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Sample records for cytochrome c-mediated apoptosis

  1. Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis.

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    Lu, Jun; Wu, Dong-mei; Zheng, Yuan-lin; Hu, Bin; Zhang, Zi-feng

    2010-05-01

    Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, protects brain function against oxidative stress induced by D-galactose (D-gal) (Sigma-Aldrich, St. Louis, MO, USA). Our data showed that PSPC enhanced open-field activity, decreased step-through latency, and improved spatial learning and memory ability in D-gal-treated old mice by decreasing advanced glycation end-products' (AGEs) formation and the AGE receptor (RAGE) expression, and by elevating Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (Sigma-Aldrich) and catalase (CAT) expression and activity. Cleavage of caspase-3 and increased terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated old mice were inhibited by PSPC, which might be attributed to its antioxidant property. PSPC also suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) and the release of cytochrome c from mitochondria that counteracted the onset of neuronal apoptosis in D-gal-treated old mice. Furthermore, it was demonstrated that phosphoinositide 3-kinase (PI3K) activation was required for PSPC to promote the neuronal survival accompanied with phosphorylation and activation of Akt and p44/42 mitogen-activated protein kinase (MAPK) by using PI3K inhibitor LY294002 (Cell Signaling Technology, Inc., Beverly, MA, USA), implicating a neuronal survival mechanism. The present results suggest that neuronal survival promoted by PSPC may be a potentially effective method to enhance resistance of neurons to age-related disease.

  2. Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

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    Berghella, Libera; Ferraro, Elisabetta

    2012-01-01

    Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

  3. Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

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    Katja C. Zimmermann

    2000-01-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

  4. Conjugation of cytochrome c with hydrogen titanate nanotubes: novel conformational state with implications for apoptosis

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    Ray, Moumita; Mazumdar, Shyamalava [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India); Chatterjee, Sriparna; Das, Tanmay; Bhattacharyya, Somnath; Ayyub, Pushan, E-mail: somnath@tifr.res.in, E-mail: pushan@tifr.res.in, E-mail: shyamal@tifr.res.in [Department of Condensed Matter Physics and Materials Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India)

    2011-10-14

    We show that hydrogen titanate (H{sub 2}Ti{sub 3}O{sub 7}) nanotubes form strongly associated reversible nano-bio-conjugates with the vital respiratory protein, cytochrome c. Resonance Raman spectroscopy along with direct electrochemical studies indicate that in this nano-bio-conjugate, cytochrome c exists in an equilibrium of two conformational states with distinctly different formal redox potentials and coordination geometries of the heme center. The nanotube-conjugated cytochrome c also showed enhanced peroxidase activity similar to the membrane-bound protein that is believed to be an apoptosis initiator. This suggests that such a nanotube-cytochrome c conjugate may be a good candidate for cancer therapy applications.

  5. Betulin induces cytochrome c release and apoptosis in colon cancer cells via NOXA.

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    Zhou, Zhiyuan; Zhu, Chenfang; Cai, Zhongfang; Zhao, Feng; He, Liu; Lou, Xiaolou; Qi, Xiaoliang

    2018-05-01

    Betulin is a common triterpene that can be readily obtained from various plants, particularly birch trees, in their natural environment. Specific tumor cells are sensitive to betulin, whereas healthy cells are not. Betulin was observed to stimulate programmed cell death of various cancer cell lines; however, the precise molecular mechanism of action of betulin remains unknown. The present study used colon cancer cells, in which mass apoptosis triggered by betulin was identified, and the apoptotic process was demonstrated to occur via the activation of caspase-3 and -9 pathways. In addition, release of cytochrome c was detected. Furthermore, the pro-apoptotic member of the Bcl-2 protein family, NOXA, was induced following treatment with betulin, and the downregulation of NOXA markedly suppressed the release of cytochrome c and apoptosis in colon cancer cells. Conversely, the overexpression of NOXA further enhanced betulin-induced apoptosis. The present study therefore offers novel insights into the mechanism of action of the natural compound betulin against tumors.

  6. Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage)

    International Nuclear Information System (INIS)

    Kim, Sang Chan; Byun, Sung Hui; Yang, Chae Ha; Kim, Chul Young; Kim, Jin Woong; Kim, Sang Geon

    2004-01-01

    Glycyrrhizae radix has been popularly used as one of the oldest and most frequently employed botanicals in herbal medicine in Asian countries, and currently occupies an important place in food products. Cadmium (Cd) induces both apoptotic and non-apoptotic cell death, in which alterations in cellular sulfhydryls participate. In the present study, we determined the effects of G. radix extract (GRE) and its representative active components on cell death induced by Cd and explored the mechanistic basis of cytoprotective effects of G. radix. Incubation of H4IIE cells with GRE inhibited cell death induced by 10 μM Cd. Also, GRE effectively blocked Cd (1 μM)-induced cell death potentiated by buthionine sulfoximine (BSO) without restoration of cellular GSH. GRE prevented both apoptotic and non-apoptotic cell injury induced by Cd (10 μM) or Cd (0.3-1 μM) + BSO. Inhibition of Cd-induced cell injury by pretreatment of cells with GRE suggested that the cytoprotective effect result from alterations in the levels of the protein(s) responsible for cell viability. GRE inhibited mitochondrial Bad translocation by Cd or Cd+BSO, and caused restoration of mitochondrial Bcl xL and cytochrome c levels. Cd-induced poly(ADP-ribose)polymerase cleavage in control cells or in cells deprived of sulfhydryls was prevented by GRE treatment. Among the major components present in GRE, liquiritigenin, but not liquiritin, isoliquiritigenin or glycyrrhizin, exerted cytoprotective effect. These results demonstrated that GRE blocked Cd-induced cell death by inhibiting the apoptotic processes involving translocation of Bad into mitochondria, decreases in mitochondrial Bcl xL and cytochrome c, and poly(ADP-ribose)polymerase cleavage

  7. Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

    International Nuclear Information System (INIS)

    Hiraoka, Yoshinori; Granja, Ana Teresa; Fialho, Arsenio M.; Schlarb-Ridley, Beatrix G.; Das Gupta, Tapas K.; Chakrabarty, Ananda M.; Yamada, Tohru

    2005-01-01

    Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c 551 from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c 551 , the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G 1 to S phase, as well as at the G 2 /M phase at higher concentrations. Unlike P. aeruginosa cytochrome c 551 which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G 2 /M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 deg. C, suggesting energy requirement in the entry process

  8. MicroRNA-661 Enhances TRAIL or STS Induced Osteosarcoma Cell Apoptosis by Modulating the Expression of Cytochrome c1

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    Lin Fan

    2017-04-01

    Full Text Available Aim: Osteosarcoma (OS is an aggressive bone malignancy that affects rapidly growing bones and is associated with a poor prognosis. Our previous study showed that cytochrome c1 (CYC1, a subunit of the cytochrome bc1 complex (complex III of the mitochondrial electron chain, is overexpressed in human OS tissues and cell lines and its silencing induces apoptosis in vitro and inhibits tumor growth in vivo. Here, we investigated the mechanism underlying the modulation of CYC1 expression in OS and its role in the resistance of OS to apoptosis. Methods: qRT-PCR, luciferase reporter assay, western blotting, fow cytometry, and animal experiments were performed in this study. Results: MicroRNA (miR-661 was identified as a downregulated miRNA in OS tissues and cells and shown to directly target CYC1. Ectopically expressed miR-661 inhibited OS cell growth, promoted apoptosis, and reduced the activity of mitochondrial complex III. miR-661 overexpression enhanced TRAIL or STS induced apoptosis and promoted the release of cytochrome c into the cytosol, which induced caspase-9 activation, and these effects were abolished by a caspase-3 inhibitor. Overexpression of CYC1 rescued the effects of miR-661 on sensitizing OS cells to TRAIL or STS induced apoptosis, indicating that the antitumor effect of miR-661 is mediated by the downregulation of CYC1. In vivo, miR-661 overexpression sensitized tumors to TRAIL or STS induced apoptosis in a xenograft mouse model, and these effects were attenuated by co-expression of CYC1. Conclusion: Taken together, our results indicate that miR-661 plays a tumor suppressor role in OS mediated by the downregulation of CYC1, suggesting a potential mechanism underlying cell death resistance in OS.

  9. Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

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    Zhang, Jiaying; Yu, Qunli; Han, Ling; Chen, Cheng; Li, Hang; Han, Guangxing

    2017-06-01

    This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

  10. Induction of apoptosis in cancer cells by NiZn ferrite nanoparticles through mitochondrial cytochrome C release

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    Al-Qubaisi MS

    2013-10-01

    Full Text Available Mothanna Sadiq Al-Qubaisi,1 Abdullah Rasedee,1,2 Moayad Husein Flaifel,3 Sahrim Hj Ahmad,3 Samer Hussein-Al-Ali,1 Mohd Zobir Hussein,4 Zulkarnain Zainal,4 Fatah H Alhassan,4 Yun H Taufiq-Yap,4 Eltayeb EM Eid,5 Ismail Adam Arbab,1 Bandar A Al-Asbahi,3 Thomas J Webster,6,7 Mohamed Ezzat El Zowalaty1,8,9 1Institute of Bioscience, 2Faculty of Veterinary Medicine, Universiti Putra Malaysia, 3Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 4Faculty of Science, Universiti Putra Malaysia, Selangor, Malaysia; 5College of Pharmacy, Qassim University, Buraidah, Saudi Arabia; 6Department of Chemical Engineering and Program in Bioengineering, Northeastern University, Boston, MA, USA; 7Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia; 8Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; 9Faculty of Public Health and Tropical Medicine, Jazan University, Jazan, Saudi Arabia Abstract: The long-term objective of the present study was to determine the ability of NiZn ferrite nanoparticles to kill cancer cells. NiZn ferrite nanoparticle suspensions were found to have an average hydrodynamic diameter, polydispersity index, and zeta potential of 254.2 ± 29.8 nm, 0.524 ± 0.013, and -60 ± 14 mV, respectively. We showed that NiZn ferrite nanoparticles had selective toxicity towards MCF-7, HepG2, and HT29 cells, with a lesser effect on normal MCF 10A cells. The quantity of Bcl-2, Bax, p53, and cytochrome C in the cell lines mentioned above was determined by colorimetric methods in order to clarify the mechanism of action of NiZn ferrite nanoparticles in the killing of cancer cells. Our results indicate that NiZn ferrite nanoparticles promote apoptosis in cancer cells via caspase-3 and caspase-9, downregulation of Bcl-2, and upregulation of Bax and p53, with cytochrome C translocation. There was a concomitant collapse of the mitochondrial membrane potential in these cancer cells when treated

  11. Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

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    Zhang YueMei

    2005-02-01

    Full Text Available Abstract Background Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic, and Bcl-2 (anti-apoptotic genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. Results In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or

  12. A novel alkaloid, evodiamine causes nuclear localization of cytochrome-c and induces apoptosis independent of p53 in human lung cancer cells

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    Mohan, Vijay [School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat (India); Agarwal, Rajesh [Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, CO (United States); Singh, Rana P., E-mail: ranaps@hotmail.com [School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat (India); Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi (India)

    2016-09-02

    Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20–40 μM evodiamine treatment for 24–48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survival of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer. - Highlights: • Evodiamine, a novel plant alkaloid, relatively selectively inhibited growth and survival of human lung cancer cells. • Increased cancer cell

  13. Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts

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    Suzana Makpol

    2012-01-01

    Full Text Available In this study, we determined the molecular mechanism of γ-tocotrienol (GTT in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs. Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P<0.05. GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P<0.05. Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P<0.05 in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

  14. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

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    Kim, Ji Young [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Lee, Seung Gee [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Chung, Jin-Yong [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Yoon-Jae [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Park, Ji-Eun [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Oh, Seunghoon [Department of Physiology, College of Medicine, Dankook University, Cheonan 330-714 (Korea, Republic of); Lee, Se Yong [Department of Obstetrics and Gynecology, Busan Medical Center, Busan 611-072 (Korea, Republic of); Choi, Hong Jo [Department of General Surgery, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Yoo, Young Hyun, E-mail: yhyoo@dau.ac.kr [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); and others

    2012-04-15

    7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system. -- Highlights: ► Cytotoxicity-mediated apoptogenic action of DMBA in human endometrial cancer cells. ► Mitochondrial pathway in DMBA-induced apoptosis of RL95-2 endometrial cancer cells. ► Requirement of ligand-selective activation of CYP1B1 in DMBA-induced apoptosis.

  15. N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

    International Nuclear Information System (INIS)

    Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-01-01

    N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

  16. Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway

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    Castranova Vincent

    2009-04-01

    Full Text Available Abstract Background Carcinogenicity of nickel compounds has been well documented. However, the carcinogenic effect of metallic nickel is still unclear. The present study investigates metallic nickel nano- and fine particle-induced apoptosis and the signal pathways involved in this process in JB6 cells. The data obtained from this study will be of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles. Results Using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, we found that metallic nickel nanoparticles exhibited higher cytotoxicity than fine particles. Both metallic nickel nano- and fine particles induced JB6 cell apoptosis. Metallic nickel nanoparticles produced higher apoptotic induction than fine particles. Western-blot analysis showed an activation of proapoptotic factors including Fas (CD95, Fas-associated protein with death domain (FADD, caspase-8, death receptor 3 (DR3 and BID in apoptotic cells induced by metallic nickel particles. Immunoprecipitation (IP western blot analysis demonstrated the formation of the Fas-related death-inducing signaling complex (DISC in the apoptotic process. Furthermore, lamin A and beta-actin were cleaved. Moreover, we found that apoptosis-inducing factor (AIF was up-regulated and released from mitochondria to cytoplasm. Interestingly, although an up-regulation of cytochrome c was detected in the mitochondria of metallic nickel particle-treated cells, no cytochrome c release from mitochondria to cytoplasm was found. In addition, activation of antiapoptotic factors including phospho-Akt (protein kinase B and Bcl-2 was detected. Further studies demonstrated that metallic nickel particles caused no significant changes in the mitochondrial membrane permeability after 24 h treatment. Conclusion In this study, metallic nickel nanoparticles caused higher cytotoxicity and apoptotic induction than fine particles in JB6 cells. Apoptotic cell death

  17. Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

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    Martínez, María-Aránzazu; Ares, Irma; Rodríguez, José-Luis; Martínez, Marta; Roura-Martínez, David; Castellano, Victor; Lopez-Torres, Bernardo; Martínez-Larrañaga, María-Rosa; Anadón, Arturo

    2018-08-01

    This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury. Copyright © 2018. Published by Elsevier B.V.

  18. Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway

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    Jia G

    2015-01-01

    Full Text Available Geng Jia,1,* Qiang Wang,2,* Rong Wang,2,* Danni Deng,2 Lian Xue,2 Naiyuan Shao,1 Yi Zhang,1 Xiwei Xia,1 Feng Zhi,2 Yilin Yang1,2 1Department of Neurosurgery, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China; 2Modern Medical Research Center, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China * These authors contributed equally to this workBackground: Tubeimoside-1 (TBMS1 is a natural compound isolated from tubeimoside, which has been widely used as a traditional Chinese herbal medicine. The purpose of the present study is to investigate the anti-tumor effect and the underling mechanism of TBMS1 on glioma cancer cells.Methods: The MTT assay was performed to evaluate the effect of TBMS1 on glioma cell proliferation. The fluorescent microscopy and flow cytometry analysis were performed to evaluate the effect of TBMS1 on glioma cell apoptosis. The Western blot analysis was used to evaluate the protein change.Results: TBMS1 inhibited glioma cancer cell proliferation in a dose- and time-dependent manner. Fluorescent microscopy and flow cytometry analysis demonstrated that TBMS1 induced glioma cell apoptosis in a concentration-dependent manner. Western blotting showed that TBMS1 induced apoptosis by increasing the expression of Bax and downregulating the level of Bcl-2. Furthermore, we found that TBMS1 induced apoptosis by increasing the concentration of reactive oxygen species through the release of Cytochrome C and activation of Caspase-3.Conclusion: These findings indicate that TBMS1 may be developed as a possible therapeutic agent for the management of glioma. Keywords: Tubeimoside-1, glioma, proliferation, apoptosis

  19. Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

    Directory of Open Access Journals (Sweden)

    Pengfei Ge

    2013-10-01

    Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

  20. A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

    Science.gov (United States)

    Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

    2014-12-05

    The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of

  1. Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly.

    Science.gov (United States)

    Shalaeva, Daria N; Dibrova, Daria V; Galperin, Michael Y; Mulkidjanian, Armen Y

    2015-05-27

    structural and sequence analyses provides hints on the evolution of the cytochrome c-mediated apoptosis.

  2. HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer

    CSIR Research Space (South Africa)

    Rangel Lopes de Campos, W

    2014-10-01

    Full Text Available - surface receptors, the CXCR4 chemokine receptor and the TNF- R1 death receptor. The predominant apoptotic pathway varied from CXCR4-triggered, mitochondrion-initiated to CD95/Fas- ligand initiated, depending on the culture conditions as previously reported... virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c- mediated apoptosis independently of Fas signaling. J Virol 75: 7637–7650. 26. Bottarel F, Feito MJ, Bragardo M, Bonissoni S, Buonfiglio D, et al. (1999...

  3. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

    Directory of Open Access Journals (Sweden)

    Fuqiang Xing

    Full Text Available Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant. Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis.

  4. The Response of Ω-Loop D Dynamics to Truncation of Trimethyllysine 72 of Yeast Iso-1-cytochrome c Depends on the Nature of Loop Deformation

    Science.gov (United States)

    McClelland, Levi J.; Seagraves, Sean M.; Khan, Khurshid Alam; Cherney, Melisa M.; Bandi, Swati; Culbertson, Justin E.; Bowler, Bruce E.

    2015-01-01

    Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70 to 85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer. PMID:25948392

  5. The reaction of neuroglobin with potential redox protein partners cytochrome b5  and cytochrome c

    DEFF Research Database (Denmark)

    Fago, Angela; Mathews, A.J.; Moens, L.

    2006-01-01

    Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b5 is relatively slow (k=6×102M-1s-1 at pH 7.0) and thus is unlikely to be of physiological...... significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2×107M-1s-1) with an apparent overall equilibrium constant of 1μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis...

  6. Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

    Directory of Open Access Journals (Sweden)

    Alejandro K. Samhan-Arias

    2018-05-01

    Full Text Available In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

  7. An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro

    Directory of Open Access Journals (Sweden)

    Xiaojing Yan

    2016-06-01

    Full Text Available Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8, inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS. In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

  8. A complex of cardiac cytochrome c1 and cytochrome c.

    Science.gov (United States)

    Chiang, Y L; Kaminsky, L S; King, T E

    1976-01-10

    The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of

  9. In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition.

    Science.gov (United States)

    Balachandran, C; Emi, N; Arun, Y; Yamamoto, N; Duraipandiyan, V; Inaguma, Yoko; Okamoto, Akinao; Ignacimuthu, S; Al-Dhabi, N A; Perumal, P T

    2016-04-05

    The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 μM with IC50 value of 0.13 μM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Aspartame-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Mitochondrial cytochrome c biogenesis: no longer an enigma.

    Science.gov (United States)

    Babbitt, Shalon E; Sutherland, Molly C; San Francisco, Brian; Mendez, Deanna L; Kranz, Robert G

    2015-08-01

    Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Aspartame-induced apoptosis in PC12 cells

    OpenAIRE

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-induc...

  13. Cytochrome oxidase assembly does not require catalytically active cytochrome C.

    Science.gov (United States)

    Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

    2003-03-14

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

  14. Tributyltin interacts with mitochondria and induces cytochrome c release.

    Science.gov (United States)

    Nishikimi, A; Kira, Y; Kasahara, E; Sato, E F; Kanno, T; Utsumi, K; Inoue, M

    2001-01-01

    Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis. PMID:11368793

  15. The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

    Science.gov (United States)

    Bickar, D; Turrens, J F; Lehninger, A L

    1986-11-05

    When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

  16. The cytochrome p450 homepage.

    Science.gov (United States)

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  17. Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

    Science.gov (United States)

    Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

    2004-12-01

    In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

  18. 7α-Hydroxy-β-Sitosterol from Chisocheton tomentosus Induces Apoptosis via Dysregulation of Cellular Bax/Bcl-2 Ratio and Cell Cycle Arrest by Downregulating ERK1/2 Activation

    Directory of Open Access Journals (Sweden)

    Mohammad Tasyriq

    2012-01-01

    Full Text Available In continuation of our interest towards the elucidation of apoptotic pathways of cytotoxic phytocompounds, we have embarked upon a study on the anticancer effects of 7α-hydroxy-β-sitosterol (CT1, a rare natural phytosterol oxide isolated from Chisocheton tomentosus. CT1 was found to be cytotoxic on three different human tumor cell lines with minimal effects on normal cell controls, where cell viability levels were maintained ≥80% upon treatment. Our results showed that cell death in MCF-7 breast tumor cells was achieved through the induction of apoptosis via downregulation of the ERK1/2 signaling pathway. CT1 was also found to increase proapoptotic Bax protein levels, while decreasing anti-apoptotic Bcl-2 protein levels, suggesting the involvement of the intrinsic pathway. Reduced levels of initiator procaspase-9 and executioner procaspase-3 were also observed following CT1 exposure, confirming the involvement of cytochrome c-mediated apoptosis via the mitochondrial pathway. These results demonstrated the cytotoxic and apoptotic ability of 7α-hydroxy-β-sitosterol and suggest its potential anti-cancer use particularly on breast adenocarcinoma cells.

  19. Caffeic Acid Induces Apoptosis in Human Cervical Cancer Cells Through the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Chun Chang

    2010-12-01

    Conclusion: Caffeic acid induces apoptosis by inhibiting Bcl-2 activity, leading to release of cytochrome c and subsequent activation of caspase-3, indicating that caffeic acid induces apoptosis via the mitochondrial apoptotic pathway. This also suggests that caffeic acid has a strong anti-tumor effect and may be a promising chemopreventive or chemotherapeutic agent.

  20. Regulatory mechanisms of apoptosis in regularly dividing cells

    Directory of Open Access Journals (Sweden)

    Ribal S Darwish

    2010-08-01

    Full Text Available Ribal S DarwishDepartment of Anesthesiology, Division of Critical Care Medicine, University of Maryland Medical Center, Baltimore, Maryland, USAAbstract: The balance between cell survival and death is essential for normal development and homeostasis of organisms. Apoptosis is a distinct type of cell death with ultrastructural features that are consistent with an active, inherently controlled process. Abnormalities and ­dysregulation of apoptosis contribute to the pathophysiology of multiple disease processes. Apoptosis is strictly regulated by several positive and negative feedback mechanisms that regulate cell death and determine the final outcome after cell exposure to apoptotic stimuli. Mitochondria and caspases are central components of the regulatory mechanisms of ­apoptosis. Recently, noncaspase pathways of apoptosis have been explored through the studies of ­apoptosis-inducing factor and endonuclease G. Multiple difficulties in the apoptosis research relate to apoptosis detection and imaging. This article reviews current understanding of the regulatory mechanisms of apoptosis.Keywords: caspases, apoptosis-inducing factor, apoptosis inhibitory proteins, cytochrome c, mitochondria 

  1. Apoptosis in Drosophila: which role for mitochondria?

    Science.gov (United States)

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  2. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

    Science.gov (United States)

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

    2015-08-11

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

  3. Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

    Science.gov (United States)

    Novak, David; Mojovic, Milos; Pavicevic, Aleksandra; Zatloukalova, Martina; Hernychova, Lenka; Bartosik, Martin; Vacek, Jan

    2018-02-01

    Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

    Science.gov (United States)

    Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

    2016-01-01

    A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

  5. JALUR MOLEKULER MEKANISME APOPTOSIS

    Directory of Open Access Journals (Sweden)

    Yani Corvianindya Rahayu

    2015-07-01

    Full Text Available Apoptosis or programmed cell death is a normal condition for development and live multicellular organism. Apoptosis is a morphological phenomenon that plays an important role in physiologic processes during fetal development and in adult. Mitochondria play an important role in apoptosis. Mitochondria can do apoptosis directly. Mitochondria has 2 family of protein Bcl-2. Bcl-2 and Bcl-XL are anti apoptosis while Bad an Bax are pro apoptosis. There are 3 different mechanism to receptors at the cell surface and a third may be triggered by dangerous agent that different from two ways before. Apoptosis also need caspase as cell death executor. Study of apoptosis still done especially in case of disease. Some disease have known related with disturbing of apoptosis mechanism for example cancer and auto immune. This article reviews about molecular mechanism of apoptosis for understanding disease and future therapy.

  6. Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

    International Nuclear Information System (INIS)

    Friesen, Claudia; Lubatschofski, Annelie; Debatin, Klaus-Michael; Kotzerke, Joerg; Buchmann, Inga; Reske, Sven N.

    2003-01-01

    Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x L , a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies. (orig.)

  7. FGFR2c-mediated ERK-MAPK activity regulates coronal suture development

    Science.gov (United States)

    Pfaff, Miles J.; Xue, Ke; Li, Li; Horowitz, Mark C.; Steinbacher, Derek M.; Eswarakumar, Jacob V.P.

    2017-01-01

    Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor’s gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. PMID:27034231

  8. Mitochondrial dysfunction in lyssavirus-induced apoptosis.

    Science.gov (United States)

    Gholami, Alireza; Kassis, Raïd; Real, Eléonore; Delmas, Olivier; Guadagnini, Stéphanie; Larrous, Florence; Obach, Dorothée; Prevost, Marie-Christine; Jacob, Yves; Bourhy, Hervé

    2008-05-01

    Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

  9. Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c

    Science.gov (United States)

    Muenzner, Julia; Toffey, Jason R.; Hong, Yuning; Pletneva, Ekaterina V.

    2014-01-01

    Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes. PMID:23713573

  10. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    Science.gov (United States)

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  11. Cytochrome P450 humanised mice

    Directory of Open Access Journals (Sweden)

    Gonzalez Frank J

    2004-05-01

    Full Text Available Abstract Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s. These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach.

  12. Cytochrome P450 humanised mice

    Science.gov (United States)

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  13. The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (Triticum aestivum L.) under waterlogging.

    Science.gov (United States)

    Qi, Yuan-Hong; Mao, Fang-Fang; Zhou, Zhu-Qing; Liu, Dong-Cheng; Min-Yu; Deng, Xiang-Yi; Li, Ji-Wei; Mei, Fang-Zhu

    2018-05-02

    It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar "huamai 8" during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that

  14. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    International Nuclear Information System (INIS)

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-01

    Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  15. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  16. Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

    Science.gov (United States)

    Tanaka, Toru; Hosoi, Fumihito; Yamaguchi-Iwai, Yuko; Nakamura, Hajime; Masutani, Hiroshi; Ueda, Shugo; Nishiyama, Akira; Takeda, Shunichi; Wada, Hiromi; Spyrou, Giannis; Yodoi, Junji

    2002-04-02

    Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

  17. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    Science.gov (United States)

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

    NARCIS (Netherlands)

    Muntyan, M.S.; Cherepanov, D.A.; Malinen, A.M.; Bloch, D.A.; Sorokin, D.Y.; Severina, I.I.; Ivashina, T.V.; Lahti, R.; Muyzer, G.; Skulachev, V.P.

    2015-01-01

    Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which

  19. Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production.

    Science.gov (United States)

    Stridh, H; Fava, E; Single, B; Nicotera, P; Orrenius, S; Leist, M

    1999-10-01

    The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

  20. [Apoptosis and pathological process].

    Science.gov (United States)

    Rami, Mukhammed Salim Iusef

    2007-01-01

    Apoptosis (programmed cell death) occurs normally for maitenance of tissue homeostasis and play an important role in morphogenesis, embriogenesis and tissue growth. On the other hand, apoptosis may be involved in different pathological processes such as malignancy, infectious diseases and autoimmune disorders. Apoptosis is regulated by various mediators. Caspases, death receptors, mitochondria, Bcl-2 protoncogenes and tumor supressor genes are considered to be the most important of them. Advance in apoptosis regulation research suggests enormouse facilities for therapy of wide range of human illnesses.

  1. Radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Ohyama, Harumi

    1995-01-01

    Apoptosis is an active process of gene-directed cellular self-destruction that can be induced in many cell types via numerous physiological and pathological stimuli. We found that interphasedeath of thymocytes is a typical apoptosis showing the characteristic features of apoptosis including cell shrinkage, chromatin condensation and DNA degradation. Moderate dose of radiation induces extensive apoptosis in rapidly proliferating cell population such as the epithelium of intestinal crypt. Recent reports indicate that the ultimate form of radiation-induced mitotic death in several cells is also apoptosis. One of the hallmarks of apoptosis is the enzymatic internucleosomal degradation of chromatin DNA. We identified an endonuclease responsible for the radiation-induced DNA degradation in rat thymocytes. The death-sparing effects of interrupting RNA and protein synthesis suggested a cell genetic program for apoptosis. Apoptosis of thymocytes initiated by DNA damage, such as radiation and radio mimetic substance, absolutely requires the protein of p53 cancer suppresser gene. The cell death induced by glucocorticoid, or aging, has no such requirement. Expression of oncogene bcl-2 rescues cells from the apoptosis. Massive apoptosis in radiosensitive cells induced by higher dose radiation may be fatal. It is suggested that selective apoptotic elimination of cells would play an important role for protection against carcinogenesis and malformation through removal of cells with unrepaired radiation-induced DNA damages. Data to evaluate the significance of apoptosis in the radiation risk are still poor. Further research should be done in order to clarify the roles of the cell death on the acute and late effects of irradiation. (author)

  2. Piroxicam and C-phycocyanin mediated apoptosis in 1,2-dimethylhydrazine dihydrochloride induced colon carcinogenesis: exploring the mitochondrial pathway.

    Science.gov (United States)

    Saini, Manpreet Kaur; Sanyal, Sankar Nath; Vaiphei, Kim

    2012-04-01

    Apoptosis is a synchronized procedure of cell death that is regulated by caspases and proapoptotic proteins. During apoptosis, translocation of cytochrome c, an electron carrier, from mitochondria into the cytosol is regulated by Bcl-2 family members. Cytochrome c in association with an apoptotic protease activating factor (Apaf), a proapoptotic protein essential for cell differentiation and procaspase-9 form the apoptosome complex, which consecutively activates effector caspase, caspase-3, and coordinate the implementation of apoptosis. In the current study, an attempt has been made to gain insight into piroxicam, a traditional nonsteroidal antiinflammatory drug and c-phycocyanin, a biliprotein from Spirulina platensis (cyanobacterium) mediated apoptosis in DMH-induced colon cancer. Male Sprague-Dawley rats were segregated into 5 groups: control, DMH, DMH + piroxicam, DMH + c-phycocyanin, and DMH + piroxicam + c-phycocyanin. Results illustrated that piroxicam and c-phycocyanin treatments stimulate cytochrome c release by downregulating the Bcl-2 (an antiapoptotic protein) expression significantly, while promoting the level of Bax (a proapoptotic protein), thereby activating caspases (caspases-9 and -3) and Apaf-1. The outcomes of the present study clearly signify that piroxicam and c-phycocyanin may mediate mitochondrial-dependent apoptosis in DMH-induced colon cancer. Moreover, apoptosis induction was more apparent in the combination regimen of piroxicam and c-phycocyanin than the individual drugs alone.

  3. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  4. Hyperthermia-induced apoptosis

    NARCIS (Netherlands)

    Nijhuis, E.H.A.

    2008-01-01

    This thesis describes a number of studies that investigated several aspects of heat-induced apoptosis in human lymphoid malignancies. Cells harbour both pro- and anti-apoptotic proteins and the balance between these proteins determines whether a cell is susceptible to undergo apoptosis. In this

  5. Apoptosis in the eye.

    OpenAIRE

    Chahory , Sabine; Torriglia , Alicia

    2006-01-01

    Apoptosis is a normal component of the development and health of multicellular organisms. Cells die during apoptosis in a controlled, regulated fashion. This form of cell death is very important in eye development as well as in eye pathology. We review in this chapter our current knowledge in this topic.

  6. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  7. Tempo enhances heat-induced apoptosis by mitochondrial targeting of Bax

    International Nuclear Information System (INIS)

    Zhao, Q.-L.; Fujiwara, Y.; Kondo, T.

    2003-01-01

    A stable membrane-permeable nitroxide, Tempo, exerts an SOD-like antioxidant activity against ROS. Reportedly, Tempo inhibits ROS-induced thymocyte apoptosis, while 10 mM Tempo activates JNK1 to induce apoptosis in breast cancer cells. We have observed that nontoxic 5 mM Tempo enhances suboptimal hyperthermia (44 deg C/10 min)-induced apoptosis in U937 cells. Here we report the enhancing mechanism, focusing on activation and targeting of Bax to mitochondria and cytochrome c release. Methods: U937 cells were treated with either Tempo (5 mM, 37 deg C/10 min), heating (44 deg C/10 min), or Tempo-plus-heating, washed and incubated for various times up to 6 h, until assessing apoptosis, mitochondrial potential (ΔΨ>), and amount of superoxide by flow cytometry using Annexin V-FITC/PI, TMRM, and dihydroethidium, respectively. Bax, Bcl-2 and Bcl-XL, and cytochrome c were detected by western blotting, activated Bax was by immunoprecipitation, and ATP was by a luciferase assay. Bax targeting to and cytochrome c release from mitochorndria were also detected immunocytochemically under fluorescent microscopy. Results and Discussion: Treatment of U937 cells with 5 mM Tempo for 10 min at 37 deg C or suboptimal heating (44 deg C/ 10 min) alone did not induce apoptosis. The combined treatment with 5 mM Tempo and 44 deg C for 10 min dramatically induced ∼90% apoptosis in 6 h, as did the 44 deg C/30 min heating. During the enhanced apoptosis, cytochrome c release progressed. Although signals of Bcl-2, Bcl-XL and Bax in cell lysates were not altered, Bax was specifically activated and translocated to mitochondria after the combined treatment. Further, loss of ΔΨ>and decreases in superoxide and ATP progressed after the combined treatment, suggesting that the treatment may disturb mitochondrial electron transport. Thus, Tempo sensitizes the heat-induced apoptosis through (1) targeting of Bax to mitochondria and releasing cytochrome c, and (2) mitochondrial dysfunction

  8. Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence.

    Science.gov (United States)

    Simon, Matthieu; Metzinger-Le Meuth, Valérie; Chevance, Soizic; Delalande, Olivier; Bondon, Arnaud

    2013-01-01

    In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV-visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.

  9. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

    Science.gov (United States)

    Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay

    2014-01-01

    Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331

  10. JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

    Science.gov (United States)

    Udupi, Vidya; Yu, Margaret; Malaviya, Swati; Saavedra, Joseph E; Shami, Paul J

    2006-10-01

    Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

  11. Heat Shock Protein 70 Neutralizes Apoptosis-Inducing Factor

    Directory of Open Access Journals (Sweden)

    Guido Kroemer

    2001-01-01

    Full Text Available Programmed cell death (apoptosis is the physiological process responsible for the demise of superfluous, aged, damaged, mutated, and ectopic cells. Its normal function is essential both for embryonic development and for maintenance of adult tissue homeostasis. Deficient apoptosis participates in cancerogenesis, whereas excessive apoptosis leads to unwarranted cell loss accounting for disparate diseases including neurodegeneration and AIDS. One critical step in the process of apoptosis consists in the permeabilization of mitochondrial membranes, leading to the release of proteins which normally are secluded behind the outer mitochondrial membrane[1]. For example, cytochrome c, which is normally confined to the mitochondrial intermembrane space, is liberated from mitochondria and interacts with a cytosolic protein, Apaf-1, causing its oligomerization and constitution of the so-called apoptosome, a protein complex which activates a specific class of cysteine proteases, the caspases[2]. Another example concerns the so-called apoptosis-inducing factor (AIF, another mitochondrial intermembrane protein which can translocate to the nucleus where it induces chromatin condensation and DNA fragmentation[3].

  12. Genetic defects of cytochrome c oxidase assembly

    Czech Academy of Sciences Publication Activity Database

    Pecina, Petr; Houšťková, H.; Hansíková, H.; Zeman, J.; Houštěk, Josef

    2004-01-01

    Roč. 53, Suppl. 1 (2004), s. S213-S223 ISSN 0862-8408 R&D Projects: GA ČR GA303/03/0749 Institutional research plan: CEZ:AV0Z5011922 Keywords : cytochrome c oxidase * mitochondrial disorders Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.140, year: 2004

  13. Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

    Science.gov (United States)

    Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

    2018-05-01

    Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

  14. Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity.

    Science.gov (United States)

    Lei, Haotian; Bowler, Bruce E

    2018-06-01

    Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648-6653) show that modest movement of Ω-loop D (residues 70-85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k cat , for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Reaper-Induced Apoptosis

    National Research Council Canada - National Science Library

    Perry, Jennifer

    2005-01-01

    Reaper is a central regulator of apoptosis in the fly, Drosophila melanogaster. At the start of this proposal our laboratory identified what was believed to be a pro-apoptotic human homolog of Reaper...

  16. Apoptosis in Pneumovirus Infection

    Directory of Open Access Journals (Sweden)

    Reinout A. Bem

    2013-01-01

    Full Text Available Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  17. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

    Science.gov (United States)

    Shukla, Sanjeev; Fu, Pingfu; Gupta, Sanjay

    2014-01-01

    Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy. PMID:24563225

  18. Thermodynamics and kinetics of reduction and species conversion at a hydrophobic surface for mitochondrial cytochromes c and their cardiolipin adducts

    International Nuclear Information System (INIS)

    Ranieri, Antonio; Di Rocco, Giulia; Millo, Diego; Battistuzzi, Gianantonio; Bortolotti, Carlo A.; Lancellotti, Lidia; Borsari, Marco; Sola, Marco

    2015-01-01

    Highlights: • Cytochrome c and its adduct with cardiolipin can be immobilized on a hydrophobic SAM. • Adsorbed cytochrome c and its adduct undergo extensive unfolding and axial ligand substitution. • An equilibrium between a six-coordinated and a five-coordinated form is observed in both cases. • The reduced five-coordinated form is stabilized by cardiolipin binding. • Immobilized cytochrome c exchanges electrons more slowly upon cardiolipin binding. - Abstract: Cytochrome c (cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface

  19. Signal pathway of hippocampal apoptosis and cognitive impairment of mice caused by cerium chloride.

    Science.gov (United States)

    Cheng, Zhe; Li, Na; Cheng, Jie; Hu, Renping; Gao, Guodong; Cui, Yaling; Gong, Xiaolan; Wang, Ling; Hong, Fashui

    2012-12-01

    Experimental studies have demonstrated that lanthanides could impair cognitive functions of children and animals, but very little is known about the hippocampal apoptosis and its molecular mechanism. The study investigated the signal pathway of hippocampal apoptosis induced by intragastric administration of CeCl(3) for 60 consecutive days. It showed that cerium had been significantly accumulated in the mouse hippocampus, and CeCl(3) caused hippocampal apoptosis and impairment of spatial recognition memory of mice. CeCl(3) effectively activated caspase-3 and -9, inhibited Bcl-2, and increased the levels of Bax and cytochrome c, promoted accumulation of reactive oxygen species in the mouse hippocampus. It implied that CeCl(3)-induced apoptosis in the mouse hippocampus could be triggered via mitochondrion-mediated pathway. Our findings suggest the need for great caution to handle the lanthanides for workers and consumers. 2011 Wiley Periodicals, Inc

  20. Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

    Science.gov (United States)

    Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

    2011-07-01

    Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    Directory of Open Access Journals (Sweden)

    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  2. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    International Nuclear Information System (INIS)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-01-01

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence

  3. Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

    Directory of Open Access Journals (Sweden)

    Fu Na

    2010-10-01

    Full Text Available Abstract Objective Heme oxygenase-1 (HO-1, the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice. Methods C57BL/6J mice were fed with methionine-choline deficient (MCD diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin, HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX and/or adenovirus carrying HO-1 gene (Ad-HO-1 were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot. Results Hepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1, inhibited cytochrome c (Cyt-c release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes. Conclusions The present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

  4. Proteomics analysis of apoptosis-regulating proteins in tissues with different radiosensitivity

    International Nuclear Information System (INIS)

    An, Jeung-Hee; Seong, Jin-Sil

    2006-01-01

    The aim of this study was to identify of radiosusceptibility proteins in tissues with different radiosensitivity. C3H/HeJ mice were exposed to 10 Gy. The tissues were processed for proteins extraction and were analyzed by 2-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionizing time-of-flight mass spectrometry and validated by immunohistochemical staining and Western blotting. The peaks of apoptosis levels were 35.3±1.7% and 0.6±0.2% in the spleen and the liver, respectively, after ionizing radiation. Analysis of liver tissue showed that the expression level of reactive oxygen species (ROS) related proteins such as cytochrome c, glutathione S transferase, NADH dehydrogenase and peroxiredoxin VI increased after radiation. The expression level of cytochrome c increased to 3-fold after ionizing radiation in both tissues. However in spleen tissue, the expression level of various kinds of apoptosis regulating proteins increased after radiation. These involved iodothyronine, CD 59A glycoprotein precursor, fas antigen and tumor necrosis factor -inducible protein TSG-6nprecursor after radiation. The difference in the apoptosis index between the liver and spleen tissues is closely associated with the expression of various kinds of apoptosis-related proteins. The result suggests that the expression of apoptosis-related protein and redox proteins play important roles in this radiosusceptibility. (author)

  5. Apoptosis signaling and radiation protection

    International Nuclear Information System (INIS)

    Morita, Akinori; Suzuki, Norio; Hosoi, Yoshio

    2005-01-01

    Radiation protection by apoptosis control is the suppression of cell death in highly radiosensitive tissues. This paper describes the outline of radiation-induced apoptosis framework, apoptosis-concerned target molecules possibly related to apoptosis by radiation and their inhibitors. Although there are intrinsic (via mitochondria) and extrinsic (via death receptor) pathways in apoptosis, this review mainly mentions the former which is more important in radiation-induced apoptosis. Those molecules known at present in the apoptosis are caspase, Bcl-2 family and p53. Caspase, a group of cystein proteases, initiates apoptosis but its inhibition is known not always to result in apoptosis suppression, suggesting the existence of caspase-independent pathways. Bcl-2 family involves apoptosis-suppressing (possessing BH domains) and -promoting (lacking BH domains or possessing BH3 domain alone/BH3-only protein) groups. Two p53-transcription-dependent and one -independent pathways in p53-induced apoptosis are known and p53 can be a most possible target molecule since it positions at the start of apoptosis. Authors have found a vanadate inactivates p53. Inhibitors affecting upstream molecules of apoptosis will be the most useful candidate for apoptosis suppression/radiation protection. (S.I.) 106 refs

  6. Silencing glypican-3 expression induces apoptosis in human hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Shiyuan [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Li, Yumin, E-mail: liym@lzu.edu.cn [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Chen, Wei [Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Lanzhou University, Lanzhou 730000, Gansu (China); Zheng, Pengfei [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Liu, Tao; He, Wenting; Zhang, Junqiang; Zeng, Xiangting [Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer RNA interference GPC3 induces apoptosis in human hepatocellular carcinoma cell. Black-Right-Pointing-Pointer Silencing GPC3 resulted in the release of cytochrome c and activation of caspase-3. Black-Right-Pointing-Pointer GPC3 modulates the Bax/Bcl-2/cytochrome c/caspase-3 apoptotic signaling pathway. Black-Right-Pointing-Pointer Knockdown of GPC3 is a novel approach to HCC treatment. -- Abstract: Hepatocellular carcinoma (HCC) is one of the most common internal malignant tumors. Glypican-3 (GPC3) is involved in the biological and molecular events in the tumorigenesis of HCC. We used RNA interference to evaluate the molecular effects of GPC3 suppression at the translational level and demonstrated for the first time that GPC3 silencing results in a significant elevation of the Bax/Bcl-2 ratio, the release of cytochrome c from mitochondria and the activation of caspase-3. The results suggest that GPC3 regulates cell proliferation by enhancing the resistance to apoptosis through the dysfunction of the Bax/Bcl-2/cytochrome c/caspase-3 signaling pathway and therefore plays a critical role in the tumorigenesis of HCC. Thus, the knockdown of GPC3 should be further investigated as an attractive novel approach for the targeted gene therapy of HCC.

  7. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

    Science.gov (United States)

    Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

    2006-09-01

    The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

  8. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

    Science.gov (United States)

    Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

    2013-01-01

    Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

  9. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    International Nuclear Information System (INIS)

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-01-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA

  10. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Directory of Open Access Journals (Sweden)

    Jingfei Huang

    Full Text Available Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS generation, activation of mitochondrial permeability transition pores (MPTPs and loss of mitochondrial membrane potential (MMP were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP inhibitor cyclosporin A (CsA, which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  11. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Science.gov (United States)

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  12. Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release.

    LENUS (Irish Health Repository)

    Huber, Heinrich J

    2011-03-01

    Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m). However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell\\'s glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

  13. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    International Nuclear Information System (INIS)

    Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

    2014-01-01

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats

  14. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Fengbo [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Zhao, Bin; Zhang, Yang; Tian, Peng [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Li, Yanjun [Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Han, Zhe [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China)

    2014-09-26

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

  15. New insight into the mechanism of mitochondrial cytochrome c function

    DEFF Research Database (Denmark)

    Chertkova, Rita V; Brazhe, Nadezda A; Bryantseva, Tatiana V

    2017-01-01

    We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76...... with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c....

  16. Cytochrome P450 monooxygenases and insecticide resistance in insects.

    OpenAIRE

    Bergé, J B; Feyereisen, R; Amichot, M

    1998-01-01

    Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

  17. Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

    Science.gov (United States)

    Qin, Guiqi; Zhao, ChuBiao; Zhang, Lili; Liu, Hongyu; Quan, Yingyao; Chai, Liuying; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

    2015-08-01

    This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway. DHA did not induce Bid cleavage and translocation from cytoplasm to mitochondria and had little effects on the expressions of Puma and Noxa, but did increase Bim and Bak expressions and decrease Mcl-1 expression. Furthermore, the cytotoxicity of DHA was remarkably reduced by silencing Bim, and modestly but significantly reduced by silencing Puma or Noxa. Silencing Bim or Noxa preferentially reduced DHA-induced Bak activation, while silencing Puma preferentially reduced DHA-induced Bax activation, demonstrating that Bim and to a lesser extent Noxa act as upstream mediators to trigger the Bak-mediated intrinsic apoptosis pathway. In addition, silencing Mcl-1 enhanced DHA-induced Bak activation and apoptosis. Taken together, our data demonstrate a crucial role of Bim in preferentially regulating the Bak/Mcl-1 rheostat to mediate DHA-induced apoptosis in HCC cells.

  18. Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury

    Directory of Open Access Journals (Sweden)

    Hao Zhou

    2017-10-01

    Full Text Available Ripk3-required necroptosis and mitochondria-mediated apoptosis are the predominant types of cell death that largely account for the development of cardiac ischemia reperfusion injury (IRI. Here, we explored the effect of Ripk3 on mitochondrial apoptosis. Compared with wild-type mice, the infarcted area in Ripk3-deficient (Ripk3-/- mice had a relatively low abundance of apoptotic cells. Moreover, the loss of Ripk3 protected the mitochondria against IRI and inhibited caspase9 apoptotic pathways. These protective effects of Ripk3 deficiency were relied on mitophagy activation. However, inhibition of mitophagy under Ripk3 deficiency enhanced cardiomyocyte and endothelia apoptosis, augmented infarcted area and induced microvascular dysfunction. Furthermore, ischemia activated mitophagy by modifying FUNDC1 dephosphorylation, which substantively engulfed mitochondria debris and cytochrome-c, thus blocking apoptosis signal. However, reperfusion injury elevated the expression of Ripk3 which disrupted FUNDC1 activation and abated mitophagy, increasing the likelihood of apoptosis. In summary, this study confirms the promotive effect of Ripk3 on mitochondria-mediated apoptosis via inhibition of FUNDC1-dependent mitophagy in cardiac IRI. These findings provide new insight into the roles of Ripk3-related necroptosis, mitochondria-mediated apoptosis and FUNDC1-required mitophagy in cardiac IRI.

  19. Chloroquine inhibits autophagy and deteriorates the mitochondrial dysfunction and apoptosis in hypoxic rat neurons.

    Science.gov (United States)

    Li, Peng; Hao, Lei; Guo, Yan-Yan; Yang, Guang-Lu; Mei, Hua; Li, Xiao-Hua; Zhai, Qiong-Xiang

    2018-06-01

    Mitochondrial dysfunction (MD) and apoptosis in the neurons are associated with neonatal hypoxic-ischemic (HI) encephalopathy (HIE). The present study was to explore the influence of autophagy on the induction of MD and apoptosis in the neurons in a neonatal HIE rats and in hypoxia-treated neurons in vitro. Ten-day-old HI rat pups were sacrificed for brain pathological examination and immunohistochemical analysis. The induction of autophagy, apoptosis and MD were also determined in the neurons under hypoxia, with or without autophagy inhibitor, chloroquine (CQ) treatment. HI treatment caused atrophy and apoptosis of neurons, with a significantly increased levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase 3 and the B subunit of autophagy-related microtubule-associated protein 1 light chain 3 (LC3-B). in vitro experiments demonstrated that the hypoxia induced autophagy in neurons, as was inhibited by CQ. The hypoxia-induced cytochrome c release, cleaved caspase 3 and cleaved caspase 9 were aggravated by CQ. Moreover, there were higher levels of reactive oxygen species, more mitochondrial superoxide and less mitochondrial membrane potential in the CQ-treated neurons under hypoxia than in the neurons singularly under hypoxia. Apoptosis and autophagy were induced in HI neonatal rat neurons, autophagy inhibition deteriorates the hypoxia-induced neuron MD and apoptosis. It implies a neuroprotection of autophagy in the hypoxic-ischemic encephalopathy. Administration of autophagy inducer agents might be promising in HIE treatment. Copyright © 2018. Published by Elsevier Inc.

  20. Prediction of cytochrome P450 mediated metabolism

    DEFF Research Database (Denmark)

    Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen

    2015-01-01

    Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard...... to rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure...

  1. Mitochondria in neutrophil apoptosis

    NARCIS (Netherlands)

    van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

    2006-01-01

    Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

  2. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  3. Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

    Science.gov (United States)

    Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

    2004-01-01

    Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

  4. Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation.

    Science.gov (United States)

    Lewis, Joan S; Meeke, Kathleen; Osipo, Clodia; Ross, Eric A; Kidawi, Noman; Li, Tianyu; Bell, Eric; Chandel, Navdeep S; Jordan, V Craig

    2005-12-07

    We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psim) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psim, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.

  5. Apoptosis and inflammation

    Directory of Open Access Journals (Sweden)

    C. Haanen

    1995-01-01

    Full Text Available During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972 introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.

  6. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

    Science.gov (United States)

    Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

    2009-12-01

    During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

  7. The SMARTCyp cytochrome P450 metabolism prediction server

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Gloriam, David Erik Immanuel; Olsen, Lars

    2010-01-01

    The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism.......The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism....

  8. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

  9. Neem oil limonoids induces p53-independent apoptosis and autophagy

    Science.gov (United States)

    Chandra, Dhyan

    2012-01-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  10. Optimization of Si–C reaction temperature and Ge thickness in C-mediated Ge dot formation

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Yuhki, E-mail: yu-ki@ecei.tohoku.ac.jp; Itoh, Yuhki; Kawashima, Tomoyuki; Washio, Katsuyoshi

    2016-03-01

    To form Ge dots on a Si substrate, the effect of thermal reaction temperature of sub-monolayer C with Si (100) was investigated and the deposited Ge thickness was optimized. The samples were prepared by solid-source molecular beam epitaxy with an electron-beam gun for C sublimation and a Knudsen cell for Ge evaporation. C of 0.25 ML was deposited on Si (100) at a substrate temperature of 200 °C, followed by a high-temperature treatment at the reaction temperature (T{sub R}) of 650–1000 °C to create Si–C bonds. Ge equivalent to 2 to 5 nm thick was subsequently deposited at 550 °C. Small and dense dots were obtained for T{sub R} = 750 °C but the dot density decreased and the dot diameter varied widely in the case of lower and higher T{sub R}. A dot density of about 2 × 10{sup 10} cm{sup −2} was achieved for Ge deposition equivalent to 3 to 5 nm thick and a standard deviation of dot diameter was the lowest of 10 nm for 5 nm thick Ge. These results mean that C-mediated Ge dot formation was strongly influenced not only by the c(4 × 4) reconstruction condition through the Si–C reaction but also the relationship between the Ge deposition thickness and the exposed Si (100)-(2 × 1) surface area. - Highlights: • The effect of Si–C reaction temperature on Ge dot formation was investigated. • Small and dense dots were obtained for T{sub R} = 750 °C. • The dot density of about 2 × 10{sup 10} cm{sup −2} was achieved for Ge = 3 to 5 nm. • The standard deviation of dot diameter was the lowest of 10 nm at Ge = 5 nm.

  11. Fullerene and apoptosis

    Directory of Open Access Journals (Sweden)

    M. A. Orlova

    2013-01-01

    Full Text Available Fullerene derivatives superfamily attracts a serious attention as antiviral and anticancer agents and drug delivery carriers as well. A large number of such fullerene С60 derivatives obtained to date. However, there is an obvious deficit of information about causes and mechanisms of immediately and long-term consequences of their effects in vivo which is a true obstacle on the way leading to practical medical use of them. First, this concerns their impact on the proliferation, apoptosis and necrosis regulation. Fullerene nanoparticle functionalization type, their sizes and surface nanopathology are of great importance to further promoting of either cytoprotective or cytotoxic effects. This lecture provides modern concept analysis regarding fullerenes effects on apoptosis pathway in normal and tumor cells.

  12. Cl- channels in apoptosis

    DEFF Research Database (Denmark)

    Wanitchakool, Podchanart; Ousingsawat, Jiraporn; Sirianant, Lalida

    2016-01-01

    A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K(+), Cl(-), and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl(-) channels LRRC8, TMEM16/anoctamin......, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also......(-) channels or as regulators of other apoptotic Cl(-) channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated...

  13. Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

    International Nuclear Information System (INIS)

    Noda, Chiseko; He, Jinsong; Takano, Tomoko; Tanaka, Chisato; Kondo, Toshinori; Tohyama, Kaoru; Yamamura, Hirohei; Tohyama, Yumi

    2007-01-01

    (-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells

  14. Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

    Science.gov (United States)

    Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

    2017-05-01

    Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Mature neurons dynamically restrict apoptosis via redundant premitochondrial brakes.

    Science.gov (United States)

    Annis, Ryan P; Swahari, Vijay; Nakamura, Ayumi; Xie, Alison X; Hammond, Scott M; Deshmukh, Mohanish

    2016-12-01

    Apoptotic cell death is critical for the early development of the nervous system, but once the nervous system is established, the apoptotic pathway becomes highly restricted in mature neurons. However, the mechanisms underlying this increased resistance to apoptosis in these mature neurons are not completely understood. We have previously found that members of the miR-29 family of microRNAs (miRNAs) are induced with neuronal maturation and that overexpression of miR-29 was sufficient to restrict apoptosis in neurons. To determine whether endogenous miR-29 alone was responsible for the inhibition of cytochrome c release in mature neurons, we examined the status of the apoptotic pathway in sympathetic neurons deficient for all three miR-29 family members. Unexpectedly, we found that the apoptotic pathway remained largely restricted in miR-29-deficient mature neurons. We therefore probed for additional mechanisms by which mature neurons resist apoptosis. We identify miR-24 as another miRNA that is upregulated in the maturing cerebellum and sympathetic neurons that can act redundantly with miR-29 by targeting a similar repertoire of prodeath BH3-only genes. Overall, our results reveal that mature neurons engage multiple redundant brakes to restrict the apoptotic pathway and ensure their long-term survival. © 2016 Federation of European Biochemical Societies.

  16. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    OpenAIRE

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

    2012-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. O...

  17. Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death.

    LENUS (Irish Health Repository)

    Huber, Heinrich J

    2010-01-01

    ABSTRACT: BACKGROUND: Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations. RESULTS: Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated. CONCLUSIONS: Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to

  18. Flower colour and cytochromes P450.

    Science.gov (United States)

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

  19. Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

    Science.gov (United States)

    Lee, Kang-In; Choi, Han-Gyu; Son, Yeo-Jin; Whang, Jake; Kim, Kwangwook; Jeon, Heat Sal; Park, Hye-Soo; Back, Yong Woo; Choi, Seunga; Kim, Seong-Woo; Choi, Chul Hee; Kim, Hwa-Jung

    2016-04-01

    Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

  20. Troglitazone induced apoptosis via PPARγ activated POX-induced ROS formation in HT29 cells.

    Science.gov (United States)

    Wang, Jing; Lv, XiaoWen; Shi, JiePing; Hu, XiaoSong; DU, YuGuo

    2011-08-01

    In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells, the effects of PPARγ and POX-induced ROS were explored. [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V and PI staining using FACS, plasmid transfection, ROS formation detected by DCFH staining, RNA interference, RT-PCR & RT-QPCR, and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells. Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis. During this process, mitochondria related pathways including ROS formation, POX expression and cytochrome c release increased, which were inhibited by pretreatment with GW9662, a specific antagonist of PPARγ. These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern. Furthermore, the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone. The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation, at least partly, via PPARγ activation. Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

  1. Arsenic induced apoptosis in rat liver following repeated 60 days exposure

    International Nuclear Information System (INIS)

    Bashir, Somia; Sharma, Yukti; Irshad, M.; Nag, T.C.; Tiwari, Monica; Kabra, M.; Dogra, T.D.

    2006-01-01

    Background: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 μM) concentration. Aim: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. Methods: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5 mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. Results: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. Conclusion: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis

  2. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    Science.gov (United States)

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  3. Effect of dicycloplatin, a novel platinum chemotherapeutical drug, on inhibiting cell growth and inducing cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Guang-quan Li

    Full Text Available Dicycloplatin, a new supramolecular platinum-based antitumor drug, has been approved by the State Food and Administration (SFDA of China. In this study, we investigated the anticancer activity of dicycloplatin in cancer cells and signaling pathways involved in dicycloplatin-induced apoptosis. Dicycloplatin inhibited the proliferation of cancer cells and increased the percentage of apoptosis in a concentration-dependent manner. Besides, some apoptosis related events were observed after treatment with dicycloplatin, including increase of reactive oxygen species (ROS, collapse of mitochondrial membrane potential (Δψm, release of cytochrome c from the mitochondria to the cytosol, upregulation of p53, which were accompanied by activation of caspase-9, caspase-3, caspase-8, and poly (ADP-ribose polymerase cleavage in a concentration-dependent manner. The role of apoptosis in dicycloplatin-mediated cell death was further confirmed by the concomitant treatment with caspase-8 or caspase-9 inhibitors, which inhibited apoptosis and PARP cleavage. Intracellular glutathione (GSH was also found to inhibit the cytotoxic effect of dicycloplatin. In conclusion, these findings suggest that dicycloplatin induces apoptosis through ROS stress-mediated death receptor pathway and mitochondrial pathway which is similar to carboplatin.

  4. Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways.

    Science.gov (United States)

    Saleh, A M; Vijayasarathy, C; Masoud, L; Kumar, L; Shahin, A; Kambal, A

    2003-07-01

    The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide

  5. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  6. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-01-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  7. NMR comparison of prokaryotic and eukaryotic cytochromes c

    International Nuclear Information System (INIS)

    Chau, Meihing; Cai, Meng Li; Timkovich, R.

    1990-01-01

    1 H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degree C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c and mutants of yeast iso-1 cytochrome reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent

  8. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N. [Univ. of Rochester, NY (United States)

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K+ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K+ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

  9. Biogenesis of the yeast cytochrome bc1 complex.

    Science.gov (United States)

    Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

    2009-01-01

    The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

  10. Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

    Science.gov (United States)

    Papa, S; Lorusso, M; Izzo, G; Capuano, F

    1981-02-15

    1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

  11. Elucidating cytochrome C release from mitochondria: insights from an in silico three-dimensional model.

    Science.gov (United States)

    Tam, Zhi Yang; Cai, Yi Hui; Gunawan, Rudiyanto

    2010-11-17

    Mitochondrial regulation of apoptosis depends on the programmed release of proapoptotic proteins such as cytochrome c (Cyt c) through the outer mitochondrial membrane (OMM). Although a few key processes involved in this release have been identified, including the liberation of inner membrane-bound Cyt c and formation of diffusible pores on the OMM, other details like the transport of Cyt c within complex mitochondrial compartments, e.g., the cristae and crista junctions, are not yet fully understood (to our knowledge). In particular, a remodeling of the inner mitochondrial membrane accompanying apoptosis seen in a few studies, in which crista junctions widen, has been hypothesized to be a necessary step in the Cyt c release. Using a three-dimensional spatial modeling of mitochondrial crista and the crista junction, model simulations and analysis illustrated how the interplay among solubilization of Cyt c, fast diffusion of Cyt c, and OMM permeabilization gives rise to the observed experimental release profile. Importantly, the widening of the crista junction was found to have a negligible effect on the transport of free Cyt c from cristae. Finally, model simulations showed that increasing the fraction of free/loosely-bound Cyt c can sensitize the cell to apoptotic stimuli in a threshold manner, which may explain increased sensitivity to cell death associated with aging. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Silencing, positive selection and parallel evolution: busy history of primate cytochromes C.

    Science.gov (United States)

    Pierron, Denis; Opazo, Juan C; Heiske, Margit; Papper, Zack; Uddin, Monica; Chand, Gopi; Wildman, Derek E; Romero, Roberto; Goodman, Morris; Grossman, Lawrence I

    2011-01-01

    Cytochrome c (cyt c) participates in two crucial cellular processes, energy production and apoptosis, and unsurprisingly is a highly conserved protein. However, previous studies have reported for the primate lineage (i) loss of the paralogous testis isoform, (ii) an acceleration and then a deceleration of the amino acid replacement rate of the cyt c somatic isoform, and (iii) atypical biochemical behavior of human cyt c. To gain insight into the cause of these major evolutionary events, we have retraced the history of cyt c loci among primates. For testis cyt c, all primate sequences examined carry the same nonsense mutation, which suggests that silencing occurred before the primates diversified. For somatic cyt c, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded the same tree topology. The evolutionary analyses show that a fast accumulation of non-synonymous mutations (suggesting positive selection) occurred specifically on the anthropoid lineage root and then continued in parallel on the early catarrhini and platyrrhini stems. Analysis of evolutionary changes using the 3D structure suggests they are focused on the respiratory chain rather than on apoptosis or other cyt c functions. In agreement with previous biochemical studies, our results suggest that silencing of the cyt c testis isoform could be linked with the decrease of primate reproduction rate. Finally, the evolution of cyt c in the two sister anthropoid groups leads us to propose that somatic cyt c evolution may be related both to COX evolution and to the convergent brain and body mass enlargement in these two anthropoid clades.

  13. Intervertebral Disc Degeneration : The Role of the Mitochondrial Pathway in Annulus Fibrosus Cell Apoptosis Induced by Overload

    OpenAIRE

    Rannou, François; Lee, Tzong-Shyuan; Zhou, Rui-Hai; Chin, Jennie; Lotz, Jeffrey C.; Mayoux-Benhamou, Marie-Anne; Barbet, Jacques Patrick; Chevrot, Alain; Shyy, John Y.-J.

    2004-01-01

    Degeneration of the intervertebral disk (IVD) is a major pathological process implicated in low back pain and is a prerequisite to disk herniation. Although mechanical stress is an important modulator of the degeneration, the underlying molecular mechanism remains unclear. The association of human IVD degeneration, assessed by magnetic resonance imaging, with annulus fibrosus cell apoptosis and anti-cytochrome c staining revealed that the activation of the mitochondria-dependent apoptosome wa...

  14. Plastocyanin/cytochrome c6 interchange in Scenedesmus vacuolatus.

    Science.gov (United States)

    Miramar, M Dolores; Inda, Luis A; Saraiva, Lígia M; Peleato, M Luisa

    2003-12-01

    Plastocyanin and cytochrome c6 from the green alga Scenedesmus vacuolatus were immunoquantified in cells grown under different concentrations of copper and iron. Plastocyanin expression was constitutive, its synthesis was not significantly affected by iron availability, and increases with copper availability. On the contrary, cytochrome c6 synthesis is repressed by copper, and only residual amounts of the protein were detected at 0.1 micromol/L copper. Under copper deficiency, cytochrome c6 is slightly dependent on iron. In natural environments, plastocyanin seems to be the predominant electron donor to P700.

  15. Reassessing apoptosis in plants.

    Science.gov (United States)

    Dickman, Martin; Williams, Brett; Li, Yurong; de Figueiredo, Paul; Wolpert, Thomas

    2017-10-01

    Cell death can be driven by a genetically programmed signalling pathway known as programmed cell death (PCD). In plants, PCD occurs during development as well as in response to environmental and biotic stimuli. Our understanding of PCD regulation in plants has advanced significantly over the past two decades; however, the molecular machinery responsible for driving the system remains elusive. Thus, whether conserved PCD regulatory mechanisms include plant apoptosis remains enigmatic. Animal apoptotic regulators, including Bcl-2 family members, have not been identified in plants but expression of such regulators can trigger or suppress plant PCD. Moreover, plants exhibit nearly all of the biochemical and morphological features of apoptosis. One difference between plant and animal PCD is the absence of phagocytosis in plants. Evidence is emerging that the vacuole may be key to removal of unwanted plant cells, and may carry out functions that are analogous to animal phagocytosis. Here, we provide context for the argument that apoptotic-like cell death occurs in plants.

  16. Esculetin Ameliorates Carbon Tetrachloride-Mediated Hepatic Apoptosis in Rats

    Directory of Open Access Journals (Sweden)

    Chuan-Sung Chiu

    2011-06-01

    Full Text Available Esculetin (ESC is a coumarin that is present in several plants such as Fraxinus rhynchophylla and Artemisia capillaris. Our previous study found that FR ethanol extract (FREtOH significantly ameliorated rats’ liver function. This study was intended to investigate the protective mechanism of ESC in hepatic apoptosis in rats induced by carbon tetrachloride. Rat hepatic apoptosis was induced by oral administration of CCl4. All rats were administered orally with CCl4 (20%, 0.5 mL/rat twice a week for 8 weeks. Rats in the ESC groups were treated daily with ESC, and silymarin group were treated daily with silymarin. Serum alanine aminotransferase (ALT, aspartate aminotransferase (AST as well as the activities of the anti-oxidative enzymes glutathione peroxidase (GPx, superoxide dismutase (SOD, and catalase in the liver were measured. In addition, expression of liver apoptosis proteins and anti-apoptotic proteins were detected. ESC (100, 500 mg/kg significantly reduced the elevated activities of serum ALT and AST caused by CCl4 and significantly increased the activities of catalase, GPx and SOD. Furthermore, ESC (100, 500 mg/kg significantly decreased the levels of the proapoptotic proteins (t-Bid, Bak and Bad and significantly increased the levels of the anti-apoptotic proteins (Bcl-2 and Bcl-xL. ESC inhibited the release of cytochrome c from mitochondria. In addition, the levels of activated caspase-9 and activated caspase-3 were significantly decreased in rats treated with ESC than those in rats treated with CCl4 alone. ESC significantly reduced CCl4-induced hepatic apoptosis in rats.

  17. Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

    Czech Academy of Sciences Publication Activity Database

    Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

    2013-01-01

    Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

  18. North African genetic variation of cytochrome and sulfotransferase ...

    African Journals Online (AJOL)

    in these genes have shown relevant ethnic differences among Sub-Saharan .... This cytochrome catalyzes a big amount of oxidative reactions of substances like ... with samples of European and African origin (because of the scarce data ...

  19. Isolation and purification of membrane-bound cytochrome c from ...

    African Journals Online (AJOL)

    Administrator

    2007-05-02

    ferrochrome and redox spectra showed the presence of heme-c. Key words: Cytochrome c, respiratory chain and Proteus mirabilis. INTRODUCTION. Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium.

  20. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  1. Cardiovascular molecular imaging of apoptosis

    International Nuclear Information System (INIS)

    Wolters, S.L.; Reutelingsperger, C.P.M.; Corsten, M.F.; Hofstra, L.; Narula, J.

    2007-01-01

    Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible. Apoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far. This review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies. (orig.)

  2. Cardiovascular molecular imaging of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wolters, S.L.; Reutelingsperger, C.P.M. [Maastricht University, Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht (Netherlands); Corsten, M.F.; Hofstra, L. [Maastricht University, Department of Cardiology, Cardiovascular Research Institute Maastricht, P.O. Box 616, Maastricht (Netherlands); Narula, J. [University of California Irvine, Department of Cardiology, Irvine (United States)

    2007-06-15

    Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible. Apoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far. This review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies. (orig.)

  3. Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients.

    Science.gov (United States)

    Elango, Tamilselvi; Thirupathi, Anand; Subramanian, Swapna; Ethiraj, Purushoth; Dayalan, Haripriya; Gnanaraj, Pushpa

    2017-08-01

    Psoriasis is a chronic inflammatory skin disease characterized by hyper proliferation of keratinocytes. Recent data show that the epidermis thickening in psoriasis may be related to imbalance of homeostasis caused by abnormal apoptotic process. Maintenance of keratinocyte apoptotic process is very important in psoriasis. Methotrexate (MTX) has been used for many years to restore the normal skin in psoriasis condition. However, the exact mechanism of MTX in psoriasis condition is poorly understood. The aim of this study was to examine the role of MTX on keratinocyte apoptosis pathway in psoriasis patients. A total of 58 psoriasis vulgaris patients were recruited for this study. Nonlesional skin biopsies served as control. Skin biopsies of psoriatic patients were collected and analyzed for cytosolic, mitochondria and total cytochrome c by ELISA. Expression of caspase-9, NFκBp65, pAkt1 by western blot, real-time PCR and immunohistochemical analysis of c-FLIP protein was analyzed in nonlesional and lesional skin biopsies before (day 0) and after (at the end of 6 and 12 weeks) MTX treatment. After MTX treatment, a significant increase in cytochrome c was observed when compared with before MTX treatment in psoriasis patients (p psoriasis by controlling the acanthosis.

  4. The biodiversity of microbial cytochromes P450.

    Science.gov (United States)

    Kelly, Steven L; Lamb, David C; Jackson, Colin J; Warrilow, Andrew G; Kelly, Diane E

    2003-01-01

    The cytochrome P450 (CYP) superfamily of genes and proteins are well known for their involvement in pharmacology and toxicology, but also increasingly for their importance and diversity in microbes. The extent of diversity has only recently become apparent with the emergence of data from whole genome sequencing projects and the coming years will reveal even more information on the diversity in microbial eukaryotes. This review seeks to describe the historical development of these studies and to highlight the importance of the genes and proteins. CYPs are deeply involved in the development of strategies for deterrence and attraction as well as detoxification. As such, there is intense interest in pathways of secondary metabolism that include CYPs in oxidative tailoring of antibiotics, sometimes influencing potency as bioactive compounds. Further to this is interest in CYPs in metabolism of xenobiotics for use as carbon sources for microbial growth and as biotransformation agents or in bioremediation. CYPs are also current and potential drug targets; compounds inhibiting CYP are antifungal and anti-protozoan agents, and potentially similar compounds may be useful against some bacterial diseases such as tuberculosis. Of note is the diversity of CYP requirements within an organism, ranging from Escherichia coli that has no CYPs as in many bacteria, to Mycobacterium smegmatis that has 40 representing 1% of coding genes. The basidiomycete fungus Phanerochaete chrysosporium surprised all when it was found to contain a hundred or more CYPs. The functional genomic investigation of these orphan CYPs is a major challenge for the future.

  5. Geometrical analysis of cytochrome c unfolding

    Science.gov (United States)

    Urie, Kristopher G.; Pletneva, Ekaterina; Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

    2011-01-01

    A geometrical model has been developed to study the unfolding of iso-1 cytochrome c. The model draws on the crystallographic data reported for this protein. These data were used to calculate the distance between specific residues in the folded state, and in a sequence of extended states defined by n = 3, 5, 7, 9, 11, 13, and 15 residue units. Exact calculations carried out for each of the 103 residues in the polypeptide chain demonstrate that different regions of the chain have different unfolding histories. Regions where there is a persistence of compact structures can be identified, and this geometrical characterization is fully consistent with analyses of time-resolved fluorescence energy-transfer (TrFET) data using dansyl-derivatized cysteine side-chain probes at positions 39, 50, 66, 85, and 99. The calculations were carried out assuming that different regions of the polypeptide chain unfold synchronously. To test this assumption, lattice Monte Carlo simulations were performed to study systematically the possible importance of asynchronicity. Calculations show that small departures from synchronous dynamics can arise if displacements of residues in the main body of the chain are much more sluggish than near-terminal residues.

  6. Epidermal CYP2 family cytochromes P450

    International Nuclear Information System (INIS)

    Du Liping; Hoffman, Susan M.G.; Keeney, Diane S.

    2004-01-01

    Skin is the largest and most accessible drug-metabolizing organ. In mammals, it is the competent barrier that protects against exposure to harmful stimuli in the environment and in the systemic circulation. Skin expresses many cytochromes P450 that have critical roles in exogenous and endogenous substrate metabolism. Here, we review evidence for epidermal expression of genes from the large CYP2 gene family, many of which are expressed preferentially in extrahepatic tissues or specifically in epithelia at the environmental interface. At least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals, and the majority of these genes are expressed in epidermis or cultured keratinocytes. Where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages. The tissue-specific transcriptional regulation of CYP2 genes in the epidermis, and in other epithelia that interface with the environment, suggests important roles for at least some CYP2 gene products in the production and disposition of molecules affecting competency of the epidermal barrier

  7. Novel extrahepatic cytochrome P450s

    International Nuclear Information System (INIS)

    Karlgren, Maria; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-01-01

    The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis

  8. Polyethylene glycol promotes autoxidation of cytochrome c.

    Science.gov (United States)

    Sato, Wataru; Uchida, Takeshi; Saio, Tomohide; Ishimori, Koichiro

    2018-06-01

    Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Goyal, Shruti; Amar, Saroj Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Dubey, Divya; Pal, Manish Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Singh, Jyoti [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Verma, Ankit; Kushwaha, Hari Narayan [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Ray, Ratan Singh, E-mail: ratanray.2011@rediffmail.com [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India)

    2015-12-30

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  10. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    International Nuclear Information System (INIS)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-01-01

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  11. A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

    Directory of Open Access Journals (Sweden)

    Sebastián Zamorano

    Full Text Available Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

  12. Electromagnetic radiation at 900 MHz induces sperm apoptosis through bcl-2, bax and caspase-3 signaling pathways in rats.

    Science.gov (United States)

    Liu, Qi; Si, Tianlei; Xu, Xiaoyun; Liang, Fuqiang; Wang, Lufeng; Pan, Siyi

    2015-08-04

    The decreased reproductive capacity of men is an important factor contributing to infertility. Accumulating evidence has shown that Electromagnetic radiation potentially has negative effects on human health. However, whether radio frequency electromagnetic radiation (RF-EMR) affects the human reproductive system still requires further investigation. Therefore, The present study investigates whether RF-EMR at a frequency of 900 MHz can trigger sperm cell apoptosis and affect semen morphology, concentration, and microstructure. Twenty four rats were exposed to 900 MHz electromagnetic radiation with a special absorption rate of 0.66 ± 0.01 W/kg for 2 h/d. After 50d, the sperm count, morphology, apoptosis, reactive oxygen species (ROS), and total antioxidant capacity (TAC), representing the sum of enzymatic and nonenzymatic antioxidants, were investigated. Western blotting and reverse transcriptase PCR were used to determine the expression levels of apoptosis-related proteins and genes, including bcl-2, bax, cytochrome c, and capase-3. In the present study, the percentage of apoptotic sperm cells in the exposure group was significantly increased by 91.42% compared with the control group. Moreover, the ROS concentration in exposure group was increased by 46.21%, while the TAC was decreased by 28.01%. Radiation also dramatically decreased the protein and mRNA expression of bcl-2 and increased that of bax, cytochrome c, and capase-3. RF-EMR increases the ROS level and decreases TAC in rat sperm. Excessive oxidative stress alters the expression levels of apoptosis-related genes and triggers sperm apoptosis through bcl-2, bax, cytochrome c and caspase-3 signaling pathways.

  13. Blockade of store-operated calcium entry alleviates ethanol-induced hepatotoxicity via inhibiting apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ruibing [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Lihui [Shandong Normal University, Jinan, Shandong Province 250012 (China); Luo, Zheng; Guo, Xiaolan [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Ming, E-mail: ymylh@163.com [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China)

    2015-08-15

    Extracellular Ca{sup 2+} influx has been suggested to play a role in ethanol-induced hepatocyte apoptosis and necrosis. Previous studies indicated that store-operated Ca{sup 2+} entry (SOCE) was involved in liver injury induced by ethanol in HepG2 cells. However, the mechanisms underlying liver injury caused by SOCE remain unclear. We aimed to investigate the effects and mechanism of SOCE inhibition on liver injury induced by ethanol in BRL cells and Sprague–Dawley rats. Our data demonstrated that ethanol (0–400 mM) dose-dependently increased hepatocyte injury and 100 mM ethanol significantly upregulated the mRNA and protein expression of SOC for at least 72 h in BRL cells. Blockade of SOCE by pharmacological inhibitors and sh-RNA knockdown of STIM1 and Orai1 attenuated intracellular Ca{sup 2+} overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and inhibited ethanol-induced apoptosis. STIM1 and Orai1 expression was greater in ethanol-treated than control rats, and the SOCE inhibitor corosolic acid ameliorated the histopathological findings and alanine transaminase and aspartate transaminase activity as well as decreased cytochrome C release and inhibited alcohol-induced cell apoptosis. These findings suggest that SOCE blockade could alleviate alcohol-induced hepatotoxicity via inhibiting apoptosis. SOCE might be a useful therapeutic target in alcoholic liver diseases. - Highlights: • Blockade of SOCE alleviated overload of Ca{sup 2+} and hepatotoxicity after ethanol application. • Blockade of SOCE inhibited mitochondrial apoptosis after ethanol application. • SOCE might be a useful therapeutic target in alcoholic liver diseases.

  14. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

    Science.gov (United States)

    Sacco, James C; Trepanier, Lauren A

    2010-01-01

    NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (Phydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

  15. Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, Alexander N., E-mail: ovolkov@vub.ac.be; Nuland, Nico A. J. van [Vrije Universiteit Brussel, Jean Jeener NMR Centre, Structural Biology Brussels (Belgium)

    2013-07-15

    Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc-CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.

  16. Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression.

    Science.gov (United States)

    Lim, Eun Jin; Heo, Jeonghoon; Kim, Young-Ho

    2015-08-01

    Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies.

  17. Bullatacin Triggered ABCB1-Overexpressing Cell Apoptosis via the Mitochondrial-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Yong-Ju Liang

    2009-01-01

    Full Text Available This paper was to explore bullatacin-mediated multidrug-resistant cell apoptosis at extremely low concentration. To investigate its precise mechanisms, the pathway of cell apoptosis induced by bullatacin was examined. Bullatacin causes an upregulation of ROS and a downregulation of ΔΨm in a concentration-dependent manner in ABCB1-overexpressing KBv200 cells. In addition, cleavers of caspase-9, caspase-3, and PARP were observed following the release of cytochrome c from mitochondria after bullatacin treatment. However, neither cleavage of caspase-8 nor change of expression level of bcl-2, bax and Fas was observed by the same treatment. Pretreating KBv200 cells with N-acetylcysteine, an antioxidant modulator, resulted in a significant reduction of ROS generation and cell apoptosis induced by bullatacin. Bullatacin-induced apoptosis was antagonized by z-LEHD-fmk, a caspase-9 inhibitor, but not by z-IETD-fmk, a caspase-8 inhibitor. These implied that apoptosis of KBv200 cells induced by bullatacin was associated with the mitochondria-dependent pathway that was limited to activation of apical caspase-9.

  18. Production and characterization of yeast cytochrome c antibodies; immunological studies of mutants with altered cytochrome c synthesis

    International Nuclear Information System (INIS)

    Matner, R.R.

    1980-01-01

    Mutations at the structural gene, CYC1, for iso-1-cytochrome c and at the structural gene, CYC7, for iso-2-cytochrome c can reduce the levels of the respective proteins by varying degrees in Saccharomyces cerevisiae. Mutations at two other loci, cyc2 and cyc3, that are unlinked to either of the structural genes, specifically reduced the levels of both iso-cytochromes c. The cyc2 mutations can cause as low as 10 to 20% of the normal level and cyc3 mutations can cause complete deficiencies. We have explored the possiblity that the CYC2 and CYC3 loci code for maturation functions in the biosynthesis of cytochrome c. The approach used to characterize the nature of the cyc2 and cyc3 induced deficiencies of cytochrome c involved four steps. The results were used to propose possible roles for the CYC2 and CYC3 encoded functions. The CYC3 encoded function is hypothesized to be enzymatic heme attachment. CYC2 may code for a protein that binds and transports apo-cytochrome c through the outer mitochondrial membrane and/or enhances the activity of the heme attachment enzyme

  19. Therapeutic assessment of cytochrome C for the prevention of obesity through endothelial cell-targeted nanoparticulate system.

    Science.gov (United States)

    Hossen, Md Nazir; Kajimoto, Kazuaki; Akita, Hidetaka; Hyodo, Mamoru; Ishitsuka, Taichi; Harashima, Hideyoshi

    2013-03-01

    Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.

  20. Cytochrome P450s and molecular epidemiology

    Science.gov (United States)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

    Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

  1. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Meili, E-mail: fumeilidrlinyi@tom.com [Department of Infectious Disease, Linyi People' s Hospital, Linyi 276000 (China); Wan, Fuqiang [Department of Head and Neck Surgery, Linyi Tumor Hospital, Linyi 276000 (China); Li, Zhengling [Department of Nursing, Tengzhou Central People' s Hospital, Tengzhou 277500 (China); Zhang, Fenghua [Department of Operating Room, Linyi People' s Hospital, Linyi 276000 (China)

    2016-03-04

    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. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.

  2. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.

  3. Lycopene inhibits regulator of calcineurin 1-mediated apoptosis by reducing oxidative stress and down-regulating Nucling in neuronal cells.

    Science.gov (United States)

    Lim, Seiyoung; Hwang, Sinwoo; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2017-05-01

    Regulator of calcineurin 1 (RCAN1) is located on the Down syndrome critical region (DSCR) locus in human chromosome 21. Oxidative stress and overexpression of RCAN1 are implicated in neuronal impairment in Down's syndrome (DS) and Alzheimer's disease (AD). Serum level of lycopene, an antioxidant pigment, is low in DS and AD patients, which may be related to neuronal damage. The present study is to investigate whether lycopene inhibits apoptosis by reducing ROS levels, NF-κB activation, expression of the apoptosis regulator Nucling, cell viability, and indices of apoptosis (cytochrome c release, caspase-3 activation) in RCAN1-overexpressing neuronal cells. Cells transfected with either pcDNA or RCAN1 were treated with or without lycopene. Lycopene decreased intracellular and mitochondrial ROS levels, NF-κB activity, and Nucling expression while it reversed decrease in mitochondrial membrane potential, mitochondrial respiration, and glycolytic function in RCAN1-overexpressing cells. Lycopene inhibited cell death, DNA fragmentation, caspase-3 activation, and cytochrome c release in RCAN1-overexpressing cells. Lycopene inhibits RCAN1-mediated apoptosis by reducing ROS levels and by inhibiting NF-κB activation, Nucling induction, and the increase in apoptotic indices in neuronal cells. Consumption of lycopene-rich foods may prevent oxidative stress-associated neuronal damage in some pathologic conditions such as DS or AD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

    Science.gov (United States)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    Energy Technology Data Exchange (ETDEWEB)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

    2013-02-15

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

  6. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    International Nuclear Information System (INIS)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

    2013-01-01

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

  7. Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

    International Nuclear Information System (INIS)

    Voorman, R.

    1987-01-01

    In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD

  8. Bcl-2 family-regulated apoptosis in health and disease

    Directory of Open Access Journals (Sweden)

    Grant Dewson

    2010-04-01

    Full Text Available Grant Dewson, Ruth M KluckMolecular Genetics of Cancer Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, AustraliaAbstract: Apoptotic cell death is essential for embryonic development, tissue homeostasis, and a well-functioning immune system, with aberrant apoptosis contributing to numerous disease conditions. Inadequate cell death is a major contributing factor to tumorigenesis, while excess cell death contributes to neurodegeneration and autoimmune disease. The major pathway of apoptotic cell death, the mitochondrial pathway, is controlled by the Bcl-2 family of proteins. The members of this family, more than 17 in humans, share significant sequence and structural homology, and fulfil either prosurvival or proapoptotic roles. Specific interactions between these functionally polar proteins, and their relative expression levels, govern the susceptibility of each cell to toxic insults. Here we review the current understanding on how apoptotic cell death is controlled by this important protein family. We also discuss how excessive or insufficient cell death can contribute to disease, and how targeting the Bcl-2 family offers novel therapeutic opportunities.Keywords: apoptosis, Bcl-2, cancer, cytochrome c, mitochondria

  9. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  10. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-01-01

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G 2 /M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC 50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G 2 /M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  11. Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

    International Nuclear Information System (INIS)

    Kresheck, G.C.; Erman, J.E.

    1988-01-01

    Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (t/sub m/) were 43.9 +- 1.4 and 63.3 +- 1.6 0 C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +- 1.3 0 C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase

  12. Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

    Science.gov (United States)

    Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

    2007-08-03

    Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

  13. Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, I-00146 Roma (Italy); Ciaccio, Chiara [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy); Sinibaldi, Federica; Santucci, Roberto [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Coletta, Massimo [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari (Italy)

    2011-01-07

    Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

  14. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2004-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of PCa cells by methyl selenium (Se)/selenol...

  15. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2008-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of prostate cancer (PCa...

  16. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2003-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of PCa cells by methyl selenium (Se...

  17. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2005-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of prostate cancer (PCa...

  18. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2007-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of prostate cancer (PCa...

  19. Methylselenium and Prostate Cancer Apoptosis

    National Research Council Canada - National Science Library

    Lu, Junxuan

    2006-01-01

    The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of prostate cancer (PCa...

  20. Blockade of store-operated calcium entry alleviates high glucose-induced neurotoxicity via inhibiting apoptosis in rat neurons.

    Science.gov (United States)

    Xu, Zhenkuan; Xu, Wenzhe; Song, Yan; Zhang, Bin; Li, Feng; Liu, Yuguang

    2016-07-25

    Altered store-operated calcium entry (SOCE) has been suggested to be involved in many diabetic complications. However, the association of altered SOCE and diabetic neuronal damage remains unclear. This study aimed to investigate the effects of altered SOCE on primary cultured rat neuron injury induced by high glucose. Our data demonstrated that high glucose increased rat neuron injury and upregulated the expression of store-operated calcium channel (SOC). Inhibition of SOCE by a pharmacological inhibitor and siRNA knockdown of stromal interaction molecule 1 weakened the intracellular calcium overload, restored mitochondrial membrane potential, downregulated cytochrome C release and inhibited cell apoptosis. As well, treatment with the calcium chelator BAPTA-AM prevented cell apoptosis by ameliorating the high glucose-increased intracellular calcium level. These findings suggest that SOCE blockade may alleviate high glucose-induced neuronal damage by inhibiting apoptosis. SOCE might be a promising therapeutic target in diabetic neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Infección por el virus de la Lengua azul: activación de señales celulares que inducen apoptosis Bluetongue virus infection: signaling pathway activated during apoptosis

    Directory of Open Access Journals (Sweden)

    E. Mortola

    2009-09-01

    Full Text Available El virus de la Lengua azul (VLA es un ARN virus de doble cadena que induce apoptosis tanto en cultivos celulares como en tejidos blanco. Con el fin de dilucidar el mecanismo de apoptosis en la infección por el VLA, en el presente trabajo examinamos en detalle, por la técnica de Western blot, las señales celulares de caspasas, Bax, citocromo c, Smac/DIABLO y factor nuclear kappa B (NF-kB que se activan en la infección viral. Hemos comprobado que luego de la infección in vitro con el VLA, se detectó la activación de la caspasa 8 y con ello el mecanismo extrínseco de la apoptosis. También detectamos por primera vez no sólo la activación de miembros de la familia Bcl-2 (Bax, sino también la liberación del citocromo c y la proteína Smac/DIABLO, confirmando que en la infección por el VLA está involucrado el mecanismo secuencial intrínseco de la apoptosis. Asimismo, demostramos que la infección por el VLA activa el NF-kB y que la apoptosis es sustancialmente reducida mediante la inhibición del mismo. La activación de las señales celulares tales como Bax, citocromo c, Smac/DIABLO y NF-kB presentados en este trabajo, esclarecen los mecanismos apoptóticos durante la infección por el VLA para una mayor comprensión del papel primario que juega la apoptosis en la patogénesis del virus.Bluetongue (BTV is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. To elucidate the apoptosis pathways in BTV infection, we have examined in detail the apoptosis mechanism by examination of caspases, Bax, cytochrome c, Smac/DIABLO and NF-kB signalling pathways. In this report, after cell infection with BTV, the activation of caspase 8 was detected, proving the extrinsic receptor binding apoptotic pathway. Apoptosis followed a sequential pathway involving the detection of activated Bcl-2 family members. Furthermore, its translocation to the mitochondria, as well as the release of cytochrome c and

  2. Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

    International Nuclear Information System (INIS)

    McCue, Jeffrey M.; Driscoll, William J.; Mueller, Gregory P.

    2008-01-01

    Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo

  3. Ouabain enhances ADPKD cell apoptosis via the intrinsic pathway

    Directory of Open Access Journals (Sweden)

    Gustavo eBlanco

    2016-03-01

    Full Text Available Progression of autosomal dominant polycystic kidney disease (ADPKD is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3nM also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells. This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key executioner caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells. Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression.

  4. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    International Nuclear Information System (INIS)

    Cho, Sung-Hee; Chung, Kyung-Sook; Choi, Jung-Hye; Kim, Dong-Hyun; Lee, Kyung-Tae

    2009-01-01

    Compound K [20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC 50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1) the activation of caspases-3, -8, and -9; (2) the loss of mitochondrial membrane potential; (3) the release of cytochrome c and Smac/DIABLO to the cytosol; (4) the translocation of Bid and Bax to mitochondria; and (5) the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation

  5. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

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    Xiao Yang

    2017-08-01

    Full Text Available Pyrrolizidine alkaloids (PAs are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1, a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs. Keywords: Senecionine, Drp1

  6. Taurine inhibits 2,5-hexanedione-induced oxidative stress and mitochondria-dependent apoptosis in PC12 cells.

    Science.gov (United States)

    Li, Shuangyue; Guan, Huai; Qian, Zhiqiang; Sun, Yijie; Gao, Chenxue; Li, Guixin; Yang, Yi; Piao, Fengyuan; Hu, Shuhai

    2017-04-07

    2,5-hexanedione (HD) is the ultimate neurotoxic metabolite of hexane, causing the progression of nerve diseases in human. It was reported that HD induced apoptosis and oxidative stress. Taurine has been shown to be a potent antioxidant. In the present study, we investigated the protection of taurine against HD-induced apoptosis in PC12 cells and the underlying mechanism. Our results showed the decreased viability and increased apoptosis in HD-exposed PC12 cells. HD also induced the disturbance of Bax and Bcl-2 expression, the loss of MMP, the release of mitochondrial cytochrome c and caspase-3 activation in PC12 cells. Moreover, HD resulted in an increase in reactive oxygen species (ROS) level and a decline in the activities of superoxidedismutase and catalase in PC12 cells. However, taurine pretreatment ameliorated the increased apoptosis and the alterations in key regulators of mitochondria-dependent pathway in PC12 exposed to HD. The increased ROS level and the decreased activities of the antioxidant enzymes in HD group were attenuated by taurine. These results indicate that pretreatment of taurine may, at least partly, prevent HD-induced apoptosis via inhibiting mitochondria-dependent pathway. It is also suggested that the potential of taurine against HD-induced apoptosis may benefit from its anti-oxidative property.

  7. Sera of patients with celiac disease and neurologic disorders evoke a mitochondrial-dependent apoptosis in vitro.

    Science.gov (United States)

    Cervio, Elisabetta; Volta, Umberto; Verri, Manuela; Boschi, Federica; Pastoris, Ornella; Granito, Alessandro; Barbara, Giovanni; Parisi, Claudia; Felicani, Cristina; Tonini, Marcello; De Giorgio, Roberto

    2007-07-01

    The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody-positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro. SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods. SH-Sy5Y cells exposed to antineuronal antibody-positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody-negative sera. Neuroblasts exposed to antineuronal antibody-negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG-depleted celiac disease sera had an apoptotic effect similar to controls. Anti-caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera. Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis.

  8. Azoxystrobin Induces Apoptosis of Human Esophageal Squamous Cell Carcinoma KYSE-150 Cells through Triggering of the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Xiao-ke Shi

    2017-05-01

    Full Text Available Recent studies indicate that mitochondrial pathways of apoptosis are potential chemotherapeutic target for the treatment of esophageal cancer. Azoxystrobin (AZOX, a methoxyacrylate derived from the naturally occurring strobilurins, is a known fungicide acting as a ubiquinol oxidation (Qo inhibitor of mitochondrial respiratory complex III. In this study, the effects of AZOX on human esophageal squamous cell carcinoma KYSE-150 cells were examined and the underlying mechanisms were investigated. AZOX exhibited inhibitory effects on the proliferation of KYSE-150 cells with inhibitory concentration 50% (IC50 of 2.42 μg/ml by 48 h treatment. Flow cytometry assessment revealed that the inhibitory effect of AZOX on KYSE-150 cell proliferation occurred with cell cycle arrest at S phase and increased cell apoptosis in time-dependent and dose-dependent manners. Cleaved poly ADP ribose polymerase (PARP, caspase-3 and caspase-9 were increased significantly by AZOX. It is worth noted that the Bcl-2/Bax ratios were decreased because of the down-regulated Bcl-2 and up-regulated Bax expression level. Meanwhile, the cytochrome c release was increased by AZOX in KYSE-150 cells. AZOX-induced cytochrome c expression and caspase-3 activation was significantly blocked by Bax Channel Blocker. Intragastric administration of AZOX effectively decreased the tumor size generated by subcutaneous inoculation of KYSE-150 cells in nude mice. Consistently, decreased Bcl-2 expression, increased cytochrome c and PARP level, and activated caspase-3 and caspase-9 were observed in the tumor samples. These results indicate that AZOX can effectively induce esophageal cancer cell apoptosis through the mitochondrial pathways of apoptosis, suggesting AZOX or its derivatives may be developed as potential chemotherapeutic agents for the treatment of esophageal cancer.

  9. Fast prediction of cytochrome P450 mediated drug metabolism

    DEFF Research Database (Denmark)

    Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris

    2009-01-01

    Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90...

  10. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Míriam Cristina Sakuragui Matuo

    2010-09-01

    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  11. Silencing, positive selection and parallel evolution: busy history of primate cytochromes C.

    Directory of Open Access Journals (Sweden)

    Denis Pierron

    Full Text Available Cytochrome c (cyt c participates in two crucial cellular processes, energy production and apoptosis, and unsurprisingly is a highly conserved protein. However, previous studies have reported for the primate lineage (i loss of the paralogous testis isoform, (ii an acceleration and then a deceleration of the amino acid replacement rate of the cyt c somatic isoform, and (iii atypical biochemical behavior of human cyt c. To gain insight into the cause of these major evolutionary events, we have retraced the history of cyt c loci among primates. For testis cyt c, all primate sequences examined carry the same nonsense mutation, which suggests that silencing occurred before the primates diversified. For somatic cyt c, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded the same tree topology. The evolutionary analyses show that a fast accumulation of non-synonymous mutations (suggesting positive selection occurred specifically on the anthropoid lineage root and then continued in parallel on the early catarrhini and platyrrhini stems. Analysis of evolutionary changes using the 3D structure suggests they are focused on the respiratory chain rather than on apoptosis or other cyt c functions. In agreement with previous biochemical studies, our results suggest that silencing of the cyt c testis isoform could be linked with the decrease of primate reproduction rate. Finally, the evolution of cyt c in the two sister anthropoid groups leads us to propose that somatic cyt c evolution may be related both to COX evolution and to the convergent brain and body mass enlargement in these two anthropoid clades.

  12. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

    Science.gov (United States)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

    2017-01-06

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    Energy Technology Data Exchange (ETDEWEB)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2016-10-07

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  14. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    International Nuclear Information System (INIS)

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

    2006-01-01

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  15. Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway.

    Science.gov (United States)

    Yan, Xiaoyan; Wang, Lu; Yang, Xia; Qiu, Yulan; Tian, Xiaolin; Lv, Yi; Tian, Fengjie; Song, Guohua; Wang, Tong

    2017-09-01

    Numerous studies have shown that chronic excessive fluoride intake can adversely affect different organ systems. In particular, the cardiovascular system is susceptible to disruption by a high concentration of fluoride. The objectives of this study were to explore the mechanism of apoptosis by detecting the toxic effects of different concentrations of sodium fluoride (NaF) in H9c2 cells exposed for up to 96 h. NaF not only inhibited H9c2 cell proliferation but also induced apoptosis and morphological damage. With increasing NaF concentrations, early apoptosis of H9c2 cells was increased while the mitochondrial membrane potential was decreased. Compared with the control group, the mRNA levels of caspase-3, caspase-9, and cytochrome c all increased with increasing concentrations of NaF. In summary, these data suggest that apoptosis is involved in NaF-induced H9c2 cell toxicity and that activation of the mitochondrial pathway may occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Anthraquinone G503 Induces Apoptosis in Gastric Cancer Cells through the Mitochondrial Pathway

    Science.gov (United States)

    Li, Shuai; Duan, Junting; Ye, Fang; Li, Hanxiang; She, Zhigang; Gao, Guoquan; Yang, Xia

    2014-01-01

    G503 is an anthraquinone compound isolated from the secondary metabolites of a mangrove endophytic fungus from the South China Sea. The present study elucidates the anti-tumor activity and the underlying mechanism of G503. Cell viability assay performed in nine cancer cell lines and two normal cell lines demonstrated that the gastric cancer cell line SGC7901 is the most G503-sensitive cancer cells. G503 induced SGC7901 cell death via apoptosis. G503 exposure activated caspases-3, -8 and -9. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not caspase-8 inbibitor Z-IETD-FMK, attenuated the effect of G503. These results suggested that the intrinsic mitochondrial apoptosis pathway, rather than the extrinsic pathway, was involved in G503-induced apoptosis. Furthermore, G503 increased the ratio of Bax to Bcl-2 in the mitochondria and decreased the ratio in the cytosol. G503 treatment resulted in mitochondrial depolarization, cytochrome c release and the subsequent cleavage of caspase -9 and -3. Moreover, it is reported that the endoplasmic reticulum apoptosis pathway may also be activated by G503 by inducing capase-4 cleavage. In consideration of the lower 50% inhibitory concentration for gastric cancer cells, G503 may serve as a promising candidate for gastric cancer chemotherapy. PMID:25268882

  17. Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse

    Directory of Open Access Journals (Sweden)

    Qianying Liu

    2018-05-01

    Full Text Available Mequindox (MEQ, belonging to quinoxaline-di-N-oxides (QdNOs, is a synthetic antimicrobial agent widely used in China. Previous studies found that the kidney was one of the main toxic target organs of the QdNOs. However, the mechanisms underlying the kidney toxicity caused by QdNOs in vivo still remains unclear. The present study aimed to explore the molecular mechanism of kidney toxicity in mice after chronic exposure to MEQ. MEQ led to the oxidative stress, apoptosis, and mitochondrial damage in the kidney of mice. Meanwhile, MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. The oxidative stress mediated by MEQ might led to mitochondria damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, upregulation of the Nrf2-Keap1 signaling pathway was also observed. Our findings revealed that the oxidative stress, mitochondrial dysfunction, and the Nrf2-Keap1 signaling pathway were associated with the kidney apoptosis induced by MEQ in vivo.

  18. The fusarium mycotoxin deoxynivalenol can inhibit plant apoptosis-like programmed cell death.

    Directory of Open Access Journals (Sweden)

    Mark Diamond

    Full Text Available The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate.

  19. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-01-01

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture...

  20. Characterization of cytochrome c as marker for retinal cell degeneration by uv/vis spectroscopic imaging

    Science.gov (United States)

    Hollmach, Julia; Schweizer, Julia; Steiner, Gerald; Knels, Lilla; Funk, Richard H. W.; Thalheim, Silko; Koch, Edmund

    2011-07-01

    Retinal diseases like age-related macular degeneration have become an important cause of visual loss depending on increasing life expectancy and lifestyle habits. Due to the fact that no satisfying treatment exists, early diagnosis and prevention are the only possibilities to stop the degeneration. The protein cytochrome c (cyt c) is a suitable marker for degeneration processes and apoptosis because it is a part of the respiratory chain and involved in the apoptotic pathway. The determination of the local distribution and oxidative state of cyt c in living cells allows the characterization of cell degeneration processes. Since cyt c exhibits characteristic absorption bands between 400 and 650 nm wavelength, uv/vis in situ spectroscopic imaging was used for its characterization in retinal ganglion cells. The large amount of data, consisting of spatial and spectral information, was processed by multivariate data analysis. The challenge consists in the identification of the molecular information of cyt c. Baseline correction, principle component analysis (PCA) and cluster analysis (CA) were performed in order to identify cyt c within the spectral dataset. The combination of PCA and CA reveals cyt c and its oxidative state. The results demonstrate that uv/vis spectroscopic imaging in conjunction with sophisticated multivariate methods is a suitable tool to characterize cyt c under in situ conditions.

  1. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    Chattha

    Economically, legumes represent the second most important family of crop plants after Poacea (grass family), accounting for ... further characterization of P450 genes with both known and unknown functions. MATERIALS AND METHODS ..... Cytochrome P450. In: Somerville CR, Meyerowitz EM (eds) .The Arabidopsis book,.

  2. Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development

    Czech Academy of Sciences Publication Activity Database

    Racca, S.; Welchen, E.; Gras, D. E.; Tarkowská, Danuše; Turečková, Veronika; Maurino, V. G.; Gonzalez, D. H.

    2018-01-01

    Roč. 94, č. 1 (2018), s. 105-121 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * cytochrome c * DELLA protein * gibberellin * mitochondrion Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 5.901, year: 2016

  3. The Role of Cytochromes P450 in Infection

    Directory of Open Access Journals (Sweden)

    Elisavet Stavropoulou

    2018-01-01

    Full Text Available Cytochromes are expressed in many different tissues of the human body. They are found mostly in intestinal and hepatic tissues. Cytochromes P450 (CYPs are enzymes that oxidize substances using iron and are able to metabolize a large variety of xenobiotic substances. CYP enzymes are linked to a wide array of reactions including and O-dealkylation, S-oxidation, epoxidation, and hydroxylation. The activity of the typical P450 cytochrome is influenced by a variety of factors, such as genus, environment, disease state, herbicide, alcohol, and herbal medications. However, diet seems to play a major role. The mechanisms of action of dietary chemicals, macro- and micronutrients on specific CYP isoenzymes have been extensively studied. Dietary modulation has effects upon the metabolism of xenobiotics. Cytochromes harbor intra- or interindividual and intra- or interethnic genetic polymorphisms. Bacteria were shown to express CYP-like genes. The tremendous metabolic activity of the microbiota is associated to its abundant pool of CYP enzymes, which catalyze phase I and II reactions in drug metabolism. Disease states, intestinal disturbances, aging, environmental toxic effects, chemical exposures or nutrition modulate the microbial metabolism of a drug before absorption. A plethora of effects exhibited by most of CYP enzymes can resemble those of proinflammatory cytokines and IFNs. Moreover, they are involved in the initiation and persistence of pathologic pain by directly activating sensory neurons and inflammatory cytokines.

  4. Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding

    NARCIS (Netherlands)

    Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.

    2009-01-01

    The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three

  5. Multivariate Modeling of Cytochrome P450 Enzymes for 4 ...

    African Journals Online (AJOL)

    Conclusion: Apart from insights into important molecular properties for CYP inhibition, the findings may also guide further investigations of novel drug candidates that are unlikely to inhibit multiple CYP sub-types. Keywords: Antimalarial, Chloroquine, Cytochrome P450, Genetic algorithm-based multiple linear regression, ...

  6. Apoptosis in unicellular organisms: mechanisms and evolution.

    Science.gov (United States)

    Gordeeva, A V; Labas, Y A; Zvyagilskaya, R A

    2004-10-01

    Data about the programmed death (apoptosis) in unicellular organisms, from bacteria to ciliates, are discussed. Firstly apoptosis appeared in lower eukaryotes, but its mechanisms in these organisms are different from the classical apoptosis. During evolution, the apoptotic process has been improving gradually, with reactive oxygen species and Ca2+ playing an essential role in triggering apoptosis. All eukaryotic organisms have apoptosis inhibitors, which might be introduced by viruses. In the course of evolution, caspases and apoptosis-inducing factor appeared before other apoptotic proteins, with so-called death receptors being the last among them. The functional analogs of eukaryotic apoptotic proteins take parts in the programmed death of bacteria.

  7. XIAP impairs mitochondrial function during apoptosis by regulating the Bcl-2 family in renal cell carcinoma.

    Science.gov (United States)

    Chen, Chao; Liu, Tian Shu; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

    2018-05-01

    Efficient apoptosis requires Bcl-2 family-mediated mitochondrial outer membrane permeabilization (MOMP), which releases pro-apoptotic proteins to the cytosol, activating apoptosis and inhibiting X-linked inhibitor of apoptosis protein (XIAP). XIAP is a member of the inhibitors of apoptosis protein family whose expression is elevated in many cancer types and participates in the release of pro-apoptotic proteins. To explore the association between XIAP and the Bcl-2 family, and the influence of XIAP on mitochondria, RNA interference of XIAP was performed in Caki-1 cells and the dynamic change in the levels of related proteins was compared with the original Caki-1 cells upon induction of apoptosis. Upon knockdown of XIAP, the release of cytochrome c (Cyt-c), second mitochondria-derived activator of caspase (Smac) and apoptotic protease activating factor 1 (Apaf-1) from mitochondria proceeded normally, whereas in Caki-1 cells, the release of these pro-apoptotic proteins was significantly prolonged, and incomplete. Downregulation of XIAP through small interfering RNA resulted in an increase of apoptosis and a marked decrease in Bcl-2 and Bcl-xl levels at 3 h. Additionally, the regulation of the level of XIAP protein affected the specific ratios of Bcl-2/Bax and Bcl-xl/Bax, which play decisive roles in cell death. In the present study, it was revealed that XIAP can feed back to mitochondria, delaying Cyt-c and Apaf-1 release. Furthermore, XIAP can limit the release of its inhibitor Smac with the involvement of Bcl-2 family proteins.

  8. Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

    Science.gov (United States)

    Chaabane, Wiem; Cieślar-Pobuda, Artur; El-Gazzah, Mohamed; Jain, Mayur V; Rzeszowska-Wolny, Joanna; Rafat, Mehrdad; Stetefeld, Joerg; Ghavami, Saeid; Los, Marek J

    2014-09-01

    The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

  9. Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells.

    Science.gov (United States)

    Chowdhury, Indrajit; Branch, Alicia; Olatinwo, Moshood; Thomas, Kelwyn; Matthews, Roland; Thompson, Winston E

    2011-08-29

    Ceramide is a key factor in inducing germ cell apoptosis by translocating from cumulus cells into the adjacent oocyte and lipid rafts through gap junctions. Therefore studies designed to elucidate the mechanistic pathways in ceramide induced granulosa cell (GC) apoptosis and follicular atresia may potentially lead to the development of novel lipid-based therapeutic strategies that will prevent infertility and premature menopause associated with chemo and/or radiation therapy in female cancer patients. Our previous studies have shown that Prohibitin (PHB) is intimately involved in GCs differentiation, atresia, and luteolysis. In the present study, we have examined the functional effects of loss-/gain-of-function of PHB using adenoviral technology in delaying apoptosis induced by the physiological ligand ceramide in rat GCs. Under these experimental conditions, exogenous ceramide C-8 (50 μM) augmented the expression of mitochondrial PHB and subsequently cause the physical destruction of GC by the release of mitochondrial cytochrome c and activation of caspase-3. In further studies, silencing of PHB expression by adenoviral small interfering RNA (shRNA) sensitized GCs to ceramide C8-induce apoptosis. In contrast, adenovirus (Ad) directed overexpression of PHB in GCs resulted in increased PHB content in mitochondria and delayed the onset of ceramide induced apoptosis in the infected GCs. Taken together, these results provide novel evidences that a critical level of PHB expression within the mitochondria plays a key intra-molecular role in GC fate by mediating the inhibition of apoptosis and may therefore, contribute significantly to ceramide induced follicular atresia. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Apoptosis: Targets in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Kalthoff Holger

    2003-01-01

    Full Text Available Abstract Pancreatic adenocarcinoma is characterized by poor prognosis, because of late diagnosis and lack of response to chemo- and/or radiation therapies. Resistance to apoptosis mainly causes this insensitivity to conventional therapies. Apoptosis or programmed cell death is a central regulator of tissue homeostasis. Certain genetic disturbances of apoptotic signaling pathways have been found in carcinomas leading to tumor development and progression. In the past few years, the knowledge about the complex pathways of apoptosis has strongly increased and new therapeutic approaches based on this knowledge are being developed. This review will focus on the role of apoptotic proteins contributing to pancreatic cancer development and progression and will demonstrate possible targets to influence this deadly disease.

  11. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation.

    Science.gov (United States)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-12-30

    Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Jaceosidin Induces Apoptosis in Human Ovary Cancer Cells through Mitochondrial Pathway

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    Wen Lv

    2008-01-01

    Full Text Available We examined the antiproliferation effect of Jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose polymerase (PARP, while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.

  13. Epothilone B induces extrinsic pathway of apoptosis in human SKOV-3 ovarian cancer cells.

    Science.gov (United States)

    Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

    2014-06-01

    The molecular mechanisms underlying epothilone B (EpoB) induced apoptosis were investigated in SKOV-3 human ovarian cancer cells. The aim of this research was to compare EpoB's, which belongs to the new class of anticancer drugs, with paclitaxel's (PTX) ability to induce apoptosis. The mode of cell death was assessed colorimetrically, fluorimetrically and by immunoblot analyses through measuring DNA fragmentation, the level of intracellular calcium, the level of cytochrome c, TRAIL, the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-9, -8 and -3. EpoB leads to an increase of the cytosolic level of cytochrome c after 4 h of cell treatment. After 24 and 48 h of cell treatment the level of intracellular calcium also increased by about 21% and 24% respectively. Moreover, EpoB, similarly to PTX, promoted the expression of TRAIL in lymphocytes, although high TRAIL expression on tumor cells was detected only after adding EpoB to SKOV-3 cells. EpoB mediates caspases-8 and -3 activation, which is independent of the reduction in the amount of caspase-9. Epitope-specific monoclonal and polyclonal antibodies revealed characteristic apoptotic changes that included cleavage of the 116 kDa PARP polypeptide to 25 kDa fragments. The results of our study show that EpoB induces mainly the extrinsic pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Bauhinia championii Flavone Attenuates Hypoxia-Reoxygenation Induced Apoptosis in H9c2 Cardiomyocytes by Improving Mitochondrial Dysfunction.

    Science.gov (United States)

    Liao, Ping; Sun, Guibo; Zhang, Chan; Wang, Min; Sun, Yao; Zhou, Yuehan; Sun, Xiaobo; Jian, Jie

    2016-11-04

    This study aimed to determine the effects of Bauhinia championii flavone (BCF) on hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cardiomyocytes and to explore potential mechanisms. The H/R model in H9c2 cardiomyocytes was established by 6 h of hypoxia and 12 h of reoxygenation. Cell viability was detected by CCK-8 assay. Apoptotic rate was measured by Annexin V/PI staining. Levels of mitochondria-associated ROS, mitochondrial transmembrane potential (∆Ψm) and mitochondrial permeability transition pores (MPTP) opening were assessed by fluorescent probes. ATP production was measured by ATP assay kit. The release of cytochrome c, translocation of Bax, and related proteins were measured by western blotting. Our results showed that pretreatment with BCF significantly improved cell viability and attenuated the cardiomyocyte apoptosis caused by H/R. Furthermore, BCF increased ATP production and inhibited ROS-generating mitochondria, depolarization of ΔΨm, and MPTP opening. Moreover, BCF pretreatment decreased Bax mitochondrial translocation, cytochrome c release, and activation of caspase-3, as well as increased the expression of p-PI3K, p-Akt, and the ratio of Bcl-2 to Bax. Interestingly, a specific inhibitor of phosphatidylinositol 3-kinase, LY294002, partly reversed the anti-apoptotic effect of BCF. These observations indicated that BCF pretreatment attenuates H/R-induced myocardial apoptosis strength by improving mitochondrial dysfunction via PI3K/Akt signaling pathway.

  15. PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    International Nuclear Information System (INIS)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-01-01

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  16. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  17. Baicalin prevents Candida albicans infections via increasing its apoptosis rate

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shulong; Fu, Yingyuan, E-mail: yingyuanfu@126.com; Wu, Xiuzhen; Zhou, Zhixing; Xu, Jing; Zeng, Xiaoping; Kuang, Nanzhen; Zeng, Yurong

    2014-08-15

    Highlights: • Baicalin increases the ratio of the G0/G1 stages and C. albicans apoptosis. • Baicalin decreases the proliferation index of C. albicans. • Baicalin inhibits the biosynthesis of DNA, RNA and protein in C. albicans. • Baicalin depresses Succinate Dehydrogenase and Ca{sup 2+}–Mg{sup 2+} ATPase in C. albicans. • Baicalin increases the endocytic free Ca{sup 2+} concentration in C. albicans. - Abstract: Background: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. Methodology and principal findings: We detected the baicalin inhibition effects on three isotope-labeled precursors of {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca{sup 2+}–Mg{sup 2+} ATPase, cytosolic Ca{sup 2+} concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca{sup 2+} concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. Innovation and significance: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase, increasing

  18. Baicalin prevents Candida albicans infections via increasing its apoptosis rate

    International Nuclear Information System (INIS)

    Yang, Shulong; Fu, Yingyuan; Wu, Xiuzhen; Zhou, Zhixing; Xu, Jing; Zeng, Xiaoping; Kuang, Nanzhen; Zeng, Yurong

    2014-01-01

    Highlights: • Baicalin increases the ratio of the G0/G1 stages and C. albicans apoptosis. • Baicalin decreases the proliferation index of C. albicans. • Baicalin inhibits the biosynthesis of DNA, RNA and protein in C. albicans. • Baicalin depresses Succinate Dehydrogenase and Ca 2+ –Mg 2+ ATPase in C. albicans. • Baicalin increases the endocytic free Ca 2+ concentration in C. albicans. - Abstract: Background: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. Methodology and principal findings: We detected the baicalin inhibition effects on three isotope-labeled precursors of 3 H-UdR, 3 H-TdR and 3 H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca 2+ –Mg 2+ ATPase, cytosolic Ca 2+ concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited 3 H-UdR, 3 H-TdR and 3 H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca 2+ –Mg 2+ ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca 2+ concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. Innovation and significance: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca 2+ –Mg 2+ ATPase, increasing cytosolic Ca 2+ content and damaging the ultrastructure of C. albicans

  19. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1.

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    Alexandr N Simonov

    Full Text Available Cytochrome P450c17 (P450 17A1, CYP17A1 is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.

  20. 2,5-hexanedione induces bone marrow mesenchymal stem cell apoptosis via inhibition of Akt/Bad signal pathway.

    Science.gov (United States)

    Sun, Jingsong; Shi, Xiaoxia; Li, Shuangyue; Piao, Fengyuan

    2018-04-01

    2,5-Hexanedione (HD) is an important bioactive metabolite of n-hexane and mediates the neurotoxicity of parent compound. Studies show that HD induces apoptotic death of neural progenitor cells. However, its underlying mechanism remains unknown. Mesenchymal stem cells (MSCs) are multipotential stem cells with the ability to differentiate into various cell types and have been used as cell model for studying the toxic effects of chemicals on stem cells. In this study, we exposed rat bone marrow MSCs to 0, 10, 20, and 40 mM HD in vitro. Apoptosis and disruption of mitochondrial transmembrane potential were estimated by immunochemistry staining. The expression of Akt, Bad, phosphorylated Akt (p-Akt), and Bad (p-Bad) as well as cytochrome c in mitochondria and cytosol were examined by Western blot. Moreover, caspase 3 activity, viability, and death of cells were measured by spectrophotometry. Our results showed that HD induced cell apoptosis and increased caspase 3 activity. HD down-regulated the expression levels of p-Akt, p-Bad and induced MMP depolarization, followed by cytochrome c release. Moreover, HD led to a concentration-dependent increase in the MSCs death, which was relative to MSCs apoptosis. However, these toxic effects of HD on the MSCs were significantly mitigated in the presence of IGF, which could activate PI3 K/Akt pathway. These results indicated that HD induced mitochondria-mediated apoptosis in the MSCs via inhibiting Akt/Bad signaling pathway and apoptotic death of MSCs via the signaling pathway. These results might provide some clues for studying further the mechanisms of HD-induced stem cell apoptosis and adverse effect on neurogenesis. © 2017 Wiley Periodicals, Inc.

  1. Single-prolonged stress induces apoptosis in dorsal raphe nucleus in the rat model of posttraumatic stress disorder

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    Liu Dongjuan

    2012-11-01

    Full Text Available Abstract Introduction Post-traumatic stress disorder (PTSD is an anxiety disorder that develops after exposure to a life-threatening traumatic experience. Meta-analyses of the brainstem showed that midsagittal area of the pons was significantly reduced in patients with PTSD, suggesting a potential apoptosis in dorsal raphe nucleus after single-prolonged stress (SPS. The aim of this study is to investigate whether SPS induces apoptosis in dorsal raphe nucleus in PTSD rats, which may be a possible mechanism of reduced volume of pons and density of gray matter. Methods In this study, rats were randomly divided into 1d, 7d and 14d groups after SPS along with the control group. The apoptosis rate was determined using annexin V-FITC/PI double-labeled flow cytometry (FCM. Levels of Cytochrome c (Cyt-C was examined by Western blotting. Expression of Cyt-C on mitochondria in the dorsal raphe nucleus neuron was determined by enzymohistochemistry under transmission electron microscopy (TEM. The change of thiamine monophosphatase (TMP levels was assessed by enzymohistochemistry under light microscope and TEM. Morphological changes of the ultrastructure of the dorsal raphe nucleus neuron were determined by TEM. Results Apoptotic morphological alterations were observed in dorsal raphe nucleus neuron for all SPS-stimulate groups of rats. The apoptosis rates were significantly increased in dorsal raphe nucleus neuron of SPS rats, along with increased release of cytochrome c from the mitochondria into the cytoplasm, increased expression of Cyt-C and TMP levels in the cytoplasm, which reached to the peak of increase 7 days of SPS. Conclusions The results indicate that SPS induced Cyt-C released from mitochondria into cytosol and apoptosis in dorsal raphe nucleus neuron of rats. Increased TMP in cytoplasm facilitated the clearance of apoptotic cells. We propose that this presents one of the mechanisms that lead to reduced volume of pons and gray matter associated

  2. Apoptosis detection in histological sections

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Dubská, Lenka; Míšek, Ivan

    2003-01-01

    Roč. 72, č. 7 (2003), s. 18-19 ISSN 0001-7213. [Congress of the European Association of Veterinary Anatomists/24./. 21.07.2002-25.07.2002, Brno] R&D Projects: GA ČR GP204/02/P112 Institutional research plan: CEZ:AV0Z5045916 Keywords : apoptosis Subject RIV: FF - HEENT, Dentistry

  3. Molecular mechanism of apoptosis and characterization of apoptosis induced by radiation

    International Nuclear Information System (INIS)

    Li Yumin; Zhang Yuguang; Li Yukun

    1999-01-01

    The major discoveries of apoptosis research in recent years were reviewed briefly. The mechanisms of caspases/ICE gene family and bcl-2 gene family on apoptosis were analyzed. And the signal transduction pathway of apoptosis found currently has been summarized. The characterizations of apoptosis induced by radiation such as time-effects, dose-effects and the radiosensibility were summed up

  4. Downregulation of β1,4-galactosyltransferase 1 inhibits CDK11p58-mediated apoptosis induced by cycloheximide

    International Nuclear Information System (INIS)

    Li Zejuan; Wang Hanzhou; Zong Hongliang; Sun Qing; Kong Xiangfei; Jiang Jianhai; Gu Jianxin

    2005-01-01

    Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34 cdc2 -related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11 p58 induces apoptosis is not clear. Some evidences suggested β1,4-galactosyltransferase 1 (β1,4-GT 1) might participate in apoptosis induced by CDK11 p58 . In this study, we demonstrated that ectopically expressed β1,4-GT 1 increased CDK11 p58 -mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of β1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11 p58 -overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of β1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11 p58 by caspase-3 was reduced. We proposed that β1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11 p58 . This may represent a new mechanism of β1,4-GT 1 in CHX-induced apoptosis of CDK11 p58 -overexpressing cells

  5. Sodium Ferulate Prevents Daunorubicin - Induced Apoptosis in H9c2 Cells via Inhibition of the ERKs Pathway

    Directory of Open Access Journals (Sweden)

    Zhi-Juan Wu

    2015-07-01

    Full Text Available Background: Daunorubicin (DNR-induced cardiotoxicity, which is closely associated with cardiomyocyte apoptosis, limits the drug's clinical application. The activation of the extracellular regulated protein kinases (ERKs pathway is responsible for the pro-apoptosis effect of DNR Sodium ferulate (SF has recently been found to attenuate both DNR-induced cardiotoxicity and mitochondrial apoptosis in juvenile rats. Nonetheless, the precise mechanism underlying SF-induced cardio-protection remains unclear. Methods: The DNR-injured H9c2 cell model was prepared by incubating the cells in 1 µM DNR for 24 h. Amounts of 15.6, 31.3 or 62.5 µM SF were simultaneously added to the cells. The effect of SF on the cytotoxic and apoptotic parameters of the cells was studied by monitoring apoptosis regulation via the ERKs pathway. Results: SF attenuated DNR-induced cell death (particularly apoptotic death, cTnI and β-tubulin degradation, and cellular morphological changes. SF reduced mitochondrial membrane potential depolarization, cytochrome c leakage, and caspase-9 and caspase-3 activation. SF also decreased ERK1/2, phospho-ERK1/2, p53 and Bax expression and increased Bcl-2 expression. These effects were similar to the results observed when using the pharmacological ERKs phosphorylation inhibitor, AZD6244. Conclusion: We determined that SF protects H9c2 cells from DNR-induced apoptosis through a mechanism that involves the interruption of the ERKs signaling pathway.

  6. SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

    Science.gov (United States)

    Jiang, Junjun; Li, Liyuan; Xie, Mingchao; Fuji, Ryosuke; Liu, Shangfeng; Yin, Xiaobei; Li, Genlin; Wang, Zhao

    2015-01-01

    Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

  7. Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine

    Directory of Open Access Journals (Sweden)

    Jianping Fan

    2015-06-01

    Full Text Available Objectives: Meconopsis integrifolia (M. integrifolia is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. Methods: 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

  8. Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells

    Science.gov (United States)

    HUA, YUAN-YUAN; WANG, XIAO-SHU; ZHANG, YU; YAO, CHEN-GUO; ZHANG, XI-MING; XIONG, ZHENG-AI

    2012-01-01

    The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway. PMID:22307872

  9. Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Sheng Chen

    2017-01-01

    Full Text Available Objective. Excessive apoptosis of nucleus pulposus cells (NPCs induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD. Mesenchymal stem cells (MSCs can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods. BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results. The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions. Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.

  10. NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

    Directory of Open Access Journals (Sweden)

    Molouki Aidin

    2012-08-01

    Full Text Available Abstract Background Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV, the matrix (M protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV. Finding In order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection. In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. Conclusion Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

  11. A novel model for rapid induction of apoptosis in spiral ganglions of mice.

    Science.gov (United States)

    Lee, Ji Eun; Nakagawa, Takayuki; Kim, Tae Soo; Iguchi, Fukuichiro; Endo, Tsuyoshi; Dong, Youyi; Yuki, Kazuo; Naito, Yasushi; Lee, Sang Heun; Ito, Juichi

    2003-06-01

    The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or "knockin" mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course. Laboratory study using experimental animals. C57BL/6 mice were used as experimental animals and were subjected to direct application of cisplatin into the inner ear. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunostaining for Neurofilament 200-kD (NF) and peripherin were used for analysis of SG degeneration. In addition, generation of peroxynitrite in affected spiral ganglions was examined by immunostaining for nitrotyrosine. Cellular location of activated caspase-9 and cytochrome-c in dying SG neurons were examined for analysis of cell death pathway. The TUNEL assay and immunohistochemical analysis for NF and peripherin indicated that type I neurons in spiral ganglions were deleted through the apoptotic pathway over time. Spiral ganglion neurons treated with cisplatin exhibited expression of nitrotyrosine, indicating induction of peroxynitrite by cisplatin. In dying SG neurons, expression of activated caspase-9 and translocation of cytochrome-c from mitochondria to cytoplasm were observed, indicating the mitochondrial pathway of apoptosis. The predictable fashion of induction of apoptosis in SG neurons over a well-defined time course in the model in the study will aid studies of the molecular mechanism of cell death and elucidation of a strategy for prevention of SG degeneration.

  12. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pan, Li; Zhang, Yuming; Zhao, Wanlu; Zhou, Xia; Wang, Chunxia; Deng, Fan

    2017-07-01

    Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca 2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca 2+ concentration, but decreased GSH concentration in the cells. The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

  13. Apoptosis Effect of Girinimbine Isolated from Murraya koenigii on Lung Cancer Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Syam Mohan

    2013-01-01

    Full Text Available Murraya koenigii Spreng has been traditionally claimed as a remedy for cancer. The current study investigated the anticancer effects of girinimbine, a carbazole alkaloid isolated from Murraya koenigii Spreng, on A549 lung cancer cells in relation to apoptotic mechanistic pathway. Girinimbine was isolated from Murraya koenigii Spreng. The antiproliferative activity was assayed using MTT and the apoptosis detection was done by annexin V and lysosomal stability assays. Multiparameter cytotoxicity assays were performed to investigate the change in mitochondrial membrane potential and cytochrome c translocation. ROS, caspase, and human apoptosis proteome profiler assays were done to investigate the apoptotic mechanism of cell death. The MTT assay revealed that the girinimbine induces cell death with an IC50 of 19.01 μM. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μM of girinimbine, decrease in the nuclear area and increase in mitochondrial membrane potential and plasma membrane permeability were readily visible. Moreover the translocation of cytochrome c also was observed. Girinimbine mediates its antiproliferative and apoptotic effects through up- and downregulation of apoptotic and antiapoptotic proteins. There was a significant involvement of both intrinsic and extrinsic pathways. Moreover, the upregulation of p53 as well as the cell proliferation repressor proteins, p27 and p21, and the significant role of insulin/IGF-1 signaling were also identified. Moreover the caspases 3 and 8 were found to be significantly activated. Our results taken together indicated that girinimbine may be a potential agent for anticancer drug development.

  14. Troxerutin abrogates mitochondrial oxidative stress and myocardial apoptosis in mice fed calorie-rich diet.

    Science.gov (United States)

    Geetha, Rajagopalan; Sathiya Priya, Chandrasekaran; Anuradha, Carani Venkatraman

    2017-12-25

    Mitochondrial oxidative stress plays a major role in the pathogenesis of myocardial apoptosis in metabolic syndrome (MS) patients. In this study, we investigated the effect of troxerutin (TX), an antioxidant on mitochondrial oxidative stress and apoptotic markers in heart of mice fed fat and fructose-rich diet. Adult male Mus musculus mice were fed either control diet or high fat, high fructose diet (HFFD) for 60 days to induce MS. Mice from each dietary group were divided into two on the 16th day and were either treated or untreated with TX (150 mg/kg bw, p.o) for the next 45 days. At the end of the study, mitochondrial reactive oxygen species (ROS) generation, oxidative stress markers, levels of intracellular calcium, cardiolipin content, cytochrome c release and apoptotic markers were examined in the myocardium. HFFD-feeding resulted in diminution of antioxidants and increased ROS production, lipid peroxidation and oxidatively modified adducts of 8-OHG, 4-HNE and 3-NT. Further increase in Ca 2+ levels, low levels of calcium transporters and decrease in cardiolipin content were noted. Changes in the mitochondrial structure were observed by electron microscopy. Furthermore, cytochrome c release, increase in proapoptotic proteins (APAF-1, BAX, caspases-9 and-3) and decrease in antiapoptotic protein (BCL-2) in HFFD-fed mice suggest myocardial apoptosis. These changes were significantly restored by TX supplementation. TX administration effectively attenuated cardiac apoptosis and exerted a protective role by increasing antioxidant potential and by improving mitochondrial function. Thus, TX could be a promising therapeutic candidate for treating cardiac disease in MS patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

    Science.gov (United States)

    Rybniker, Jan; Vocat, Anthony; Sala, Claudia; Busso, Philippe; Pojer, Florence; Benjak, Andrej; Cole, Stewart T

    2015-07-09

    Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

  16. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

  17. Targeting Apoptosis Signaling in Pancreatic Cancer

    International Nuclear Information System (INIS)

    Fulda, Simone

    2011-01-01

    The ability to escape apoptosis or programmed cell death is a hallmark of human cancers, for example pancreatic cancer. This can promote tumorigenesis, since too little cell death by apoptosis disturbs tissue homeostasis. Additionally, defective apoptosis signaling is the underlying cause of failure to respond to current treatment approaches, since therapy-mediated antitumor activity requires the intactness of apoptosis signaling pathways in cancer cells. Thus, the elucidation of defects in the regulation of apoptosis in pancreatic carcinoma can result in the identification of novel targets for therapeutic interference and for exploitation for cancer drug discovery

  18. Targeting Apoptosis Signaling in Pancreatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Fulda, Simone [Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt (Germany)

    2011-01-11

    The ability to escape apoptosis or programmed cell death is a hallmark of human cancers, for example pancreatic cancer. This can promote tumorigenesis, since too little cell death by apoptosis disturbs tissue homeostasis. Additionally, defective apoptosis signaling is the underlying cause of failure to respond to current treatment approaches, since therapy-mediated antitumor activity requires the intactness of apoptosis signaling pathways in cancer cells. Thus, the elucidation of defects in the regulation of apoptosis in pancreatic carcinoma can result in the identification of novel targets for therapeutic interference and for exploitation for cancer drug discovery.

  19. Sulphamoylated 2-methoxyestradiol analogues induce apoptosis in adenocarcinoma cell lines.

    Directory of Open Access Journals (Sweden)

    Michelle Visagie

    Full Text Available 2-Methoxyestradiol (2ME2 is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1-25 μM was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 μM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.

  20. Interaction of rocuronium with human liver cytochromes P450

    OpenAIRE

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-01-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

  1. Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

    Science.gov (United States)

    Vanlerberghe, G C; McIntosh, L

    1992-12-01

    In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

  2. Monoclonal antibodies to drosophila cytochrome P-450's

    International Nuclear Information System (INIS)

    Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

    1987-01-01

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

  3. Covalent modification of cytochrome c by reactive metabolites of furan.

    Science.gov (United States)

    Phillips, Martin B; Sullivan, Mathilde M; Villalta, Peter W; Peterson, Lisa A

    2014-01-21

    Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.

  4. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    International Nuclear Information System (INIS)

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs

  5. Identification of human cytochrome P450s as autoantigens.

    Science.gov (United States)

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  6. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    International Nuclear Information System (INIS)

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-01-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

  7. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

    DEFF Research Database (Denmark)

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR und...... to serve as an effective electron transferring "nano-machine"....

  8. One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

    NARCIS (Netherlands)

    Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

    1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

  9. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

    Science.gov (United States)

    Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

    2008-01-15

    In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

  10. Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

    Science.gov (United States)

    Yadav, N; Kumar, S; Marlowe, T; Chaudhary, A K; Kumar, R; Wang, J; O'Malley, J; Boland, P M; Jayanthi, S; Kumar, T K S; Yadava, N; Chandra, D

    2015-11-05

    Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency

  11. Oxidative stress and apoptosis after acute respiratory hypoxia and reoxygenation in rat brain

    Directory of Open Access Journals (Sweden)

    Debora Coimbra-Costa

    2017-08-01

    Full Text Available Acute hypoxia increases the formation of reactive oxygen species (ROS in the brain. However, the effect of reoxygenation, unavoidable to achieve full recovery of the hypoxic organ, has not been clearly established. The aim of the present study was to evaluate the effects of exposition to acute severe respiratory hypoxia followed by reoxygenation on the evolution of oxidative stress and apoptosis in the brain. We investigated the effect of in vivo acute severe normobaric hypoxia (rats exposed to 7% O2 for 6 h and reoxygenation in normoxia (21% O2 for 24 h or 48 h on oxidative stress markers, the antioxidant system and apoptosis in the brain. After respiratory hypoxia we found increased levels of HIF-1α expression, lipid peroxidation, protein oxidation and nitric oxide in brain extracts. Antioxidant defence systems such as superoxide dismutase (SOD, reduced glutathione (GSH and glutathione peroxidase (GPx and the reduced/oxidized glutathione (GSH/GSSG ratio were significantly decreased in the brain. After 24 h of reoxygenation, oxidative stress parameters and the anti-oxidant system returned to control values. Regarding the apoptosis parameters, acute hypoxia increased cytochrome c, AIF and caspase 3 activity in the brain. The apoptotic effect is greatest after 24 h of reoxygenation. Immunohistochemistry suggests that CA3 and dentate gyrus in the hippocampus seem more susceptible to hypoxia than the cortex. Severe acute hypoxia increases oxidative damage, which in turn could activate apoptotic mechanisms. Our work is the first to demonstrate that after 24 h of reoxygenation oxidative stress is attenuated, while apoptosis is maintained mainly in the hippocampus, which may, in fact, be the cause of impaired brain function. Keywords: Antioxidants, Apoptosis, Normobaric hypoxia, Oxidative stress, Reoxygenation

  12. Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action.

    Directory of Open Access Journals (Sweden)

    Alka Jaudan

    Full Text Available Pinostrobin (PN is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6 and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3 was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.

  13. Taurine ameliorated homocysteine-induced H9C2 cardiomyocyte apoptosis by modulating endoplasmic reticulum stress.

    Science.gov (United States)

    Zhang, Zhimin; Zhao, Lianyou; Zhou, Yanfen; Lu, Xuanhao; Wang, Zhengqiang; Wang, Jipeng; Li, Wei

    2017-05-01

    Homocysteine (Hcy)-triggered endoplasmic reticulum (ER) stress-mediated endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury. However, whether ER stress is the molecular mechanism linking Hcy and cardiomyocytes death is unclear. Taurine has been reported to exert cardioprotective effects via various mechanisms. However, whether taurine protects against Hcy-induced cardiomyocyte death by attenuating ER stress is unknown. This study aimed to evaluate the opposite effects of taurine on Hcy-induced cardiomyocyte apoptosis and their underlying mechanisms. Our results demonstrated that low-dose or short-term Hcy treatment increased the expression of glucose-regulated protein 78 (GRP78) and activated protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), which in turn prevented apoptotic cell death. High-dose Hcy or prolonged Hcy treatment duration significantly up-regulated levels of C/EBP homologous protein (CHOP), cleaved caspase-12, p-c-Jun N-terminal kinase (JNK), and then triggered apoptotic events. High-dose Hcy also resulted in a decrease in mitochondrial membrane potential (Δψm) and an increase in cytoplasmic cytochrome C and the expression of cleaved caspase-9. Pretreatment of cardiomyocytes with sodium 4-phenylbutyric acid (an ER stress inhibitor) significantly inhibited Hcy-induced apoptosis. Furthermore, blocking the PERK pathway partly alleviated Hcy-induced ER stress-modulated cardiomyocyte apoptosis, and down-regulated the levels of Bax and cleaved caspase-3. Experimental taurine pretreatment inhibited the expression of ER stress-related proteins, and protected against apoptotic events triggered by Hcy-induced ER stress. Taken together, our results suggest that Hcy triggered ER stress in cardiomyocytes, which was the crucial molecular mechanism mediating Hcy-induced cardiomyocyte apoptosis, and the adverse effect of Hcy could be prevented by taurine.

  14. Isoalantolactone inhibits UM-SCC-10A cell growth via cell cycle arrest and apoptosis induction.

    Directory of Open Access Journals (Sweden)

    Minjun Wu

    Full Text Available Isoalantolactone is a sesquiterpene lactone compound isolated from the roots of Inula helenium L. Previous studies have demonstrated that isoalantolactone possesses antifungal, anti-bacterial, anti-helminthic and anti-proliferative properties in a variety of cells, but there are no studies concerning its effects on head and neck squamous cell carcinoma (HNSCC. In the present study, an MTT assay demonstrated that isoalantolactone has anti-proliferative activity against the HNSCC cell line (UM-SCC-10A. Immunostaining identified that this compound induced UM-SCC-10A cell apoptosis but not necrosis. To explain the molecular mechanisms underlying its effects, flow cytometry and western blot analysis showed that the apoptosis was associated with cell cycle arrest during the G1 phase, up-regulation of p53 and p21, and down-regulation of cyclin D. Furthermore, our results revealed that induction of apoptosis through a mitochondrial pathway led to up-regulation of pro-apoptotic protein expression (Bax, down-regulation of anti-apoptotic protein expression (Bcl-2, mitochondrial release of cytochrome c (Cyto c, reduction of mitochondrial membrane potential (MMP and activation of caspase-3 (Casp-3. Involvement of the caspase apoptosis pathway was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. Together, our findings suggest that isoalantolactone induced caspase-dependent apoptosis via a mitochondrial pathway and was associated with cell cycle arrest in the G1 phase in UM-SCC-10A cells. Therefore, isoalantolactone may become a potential drug for treating HNSCC.

  15. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Directory of Open Access Journals (Sweden)

    Yiwen Li

    2012-11-01

    Full Text Available Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  16. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yiwen; Xu, Yuqing [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Lei, Bo [Department of Breast Surgery, Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang (China); Wang, Wenxiu [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Ge, Xin; Li, Jingrui [Department of General Surgery, Heilongjiang Province Hospital, Harbin, Heilongjiang (China)

    2012-08-03

    Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM) for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  17. Cadmium-induced apoptosis through the mitochondrial pathway in rainbow trout hepatocytes: involvement of oxidative stress

    International Nuclear Information System (INIS)

    Risso-de Faverney, C.; Orsini, N.; Sousa, G. de; Rahmani, R.

    2004-01-01

    Cadmium (Cd) induces oxidative stress and apoptosis in trout hepatocytes. We therefore investigated the involvement of the mitochondrial pathway in the initiation of apoptosis and the possible role of oxidative stress in that process. This study demonstrates that hepatocyte exposure to Cd (2, 5 and 10 μM) triggers significant caspase-3, but also caspase-8 and -9 activation in a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial and cytosolic fractions revealed that cytochrome c (Cyt c) was released in the cytosol in a dose-dependent manner, whereas the pro-apoptotic protein Bax was redistributed to mitochondria after 24 and 48 h exposure. We also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulated under mild oxidative stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24 and 48 h exposure to 10 μM Cd. In the second part of this work, two antioxidant agents, 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) (100 μM) and N-acetylcysteine (NAC, 100 μM) were used to determine the involvement of reactive oxygen species (ROS) in Cd-induced apoptosis. Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reduced caspase-3 activation after 48 h and had a suppressive effect on caspase-8 and -9 also, mostly after 24 h. Lastly, the presence of either one of these antioxidants in the treatment medium also attenuated Cd-induced Cyt c release in cytosol and the level of Bax in the mitochondria after 24 and 48 h, while high Bcl-xL expression was observed. Taken together, these data clearly evidenced the key role of mitochondria in the cascade of events leading to trout hepatocyte apoptosis in response to Cd and the relationship that exists between oxidative stress and cell death

  18. In-silico assessment of protein-protein electron transfer. a case study: cytochrome c peroxidase--cytochrome c.

    Directory of Open Access Journals (Sweden)

    Frank H Wallrapp

    Full Text Available The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47 × 10(6 s(-1 for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.

  19. Effects of intrauterine growth restriction during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver.

    Science.gov (United States)

    Liu, Yingchun; Ma, Chi; Li, Hui; Li, Lingyao; Gao, Feng; Ao, Changjin

    2017-03-01

    This study investigated the effect of intrauterine growth restriction (IUGR) during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver. Eighteen time-mated Mongolian ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.18 MJ ME kg BW -0.75  d -1 , n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW -0.75  d -1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW -0.75  d -1 , n = 6). Fetuses were recovered at slaughter on d 140. Fetal liver weight, DNA content and protein/DNA ratio, proliferation index, cytochrome c, activities of Caspase-3, 8, and 9 were examined, along with relative expression of genes related to apoptosis. Fetuses in both restricted groups exhibited decreased BW, hepatic weight, DNA content, and protein/DNA ratio when compared to CG (P restricted groups (P  0.05). Hepatic expression of gene related to apoptosis showed reduced protein 21 (P21), B-cell lymphoma 2 (Bcl-2) and apoptosis antigen 1 ligand (FasL) expression in RG1 and RG2 (P < 0.05). In contrast, the increased hepatic expression of protein 53 (P53), Bcl-2 associated X protein (Bax) and apoptosis antigen 1 (Fas) in both IUGR fetuses were found (P < 0.05). These results indicate that the fetal hepatocyte proliferation were arrested in G1 cell cycle, and the fetal hepatocyte apoptosis was sensitive to the IUGR resulted from maternal undernutrition. The cell apoptosis in IUGR fetal liver were the potential mechanisms for its retarded proliferation and impaired development. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    International Nuclear Information System (INIS)

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-01-01

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  1. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Wei-Jie; Wang, Sheng [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Hu, Zhuang, E-mail: zhuanghu475000@sina.com [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China); Zhou, Zhen-Yu; Song, Cai-Juan [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China)

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  2. Transcutaneous application of carbon dioxide (CO2 induces mitochondrial apoptosis in human malignant fibrous histiocytoma in vivo.

    Directory of Open Access Journals (Sweden)

    Yasuo Onishi

    Full Text Available Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO(2 to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO(2 exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO(2 induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO(2 treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO(2 treated tumors, highlighting the involvement of mitochondria in apoptosis

  3. Structure and expression of cytochrome f in an Oenothera plastome mutant.

    Science.gov (United States)

    Johnson, E M; Sears, B B

    1990-06-01

    The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

  4. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    International Nuclear Information System (INIS)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-01-01

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  5. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan, E-mail: moonsonlife@yahoo.com; Xian, Shulin; Lu, Yunfei, E-mail: doctorlife@126.com

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  6. Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced rhodobacter capsulatus cytochrome c(2) to the cytochrome bc(1) complex mediated by the conformation of the rieske iron-sulfur protein

    International Nuclear Information System (INIS)

    Devanathan, S.; Salamon, Z.; Tollin, G.; Fitch, J.C.; Meyer, T.E.; Berry, E.A.; Cusanovich, M.A.

    2007-01-01

    The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to the oxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 M. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 binds less strongly (Kd = 0.11 M) but ∼30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c

  7. Curcumin increases the sensitivity of Paclitaxel-resistant NSCLC cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction.

    Science.gov (United States)

    Lu, Yimin; Wang, Jun; Liu, Lei; Yu, Lequn; Zhao, Nian; Zhou, Xingju; Lu, Xudong

    2017-04-01

    Non-small-cell lung cancer is one of the most lethal cancers in the worldwide. Although Paclitaxel-based combinational therapies have long been used as a standard treatment in aggressive non-small-cell lung cancers, Paclitaxel resistance emerges as a major clinical problem. It has been demonstrated that Curcumin from Curcuma longa as a traditional Chinese medicine can inhibit cancer cell proliferation. However, the role of Curcumin in Paclitaxel-resistant non-small-cell lung cancer cells is not clear. In this study, we investigated the effect of Curcumin on the Paclitaxel-resistant non-small-cell lung cancer cells and found that Curcumin treatment markedly increased the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel. Mechanically, the study revealed that Curcumin could reduce the expression of metastasis-associated gene 1 (MTA1) gene through upregulation of microRNA-30c in Paclitaxel-resistant non-small-cell lung cancer cells. During the course, MTA1 reduction sensitized Paclitaxel-resistant non-small-cell lung cancer cells and enhanced the effect of Paclitaxel. Taken together, our studies indicate that Curcumin increases the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction. Curcumin might be a potential adjuvant for non-small-cell lung cancer patients during Paclitaxel treatment.

  8. In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (−)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase

    International Nuclear Information System (INIS)

    Çelik, Haydar; Koşar, Müberra; Arinç, Emel

    2013-01-01

    Highlights: • We assessed inhibitory effects of 8 dietary flavonoids on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase. • The flavonol myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. • We investigated kinetics of myricetin-induced inhibition in detail. • We explored the structure–inhibitory activity relationship of compounds. • Modulation of cytochrome b5 reduction indicates a potential for myricetin to lead to some food–drug/xenobiotic interactions. - Abstract: The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. Myricetin inhibited b5 reductase noncompetitively with a K i of 0.21 μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis–Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC 50 = 9.8 μM). The remaining flavonoids were inactive within the concentration range tested (1–50 μM). Analysis of structure–activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics

  9. Chromium picolinate induced apoptosis of lymphocytes and the signaling mechanisms thereof

    International Nuclear Information System (INIS)

    Jana, Mahadevan; Rajaram, Anantanarayanan; Rajaram, Rama

    2009-01-01

    Cr(III)(picolinate) 3 [Cr(III)(pic) 3 ] is currently used as a nutritional supplement and for treating Type-2 diabetes. The effect of Cr(III)(pic) 3 uptake in peripheral blood lymphocytes is investigated in this study. From the cytotoxicity data, DNA fragmentation pattern, Annexin V staining, TUNEL positivity and the ultrastructural characteristics such as chromatin condensation and formation of apoptotic bodies, it is clear that Cr(III)(pic) 3 induces a concentration dependent apoptosis. It is shown that reactive oxygen species (ROS) produced by treatment with Cr(III)(pic) 3 leads to apoptosis, since we find that pretreatment with N-acetyl cysteine inhibits the process. Using Western blotting technique and fluorescence measurements, the downstream signaling molecules have also been identified. Cr(III)(pic) 3 treatment leads to collapse of the mitochondrial membrane potential, Bax expression, increase in cytosolic cytochrome c content and active caspase-3 and DNA fragmentation and all these manifestations are reduced by pretreating the lymphocytes with N-acetyl cysteine. Thus, it is shown that Cr(III)(pic) 3 is cytotoxic to lymphocytes with ROS and mitochondrial events playing a role in bringing about apoptosis.

  10. Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

    Science.gov (United States)

    Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

    2009-01-01

    The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

  11. Solena amplexicaulis induces cell cycle arrest, apoptosis and inhibits angiogenesis in hepatocarcinoma cells and HUVECs.

    Science.gov (United States)

    Ren, Jie; Xu, Yuan Yuan; Jiang, He Fei; Yang, Meng; Huang, Qian Hui; Yang, Jie; Hu, Kun; Wei, Kun

    2014-01-01

    Solena amplexicaulis (Lam.) Gandhi (SA) has been used as a traditional medicine for the treatment of dysentery, multiple abscess, gastralgia, urethritis, and eczema in the minority area of China. This study was aimed to examine the cell proliferation inhibitory activity of the SA extract (SACE) and its mechanism of action in human hepatoma cell line (HepG2) and evaluate its anti-angiogenesis activity in human umbilical vein endothelial cell line (HUVEC). SACE could inhibit the growth of HepG2 cells in a dose- and time-dependent manner. FCM analysis showed that SACE could induce G2/M phase arrest, cell apoptosis, the mitochondrial membrane potential loss (ΔΨm) and increase the production of intracellular ROS of HepG2 cells. After treatment with SACE, topical morphological changes of apoptotic body formation, obvious increase of apoptosis-related protein expressions, such as Bax, cytochrome c, caspase-3, PARP-1, and decrease of Bcl-2, procaspase-9 protein expressions were observed at the same time. Moreover, SACE caused the significant inhibition of endothelial cell migration and tube formation in HUVEC cells. The results suggested that SACE could act as an angiogenesis inhibitor and induce cell apoptosis via a caspase-dependent mitochondrial pathway. Therefore, SACE could be a potent candidate for the prevention and treatment of liver cancer.

  12. Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway.

    Science.gov (United States)

    Wagatsuma, Akira; Takayama, Yuzo; Hoshino, Takayuki; Shiozuka, Masataka; Yamada, Shigeru; Matsuda, Ryoichi; Mabuchi, Kunihiko

    2017-12-16

    We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

  13. Ginsenoside Rh2 Induces Human Hepatoma Cell Apoptosisvia Bax/Bak Triggered Cytochrome C Release and Caspase-9/Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Xiao-Xi Guo

    2012-11-01

    Full Text Available Ginsenoside Rh2 (G-Rh2 has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

  14. Autoregulatory Feedback Mechanism of P38MAPK/Caspase-8 in Photodynamic Therapy-Hydrophilic/Lipophilic Tetra-α-(4-carboxyphenoxy Phthalocyanine Zinc-Induced Apoptosis of Human Hepatocellular Carcinoma Bel-7402 Cells

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Photodynamic therapy (PDT is a novel and promising antitumor treatment. Our previous study showed that hydrophilic/lipophilic tetra-α-(4-carboxyphenoxy phthalocyanine zinc- (TαPcZn- mediated PDT (TαPcZn-PDT inhibits the proliferation of human hepatocellular carcinoma Bel-7402 cells by triggering apoptosis and arresting cell cycle. However, mechanisms of TαPcZn-PDT-induced apoptosis of Bel-7402 cells have not been fully clarified. In the present study, therefore, effect of TαPcZn-PDT on apoptosis, P38MAPK, p-P38MAPK, Caspase-8, Caspase-3, Bcl-2, Bid, Cytochrome c, and mitochondria membrane potential in Bel-7402 cells without or with P38MAPK inhibitor SB203580 or Caspase-8 inhibitor Ac-IEFD-CHO was investigated by haematoxylin and eosin (HE staining assay, flow cytometry analysis of annexin V-FITC/propidium iodide (PI double staining cells and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, and immunoblot assay. We found that TαPcZn-PDT resulted in apoptosis induction, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. In contrast, SB203580 or Ac-IEFD-CHO attenuated induction of apoptosis, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. Taken together, we conclude that Caspase-3, Bcl-2, Bid, and mitochondria are involved in autoregulatory feedback of P38MAPK/Caspase-8 during TαPcZn-PDT-induced apoptosis of Bel-7402 cells.

  15. Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

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    Wen-Xue Li

    Full Text Available BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic

  16. The influence of very low doses of N-nitrosodimethylamine (NDMA) on the apoptosis of rat neutrophils in vivo. The role of reactive oxygen species.

    Science.gov (United States)

    Jablonski, J; Jablonska, E; Chojnowski, M

    2001-08-13

    N-nitrosodimethylamine (NDMA) causes the apoptosis of neutrophils in vitro experiments. This compound also has the ability to stimulate neutrophils for the production of reactive oxygen species. It has been decided to examine more closely whether the apoptosis of neutrophils by NDMA is caused by the influence of the radicals produced by these cells and whether the stimulation to undergo apoptosis of neutrophils is caused by NDMA in either the original form or by its metabolites. The experiment was conducted on rats. The animals were administered a one-time dose of NDMA intragastrically, 1.5 mg/kg. The research was conducted 1,2,4,12 h consecutively following NDMA administration. The concentration of NDMA in blood was evaluated by means of the gas chromatography method. The neutrophils were isolated from blood by means of differential centrifugation. Respiratory burst was assessed in cells, by means of the cytochrome c reduction method. The percentage of cells revealing morphological properties of apoptosis was determined under the fluorescent microscope. It has been observed that the activation of the respiratory burst is caused mainly by non-metabolised NDMA. Probably the non-metabolised molecules of this compound also have a decisive role in the initiation of apoptosis of neutrophils. It can be assumed that the main factor responsible for the apoptosis of neutrophil rats following a one-time NDMA administration is the induction of respiratory burst in neutrophils by this compound.

  17. Activating Nrf-2 signaling depresses unilateral ureteral obstruction-evoked mitochondrial stress-related autophagy, apoptosis and pyroptosis in kidney.

    Directory of Open Access Journals (Sweden)

    Shue Dong Chung

    Full Text Available Exacerbated oxidative stress and inflammation may induce three types of programmed cell death, autophagy, apoptosis and pyroptosis in unilateral ureteral obstruction (UUO kidney. Sulforaphane activating NF-E2-related nuclear factor erythroid-2 (Nrf-2 signaling may ameliorate UUO-induced renal damage. UUO was induced in the left kidney of female Wistar rats. The level of renal blood flow, cortical and medullary oxygen tension and reactive oxygen species (ROS was evaluated. Fibrosis, ED-1 (macrophage/monocyte infiltration, oxidative stress, autophagy, apoptosis and pyroptosis were evaluated by immunohistochemistry and Western blot in UUO kidneys. Effects of sulforaphane, an Nrf-2 activator, on Nrf-2- and mitochondrial stress-related proteins and renal injury were examined. UUO decreased renal blood flow and oxygen tension and increased renal ROS, 3-nitrotyrosine stain, ED-1 infiltration and fibrosis. Enhanced renal tubular Beclin-1 expression started at 4 h UUO and further enhanced at 3d UUO, whereas increased Atg-5-Atg12 and LC3-II expression were found at 3d UUO. Increased renal Bax/Bcl-2 ratio, caspase 3 and PARP fragments, apoptosis formation associated with increased caspase 1 and IL-1β expression for pyroptosis formation were started from 3d UUO. UUO reduced nuclear Nrf-2 translocation, increased cytosolic and inhibitory Nrf-2 expression, increased cytosolic Bax translocation to mitochondrial and enhanced mitochondrial Cytochrome c release into cytosol of the UUO kidneys. Sulforaphane significantly increased nuclear Nrf-2 translocation and decreased mitochondrial Bax translocation and Cytochrome c release into cytosol resulting in decreased renal injury. In conclusion, sulforaphane via activating Nrf-2 signaling preserved mitochondrial function and suppressed UUO-induced renal oxidative stress, inflammation, fibrosis, autophagy, apoptosis and pyroptosis.

  18. Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

    Science.gov (United States)

    Yu, Jian-Qing; Yin, Yan; Lei, Jia-Chuan; Zhang, Xiu-Qiao; Chen, Wei; Ding, Cheng-Li; Wu, Shan; He, Xiao-Yu; Liu, Yan-Wen; Zou, Guo-Lin

    2012-02-01

    Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 μg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 μg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 μg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. ACS6, a Hydrogen sulfide-donating derivative of sildenafil, inhibits homocysteine-induced apoptosis by preservation of mitochondrial function

    Directory of Open Access Journals (Sweden)

    Tang Xiao-Qing

    2011-08-01

    Full Text Available Abstract Background The hydrogen sulfide-releasing sildenafil, ACS6, has been demonstrated to inhibit superoxide formation through donating hydrogen sulfide (H2S. We have found that H2S antagonizes homocysteine-induced oxidative stress and neurotoxicity. The aim of the present study is to explore the protection of ACS6 against homocysteine-triggered cytotoxicity and apoptosis and the molecular mechanisms underlying in PC12 cells. Methods Cell viability was determined by Cell Counting Kit-8 assay. Cell apoptosis was observed using the chromatin dye Hoechst 33258 and analyzed by Flow Cytometry after propidium iodide staining. Mitochondrial membrane potential was monitored using the fluorescent dye Rh123. Intracellular reactive oxygen species were determined by oxidative conversion of cell permeable 2',7'-dichlorfluorescein-diacetate to fluorescent 2',7'-dichlorfluorescein. The expression of cleaved caspase-3 and bcl-2 and the accumulation of cytosolic cytochrome c were analyzed by Western blot. Results We show that ACS6 protects PC12 cells against cytotoxicity and apoptosis induced by homocysteine and blocks homocysteine-triggered cytochrome c release and caspase-3 activation. ACS6 treatment results in not only prevention of homocysteine-caused mitochondrial membrane potential (Δψ loss and reactive oxygen species (ROS overproduction but also reversal of Bcl-2 down-expression. Conclusions These results indicate that ACS6 protects PC12 cells against homocysteine-induced cytotoxicity and apoptosis by preservation of mitochondrial function though inhibiting both loss of Δψ and accumulation of ROS as well as modulating the expression of Bcl-2. Our study provides evidence both for a neuroprotective effect of ACS6 and for further evaluation of ACS6 as novel neuroprotectants for Alzheimer's disease associated with homocysteine.

  20. Oxidative modification of methionine80 in cytochrome c by reaction with peroxides.

    Science.gov (United States)

    Nugraheni, Ari Dwi; Ren, Chunguang; Matsumoto, Yorifumi; Nagao, Satoshi; Yamanaka, Masaru; Hirota, Shun

    2018-05-01

    The Met80-heme iron bond of cytochrome c (cyt c) is cleaved by the interaction of cyt c with cardiolipin (CL) in membranes. The Met80 dissociation enhances the peroxidase activity of cyt c and triggers cyt c release from mitochondrion to the cytosol at the early stage of apoptosis. This paper demonstrates the selective oxidation of Met80 for the reaction of ferric cyt c with a peroxide, meta-chloroperbenzoic acid (mCPBA), in the presence of CL-containing liposomes by formation of a ferryl species (Compound I). After the reaction of cyt c with mCPBA in the presence of 1,2-dioloeyl-sn-glycero-3-phosphocholine (DOPC) liposomes containing CL, the electrospray ionization mass spectrum of the peptide fragments, obtained by digestion of cyt c with lysyl endopeptidase, exhibited a peak at m/z = 795.45; whereas, this peak was not observed for the peptide fragments obtained after the reaction in the presence of DOPC liposomes not containing CL. According to the tandem mass spectrum of the m/z = 795.45 peptide fragment, Met80 was modified with a 16 Da mass increase. The purified Met80-modified cyt c exhibited a peroxidase activity more than 5-fold higher than that of the unmodified protein. Transient absorption bands around 650 nm were generated by the reactions with mCPBA for ferric wild-type cyt c in the presence of CL-containing DOPC liposomes and ferric Y67F cyt c in the absence of liposomes. The formation and decomposition rates of the 650-nm absorption species increased and decreased, respectively, by increasing the mCPBA concentration in the reaction, indicating transient formation of Compound I. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Suppression of cytochrome p450 reductase enhances long-term hematopoietic stem cell repopulation efficiency in mice.

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    Yan Zhang

    Full Text Available BACKGROUND: Bone marrow microenvironment (niche plays essential roles in the fate of hematopoietic stem cells (HSCs. Intracellular and extracellular redox metabolic microenvironment is one of the critical factors for the maintenance of the niche. Cytochrome P450 reductase (CPR is an obligate electron donor to all microsomal cytochrome P450 enzymes (P450 or CYP, and contributes to the redox metabolic process. However, its role in maintaining HSCs is unknown. OBJECTIVE: To examine the effects of low CPR expression on HSCs function using a mouse model of globally suppressed Cpr gene expression (Cpr Low, CL mice. METHODS: Hematopoietic cell subpopulations in bone marrow (BM and peripheral blood (PB from WT and CL mice were examined for their repopulation and differentiation ability upon BM competitive transplantation and enriched HSC (LKS(+ transplantation. Effects of low CPR expression on hematopoiesis were examined by transplanting normal BM cells into CL recipients. Reactive oxygen species (ROS, cell cycle, and apoptosis in CL mice were analyzed by flow cytometry for DCF-DA fluorescence intensity, Ki67 protein, and Annexin-V, respectively. RESULTS: The levels of ROS in BM cells, HPCs and HSCs were comparable between CL and WT mice. In comparison to WT mice, the number of LT-HSCs or ST-HSCs was lower in CL mice while CMPs, GMPs and MEPs in CL mice were higher than that in WT control. Competitive transplantation assay revealed enhanced repopulation capacity of HSCs with low CPR expression, but no difference in differentiation potential upon in vitro experiments. Furthermore, lymphoid differentiation of donor cells decreased while their myeloid differentiation increased under CL microenvironment although the overall level of donor hematopoietic repopulation was not significantly altered. CONCLUSIONS: Our studies demonstrate that suppressing CPR expression enhances the repopulation efficiency of HSCs and a low CPR expression microenvironment favors

  2. The amino acid sequence of cytochrome c from Cucurbita maxima L. (pumpkin)

    Science.gov (United States)

    Thompson, E. W.; Richardson, M.; Boulter, D.

    1971-01-01

    The amino acid sequence of pumpkin cytochrome c was determined on 2μmol of protein. Some evidence was found for the occurrence of two forms of cytochrome c, whose sequences differed in three positions. Pumpkin cytochrome c consists of 111 residues and is homologous with mitochondrial cytochromes c from other plants. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7. PMID:5131733

  3. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Park, Jae Hyeon; Lee, Jeong Eun; Shin, In Chul; Koh, Hyun Chul

    2013-01-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  4. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  5. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    Hao, Hongying; Dong, Yanbin; Bowling, Maria T; Gomez-Gutierrez, Jorge G; Zhou, H Sam; McMasters, Kelly M

    2007-01-01

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  6. Covalent Modification of Cytochrome C by Reactive Metabolites of Furan

    OpenAIRE

    Phillips, Martin B.; Sullivan, Mathilde M.; Villalta, Peter W.; Peterson, Lisa A.

    2013-01-01

    Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivit...

  7. Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding

    OpenAIRE

    Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.

    2009-01-01

    The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three protein forms which were assigned to a low-temperature and a high-temperature His-Met intermediate species and a bis-histidinate form (although the presence of a His-Lys form cannot be excluded). The muc...

  8. Enzymes activities involving bacterial cytochromes incorporated in clays

    International Nuclear Information System (INIS)

    Lojou, E.; Giudici-Orticoni, M.Th.; Bianco, P.

    2005-01-01

    With the development of bio electrochemistry, researches appeared on the enzymes immobilization at the surface of electrodes for the realization of bioreactors and bio sensors. One of the main challenges is the development of host matrix able to immobilize the protein material preserving its integrity. In this framework the authors developed graphite electrodes modified by clay films. These electrodes are examined for two enzyme reactions involving proteins of sulfate-reduction bacteria. Then in the framework of the hydrogen biological production and bioreactors for the environmental pollution de-pollution, the electrochemical behavior of the cytochrome c3 in two different clays deposed at the electrode is examined

  9. Apoptosis and Molecular Targeting Therapy in Cancer

    Science.gov (United States)

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  10. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  11. Mechanisms of Neuronal Apoptosis In Vivo

    National Research Council Canada - National Science Library

    Martin, Lee J

    2004-01-01

    .... Neuronal cell death in the form of apoptosis or necrosis occurs after exposure to neurotoxins, chemical warfare agents, radiation, viruses, and after seizures, trauma, limb amputation, and hypoxic...

  12. Reduction of reversed micelle entrapped cytochrome c and cytochrome c3 by electrons generated by pulse radiolysis or by pyrene photoionization

    International Nuclear Information System (INIS)

    Vlsser, A.J.W.G.; Fendler, J.H.

    1982-01-01

    Horse heart cytochrome c and cytochrome c 3 , isolated from Desulfovibrio vulgaris, have been incorporated in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) entrapped water pools in heptane. The absorption spectra of the cytochromes have been found to be strongly dependent on the water to AOT concentration ratios. The proteins solubilized in heptane by the AOT reversed micelles have retained their ability to mediate electron transfer. They reacted very rapidly with hydrated electrons, generated pulse radiolytically or, alternatively, formed in the laser photoionization of pyrene

  13. Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung

    Energy Technology Data Exchange (ETDEWEB)

    Ghanem, M.M.; Battelli, L.A.; Mercer, R.R.; Scabilloni, J.F.; Kashon, M.L.; Ma, J.Y.C.; Nath, J.; Hubbs, A.F.

    2006-09-15

    Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CID was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal P-naphthoflavone (BNF), a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH (quinoline-Val-Asp (OMe)-CH{sub 2}-OPH). In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. It is concluded that combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.

  14. A novel benzofuran derivative, ACDB, induces apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress.

    Science.gov (United States)

    Su, Chen-Ming; Chen, Chien-Yu; Lu, Tingting; Sun, Yi; Li, Weimin; Huang, Yuan-Li; Tsai, Chun-Hao; Chang, Chih-Shiang; Tang, Chih-Hsin

    2016-12-13

    Chondrosarcoma is one of the bone tumor with high mortality in respond to poor radiation and chemotherapy treatment. Here, we analyze the antitumor activity of a novel benzofuran derivative, 2-amino-3-(2-chlorophenyl)-6-(4-dimethylaminophenyl)benzofuran-4-yl acetate (ACDB), in human chondrosarcoma cells. ACDB increased the cell apoptosis of human chondrosarcomas without harm in chondrocytes. ACDB also enhanced endoplasmic reticulum (ER) stress, which was characterized by varieties in the cytosolic calcium levels and induced the expression of glucose-regulated protein (GRP) and calpain. Furthermore, the ACDB-induced chondrosarcoma apoptosis was associated with the upregulation of the B cell lymphoma-2 (Bcl-2) family members including pro- and anti-apoptotic proteins, downregulation of dysfunctional mitochondria that released cytochrome C, and subsequent activation of caspases-3. In addition, the ACDB-mediated cellular apoptosis was suppressed by transfecting cells with glucose-regulated protein (GRP) and calpain siRNA or treating cells with ER stress chelators and caspase inhibitors. Interestingly, animal experiments illustrated a reduction in the tumor volume following ACDB treatment. Together, these results suggest that ACDB may be a novel tumor suppressor of chondrosarcoma, and this study demonstrates that the novel antitumor agent, ACDB, induced apoptosis by mitochondrial dysfunction and ER stress in human chondrosarcoma cells in vitro and in vivo.

  15. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  16. Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells.

    Science.gov (United States)

    Shao, Xuehua; Lai, Duo; Zhang, Ling; Xu, Hanhong

    2016-10-18

    Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5.

  17. Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Dae Sung Kim

    2012-01-01

    Full Text Available Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

  18. Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-κB-mediated survival signaling

    International Nuclear Information System (INIS)

    Leskinen, Markus J.; Heikkilae, Hanna M.; Speer, Mei Y.; Hakala, Jukka K.; Laine, Mika; Kovanen, Petri T.; Lindstedt, Ken A.

    2006-01-01

    Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-κB-mediated survival signaling. Following chymase treatment, the translocation of active NF-κB/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1β-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-κB-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-κB-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques

  19. Dietary flavonoid fisetin regulates aluminium chloride-induced neuronal apoptosis in cortex and hippocampus of mice brain.

    Science.gov (United States)

    Prakash, Dharmalingam; Sudhandiran, Ganapasam

    2015-12-01

    Dietary flavonoids have been suggested to promote brain health by protecting brain parenchymal cells. Recently, understanding the possible mechanism underlying neuroprotective efficacy of flavonoids is of great interest. Given that fisetin exerts neuroprotection, we have examined the mechanisms underlying fisetin in regulating Aβ aggregation and neuronal apoptosis induced by aluminium chloride (AlCl3) administration in vivo. Male Swiss albino mice were induced orally with AlCl3 (200 mg/kg. b.wt./day/8 weeks). Fisetin (15 mg/Kg. b.wt. orally) was administered for 4 weeks before AlCl3-induction and administered simultaneously for 8 weeks during AlCl3-induction. We found aggregation of Amyloid beta (Aβ 40-42), elevated expressions of Apoptosis stimulating kinase (ASK-1), p-JNK (c-Jun N-terminal Kinase), p53, cytochrome c, caspases-9 and 3, with altered Bax/Bcl-2 ratio in favour of apoptosis in cortex and hippocampus of AlCl3-administered mice. Furthermore, TUNEL and fluoro-jade C staining demonstrate neurodegeneration in cortex and hippocampus. Notably, treatment with fisetin significantly (Pfisetin treatment. We have identified the involvement of fisetin in regulating ASK-1 and p-JNK as possible mediator of Aβ aggregation and subsequent neuronal apoptosis during AlCl3-induced neurodegeneration. These findings define the possibility that fisetin may slow or prevent neurodegneration and can be utilised as neuroprotective agent against Alzheimer's and Parkinson's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt

    Directory of Open Access Journals (Sweden)

    Yanfang Zong

    2015-01-01

    Full Text Available Background. The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC apoptosis. Methods. Cultured human umbilical vein endothelial cells (HUVECs were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE, cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc. Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL methods. Results. High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt. Conclusion. H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

  1. Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species

    Science.gov (United States)

    Quan, Juan-Hua; Kang, Byung-Hun; Yang, Jung-Bo; Rhee, Yun-Ee; Noh, Heung-Tae; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min

    2017-01-01

    Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS), and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5) for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC), reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway. PMID:29410962

  2. Ganoderma atrum polysaccharide ameliorates anoxia/reoxygenation-mediated oxidative stress and apoptosis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Zhang, Yan-Song; Li, Wen-Juan; Zhang, Xian-Yi; Yan, Yu-Xin; Nie, Shao-Ping; Gong, De-Ming; Tang, Xiao-Fang; He, Ming; Xie, Ming-Yong

    2017-05-01

    Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant capacity and cardiovascular benefits. The aim of this study was to investigate the role of PSG-1 in oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs) under anoxia/reoxygenation (A/R) injury conditions. The results showed that exposure of HUVECs to A/R triggered cell death and apoptosis. Administration of PSG-1 significantly inhibited A/R-induced cell death and apoptosis in HUVECs. PSG-1-reduced A/R injury was mediated via mitochondrial apoptotic pathway, as evidenced by elevation of mitochondrial Bcl-2 protein and mitochondrial membrane potential, and attenuation of Bax translocation, cytochrome c release and caspases activation. Furthermore, PSG-1 enhanced the activities of superoxide dismutase, catalase and glutathione peroxidase and glutathione content, and concomitantly attenuated reactive oxygen species generation, lipid peroxidation and glutathione disulfide content. The antioxidant, N-acetyl-l-cysteine, significantly ameliorated all of these endothelial injuries caused by A/R, suggesting that antioxidant activities might play a key role in PSG-1-induced endothelial protection. Taken together, these findings suggested that PSG-1 could be as a promising adjuvant against endothelial dysfunction through ameliorating oxidative stress and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Primary bovine skeletal muscle cells enters apoptosis rapidly via the intrinsic pathway when available oxygen is removed.

    Directory of Open Access Journals (Sweden)

    Sissel Beate Rønning

    Full Text Available Muscle cells undergo changes post-mortem during the process of converting muscle into meat, and this complex process is far from revealed. Recent reports have suggested programmed cell death (apoptosis to be important in the very early period of converting muscle into meat. The dynamic balance that occurs between anti-apoptotic members, such as Bcl-2, and pro-apoptotic members (Bid, Bim helps determine whether the cell initiates apoptosis. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to investigate if apoptosis is induced when oxygen is removed from the growth medium. Primary bovine muscle cells were differentiated to form myotubes, and anoxia was induced for 6h. The anoxic conditions significantly increased (P<0.05 the relative gene expression of anti- and pro-apoptotic markers (Aif, Bcl-2, Bid and Bim, and the PARK7 (P<0.05 and Grp75 (Hsp70 protein expressions were transiently increased. The anoxic conditions also led to a loss of mitochondrial membrane potential, which is an early apoptotic event, as well as cytochrome c release from the mitochondria. Finally, reorganization and degradation of cytoskeletal filaments occurred. These results suggest that muscle cells enters apoptosis via the intrinsic pathway rapidly when available oxygen in the muscle diminishes post-mortem.

  4. A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera.

    Science.gov (United States)

    Bivén, K; Erdal, H; Hägg, M; Ueno, T; Zhou, R; Lynch, M; Rowley, B; Wood, J; Zhang, C; Toi, M; Shoshan, M C; Linder, S

    2003-06-01

    We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

  5. Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species

    Directory of Open Access Journals (Sweden)

    Juan-Hua Quan

    2017-01-01

    Full Text Available Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS, and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5 for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC, reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway.

  6. 8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

    International Nuclear Information System (INIS)

    Ma, Jun; Zhang, Lei; Li, Shanshan; Liu, Shulin; Ma, Cui; Li, Weiyang; Falck, J.R.; Manthati, Vijay L.; Reddy, D. Sudarshan; Medhora, Meetha; Jacobs, Elizabeth R.; Zhu, Daling

    2010-01-01

    Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.

  7. 8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Jun; Zhang, Lei; Li, Shanshan [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Liu, Shulin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Cui [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Li, Weiyang [Mudanjiang Medical College, Mudanjiang 157011 (China); Falck, J.R.; Manthati, Vijay L.; Reddy, D. Sudarshan [University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Medhora, Meetha; Jacobs, Elizabeth R. [Division of Pulmonary and Critical Care, Department of Medicine, Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081 (China); Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2010-08-15

    Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.

  8. Activation of mPTP-dependent mitochondrial apoptosis pathway by a novel pan HDAC inhibitor resminostat in hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Meili [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Shi, Wenhong [Department of Radiotherapy, Linyi Tumor Hospital, Linyi (China); Li, Zhengling [Department of Nursing, Tengzhou Central People’s Hospital, Tengzhou (China); Liu, Haiyan, E-mail: liuhaiyanlinyi5@sina.com [Department of Nursing, Linyi People’s Hospital, No. 27 Jiefang Road, Linyi 276000, Shandong (China)

    2016-09-02

    Over-expression and aberrant activation of histone deacetylases (HDACs) are often associated with poor prognosis of hepatocellular carcinoma (HCC). Here, we evaluated the potential anti-hepatocellular carcinoma (HCC) cell activity by resminostat, a novel pan HDAC inhibitor (HDACi). We demonstrated that resminostat induced potent cytotoxic and anti-proliferative activity against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, resminostat treatment in HCC cells activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway, which was evidenced by physical association of cyclophilin-D and adenine nucleotide translocator 1 (ANT-1), mitochondrial depolarization, cytochrome C release and caspase-9 activation. Intriguingly, the mPTP blockers (sanglifehrin A and cyclosporine A), shRNA knockdown of cyclophilin-D or the caspase-9 inhibitor dramatically attenuated resminostat-induced HCC cell apoptosis and cytotoxicity. Reversely, HCC cells with exogenous cyclophilin-D over-expression were hyper-sensitive to resminostat. Intriguingly, a low concentration of resminostat remarkably potentiated sorafenib-induced mitochondrial apoptosis pathway activation, leading to a profound cytotoxicity in HCC cells. The results of this preclinical study indicate that resminostat (or plus sorafenib) could be further investigated as a valuable anti-HCC strategy. - Highlights: • Resminostat inhibits human HCC cell survival and proliferation. • Resminostat activates mPTP-dependent mitochondrial apoptosis pathway in HCC cells. • Resminostat potentiates sorafenib-induced mitochondrial apoptosis pathway activation. • mPTP or caspase-9 inhibition attenuates apoptosis by resminostat or plus sorafenib.

  9. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

    International Nuclear Information System (INIS)

    Su, Miaoxian; Chung, Hau Yin; Li, Yaolan

    2011-01-01

    Highlights: → Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. → ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. → ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. → ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential (ΔΨm), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

  10. BAD overexpression inhibits cell growth and induces apoptosis via mitochondrial-dependent pathway in non-small cell lung cancer.

    Science.gov (United States)

    Jiang, Li; Luo, Man; Liu, Dan; Chen, Bojiang; Zhang, Wen; Mai, Lin; Zeng, Jing; Huang, Na; Huang, Yi; Mo, Xianming; Li, Weimin

    2013-06-01

    The pro-apoptotic Bcl-2 protein BAD initiated apoptosis in human cells and has been identified as a prognostic marker in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the functions of BAD in NSCLC. Overexpression of BAD was performed by transfecting different NSCLC cell lines with wild-type BAD. Cell proliferation, cell cycle, apoptosis, and invasion were characterized in vitro. Tumorigenicity was analyzed in vivo. Western blot was performed to determine the effects of BAD overexpression on the Bcl-2 family proteins and apoptosis-related proteins. Overexpression of BAD significantly inhibited cell proliferation in H1299, H292, and SPC-A1 but not in SK-MES-1 and H460 cell lines in vitro. BAD overexpression also reduced the tumorigenicity of H1299/SPC-A1 cell in vivo. However, no appreciable effects on cell cycle distribution and invasion were observed in all these cell lines. BAD overexpression also induced apoptosis in all cell types, in which process expression of mitochondrial cytochrom c (cyto-c) and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax and caspase 8 expressions did not changed. These findings indicated that a mitochondrial pathway, in which process cyto-c was released from mitochondrial to activate caspase 3, was involved in BAD overexpression-mediated apoptosis. Our data suggested that increased expression of BAD enhance apoptosis and has negative influence on cell proliferation and tumor growth in NSCLC. Bad is a new potential target for tumor interventions.

  11. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Miaoxian [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Chung, Hau Yin, E-mail: anthonychung@cuhk.edu.hk [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Li, Yaolan [Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou (China); Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Guangzhou (China)

    2011-07-29

    Highlights: {yields} Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. {yields} ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. {yields} ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. {yields} ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential ({Delta}{Psi}m), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

  12. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  13. Suppression of postmitochondrial signaling and delayed response to UV-induced nuclear apoptosis in HeLa cells

    International Nuclear Information System (INIS)

    Sasai, Kaori; Yajima, Hirohiko; Suzuki, Fumio

    2002-01-01

    Activation of postmitochondrial pathways by UV irradiation was examined using mouse lymphoma 3SB and human leukemic Jurkat cells and two human carcinoma cell lines (HeLa and MCF-7). Exposure of 3SB and Jurkat cells resulted in large amounts of cytochrome c and apoptosis-inducing factor (AIF) being released into the cytosol, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase-9 and its downstream caspases was detected. HeLa and MCF-7 cells also showed extensive release of mitochondrial factors and caspase-9 activation at 4 to 6 h after exposure, but apoptotic nuclear changes appeared much later. Compared with 3SB and Jurkat cells, these carcinoma cell lines exhibited reduced activation of caspase-9-like proteolytic activity by UV radiation, and levels of caspase-3-like activity in HeLa cells were extremely low, similar to those in caspase-3-deficient MCF-7 cells. These results suggest that the delayed response to UV-induced nuclear apoptosis in HeLa cells is due to a reduced activation of the caspase cascade downstream of cytochrome c release and suppression of caspase-3 activity. (author)

  14. Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells.

    Science.gov (United States)

    Gilardini Montani, Maria Saveria; Granato, Marisa; Santoni, Claudio; Del Porto, Paola; Merendino, Nicolò; D'Orazi, Gabriella; Faggioni, Alberto; Cirone, Mara

    2017-04-01

    Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations. Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy. We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells. From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.

  15. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing; Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-01

    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 β 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

  16. MicroRNA-132 protects hippocampal neurons against oxygen-glucose deprivation-induced apoptosis.

    Science.gov (United States)

    Sun, Zu-Zhen; Lv, Zhan-Yun; Tian, Wen-Jing; Yang, Yan

    2017-09-01

    Hypoxic-ischemic brain injury (HIBI) results in death or long-term neurologic impairment in both adults and children. In this study, we investigated the effects of microRNA-132 (miR-132) dysregulation on oxygen-glucose deprivation (OGD)-induced apoptosis in fetal rat hippocampal neurons, in order to reveal the therapeutic potential of miR-132 on HIBI. MiR-132 dysregulation was induced prior to OGD exposure by transfection of primary fetal rat hippocampal neurons with miR-132 mimic or miR-132 inhibitor. The effects of miR-132 overexpression and suppression on OGD-stimulated hippocampal neurons were evaluated by detection of cell viability, apoptotic cells rate, and the expression of apoptosis-related proteins. Besides, TargetScan database and dual luciferase activity assay were used to seek a target gene of miR-132. As a result, miR-132 was highly expressed in hippocampal neurons following 2 h of OGD exposure. MiR-132 overexpression significantly increased OGD-diminished cell viability and reduced OGD-induced apoptosis at 12, 24, and 48 h post-OGD. MiR-132 overexpression significantly down-regulated the expressions of Bax, cytochrome c, and caspase-9, but up-regulated BCl-2. Caspase-3 activity was also significantly decreased by miR-132 overexpression. Furthermore, FOXO3 was a direct target of miR-132, and it was negatively regulated by miR-132. To conclude, our results provide evidence that miR-132 protects hippocampal neurons against OGD injury by inhibiting apoptosis.

  17. Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

    Science.gov (United States)

    Balodite, Elina; Strazdina, Inese; Galinina, Nina; McLean, Samantha; Rutkis, Reinis; Poole, Robert K; Kalnenieks, Uldis

    2014-09-01

    The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase. © 2014 The Authors.

  18. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    Science.gov (United States)

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  19. A cytosolic cytochrome b 5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme

    International Nuclear Information System (INIS)

    Nakagawa, Manabu; Yamano, Toshio; Kuroda, Kiyo; Nonaka, Yasuki; Tojo, Hiromasa; Fujii, Shigeru

    2005-01-01

    A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b 5 . At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b 5 , but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b 5 -like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6 x 10 -8 M of the protein

  20. Involvement of phorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and -independent apoptosis in hepatocellular carcinoma-derived cells

    International Nuclear Information System (INIS)

    Kuo, Kung-Kai; Chen, Yi-Ling; Chen, Lih-Ren; Li, Chien-Feng; Lan, Yu-Hsuan; Chang, Fang-Rong; Wu, Yang-Chang; Shiue, Yow-Ling

    2011-01-01

    The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation of reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene. - Highlights: → Goniothalamin (GTN) induced apoptosis in hepatocellular carcinomas-derived cells. → The apoptosis induced by GTN is PMAIP1-dependent, regardless of TP53 status. → The apoptosis induced by GTN might be TP53 transcription-dependent or -independent. → GTN-induced apoptosis is mitochondria- and caspases-mediated.

  1. Fas-induced apoptosis in malnourished infants

    African Journals Online (AJOL)

    EL-HAKIM

    deprivation in animals, including man11. Factor of apoptosis signal (Fas) induces apoptosis in activated T cells when they are repeatedly stimulated by antigen and functions to maintain T cell tolerance by deleting auto reactive cells12. The functional role of Fas (CD95) in the immune system has been examined in a variety ...

  2. Apoptosis in mammalian oocytes: a review.

    Science.gov (United States)

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  3. Targeted induction of apoptosis for cancer therapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2006-01-01

    Introduction to the thesis Programmed cell death, known as apoptosis, is an essential cellular homeostasis mechanism that ensures correct development and function of multi-cellular organisms. The pivotal importance of correct execution of apoptosis is apparent from the many human diseases with

  4. Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model

    Science.gov (United States)

    Mano, Camila M.; Cardozo, Karina H. M.; Colepicolo, Pio; Bechara, Etelvino J. H.

    2018-01-01

    Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a “mitochondrial-targeted” action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl)ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes. PMID:29649159

  5. Protein alkylation, transcriptional responses and cytochrome c release during acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents.

    Science.gov (United States)

    Thompson, Colin A; Burcham, Philip C

    2008-06-01

    Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.

  6. Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Marres, C A; Slater, Conor

    1975-01-01

    1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reo...

  7. Electrochemical determination of hydrogen peroxide using Rhodobacter capsulatus cytochrome c peroxidase at a gold electrode

    NARCIS (Netherlands)

    De Wael, K.; Buschop, H.; Heering, H.A.; De Smet, L.; Van Beeumen, J.; Devreese, B.; Adriaens, A.

    2007-01-01

    We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4?-bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it

  8. Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

    Science.gov (United States)

    Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco

    2011-10-21

    Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

  9. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

  10. The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

    NARCIS (Netherlands)

    Van Wielink, J E; Reijnders, W N; Van Spanning, R J; Oltmann, L F; Stouthamer, A.H.

    1986-01-01

    The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between

  11. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

    Full Text Available Abstract Background Compound K [20-O-β-(D-glucopyranosyl-20(S-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. Methods We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Results Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1 the activation of caspases-3, -8, and -9; (2 the loss of mitochondrial membrane potential; (3 the release of cytochrome c and Smac/DIABLO to the cytosol; (4 the translocation of Bid and Bax to mitochondria; and (5 the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. Conclusions The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through

  12. Sinoporphyrin sodium, a novel sensitizer, triggers mitochondrial-dependent apoptosis in ECA-109 cells via production of reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Wang H

    2014-06-01

    Full Text Available Haiping Wang,1 Xiaobing Wang,1 Shaoliang Zhang,2 Pan Wang,1 Kun Zhang,1 Quanhong Liu1 1Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, 2Qinglong High-Tech Co, Ltd, Yichun, Jiangxi, People's Republic of China Background: Sonodynamic therapy (SDT is a promising method that uses ultrasound to activate certain chemical sensitizers for the treatment of cancer. The purpose of this study was to investigate the sonoactivity of a novel sensitizer, sinoporphyrin sodium (DVDMS, and its sonotoxicity in an esophageal cancer (ECA-109 cell line. Methods: The fluorescence intensity of DVDMS, hematoporphyrin, protoporphyrin IX, and Photofrin II was detected by fluorescence microscopy and flow cytometry. Generation of singlet oxygen was measured using a 1, 3-diphenylisobenzofuran experiment. A 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide assay was used to examine cell viability. Production of reactive oxygen species (ROS and destabilization of the mitochondrial membrane potential were assessed by flow cytometry. Apoptosis was analyzed using Annexin-PE/7-amino-actinomycin D staining. Confocal microscopy was performed to assess mitochondrial damage and identify release of cytochrome C after treatment. Western blots were used to determine expression of oxidative stress-related and apoptosis-associated protein. Ultrastructural changes in the cell were studied by scanning electron microscopy. Results: DVDMS showed higher autofluorescence intensity and singlet oxygen production efficiency compared with other photosensitizers in both cancerous and normal cells. Compared with hematoporphyrin, DVDMS-mediated SDT was more cytotoxic in ECA-109 cells. Abundant intracellular ROS was found in the SDT groups, and the cytotoxicity

  13. In vivo nuclear imaging of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2004-04-01

    Apoptosis plays a role in the pathophysiology of many kinds of diseases and in the response of treatment. Compared to the necrosis, the apoptosis a genetically controlled and energy-dependent process which removes the unwanted cells from the body; programmed cell death or cell suicide. During the apoptosis, phosphatidylserine is expressed in the cytoplasmic outer membrane in the early phase. Annexin V, an endogenous human protein (MW=35 kD), has an affinity of about 10{sup -9} M for the phosphatidylserine exposed on the outer membrane of apoptotic cells. Annexin V can be radiolabeled with {sup 99}mTc by HYNIC or EC chelators, which can be used as an radiotracer for the in vivo imaging of apoptosis. In this article, we reviewed the apoptosis, radiolabeling of annexin V, and the experimental and clinical data using annexin V imaging.

  14. SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation of ASK-1 and dissociation of the ASK-1-Trx complex.

    Science.gov (United States)

    Madan, Esha; Gogna, Rajan; Kuppusamy, Periannan; Bhatt, Madan; Mahdi, Abbas Ali; Pati, Uttam

    2013-04-01

    p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

  15. Effectiveness of cytochrome C and cepharanthin for leukopenia following multidisciplinary treatment

    International Nuclear Information System (INIS)

    Tabata, Kumiko; Endow, Masaru; Suzuki, Hirotoshi

    1986-01-01

    Leukopenia is one of important problems for multidisciplinary treatment of malignant tumor. We could not be able to take a continuous cancer therapy because of leukopenia. And then we had a study of effectiveness combination treatment of cytochrome C with cepharanthin for leukopenia of cancer patient. We carried on the study of 3 classifications of treatment as follows, a) cytochrome C only, b) combined cytochrome C with cepharanthin, and c) control group without drugs. Bone marrow potentiality is individual differentiation and then the group was administrated both cytochrome C and cepharanthin following radiotherapy associated with postoperative breast cancer. The above description lead to conclusion that combination treatment of cytochrome C and cepharanthin was available for protective drugs from multidisciplinary treatment induced leukemia. (author)

  16. Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Jung Hoon [Cheongju Univ., Cheongju (Korea, Republic of)

    2013-11-15

    Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD.

  17. Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

    International Nuclear Information System (INIS)

    Kang, Jung Hoon

    2013-01-01

    Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD

  18. Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

    Science.gov (United States)

    Hough, Michael A; Andrew, Colin R

    2015-01-01

    Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins. © 2015 Elsevier Ltd. All rights reserved.

  19. Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

    Science.gov (United States)

    Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

    Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride

    International Nuclear Information System (INIS)

    Zhang, Shun; Niu, Qiang; Gao, Hui; Ma, Rulin; Lei, Rongrong; Zhang, Cheng; Xia, Tao; Li, Pei; Xu, Chunyan; Wang, Chao; Chen, Jingwen; Dong, Lixing; Zhao, Qian; Wang, Aiguo

    2016-01-01

    Fluoride, a ubiquitous environmental contaminant, is known to impair testicular functions and fertility; however the underlying mechanisms remain obscure. In this study, we used a rat model to mimic human exposure and sought to investigate the roles of apoptosis and autophagy in testicular toxicity of fluoride. Sprague–Dawley rats were developmentally exposed to 25, 50, or 100 mg/L sodium fluoride (NaF) via drinking water from pre-pregnancy to post-puberty, and then the testes of offspring were excised on postnatal day 56. Our results demonstrated that developmental NaF exposure induced an enhanced testicular apoptosis, as manifested by a series of hallmarks such as caspase-3 activation, chromatin condensation and DNA fragmentation. Further study revealed that fluoride exposure elicited significant elevations in the levels of cell surface death receptor Fas with a parallel increase in cytoplasmic cytochrome c, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Intriguingly, fluoride treatment also simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II but not Beclin1. Unexpectedly, the expression of p62, a substrate that is degraded by autophagy, was also significantly elevated, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation rather than increased formation. Importantly, these were associated with marked histopathological lesions including spermatogenic failure and germ cell loss, along with severe ultrastructural abnormalities in testes. Taken together, our findings provide deeper insights into roles of excessive apoptosis and defective autophagy in the aggravation of testicular damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity. - Highlights: • Rats were developmentally exposed to fluoride from pre-pregnancy to post-puberty. • Both excessive apoptosis and defective autophagy are involved in

  1. Correlation between the potency of flavonoids for cytochrome c reduction and inhibition of cardiolipin-induced peroxidase activity.

    Science.gov (United States)

    Lagoa, Ricardo; Samhan-Arias, Alejandro K; Gutierrez-Merino, Carlos

    2017-05-06

    There are large differences between flavonoids to protect against apoptosis, a process in which cytochrome c (Cyt c) plays a key role. In this work, we show that 7 of 13 flavonoids studied have a capacity to reduce Cyt c similar or higher than ascorbate, the flavonols quercetin, kaempferol and myricetin, flavanol epigallocatechin-gallate, anthocyanidins cyanidin and malvidin, and the flavone luteolin. In contrast, the kaempferol 3(O)- and 3,4'(O)-methylated forms, the flavanone naringenin, and also apigenin and chrysin, had a negligible reducing capacity. Equilibrium dialysis and quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence experiments showed that flavonoids did not interfere with Cyt c binding to cardiolipin (CL)/phosphatidylcholine (PC) vesicles. However, the CL-induced loss of Cyt c Soret band intensity was largely attenuated by flavonoids, pointing out a stabilizing action against Cyt c unfolding in the complex. Moreover, flavonoids that behave as Cyt c reductants also inhibited the pro-apoptotic CL-induced peroxidase activity of Cyt c, indicating that modulation of Cyt c signaling are probable mechanisms behind the protective biological activities of flavonoids. © 2016 BioFactors, 43(3):451-468, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  2. Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

    Science.gov (United States)

    Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

    2017-12-01

    Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

  3. The Nitric Oxide Prodrug JS-K Induces Ca(2+)-Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

    Science.gov (United States)

    Liu, Ling; Wang, Dongmei; Wang, Jiangang; Wang, Shuying

    2016-04-01

    Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS-K, O(2)-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS-K inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner and significantly induced apoptosis. JS-K enhanced the ratio of Bax-to-Bcl-2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase-9/3. JS-K caused an increasing cytosolic Ca(2+) and the loss of mitochondrial membrane potential. Carboxy-PTIO (a NO scavenger) and BAPTA-AM (an intracellular Ca(2+) chelator) significantly blocked an increasing cytosolic Ca(2+) in JS-K-induced HepG2 cells apoptosis, especially Carboxy-PTIO. Meanwhile, Carboxy-PTIO and BAPTA-AM treatment both attenuate JS-K-induced apoptosis through upregulation of Bcl-2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase-9/3. In summary, JS-K induced HepG2 cells apoptosis via Ca(2+)/caspase-3-mediated mitochondrial pathway. © 2015 Wiley Periodicals, Inc.

  4. Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

    Science.gov (United States)

    Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

    2016-01-01

    The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

  5. E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

    Directory of Open Access Journals (Sweden)

    Kuan-Ho Lin

    2017-04-01

    Full Text Available Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2, for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs. In addition, protein phosphatase such as protein phosphatase 1 (PP1 and calcineurin (PP2B are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

  6. Epothilones Suppress Neointimal Thickening in the Rat Carotid Balloon-Injury Model by Inducing Vascular Smooth Muscle Cell Apoptosis through p53-Dependent Signaling Pathway.

    Science.gov (United States)

    Son, Dong Ju; Jung, Jae Chul; Hong, Jin Tae

    2016-01-01

    Microtubule stabilizing agents (MTSA) are known to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and effectively reduce neointimal hyperplasia and restenosis. Epothilones (EPOs), non-taxane MTSA, have been found to be effective in the inhibition of VSMC proliferation and neointimal formation by cell cycle arrest. However, effect of EPOs on apoptosis in hyper-proliferated VSMCs as a possible way to reduce neointimal formation and its action mechanism related to VSMC viability has not been suited yet. Thus, the purposes of the present study was to investigate whether EPOs are able to inhibit neointimal formation by inducing apoptosis within the region of neointimal hyperplasia in balloon-injured rat carotid artery, as well as underlying action mechanism. Treatment of EPO-B and EPO-D significantly induced apoptotic cell death and mitotic catastrophe in hyper-proliferated VSMCs, resulting in cell growth inhibition. Further, EPOs significantly suppressed VSMC proliferation and induced apoptosis by activation of p53-dependent apoptotic signaling pathway, Bax/cytochrome c/caspase-3. We further demonstrated that the local treatment of carotid arteries with EPOs potently inhibited neointimal lesion formation by induction of apoptosis in rat carotid injury model. Our findings demonstrate a potent anti-neointimal hyperplasia property of EPOs by inducing p53-depedent apoptosis in hyper-proliferated VSMCs.

  7. The production of ammonia by multiheme cytochromes C.

    Science.gov (United States)

    Simon, Jörg; Kroneck, Peter M H

    2014-01-01

    The global biogeochemical nitrogen cycle is essential for life on Earth. Many of the underlying biotic reactions are catalyzed by a multitude of prokaryotic and eukaryotic life forms whereas others are exclusively carried out by microorganisms. The last century has seen the rise of a dramatic imbalance in the global nitrogen cycle due to human behavior that was mainly caused by the invention of the Haber-Bosch process. Its main product, ammonia, is a chemically reactive and biotically favorable form of bound nitrogen. The anthropogenic supply of reduced nitrogen to the biosphere in the form of ammonia, for example during environmental fertilization, livestock farming, and industrial processes, is mandatory in feeding an increasing world population. In this chapter, environmental ammonia pollution is linked to the activity of microbial metalloenzymes involved in respiratory energy metabolism and bioenergetics. Ammonia-producing multiheme cytochromes c are discussed as paradigm enzymes.

  8. The Membrane Modulates Internal Proton Transfer in Cytochrome c Oxidase

    DEFF Research Database (Denmark)

    Öjemyr, Linda Nasvik; Ballmoos, Christoph von; Faxén, Kristina

    2012-01-01

    The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O-2 reduction...... DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.......-glycerol) (DOPG). In addition, a small Change in the internal Cu-A-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic...

  9. Cytochrome P450 polymorphism and postoperative cognitive dysfunction

    DEFF Research Database (Denmark)

    Steinmetz, J; Jespersgaard, Cathrine; Dalhoff, Kim Peder

    2012-01-01

    neuropsychological testing at one week had POCD, and 24 out of 307 (7.8%) had POCD at three months. None of the examined CYP2C19, 2D6 alleles, or various phenotypes were significantly associated with POCD. CONCLUSION: Polymorphisms in CYP2C19, or 2D6 genes do not seem to be related to the occurrence of cognitive......BACKGROUND:The etiology of postoperative cognitive dysfunction (POCD) remains unclear but toxicity of anesthetic drugs and their metabolites could be important. We aimed to assess the possible association between POCD after propofol anesthesia and various phenotypes owing to polymorphisms...... in cytochrome P450 encoding genes. METHODS:We included patients who underwent non-cardiac surgery under total intravenous anesthesia with propofol. POCD was identified using a neuropsychological test-battery administered preoperatively, one week, and three months after surgery. Genotyping of CYP2C19*2, *3, CYP2...

  10. Role of cytochrome P450 in drug interactions

    Directory of Open Access Journals (Sweden)

    Bibi Zakia

    2008-10-01

    Full Text Available Abstract Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events.

  11. Advances in molecular modeling of human cytochrome P450 polymorphism.

    Science.gov (United States)

    Martiny, Virginie Y; Miteva, Maria A

    2013-11-01

    Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined. © 2013.

  12. Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

    International Nuclear Information System (INIS)

    Koopmann, E.; Hahlbrock, K.

    1997-01-01

    Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H

  13. Stability enhancement of cytochrome c through heme deprotonation and mutations

    OpenAIRE

    Sonoyama, Takafumi; Hasegawa, Jun; Uchiyama, Susumu; Nakamura, Shota; Kobayashi, Yuji; Sambongi, Yoshihiro

    2009-01-01

    The chemical denaturation of Pseudomonas aeruginosa cytochrome c551 variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0 – 4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond form...

  14. Ligand Access Channels in Cytochrome P450 Enzymes: A Review

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2018-05-01

    Full Text Available Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents different in silico tools that were developed to uncover such channels in P450 crystal structures. It also lists some of the experimental evidence that actually suggest that these predicted channels might indeed play a critical role in modulating P450 functions. Amino acid residues at the entrance of the channels may participate to a first global ligand recognition of ligands by P450 enzymes before they reach the buried active site. Moreover, different P450 enzymes show different networks of predicted channels. The plasticity of P450 structures is also important to take into account when looking at how channels might play their role.

  15. Prognostic relevance of cytochrome C oxidase in primary glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Corinne E Griguer

    Full Text Available Patients with primary glioblastoma multiforme (GBM have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%, and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.

  16. Mode of Antifungal Drugs Interaction with Cytochrome P- 450

    Directory of Open Access Journals (Sweden)

    M- Mahmodian

    1991-07-01

    Full Text Available Computer was used to identify the interactions of substrates and antifungal drugs with the enzyme, Cytochrome P-450; and then Molplot.bas computer program was applied to get three dimensional figures of 5-hydroxy camphor.oxidation products of camphor analogues, and antifungal drugs.Cartesian characteristics of atoms building molecules, are taken from Buildz. for program, which can calculate X,Y,Z coordinates of atoms by Zmatrix data. The other program which can calculate X,Y,Z coordinates, using fractional characteristics, is the Coord, for program that, gives our cartesian characteristics of the atoms of molecule, then by using these data, we obtain three dimensional figures and distance between active atoms in compounds under consideration. Results show that distance between two oxygen atoms in 5-exo-hydroxy- camphor and the other compounds obtained from oxidation of camphor analogues, with the distance of two oxygen atoms in antifungal compounds under discussion are equal. Therefore, we can conclude that, the antifungal molecule also interacts with enzyme's active site, by its own sites, in a similar manner to the 5-hydroxy camphor molecule, which is:"n1. Nitrogen atom (N of Imidazole and Triazole ring in antifungal molecule with Iron atom in heam molecule belonging to Cytochrome P-450 enzyme, are coordinated."n2. The other atoms such as : 0,S or N in structure of the antifungal drug are coordinated with hydrogen atom of hydroxyl group belong ing to Tyr-96 in the structure of enzyme, forming hydrogen bonding.

  17. Honey and Apoptosis in Human Gastric Mucosa

    Directory of Open Access Journals (Sweden)

    Alireza Ostadrahimi

    2012-07-01

    Full Text Available Background: Gastric cancer is the fourth most common malignancy in the world. Honey is acomplex mixture of special biological active constituents. Honey possesses antioxidant and antitumorproperties. Nutritional studies have indicated that consumption of honey modulates therisk of developing gastric cancer. On the other hand, apoptosis has been reported to play a decisiverole in precancerous changes. Our chief study was conducted to assess the relationship betweenconsumption of honey and apoptosis in human gastric mucosa.Method: This cross-sectional study was conducted on 98 subjects over 18 years old, referred totwo hospitals in Tabriz, Iran. Subjects were undergone an upper gastrointestinal endoscopy, 62subjects were finally enrolled. Honey consumption was assessed by a Food Frequency Questionnaire(FFQ and apoptosis was detected by TUNEL technique. We tested polynomial curve tofind the best fit between honey consumption and apoptosis.Results: A positive relation between honey consumption and apoptosis was found (P=0.024.Our results indicated that the final and the best fit curve was: apoptosis = 1.714+1.648(honeyamount - 0.533(honey amount2 +1.833×10-5(honey amount7.Conclusion: Honey consumption had positive effects on gastric cancer by inducing apoptosis ingastric mucosa.

  18. Chk2 mediates RITA-induced apoptosis.

    Science.gov (United States)

    de Lange, J; Verlaan-de Vries, M; Teunisse, A F A S; Jochemsen, A G

    2012-06-01

    Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.

  19. Critical role of heat shock protein 27 in bufalin-induced apoptosis in human osteosarcomas: a proteomic-based research.

    Directory of Open Access Journals (Sweden)

    Xian-biao Xie

    Full Text Available Bufalin is the primary component of the traditional Chinese herb "Chan Su". Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27, decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27 were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

  20. Inhibition of VDAC1 prevents Ca²⁺-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.

    Science.gov (United States)

    Chen, Haibo; Gao, Weiwei; Yang, Yang; Guo, Shuyuan; Wang, Huan; Wang, Wei; Zhang, Shuisheng; Zhou, Qi; Xu, Haobo; Yao, Jianting; Tian, Zhen; Li, Bicheng; Cao, Wenwu; Zhang, Zhiguo; Tian, Ye

    2014-12-01

    Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca(2+) [Ca(2+)]i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca(2+)]i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca(2+) chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these

  1. Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states.

    Science.gov (United States)

    Ogura, T; Yoshikawa, S; Kitagawa, T

    1985-12-17

    Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).

  2. Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

    Science.gov (United States)

    Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

    1990-12-01

    We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

  3. MicroRNA-1 promotes apoptosis of hepatocarcinoma cells by targeting apoptosis inhibitor-5 (API-5).

    Science.gov (United States)

    Li, Dong; Liu, Yu; Li, Hua; Peng, Jing-Jing; Tan, Yan; Zou, Qiang; Song, Xiao-Feng; Du, Min; Yang, Zheng-Hui; Tan, Yong; Zhou, Jin-Jun; Xu, Tao; Fu, Zeng-Qiang; Feng, Jian-Qiong; Cheng, Peng; chen, Tao; Wei, Dong; Su, Xiao-Mei; Liu, Huan-Yi; Qi, Zhong-Chun; Tang, Li-Jun; Wang, Tao; Guo, Xin; Hu, Yong-He; Zhang, Tao

    2015-01-02

    Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Apoptosis: its pathophysiology and monitoring. The role of apoptosis in the radioiodine therapy of hyperthyroidism

    International Nuclear Information System (INIS)

    Sopotyk, J.; Rogowski, F.; Parfienczyk, A.

    2004-01-01

    The review aims to give an up to date understanding of the mechanisms of apoptosis (programmed cell death), the methods of detecting apoptosis, in particular with regard to imaging such changes non-invasively. Radioiodine (I-131) is a gamma and beta emitting radionuclide and is commonplace in the treatment of hyperthyroidism. I-131 therapy relies on the destruction of thyroid tissue by beta radiation, and such destruction is proposed to be partly as a result of apoptosis. The review undertakes to explore and provoke research into the mechanisms of thyroid cell destruction by I-131, and whether such changes are able to be detected or monitored. Current knowledge concerning apoptosis in the thyroid gland in diseased states (including cancer) are described. The clinical significance of monitoring and modifying apoptosis are emphasized. Furthermore, overt and late destruction of thyroid tissue following I-131 therapy requires elaboration, and the relevance of detecting and modifying thyroid cell apoptosis following I-131 are questioned.(author)

  5. Cudraflavone C Induces Apoptosis of A375.S2 Melanoma Cells through Mitochondrial ROS Production and MAPK Activation.

    Science.gov (United States)

    Lee, Chiang-Wen; Yen, Feng-Lin; Ko, Horng-Huey; Li, Shu-Yu; Chiang, Yao-Chang; Lee, Ming-Hsueh; Tsai, Ming-Horng; Hsu, Lee-Fen

    2017-07-13

    Melanoma is the most malignant form of skin cancer and is associated with a very poor prognosis. The aim of this study was to evaluate the apoptotic effects of cudraflavone C on A375.S2 melanoma cells and to determine the underlying mechanisms involved in apoptosis. Cell viability was determined using the MTT and real-time cytotoxicity assays. Flow cytometric evaluation of apoptosis was performed after staining the cells with Annexin V-FITC and propidium iodide. The mitochondrial membrane potential was evaluated using the JC-1 assay. Cellular ROS production was measured using the CellROX assay, while mitochondrial ROS production was evaluated using the MitoSOX assay. It was observed that cudraflavone C inhibited growth in A375.S2 melanoma cells, and promoted apoptosis via the mitochondrial pathway mediated by increased mitochondrial ROS production. In addition, cudraflavone C induced phosphorylation of MAPKs (p38, ERK, and JNK) and up-regulated the expression of apoptotic proteins (Puma, Bax, Bad, Bid, Apaf-1, cytochrome C, caspase-9, and caspase-3/7) in A375.S2 cells. Pretreatment of A375.S2 cells with MitoTEMPOL (a mitochondria-targeted antioxidant) attenuated the phosphorylation of MAPKs, expression of apoptotic proteins, and the overall progression of apoptosis. In summary, cudraflavone C induced apoptosis in A375.S2 melanoma cells by increasing mitochondrial ROS production; thus, activating p38, ERK, and JNK; and increasing the expression of apoptotic proteins. Therefore, cudraflavone C may be regarded as a potential form of treatment for malignant melanoma.

  6. Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Goyal, Shruti; Amar, Saroj Kumar [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research (AcSIR), CSIR — IITR, Lucknow 226001 (India); Dwivedi, Ashish; Mujtaba, Syed Faiz; Kushwaha, Hari Narayan; Chopra, Deepti; Pal, Manish Kumar; Singh, Dhirendra [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Chaturvedi, Rajnish Kumar [Developmental Toxicology Division, CSIR — Indian Institute of Toxicology Research, P. O. Box 80, M.G. Marg, Lucknow 226001 (India); Ray, Ratan Singh, E-mail: ratanray.2011@rediffmail.com [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research (AcSIR), CSIR — IITR, Lucknow 226001 (India)

    2016-04-15

    The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles. - Highlights: • Photodegradation of A132 and formation of novel photoproduct • Involvement of ROS in A132 phototoxicity • Role of ROS in DNA damage, CPD and micronuclei formation • Release of

  7. Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells

    International Nuclear Information System (INIS)

    Goyal, Shruti; Amar, Saroj Kumar; Dwivedi, Ashish; Mujtaba, Syed Faiz; Kushwaha, Hari Narayan; Chopra, Deepti; Pal, Manish Kumar; Singh, Dhirendra; Chaturvedi, Rajnish Kumar; Ray, Ratan Singh

    2016-01-01

    The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles. - Highlights: • Photodegradation of A132 and formation of novel photoproduct • Involvement of ROS in A132 phototoxicity • Role of ROS in DNA damage, CPD and micronuclei formation • Release of

  8. Allicin protects against H2O2-induced apoptosis of PC12 cells via the mitochondrial pathway.

    Science.gov (United States)

    Lv, Runxiao; Du, Lili; Lu, Chunwen; Wu, Jinhui; Ding, Muchen; Wang, Chao; Mao, Ningfang; Shi, Zhicai

    2017-09-01

    Allicin is a major bioactive ingredient of garlic and has a broad range of biological activities. Allicin has been reported to protect against cell apoptosis induced by H 2 O 2 in human umbilical vein endothelial cells. The present study evaluated the neuroprotective effect of allicin on the H 2 O 2 -induced apoptosis of rat pheochromocytoma PC12 cells in vitro and explored the underlying mechanism involved. PC12 cells were incubated with increasing concentrations of allicin and the toxic effect of allicin was measured by MTT assay. The cells were pretreated for 24 h with low dose (L-), medium dose (M-) and high dose (H-) of allicin, followed by exposure to 200 µM H 2 O 2 for 2 h, and the cell viability was examined by MTT assay. In addition, cell apoptosis rate was analyzed by Annexin V-FITC/PI assay, while intracellular reactive oxygen species (ROS) and mitochondrial transmembrane potential (∆ψm) were measured by flow cytometry. Bcl-2, Bax, cleaved-caspase-3 and cytochrome c (Cyt C) in the mitochondria were also examined by western blotting. The results demonstrated that 0.01 µg/ml (L-allicin), 0.1 µg/ml (M-allicin) and 1 µg/ml (H-allicin) were non-toxic doses of allicin. Furthermore, H 2 O 2 reduced cell viability, promoted cell apoptosis, induced ROS production and decreased ∆ψm. However, allicin treatment reversed the effect of H 2 O 2 in a dose-dependent manner. It was also observed that H 2 O 2 exposure significantly decreased Bcl-2 and mitochondrial Cyt C, while it increased Bax and cleaved-caspase-3, which were attenuated by allicin pretreatment. The results revealed that allicin protected PC12 cells from H 2 O 2 -induced cell apoptosis via the mitochondrial pathway, suggesting the potential neuroprotective effect of allicin against neurological diseases.

  9. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-01

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection

  10. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  11. Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Dong-Hyun Shin

    2018-04-01

    Full Text Available Background: Extended endoplasmic reticulum (ER stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular Ca2+ chelator or dantrolene (an RyR channel antagonist. These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12 partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular Ca2+ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway. Keywords: apoptosis, calcium, compound K, ER stress, lung cancer cells

  12. Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest

    International Nuclear Information System (INIS)

    Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge

    2015-01-01

    Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca 2+ , loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest

  13. Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways

    Directory of Open Access Journals (Sweden)

    Gu Bin

    2011-12-01

    Full Text Available Abstract Background Human herpesvirus 6 (HHV-6 is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs during productive HHV-6A infection and the underlying mechanisms. Results HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP, which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs. Conclusion This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

  14. Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway.

    Science.gov (United States)

    Deng, Lin; Adachi, Tetsuya; Kitayama, Kikumi; Bungyoku, Yasuaki; Kitazawa, Sohei; Ishido, Satoshi; Shoji, Ikuo; Hotta, Hak

    2008-11-01

    We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

  15. Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn.

    Science.gov (United States)

    Wang, Qiang; Du, Xia; Zhou, Bingjie; Li, Jing; Lu, Wenlong; Chen, Qiuyun; Gao, Jing

    2017-12-01

    Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate

  16. Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

    Science.gov (United States)

    Yang, Deng-Liang; Xu, Jia-Wen; Zhu, Jian-Guo; Zhang, Yi-Lin; Xu, Jian-Bang; Sun, Qing; Cao, Xiao-Nian; Zuo, Wu-Lin; Xu, Ruo-Shui; Huang, Jie-Hong; Jiang, Fu-Neng; Zhuo, Yang-Jia; Xiao, Bai-Quan; Liu, Yun-Zhong; Yuan, Dong-Bo; Sun, Zhao-Lin; He, Hui-Chan; Lun, Zhao-Rong; Zhong, Wei-De; Zhou, Wen-Liang

    2017-06-03

    Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca 2+ release from the endoplasmic reticulum, increased the mitochondrial Ca 2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca 2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP 3 ) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    International Nuclear Information System (INIS)

    Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  18. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  19. Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells.

    Directory of Open Access Journals (Sweden)

    Fabien J Cousin

    Full Text Available Gastric cancer is one of the most common cancers in the world. The "economically developed countries" life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.

  20. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yanwei Li

    Full Text Available An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

  1. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

    Science.gov (United States)

    Li, Yanwei; Liu, Haifeng; Zeng, Wei; Wei, Jing

    2017-01-01

    An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

  2. Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

    International Nuclear Information System (INIS)

    Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H.

    2006-01-01

    The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis

  3. Acacia catechu ethanolic bark extract induces apoptosis in human oral squamous carcinoma cells

    Directory of Open Access Journals (Sweden)

    Thangavelu Lakshmi

    2017-01-01

    Full Text Available Oral cancer is in approximately 30% of all cancers in India. This study was conducted to evaluate the cytotoxic activity of ethanolic extract of Acacia catechu bark (ACB against human squamous cell carcinoma cell line-25 (SCC-25. Cytotoxic effect of ACB extract was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium Bromide assay. A. catechu extract was treated SCC-25 cells with 25 and 50 μg/mL for 24 h. Apoptosis markers such as caspases-8 and 9, bcl-2, bax, and cytochrome c (Cyt-c were done by RT-PCR. Morphological changes of ACB treated cells were evaluated using acridine orange/ethidium bromide (AO/EB dual staining. Nuclear morphology and DNA fragmentation were evaluated using propidium iodide (PI staining. Further, cell cycle analysis was performed using flow cytometry. A. catechu treatment caused cytotoxicity in SCC-25 cells with an IC50 of 52.09 μg/mL. Apoptotic marker gene expressions were significantly increased on ACB treatment. Staining with AO/EB and PI shows membrane blebbing and nuclear membrane distortion, respectively, and it confirms the apoptosis induction in SCC-25 cells. These results suggest that ACB extract can be used as a modulating agent in oral squamous cell carcinoma.

  4. Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut.

    Science.gov (United States)

    Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Yi, Xin; Zhong, Guohua

    2018-01-01

    Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura , but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE) analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK) and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR). In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1) on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae.

  5. Azadirachtin Affects the Growth of Spodoptera litura Fabricius by Inducing Apoptosis in Larval Midgut

    Directory of Open Access Journals (Sweden)

    Benshui Shu

    2018-02-01

    Full Text Available Azadirachtin, the environmentally friendly botanical pesticide, has been used as an antifeedant and pest growth regulator in integrated pest management for decades. It has shown strong biological activity against Spodoptera litura, but the mechanism of toxicity remains unclear. The present study showed that azadirachtin inhibited the growth of S. litura larvae, which was resulted by structure destroy and size inhibition of the midgut. Digital gene expression (DGE analysis of midgut suggested that azadirachtin regulated the transcriptional level of multiple unigenes involved in mitogen-activated protein kinase (MAPK and calcium apoptotic signaling pathways. Simultaneously, the expression patterns of some differentially expressed unigenes were verified by quantitative real time-PCR (qRT-PCR. In addition, the enhanced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL staining, the increased expression of caspase family members and apoptosis-binding motif 1 (IBM1 on both gene and protein level and the release of cytochrome c from mitochondria to cytoplasm were induced in midgut after azadirachtin treatment. These results demonstrated that azadirachtin induced structural alteration in S. litura larval midgut by apoptosis activation. These alterations may affect the digestion and absorption of nutrients and eventually lead to the growth inhibition of larvae.

  6. Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

    International Nuclear Information System (INIS)

    Kondo-Nakamura, Mihoko; Shintani-Ishida, Kaori; Uemura, Koichi; Yoshida, Ken-ichi

    2010-01-01

    We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced O 2 - generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O 2 - scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O 2 - , which is then converted by SOD to H 2 O 2 , and subsequent Akt activation by H 2 O 2 attenuates apoptosis in ischemia-reperfusion.

  7. Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

    Energy Technology Data Exchange (ETDEWEB)

    Kondo-Nakamura, Mihoko [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shintani-Ishida, Kaori, E-mail: kaori@m.u-tokyo.ac.jp [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Uemura, Koichi; Yoshida, Ken-ichi [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2010-03-12

    We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced O{sub 2}{sup -} generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O{sub 2}{sup -} scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O{sub 2}{sup -}, which is then converted by SOD to H{sub 2}O{sub 2}, and subsequent Akt activation by H{sub 2}O{sub 2} attenuates apoptosis in ischemia-reperfusion.

  8. Mechanisms underlying apoptosis-inducing effects of Kaempferol in HT-29 human colon cancer cells.

    Science.gov (United States)

    Lee, Hyun Sook; Cho, Han Jin; Yu, Rina; Lee, Ki Won; Chun, Hyang Sook; Park, Jung Han Yoon

    2014-02-17

    We previously noted that kaempferol, a flavonol present in vegetables and fruits, reduced cell cycle progression of HT-29 cells. To examine whether kaempferol induces apoptosis of HT-29 cells and to explore the underlying molecular mechanisms, cells were treated with various concentrations (0-60 μmol/L) of kaempferol and analyzed by Hoechst staining, Annexin V staining, JC-1 labeling of the mitochondria, immunoprecipitation, in vitro kinase assays, Western blot analyses, and caspase-8 assays. Kaempferol increased chromatin condensation, DNA fragmentation and the number of early apoptotic cells in HT-29 cells in a dose-dependent manner. In addition, kaempferol increased the levels of cleaved caspase-9, caspase-3 and caspase-7 as well as those of cleaved poly (ADP-ribose) polymerase. Moreover, it increased mitochondrial membrane permeability and cytosolic cytochrome c concentrations. Further, kaempferol decreased the levels of Bcl-xL proteins, but increased those of Bik. It also induced a reduction in Akt activation and Akt activity and an increase in mitochondrial Bad. Additionally, kaempferol increased the levels of membrane-bound FAS ligand, decreased those of uncleaved caspase-8 and intact Bid and increased caspase-8 activity. These results indicate that kaempferol induces the apoptosis of HT-29 cells via events associated with the activation of cell surface death receptors and the mitochondrial pathway.

  9. 3'-Azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

    International Nuclear Information System (INIS)

    Collier, Abby C.; Helliwell, Rachel J.A.; Keelan, Jeffrey A.; Paxton, James W.; Mitchell, Murray D.; Tingle, Malcolm D.

    2003-01-01

    The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, β-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, β-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier

  10. Milk Fermented by Propionibacterium freudenreichii Induces Apoptosis of HGT-1 Human Gastric Cancer Cells

    Science.gov (United States)

    Cousin, Fabien J.; Jouan-Lanhouet, Sandrine; Dimanche-Boitrel, Marie-Thérèse; Corcos, Laurent; Jan, Gwénaël

    2012-01-01

    Background Gastric cancer is one of the most common cancers in the world. The “economically developed countries” life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate. Methodology/Principal Findings A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy. Conclusions/Significance Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments. PMID:22442660

  11. 14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in Head and Neck cancer cells

    International Nuclear Information System (INIS)

    Macha, Muzafar A; Matta, Ajay; Chauhan, SS; Siu, KW Michael; Ralhan, Ranju

    2010-01-01

    The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP). GS targets 14-3-3 zeta associated cellular pathways for reducing

  12. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

    Science.gov (United States)

    Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.E.; Di Giulio, R.T.

    2001-01-01

    Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ???19.7??9.5 pg TCDD/??l (water bath) and 0.25??0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction. Copyright ?? 2001 Elsevier Science B.V.

  13. Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

    Energy Technology Data Exchange (ETDEWEB)

    Tomita, Hiroshi, E-mail: htomita@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan); Clinical Research, Innovation and Education Center, Tohoku University Hospital, 1-1 Seiryo, Aoba, Sendai, Miyagi 980-8574 (Japan); Tabata, Kitako, E-mail: ktabata@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Takahashi, Maki, E-mail: mqdelta@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Nishiyama, Fumiaki, E-mail: t2114018@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Sugano, Eriko, E-mail: sseriko@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan)

    2016-05-13

    The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.

  14. Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

    International Nuclear Information System (INIS)

    Tomita, Hiroshi; Tabata, Kitako; Takahashi, Maki; Nishiyama, Fumiaki; Sugano, Eriko

    2016-01-01

    The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.

  15. Regulation of apoptosis-inducing factor-mediated, cisplatin-induced apoptosis by Akt

    OpenAIRE

    Yang, X; Fraser, M; Abedini, M R; Bai, T; Tsang, B K

    2008-01-01

    Cisplatin is a first-line chemotherapeutic for ovarian cancer, although chemoresistance limits treatment success. Apoptosis, an important determinant of cisplatin sensitivity, occurs via caspase-dependent and -independent mechanisms. Activation of the protein kinase Akt, commonly observed in ovarian tumours, confers resistance to ovarian cancer cells via inhibition of caspase-dependent apoptosis. However, the effect of Akt on cisplatin-induced, caspase-independent apoptosis remains unclear. W...

  16. Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sangsoo Daniel; Antenos, Monica [Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Squires, E. James [Department of Animal and Poultry Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Kirby, Gordon M., E-mail: gkirby@uoguelph.ca [Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)

    2013-07-15

    Bilirubin (BR) has recently been identified as the first endogenous substrate for cytochrome P450 2A5 (CYP2A5) and it has been suggested that CYP2A5 plays a major role in BR clearance as an alternative mechanism to BR conjugation by uridine-diphosphate glucuronyltransferase 1A1. This study investigated the mechanisms of Cyp2a5 gene regulation by BR and the cytoprotective role of CYP2A5 in BR hepatotoxicity. BR induced CYP2A5 expression at the mRNA and protein levels in a dose-dependent manner in primary mouse hepatocytes. BR treatment also caused nuclear translocation of Nuclear factor-E2 p45-related factor 2 (Nrf2) in hepatocytes. In reporter assays, BR treatment of primary hepatocytes transfected with a Cyp2a5 promoter-luciferase reporter construct resulted in a 2-fold induction of Cyp2a5 reporter activity. Furthermore, cotransfection of the hepatocytes with a Nrf2 expression vector without BR treatment resulted in an increase in Cyp2a5 reporter activity of approximately 2-fold and BR treatment of Nrf2 cotransfectants further increased reporter activity by 4-fold. In addition, site-directed mutation of the ARE in the reporter construct completely abolished both the BR- and Nrf2-mediated increases in reporter activity. The cytoprotective role of CYP2A5 against BR-mediated apoptosis was also examined in Hepa 1–6 cells that lack endogenous CYP2A5. Transient overexpression of CYP2A5 partially blocked BR-induced caspase-3 cleavage in Hepa 1–6 cells. Furthermore, in vitro degradation of BR was increased by microsomes from Hepa 1–6 cells overexpressing CYP2A5 compared to control cells transfected with an empty vector. Collectively, these results suggest that Nrf2-mediated CYP2A5 transactivation in response to BR may provide an additional mechanism for adaptive cytoprotection against BR hepatotoxicity. - Highlights: • The mechanism of Cyp2a5 gene regulation by BR was investigated. • The cytoprotective role of CYP2A5 in BR hepatotoxicity was determined. • BR

  17. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chun-Ru [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Liao, Wei-Siang [Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Wu, Ya-Hui [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Murugan, Kaliyappan [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chen, Chinpiao, E-mail: chinpiao@mail.ndhu.edu.tw [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chao, Jui-I, E-mail: jichao@faculty.nctu.edu.tw [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China)

    2013-12-15

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy.

  18. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

    International Nuclear Information System (INIS)

    Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

    2013-01-01

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy

  19. 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.

    Science.gov (United States)

    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

  20. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    Directory of Open Access Journals (Sweden)

    Antonio Serapio-Palacios

    2016-06-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS, but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK, which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii cytochrome c release from mitochondria to the cytoplasm, (iv loss of mitochondrial membrane potential, (v caspase-9 activation, (vi cleavage of procaspase-3 and (vii an increase in caspase-3 activity, (viii PARP proteolysis, and (ix nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.

  1. Glechoma longituba (Lamiaceae) alleviates apoptosis in calcium ...

    African Journals Online (AJOL)

    Glechoma longituba pre-treatment on cell oxidative stress and apoptosis induced by CaOx. Conclusion: ... reproduction in any medium, provided the original work is properly credited. Tropical .... kit (MaiBio, Hong Kong, China) according to the.

  2. Apoptosis and Tumor Progressionin Prostate Cancer

    National Research Council Canada - National Science Library

    Tenniswood, Martin P

    2005-01-01

    ... (as measured by BrdU incorporation) and apoptosis as measured by TUNEL staining. We have standardized an efficient methodologies for isolating cells from primary tumors expressing REP by fluorescence activated cell sorting (FACS...

  3. Molecular imaging of apoptosis in cancer

    International Nuclear Information System (INIS)

    Hakumaeki, Juhana M.; Liimatainen, Timo

    2005-01-01

    Apoptosis plays an important role in cancer. Mechanisms hindering its action are implicated in a number of malignancies. Also, the induction of apoptosis plays a pivotal role in non-surgical cancer treatment regimes such as irradiation, chemotherapy, or hormones. Recent advanced in imaging science have made it now possible for us to detect and visualize previously inaccessible and even unrecognized biological phenomena in cells and tissue undergoing apoptosis in vivo. Not only are these imaging techniques painting an intriguing picture of the spatiotemporal characteristics and metabolic and biophysical of apoptosis in situ, but they are expected to have an ever increasing impact in preclinical testing and design of new anticancer agents as well. Rapid and accurate visualization of apoptotic response in the clinical settings can also be of significant diagnostic and prognostic worth. With the advent of molecular medicine and patient-tailored treatment options and therapeutic agents, such monitoring techniques are becoming paramount

  4. Apoptosis – is it good or bad?

    Directory of Open Access Journals (Sweden)

    Bakir Mehić

    2012-08-01

    Full Text Available The most widely used classification of mammalian cell death recognizes two types: apoptosis and necrosis. Autophagy, which has been proposed as a third mode of cell death allows a starving cell, or in situations when cell is deprived of growth factors, to survive. Apoptosis, autophagy and necrosis, a particular mode of cell death may predominate, depending of the injury and the type of cell. [1] One very important characteristic of all multicellular organisms is apoptosis, the controlled death of cells. In necrosis, early loss of integrity of the plasma membrane resultant with swelling of the cell and its organelles. A key morphologic feature of apoptosis is collapses of cell and its subcellular components.[2] The distinction between apoptosis and necrosis is due in part to differences in how the plasma membrane participates in these processes. In apoptosis, plasma membrane integrity persists until late in the process. In necrosis, early loss of integrity of the plasma membrane allows an influx of extracellular ions and fluid, with resultant swelling of the cell and its organelles. During that time, on the inside of cell there occurs the cleavage of cytoskeletal proteins by aspartate specific proteases, which thereby collapses subcellular components. Other characteristic features are chromatin condensation, nuclear fragmentation and the formation of plasma membrane blebs. The type and intensity of noxious signals, ATP concentration, cell type, and other factors determine how cell death occurs. Acute myocardial ischemia induces necrosis (because the ischemia precipitates rapid and profound decreases of ATP, whereas chronic congestive heart failure induces apoptosis (with more modest and chronic decreases of ATP. The blockade of a particular pathway of cell death may not prevent the destruction of the cell but may instead recruit an alternative path: antiapoptotic caspase inhibitors cause hyperacute necrosis of hepatocytes and kidney tubular cells

  5. Mitochondrial disfunction and apoptosis in leukemia cells

    Directory of Open Access Journals (Sweden)

    Annamaria PALLAG

    2008-05-01

    Full Text Available Apoptosis or programmed cell death is a process which involves the intentional degradation of the cell from the inside, the participation of the mitochondria to propagate the apoptotic signal, the alteration of the phospholipid cell membrane composition, the perturbation and alteration of the cell metabolism.The antineoplastic drugs is inducing the apoptotic process in the sensitive cells.It have been studied acute lymphoblastic leukemia cells. Using Annexin V-PE Apoptosis Detection Kit and flow cytometer, the amount of cells undergoing apoptosis, in various stages of the antineoplasic treatment, was detected. At the same time, were monitored, the serum level of malondialdehyde. The results obtained confirm the alteration of the mitochondrial metabolism. We can observed the mitochondrial dysfunction role in cell apoptosis.

  6. Norcantharidin (NCTD) induces mitochondria mediated apoptosis in ...

    African Journals Online (AJOL)

    Administrator

    2011-06-15

    Jun 15, 2011 ... cancer deaths for both sexes being attributable to hepatoma. However ..... Resveratrol induces apoptosis and cell cycle arrest of human T24 bladder cancer cells in ... involvement of the CD95 receptor/ligand. J. Cancer. Res.

  7. Molecular Analysis of Neurotoxin-Induced Apoptosis

    National Research Council Canada - National Science Library

    D'Mello, Santosh R

    2006-01-01

    Apoptosis is a cell-suicide process that is required for the normal development of the nervous system, but that can be aberrantly activated in neurodegenerative diseases and following exposure to neurotoxins...

  8. In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart

    DEFF Research Database (Denmark)

    Brazhe, Nadezda; Treiman, Marek; Faricelli, Barbara

    2013-01-01

    We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial...... surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling...

  9. Removal of Bound Triton X-100 from Purified Bovine Heart Cytochrome bc1

    OpenAIRE

    Varhač, Rastislav; Robinson, Neal C.; Musatov, Andrej

    2009-01-01

    Cytochrome bc1 isolated from Triton X-100 solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nmol of the enzyme. Purified cytochrome bc1 is fully active; however, protein bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc1 was accomplished by incubation with Bio-Beads SM-2 in presence of sodium cholate. Sodium cholate is critical since it does not interfere with the adsorpt...

  10. The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

    Directory of Open Access Journals (Sweden)

    Julia Leclerc

    Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

  11. Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b/sub 5/ and cytochrome P-450/sub cam/

    Energy Technology Data Exchange (ETDEWEB)

    Vickery, L; Salmon, A; Sauer, K

    1975-01-01

    Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b/sub 5/ and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome b/sub 5/ from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450/sub cam/ from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome b/sub 5/ are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450/sub cam/ also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.

  12. Activation of human herpesvirus replication by apoptosis.

    Science.gov (United States)

    Prasad, Alka; Remick, Jill; Zeichner, Steven L

    2013-10-01

    A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

  13. Effect of sevoflurane on human neutrophil apoptosis.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

  14. Involvement of TR3/Nur77 translocation to the endoplasmic reticulum in ER stress-induced apoptosis

    International Nuclear Information System (INIS)

    Liang Bin; Song Xuhong; Liu Gefei; Li Rui; Xie Jianping; Xiao Lifeng; Du Mudan; Zhang Qiaoxia; Xu Xiaoyuan; Gan Xueqiong; Huang Dongyang

    2007-01-01

    Nuclear orphan receptor TR3/Nur77/NGFI-B is a novel apoptotic effector protein that initiates apoptosis largely by translocating from the nucleus to the mitochondria, causing the release of cytochrome c. However, it is possible that TR3 translocates to other organelles. The present study was designed to determine the intracellular localization of TR3 following CD437-induced nucleocytoplasmic translocation and the mechanisms involved in TR3-induced apoptosis. In human neuroblastoma SK-N-SH cells and human esophageal squamous carcinoma EC109 and EC9706 cells, 5 μM CD437 induced translocation of TR3 to the endoplasmic reticulum (ER). This distribution was confirmed by immunofluorescence analysis, subcellular fractionation analysis and coimmunoprecipitation analysis. The translocated TR3 interacted with ER-targeting Bcl-2; initiated an early release of Ca 2+ from ER; resulted in ER stress and induced apoptosis through ER-specific caspase-4 activation, together with induction of mitochondrial stress and subsequent activation of caspase-9. Our results identified a novel distribution of TR3 in the ER and defined two parallel mitochondrial- and ER-based pathways that ultimately result in apoptotic cell death

  15. Troxerutin Reduces Kidney Damage against BDE-47-Induced Apoptosis via Inhibiting NOX2 Activity and Increasing Nrf2 Activity

    Directory of Open Access Journals (Sweden)

    Qun Shan

    2017-01-01

    Full Text Available 2,2,4,4-Tetrabromodiphenyl ether (BDE-47, one of the persistent organic pollutants, seriously influences the quality of life; however, its pathological mechanism remains unclear. Troxerutin is a flavonoid with pharmacological activity of antioxidation and anti-inflammation. In the present study, we investigated troxerutin against BDE-47-induced kidney cell apoptosis and explored the underlying mechanism. The results show that troxerutin reduced renal cell apoptosis and urinary protein secretion in BDE-47-treated mice. Western blot analysis shows that troxerutin supplement enhanced the ratio of Bcl-2/Bax; inhibited the release of cytochrome c from mitochondria, the activation of procaspase-9 and procaspase-3, and the cleavage of PARP; and reduced FAS, FASL, and caspase-8 levels induced by BDE-47. In addition, troxerutin decreased the production of reactive oxygen species (ROS and increased the activities of antioxidative enzymes. Furthermore, troxerutin blunted Nrf2 ubiquitylation, enhanced the activity of Nrf2, decreased the activity of NOX2, and ameliorated kidney oxidant status of BDE-47-treated mice. Together, these results confirm that troxerutin could alleviate the cytotoxicity of BDE-47 through antioxidation and antiapoptosis, which suggests that its protective mechanism is involved in the inhibition of apoptosis via suppressing NOX2 activity and increasing Nrf2 signaling pathway.

  16. Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

    Science.gov (United States)

    Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

    2018-02-01

    Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

  17. The neem limonoids azadirachtin and nimbolide inhibit cell proliferation and induce apoptosis in an animal model of oral oncogenesis.

    Science.gov (United States)

    Harish Kumar, G; Vidya Priyadarsini, R; Vinothini, G; Vidjaya Letchoumy, P; Nagini, S

    2010-08-01

    Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention for their cytotoxicity against human cancer cell lines. However, the antiproliferative and apoptosis inducing effects of neem limonoids have not been tested in animal tumour models. The present study was therefore designed to evaluate the relative chemopreventive potential of the neem limonoids azadirachtin and nimbolide in the hamster buccal pouch (HBP) carcinogenesis model by analyzing the expression of proliferating cell nuclear antigen (PCNA), p21(waf1), cyclin D1, glutathione S-transferase pi (GST-P), NF-kappaB, inhibitor of kappaB (IkappaB), p53, Fas, Bcl-2, Bax, Bid, Apaf-1, cytochrome C, survivin, caspases-3, -6, -8 and -9, and poly(ADP-ribose) polymerase (PARP) by RT-PCR, immunohistochemical, and Western blot analyses. The results provide compelling evidence that azadirachtin and nimbolide mediate their antiproliferative effects by downregulating proteins involved in cell cycle progression and transduce apoptosis by both the intrinsic and extrinsic pathways. On a comparative basis, nimbolide was found to be a more potent antiproliferative and apoptosis inducing agent and offers promise as a candidate agent in multitargeted prevention and treatment of cancer.

  18. Extracts of Artocarpus communis Induce Mitochondria-Associated Apoptosis via Pro-oxidative Activity in Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Chiang-Wen Lee

    2018-05-01

    Full Text Available Glioblastoma multiforme (GBM is an extremely aggressive and devastating malignant tumor in the central nervous system. Its incidence is increasing and the prognosis is poor. Artocarpin is a natural prenylated flavonoid with various anti-inflammatory and anti-tumor properties. Studies have shown that artocarpin is associated with cell death of primary glioblastoma cells. However, the in vivo effects and the cellular and molecular mechanisms modulating the anticancer activities of artocarpin remain unknown. In this study, we demonstrated that treating the glioblastoma cell lines U87 and U118 cells with artocarpin induced apoptosis. Artocarpin-induced apoptosis is associated with caspase activation and poly (ADP-ribose polymerase (PARP cleavage and is mediated by the mitochondrial pathway. This is associated with mitochondrial depolarization, mitochondrial-derived reactive oxidative species (ROS production, cytochrome c release, Bad and Bax upregulations, and Bcl-2 downregulation. Artocarpin induced NADPH oxidase/ROS generation plays an important role in the mitochondrial pathway activation. Furthermore, we found artocarpin-induced ROS production in mitochondria is associated with Akt- and ERK1/2 activation. After treatment with artocarpin, ROS causes PI3K/Akt/ERK1/2-induced cell death of these tumor cells. These observations were further verified by the results from the implantation of both U87 and U118 cells into in vivo mouse. In conclusion, our findings suggest that artocarpin induces mitochondria-associated apoptosis of glioma cells, suggesting that artocarpine can be a potential chemotherapeutic agent for future GBM treatment.

  19. Curcumin-Induced Apoptosis in Human Hepatocellular Carcinoma J5 Cells: Critical Role of Ca+2-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Hsun Wang

    2012-01-01

    Full Text Available The antitumor effects of curcumin, a natural biologically active compound extracted from rhizomes of Curcuma longa, have been studied in many cancer cell types including human hepatocellular carcinoma (HCC. Here, we investigated the effects of Ca2+ on curcumin-induced apoptosis in human HCC J5 cells. The abrogation of mitochondrial membrane potential (ΔΨm, the increase of reactive oxygen species (ROS production, and calcium release were demonstrated with flow cytometry as early as 15 minutes after curcumin treatment. In addition, an increase level of cytochrome c in the cytoplasm which led to DNA fragmentation was observed. To verify the role of Ca2+ in curcumin-induced apoptosis, 1,2-bis(o-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA, an intracellular calcium chelator, was applied. Cell viability was increased, but ΔΨm, ROS production, activation of caspase 3, and cell death were decreased in J5 cells pretreated with BAPTA for 2 h followed by the treatment of 25 μM curcumin. These results suggest that the curcumin-induced apoptosis in human HCC J5 cells is via mitochondria-dependent pathway and is closely related to the level of intracellular accumulation of calcium.

  20. Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

    Science.gov (United States)

    Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

    2017-06-01

    Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

  1. Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

    Science.gov (United States)

    Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

    2014-01-01

    Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

  2. Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1.

    Science.gov (United States)

    Guo, L-J; Luo, X-H; Xie, H; Zhou, H-D; Yuan, L-Q; Wang, M; Liao, E-Y

    2006-05-01

    We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

  3. Identification of proteins that regulate radiation-induced apoptosis in murine tumors with wild type p53

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Jinsil; Oh, Hae Jin; Kim, Jiyoung; An, Jeung Hee; Kim, Wonwoo [Dept. of Radiation Oncology, Yonsei Univ. Medical College, Seoul (Korea, Republic of)

    2007-09-15

    In this study, we investigated the molecular factors determining the induction of apoptosis by radiation. Two murine tumors syngeneic to C3H/HeJ mice were used: an ovarian carcinoma OCa-I, and a hepatocarcinoma HCa-I. Both have wild type p53, but display distinctly different radiosensitivity in terms of specific growth delay (12.7 d in OCa-I and 0.3 d in HCa-I) and tumor cure dose 50% (52.6 Gy in OCa-I and >80 Gy in HCa-I). Eight-mm tumors on the thighs of mice were irradiated with 25 Gy and tumor samples were collected at regular time intervals after irradiation. The peak levels of apoptosis were 16.1{+-}0.6% in OCa-I and 0.2{+-}0.0% in HCa-I at 4 h after radiation, and this time point was used for subsequent proteomics analysis. Protein spots were identified by peptide mass fingerprinting with a focus on those related to apoptosis. In OCa-I tumors, radiation increased the expression of cytochrome c oxidase and Bcl2/adenovirus E1B-interacting 2 (Nip 2) protein higher than 3-fold. However in HCa-I, these two proteins showed no significant change. The results suggest that radiosensitivity in tumors with wild type p53 is regulated by a complex mechanism. Furthermore, these proteins could be molecular targets for a novel therapeutic strategy involving the regulation of radiosensitivity. (author)

  4. Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Murugesan Velayutham

    2016-03-01

    Full Text Available In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+cyt c in the absence and presence of H2O2. Electron paramagnetic resonance (EPR spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO were performed with sulfite, Fe3+cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3- was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which

  5. Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride.

    Science.gov (United States)

    Zhang, Shun; Niu, Qiang; Gao, Hui; Ma, Rulin; Lei, Rongrong; Zhang, Cheng; Xia, Tao; Li, Pei; Xu, Chunyan; Wang, Chao; Chen, Jingwen; Dong, Lixing; Zhao, Qian; Wang, Aiguo

    2016-05-01

    Fluoride, a ubiquitous environmental contaminant, is known to impair testicular functions and fertility; however the underlying mechanisms remain obscure. In this study, we used a rat model to mimic human exposure and sought to investigate the roles of apoptosis and autophagy in testicular toxicity of fluoride. Sprague-Dawley rats were developmentally exposed to 25, 50, or 100 mg/L sodium fluoride (NaF) via drinking water from pre-pregnancy to post-puberty, and then the testes of offspring were excised on postnatal day 56. Our results demonstrated that developmental NaF exposure induced an enhanced testicular apoptosis, as manifested by a series of hallmarks such as caspase-3 activation, chromatin condensation and DNA fragmentation. Further study revealed that fluoride exposure elicited significant elevations in the levels of cell surface death receptor Fas with a parallel increase in cytoplasmic cytochrome c, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Intriguingly, fluoride treatment also simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II but not Beclin1. Unexpectedly, the expression of p62, a substrate that is degraded by autophagy, was also significantly elevated, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation rather than increased formation. Importantly, these were associated with marked histopathological lesions including spermatogenic failure and germ cell loss, along with severe ultrastructural abnormalities in testes. Taken together, our findings provide deeper insights into roles of excessive apoptosis and defective autophagy in the aggravation of testicular damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis.

    Science.gov (United States)

    Shirazi, F; Kontoyiannis, D P

    2013-09-01

    The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales. We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca(2+), phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae, Cunninghamella bertholletiae, and Mucor circinelloides. Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca(2+) and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies.

  7. Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells

    International Nuclear Information System (INIS)

    Jayaraj, R.; Gupta, Nimesh; Rao, P.V. Lakshmana

    2009-01-01

    Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100 nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AIF mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with Ac-DEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis

  8. Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and mitochondria

    International Nuclear Information System (INIS)

    Servais, Helene; Van Der Smissen, Patrick; Thirion, Gaetan; Van der Essen, Gauthier; Van Bambeke, Francoise; Tulkens, Paul M.; Mingeot-Leclercq, Marie-Paule

    2005-01-01

    Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubules and renal cell lines. Using LLC-PK1 cells, we have examined the concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM; 0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway; (iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive necrosis as assessed by the measurement of lactate dehydrogenase release). Within 2 h, gentamicin induced a partial relocalization [from lysosomes to cytosol] of the weak organic base acridine orange. We thereafter observed (a) a loss of mitochondrial membrane potential (as from 10 h, based on spectrophotometric and confocal microscopy using JC1 probe) and (b) the release of cytochrome c from granules to cytosol, and the activation of caspase-9 (as from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity (assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of calcein from negatively-charged liposomes at pH 5.4, which was slowed down by raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin (to mimic the inner mitochondrial membrane). The present data provide temporal evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration of the permeability of lysosomes, triggering of the mitochondrial pathway, and activation of caspase-3

  9. RBFOX3 Regulates the Chemosensitivity of Cancer Cells to 5-Fluorouracil via the PI3K/AKT, EMT and Cytochrome-C/Caspase Pathways

    Directory of Open Access Journals (Sweden)

    Tianze Liu

    2018-04-01

    Full Text Available Background/Aims: RBFOX3, an RNA-binding fox protein, plays an important role in the differentiation of neuronal development, but its role in the chemosensitivity of hepatocellular carcinoma (HCC to 5-FU is unknown. Methods: In this study, we examined the biological functions of RBFOX3 and its effect on the chemosensitivity of HCC cells to 5-FU in vitro and in a mouse xenograft model. Results: RBFOX3 was found to have elevated expression in HCC cell lines and tissue samples, and its knockdown inhibited HCC cell proliferation. Moreover, knockdown of RBFOX3 improved the inhibitory effect of 5-fluorouracil (5-FU on cell proliferation, migration and invasion, and enhanced the apoptosis induced by 5-FU. However, overexpression of RBFOX3 reduced the inhibitory effect of 5-fluorouracil (5-FU on cell proliferation, migration and invasion, and decreased the apoptosis induced by 5-FU. We further elucidated that RBFOX3 knockdown synergized with 5-FU to inhibit the growth and invasion of HCC cells through PI3K/AKT and epithelial-mesenchymal transition (EMT signaling, and promote apoptosis by activating the cytochrome-c/caspase signaling pathway. Finally, we validated that RBFOX3 regulated 5-FU-mediated cytotoxicity in HCC in mouse xenograft models. Conclusions: The findings from this study indicate that RBFOX3 regulates the chemosensitivity of HCC to 5-FU in vitro and in vivo. Therefore, targeting RBFOX3 may improve the inhibition of HCC growth and progression by 5-FU, and provide a novel potential therapeutic strategy for HCC.

  10. Prognostic Value of Cytochrome C and Cytokines in Acute Viral Encephalopathy

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2006-06-01

    Full Text Available Serum cytochrome c and cytokines were evaluated as prognostic predictors in 29 children (ages 9 mos to 9 yrs 11 mos with viral acute encephalopathies and multiple organ failure at Fukushima Medical University School of Medicine, Japan.

  11. Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

    International Nuclear Information System (INIS)

    Kim, Seong K.; Stark, Benjamin C.; Webster, Dale A.

    2005-01-01

    The operon (cyo) encoding the Na + -pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H + transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na + -pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na + against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na + through the D-channel to the exterior and transports four H + through the K-channel for the reduction of each O 2

  12. A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

    NARCIS (Netherlands)

    Blakely, E.L.; Mitchell, A.L.; Fisher, N.; Meunier, B.; Nijtmans, L.G.J.; Schaefer, A.M.; Jackson, M.J.; Turnbull, D.M.; Taylor, R.W.

    2005-01-01

    Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical

  13. The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF is separated from its N-terminal

    Directory of Open Access Journals (Sweden)

    YONG ZHANG

    2009-01-01

    Full Text Available Apoptosis-inducing factor (AIF is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

  14. Physalis peruviana extract induces apoptosis in human Hep G2 cells through CD95/CD95L system and the mitochondrial signaling transduction pathway.

    Science.gov (United States)

    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.

  15. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan; Han, Lirong [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Qi, Wentao [Academy of State Administration of Grain, No.11 Baiwanzhuang Avenue, Xicheng District, Beijing, 100037 (China); Cheng, Dai; Ma, Xiaolei; Hou, Lihua [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Cao, Xiaohong, E-mail: caoxh@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Wang, Chunling, E-mail: wangchunling@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China)

    2015-01-24

    Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP

  16. Induction of p53-mediated apoptosis in splenocytes and thymocytes of C57BL/6 mice exposed to perfluorooctane sulfonate (PFOS)

    International Nuclear Information System (INIS)

    Dong, Guang-Hui; Wang, Jing; Zhang, Ying-Hua; Liu, Miao-Miao; Wang, Da; Zheng, Li; Jin, Yi-He

    2012-01-01

    Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant found in human and wildlife tissues. It has been reported that PFOS can cause atrophy of the immune organs and apoptosis of immunocytes in rodents. However, the mechanism behind such cause is still unclear. To understand the model of cell death and its mechanism on lymphoid cells in vivo, we conducted a dose/response experiment in which 4 groups of male adult C57BL/6 mice (12 mice per group) were dosed daily by oral gavage with PFOS at 0, 0.0167, 0.0833, or 0.8333 mg/kg/day, yielding targeted Total Administered Dose (TAD) of 0, 1, 5, or 50 mg PFOS/kg, respectively, over 60 days. The results showed that spleen and thymus weight were significantly reduced in the highest PFOS-dose-group (TAD 50 mg PFOS/kg) compared to the control group, whereas liver weight was significantly increased. We analyzed the cell death via apoptosis with an annexin-V/propidium iodide assay by flow cytometry, and observed that both the percentage of apoptosis and the expression of the pro-apoptotic proteins p53 in splenocytes and thymocytes increased in a dose-related manner after PFOS treatment. We also observed that PFOS induced p53-dependent apoptosis through the cooperation between the Bcl-xl down regulation without changing the Bcl-2 and Bax expression. The down regulation of Bcl-xl was strongly indicating mitochondrial involvement in apoptosis. It is confirmed by the release of cytochrome c and activation of caspase-3. All of these findings establish an important role of p53 and mitochondrial function in PFOS induced toxic environment in the host. -- Highlights: ► PFOS immunotoxicity is caused by induction of apoptosis via the p53 activation. ► PFOS exposure can induce down regulation of Bcl-xl. ► Mitochondria are involved in PFOS-induced apoptosis. ► PFOS exposure can cause the release of cytochrome c and activation of caspase-3.

  17. Induction of p53-mediated apoptosis in splenocytes and thymocytes of C57BL/6 mice exposed to perfluorooctane sulfonate (PFOS)

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Guang-Hui, E-mail: ghdong@mail.cmu.edu.cn [School of Public Health, China Medical University, Shenyang 110001 (China); Wang, Jing [Department of Biostatistics, School of Public Health, Saint Louis University, Saint Louis, MO 63104 (United States); Zhang, Ying-Hua; Liu, Miao-Miao; Wang, Da [School of Public Health, China Medical University, Shenyang 110001 (China); Zheng, Li [Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang 110001 (China); Jin, Yi-He [School of Public Health, China Medical University, Shenyang 110001 (China); School of Environmental Science and Technology, Dalian University of Technology, Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, Dalian 116024 (China)

    2012-10-15

    Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant found in human and wildlife tissues. It has been reported that PFOS can cause atrophy of the immune organs and apoptosis of immunocytes in rodents. However, the mechanism behind such cause is still unclear. To understand the model of cell death and its mechanism on lymphoid cells in vivo, we conducted a dose/response experiment in which 4 groups of male adult C57BL/6 mice (12 mice per group) were dosed daily by oral gavage with PFOS at 0, 0.0167, 0.0833, or 0.8333 mg/kg/day, yielding targeted Total Administered Dose (TAD) of 0, 1, 5, or 50 mg PFOS/kg, respectively, over 60 days. The results showed that spleen and thymus weight were significantly reduced in the highest PFOS-dose-group (TAD 50 mg PFOS/kg) compared to the control group, whereas liver weight was significantly increased. We analyzed the cell death via apoptosis with an annexin-V/propidium iodide assay by flow cytometry, and observed that both the percentage of apoptosis and the expression of the pro-apoptotic proteins p53 in splenocytes and thymocytes increased in a dose-related manner after PFOS treatment. We also observed that PFOS induced p53-dependent apoptosis through the cooperation between the Bcl-xl down regulation without changing the Bcl-2 and Bax expression. The down regulation of Bcl-xl was strongly indicating mitochondrial involvement in apoptosis. It is confirmed by the release of cytochrome c and activation of caspase-3. All of these findings establish an important role of p53 and mitochondrial function in PFOS induced toxic environment in the host. -- Highlights: ► PFOS immunotoxicity is caused by induction of apoptosis via the p53 activation. ► PFOS exposure can induce down regulation of Bcl-xl. ► Mitochondria are involved in PFOS-induced apoptosis. ► PFOS exposure can cause the release of cytochrome c and activation of caspase-3.

  18. Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Leang Ching

    2007-03-01

    Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

  19. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

    OpenAIRE

    Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

    2008-01-01

    The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

  20. [Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

    Science.gov (United States)

    Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

    1990-01-01

    Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

  1. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

    DEFF Research Database (Denmark)

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

    2016-01-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

  2. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Shilpi Khare

    2015-07-01

    Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

  3. Glaucoma and Cytochrome P4501B1 Gene Mutations

    Directory of Open Access Journals (Sweden)

    Mukesh Tanwar

    2010-01-01

    Full Text Available Developmental anomalies of the ocular anterior chamber angle may lead to an incomplete development of the structures that form the conventional aqueous outflow pathway. Thus, disorders that present with such dysfunction tend to be associated with glaucoma. Among them, Axenfeld-Rieger (ARS malformation is a rare clinical entity with an estimated prevalence of one in every 200,000 individuals. The changes in eye morphogenesis in ARS are highly penetrant and are associated with 50% risk of development of glaucoma. Mutations in the cytochrome P4501B1 (CYP1B1 gene have been reported to be associated with primary congenital glaucoma and other forms of glaucoma and mutations in pituitary homeobox 2 (PITX2 gene have been identified in ARS in various studies. This case was negative for PITX2 mutations and compound heterozygote for CYP1B1 mutations. Clinical manifestations of this patient include bilateral elevated intraocular pressure (>40 mmHg with increased corneal diameter (>14 mm and corneal opacity. Patient also had iridocorneal adhesions, anteriorly displaced Schwalbe line, anterior insertion of iris, broad nasal bridge and protruding umbilicus. This is the first study from north India reporting CYP1B1 mutations in Axenfeld-Rieger syndrome with bilateral buphthalmos and early onset glaucoma. Result of this study supports the role of CYP1B1 as a causative gene in ASD disorders and its role in oculogenesis.

  4. Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

    Science.gov (United States)

    Chen, Xi; Moerschell, Richard P; Pearce, David A; Ramanan, Durga D; Sherman, Fred

    2005-02-01

    The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

  5. Clinical Pharmacogenetics of Cytochrome P450-Associated Drugs in Children

    Directory of Open Access Journals (Sweden)

    Ida Aka

    2017-11-01

    Full Text Available Cytochrome P450 (CYP enzymes are commonly involved in drug metabolism, and genetic variation in the genes encoding CYPs are associated with variable drug response. While genotype-guided therapy has been clinically implemented in adults, these associations are less well established for pediatric patients. In order to understand the frequency of pediatric exposures to drugs with known CYP interactions, we compiled all actionable drug–CYP interactions with a high level of evidence using Clinical Pharmacogenomic Implementation Consortium (CPIC data and surveyed 10 years of electronic health records (EHR data for the number of children exposed to CYP-associated drugs. Subsequently, we performed a focused literature review for drugs commonly used in pediatrics, defined as more than 5000 pediatric patients exposed in the decade-long EHR cohort. There were 48 drug–CYP interactions with a high level of evidence in the CPIC database. Of those, only 10 drugs were commonly used in children (ondansetron, oxycodone, codeine, omeprazole, lansoprazole, sertraline, amitriptyline, citalopram, escitalopram, and risperidone. For these drugs, reports of the drug–CYP interaction in cohorts including children were sparse. There are adequate data for implementation of genotype-guided therapy for children for three of the 10 commonly used drugs (codeine, omeprazole and lansoprazole. For the majority of commonly used drugs with known CYP interactions, more data are required to support pharmacogenomic implementation in children.

  6. Force modulation and electrochemical gating of conductance in a cytochrome

    Science.gov (United States)

    Davis, Jason J.; Peters, Ben; Xi, Wang

    2008-09-01

    Scanning probe methods have been used to measure the effect of electrochemical potential and applied force on the tunnelling conductance of the redox metalloprotein yeast iso-1-cytochrome c (YCC) at a molecular level. The interaction of a proximal probe with any sample under test will, at this scale, be inherently perturbative. This is demonstrated with conductive probe atomic force microscopy (CP-AFM) current-voltage spectroscopy in which YCC, chemically adsorbed onto pristine Au(111) via its surface cysteine residue, is observed to become increasingly compressed as applied load is increased, with concomitant decrease in junction resistance. Electrical contact at minimal perturbation, where probe-molecule coupling is comparable to that in scanning tunnelling microscopy, brings with it the observation of negative differential resistance, assigned to redox-assisted probe-substrate tunnelling. The role of the redox centre in conductance is also resolved in electrochemical scanning tunnelling microscopy assays where molecular conductance is electrochemically gateable through more than an order of magnitude.

  7. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver.

    Directory of Open Access Journals (Sweden)

    Natalia Moskaleva

    Full Text Available Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM. Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.

  8. Regulation of the cytochrome P450 2A genes

    International Nuclear Information System (INIS)

    Su Ting; Ding Xinxin

    2004-01-01

    Cytochrome P450 monooxygenases of the CYP2A subfamily play important roles in xenobiotic disposition in the liver and in metabolic activation in extrahepatic tissues. Many of the CYP2A transcripts and enzymes are inducible by xenobiotic compounds, and the expression of at least some of the CYP2A genes is influenced by physiological status, such as circadian rhythm, and pathological conditions, such as inflammation, microbial infection, and tumorigenesis. Variability in the expression of the CYP2A genes, which differs by species, animal strain, gender, and organ, may alter the risks of chemical toxicity for numerous compounds that are CYP2A substrates. The mechanistic bases of these variabilities are generally not well understood. However, recent studies have yielded interesting findings in several areas, such as the role of nuclear factor 1 in the tissue-selective expression of CYP2A genes in the olfactory mucosa (OM); the roles of constitutive androstane receptor, pregnane X receptor (PXR), and possibly, peroxisome proliferator-activated receptors in transcriptional regulation of the Cyp2a5 gene; and the involvement of heterogeneous nuclear ribonucleoprotein A1 in pyrazole-induced stabilization of CYP2A5 mRNA. The aims of this minireview are to summarize current knowledge of the regulation of the CYP2A genes in rodents and humans, and to stimulate further mechanistic studies that will ultimately improve our ability to determine, and to understand, these variabilities in humans

  9. Expression of cytochrome P450 regulators in cynomolgus macaque.

    Science.gov (United States)

    Uno, Yasuhiro; Yamazaki, Hiroshi

    2017-09-11

    1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs.

  10. Interaction of rocuronium with human liver cytochromes P450.

    Science.gov (United States)

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  11. Direct observation of vibrational energy flow in cytochrome c.

    Science.gov (United States)

    Fujii, Naoki; Mizuno, Misao; Mizutani, Yasuhisa

    2011-11-10

    Vibrational energy flow in ferric cytochrome c has been examined by picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) measurements. By taking advantage of the extremely short nonradiative excited state lifetime of heme in the protein (energy of 20000-25000 cm(-1) was optically deposited selectively at the heme site. Subsequent energy relaxation in the protein moiety was investigated by monitoring the anti-Stokes UVRR intensities of the Trp59 residue, which is a single tryptophan residue involved in the protein that is located close to the heme group. It was found from temporal changes of the anti-Stokes UVRR intensities that the energy flow from the heme to Trp59 and the energy release from Trp59 took place with the time constants of 1-3 and ~8 ps, respectively. These data are consistent with the time constants for the vibrational relaxation of the heme and heating of water reported for hemeproteins. The kinetics of the energy flow were not affected by the amount of excess energy deposited at the heme group. These results demonstrate that the present technique is a powerful tool for studying the vibrational energy flow in proteins.

  12. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    Science.gov (United States)

    Rao, P. S. S.; Kumar, Santosh

    2015-01-01

    High prevalence of cigarette smoking in HIV patients is associated with increased HIV pathogenesis and disease progression. While the effect of smoking on the occurrence of lung cancer has been studied extensively, the association between smoking and HIV pathogenesis is poorly studied. We have recently shown the possible role of cytochrome P450 (CYP) in smoking/nicotine-mediated viral replication. In this review, we focus on the potential role of CYP pathway in polycyclic aromatic hydrocarbons (PAH), important constituents of cigarette smoke, mediated HIV pathogenesis. More specifically, we will discuss the role of CYP1A1 and CYP1B1, which are the major PAH-activating CYP enzymes. Our results have shown that treatment with cigarette smoke condensate (CSC) increases viral replication in HIV-infected macrophages. CSC contains PAH, which are known to be activated by CYP1A1 and CYP1B1 into procarcinogens/toxic metabolites. The expression of these CYPs is regulated by aryl hydrocarbon receptors (AHR), the cellular target of PAH, and an important player in various diseases including cancer. We propose that PAH/AHR-mediated CYP pathway is a novel target to develop new interventions for HIV positive smokers. PMID:26082767

  13. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

    Science.gov (United States)

    Cook, D J; Finnigan, J D; Cook, K; Black, G W; Charnock, S J

    Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s. © 2016 Elsevier Inc. All rights reserved.

  14. Differences in renal metabolism of insulin and cytochrome c

    International Nuclear Information System (INIS)

    Herrman, J.; Simmons, R.E.; Frank, B.H.; Rabkin, R.

    1988-01-01

    Kidneys degrade small proteins such as cytochrome c (CYT c) by the classic lysosomal pathway. However, because alternate routes for the transport and degradation of protein hormones have been identified in other tissues, the authors set out to determine whether extralysosomal sites might participate in the renal degradation of insulin. First, they compared the effect of the lysosomal inhibitor NH 4 Cl on insulin and CYT c degradation by isolated perfused rat kidneys. After kidneys were loaded with radiolabeled proteins to allow for absorption and transport to lysosomes, degradation was measured in the presence or absence of inhibitors. Next they followed the subcellular distribution of 125 I-labeled insulin in kidneys exposed to 125 I-labeled insulin in vivo or when isolated and perfused. Under both circumstances the distribution of insulin on a linear sucrose gradient differed from that of the lysosomal enzyme N-acetyl-β-glucosaminidase. In contrast, [ 14 CH 3 ]CYT c, injected in vivo, distributed over a density similar to the lysosomal marker. Thus important differences exist between the renal metabolism of CYT c, which proceeds in lysosomes, and the renal metabolism of insulin. These include rate of degradation, sensitivity to NH 4 Cl, and subcellular sites of localization. Accordingly, they suggest that insulin degradation may occur, at least in part, in a d