP) levels, and analyzing reactive oxygen species (ROS) concentrations were analyzed by flow cytometry. Cytochrome C (Cyt C), apoptosis-inducing factor (AIF), endonuclease G (Endo G), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point (Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits. RESULTS: Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/mL for 1, 2, 4, 6, or 8 h and showed a time- and concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/mL melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h (n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99% (n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42 (n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control (5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor (Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group (1067.0 ± 132.5 U/g vs

  9. Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells*

    PubMed Central

    Wang, Jin-bo; Qi, Li-li; Zheng, Shui-di; Wu, Tian-xing

    2009-01-01

    Objective: To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells. Methods: HT-29 cells were treated with curcumin (0~80 μmol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: Curcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10~80 μmol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner. Conclusion: These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway. PMID:19235267

  10. Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells*

    PubMed Central

    Mazar, Julia; Thomas, Molly; Bezrukov, Ludmila; Chanturia, Alexander; Pekkurnaz, Gulcin; Yin, Shurong; Kuznetsov, Sergei A.; Robey, Pamela G.; Zimmerberg, Joshua

    2009-01-01

    Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(−/−) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. PMID:19531476

  11. Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import.

    PubMed

    Yamano, Koji; Yatsukawa, Yoh-Ichi; Esaki, Masatoshi; Hobbs, Alyson E Aiken; Jensen, Robert E; Endo, Toshiya

    2008-02-15

    Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import. PMID:18063580

  12. Apoptotic effect of gambogic acid in esophageal squamous cell carcinoma cells via suppression of the NF-κB pathway

    PubMed Central

    LIU, WEN-YUE; WU, XU; LIAO, CHENG-QUAN; SHEN, JIE; LI, JUN

    2016-01-01

    Despite extensive investigations of therapeutic improvements for surgical techniques, chemotherapy and chemoradiotherapy, esophageal squamous cell carcinoma (ESCC) remains one of the most aggressive forms of cancer, and the prognosis for patients with advanced ESCC remains poor. Therefore, effective therapies are urgently required in order to improve the prognosis of patients with ESCC. TE-1 cells were treated with gambogic acid (GA), and then subjected to western blot analysis, TUNEL assay and caspase activity analysis. GA significantly induced apoptosis in ESCC TE-1 cells. In addition, the antitumor activity of GA was accompanied by the decreased expression of phosphorylated-protein kinase B (p-AKT) and nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (NF-κB). The inhibition of protein kinase B (AKT) and NF-κB activation by chemical inhibitors augmented the apoptotic effect responses to GA in the TE-1 cells. The pan-caspase inhibitor z-VAD-fmk (zVAD) decreased GA-induced apoptosis. Furthermore, zVAD attenuated GA-induced growth inhibition in TE-1 cells. GA induced apoptosis in ESCC TE-1 via suppression of NF-κB pathway. The findings of the present study may provide a novel insight into ESCC treatment. PMID:27284372

  13. Helium-based cold atmospheric plasma-induced reactive oxygen species-mediated apoptotic pathway attenuated by platinum nanoparticles.

    PubMed

    Jawaid, Paras; Rehman, Mati Ur; Zhao, Qing Li; Takeda, Keigo; Ishikawa, Kenji; Hori, Masaru; Shimizu, Tadamichi; Kondo, Takashi

    2016-09-01

    Plasma is generated by ionizing gas molecules. Helium (He)-based cold atmospheric plasma (CAP) was generated using a high-voltage power supply with low-frequency excitation (60 Hz at 7 kV) and He flow at 2 l/min. Platinum nanoparticles (Pt-NPs) are potent antioxidants due to their unique ability to scavenge superoxides and peroxides. These features make them useful for the protection against oxidative stress-associated pathologies. Here, the effects of Pt-NPs on He-CAP-induced apoptosis and the underlying mechanism were examined in human lymphoma U937 cells. Apoptosis was measured after cells were exposed to He-CAP in the presence or absence of Pt-NPs. The effects of combined treatment were determined by observing the changes in intracellular reactive oxygen species (ROS) and both mitochondrial and Fas dependent pathway. The results indicate that Pt-NPs substantially scavenge He-CAP-induced superoxides and peroxides and inhibit all the pathways involved in apoptosis execution. This might be because of the SOD/catalase mimetic effects of Pt-NPs. These results showed that the Pt-NPs can induce He-CAP desensitization in human lymphoma U937 cells. PMID:27256594

  14. Isoledene from Mesua ferrea oleo-gum resin induces apoptosis in HCT 116 cells through ROS-mediated modulation of multiple proteins in the apoptotic pathways: A mechanistic study.

    PubMed

    Asif, Muhammad; Shafaei, Armaghan; Jafari, Seyedeh Fatemeh; Mohamed, Shazmin Kithur; Ezzat, Mohammed Oday; Abdul Majid, Aman Shah; Oon, Chern Ein; Petersen, Sven H; Kono, Koji; Abdul Majid, Amin Malik Shah

    2016-08-22

    Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as KRAS and APC. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis-inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, showed that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in the treated cells. In addition, an increment in the levels of ROS, caspase-8, -9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bim and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-survival proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways. PMID:27268964

  15. Overexpression of amyloid-β protein precursor induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade.

    PubMed

    Bartley, Matthew G; Marquardt, Kristin; Kirchhof, Danielle; Wilkins, Heather M; Patterson, David; Linseman, Daniel A

    2012-01-01

    Aberrant processing of amyloid-β protein precursor (AβPP) into amyloid-β (Aβ) fragments underlies the formation of senile plaques in Alzheimer's disease (AD). Moreover, Aβ fragments, particularly Aβ(42), exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces caspase-3 activation and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and induces glutathione-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP are significantly inhibited by co-expression of Bcl-2 or treatment with either glutathione or a pan-caspase inhibitor. The mPTP inhibitor, cyclosporin A, also significantly attenuates AβPP-induced apoptosis. AβPP-induced apoptosis is unaffected by a β-secretase inhibitor and is independent of detectable Aβ(42); however, a γ-secretase inhibitor significantly protects against AβPP overexpression, suggesting a possible role of the AβPP intracellular domain in cell death. These data indicate that overexpression of WT AβPP is sufficient to induce MOS and intrinsic apoptosis, suggesting a novel pro-oxidant role for AβPP at mitochondria which may be relevant in AD and DS disease pathologies. PMID:22133762

  16. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario.

    PubMed

    Arrázola, Macarena S; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C

    2015-01-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as "mitochondrial dynamics" is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

  17. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario

    PubMed Central

    Arrázola, Macarena S.; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C.

    2015-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as “mitochondrial dynamics” is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

  18. Photodynamic therapy activated STAT3 associated pathways: Targeting intrinsic apoptotic pathways to increase PDT efficacy in human squamous carcinoma cells.

    PubMed

    Qiao, Li; Xu, Chengshan; Li, Qiang; Mei, Zhusong; Li, Xinji; Cai, Hong; Liu, Wei

    2016-06-01

    5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) has been used for part of squamous cell carcinoma (premalignant conditions or in situ cutaneous SCC-Bowen disease). However, mechanism of ALA-PDT is not fully understood yet on the cell apoptosis pathway. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on human squamous carcinoma A431cells. Apoptosis and cell viability after PDT were evaluated using Annexin V-FITC apoptosis detection kit and MTT assay. The mRNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Our data showed that 5-ALA-PDT significantly inhibited cell proliferation (p<0.05), but there was no significant difference when the photosensitizer reached to 4.8mM. The inhibition in cell proliferation after 5-ALA-PDT treatment was correlated to more cells being arrested in the G0/G1 phase of the cell cycle (p<0.01). Immunocytochemical observations using anti-active caspase-3 antibodies showed active caspase-3 was translocated from cytoplasm to nuclear during apoptosis. STAT3 and its downstream gene Bax and BCL-2 were changed after 5-ALA-PDT treatment for the mRNA and protein expression. Our studies confirmed that 5-ALA-PDT might be an effective treatment for human squamous carcinoma by inhibiting the tumor cell A431growth and for the first time demonstrated that the expression of STAT3 was significantly reduced at 24h after 5-ALA-PDT treatment. PMID:26607555

  19. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

    PubMed Central

    McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

    2014-01-01

    Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

  20. PI3K inhibitors prime neuroblastoma cells for chemotherapy by shifting the balance towards pro-apoptotic Bcl-2 proteins and enhanced mitochondrial apoptosis.

    PubMed

    Bender, A; Opel, D; Naumann, I; Kappler, R; Friedman, L; von Schweinitz, D; Debatin, K-M; Fulda, S

    2011-01-27

    We recently identified activation of phosphatidylinositol 3'-kinase (PI3K)/Akt as a novel predictor of poor outcome in neuroblastoma. Here, we investigated the effect of small-molecule PI3K inhibitors on chemosensitivity. We provide first evidence that PI3K inhibitors, for example PI103, synergize with various chemotherapeutics (Doxorubicin, Etoposide, Topotecan, Cisplatin, Vincristine and Taxol) to trigger apoptosis in neuroblastoma cells (combination index: high synergy). Mechanistic studies reveal that PI103 cooperates with Doxorubicin to reduce Mcl-1 expression and Bim(EL) phosphorylation and to upregulate Noxa and Bim(EL) levels. This shifted ratio of pro- and antiapoptotic Bcl-2 proteins results in increased Bax/Bak conformational change, loss of mitochondrial membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Although Mcl-1 knockdown enhances Doxorubicin- and PI103-induced apoptosis, silencing of Noxa, Bax/Bak or p53 reduces apoptosis, underscoring the functional relevance of the Doxorubicin- and PI103-mediated modulation of these proteins for chemosensitization. Bcl-2 overexpression inhibits Bax activation, mitochondrial perturbations, cleavage of caspases and Bid, and apoptosis, confirming the central role of the mitochondrial pathway for chemosensitization. Interestingly, the broad-range caspase inhibitor zVAD.fmk does not interfere with Bax activation or mitochondrial outer membrane permeabilization, whereas it blocks caspase activation and apoptosis, thus placing mitochondrial events upstream of caspase activation. Importantly, PI103 and Doxorubicin cooperate to induce apoptosis and to suppress tumor growth in patients' derived primary neuroblastoma cells and in an in vivo neuroblastoma model, underlining the clinical relevance of the results. Thus, targeting PI3K presents a novel and promising strategy to sensitize neuroblastoma cells for chemotherapy-induced apoptosis, which has important implications for the

  1. Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells.

    PubMed

    Gerges, Steve; Rohde, Katharina; Fulda, Simone

    2016-05-28

    Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that small-molecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2'-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as Receptor-Interacting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK'872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies

  2. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

    SciTech Connect

    Ma, Gui-Fen; Chen, Shi-Yao; Sun, Zhi-Rong; Miao, Qing; Liu, Yi-Mei; Zeng, Xiao-Qing; Luo, Tian-Cheng; Ma, Li-Li; Lian, Jing-Jing; Song, Dong-Li

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer The article revealed FoxP3 gene function in gastric cancer firstly. Black-Right-Pointing-Pointer Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. Black-Right-Pointing-Pointer Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. Black-Right-Pointing-Pointer Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Black-Right-Pointing-Pointer FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis

  3. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the RU486-treated rat uterus during early pregnancy.

    PubMed

    Theron, Kathrine E; Penny, Clement B; Hosie, Margot J

    2013-12-01

    An increase in apoptotic activity has been observed in both the rabbit and the rat endometria following treatment with RU486. The aim of this study was to assess whether Bax and Bcl-2 signaling, in response to RU486, could be crucial role players mediating apoptosis in the rat uterus during early pregnancy. RU486 is a partial progesterone (P4) and estrogen receptor antagonist, functioning to actively silence P4 receptor gene-associated transcription. Although an increase in apoptosis as a result of RU486 administration has been previously reported in rabbits, the specific apoptotic factors and pathways involved in driving this process have not yet been established. Immunofluorescent techniques were used to determine protein expression levels of both Bax and Bcl-2 in RU486-treated endometria at days 4.5, 5.5 and 6.5 of pregnancy. The Bax/Bcl-2 index was used to determine the overall pro- or anti-apoptotic setting at each day of pregnancy, following RU486 administration. Changes in the Bax and Bcl-2 gene expression levels as a consequence of RU486 administration were evaluated using RT-qPCR. Both the protein and gene expression analyses suggest that RU486 induces a change toward an overall anti-apoptotic signal within the Bax/Bcl-2 pathway. These results suggest that the observed increase in apoptosis following RU486 administration is not driven by a shift in the Bax/Bcl-2 ratio toward cell death, when the P4 and estrogen receptors are partially inactivated by RU486, but is possibly regulated by another apoptotic pathway. PMID:24287037

  4. Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways

    PubMed Central

    Huang, Guang-Ming; Sun, Yu; Ge, Xin; Wan, Xin; Li, Chun-Bo

    2015-01-01

    of pro-caspase-8, -9 and -3 were significantly decreased (P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model (P < 0.05). CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor (extrinsic) and mitochondrial (intrinsic) pathways. PMID:26034354

  5. Cytochrome c modulates the mitochondrial signaling pathway and polymorphonuclear neutrophil apoptosis in bile duct-ligated rats

    PubMed Central

    DENG, XUESONG; DENG, TONGMING; NI, YONG; ZHAN, YONGQIANG; HUANG, WENLONG; LIU, JIANFENG; LIAO, CAIXIAN

    2016-01-01

    increase. Morphological changes in PMN mitochondria were marginal in BDL rats and marked in the BDL + Cytc group. In the BDL rats, PMN apoptosis was inhibited, a process induced by the mitochondrial apoptotic signaling pathway in which Cytc has an important role. High ΔΨm in the mitochondria and decreased Cytc expression levels in the cytoplasm result in PMN apoptosis inhibition. Intravenous injection of Cytc may help compensate for the lack of Cytc proteins in the cytoplasm, inducing PMN apoptosis following BDL. PMID:27347059

  6. E. adenophorum induces Cell Cycle Arrest and Apoptosis of Splenocytes through the Mitochondrial Pathway and Caspase Activation in Saanen Goats

    PubMed Central

    He, Yajun; Mo, Quan; Hu, Yanchun; Chen, Weihong; Luo, Biao; Wu, Lei; Qiao, Yan; Xu, Ruiguang; Zhou, Yancheng; Zuo, Zhicai; Deng, Junliang; He, Wei; Wei, Yahui

    2015-01-01

    The precise cytotoxicity of E. Adenophorum in relation to the cell cycle and apoptosis of splenocytes in Saanen goats remains unclear. In the present study, 16 Saanen goats were randomly divided into four groups, which were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The results of TUNEL, DAPI and AO/EB staining, flow cytometry analysis and DNA fragmentation assays showed that E. adenophorum induced typical apoptotic features in splenocytes, suppressed splenocyte viability, and caused cell cycle arrest in a dose-dependent manner. However, westernblot, ELISA, qRT-PCR and caspase activity analyses showed that E. adenophoruminhibited Bcl-2 expression, promoted Bax translocation to the mitochondria, triggered the release of Cytc from the mitochondria into the cytosol, and activated caspase-9 and -3 and the subsequent cleavage of PARP. Moreover, in E. adenophorum-induced apoptosis, the protein levels of Fas, Bid, FasL and caspase-8 showed no significant changes. E. adenophorum treatment induced the collapse of ΔΨm. Moreover, these data suggested that E. adenophorum induces splenocyte apoptosis via the activation of the mitochondrial apoptosis pathway in splenocytes. These findings provide new insights into the mechanisms underlying the effects of E. adenophorum cytotoxicity on splenocytes. PMID:26527166

  7. The P66Shc/mitochondrial permeability transition pore pathway determines neurodegeneration.

    PubMed

    Savino, Costanza; Pelicci, PierGiuseppe; Giorgio, Marco

    2013-01-01

    Mitochondrial-mediated oxidative stress and apoptosis play a crucial role in neurodegenerative disease and aging. Both mitochondrial permeability transition (PT) and swelling of mitochondria have been involved in neurodegeneration. Indeed, knockout mice for cyclophilin-D (Cyc-D), a key regulatory component of the PT pore (PTP) that triggers mitochondrial swelling, resulted to be protected in preclinical models of multiple sclerosis (MS), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, how neuronal stress is transduced into mitochondrial oxidative stress and swelling is unclear. Recently, the aging determinant p66Shc that generates H2O2 reacting with cytochrome c and induces oxidation of PTP and mitochondrial swelling was found to be involved in MS and ALS. To investigate the role of p66Shc/PTP pathway in neurodegeneration, we performed experimental autoimmune encephalomyelitis (EAE) experiments in p66Shc knockout mice (p66Shc-/-), knock out mice for cyclophilin-D (Cyc-D-/-), and p66Shc Cyc-D double knock out (p66Shc/Cyc-D-/-) mice. Results confirm that deletion of p66Shc protects from EAE without affecting immune response, whereas it is not epistatic to the Cyc-D mutation. These findings demonstrate that p66Shc contributes to EAE induced neuronal damage most likely through the opening of PTP suggesting that p66Shc/PTP pathway transduces neurodegenerative stresses. PMID:23766859

  8. Anticancer effect of celastrol on human triple negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways.

    PubMed

    Shrivastava, Shweta; Jeengar, Manish Kumar; Reddy, V Sudhakar; Reddy, G Bhanuprakash; Naidu, V G M

    2015-06-01

    Signaling via the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is crucial for divergent physiological processes including transcription, translation, cell-cycle progression and apoptosis. The aim of work was to elucidate the anti-cancer effect of celastrol and the signal transduction pathways involved. Cytotoxic effect of celastrol was assessed by MTT assay on human triple negative breast cancer cells (TNBCs) and compared with that of MCF-7. Apoptosis induction was determined by AO/EtBr staining, mitochondrial membrane potential by JC-1, Annexin binding assays and modulation of apoptotic proteins and its effect on PI3K/Akt/mTOR pathway by western blotting. Celastrol induced apoptosis in TNBC cells, were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, celastrol triggered reactive oxygen species production with collapse of mitochondrial membrane potential, down-regulation of Bcl-2 and up-regulation of Bax expression. Celastrol effectively decreased PI3K 110α/85α enzyme activity, phosphorylation of Akt(ser473) and p70S6K1 and 4E-BP1. Although insulin treatment increased the phosphorylation of Akt(ser473), p70S6K1, 4E-BP1, celastrol abolished the insulin mediated phosphorylation. It clearly indicates that celastrol acts through PI3k/Akt/mTOR axis. We also found that celastrol inhibited the Akt/GSK3β and Akt/NFkB survival pathway. PI3K/Akt/mTOR inhibitor, PF-04691502 and mTOR inhibitor rapamycin enhanced the apoptosis-inducing effect of celastrol. These data demonstrated that celastrol induces apoptosis in TNBC cells and indicated that apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway. PMID:25818165

  9. Syringaresinol induces mitochondrial biogenesis through activation of PPARβ pathway in skeletal muscle cells.

    PubMed

    Thach, Trung Thanh; Lee, Chan-Kyu; Park, Hyun Woo; Lee, Sang-Jun; Lee, Sung-Joon

    2016-08-15

    Activation of peroxisome proliferator-activated receptors (PPARs) plays a crucial role in cellular energy metabolism that directly impacts mitochondrial biogenesis. In this study, we demonstrate that syringaresinol, a pharmacological lignan extracted from Panax ginseng berry, moderately binds to and activates PPARβ with KD and EC50 values of 27.62±15.76μM and 18.11±4.77μM, respectively. Subsequently, the expression of peroxisome proliferator-activated receptor γ coactivator-1α together with PPARβ transcriptional targets, mitochondrial carnitine palmitoyltransferase 1 and uncoupling protein 2, was also enhanced in terms of both mRNA and protein levels. The activation of these proteins induced mitochondrial biogenesis by enrichment of mitochondrial replication and density within C2C12 myotubes. Importantly, knockdown of PPARβ reduced the syringaresinol-induced protein expression followed by the significant reduction of mitochondrial biogenesis. Taken together, our results indicate that syringaresinol induces mitochondrial biogenesis by activating PPARβ pathway. PMID:27450788

  10. A mitochondrial pathway for biosynthesis of lipid mediators

    NASA Astrophysics Data System (ADS)

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

  11. A mitochondrial pathway for biosynthesis of lipid mediators.

    PubMed

    Tyurina, Yulia Y; Poloyac, Samuel M; Tyurin, Vladimir A; Kapralov, Alexander A; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I; Vikulina, Anna S; Jung, Mi-Yeon; Epperly, Michael W; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C; Kochanek, Patrick M; Pitt, Bruce R; Greenberger, Joel S; Vladimirov, Yury A; Bayır, Hülya; Kagan, Valerian E

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury. PMID:24848241

  12. Molecular and Computational Studies on Apoptotic Pathway Regulator, Bcl-2 Gene from Breast Cancer Cell Line MCF-7.

    PubMed

    Tiwari, Pragya; Khan, M J

    2016-01-01

    Cancer is a dreadful disease constituting abnormal growth and proliferation of malignant cells in the body. Next to lung cancer, breast cancer is the most common form of cancer affecting women. The apoptotic pathway regulators, B cell lymphoma family of protein, play a key role in various malignancies defining cancer and their constitutive expression plays an integral role in breast cancer chemotherapy. The research work discusses the identification and molecular cloning of a B cell lymphoma like gene from human breast cancer cell line. The open reading frame of the gene consisted of 965 nucleotides, encoding a protein of 380 amino acids with a predicted molecular weight of 42.5 kilodalton. The predicted physiochemical properties of the gene were as follows: Isoelectric point - 9.49, molecular formula - C1893H3004N534O548S16, total number of negatively charged residues, (Aspartate+Glutamate) - 26, total number of positively charged residues, (Arginine+Lysine)-39, instability index-42.08 (unstable protein) and grand average of hydropathicity is -0.202. Additionally, phobius prediction suggested non-cytoplasmic localization of the putative protein. The presence of secondary structure in the protein was determined by Memsat program. A 3 dimensional protein homology model was generated using threading based method of protein modeling for structural and functional annotation of the putative protein. Future prospects accounts for the biochemical characterization of the enzyme including in vitro assays on breast cancer cell line would establish the functional characteristics of the protein and its physiological mechanisms in breast cancer development and its therapeutic-target role in future. PMID:27168686

  13. c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway[S

    PubMed Central

    Mollica, Maria Pina; Trinchese, Giovanna; Cavaliere, Gina; De Filippo, Chiara; Cocca, Ennio; Gaita, Marcello; Della-Gatta, Antonio; Marano, Angela; Mazzarella, Giuseppe; Bergamo, Paolo

    2014-01-01

    Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis. PMID:24634500

  14. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

    PubMed Central

    Wang, Z; Liu, D; Varin, A; Nicolas, V; Courilleau, D; Mateo, P; Caubere, C; Rouet, P; Gomez, A-M; Vandecasteele, G; Fischmeister, R; Brenner, C

    2016-01-01

    Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies. PMID:27100892

  15. Apoptotic cell death in rat epididymis following epichlorohydrin treatment.

    PubMed

    Lee, I-C; Kim, K-H; Kim, S-H; Baek, H-S; Moon, C; Kim, S-H; Yun, W-K; Nam, K-H; Kim, H-C; Kim, J-C

    2013-06-01

    Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8-24 h. Caspase-3 and caspase-8 activities also increased at 8-48 h and 12-48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway. PMID:23386780

  16. Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro

    PubMed Central

    Chen, Jiezhong; Lin, Chao; Shao, Renfu; Yan, Chunxia; Chen, Chen

    2016-01-01

    Mitochondrial functions are crucial for pancreatic β-cell survival and glucose-induced insulin secretion. Hexarelin (Hex) is a synthetic small peptide ghrelin analogue, which has been shown to protect cardiomyocytes from the ischemia-reperfusion process. In this study, we used in vitro and in vivo models of streptozotocin (STZ)-induced β-cell damage to study the protective effect of Hex and the associated mechanisms. We found that STZ produced a cytotoxic effect in a dose- and time-dependent manner in MIN6 cells (a mouse β-cell line). Hex (1.0 μM) decreased the STZ-induced damage in β-cells. Rhodamine 123 assay and superoxide DHE production assay revealed that Hex ameliorated STZ-induced mitochondrial damage and excessive superoxide activity in β-cells. In addition, Hex significantly reduced STZ-induced expression of cleaved Caspases-3, Caspases-9 and the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 in MIN6 cells. We further examined the in vivo effect of Hex in a rat model of type 1 diabetes induced by STZ injection. Hex ameliorated STZ-induced decrease in plasma insulin and protected the structure of islets from STZ-induced disruption. Hex also ameliorated STZ-induced expression of cleaved Caspase-9 and the Bax in β-cells. In conclusion, our data indicate that Hex is able to protects β-cell mass from STZ-caused cytotoxic effects involving mitochondrial pathways in vitro and in vivo. Hex may serve as a potential protective agent for the management of diabetes. PMID:26918825

  17. Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae.

    PubMed

    Mutlu, Nebibe; Garipler, Görkem; Akdoğan, Emel; Dunn, Cory D

    2014-07-01

    Genetic and microscopic approaches using Saccharomyces cerevisiae have identified many proteins that play a role in mitochondrial dynamics, but it is possible that other proteins and pathways that play a role in mitochondrial division and fusion remain to be discovered. Mutants lacking mitochondrial fusion are characterized by rapid loss of mitochondrial DNA. We took advantage of a petite-negative mutant that is unable to survive mitochondrial DNA loss to select for mutations that allow cells with fusion-deficient mitochondria to maintain the mitochondrial genome on fermentable medium. Next-generation sequencing revealed that all identified suppressor mutations not associated with known mitochondrial division components were localized to PDR1 or PDR3, which encode transcription factors promoting drug resistance. Further studies revealed that at least one, if not all, of these suppressor mutations dominantly increases resistance to known substrates of the pleiotropic drug resistance pathway. Interestingly, hyperactivation of this pathway did not significantly affect mitochondrial shape, suggesting that mitochondrial division was not greatly affected. Our results reveal an intriguing genetic connection between pleiotropic drug resistance and mitochondrial dynamics. PMID:24807265

  18. Epoxyeicosatrienoic acids prevent cisplatin-induced renal apoptosis through a p38 mitogen-activated protein kinase-regulated mitochondrial pathway.

    PubMed

    Liu, Yingmei; Lu, Xiaodan; Nguyen, Sinh; Olson, Jean L; Webb, Heather K; Kroetz, Deanna L

    2013-12-01

    Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2(-/-) mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage. PMID:24092818

  19. Epoxyeicosatrienoic Acids Prevent Cisplatin-Induced Renal Apoptosis through a p38 Mitogen-Activated Protein Kinase–Regulated Mitochondrial Pathway

    PubMed Central

    Liu, Yingmei; Lu, Xiaodan; Nguyen, Sinh; Olson, Jean L.; Webb, Heather K.

    2013-01-01

    Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2−/− mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage. PMID:24092818

  20. Antihepatocellular Carcinoma Potential of Tetramethylpyrazine Induces Cell Cycle Modulation and Mitochondrial-Dependent Apoptosis: Regulation of p53 Signaling Pathway in HepG2 Cells In Vitro.

    PubMed

    Bi, Lei; Yan, Xiaojing; Chen, Weiping; Gao, Jing; Qian, Lei; Qiu, Shuang

    2016-06-01

    Tetramethylpyrazine (TMP) was originally isolated from a traditional Chinese herbal medicine, Ligusticum chuanxiong In the present study, TMP exhibits potent antitumor activities in vitro. However, the molecular mechanisms remain to be defined. Hence, this study aims to investigate the antiproliferative and apoptotic effects of TMP on HepG2 and elucidate the underlying mechanisms. Analyses using Cell Counting Kit-8 and real-time cell analyzer indicated that TMP significantly inhibited HepG2 cell proliferation. We also observed that TMP induced cell cycle arrest at the G0/G1 checkpoint and apoptosis, using flow cytometry and high-content screening. Furthermore, our results predicted that TMP could directly decrease mitochondrial membrane potential (Δψm), increase the release of cytochrome c, and increase caspase activation, indicating that mitochondrial pathway apoptosis could be the mechanism for TMP within HepG2 cells. Moreover, TMP altered expression of p53 and the Bcl-2/Bax protein ratio, which revealed that TMP induced cell cycle arrest and caspase-dependent mitochondrial apoptosis in HepG2 cells in vitro. These studies provided mechanistic insights into the antitumor properties of TMP, which may be explored as a potential option for treatment of hepatocellular carcinoma. PMID:27179035

  1. Insulin Stimulates Mitochondrial Fusion and Function in Cardiomyocytes via the Akt-mTOR-NFκB-Opa-1 Signaling Pathway

    PubMed Central

    Parra, Valentina; Verdejo, Hugo E.; Iglewski, Myriam; del Campo, Andrea; Troncoso, Rodrigo; Jones, Deborah; Zhu, Yi; Kuzmicic, Jovan; Pennanen, Christian; Lopez‑Crisosto, Camila; Jaña, Fabián; Ferreira, Jorge; Noguera, Eduard; Chiong, Mario; Bernlohr, David A.; Klip, Amira; Hill, Joseph A.; Rothermel, Beverly A.; Abel, Evan Dale; Zorzano, Antonio; Lavandero, Sergio

    2014-01-01

    Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway. PMID:24009260

  2. Nuclear export and mitochondrial and endoplasmic reticulum localization of IGF-binding protein 3 regulate its apoptotic properties

    PubMed Central

    Paharkova-Vatchkova, Vladislava; Lee, Kuk-Wha

    2011-01-01

    Tumor suppression by IGF-binding protein 3 (IGFBP3) may occur in an IGF-independent manner, in addition to its role as a regulator of IGF bioavailability. After secretion, IGFBP3 is internalized, rapidly localized to the nucleus, and is later detected in the cytoplasm. We identified a putative nuclear export sequence (NES) in IGFBP3 between amino acids 217 and 228, analogous to the leucine-rich NES sequence of p53 and HIV Rev. Mutation of the NES prevents nucleocytoplasmic shuttling of IGFBP3 and blocks its ability to induce apoptosis. Targeting of IGFBP3 to the mitochondria and endoplasmic reticulum (ER) was confirmed by co-localization with organelle markers using fluorescence confocal microscopy and subcellular fractionation. Mitochondrial targeting was also demonstrated in vivo in IGFBP3-treated prostate cancer xenografts. These results show that IGFBP3 shuttles from the nucleus to the mitochondria and ER, and that nuclear export is essential for its effects on prostate cancer apoptosis. PMID:20228135

  3. The Infertility of Repeat-Breeder Cows During Summer Is Associated with Decreased Mitochondrial DNA and Increased Expression of Mitochondrial and Apoptotic Genes in Oocytes.

    PubMed

    Ferreira, Roberta Machado; Chiaratti, Marcos Roberto; Macabelli, Carolina Habermann; Rodrigues, Carlos Alberto; Ferraz, Márcio Leão; Watanabe, Yeda Fumie; Smith, Lawrence Charles; Meirelles, Flávio Vieira; Baruselli, Pietro Sampaio

    2016-03-01

    Oocyte quality is known to be a major cause of infertility in repeat-breeder (RB) and heat-stressed dairy cows. However, the mechanisms by which RB oocytes become less capable of supporting embryo development remain largely unknown. Thus, the aim of this study was to investigate whether the decreased oocyte competence of RB cows (RBs) during summer is associated with an altered gene expression profile and a decrease in mitochondrial DNA (mtDNA) copy number. Therefore, oocytes collected from heifers, non-RBs in peak lactation (PLs), and RBs were used to evaluate mtDNA amounts as well as the expression levels of genes associated with the mitochondria (MT-CO1,NRF1,POLG,POLG2,PPARGC1A, andTFAM), apoptosis (BAX,BCL2, andITM2B), and oocyte maturation (BMP15,FGF8,FGF10,FGF16,FGF17, andGDF9). The oocytes retrieved from RBs during winter contained over eight times more mtDNA than those retrieved from RBs during summer. They also contained significantly less mtDNA than oocytes retrieved from heifers and PLs during summer. Moreover, the expression of mitochondria- (NRF1,POLG,POLG2,PPARGC1A, andTFAM) and apoptosis-related (BAXandITM2B) genes, as well as ofGDF9, in RB oocytes collected during summer was significantly greater than that in oocytes collected from heifers and PLs during the same season. In oocytes from heifers and PLs, the expression levels of these genes were lower in those collected during summer compared with winter, but this difference was not observed in oocytes collected from RBs. Altogether, these data provide evidence of altered gene expression and reduced mtDNA copy number in the oocytes collected from RBs during summer. This indicates a loss of fertility in RBs during summer, which might be caused by a possible mitochondrial dysfunction associated with a greater chance of oocytes to undergo apoptosis. PMID:26843447

  4. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    SciTech Connect

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  5. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1.

    PubMed

    Peng, Xi; Yu, Zhengqiang; Liang, Na; Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-03-15

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  6. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1

    PubMed Central

    Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-01-01

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  7. Dracorhodin Perchlorate Induced Human Breast Cancer MCF-7 Apoptosis through Mitochondrial Pathways

    PubMed Central

    Yu, Jing-hua; Zheng, Gui-bin; Liu, Chun-yu; Zhang, Li-ying; Gao, Hong-mei; Zhang, Ya-hong; Dai, Chun-yan; Huang, Lin; Meng, Xian-ying; Zhang, Wen-yan; Yu, Xiao-fang

    2013-01-01

    Objective: Dracorhodin perchlorate (DP) was a synthetic analogue of the antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could induce apoptosis in human prostate cancer, human gastric tumor cells and human melanoma, but the cytotoxic effect of DP on human breast cancer was not investigated. This study would investigate whether DP was a candidate chemical of anti-human breast cancer. Methods: The MTT assay reflected the number of viable cells through measuring the activity of cellular enzymes. Phase contrast microscopy visualized cell morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst 33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine 123 staining was used to detect cell apoptosis and mitochondrial membrane potential (MMP). Real time PCR detected mRNA level. Western blot examined protein expression. Results: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing factor (AIF) and cytochrome c which localized in mitochondrial in physiological condition were released into cytoplasm when MMP was decreased. DP activated caspase-9, which was the downstream of mitochondrial pathway. Therefore DP decreased MMP to release AIF and cytochrome c into cytoplasm, further activating caspase 9, lastly led to apoptosis. Conclusion: Therefore DP was a candidate for anti-breast cancer, DP induced apoptosis of MCF-7 through mitochondrial pathway. PMID:23869191

  8. Putting the pieces together: How is the mitochondrial pathway of apoptosis regulated in cancer and chemotherapy?

    PubMed Central

    2014-01-01

    In order to solve a jigsaw puzzle, one must first have the complete picture to logically connect the pieces. However, in cancer biology, we are still gaining an understanding of all the signaling pathways that promote tumorigenesis and how these pathways can be pharmacologically manipulated by conventional and targeted therapies. Despite not having complete knowledge of the mechanisms that cause cancer, the signaling networks responsible for cancer are becoming clearer, and this information is serving as a solid foundation for the development of rationally designed therapies. One goal of chemotherapy is to induce cancer cell death through the mitochondrial pathway of apoptosis. Within this review, we present the pathways that govern the cellular decision to undergo apoptosis as three distinct, yet connected puzzle pieces: (1) How do oncogene and tumor suppressor pathways regulate apoptosis upstream of mitochondria? (2) How does the B-cell lymphoma 2 (BCL-2) family influence tumorigenesis and chemotherapeutic responses? (3) How is post-mitochondrial outer membrane permeabilization (MOMP) regulation of cell death relevant in cancer? When these pieces are united, it is possible to appreciate how cancer signaling directly impacts upon the fundamental cellular mechanisms of apoptosis and potentially reveals novel pharmacological targets within these pathways that may enhance chemotherapeutic success. PMID:25621172

  9. Divergence of Erv1-Associated Mitochondrial Import and Export Pathways in Trypanosomes and Anaerobic Protists

    PubMed Central

    Basu, Somsuvro; Leonard, Joanne C.; Desai, Nishal; Mavridou, Despoina A. I.; Tang, Kong Ho; Goddard, Alan D.

    2013-01-01

    In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O2, unexpectedly find O2 and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O2 by TbERV1 is not dependent upon a simple O2 channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes. PMID:23264646

  10. Antiproliferative Activity of Cinnamomum cassia Constituents and Effects of Pifithrin-Alpha on Their Apoptotic Signaling Pathways in Hep G2 Cells

    PubMed Central

    Ng, Lean-Teik; Wu, Shu-Jing

    2011-01-01

    Cinnamaldehyde (Cin), cinnamic acid (Ca) and cinnamyl alcohol (Cal), major constituents of Cinnamomum cassia, have been shown to possess antioxidant, anti-inflammatory, anticancer and other activities. In this study, our aim was to evaluate the antiproliferative activity of these compounds in human hepatoma Hep G2 cells and examine the effects of pifithrin-alpha (PFTα; a specific p53 inhibitor) on their apoptotic signaling transduction mechanism. The antiproliferative activity was measured by XTT assay. Expression of apoptosis-related proteins was detected by western blotting. Results showed that at a concentration of 30 μM, the order of antiproliferative activity in Hep G2 cells was Cin > Ca > Cal. Cin (IC50 9.76 ± 0.67 μM) demonstrated an antiproliferative potency as good as 5-fluorouracil (an anti-cancer drug; IC50 9.57 ± 0.61 μM). Further studies on apoptotic mechanisms of Cin showed that it downregulated the expression of Bcl-XL, upregulated CD95 (APO-1), p53 and Bax proteins, as well as cleaving the poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. PFTα pre-incubation significantly diminished the effect of Cin-induced apoptosis. It markedly upregulated the anti-apoptotic (Bcl-XL) expression and downregulated the pro-apoptotic (Bax) expression, as well as effectively blocking the CD95 (APO-1) and p53 expression, and PARP cleavage in Cin-treated cells. This study indicates that Cin was the most potent antiproliferative constituent of C. cassia, and its apoptotic mechanism in Hep G2 cells could be mediated through the p53 induction and CD95 (APO-1) signaling pathways. PMID:20038571

  11. Yeast as a tool to study mitochondrial retrograde pathway en route to cell stress response.

    PubMed

    Ždralević, Maša; Guaragnella, Nicoletta; Giannattasio, Sergio

    2015-01-01

    Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, such as those encoding for peroxisomal citrate synthase, aconitase, and NAD(+)-specific isocitrate dehydrogenase subunit 1, as well as the phosphorylation status of Rtg1/3p transcriptional factor which controls RTG target gene transcription. PMID:25634284

  12. Oroxylin A modulates mitochondrial function and apoptosis in human colon cancer cells by inducing mitochondrial translocation of wild-type p53

    PubMed Central

    Zhou, Yuxin; Ni, Ting; Dai, Yuanyuan; Li, Zhiyu; Guo, Qinglong; Wei, Libin

    2016-01-01

    Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. We previously demonstrated that oroxylin A induced apoptosis in human colon cancer cells via the mitochondrial pathway. In the present study, we investigated the underlying mechanisms responsible for the mitochondrial apoptotic pathway triggered by oroxylin A. p53 regulates mitochondrial survival, mitochondrial DNA integrity, and protection from oxidative stress. We determined that oroxylin A induces p53 mitochondrial translocation and inhibits SOD2 activity. Additionally, our studies demonstrate that oroxylin A promotes the formation and mitochondrial translocation of the p53-Recql4 complex in HCT-116 cells. Finally, we showed that oroxylin A triggers cytosolic p53 activation, thereby promoting apoptosis. Mitochondrial translocation of p53 was also validated in vivo. Thus, oroxylin A induces mitochondrial translocation of p53 and leads to mitochondrial dysfunction in human colon cancer cells. PMID:26958937

  13. Oroxylin A modulates mitochondrial function and apoptosis in human colon cancer cells by inducing mitochondrial translocation of wild-type p53.

    PubMed

    Qiao, Chen; Lu, Na; Zhou, Yuxin; Ni, Ting; Dai, Yuanyuan; Li, Zhiyu; Guo, Qinglong; Wei, Libin

    2016-03-29

    Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. We previously demonstrated that oroxylin A induced apoptosis in human colon cancer cells via the mitochondrial pathway. In the present study, we investigated the underlying mechanisms responsible for the mitochondrial apoptotic pathway triggered by oroxylin A. p53 regulates mitochondrial survival, mitochondrial DNA integrity, and protection from oxidative stress. We determined that oroxylin A induces p53 mitochondrial translocation and inhibits SOD2 activity. Additionally, our studies demonstrate that oroxylin A promotes the formation and mitochondrial translocation of the p53-Recql4 complex in HCT-116 cells. Finally, we showed that oroxylin A triggers cytosolic p53 activation, thereby promoting apoptosis. Mitochondrial translocation of p53 was also validated in vivo. Thus, oroxylin A induces mitochondrial translocation of p53 and leads to mitochondrial dysfunction in human colon cancer cells. PMID:26958937

  14. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    SciTech Connect

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B. . E-mail: dbweiner@mail.med.upenn.edu

    2006-02-05

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

  15. A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

    PubMed Central

    Otera, Hidenori; Taira, Yohsuke; Horie, Chika; Suzuki, Yurina; Suzuki, Hiroyuki; Setoguchi, Kiyoko; Kato, Hiroki; Oka, Toshihiko; Mihara, Katsuyoshi

    2007-01-01

    The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs. PMID:18158327

  16. A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments.

    PubMed

    Otera, Hidenori; Taira, Yohsuke; Horie, Chika; Suzuki, Yurina; Suzuki, Hiroyuki; Setoguchi, Kiyoko; Kato, Hiroki; Oka, Toshihiko; Mihara, Katsuyoshi

    2007-12-31

    The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component-depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail-anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs. PMID:18158327

  17. Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

    SciTech Connect

    Meng, Xiangbao; Wang, Min; Sun, Guibo; Ye, Jingxue; Zhou, Yanhui; Dong, Xi; Wang, Tingting; Lu, Shan; Sun, Xiaobo

    2014-08-15

    Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio. Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or

  18. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane.

    PubMed

    Westphal, Dana; Dewson, Grant; Menard, Marie; Frederick, Paul; Iyer, Sweta; Bartolo, Ray; Gibson, Leonie; Czabotar, Peter E; Smith, Brian J; Adams, Jerry M; Kluck, Ruth M

    2014-09-30

    The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores. PMID:25228770

  19. Integrin α5β1 and p53 convergent pathways in the control of anti-apoptotic proteins PEA-15 and survivin in high-grade glioma.

    PubMed

    Renner, G; Janouskova, H; Noulet, F; Koenig, V; Guerin, E; Bär, S; Nuesch, J; Rechenmacher, F; Neubauer, S; Kessler, H; Blandin, A-F; Choulier, L; Etienne-Selloum, N; Lehmann, M; Lelong-Rebel, I; Martin, S; Dontenwill, M

    2016-04-01

    Integrin α5β1 expression is correlated with a worse prognosis in high-grade glioma. We previously unraveled a negative crosstalk between integrin α5β1 and p53 pathway, which was proposed to be part of the resistance of glioblastoma to chemotherapies. The restoration of p53 tumor-suppressor function is under intensive investigations for cancer therapy. However, p53-dependent apoptosis is not always achieved by p53-reactivating compounds such as Nutlin-3a, although full transcriptional activity of p53 could be obtained. Here we investigated whether integrin α5β1 functional inhibition or repression could sensitize glioma cells to Nutlin-3a-induced p53-dependent apoptosis. We discovered that α5β1 integrin-specific blocking antibodies or small RGD-like antagonists in association with Nutlin-3a triggered a caspase (Casp) 8/Casp 3-dependent strong apoptosis in glioma cells expressing a functional p53. We deciphered the molecular mechanisms involved and we showed the crucial role of two anti-apoptotic proteins, phosphoprotein enriched in astrocytes 15 (PEA-15) and survivin in glioma cell apoptotic outcome. PEA-15 is under α5β1 integrin/AKT (protein kinase B) control and survivin is a p53-repressed target. Moreover, interconnections between integrin and p53 pathways were revealed. Indeed PEA-15 repression by specific small-interfering RNA (siRNA)-activated p53 pathway to repress survivin and conversely survivin repression by specific siRNA decreased α5β1 integrin expression. This pro-apoptotic loop could be generalized to several glioma cell lines, whatever their p53 status, inasmuch PEA-15 and survivin protein levels were decreased. Our findings identify a novel mechanism whereby inhibition of α5β1 integrin and activation of p53 modulates two anti-apoptotic proteins crucially involved in the apoptotic answer of glioma cells. Importantly, our results suggest that high-grade glioma expressing high level of α5β1 integrin may benefit from associated therapies

  20. Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway.

    PubMed

    Altonsy, Mohammed O; Andrews, Simon C; Tuohy, Kieran M

    2010-02-28

    The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytochrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses. PMID:20036023

  1. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways.

    PubMed

    Xia, Tian; Kovochich, Michael; Liong, Monty; Zink, Jeffrey I; Nel, Andre E

    2008-01-01

    The exponential increase in the number of new nanomaterials that are being produced increases the likelihood of adverse biological effects in humans and the environment. In this study we compared the effects of cationic nanoparticles in five different cell lines that represent portal-of-entry or systemic cellular targets for engineered nanoparticles. Although 60 nm NH(2)-labeled polystyrene (PS) nanospheres were highly toxic in macrophage (RAW 264.7) and epithelial (BEAS-2B) cells, human microvascular endothelial (HMEC), hepatoma (HEPA-1), and pheochromocytoma (PC-12) cells were relatively resistant to particle injury. While the death pathway in RAW 264.7 cells involves caspase activation, the cytotoxic response in BEAS-2B cells is more necrotic in nature. Using fluorescent-labeled NH(2)-PS, we followed the routes of particle uptake. Confocal microscopy showed that the cationic particles entered a LAMP-1 positive lysosomal compartment in RAW 264.7 cells from where the particles could escape by lysosomal rupture. A proton pump inhibitor interfered in this pathway. Subsequent deposition of the particles in the cytosol induced an increase in mitochondrial Ca(2+) uptake and cell death that could be suppressed by cyclosporin A (CsA). In contrast, NH(2)-PS toxicity in BEAS-2B cells did not involve the LAMP-1 endosomal compartment, stimulation of proton pump activity, or an increase in mitochondrial Ca(2+). Particles were taken up by caveolae, and their toxicity could be disrupted by cholesterol extraction from the surface membrane. Although the particles induced mitochondrial damage and ATP depletion, CsA did not affect cytotoxicity. Cationic particles were taken up into HEPA-1, HMEC, and PC-12 cells, but this did not lead to lysosomal permeabilization, increased Ca(2+) flux, or mitochondrial damage. Taken together, the results of this study demonstrate the importance of cell-specific uptake mechanisms and pathways that could lead to sensitivity or resistance to cationic

  2. A role for transforming growth factor-{beta} apoptotic signaling pathway in liver injury induced by ingestion of water contaminated with high levels of Cr(VI)

    SciTech Connect

    Rafael, A.I.; Almeida, A.; Santos, P.; Parreira, I.; Madeira, V.M.S.; Alves, R.; Cabrita, A.M.S.; Alpoim, M.C.

    2007-10-15

    Hexavalent chromium [Cr(VI)] exposure is commonly associated with lung cancer. Although other adverse health effects have been reported, some authors, on assuming that orally ingested Cr(VI) is efficiently detoxified upon reduction by body fluids, believe that Cr(VI) do not target cells other than respiratory tract cells. In rodents, ingested Cr(VI)-contaminated water was reported to induce, in the liver, increases in TGF-{beta} transcripts. As TGF-{beta} dependent signaling pathways are closely associated with hepatic injury, the present study was undertaken addressing two specific issues: the effects of ingestion of water contaminated with high levels of Cr(VI) in rat liver structure and function; and the role of the TGF-{beta} pathway in Cr(VI)-induced liver injury. Examination of Wistar rats exposed to 20 ppm Cr(VI)-contaminated water for 10 weeks showed increased serum glucose and alanine aminotransferase (ALT) levels. Liver histological examination revealed hepatocellular apoptosis, further confirmed by immunohystochemical study of Caspase 3 expression. Liver gene expression analysis revealed increased expression of Smad2/Smad4 and Dapk, suggesting the involvement of the TGF-{beta} pathway in the apoptotic process. Since no changes in Smad3 expression were observed it appears apoptosis is using a Smad3-independent pathway. Increased expression of both Caspase 8 and Daxx genes suggests also the involvement of the Fas pathway. Gene expression analysis also revealed that a p160{sup ROCK}-Rho-independent pathway operates, leading to cell contraction and membrane blebbing, characteristic apoptotic features. These findings suggest that either the amount of Cr(VI) ingested overwhelmed the body fluids reductive capacity or some Cr(VI) escapes the reductive protection barrier, thus targeting the liver and inducing apoptosis.

  3. Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

    PubMed

    Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

    2015-01-01

    Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression

  4. Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression

    PubMed Central

    Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

    2015-01-01

    Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression

  5. Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

    PubMed

    Motz, Christian; Martin, Heiko; Krimmer, Thomas; Rassow, Joachim

    2002-11-01

    The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane. PMID:12419260

  6. Acetylcholinesterase (AChE) is an important link in the apoptotic pathway induced by hyperglycemia in Y79 retinoblastoma cell line.

    PubMed

    Masha'our, R Shehadeh; Heinrich, R; Garzozi, H J; Perlman, I

    2012-01-01

    Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM) shRNA for AChE was used to inhibit AChE protein expression. Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the mRNA and protein level. Inhibition of AChE protein expression by shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R isoform, is involved in the apoptotic pathway caused by hyperglycemia in Y79 cells. PMID:22685426

  7. Methyl jasmonate induces apoptosis and pro-apoptotic autophagy via the ROS pathway in human non-small cell lung cancer

    PubMed Central

    Zhang, Mutian; Su, Ling; Xiao, Zhenna; Liu, Xianfang; Liu, Xiangguo

    2016-01-01

    Methyl jasmonate (MJ) is a botanical hormone that serves as a signal transduction intermediate and regulates cell death in stressed plants. MJ induces cell cycle arrest, apoptosis and non-apoptotic cell death selectively in cancer cells. However, the underlying mechanism of MJ-induced apoptosis remains unclear. In this study, we examined the molecular mechanism through which MJ induces apoptosis in human non-small cell lung cancer (NSCLC). We found that MJ triggered apoptosis via the DDIT3-TNFRSF10B-CASP axis. MJ treatment significantly decreased the expression of CFLAR (CASP8 and FADD-like apoptosis regulator, an inhibitor of CASP8) in NSCLC cells, and ectopic expression of CFLAR partly protected cells from MJ-induced apoptosis. MJ also induced pro-apoptotic autophagy in NSCLC cells. Importantly, inhibition of ROS suppressed both MJ-induced apoptosis and autophagy. Taken together, MJ induces apoptosis and pro-apoptotic autophagy in NSCLC cells through the ROS pathway. Thus, MJ and its derivative treatment may serve as a novel chemotherapeutic strategy for cancer therapy.

  8. Enhanced Anticancer Activity of Gemcitabine in Combination with Noscapine via Antiangiogenic and Apoptotic Pathway against Non-Small Cell Lung Cancer

    PubMed Central

    Chougule, Mahavir B.; Patel, Apurva; Sachdeva, Pratik; Jackson, Tanise; Singh, Mandip

    2011-01-01

    Background The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos) and Gemcitabine (Gem) combination (NGC) against non-small cell lung cancer (NSCLC) and to elucidate the underlying mechanism of action. Methods Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues. Results The CI values (<0.59) were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups. Conclusion Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer. PMID:22102891

  9. Methyl jasmonate induces apoptosis and pro-apoptotic autophagy via the ROS pathway in human non-small cell lung cancer.

    PubMed

    Zhang, Mutian; Su, Ling; Xiao, Zhenna; Liu, Xianfang; Liu, Xiangguo

    2016-01-01

    Methyl jasmonate (MJ) is a botanical hormone that serves as a signal transduction intermediate and regulates cell death in stressed plants. MJ induces cell cycle arrest, apoptosis and non-apoptotic cell death selectively in cancer cells. However, the underlying mechanism of MJ-induced apoptosis remains unclear. In this study, we examined the molecular mechanism through which MJ induces apoptosis in human non-small cell lung cancer (NSCLC). We found that MJ triggered apoptosis via the DDIT3-TNFRSF10B-CASP axis. MJ treatment significantly decreased the expression of CFLAR (CASP8 and FADD-like apoptosis regulator, an inhibitor of CASP8) in NSCLC cells, and ectopic expression of CFLAR partly protected cells from MJ-induced apoptosis. MJ also induced pro-apoptotic autophagy in NSCLC cells. Importantly, inhibition of ROS suppressed both MJ-induced apoptosis and autophagy. Taken together, MJ induces apoptosis and pro-apoptotic autophagy in NSCLC cells through the ROS pathway. Thus, MJ and its derivative treatment may serve as a novel chemotherapeutic strategy for cancer therapy. PMID:27186395

  10. Noscapine Increases the Sensitivity of Drug-Resistant Ovarian Cancer Cell Line SKOV3/DDP to Cisplatin by Regulating Cell Cycle and Activating Apoptotic Pathways.

    PubMed

    Shen, Wei; Liang, Bingfeng; Yin, Jie; Li, Xiurong; Cheng, Jianxin

    2015-05-01

    Cisplatin is a first-line chemotherapy drug against ovarian cancer. However, its strong toxic side effects and the development of cisplatin resistance in human cancer cells seriously influence the effects of chemotherapy and quality of life in patients. Noscapine (Nos), a non-toxic benzylisoquinoline alkaloid extracted from opium, has been recently reported to have anti-cancer activity, but the mechanism of that effect has not been clearly established. In the present study, we investigated cytotoxicity of Nos in combination with cisplatin (DDP) in drug-resistant human ovarian cancer cell line SKOV3/DDP in vitro and in vivo null mice xenograft model. Cell proliferation was measured by MTT assay, flow cytometry was used to analyze cell cycle and apoptosis, protein expression of several apoptotic factors was investigated by flow cytometry and immunohistochemical method, and their mRNA expression levels were determined by real-time PCR. In vitro experiments showed that Nos significantly inhibited proliferation of SKOV3/DDP cells. DDP/Nos-combined treatment notably enhanced DDP-induced inhibition of cell proliferation and increased the pro-apoptotic effect of DDP in SKOV3/DDP cells. DDP/Nos administration increased the proportion of G2/M cells, reduced both protein and mRNA expression of anti-apoptotic factors XIAP, surviving and NF-kB, and augmented protein and mRNA levels of pro-apoptotic caspase-3. In vivo experiments revealed that Nos/DDP treatment increased the apoptotic rate of xenograft tumors in null mice. Tumor volume decreased from 1.733 ± 0.155 g in mice treated with DDP alone to 1.191 ± 0.106 g in animals treated with Nos/DDP. These observations suggest that Nos increases the anti-cancer activity of DDP against the drug-resistant ovarian cancer cell line SKOV3/DDP by modulating the cell cycle and activating apoptotic pathways. The study provides a new chemotherapy strategy for the treatment of DDP-resistant human ovarian cancer. PMID:25510462

  11. Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

    PubMed

    Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

    2014-02-01

    Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death. PMID:24316735

  12. Ethanolic extract of fermented Thunb induces human leukemic HL-60 and Molt-4 cell apoptosis via oxidative stress and a mitochondrial pathway.

    PubMed

    Banjerdpongchai, Ratana; Kongtawelert, Prachya

    2011-01-01

    Houttuynia cordata Thunb (HCT) is a medicinal plant of the Saururaceae family which features antimutagenic and antiviral properties. For extraction, the whole plants were fermented or non-fermented with yeast and ethanol then the whole plants were dried, ground and extracted with 95% ethanol or water. The aims of this study were to compare cytotoxic effects, apoptosis induction, and mechanism(s) with the ethanolic and water extracts of fermented and non-fermented HCT. Cytotoxicity was assessed using the MTT assay in human leukemic HL-60, Molt-4 and peripheral blood mononuclear cells (PBMCs). Apoptotic death was characterized by staining with propidium iodide and examined under a fluorescence microscope. Peroxide radical production and reduction of mitochondrial transmembrane potential (MTP) were determined using 2',7'-dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. The expression of caspase-9 was identified by immunoblotting. The ethanolic extract of fermented HCT was cytotoxic to HL-60 >Molt- 4 > PBMCs, to a greater extent than the non-fermented preparation and the number of apoptotic cells was higher. The alcoholic (fermented) extract produced more radicals than the non-fermented in HL-60 cells but the converse was observed in Molt-4 cells. Reduction of MTP was found in HL-60 and Molt-4 cells treated with the alcoholic (fermented) extract and caspase-9 was cleaved dose-dependently in both cells. In conclusion, the alcoholic extract of fermented HCT was more toxic to human leukemic cells than the non-fermented and both cell lines underwent apoptosis via oxidative stress and a mitochondrial pathway. PMID:22393956

  13. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

    PubMed Central

    Xiong, Xin-xin; Liu, Ju-mei; Qiu, Xin-yao; Pan, Feng; Yu, Shang-bin; Chen, Xiao-qian

    2015-01-01

    Aim: To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action. Methods: Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting. Results: PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity. Conclusion: Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways. PMID:25619389

  14. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    SciTech Connect

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  15. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

    NASA Astrophysics Data System (ADS)

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-11-01

    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

  16. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway.

    PubMed

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-11-01

    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5±1.2μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway. PMID:27294548

  17. Hematoporphyrin monomethyl ether combined with He–Ne laser irradiation-induced apoptosis in canine breast cancer cells through the mitochondrial pathway

    PubMed Central

    Li, Huatao; Tong, Jinjin; Bao, Jun; Tang, Damu; Tian, Wenru

    2016-01-01

    Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis that can be applied in vitro on canine breast cancer cells. However, the exact pathway responsible for HMME-PDT in canine breast cancer cells remains unknown. CHMm cells morphology and apoptosis were analyzed using optical microscope, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescein staining and DNA ladder assays. Apoptotic pathway was further confirmed by Real-time-polymerase chain reaction and Western blotting assays. Our results showed that HMME-PDT induced significant changes in cell morphology, such as formation of cytoplasmic vacuoles and the gradual rounding of cells coupled with decreased size and detachment. DNA fragmentation and cell death was shown to occur in a time-dependent manner. Furthermore, HMME-PDT increased the activities of caspase-9 and caspase-3, and released cytochrome c from mitochondria into the cytoplasm. HMME-PDT also significantly increased both mRNA and protein levels of Bax and decreased P53 gene expression in a time-dependent manner, while the mRNA and protein expression of Bcl-2 were repressed. These alterations suggest that HMME-PDT induced CHMm cell apoptosis via the mitochondrial apoptosis pathway and had anti-canine breast cancer effects in vitro. PMID:26645330

  18. Hematoporphyrin monomethyl ether combined with He-Ne laser irradiation-induced apoptosis in canine breast cancer cells through the mitochondrial pathway.

    PubMed

    Li, Huatao; Tong, Jinjin; Bao, Jun; Tang, Damu; Tian, Wenru; Liu, Yun

    2016-06-30

    Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis that can be applied in vitro on canine breast cancer cells. However, the exact pathway responsible for HMME-PDT in canine breast cancer cells remains unknown. CHMm cells morphology and apoptosis were analyzed using optical microscope, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescein staining and DNA ladder assays. Apoptotic pathway was further confirmed by Real-time-polymerase chain reaction and Western blotting assays. Our results showed that HMME-PDT induced significant changes in cell morphology, such as formation of cytoplasmic vacuoles and the gradual rounding of cells coupled with decreased size and detachment. DNA fragmentation and cell death was shown to occur in a time-dependent manner. Furthermore, HMME-PDT increased the activities of caspase-9 and caspase-3, and released cytochrome c from mitochondria into the cytoplasm. HMME-PDT also significantly increased both mRNA and protein levels of Bax and decreased P53 gene expression in a time-dependent manner, while the mRNA and protein expression of Bcl-2 were repressed. These alterations suggest that HMME-PDT induced CHMm cell apoptosis via the mitochondrial apoptosis pathway and had anti-canine breast cancer effects in vitro. PMID:26645330

  19. Cordyceps militaris induces tumor cell death via the caspase‑dependent mitochondrial pathway in HepG2 and MCF‑7 cells.

    PubMed

    Song, Jingjing; Wang, Yingwu; Teng, Meiyu; Zhang, Shiqiang; Yin, Mengya; Lu, Jiahui; Liu, Yan; Lee, Robert J; Wang, Di; Teng, Lesheng

    2016-06-01

    Cordyceps militaris (CM), an entomopathogenic fungus belonging to the class ascomycetes, possesses various pharmacological activities, including cytotoxic effects, on various types of human tumor cells. The present study investigated the anti‑hepatocellular carcinoma (HCC) and anti‑breast cancer effects of CM in in vitro and in vivo models. CM aqueous extract reduced cell viability, suppressed cell proliferation, inhibited cell migration ability, caused the over-release of lactate dehydrogenase, induced mitochondrial dysfunction and enhanced apoptotic rates in MCF‑7 and HepG2 cells. The expression levels of cleaved poly (ADP ribose) polymerase and caspase‑3, biomarkers of apoptosis, were increased following treatment with CM aqueous extract for 24 h. Furthermore, in the MCF‑7 and HepG2 cells, enhanced levels of B cell‑associated X protein and cleaved caspase‑8 were observed in the CM‑treated cells. Finally, the antitumor activities of CM in HCC and breast cancer were also confirmed in MCF‑7‑ and HepG2‑xengraft nude mice models. Collectively, the data obtained in the present study suggested that the cytotoxic effects of CM aqueous extract on HCC and breast cancer are associated with the caspase‑dependent mitochondrial pathway. PMID:27109250

  20. Cordyceps militaris induces tumor cell death via the caspase-dependent mitochondrial pathway in HepG2 and MCF-7 cells

    PubMed Central

    SONG, JINGJING; WANG, YINGWU; TENG, MEIYU; ZHANG, SHIQIANG; YIN, MENGYA; LU, JIAHUI; LIU, YAN; LEE, ROBERT J; WANG, DI; TENG, LESHENG

    2016-01-01

    Cordyceps militaris (CM), an entomopathogenic fungus belonging to the class ascomycetes, possesses various pharmacological activities, including cytotoxic effects, on various types of human tumor cells. The present study investigated the anti-hepatocellular carcinoma (HCC) and anti-breast cancer effects of CM in in vitro and in vivo models. CM aqueous extract reduced cell viability, suppressed cell proliferation, inhibited cell migration ability, caused the over-release of lactate dehydrogenase, induced mitochondrial dysfunction and enhanced apoptotic rates in MCF-7 and HepG2 cells. The expression levels of cleaved poly (ADP ribose) polymerase and caspase-3, biomarkers of apoptosis, were increased following treatment with CM aqueous extract for 24 h. Furthermore, in the MCF-7 and HepG2 cells, enhanced levels of B cell-associated X protein and cleaved caspase-8 were observed in the CM-treated cells. Finally, the antitumor activities of CM in HCC and breast cancer were also confirmed in MCF-7- and HepG2-xengraft nude mice models. Collectively, the data obtained in the present study suggested that the cytotoxic effects of CM aqueous extract on HCC and breast cancer are associated with the caspase-dependent mitochondrial pathway. PMID:27109250

  1. Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

    PubMed Central

    Dai, Zhi-Jun; Wang, Xi-Jing; Li, Zong-Fang; Ji, Zong-Zheng; Ren, Hong-Tao; Tang, Wei; Liu, Xiao-Xu; Kang, Hua-Feng; Guan, Hai-Tao; Song, Ling-Qin

    2008-01-01

    AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G1 phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3. PMID:19109865

  2. Apoptotic effects of extract from Cnidium monnieri (L.) Cusson by adenosine monosphosphate-activated protein kinase-independent pathway in HCT116 colon cancer cells.

    PubMed

    Lim, Eun Gyeong; Kim, Guen Tae; Lee, Se Hee; Kim, Sang-Yong; Kim, Young Min

    2016-06-01

    Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are well‑known as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphate‑activated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)‑Akt and control of B‑cell lymphoma 2 (Bcl‑2) family members. The pro‑apoptotic proteins Bcl‑2‑associated X protein (Bax) and Bcl‑2‑homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochrome C release and caspase activity. In the present study, the apoptotic effects and influence on mitochondria‑mediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of p‑Akt in an AMPK‑independent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of p‑Akt to control Bcl‑2 and activate Bax and Bak. Co‑treatment with CME and AMPK inhibitors showed that CME‑induced apoptosis does not occurr through a AMPK‑dependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the de‑phosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116

  3. Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells.

    PubMed

    Li, Yan-Cong; He, Shu-Ming; He, Zhi-Xu; Li, Minghua; Yang, Yinxue; Pang, Jian-Xin; Zhang, Xueji; Chow, Kevin; Zhou, Qingyu; Duan, Wei; Zhou, Zhi-Wei; Yang, Tianxin; Huang, Gui-Hua; Liu, Aibing; Qiu, Jia-Xuan; Liu, Jun-Ping; Zhou, Shu-Feng

    2014-03-28

    Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways. PMID:24280585

  4. Assessment of the Cytotoxic and Apoptotic Effects of Chaetominine in a Human Leukemia Cell Line

    PubMed Central

    Yao, Jingyun; Jiao, Ruihua; Liu, Changqing; Zhang, Yupeng; Yu, Wanguo; Lu, Yanhua; Tan, Renxiang

    2016-01-01

    Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an IC50 value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia. PMID:26902083

  5. Lycopene protects human SH‑SY5Y neuroblastoma cells against hydrogen peroxide‑induced death via inhibition of oxidative stress and mitochondria‑associated apoptotic pathways.

    PubMed

    Feng, Chunsheng; Luo, Tianfei; Zhang, Shuyan; Liu, Kai; Zhang, Yanhong; Luo, Yinan; Ge, Pengfei

    2016-05-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)‑induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH‑SY5Y neuroblastoma cells against H2O2‑induced death via inhibition of apoptosis resulting from activation of caspase‑3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over‑produced ROS, as well as the reduced activities of anti‑oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2‑induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl‑2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  6. Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

    PubMed Central

    FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

    2016-01-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  7. Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

    PubMed Central

    Kretschmer, Matthias; Wang, Joyce

    2012-01-01

    An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration. PMID:22707485

  8. Leptin exerts proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway.

    PubMed

    Wen, Rui; Hu, Shenqiang; Xiao, Qihai; Han, Chunchun; Gan, Chao; Gou, Hua; Liu, Hehe; Li, Liang; Xu, Hengyong; He, Hua; Wang, Jiwen

    2015-05-01

    Leptin was known as a pivotal regulator for the control of food intake and energy expenditure. However, leptin has also been found to be involved in the regulation of female reproductive system through interactions with pathways in the hypothalamic-hypophyseal axis and direct action at the ovarian level. In the present study, granulosa cells from goose ovarian preovulatory (F1-F3) follicles were cultured with leptin (0, 1, 10 or 100ng/ml). The proliferative and anti-apoptotic actions of leptin in granulosa cells were revealed by CCK-8, BrdU and TUNEL assays. Quantitative real-time PCR and Western blot analyses further indicated that leptin treatment led to increased expression of cyclin D1, cyclin D2, cyclin D3 and bcl-2, and decreased expression of p21 and caspase-3. The effects were involved in the activation of the PI3K/Akt/mTOR signaling pathway, as leptin treatment enhanced the expression of PI3K, Akt1, Akt2, Raptor, mTOR, S6K and p-S6K. Moreover, blockade of the PI3K/Akt/mTOR pathway attenuated the influences of leptin on proliferation and apoptosis of granulosa cells, considering that activated factors by leptin were inhibited in the presence of either 20μM LY294002 (a PI3K inhibitor) or 10μM rapamycin (an mTOR inhibitor). In addition, leptin had a modulatory effect on the expression of its receptor at the transcriptional and translational levels, and blockade of PI3K/Akt/mTOR inhibited both basal and leptin-induced Lepr gene and protein expression. These findings suggest that leptin exerts its proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway via interaction with its receptor. PMID:25576904

  9. Kaempferol protects cardiomyocytes against anoxia/reoxygenation injury via mitochondrial pathway mediated by SIRT1.

    PubMed

    Guo, Zhen; Liao, Zhangping; Huang, Liqing; Liu, Dan; Yin, Dong; He, Ming

    2015-08-15

    Mitochondria-mediated apoptosis is a critical mechanism of anoxia/ reoxygenation (A/R)-induced injury in cardiomyocytes. Kaempferol (Kae) is a natural polyphenol and a type of flavonoid, which has been demonstrated to protect myocardium against ischemia/reperfusion (I/R) injury. However, the mechanism is still not fully elucidated. We hypothesize that Kae may improve the mitochondrial function during I/R injury via a potential signal pathway. In this study, an in vitro I/R model was replicated on neonatal rat primary cardiomyocytes by A/R treatment. Cell viability was monitored by the 3-(4,5-dimethylthiazol- 2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. The levels of intracellular reactive oxygen species, mitochondrial membrane potential (Δψm) and apoptosis were determined by flow cytometry. Protein expression was detected by Western Blotting. mPTP opening and the activity of caspase-3 were measured by colorimetric method. The results showed that Kae effectively enhanced the cell viability and decreased the LDH release in cardiomyocytes subjected to A/R injury. Kae reduced the A/R-induced reactive oxygen species generation, the loss of Δψm, and the release of cytochrome c from mitochondria into cytosol. Kae inhibited the A/R-stimulated mPTP opening and activation of caspase-3, and ultimate decrease in cardiomyocytes apoptosis. Furthermore, we found Kae up-regulated Human Silent Information Regulator Type 1 (SIRT1) expression, indicating SIRT1 signal pathway likely involved the cardioprotection of Kae. Sirtinol, a SIRT1 inhibitor, abolished the protective effect of Kae in cardiomyocytes subjected to A/R. Additionally, Kae significantly increased the expression of Bcl-2. Thus, we firstly demonstrate that Kae protects cardiomyocytes against A/R injury through mitochondrial pathway mediated by SIRT1. PMID:26086862

  10. Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

    PubMed Central

    Gandhi, Vishal V.; Samuels, David C.

    2011-01-01

    Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides. PMID:21829339

  11. Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.

    PubMed

    Nichols, M; Zhang, J; Polster, B M; Elustondo, P A; Thirumaran, A; Pavlov, E V; Robertson, G S

    2015-11-12

    In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10μM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3μM) increased spare respiratory capacity but only E+Q (0.3μM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0μM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0μM), but not E (3.0μM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3μM), but not Q (0.1 and 0.3μM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0μM) blocked neuroprotection by E (0.3μM) or Q (1.0μM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury. PMID:26363153

  12. The BCL-2 family: key mediators of the apoptotic response to targeted anti-cancer therapeutics

    PubMed Central

    Hata, Aaron N.; Engelman, Jeffrey A.; Faber, Anthony C.

    2016-01-01

    The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The BCL-2 family proteins comprise the sentinel network that regulates the mitochondrial or intrinsic apoptotic response. Recent advances in our understanding of apoptotic signaling pathways have enabled methods to identify cancers that are “primed” to undergo apoptosis, and have revealed potential biomarkers that may predict which cancers will undergo apoptosis in response to specific therapies. Complementary efforts have focused on developing novel drugs that directly target anti-apoptotic BCL-2 family proteins. In this review, we summarize the current knowledge of the role of BCL-2 family members in cancer development and response to therapy, focusing on targeted therapeutics, recent progress in the development of apoptotic biomarkers, and therapeutic strategies designed to overcome deficiencies in apoptosis. PMID:25895919

  13. A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4

    PubMed Central

    Evstafieva, A G; Garaeva, A A; Khutornenko, A A; Klepikova, A V; Logacheva, M D; Penin, A A; Novakovsky, G E; Kovaleva, I E; Chumakov, P M

    2014-01-01

    Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13–17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency. PMID:25375376

  14. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    PubMed Central

    Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  15. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    PubMed

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  16. Redox Regulation of Mitochondrial Function

    PubMed Central

    Handy, Diane E.

    2012-01-01

    Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

  17. Panax notoginseng saponins attenuates cisplatin-induced nephrotoxicity via inhibiting the mitochondrial pathway of apoptosis.

    PubMed

    Liu, Xinwen; Huang, Zhenguang; Zou, Xiaoqin; Yang, Yufang; Qiu, Yue; Wen, Yan

    2014-01-01

    The goal of this experiment was to investigate the protective effect and the molecular mechanism of Panax Notoginseng Saponins (PNS) on cisplatin-induced nephrotoxicity through mitochondrial pathway of apoptosis. The rats underwent intraperitoneal injection with a single dose of cisplatin, a subset of rats were also intraperitoneally injected with 31.35 mg/kg PNS once a day for 8 days. At day 1, 4 and 8 after exposure to cisplatin, the concentrations of blood urea nitrogen (BUN), serum creatinine (Scr) and urinary N-acetyl-β-D-Glucosaminidase (NAG) were determined using commercial kits. The pathological change of renal tissue were examined using H & E staining and transmission electron microscopy. The rate of apoptosis and the expression of Bcl-2 in rat renal tissue were detected by using TUNEL staining and Western bloting, respectively. And the expressions of Bax and caspases 9 were detected by immunnohistochemistry. The results showed that PNS significantly protected against cisplatin-induced nephrotoxicity, as evidenced by the decrease in concentration of blood BUN, Scr and urinary NAG, as well as the attenuation of renal histopathological damage. Furthermore, PNS reduced the rate of apoptosis, and the mechanism studies showed that PNS inhibited the expression of Bax and caspase 9, while increased the expression of Bcl-2. This study first demonstrated that PNS can protect against cisplatin-induced nephrotoxicity and reduce renal tissue apoptosis via inhibiting the mitochondrial pathway. PMID:25674203

  18. Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability

    PubMed Central

    Ahmed, Naghia; Ronchi, Dario; Comi, Giacomo Pietro

    2015-01-01

    Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability. PMID:26251896

  19. Curcumin attenuates Mancozeb-induced toxicity in rat thymocytes through mitochondrial survival pathway.

    PubMed

    Pavlovic, Voja; Cekic, Snezana; Ciric, Milan; Krtinic, Dane; Jovanovic, Jelena

    2016-02-01

    The widely used fungicide Mancozeb (Man) has been shown to cause genotoxic effects in rodents and toxicological manifestations in different cells, mainly by altering the antioxidant defense in cells. On the other hand, curcumin (Cur), a natural phenolic compound, is thought to possess anti-inflammatory and antioxidant properties. Here, we investigated the possible protective role of Cur on Man-induced toxicity in rat thymocytes and potential mechanism involved. Rat thymocytes were treated with Man(0.01 μg/ml) and/or increasing Cur(0.3, 1, 3 μM) concentrations and levels of cell viability, apoptosis, mitochondrial membrane potential (MMP),Bcl-2, Bax protein expression, caspase-3 and -9 activity and p38 MAPK signaling involvement were examined. Cells treated with Man displayed increased cell toxicity, hypodiploid cells, caspase-3 and -9 activity, Bax protein expression, followed with decreased MMP and Bcl-2 protein expression. Inhibition of p38 MAPK signaling pathway markedly reduced apoptosis rate and caspase-3 activity in thymocytes exposed to Man. Application of increasing Cur (1, 3 μM) concentrations resulted with significantly reduced cytotoxicity, apoptosis, caspase-3, -9 activity, Bax protein expression, together with increased MMP and Bcl-2 protein expression in rat thymocytes. These result suggest that certain Cur concentrations may mediate Man-induced rat thymocytes toxicity through mitochondrial survival pathway, which may be useful in preventing possible secondary immunological consequences induced by Man. PMID:26763609

  20. Bruceine D induces apoptosis in human chronic myeloid leukemia K562 cells via mitochondrial pathway

    PubMed Central

    Zhang, Jian-Ye; Lin, Min-Ting; Tung, Ho-Yi; Tang, Si-Li; Yi, Tao; Zhang, Ya-Zhou; Tang, Yi-Na; Zhao, Zhong-Zhen; Chen, Hu-Biao

    2016-01-01

    Chronic myeloid leukemia (CML), an acquired malignant myeloproliferative disorder of hematopoietic stem cells, is one of the three most common forms of leukemia. In this study, we investigated the effects of bruceine D, which have been isolated from Brucea javanica (L.) Merr. on human chronic myeloid leukemia K562 cells. MTT assay was used to evaluate cell growth inhibition. Flow cytometry was performed to analyze mitochondrial membrane potential (ΔΨm). Western blot was applied to detect expression of cytochrome c, caspases-9, -3, PARP and other proteins. Bruceine D exhibited potent cytotoxicity to K562 cells with IC50 of 6.37 ± 0.39 μM. It led to loss of ΔΨm, release of cytochrome c, activation of caspases-9, -3 and cleavage of PARP, which suggested that bruceine D induced apoptosis of K562 cells through mitochondrial pathway. In addition, bruceine D inhibited the phosphorylation of AKT and ERK. It’s indicative that the potent anticancer activity of bruceine D be related to MAPK and PI3K pathways. PMID:27186433

  1. Shikonin induces apoptosis of HaCaT cells via the mitochondrial, Erk and Akt pathways

    PubMed Central

    JING, HUILING; SUN, WENYAN; FAN, JINGHUA; ZHANG, YANMIN; YANG, JIAO; JIA, JINJING; LI, JICHANG; GUO, JIAQI; LUO, SUJU; ZHENG, YAN

    2016-01-01

    Shikonin, which is a major ingredient of the traditional Chinese herb Lithospermum erythrorhizon, possesses various biological functions, including antimicrobial, anti-inflammatory, and antitumor activities. The present study aimed to determine the molecular mechanisms underlying the effects of shikonin on HaCaT cell apoptosis. Treatment with shikonin significantly inhibited the viability of HaCaT cells in a dose- and time-dependent manner, and promoted cell cycle arrest at G0/G1 phase and apoptosis. In addition, shikonin treatment reduced the mitochondrial membrane potential and induced reactive oxygen species generation. The results of a western blot analysis demonstrated that shikonin significantly activated caspase 3 expression, downregulated B-cell lymphoma 2 (Bcl-2) expression, and upregulated Bcl-2-associated X protein and Bcl-2 homologous antagonist killer expression in a dose-dependent manner in HaCaT cells. Furthermore, shikonin decreased extracellular signal-regulated kinase (Erk) and Akt phosphorylation. These results indicated that shikonin may exert its anti-proliferative effects by inducing apoptosis via activation of the mitochondrial signaling pathway and inactivation of the Akt and Erk pathways in HaCaT cells. Therefore, the present study suggested that shikonin may have potential as a component of therapeutic strategies for the treatment of skin diseases. PMID:26935874

  2. A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells.

    PubMed

    Chiruvella, Kishore K; Raghavan, Sathees C

    2011-08-01

    Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells. PMID:20169399

  3. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  4. Interleukin-24 mediates apoptosis in human B-cells through early activation of cell cycle arrest followed by late induction of the mitochondrial apoptosis pathway.

    PubMed

    Hadife, Nader; Nemos, Christophe; Frippiat, Jean-Pol; Hamadé, Tala; Perrot, Aurore; Dalloul, Ali

    2013-03-01

    Interleukin (IL)-24 has death-promoting effects on various proliferating cells including B-cells from chronic lymphocytic leukemia (CLL) and germinal center B-cells, but its molecular mechanisms are poorly understood. Using a B-cell differentiation model and mRNA profiling, we found that recombinant (r)IL-24 stimulated genes of the mitochondrial apoptotic pathway (Bax, Bid, Casp8, COX6C, COX7B) after 36 h, whereas the transcription of genes involved in DNA replication and metabolism was inhibited within 6 h. Unexpectedly, insulin-like growth factor 1 (IGF1), a hormone known to promote cell growth, was stimulated by IL-24. Activated B-cells express receptor for IGF1, to which they become sensitized and undergo apoptosis, a mechanism similar in this respect to IL-24-induced cell death. Furthermore, inhibition of the IGF1 pathway reversed the effects of IL-24. IL-24-mediated apoptosis was also antagonized by pifithrin-alpha, an inhibitor of p53 transactivation. Altogether, these results disclose sequential molecular signals generated by IL-24 in activated B-cells. PMID:22860893

  5. Investigation of anticancer mechanism of oleuropein via cell cycle and apoptotic pathways in SH-SY5Y neuroblastoma cells.

    PubMed

    Seçme, Mücahit; Eroğlu, Canan; Dodurga, Yavuz; Bağcı, Gülseren

    2016-07-01

    Neuroblastoma is one of the most common types of pediatric tumors that can spread quickly in neuronal tissues. Oleuropein which is active compound of olive leaves, belongs to polyphenols group and has antioxidant, anti-microbial, anti-inflammatory, anti-hypertensive and anti-carcinogenic effects. The aim of the study is to determine the therapeutic effects of oleuropein on cell proliferation, invasion, colony formation, cell cycle and apoptotic mechanisms in SH-SY5Y neuroblastoma cell line under in vitro conditions. The effect of oleuropein on cell viability was determined by XTT method. 84 cell cycle control and 84 apoptosis related genes were evaluated by RT-PCR. Effects of oleuropein on apoptosis were researched by TUNEL assay. Protein expressions were determined by western blot analysis. Effects of oleuropein on cell invasion, colony formation and migration were detected by matrigel-chamber, colony formation assay and wound-healing assay, respectively. IC50 value of oleuropein in SH-SY5Y cells was detected as 350μM at 48th hours. It is determined that oleuropein causes cell cycle arrest by down-regulating of CylinD1,CylinD2,CyclinD3,CDK4,CDK6 and up-regulating of p53 and CDKN2A, CDKN2B, CDKN1A gene expressions. Oleuropein also induces apoptosis by inhibiting of Bcl-2 and activating of Bax,caspase-9 and caspase-3 gene expressions. Apoptotic cell ratio was found 36.4±3.27% in oleuropein dose group. Oleuropein decreased invasion in SH-SY5Y cells and suppressed colony numbers in ratio of 53.6±4.71%.Our results demonstrated that oleuropein can be a therapeutic agent in the treatment of neuroblastoma. PMID:27032461

  6. Amelioration of nandrolone decanoate-induced testicular and sperm toxicity in rats by taurine: Effects on steroidogenesis, redox and inflammatory cascades, and intrinsic apoptotic pathway

    SciTech Connect

    Ahmed, Maha A.E.

    2015-02-01

    The wide abuse of the anabolic steroid nandrolone decanoate by athletes and adolescents for enhancement of sporting performance and physical appearance may be associated with testicular toxicity and infertility. On the other hand, taurine; a free β-amino acid with remarkable antioxidant activity, is used in taurine-enriched beverages to boost the muscular power of athletes. Therefore, the purpose of this study was to investigate the mechanisms of the possible protective effects of taurine on nandrolone decanoate-induced testicular and sperm toxicity in rats. To achieve this aim, male Wistar rats were randomly distributed into four groups and administered either vehicle, nandrolone decanoate (10 mg/kg/week, I.M.), taurine (100 mg/kg/day, p.o.) or combination of taurine and nandrolone decanoate, for 8 successive weeks. Results of the present study showed that taurine reversed nandrolone decanoate-induced perturbations in sperm characteristics, normalized serum testosterone level, and restored the activities of the key steroidogenic enzymes; 3β-HSD, and 17β-HSD. Moreover, taurine prevented nandrolone decanoate-induced testicular toxicity and DNA damage by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic effects. This was evidenced by taurine-induced modulation of testicular LDH-x activity, redox markers (MDA, NO, GSH contents, and SOD activity), inflammatory indices (TNF-α, ICAM-1 levels, and MMP-9 gene expression), intrinsic apoptotic pathway (cytochrome c gene expression and caspase-3 content), and oxidative DNA damage markers (8-OHdG level and comet assay). In conclusion, at the biochemical and histological levels, taurine attenuated nandrolone decanoate-induced poor sperm quality and testicular toxicity in rats. - Highlights: • Nandrolone decanoate (ND) disrupts sperm profile and steroidogenesis in rats. • ND upregulates gene expression of inflammatory and apoptotic markers. • Taurine normalizes sperm profile and serum testosterone level

  7. Association of Genes, Pathways, and Haplogroups of the Mitochondrial Genome with the Risk of Colorectal Cancer: The Multiethnic Cohort

    PubMed Central

    Li, Yuqing; Beckman, Kenneth B.; Caberto, Christian; Kazma, Remi; Lum-Jones, Annette; Haiman, Christopher A.; Marchand, Loïc Le; Stram, Daniel O.; Saxena, Richa; Cheng, Iona

    2015-01-01

    The mitochondrial genome encodes for the synthesis of 13 proteins that are essential for the oxidative phosphorylation (OXPHOS) system. Inherited variation in mitochondrial genes may influence cancer development through changes in mitochondrial proteins, altering the OXPHOS process, and promoting the production of reactive oxidative species. To investigate the role of the OXPHOS pathway and mitochondrial genes in colorectal cancer (CRC) risk, we tested 185 mitochondrial SNPs (mtSNPs), located in 13 genes that comprise four complexes of the OXPHOS pathway and mtSNP groupings for rRNA and tRNA, in 2,453 colorectal cancer cases and 11,930 controls from the Multiethnic Cohort Study. Using the sequence kernel association test, we examined the collective set of 185 mtSNPs, as well as subsets of mtSNPs grouped by mitochondrial pathways, complexes, and genes, adjusting for age, sex, principal components of global ancestry, and self-reported maternal race/ethnicity. We also tested for haplogroup associations using unconditional logistic regression, adjusting for the same covariates. Stratified analyses were conducted by self-reported maternal race/ethnicity. In European Americans, a global test of all genetic variants of the mitochondrial genome identified an association with CRC risk (P = 0.04). In mtSNP-subset analysis, the NADH dehydrogenase 2 (MT-ND2) gene in Complex I was associated with CRC risk at a P-value of 0.001 (q = 0.015). In addition, haplogroup T was associated with CRC risk (OR = 1.66, 95% CI: 1.19–2.33, P = 0.003). No significant mitochondrial pathway and gene associations were observed in the remaining four racial/ethnic groups—African Americans, Asian Americans, Latinos, and Native Hawaiians. In summary, our findings suggest that variations in the mitochondrial genome and particularly in the MT-ND2 gene may play a role in CRC risk among European Americans, but not in other maternal racial/ethnic groups. Further replication is warranted and future

  8. Association of Genes, Pathways, and Haplogroups of the Mitochondrial Genome with the Risk of Colorectal Cancer: The Multiethnic Cohort.

    PubMed

    Li, Yuqing; Beckman, Kenneth B; Caberto, Christian; Kazma, Remi; Lum-Jones, Annette; Haiman, Christopher A; Le Marchand, Loïc; Stram, Daniel O; Saxena, Richa; Cheng, Iona

    2015-01-01

    The mitochondrial genome encodes for the synthesis of 13 proteins that are essential for the oxidative phosphorylation (OXPHOS) system. Inherited variation in mitochondrial genes may influence cancer development through changes in mitochondrial proteins, altering the OXPHOS process, and promoting the production of reactive oxidative species. To investigate the role of the OXPHOS pathway and mitochondrial genes in colorectal cancer (CRC) risk, we tested 185 mitochondrial SNPs (mtSNPs), located in 13 genes that comprise four complexes of the OXPHOS pathway and mtSNP groupings for rRNA and tRNA, in 2,453 colorectal cancer cases and 11,930 controls from the Multiethnic Cohort Study. Using the sequence kernel association test, we examined the collective set of 185 mtSNPs, as well as subsets of mtSNPs grouped by mitochondrial pathways, complexes, and genes, adjusting for age, sex, principal components of global ancestry, and self-reported maternal race/ethnicity. We also tested for haplogroup associations using unconditional logistic regression, adjusting for the same covariates. Stratified analyses were conducted by self-reported maternal race/ethnicity. In European Americans, a global test of all genetic variants of the mitochondrial genome identified an association with CRC risk (P = 0.04). In mtSNP-subset analysis, the NADH dehydrogenase 2 (MT-ND2) gene in Complex I was associated with CRC risk at a P-value of 0.001 (q = 0.015). In addition, haplogroup T was associated with CRC risk (OR = 1.66, 95% CI: 1.19-2.33, P = 0.003). No significant mitochondrial pathway and gene associations were observed in the remaining four racial/ethnic groups--African Americans, Asian Americans, Latinos, and Native Hawaiians. In summary, our findings suggest that variations in the mitochondrial genome and particularly in the MT-ND2 gene may play a role in CRC risk among European Americans, but not in other maternal racial/ethnic groups. Further replication is warranted and future studies

  9. BCL-2 Antagonism to Target the Intrinsic Mitochondrial Pathway of Apoptosis.

    PubMed

    Gibson, Christopher J; Davids, Matthew S

    2015-11-15

    Despite significant improvements in treatment, cure rates for many cancers remain suboptimal. The rise of cytotoxic chemotherapy has led to curative therapy for a subset of cancers, though intrinsic treatment resistance is difficult to predict for individual patients. The recent wave of molecularly targeted therapies has focused on druggable-activating mutations, and is thus limited to specific subsets of patients. The lessons learned from these two disparate approaches suggest the need for therapies that borrow aspects of both, targeting biologic properties of cancer that are at once distinct from normal cells and yet common enough to make the drugs widely applicable across a range of cancer subtypes. The intrinsic mitochondrial pathway of apoptosis represents one such promising target for new therapies, and successfully targeting this pathway has the potential to alter the therapeutic landscape of therapy for a variety of cancers. Here, we discuss the biology of the intrinsic pathway of apoptosis, an assay known as BH3 profiling that can interrogate this pathway, early attempts to target BCL-2 clinically, and the recent promising results with the BCL-2 antagonist venetoclax (ABT-199) in clinical trials in hematologic malignancies. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy." PMID:26567361

  10. Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus

    PubMed Central

    English, Jane A.; Harauma, Akiko; Föcking, Melanie; Wynne, Kieran; Scaife, Caitriona; Cagney, Gerard; Moriguchi, Toru; Cotter, David R.

    2013-01-01

    Omega-3 fatty acid (n-3 FA) deficiency is an environmental risk factor for schizophrenia, yet characterization of the consequences of deficiency at the protein level in the brain is limited. We aimed to identify the protein pathways disrupted as a consequence of chronic n-3 deficiency in the hippocampus of mice. Fatty acid analysis of the hippocampus following chronic dietary deficiency revealed a 3-fold decrease (p < 0.001) in n-3 FA levels. Label free LC-MS/MS analysis identified and profiled 1008 proteins, of which 114 were observed to be differentially expressed between n-3 deficient and control groups (n = 8 per group). The cellular processes that were most implicated were neuritogenesis, endocytosis, and exocytosis, while specific protein pathways that were most significantly dysregulated were mitochondrial dysfunction and clathrin mediated endocytosis (CME). In order to characterize whether these processes and pathways are ones influenced by antipsychotic medication, we used LC-MS/MS to test the differential expression of these 114 proteins in the hippocampus of mice chronically treated with the antipsychotic agent haloperidol. We observed 23 of the 114 proteins to be differentially expressed, 17 of which were altered in the opposite direction to that observed following n-3 deficiency. Overall, our findings point to disturbed synaptic function, neuritogenesis, and mitochondrial function as a consequence of dietary deficiency in n-3 FA. This study greatly aids our understanding of the molecular mechanism by which n-3 deficiency impairs normal brain function, and provides clues as to how n-3 FA exert their therapeutic effect in early psychosis. PMID:24194745

  11. Targeting RING domains of Mdm2–MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells

    PubMed Central

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-01-01

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development. PMID:26720344

  12. RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing

    NASA Astrophysics Data System (ADS)

    Conde, João; Tian, Furong; Hernandez, Yulan; Bao, Chenchen; Baptista, Pedro V.; Cui, Daxiang; Stoeger, Tobias; de La Fuente, Jesus M.

    2015-05-01

    Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ~80% reduction in tumour size without associated inflammation.Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and

  13. Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells.

    PubMed

    Seth, Rohit; Corniola, Rikki S; Gower-Winter, Shannon D; Morgan, Thomas J; Bishop, Brian; Levenson, Cathy W

    2015-04-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency. PMID:25467851

  14. Protective Efficacy of Vitamins C and E on p,p′-DDT-Induced Cytotoxicity via the ROS-Mediated Mitochondrial Pathway and NF-κB/FasL Pathway

    PubMed Central

    Jin, Xiaoting; Song, Li; Liu, Xiangyuan; Chen, Meilan; Li, Zhuoyu; Cheng, Long; Ren, Hua

    2014-01-01

    Dichlorodiphenoxytrichloroethane (DDT) is a known persistent organic pollutant and liver damage toxicant. However, there has been little emphasis on the mechanism underlying liver damage toxicity of DDT and the relevant effective inhibitors. Hence, the present study was conducted to explore the protective effects of vitamin C (VC) and vitamin E (VE) on the cytotoxicity of DDT in HL-7702 cells and elaborate the specific molecular mechanisms. The results demonstrated that p,p′-DDT exposure at over 10 µM depleted cell viability of HL-7702 cells and led to cell apoptotic. p,p′-DDT treatment elevated the level of reactive oxygen species (ROS) generation, induced mitochondrial membrane potential, and released cytochrome c into the cytosol, with subsequent elevations of Bax and p53, along with suppression of Bcl-2. In addition, the activations of caspase-3 and -8 were triggered. Furthermore, p,p′-DDT promoted the expressions of NF-κB and FasL. When the cells were exposed to the NF-κB inhibitor (PDTC), the up-regulated expression of FasL was attenuated. Strikingly, these alterations caused by DDT treatment were prevented or reversed by the addition of VC or VE, and the protective effects of co-treatment with VC and VE were higher than the single supplement with p,p′-DDT. Taken together, these findings provide novel experimental evidences supporting that VC or/and VE could reduce p,p′-DDT-induced cytotoxicity of HL-7702 cells via the ROS-mediated mitochondrial pathway and NF-κB/FasL pathway. PMID:25464339

  15. Pim-1 Kinase Protects Mitochondrial Integrity in Cardiomyocytes

    PubMed Central

    Borillo, Gwynngelle A.; Mason, Matt; Quijada, Pearl; Völkers, Mirko; Cottage, Christopher; McGregor, Michael; Din, Shabana; Fischer, Kimberlee; Gude, Natalie; Avitable, Daniele; Barlow, Steven; Gustafsson, Asa B.; Glembotski, Christopher; Gottlieb, Roberta A.; Brown, Joan Heller; Sussman, Mark A.

    2010-01-01

    Rationale Cardioprotective signaling mediates anti-apoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details anti-apoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity. Objective The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes. Methods and Results A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 upon mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of anti-apoptotic Bcl-XL and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge Pim-1 preserves the inner mitochondrial membrane potential (ΔΨm). Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of pro-apoptotic Bid. Conclusion Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing pro-apoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway. PMID:20203306

  16. Assembly of the Bak Apoptotic Pore

    PubMed Central

    Ma, Stephen; Hockings, Colin; Anwari, Khatira; Kratina, Tobias; Fennell, Stephanie; Lazarou, Michael; Ryan, Michael T.; Kluck, Ruth M.; Dewson, Grant

    2013-01-01

    Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins. PMID:23893415

  17. Mesenchymal stem cells from rat bone marrow down regulate Caspase-3 mediated apoptotic pathway after spinal cord injury in rats

    PubMed Central

    Dasari, Venkata Ramesh; Spomar, Daniel G.; Cady, Craig; Gujrati, Meena; Rao, Jasti S.; Dinh, Dzung H.

    2007-01-01

    Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in caudal segments 2mm away from the lesion site. Expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly downregulated by rMSC. Caspase-3 downregulation by rMSC involves increased expression of FLIP and XIAP in the cytosol and inhibition of PARP cleavage in the nucleus. Animals treated with rMSC had higher BBB scores and better recovery of hind limb sensitivity. Treatment with rMSC had a positive effect on behavioral outcome and histopathological assessment after SCI. The ability of rMSC to incorporate into the spinal cord, differentiate and to improve locomotor recovery hold promise for a potential cure after SCI. PMID:17564836

  18. RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing.

    PubMed

    Conde, João; Tian, Furong; Hernandez, Yulan; Bao, Chenchen; Baptista, Pedro V; Cui, Daxiang; Stoeger, Tobias; de la Fuente, Jesus M

    2015-05-21

    Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ∼80% reduction in tumour size without associated inflammation. PMID:25924183

  19. The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells

    PubMed Central

    Xu, Xia; Cowley, Sally; Flaim, Christopher J; James, William; Seymour, Leonard; Cui, Zhanfeng

    2010-01-01

    Human embryonic stem (hES) cells have enormous potential for clinical applications. However, one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis, which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased, F-actin content and distribution is altered, and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However, exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 PMID:20077485

  20. Luffa echinata Roxb. Induced Apoptosis in Human Colon Cancer Cell (SW-480) in the Caspase-dependent Manner and Through a Mitochondrial Apoptosis Pathway

    PubMed Central

    Shang, Li-Hua; Yu, Yan; Che, De-Hai; Pan, Bo; Jin, Shi; Zou, Xiao-Long

    2016-01-01

    Background: Luffa echinata Roxb. (LER) (Cucurbitaceae) showed tremendous medicinal importance and are being used for the treatment of different ailments. Objective: In this study, the antiproliferative properties and cell death mechanism induced by the extract of the fruits of LER were investigated. Materials and Methods: MTT and LDH assay were used to test the antiproliferative and cytotoxicity of LER extract, respectively. The intracellular ROS were measured by a fluorometric assay. The expression of several apoptotic-related proteins in SW-480 cells treated by LER was evaluated by Western blot analysis. Results: The methanolic extract of LER fruits inhibited the proliferation of human colon cancer cells (SW-480) in both dose- and time-dependent manners. The LER-treated cells showed obvious characteristics of cell apoptosis, including cell shrinkage, destruction of the monolayer, and condensed chromatin. In addition, treatments of various concentrations of LER extracts caused the release of lactate dehydrogenase as a dose-dependent manner via stimulation of the intracellular metabolic system. LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL. Conclusions: These results indicated that treatment with LER-induced cell death in mitochondrial apoptosis pathway by regulating pro-apoptotic proteins via the up regulation of the p53 protein. These findings highlight the potentials of LER in the treatment of human colon cancer. SUMMARY LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL. PMID:27019558

  1. Flavonoids isolated from Citrus platymamma induce mitochondrial-dependent apoptosis in AGS cells by modulation of the PI3K/AKT and MAPK pathways.

    PubMed

    Lee, Ho Jeong; Nagappan, Arulkumar; Park, Hyeon Soo; Hong, Gyeong Eun; Yumnam, Silvia; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-09-01

    Citrus platymamma hort. ex Tanaka (Rutaceae family) has been widely used in Korean folk medicine for its wide range of medicinal benefits including an anticancer effect. In the present study, we aimed to investigate the molecular mechanism of the anticancer effects of flavonoids isolated from Citrus platymamma (FCP) on AGS cells. FCP treatment significantly inhibited AGS cell growth in a dose‑dependent manner. Furthermore, FCP significantly increased the percentage of cells in the sub-G1 phase (apoptotic cell population), and apoptosis was confirmed by Annexin V double staining. Chromatin condensation and apoptotic bodies were also noted in the FCP-treated AGS cells. Moreover, immunoblotting results showed that FCP treatment significantly decreased the expression of procaspase-3, -6, -8 and -9, and PARP and increased cleaved caspase-3, cleaved PARP and the Bax/Bcl-xL ratio in a dose-dependent manner. In addition, the phosphorylation of AKT was significantly decreased, whereas extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were significantly increased in the FCP-treated AGS cells. Taken together, the cell death of AGS cells in response to FCP was mitochondrial-dependent via modulation of the PI3K/AKT and MAPK pathways. These findings provide new insight for understanding the mechanism of the anticancer effects of FCP. Thus, FCP may be a potential chemotherapeutic agent for the treatment of gastric cancer. PMID:26165353

  2. Physalis peruviana extract induces apoptosis in human Hep G2 cells through CD95/CD95L system and the mitochondrial signaling transduction pathway.

    PubMed

    Wu, Shu-Jing; Ng, Lean-Teik; Lin, Doung-Liang; Huang, Shan-Ney; Wang, Shyh-Shyan; Lin, Chun-Ching

    2004-11-25

    Physalis species is a popular folk medicine used for treating cancer, leukemia, hepatitis and other diseases. Studies have shown that the ethanol extract of Physalis peruviana (EEPP) inhibits growth and induces apoptotic death of human Hep G2 cells in culture, whereas proliferation of the mouse BALB/C normal liver cells was not affected. In this study, we performed detailed studies to define the molecular mechanism of EEPP-induced apoptosis in Hep G2 cells. The results further confirmed that EEPP inhibited cell proliferation in a dose- and time-dependent manner. At 50 microg/ml, EEPP significantly increased the accumulation of the sub-G1 peak (hypoploid) and the portion of apoptotic annexin V positive cells. EEPP was found to trigger apoptosis through the release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria to cytosol and consequently resulted in caspase-3 activation. Pre-treatment with a general caspase inhibitor (z-VAD-fmk) prevented cytochrome c release. After 48 h of EEPP treatment, the apoptosis of Hep G2 cells was found to associate with an elevated p53, and CD95 and CD95L proteins expression. Furthermore, a marked down-regulation of the expression of the Bcl-2, Bcl-XL and XIAP, and up-regulation of the Bax and Bad proteins were noted. Taken together, the present results suggest that EEPP-induced Hep G2 cell apoptosis was possibly mediated through the CD95/CD95L system and the mitochondrial signaling transduction pathway. PMID:15488639

  3. Folate deficiency decreases apoptosis of endometrium decidual cells in pregnant mice via the mitochondrial pathway.

    PubMed

    Liao, Xing Gui; Li, Yan Li; Gao, Ru Fei; Geng, Yan Qing; Chen, Xue Mei; Liu, Xue Qing; Ding, Yu Bin; Mu, Xin Yi; Wang, Ying Xiong; He, Jun Lin

    2015-03-01

    It is well known that maternal folate deficiency results in adverse pregnancy outcomes. In addition to aspects in embryonic development, maternal uterine receptivity and the decidualization of stromal cells is also very important for a successful pregnancy. In this study, we focused on endometrium decidualization and investigated whether apoptosis, which is essential for decidualization, was impaired. Flow cytometry and TUNEL detection revealed that apoptosis of mouse endometrium decidual cells was suppressed in the dietary folate-deficient group on Days 7 and 8 of pregnancy (Day 1 = vaginal plug) when decidua regression is initiated. The endometrium decidual tissue of the folate deficiency group expressed less Bax compared to the normal diet group while they had nearly equal expression of Bcl2 protein. Further examination revealed that the mitochondrial transmembrane potential (ΔΨm) decreased, and the fluorescence of diffuse cytoplasmic cytochrome c protein was detected using laser confocal microscopy in normal decidual cells. However, no corresponding changes were observed in the folate-deficient group. Western blotting analyses confirmed that more cytochrome c was released from mitochondria in normal decidual cells. Taken together, these results demonstrated that folate deficiency could inhibit apoptosis of decidual cells via the mitochondrial apoptosis pathway, thereby restraining decidualization of the endometrium and further impairing pregnancy. PMID:25781218

  4. γ-Synuclein antibodies have neuroprotective potential on neuroretinal cells via proteins of the mitochondrial apoptosis pathway.

    PubMed

    Wilding, Corina; Bell, Katharina; Beck, Sabine; Funke, Sebastian; Pfeiffer, Norbert; Grus, Franz H

    2014-01-01

    The family of synuclein proteins (α, β and γ) are related to neurodegenerative disease e.g. Parkinson disease and Morbus Alzheimer. Additionally, a connection between γ-synuclein and glaucoma, a neurodegenerative disease characterized by a progressive loss of retinal ganglion cells, which finally leads to blindness, exists. The reason for the development of glaucoma is still unknown. Recent studies evaluating the participation of immunological components, demonstrate complex changed antibody reactivities in glaucoma patients in comparison to healthy people, showing not only up-regulations (e.g. alpha-fodrin antibody) but also down-regulations (e.g. γ-synuclein antibody) of antibodies in glaucoma patients. Up-regulated antibodies could be auto-aggressive, but the role of down-regulated antibodies is still unclear. Previous studies show a significant influence of the serum and the antibodies of glaucoma patients on protein expression profiles of neuroretinal cells. The aim of this study was to investigate the effect of γ-synuclein antibody on the viability and reactive oxygen species levels of a neuroretinal cell line (RGC-5) as well as their interaction with cellular proteins. We found a protective effect of γ-synuclein antibody resulting in an increased viability (up to 15%) and decreased reactive oxygen species levels (up to -12%) of glutamate and oxidative stressed RGC-5. These can be traced back to anti-apoptotic altered protein expressions in the mitochondrial apoptosis pathway indicated by mass spectrometry and validated by microarray analysis such as active caspase 3, bcl-2 associated-x-protein, S100A4, voltage-dependent anion channel, extracellular-signal-regulated-kinase (down-regulated) and baculoviral IAP repeat-containing protein 6, phosphorylated extracellular-signal-regulated-kinase (up-regulated). These changed protein expression are triggered by the γ-synuclein antibody internalization of RGC-5 we could see in immunohistochemical stainings

  5. Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway

    PubMed Central

    Abdelwahab, Siddig Ibrahim; Mohan, Syam; Mohamed Elhassan, Manal; Al-Mekhlafi, Nabil; Mariod, Abdelbasit Adam; Abdul, Ahmad Bustamam; Abdulla, Mahmood Ameen; Alkharfy, Khalid M.

    2011-01-01

    Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, β-carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for different extract/fractions were 126.2 ± 23, 31.25 ± 1.2, 15.25 ± 2.3, 13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 μg/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of β-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H2O2-induced apoptosis. The current results showed that phenolics may provide health and nutritional benefits. PMID:21234328

  6. Stimulation of apoptotic pathways in liver cancer cells: An alternative perspective on the biocompatibility and the utility of biomedical glasses.

    PubMed

    Kilcup, Nancy; Gaynard, Seán; Werner-Zwanziger, Ulrike; Tonkopi, Elena; Hayes, Jessica; Boyd, Daniel

    2016-05-01

    A host of research opportunities with innumerable clinical applications are open to biomedical glasses if one considers their potential as therapeutic inorganic ion delivery systems. Generally, applications have been limited to repair and regeneration of hard tissues while compositions are largely constrained to the original bioactive glass developed in the 1960s. However, in oncology applications the therapeutic paradigm shifts from repair to targeted destruction. With this in mind, the composition-structure-property-function relationships of vanadium-containing zinc-silicate glasses (0.51SiO2-0.29Na2O-(0.20-X)ZnO-XV2O5, 0 ≤ X ≤ 0.09) were characterized in order to determine their potential as therapeutic inorganic ion delivery systems. Increased V2O5mole fraction resulted in a linear decrease in density and glass transition temperature (Tg).(29)Si MAS NMR peak maxima shifted upfield while(51)V MAS NMR peak maxima were independent of V2O5content and overlapped well with the spectra NaVO3 Increased V2O5mole fraction caused ion release to increase. When human liver cancer cells, HepG2, were exposed to these ions they demonstrated a concentration-dependent cytotoxic response, mediated by apoptosis. This work demonstrates that the zinc-silicate system studied herein is capable of delivering therapeutic inorganic ions at concentrations that induce apoptotic cell death and provide a simple means to control therapeutic inorganic ion delivery. PMID:26675751

  7. Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV⁺ cells.

    PubMed

    Yuan, C-H; Filippova, M; Krstenansky, J L; Duerksen-Hughes, P J

    2016-01-01

    High-risk human papillomaviruses (HR-HPVs) cause nearly all cases of cervical cancer, as well as approximately 30% of head and neck cancers. HPV 16 E6, one of two major viral oncogenes, protects cells from apoptosis by binding to and accelerating the degradation of several proteins important in apoptotic signaling, including caspase 8 and p53. We proposed that blocking the interactions between HPV E6 and its partners using small molecules had the potential to re-sensitize HPV(+) cells to apoptosis. To test this idea, we screened libraries of small molecules for candidates that could block E6/caspase 8 binding and identified several candidates from different chemical classes. We tested hits for dose-dependency and specificity in vitro and for toxicity in a cell-based assay and then used this information to select the two best candidates for further testing: myricetin, a flavonol, and spinacine, an imidazole amino-acid derivative of histidine. Both compounds clearly inhibited the ability of E6 to bind in vitro to both caspase 8 and E6AP, the protein that mediates p53 degradation. In addition, both compounds were able to increase the level of caspase 8 and p53 in SiHa cervical cancer cells, resulting in an increase of caspase 3/7 activity. Finally, both myricetin and spinacine sensitized HPV(+) cervical and oral cancer cells, but not HPV(-) cervical and oral cancer cells, to apoptosis induced by the cancer-specific ligand TRAIL, as well as the chemotherapeutic agents doxorubicin and cisplatin. New therapies based on this work may improve treatment for HPV(+) cancer patients. PMID:26794656

  8. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    SciTech Connect

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-15

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the {beta}{sub 1} integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes.

  9. Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I

    PubMed Central

    Imaizumi, Naoki; Kwang Lee, Kang; Zhang, Carmen; Boelsterli, Urs A.

    2015-01-01

    Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD+ ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30 µM EFV and submaximal effects at 50 µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD+ ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40 µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical. PMID:25625582

  10. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis.

    PubMed

    Jourdain, Alexis; Martinou, Jean-Claude

    2009-10-01

    Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP. PMID:19439192

  11. Apoptotic Effects of Cordycepin Through the Extrinsic Pathway and p38 MAPK Activation in Human Glioblastoma U87MG Cells.

    PubMed

    Baik, Ji-Sue; Mun, Seo-Won; Kim, Kyoung-Sook; Park, Shin-Ji; Yoon, Hyun-Kyoung; Kim, Dong-Hyun; Park, Min-Kyu; Kim, Cheorl-Ho; Lee, Young-Choon

    2016-02-28

    We first demonstrated that cordycepin inhibited cell growth and triggered apoptosis in U87MG cells with wild-type p53, but not in T98G cells with mutant-type p53. Western blot data revealed that the levels of procaspase-8, -3, and Bcl-2 were downregulated in cordycepintreated U87MG cells, whereas the levels of Fas, FasL, Bak, cleaved caspase-3, -8, and cleaved PARP were upregulated, indicating that cordycepin induces apoptosis by activating the death receptor-mediated pathway in U87MG cells. Cordycepin-induced apoptosis could be suppressed by only SB203580, a p38 MAPK-specific inhibitor. These results suggest that cordycepin triggered apoptosis in U87MG cells through p38 MAPK activation and inhibition of the Akt survival pathway. PMID:26597532

  12. Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

    PubMed Central

    Lee, Moo-Seung; Kwon, Haenaem; Lee, Eun-Young; Kim, Dong-Jae; Park, Jong-Hwan; Tesh, Vernon L.; Oh, Tae-Kwang

    2015-01-01

    Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death. PMID:26502906

  13. Ubiquitination of the Transcription Factor IRF-3 Activates RIPA, the Apoptotic Pathway that Protects Mice from Viral Pathogenesis.

    PubMed

    Chattopadhyay, Saurabh; Kuzmanovic, Teodora; Zhang, Ying; Wetzel, Jaime L; Sen, Ganes C

    2016-05-17

    The transcription factor IRF-3 mediates cellular antiviral response by inducing the expression of interferon and other antiviral proteins. In RNA-virus infected cells, IRF-3's transcriptional activation is triggered primarily by RIG-I-like receptors (RLR), which can also activate the RLR-induced IRF-3-mediated pathway of apoptosis (RIPA). Here, we have reported that the pathway of IRF-3 activation in RIPA was independent of and distinct from the known pathway of transcriptional activation of IRF-3. It required linear polyubiquitination of two specific lysine residues of IRF-3 by LUBAC, the linear polyubiquitinating enzyme complex, which bound IRF-3 in signal-dependent fashion. To evaluate the role of RIPA in viral pathogenesis, we engineered a genetically targeted mouse, which expressed a mutant IRF-3 that was RIPA-competent but transcriptionally inert; this single-action IRF-3 could protect mice from lethal viral infection. Our observations indicated that IRF-3-mediated apoptosis of virus-infected cells could be an effective antiviral mechanism, without expression of the interferon-stimulated genes. PMID:27178468

  14. Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress.

    PubMed

    Baker, Simon C; Shabir, Saqib; Georgopoulos, Nikolaos T; Southgate, Jennifer

    2016-05-01

    Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca(2+) concentration that was unresolved. The sustained elevation in cytosolic Ca(2+) concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway. PMID:27001627

  15. Punica granatum (pomegranate) leaves extract induces apoptosis through mitochondrial intrinsic pathway and inhibits migration and invasion in non-small cell lung cancer in vitro.

    PubMed

    Li, Yali; Yang, Fangfang; Zheng, Weidong; Hu, Mingxing; Wang, Juanxiu; Ma, Sisi; Deng, Yuanle; Luo, Yi; Ye, Tinghong; Yin, Wenya

    2016-05-01

    Most conventional treatments on non-small cell lung carcinoma always accompany with awful side effects, and the incidence and mortality rates of this cancer are increasing rapidly worldwide. The objective of this study was to examine the anticancer effects of extract of Punica granatum (pomegranate) leaves extract (PLE) on the non-small cell lung carcinoma cell line A549, H1299 and mouse Lewis lung carcinoma cell line LL/2 in vitro, and explore its mechanisms of action. Our results have shown that PLE inhibited cell proliferation in non-small cell lung carcinoma cell line in a concentration- and time-dependent manner. Flow cytometry (FCM) assay showed that PLE affected H1299 cell survival by arresting cell cycle progression in G2/M phase in a dose-dependent manner and inducing apoptosis. Moreover, PLE could also decrease the reactive oxygen species (ROS) and the mitochondrial membrane potential (ΔYm), indicating that PLE may induce apoptosis via mitochondria-mediated apoptotic pathway. Furthermore, PLE blocked H1299 cell migration and invasion, and the reduction of matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression were also observed in vitro. These results suggested that PLE could be an effective and safe chemotherapeutic agent in non-small cell lung carcinoma treatment by inhibiting proliferation, inducing apoptosis, cell cycle arrest and impairing cell migration and invasion. PMID:27133061

  16. Swainsonine Induces Apoptosis through Mitochondrial Pathway and Caspase Activation in Goat Trophoblasts

    PubMed Central

    Huang, Yong; Dong, Feng; Du, Qian; Zhang, Hongling; Luo, Xiaomao; Song, Xiangjun; Zhao, Xiaomin; Zhang, Wenlong; Tong, Dewen

    2014-01-01

    The indolizidine alkaloid swainsonine (SW) has been reported to impair placentae and ultimately cause abortion in pregnant goats. Up to now, however, the precise effects of SW on goat trophoblast cells (GTCs) are still unclear. In this study, the cytotoxicity effects of SW on GTCs were detected and evaluated by MTT assay, AO/EB double staining, DNA fragmentation assay and flow cytometry analysis. Results showed that SW treatment significantly suppressed GTCs viability and induced typical apoptotic features in a time- and concentration-dependent manner. SW treatment increased Bax protein levels, reduced Bcl-2 protein levels, induced Bax translocation to mitochondria, and triggered the release of cytochrome c from mitochondria into cytosol, which in turn activated caspase-9 and caspase-3, and cleaved PARP, resulting in GTCs apoptosis. However, caspase-8 activity and the level of Bid did not exhibit significant changes in the process of SW-induced apoptosis. In addition, TUNEL assay suggested that SW induced GTCs apoptosis but not other cells in goat placenta cotyledons. Taken together, these data suggest that SW selectively induces GTCs apoptosis via the activation of mitochondria-mediated apoptosis pathway in goat placenta cotyledons, which might contribute to placentae impairment and abortion in pregnant goats fed with SW-containing plants. These findings may provide new insights to understand the mechanisms involved in SW-caused goat's abortion. PMID:25076855

  17. Carvedilol promotes mitochondrial biogenesis by regulating the PGC-1/TFAM pathway in human umbilical vein endothelial cells (HUVECs).

    PubMed

    Yao, Kai; Zhang, Wayne W; Yao, Luyu; Yang, Shu; Nie, Wanpin; Huang, Feizhou

    2016-02-19

    Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics. It has been shown that Carvedilol protects cells against mitochondrial dysfunction. However, it's unknown whether Carvedilol affects mitochondrial biogenesis. In this study, we found that treatment with Carvedilol in HUVECs resulted in a significant increase of PGC-1α, NRF1, and TFAM. Notably, Carvedilol significantly increased mtDNA contents and the two mitochondrial proteins, cytochrome C and COX IV. In addition, MitoTracker Red staining results indicated that treatment with Carvedilol increased mitochondria mass. Mechanistically, we found that the effect of Carvedilol on the expression of PGC-1α is mediated by the PKA-CREB pathway. Importantly, our results revealed that stimulation of mitochondrial biogenesis by carvedilol resulted in functional gain of the mitochondria by showing increased oxygen consumption and mitochondrial respiratory rate. The increased expression of PGC-1α and mitochondrial biogenesis induced by Carvedilol might suggest a new mechanism of the therapeutic effects of Carvedilol in heart failure. PMID:26797282

  18. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells

    PubMed Central

    Arbon, Kate S.; Christensen, Cody M.; Harvey, Wendy A.; Heggland, Sara J.

    2012-01-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10 μM CdCl2 for 2–72 hours. We detected significant ERK activation in response to CdCl2 and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl2 and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl2 exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl2. Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity. PMID:22019892

  19. The mitochondrial unfolded protein response, a conserved stress response pathway with implications in health and disease

    PubMed Central

    Jovaisaite, Virginija; Mouchiroud, Laurent; Auwerx, Johan

    2014-01-01

    The ability to respond to various intracellular and/or extracellular stresses allows the organism to adapt to changing environmental conditions and drives evolution. It is now well accepted that a progressive decline of the efficiency of stress response pathways occurs with aging. In this context, a correct proteostasis is essential for the functionality of the cell, and its dysfunction has been associated with protein aggregation and age-related degenerative diseases. Complex response mechanisms have evolved to deal with unfolded protein stress in different subcellular compartments and their moderate activation translates into positive effects on health. In this review, we focus on the mitochondrial unfolded protein response (UPRmt), a response to proteotoxic stress specifically in mitochondria, an organelle with a wide array of fundamental functions, most notably the harvesting of energy from food and the control of cell death. We compare UPRmt with the extensively characterized cytosolic heat shock response (HSR) and the unfolded protein response in endoplasmic reticulum (UPRER), and discuss the current knowledge about UPRmt signaling pathways as well as their potential involvement in physiology. PMID:24353213

  20. High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer

    PubMed Central

    Fitzgerald, Seán; Sheehan, Katherine M; Espina, Virginia; O'Grady, Anthony; Cummins, Robert; Kenny, Dermot; Liotta, Lance; O'Kennedy, Richard; Kay, Elaine W

    2014-01-01

    Abstract Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico‐pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin‐fixed and paraffin‐embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5‐year overall survival (p = 0.001) and 5‐year recurrence‐free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection‐enriched carcinoma cells from 19 fresh‐frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer‐related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub‐network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing

  1. Systems Modeling of Anti-apoptotic Pathways in Prostate Cancer: Psychological Stress Triggers a Synergism Pattern Switch in Drug Combination Therapy

    PubMed Central

    Sun, Xiaoqiang; Bao, Jiguang; Nelson, Kyle C.; Li, King Chuen; Kulik, George; Zhou, Xiaobo

    2013-01-01

    Prostate cancer patients often have increased levels of psychological stress or anxiety, but the molecular mechanisms underlying the interaction between psychological stress and prostate cancer as well as therapy resistance have been rarely studied and remain poorly understood. Recent reports show that stress inhibits apoptosis in prostate cancer cells via epinephrine/beta2 adrenergic receptor/PKA/BAD pathway. In this study, we used experimental data on the signaling pathways that control BAD phosphorylation to build a dynamic network model of apoptosis regulation in prostate cancer cells. We then compared the predictive power of two different models with or without the role of Mcl-1, which justified the role of Mcl-1 stabilization in anti-apoptotic effects of emotional stress. Based on the selected model, we examined and quantitatively evaluated the induction of apoptosis by drug combination therapies. We predicted that the combination of PI3K inhibitor LY294002 and inhibition of BAD phosphorylation at S112 would produce the best synergistic effect among 8 interventions examined. Experimental validation confirmed the effectiveness of our predictive model. Moreover, we found that epinephrine signaling changes the synergism pattern and decreases efficacy of combination therapy. The molecular mechanisms responsible for therapeutic resistance and the switch in synergism were explored by analyzing a network model of signaling pathways affected by psychological stress. These results provide insights into the mechanisms of psychological stress signaling in therapy-resistant cancer, and indicate the potential benefit of reducing psychological stress in designing more effective therapies for prostate cancer patients. PMID:24339759

  2. IMMUNE TOLERANCE INDUCTION BY APOPTOTIC CELLS REQUIRES CASPASE-DEPENDENT OXIDATION OF HMGB1

    PubMed Central

    Kazama, Hirotaka; Ricci, Jean-Ehrland; Herndon, John M.; Hoppe, George; Green, Douglas R.; Ferguson, Thomas A.

    2009-01-01

    SUMMARY The mammalian immune system discriminates between modes of cell death, with necrosis often resulting in inflammation and adaptive immunity, while apoptosis tends to be anti-inflammatory, promoting immune tolerance. In many systems immune tolerance can be established through cross presentation of antigens derived from apoptotic cells via the MHC class I pathway to CD8+ T cells. We have examined the features of apoptosis responsible for tolerance to cell-mediated immune responses in vivo, specifically the roles of caspases and the mitochondria. Our results show that caspase activation targets the mitochondria to produce ROS, which are critical to tolerance induction by apoptotic cells. ROS oxidizes the potential danger signal HMGB1 released from dying cells, thereby neutralizing its stimulatory activity and promoting tolerance. Apoptotic cells failed to induce tolerance and instead stimulated immune responses when caspase-dependent ROS activity was prohibited by scavenging or by mutation of a mitochondrial caspase target, p75 NDUSF1. Similarly blocking sites of oxidation in HMGB1 prevented tolerance induction by apoptotic cells. These results suggest that caspase orchestrated mitochondrial events determine the impact of apoptotic cells on the immune response. PMID:18631454

  3. Goniothalamin induces cell cycle arrest and apoptosis in H400 human oral squamous cell carcinoma: A caspase-dependent mitochondrial-mediated pathway with downregulation of NF-κβ.

    PubMed

    Li, Lim K; Rola, Ali-Saeed; Kaid, Fahme A; Ali, Abdul Manaf; Alabsi, Aied M

    2016-04-01

    Goniothalamin is a natural occurring styryl-lactone compound isolated from Goniothalamus macrophyllus. It had been demonstrated to process promising anticancer activity on various cancer cell lines. However, little study has been carried out on oral cancer. The aim of this study was to determine the cytotoxic effects of goniothalamin against H400 oral cancer cells and its underlying molecular pathways. Results from MTT assay demonstrated that goniothalamin exhibited selective cytotoxicity as well as inhibited cells growth of H400 in dose and time-dependent manner. This was achieved primarily via apoptosis where apoptotic bodies and membrane blebbing were observed using AO/PI and DAPI/Annexin V-FITC fluorescence double staining. In order to understand the apoptosis mechanisms induced by goniothalamin, apoptosis assessment based on mitochondrial membrane potential assay and cytochrome c enzyme-linked immunosorbent assay were carried out. Results demonstrated that the depolarization of mitochondrial transmembrane potential facilitated the release of mitochondrial cytochrome c into cytosol. Caspases assays revealed the activation of initiator caspase-9 and executioner caspase-3/7 in dose-dependent manners. This form of apoptosis was closely associated with the regulation on Bcl-2 family proteins, cell cycle arrest at S phase and inhibition of NF-κβ translocation from cytoplasm to nucleus. Conclusion, goniothalamin has the potential to act as an anticancer agent against human oral squamous cell carcinoma (H400 cells). PMID:26752226

  4. The involvement of the mitochondrial pathway in manganese-induced apoptosis of chicken splenic lymphocytes.

    PubMed

    Zhu, Yihao; Li, Shu; Teng, Xiaohua

    2016-06-01

    The purpose of this study was to investigate the effect of excess manganese (Mn)-induced cytotoxicity on apoptosis in chicken splenic lymphocytes. Chicken splenic lymphocytes were cultured in medium in the absence and presence of manganese (II) chloride (MnCl2) (2 × 10(-4), 4 × 10(-4), 6 × 10(-4), 8 × 10(-4), 10 × 10(-4), and 12 × 10(-4) mM), in N-acetyl-l-cysteine (NAC) (1 mM), and the combination of MnCl2 and NAC for 12, 24, 36, and 48 h. Tests were performed on morphologic observation, reactive oxygen species (ROS) and malondialdehyde (MDA) content, manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase (GSH-Px) activities, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), p53, and calmodulin (CaM) messenger RNA (mRNA) expression, Caspase-3 mRNA and protein expression, intracellular free Ca(2+) ([Ca(2+)]i), and mitochondrial transmembrane potential (ΔΨm). Our research indicated that excess Mn induced ROS and MDA content, inhibited Mn-SOD and GSH-Px activities, induced Bax and p53 mRNA expression, inhibited Bcl-2 and CaM mRNA expression, induced Caspase-3 mRNA and protein expression, upregulated [Ca(2+)]i, inhibited ΔΨm, and induced apoptosis in a dose effect. NAC relieved excess Mn-caused the changes of all above factors. Mn-induced oxidative injuries were alleviated by treatment with NAC, an ROS scavenger. The above results demonstrated that excess Mn caused oxidative stress and apoptosis via mitochondrial pathway in chicken splenic lymphocytes. PMID:27035383

  5. Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways

    PubMed Central

    Weiss, Martin; Gümbel, Denis; Hanschmann, Eva-Maria; Mandelkow, Robert; Gelbrich, Nadine; Zimmermann, Uwe; Walther, Reinhard; Ekkernkamp, Axel; Sckell, Axel; Kramer, Axel; Burchardt, Martin; Lillig, Christopher H.; Stope, Matthias B.

    2015-01-01

    One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment. PMID:26132846

  6. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

    PubMed

    Tao, Weikang; South, Victoria J; Diehl, Ronald E; Davide, Joseph P; Sepp-Lorenzino, Laura; Fraley, Mark E; Arrington, Kenneth L; Lobell, Robert B

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors. PMID:17101792

  7. ESCRT-0 dysfunction compromises autophagic degradation of protein aggregates and facilitates ER stress-mediated neurodegeneration via apoptotic and necroptotic pathways

    PubMed Central

    Oshima, Ryuji; Hasegawa, Takafumi; Tamai, Keiichi; Sugeno, Naoto; Yoshida, Shun; Kobayashi, Junpei; Kikuchi, Akio; Baba, Toru; Futatsugi, Akira; Sato, Ikuro; Satoh, Kennichi; Takeda, Atsushi; Aoki, Masashi; Tanaka, Nobuyuki

    2016-01-01

    Endosomal sorting required for transport (ESCRT) complexes orchestrate endo-lysosomal sorting of ubiquitinated proteins, multivesicular body formation and autophagic degradation. Defects in the ESCRT pathway have been implicated in many neurodegenerative diseases, but the underlying molecular mechanisms that link them to neurodegeneration remain unknown. In this study, we showed that forebrain-specific ablation of ESCRT-0/Hrs induced marked hippocampal neuronal cell loss accompanied by the accumulation of ubiquitinated proteins, including α-synuclein, TDP-43 and huntingtin as well as the autophagic substrate SQSTM1/p62. Consistent with this, silencing of Hrs in cultured cells not only led to α-synuclein and TDP-43 accumulation in addition to impaired autophagic flux but also suppressed cell viability through the induction of ER stress followed by the activation of JNK and RIPK1, a key regulator of necroptosis. Moreover, necrostatin-1, a specific inhibitor of RIPK1, and pan-caspase inhibitors partially reduced the neurotoxicity in the Hrs-silenced cells. Altogether, these findings suggest that the disruption of ESCRT-0/Hrs in the nervous system compromises autophagic/lysosomal degradation of neurodegenerative disease-related proteins, which thereby triggers ER stress-mediated apoptotic and necroptotic cell death. PMID:27112194

  8. Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    PubMed Central

    Korashy, Hesham M.; Maayah, Zaid H.; Abd-Allah, Adel R.; El-Kadi, Ayman O. S.; Alhaider, Abdulqader A.

    2012-01-01

    Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2) and human breast (MCF7) cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways. PMID:22654482

  9. The Apoptotic Effect of Ursolic Acid on SK-Hep-1 Cells is Regulated by the PI3K/Akt, p38 and JNK MAPK Signaling Pathways.

    PubMed

    Chuang, Wan-Ling; Lin, Ping-Yi; Lin, Hui-Chuan; Chen, Yao-Li

    2016-01-01

    Ursolic acid (UA) is a pentacyclic triterpene acid that is present in a wide variety of medicinal herbs and edible plants. This study investigated the effect of UA on apoptosis and proliferation of hepatocellular carcinoma SK-Hep-1 cells. After treatment of SK-Hep-1 cells with different concentrations of UA, we observed that cell viability was reduced in a dose- and time-dependent manner. Furthermore, there was a dose-dependent increase in the percentage of cells in the sub-G1 and G2/M phases, with cells treated with 60 μM showing the highest percentages of cells in those phases. UA-induced chromatin condensation of nuclei was observed by using DAPI staining. The western blot results revealed that exposure to UA was associated with decreased expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, and TCTP and increased expression of apoptosis-related proteins TNF-α, Fas, FADD, Bax, cleaved caspase-3, caspase-8, caspase-9, and PARP. Immunocytochemistry staining showed that treatment with UA resulted in increased expression of caspase-3. Moreover, exposure to UA resulted in the inhibition of the PI3K/Akt and p38 MAPK signaling pathways. These findings suggest that UA inhibits the proliferation of SK-Hep-1 cells and induces apoptosis. PMID:27104510

  10. Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation.

    PubMed

    Zhang, Litao; Liu, Jianguo

    2016-07-01

    The purpose of this study was to clarify the interrelation between the mitochondrial alternative oxidase (AOX) pathway and fatty acid accumulation in marine microalga Isochrysis galbana. Under normal conditions, the activity of the AOX pathway was maintained at a low level in I. galbana. Compared with the normal condition, nitrogen deprivation significantly increased the AOX pathway activity and fatty acid accumulation. Under nitrogen deprivation, the inhibition of the AOX pathway by salicylhydroxamic acid caused the accumulation of reducing equivalents and the over-reduction of chloroplasts in I. galbana cells, leading to a decrease in the photosynthetic O2 evolution rate. The over-production of reducing equivalents due to the inhibition of the AOX pathway under nitrogen deprivation further enhanced the accumulation of fatty acids in I. galbana cells. PMID:27068057

  11. Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway

    PubMed Central

    Somarajan, Bindu I.; Khanday, Mudasir A.; Mallick, Birendra N.

    2016-01-01

    Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate

  12. Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway.

    PubMed

    Somarajan, Bindu I; Khanday, Mudasir A; Mallick, Birendra N

    2016-01-01

    Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate

  13. Grape seed extract targets mitochondrial electron transport chain complex III and induces oxidative and metabolic stress leading to cytoprotective autophagy and apoptotic death in human head and neck cancer cells.

    PubMed

    Shrotriya, Sangeeta; Deep, Gagan; Lopert, Pamela; Patel, Manisha; Agarwal, Rajesh; Agarwal, Chapla

    2015-12-01

    Head and neck squamous cell carcinoma (HNSCC) is a major killer worldwide and innovative measures are urgently warranted to lower the morbidity and mortality caused by this malignancy. Aberrant redox and metabolic status in HNSCC cells offer a unique opportunity to specifically target cancer cells. Therefore, we investigated the efficacy of grape seed extract (GSE) to target the redox and bioenergetic alterations in HNSCC cells. GSE treatment decreased the mitochondrial electron transport chain complex III activity, increased the mitochondrial superoxide levels and depleted the levels of cellular antioxidant (glutathione), thus resulting in the loss of mitochondrial membrane potential in human HNSCC Detroit 562 and FaDu cells. Polyethylene glycol-SOD addition reversed the GSE-mediated apoptosis without restoring complex III activity. Along with redox changes, GSE inhibited the extracellular acidification rate (representing glycolysis) and oxygen consumption rate (indicating oxidative phosphorylation) leading to metabolic stress in HNSCC cells. Molecular studies revealed that GSE activated AMP-activated protein kinase (AMPK), and suppressed Akt/mTOR/4E-BP1/S6K signaling in both Detroit 562 and FaDu cells. Interestingly, GSE increased the autophagic load specifically in FaDu cells, and autophagy inhibition significantly augmented the apoptosis in these cells. Consistent with in vitro results, in vivo analyses also showed that GSE feeding in nude mice activated AMPK and induced-autophagy in FaDu xenograft tumor tissues. Overall, these findings are innovative as we for the first time showed that GSE targets ETC complex III and induces oxidative and metabolic stress, thereby, causing autophagy and apoptotic death in HNSCC cells. PMID:25557495

  14. Amyloid β oligomers elicit mitochondrial transport defects and fragmentation in a time-dependent and pathway-specific manner.

    PubMed

    Rui, Yanfang; Zheng, James Q

    2016-01-01

    Small oligomeric forms of amyloid-β (Aβ) are believed to be the culprit for declined brain functions in AD in part through their impairment of neuronal trafficking and synaptic functions. However, the precise cellular actions of Aβ oligomers and underlying mechanisms in neurons remain to be fully defined. Previous studies have identified mitochondria as a major target of Aβ toxicity contributing to early cognitive decline and memory loss in neurodegenerative diseases including Alzheimer's disease (AD). In this study, we report that Aβ oligomers acutely elicit distinct effects on the transport and integrity of mitochondria. We found that acute exposure of hippocampal neurons to Aβ oligomers from either synthetic peptides or AD brain homogenates selectively impaired fast transport of mitochondria without affecting the movement of late endosomes and lysosomes. Extended exposure of hipoocampal neurons to Aβ oligomers was found to result in mitochondrial fragmentation. While both mitochondrial effects induced by Aβ oligomers can be abolished by the inhibition of GSK3β, they appear to be independent from each other. Aβ oligomers impaired mitochondrial transport through HDAC6 activation whereas the fragmentation involved the GTPase Drp-1. These results show that Aβ oligomers can acutely disrupt mitochondrial transport and integrity in a time-dependent and pathway-specific manner. These findings thus provide new insights into Aβ-induced mitochondrial defects that may contribute to neuronal dysfunction and AD pathogenesis. PMID:27535553

  15. Synergism between PGC-1α and estrogen in the survival of endometrial cancer cells via the mitochondrial pathway

    PubMed Central

    Yang, Hui; Yang, Rui; Liu, Hao; Ren, Zhongqian; Kong, Fanfei; Li, Da; Ma, Xiaoxin

    2016-01-01

    Accumulating evidence shows that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is involved in the progression of hormone-related cancers, and there may exist an association between estrogen and PGC-1α. Notably, emerging evidence has led to considerable interest in the role of PGC-1α in endometrial cancer development. However, whether the synergism exists between PGC-1α and estrogen for regulating mitochondrial function to promote the development of endometrial cancer remains largely unknown. Here, we show that: 1) knockdown of PGC-1α attenuates the survival of endometrial cancer cells by inducing cell apoptosis through the mitochondrial pathway; 2) estrogen remedies the PGC-1α efficiency-induced decline of endometrial cancer cell viability; and 3) estrogen modulates the mitochondrial function to inhibit the PGC-1α deficiency-induced apoptosis in endometrial cancer cells. Collectively, these results demonstrated that the synergism between PGC-1α and estrogen was required for the survival of endometrial cancer cells, which was dependent on the mitochondrial pathway. PMID:27418839

  16. Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway.

    PubMed

    Garcia-Diaz, Beatriz; Barca, Emanuele; Balreira, Andrea; Lopez, Luis C; Tadesse, Saba; Krishna, Sindhu; Naini, Ali; Mariotti, Caterina; Castellotti, Barbara; Quinzii, Catarina M

    2015-08-15

    Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases. PMID:25976310

  17. Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway

    PubMed Central

    LIN, RUHUI; LI, ZUANFANG; LIN, JIUMAO; YE, JINXIA; CAI, QIAOYAN; CHEN, LIDIAN; PENG, JUN

    2015-01-01

    Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis. PMID:26622854

  18. Suppression of epithelial–mesenchymal transition and apoptotic pathways by miR-294/302 family synergistically blocks let-7-induced silencing of self-renewal in embryonic stem cells

    PubMed Central

    Guo, W-T; Wang, X-W; Yan, Y-L; Li, Y-P; Yin, X; Zhang, Q; Melton, C; Shenoy, A; Reyes, N A; Oakes, S A; Blelloch, R; Wang, Y

    2015-01-01

    The embryonic stem cell (ESC)-enriched miR-294/302 family and the somatic cell-enriched let-7 family stabilizes the self-renewing and differentiated cell fates, respectively. The mechanisms underlying these processes remain unknown. Here we show that among many pathways regulated by miR-294/302, the combinatorial suppression of epithelial–mesenchymal transition (EMT) and apoptotic pathways is sufficient in maintaining the self-renewal of ESCs. The silencing of ESC self-renewal by let-7 was accompanied by the upregulation of several EMT regulators and the induction of apoptosis. The ectopic activation of either EMT or apoptotic program is sufficient in silencing ESC self-renewal. However, only combined but not separate suppression of the two programs inhibited the silencing of ESC self-renewal by let-7 and several other differentiation-inducing miRNAs. These findings demonstrate that combined repression of the EMT and apoptotic pathways by miR-294/302 imposes a synergistic barrier to the silencing of ESC self-renewal, supporting a model whereby miRNAs regulate complicated cellular processes through synergistic repression of multiple targets or pathways. PMID:25501598

  19. DRP1-dependent apoptotic mitochondrial fission occurs independently of BAX, BAK and APAF1 to amplify cell death by BID and oxidative stress.

    PubMed

    Oettinghaus, Björn; D'Alonzo, Donato; Barbieri, Elisa; Restelli, Lisa Michelle; Savoia, Claudia; Licci, Maria; Tolnay, Markus; Frank, Stephan; Scorrano, Luca

    2016-08-01

    During apoptosis mitochondria undergo cristae remodeling and fragmentation, but how the latter relates to outer membrane permeabilization and downstream caspase activation is unclear. Here we show that the mitochondrial fission protein Dynamin Related Protein (Drp) 1 participates in cytochrome c release by selected intrinsic death stimuli. While Bax, Bak double deficient (DKO) and Apaf1(-/-) mouse embryonic fibroblasts (MEFs) were less susceptible to apoptosis by Bcl-2 family member BID, H(2)O(2), staurosporine and thapsigargin, Drp1(-/-) MEFs were protected only from BID and H(2)O(2). Resistance to cell death of Drp1(-/-) and DKO MEFs correlated with blunted cytochrome c release, whereas mitochondrial fragmentation occurred in all cell lines in response to all tested stimuli, indicating that other mechanisms accounted for the reduced cytochrome c release. Indeed, cristae remodeling was reduced in Drp1(-/-) cells, potentially explaining their resistance to apoptosis. Our results indicate that caspase-independent mitochondrial fission and Drp1-dependent cristae remodeling amplify apoptosis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26997499

  20. Resveratrol induces apoptosis in K562 cells via the regulation of mitochondrial signaling pathways.

    PubMed

    Wang, Binghua; Liu, Jiao; Gong, Zhanfeng

    2015-01-01

    Resveratrol, an edible polyphenolic phytoalexin obtained primarily from root extracts of the oriental plant, Polygonum cuspidatum and from grapes and red wine, has been reported as an anticancer compound against several types of cancer, the accurate molecular mechanisms of by which it induces apoptosis are limited. In the present study, the molecular mechanisms of resveratrol on human leukemia K562 cells apoptosis was examined. Our results showed that resveratrol significantly decreased cell viability and triggered cell apoptosis in K562 cells. Resveratrol-induced apoptosis of K562 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the up-regulation of Bax/Bcl-2 ratio, the activation of caspase-3 and increased cleaved PARP was also observed in K562 cells treated with resveratrol. Thus, we considered that the resveratrol-induced apoptosis of K562 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of resveratrol as a potential cancer therapeutic compound. PMID:26629245

  1. Resveratrol induces apoptosis in K562 cells via the regulation of mitochondrial signaling pathways

    PubMed Central

    Wang, Binghua; Liu, Jiao; Gong, Zhanfeng

    2015-01-01

    Resveratrol, an edible polyphenolic phytoalexin obtained primarily from root extracts of the oriental plant, Polygonum cuspidatum and from grapes and red wine, has been reported as an anticancer compound against several types of cancer, the accurate molecular mechanisms of by which it induces apoptosis are limited. In the present study, the molecular mechanisms of resveratrol on human leukemia K562 cells apoptosis was examined. Our results showed that resveratrol significantly decreased cell viability and triggered cell apoptosis in K562 cells. Resveratrol-induced apoptosis of K562 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the up-regulation of Bax/Bcl-2 ratio, the activation of caspase-3 and increased cleaved PARP was also observed in K562 cells treated with resveratrol. Thus, we considered that the resveratrol-induced apoptosis of K562 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of resveratrol as a potential cancer therapeutic compound. PMID:26629245

  2. Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway.

    PubMed

    Kawiak, Anna; Zawacka-Pankau, Joanna; Lojkowska, Ewa

    2012-04-27

    Breast cancer is the leading cause of death-related cancers in women. Approximately 30% of breast cancers overexpress the Her2 oncogene, which is associated with a poor prognosis and increased resistance to chemotherapy. Plumbagin (1), a constituent of species in the plant genera Drosera and Plumbago, displays antineoplastic activity toward various cancers. The present study was aimed at determining the anticancer potential of 1 toward Her2-overexpressing breast cancer cells and defining the mode of cell death induced in these cells. The results showed that 1 exhibited high antiproliferative activity toward the Her2-overexpressing cell lines SKBR3 and BT474. The antiproliferative activity of 1 was associated with apoptosis-mediated cell death, as revealed by caspase activation and an increase in the sub-G1 fraction of the cell cycle. Compound 1 increased the levels of the proapoptotic Bcl-2 family of proteins and decreased the level of the antiapoptotic Bcl-2 protein in SKBR3 and BT474 cells. Thus, these findings indicate that 1 induces apoptosis in Her2-overexpressing breast cancers through the mitochondrial-mediated pathway and suggest its potential for further investigation for the treatment of Her2-overexpressing breast cancer. PMID:22512718

  3. Apoptosis Cell Death Effect of Scrophularia Variegata on Breast Cancer Cells via Mitochondrial Intrinsic Pathway

    PubMed Central

    Azadmehr, Abbas; Hajiaghaee, Reza; Baradaran, Behzad; Haghdoost-Yazdi, Hashem

    2015-01-01

    Purpose: Scrophularia variegata M. Beib. (Scrophulariaceae) is an Iranian medicinal plant which is used for various inflammatory disorders in traditional medicine. In this study we evaluated the anti-cancer and cytotoxic effects of the Scrophularia variegata (S. variegata) ethanolic extract on the human breast cancer cell line. Methods: The cytotoxicity effect of the extract on MCF-7 cells was evaluated by MTT assay. In addition, Caspase activity, DNA ladder and Cell death were evaluated by ELISA, gel electrophoresis and Annexin V-FITC/PI staining, respectively. Results: The S. variegata extract showed significant effect cytotoxicity on MCF-7 human breast cancer cell line. Treatment with the extract induced apoptosis on the breast cancer cells by cell cycle arrest in G2/M phase. The results indicated that cytotoxicity activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation as well as an increase of the amount of caspase 3 and caspase 9. In addition, the phytochemical assay showed that the extract had antioxidant capacity and also flavonoids, phenolic compounds and phenyl propanoids were presented in the extract. Conclusion: Our findings indicated that S. variegata extract induced apoptosis via mitochondrial intrinsic pathway on breast cancer by cell cycle arrest in G2/M phase and an increase of caspase 3 and caspase 9. However future studies are needed. PMID:26504768

  4. Response rate of fibrosarcoma cells to cytotoxic drugs on the expression level correlates to the therapeutic response rate of fibrosarcomas and is mediated by regulation of apoptotic pathways

    PubMed Central

    Lehnhardt, Marcus; Klein-Hitpass, Ludger; Kuhnen, Cornelius; Homann, Heinz Herbert; Daigeler, Adrien; Steinau, Hans Ulrich; Roehrs, Sonja; Schnoor, Laura; Steinstraesser, Lars; Mueller, Oliver

    2005-01-01

    Background Because of the high resistance rate of fibrosarcomas against cytotoxic agents clinical chemotherapy of these tumors is not established. A better understanding of the diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new chemotherapeutic strategies. In this study we elucidated the response of a fibrosarcoma cell line to clinically used cytostatic agents on the level of gene expression. Methods HT1080 fibrosarcoma cells were exposed to the chemotherapeutic agents doxorubicin, actinomycin D or vincristine. Total RNA was isolated and the gene expression patterns were analyzed by microarray analysis. Expression levels for 46 selected candidate genes were validated by quantitative real-time PCR. Results The analysis of the microarray data resulted in 3.309 (actinomycin D), 1.019 (doxorubicin) and 134 (vincristine) probesets that showed significant expression changes. For the RNA synthesis blocker actinomycin D, 99.4% of all differentially expressed probesets were under-represented. In comparison, probesets down-regulated by doxorubicin comprised only 37.4% of all genes effected by this agent. Closer analysis of the differentially regulated genes revealed that doxorubicin induced cell death of HT1080 fibrosarcoma cells mainly by regulating the abundance of factors mediating the mitochondrial (intrinsic) apoptosis pathway. Furthermore doxorubicin influences other pathways and crosstalk to other pathways (including to the death receptor pathway) at multiple levels. We found increased levels of cytochrome c, APAF-1 and members of the STAT-family (STAT1, STAT3), while Bcl-2 expression was decreased. Caspase-1, -3, -6, -8, and -9 were increased indicating that these proteases are key factors in the execution of doxorubicin mediated apoptosis. Conclusion This study demonstrates that chemotherapy regulates the expression of apoptosis-related factors in fibrosarcoma cells. The number and the specific pattern of the genes

  5. Upstream Pathways Controlling Mitochondrial Function in Major Psychosis: A Focus on Bipolar Disorder.

    PubMed

    Machado, Alencar Kolinski; Pan, Alexander Yongshuai; da Silva, Tatiane Morgana; Duong, Angela; Andreazza, Ana Cristina

    2016-08-01

    Mitochondrial dysfunction is commonly observed in bipolar disorder (BD) and schizophrenia (SCZ) and may be a central feature of psychosis. These illnesses are complex and heterogeneous, which is reflected by the complexity of the processes regulating mitochondrial function. Mitochondria are typically associated with energy production; however, dysfunction of mitochondria affects not only energy production but also vital cellular processes, including the formation of reactive oxygen species, cell cycle and survival, intracellular Ca(2+) homeostasis, and neurotransmission. In this review, we characterize the upstream components controlling mitochondrial function, including 1) mutations in nuclear and mitochondrial DNA, 2) mitochondrial dynamics, and 3) intracellular Ca(2+) homeostasis. Characterizing and understanding the upstream factors that regulate mitochondrial function is essential to understand progression of these illnesses and develop biomarkers and therapeutics. PMID:27310240

  6. FAM3A attenuates ER stress-induced mitochondrial dysfunction and apoptosis via CHOP-Wnt pathway.

    PubMed

    Song, Qing; Gou, Wen-Li; Zhang, Rong

    2016-03-01

    Endoplasmic reticulum (ER) stress is linked to several neurological disorders, and neuronal injury cascades initiated by excessive ER stress are mediated, in part, via mitochondrial dysfunction. In the present study, we identified FAM3A as an important regulator of ER stress-induced cell death in neuronal HT22 cells. The ER stress inductor tunicamycin (TM) significantly decreased the expression of FAM3A at both mRNA and protein levels, which was shown to be dependent on the induction of reactive oxygen species (ROS). Overexpression of FAM3A attenuated TM-induced apoptosis and activation of ER stress factors, but had no effect on ER calcium metabolism in HT22 cells. We also found decreased mitochondrial ROS generation, inhibited cytochrome c release and preserved mitochondrial membrane potential (MMP) in FAM3A overexpressed cells. In addition, the experiments using isolated mitochondria showed that overexpression of FAM3A attenuated mitochondrial swelling and loss of mitochondrial Ca(2+) buffering capacity after TM exposure. By using specific targeted small interfering RNA (siRNA) to knockdown the expression of the C/EBP homologous protein (CHOP), we found that FAM3A-induced protection and inhibition of ER stress was mediated by inverting TM-induced decrease of Wnt through the CHOP pathway. Our study demonstrates a pivotal role of FAM3A in protecting against TM-induced cytotoxicity via regulating CHOP-Wnt pathway, and suggests the therapeutic values of FAM3A overexpression against ER stress-associated neuronal injury. PMID:26939760

  7. Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptotic pathway.

    PubMed

    Yu, Zhiqi; Zhang, Chang; Wang, Hao; Xing, Junjie; Gong, Haifeng; Yu, Enda; Zhang, Wei; Zhang, Xiaoqing; Cao, Guangwen; Fu, Chuangang

    2014-10-28

    This study aimed to clarify the role of multidrug resistance-associated protein 3 (MRP3) in resistance to neoadjuvant chemoradiotherapy and long-term prognosis of advanced rectal cancer. Immunohistochemistry was used to measure MRP3 expression in biopsy specimens of 144 stage II-III rectal cancer patients who received preoperative chemoradiotherapy. The effect of MRP3 expression on short-term pathological response and postoperative long-term prognosis were assessed using the Cox proportional hazards model. Short interfering RNAs targeting MRP3 were synthesized and used to transfect human colorectal carcinoma cell lines. The effect of MRP3 down-regulation on cell proliferation and apoptosis in response to 5-fluorouracil and/or irradiation were examined in vitro and in xenograft mouse models, respectively. The content of intracellular reactive oxygen species and the activity of caspase-3-dependent apoptotic pathway in response to irradiation were further evaluated. High expression (immunoreactive score > 6) of MRP3 significantly predicted poor pathological response to chemoradiotherapy (tumor regression grade ≤ 2 vs. ≥3, p = 0.002) in univariate analysis and unfavorable long-term prognosis (5-year overall survival: HR = 1.612, 95% CI, 1.094-2.375, p = 0.016; 5-year disease-free survival: HR = 1.513, 95% CI, 1.041-2.200, p = 0.030) in multivariate Cox analysis. MRP3 down-regulation significantly increased 5-fluorouracil or irradiation-induced cell apoptosis and attenuated tumor growth following irradiation in animal models. MRP3 inhibition significantly reduced intracellular reactive oxygen species exporting from cells following irradiation, and increased expression of cleaved poly ADP-ribose polymerase and caspase-3. Aberrant expression of MRP3 in rectal cancer confers chemo-radioresistance. MRP3 might be a predictive factor and an attractive target in treating advanced rectal cancer. PMID:25088576

  8. An apoptotic signaling pathway in the interferon antiviral response mediated by RNase L and c-Jun NH2-terminal kinase.

    PubMed

    Li, Geqiang; Xiang, Ying; Sabapathy, Kanaga; Silverman, Robert H

    2004-01-01

    Cellular stress responses induced during viral infections are critical to the health and survival of organisms. In higher vertebrates, interferons (IFNs) mediate the innate antiviral response in part through the action of RNase L, a uniquely regulated enzyme. RNase L is activated by 5'-phosphorylated, 2'-5' oligoadenylates (2-5A) produced from IFN-inducible and double stranded RNA-dependent synthetases. We show that viral activation of the c-Jun NH2-terminal kinases (JNK) family of MAP kinases and viral induction of apoptosis are both deficient in mouse cells lacking RNase L. Also, JNK phosphorylation in response to 2-5A was greatly reduced in RNase L-/- mouse cells. In addition, 2-5A treatment of the human ovarian carcinoma cell line, Hey1b, resulted in specific ribosomal RNA cleavage products coinciding with JNK activation. Furthermore, suppression of JNK activity with the chemical inhibitor, SP600125, prevented apoptosis induced by 2-5A. In contrast, inhibition of alternative MAP kinases, p38 and ERK, failed to prevent 2-5A-mediated apoptosis. Short interfering RNA to JNK1/JNK2 mRNAs resulted in JNK ablation while also suppressing 2-5A-mediated apoptosis. Moreover, Jnk1-/- Jnk2-/- cells were highly resistant to the apoptotic effects of IFN and 2-5A. These findings suggest that JNK and RNase L function in an integrated signaling pathway during the IFN response that leads to elimination of virus-infected cells through apoptosis. PMID:14570908

  9. Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

    PubMed Central

    BOUSSEROUEL, SOUAD; LE GRANDOIS, JULIE; GOSSÉ, FRANCINE; WERNER, DALAL; BARTH, STEPHAN W.; MARCHIONI, ERIC; MARESCAUX, JACQUES; RAUL, FRANCIS

    2013-01-01

    Shoots of white asparagus are a popular vegetable dish, known to be rich in many bioactive phytochemicals reported to possess antioxidant, and anti-inflammatory and antitumor activities. We evaluated the anticancer mechanisms of a methanolic extract of Asparagus officinalis L. shoots (Asp) on human colon carcinoma cells (SW480) and their derived metastatic cells (SW620), and Asp chemopreventive properties were also assessed in a model of colon carcinogenesis. SW480 and SW620 cell proliferation was inhibited by 80% after exposure to Asp (80 μg/ml). We demonstrated that Asp induced cell death through the activation of TRAIL DR4/DR5 death receptors leading to the activation of caspase-8 and caspase-3 and to cell apoptosis. By specific blocking agents of DR4/DR5 receptors we were able to prevent Asp-triggered cell death confirming the key role of DR4/DR5 receptors. We found also that Asp (80 μg/ml) was able to potentiate the effects of the cytokine TRAIL on cell death even in the TRAIL-resistant metastatic SW620 cells. Colon carcinogenesis was initiated in Wistar rats by intraperitoneal injections of azoxymethane (AOM), once a week for two weeks. One week after (post-initiation) rats received daily Asp (0.01%, 14 mg/kg body weight) in drinking water. After 7 weeks of Asp-treatment the colon of rats exhibited a 50% reduction of the number of preneoplastic lesions (aberrant crypt foci). In addition Asp induced inhibition of several pro-inflammatory mediators, in association with an increased expression of host-defense mediators. In the colonic mucosa of Asp-treated rats we also confirmed the pro-apoptotic effects observed in vitro including the activation of the TRAIL death-receptor signaling pathway. Taken together, our data highlight the chemopreventive effects of Asp on colon carcinogenesis and its ability to promote normal cellular homeostasis. PMID:23754197

  10. Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model.

    PubMed

    Bousserouel, Souad; Le Grandois, Julie; Gossé, Francine; Werner, Dalal; Barth, Stephan W; Marchioni, Eric; Marescaux, Jacques; Raul, Francis

    2013-08-01

    Shoots of white asparagus are a popular vegetable dish, known to be rich in many bioactive phytochemicals reported to possess antioxidant, and anti-inflammatory and antitumor activities. We evaluated the anticancer mechanisms of a methanolic extract of Asparagus officinalis L. shoots (Asp) on human colon carcinoma cells (SW480) and their derived metastatic cells (SW620), and Asp chemopreventive properties were also assessed in a model of colon carcinogenesis. SW480 and SW620 cell proliferation was inhibited by 80% after exposure to Asp (80 µg/ml). We demonstrated that Asp induced cell death through the activation of TRAIL DR4/DR5 death receptors leading to the activation of caspase-8 and caspase-3 and to cell apoptosis. By specific blocking agents of DR4/DR5 receptors we were able to prevent Asp-triggered cell death confirming the key role of DR4/DR5 receptors. We found also that Asp (80 µg/ml) was able to potentiate the effects of the cytokine TRAIL on cell death even in the TRAIL-resistant metastatic SW620 cells. Colon carcinogenesis was initiated in Wistar rats by intraperitoneal injections of azoxymethane (AOM), once a week for two weeks. One week after (post-initiation) rats received daily Asp (0.01%, 14 mg/kg body weight) in drinking water. After 7 weeks of Asp-treatment the colon of rats exhibited a 50% reduction of the number of preneoplastic lesions (aberrant crypt foci). In addition Asp induced inhibition of several pro-inflammatory mediators, in association with an increased expression of host-defense mediators. In the colonic mucosa of Asp-treated rats we also confirmed the pro-apoptotic effects observed in vitro including the activation of the TRAIL death‑receptor signaling pathway. Taken together, our data highlight the chemopreventive effects of Asp on colon carcinogenesis and its ability to promote normal cellular homeostasis. PMID:23754197

  11. The S-Nitrosylation Status of PCNA Localized in Cytosol Impacts the Apoptotic Pathway in a Parkinson’s Disease Paradigm

    PubMed Central

    Yin, Liang; Xie, Yingying; Yin, Songyue; Lv, Xiaolei; Zhang, Jia; Gu, Zezong; Sun, Haidan; Liu, Siqi

    2015-01-01

    It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson’s disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the