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Activation of Apoptosis by Hydrogen Peroxide through Death Receptor Signaling is Inhibited by Mild Heat Preconditioning at 40°C
Cancer 115 Mediation of Doxorubicin Toxicity in Cancer Cells by Coupled Redox Reactions Nnenna Jacinta Adimora1, John Charles Vaughns2, and Melissa Lambeth Kemp1 1 2 Georgia Institute of Technology and Emory University, Georgia Institute of Technology The mechanism of doxorubicin toxicity in cancer cells is poorly understood; recent evidence implicates the bioreductive activation of the drug as a major contributor to its overall toxicity. The aim of this study is to determine what factors promote reductive conversion of doxorubicin, a process that leads to the increased toxicity of the drug. To this end, we characterized two acute lymphoblastic leukemia cell lines that display different sensitivities to doxorubicin. Doxorubicin treatment resulted in increased accumulation of the non-toxic, quinone form of the drug in the resistant cell line alone, despite the fact that both cell lines have negligible drug efflux capabilities. The bioreductive activation of doxorubicin involves the enzymatic reduction of the drug by NADPH cytochrome P450 reductase (CPR), which utilizes NADPH as a source of reducing equivalents. We assayed the effect of doxorubicin treatment on CPR activity and glucose-6 phosphate dehydrogenase (G6PD) activity in both cell lines. Doxorubicin-induced CPR activity was comparable between cell lines; yet drug-induced G6PD activity was observed only in the doxorubicin-sensitive cell line. Doxorubicin caused equivalent superoxide production in both cell lines; however only the drugresistant cell line displayed an increase in hydrogen peroxide. Further assessment of the effect of doxorubicin treatment on superoxide dismutase activity (SOD) revealed drug-induced inhibition of SOD activity in the doxorubicin-sensitive cell line. Based on these findings, we hypothesize that increased G6PD activity paired with decreased SOD activity promotes the reductive conversion of doxorubicin, thereby leading to increased toxicity in cancer cells. Our results suggest that in cancer cells, increased levels of hydrogen peroxide are indicative of protection against anthracycline drugs. doi: 10.1016/j.freeradbiomed.2010.10.119
116 Activation of Apoptosis by Hydrogen Peroxide through Death Receptor Signaling is Inhibited by Mild Heat Preconditioning at 40°C Diana A Averill-Bates1, and Pragathi Pallepati1 1 Université du Québec à Montréal Low dose exposure to stresses such as heat shock, ROS and radiation can induce adaptive responses, which allow cells and organisms to continue normal function in the face of an adverse stimulus. Adaptive responses involve multiple changes in gene and protein expression, including induction of cellular defences (antioxidants, heat shock proteins (Hsps), etc.) to enable the cell to survive. If an adaptive response cannot counteract an adverse stress exposure, then cells are eliminated by death processes such as apoptosis or necrosis. Pre-exposure to mild temperatures such as 40°C induces an adaptive response (thermotolerance), whereby cells resist subsequent exposure to a toxic insult. This study demonstrates the protective effect of mild heat preconditioning at 40°C against activation of death receptormediated apoptosis by H2O2 in HeLa cells. Pre-exposure to 40°C increased the expression of major Hsps, catalase, MnSOD and glutathione. H2O2 activated the Fas death receptor pathway of apoptosis, which was evident by up-regulation of death ligand FasL and recruitment of adaptor protein Fas-associated death
S54
domain (FADD) to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, tBid translocation to mitochondria, caspase-9 activation and apoptosis. These changes were all diminished in mild thermotolerant cells. Polyethylene glycol-catalase abolished FasL induction and caspase-8 activation due to H2O2. FasL up-regulation, activation of caspases and apoptosis were decreased by p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in activation of death receptor-mediated apoptosis by H2O2. This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against the induction of apoptosis by pro-oxidants. Financial support: Natural Sciences and Engineering Council of Canada doi: 10.1016/j.freeradbiomed.2010.10.120
117 Exploring the Role of Cancer Cell Mitochondria in Malignant Phenotype Nukhet Aykin-Burns1, Yueming Zhu1, Anthony R. Cyr1, Aloysius J. Klingelhutz1, and Frederick E. Domann1 1 The University of Iowa There is growing evidence that reactive oxygen species (ROS) can promote carcinogenesis through diverse cellular processes. It has been reported that higher glycolytic rates seen in cancer cells (Warburg effect) may occur to compensate for increased steadystate levels of mitochondrial superoxide and hydroperoxides in tumor vs. normal cells. Furthermore, structural and functional deficiencies in mitochondria, and mutations in human mitochondrial DNA genome, have been associated with various types of human cancers in different studies but whether a causal relationship exists between ROS production and transformation is currently being debated. Telomere maintenance is required during immortalization and has also been suggested to be a rate-limiting step in cancer progression through reactivation or upregulation of telomerase activity and its template RNA and hTERT. The human tonsil epithelial cells used in this study have been immortalized via simultaneous hTERT overexpression and p16 downregulation. The cells were treated with 50 ng/ml ethidium bromide for 6 weeks and the generation of Rho 0 cells was confirmed via real time PCR using primers specific for COX I. A mixed population as well as clones isolated from the mixed population lost approximately 30% of their mitochondrial membrane potential. Rho 0 cells also had 85% less dihydroethidium oxidation compared to HTEChTERT/p16-/-. Moreover, transmitochondrial cybrid cells were generated by fusing Rho 0 cells with chemically enucleated FaDu and Cal27 head and neck carcinoma cells. Both cybrids restored their mitochondrial potential and showed significant decreases in doubling times, increases in plating efficiency, and increases in glucose consumption compared to parental cell line. Cybrids, which contain mitochondria from head and neck cancer cells, have also demonstrated significant increases in aldehyde dehydrogenase expression which has been suggested to be elevated in tumor initiating cells in head and neck cancers. These results imply a role for cancer cell mitochondria towards malignant phenotype in epithelial cells, while a concrete connection between mitochondrial ROS and tumorigenesis still needs to be investigated. doi: 10.1016/j.freeradbiomed.2010.10.121
SFRBM/SFRRI 2010
116 Activation of Apoptosis by Hydrogen Peroxide through Death Receptor Signaling is Inhibited by Mild Heat Preconditioning at 40°C Diana A Averill-Bates1, and Pragathi Pallepati1 1 Université du Québec à Montréal Low dose exposure to stresses such as heat shock, ROS and radiation can induce adaptive responses, which allow cells and organisms to continue normal function in the face of an adverse stimulus. Adaptive responses involve multiple changes in gene and protein expression, including induction of cellular defences (antioxidants, heat shock proteins (Hsps), etc.) to enable the cell to survive. If an adaptive response cannot counteract an adverse stress exposure, then cells are eliminated by death processes such as apoptosis or necrosis. Pre-exposure to mild temperatures such as 40°C induces an adaptive response (thermotolerance), whereby cells resist subsequent exposure to a toxic insult. This study demonstrates the protective effect of mild heat preconditioning at 40°C against activation of death receptormediated apoptosis by H2O2 in HeLa cells. Pre-exposure to 40°C increased the expression of major Hsps, catalase, MnSOD and glutathione. H2O2 activated the Fas death receptor pathway of apoptosis, which was evident by up-regulation of death ligand FasL and recruitment of adaptor protein Fas-associated death
S54
domain (FADD) to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, tBid translocation to mitochondria, caspase-9 activation and apoptosis. These changes were all diminished in mild thermotolerant cells. Polyethylene glycol-catalase abolished FasL induction and caspase-8 activation due to H2O2. FasL up-regulation, activation of caspases and apoptosis were decreased by p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in activation of death receptor-mediated apoptosis by H2O2. This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against the induction of apoptosis by pro-oxidants. Financial support: Natural Sciences and Engineering Council of Canada doi: 10.1016/j.freeradbiomed.2010.10.120
117 Exploring the Role of Cancer Cell Mitochondria in Malignant Phenotype Nukhet Aykin-Burns1, Yueming Zhu1, Anthony R. Cyr1, Aloysius J. Klingelhutz1, and Frederick E. Domann1 1 The University of Iowa There is growing evidence that reactive oxygen species (ROS) can promote carcinogenesis through diverse cellular processes. It has been reported that higher glycolytic rates seen in cancer cells (Warburg effect) may occur to compensate for increased steadystate levels of mitochondrial superoxide and hydroperoxides in tumor vs. normal cells. Furthermore, structural and functional deficiencies in mitochondria, and mutations in human mitochondrial DNA genome, have been associated with various types of human cancers in different studies but whether a causal relationship exists between ROS production and transformation is currently being debated. Telomere maintenance is required during immortalization and has also been suggested to be a rate-limiting step in cancer progression through reactivation or upregulation of telomerase activity and its template RNA and hTERT. The human tonsil epithelial cells used in this study have been immortalized via simultaneous hTERT overexpression and p16 downregulation. The cells were treated with 50 ng/ml ethidium bromide for 6 weeks and the generation of Rho 0 cells was confirmed via real time PCR using primers specific for COX I. A mixed population as well as clones isolated from the mixed population lost approximately 30% of their mitochondrial membrane potential. Rho 0 cells also had 85% less dihydroethidium oxidation compared to HTEChTERT/p16-/-. Moreover, transmitochondrial cybrid cells were generated by fusing Rho 0 cells with chemically enucleated FaDu and Cal27 head and neck carcinoma cells. Both cybrids restored their mitochondrial potential and showed significant decreases in doubling times, increases in plating efficiency, and increases in glucose consumption compared to parental cell line. Cybrids, which contain mitochondria from head and neck cancer cells, have also demonstrated significant increases in aldehyde dehydrogenase expression which has been suggested to be elevated in tumor initiating cells in head and neck cancers. These results imply a role for cancer cell mitochondria towards malignant phenotype in epithelial cells, while a concrete connection between mitochondrial ROS and tumorigenesis still needs to be investigated. doi: 10.1016/j.freeradbiomed.2010.10.121
SFRBM/SFRRI 2010