Zinc Depletion by TPEN induces Apoptosis Involving P38 Protein and Lysosomal Disruption in Neuroblastoma Cell

  Modulation of Erk Activity by Reversible Cysteine Modification Jeremiah David Keyes 1, Chananat Klomsiri1, Kimberly Nelson1, and Leslie Poole1 1 Wake Forest University, United States In order to elicit an appropriate response toward a stimulus, cells have developed intricate systems to control signaling pathways. These pathways are often centered on kinase cascades, and are modulated by 2nd messengers and protein-protein interactions. Pathway modulation is necessary for the spatial-temporal control of kinase signal exertion. It is through such mechanisms that a signal can induce a specific response through a core kinase cascade that is shared by other signals to elicit different responses. Recently recognized 2nd messengers are Reactive Oxygen Species (ROS) such as H2O2. These ROS oxidize cysteine residues of proteins, which has the potential to modify the surface and configuration of a protein, thereby affecting interactions with small molecules, proteins, lipids, or nucleotides. Our collaborative team recently identified that ROS are necessary for the survival and proliferative response of ovarian and prostate cancer cells to lysophosphatidic acid (LPA), a signaling lipid found in the ascites fluid of ovarian and prostate tumors. We report here that Extracellular-regulated kinase (Erk) is oxidized in LPAstimulated, cancer-derived cells in culture. the oxidation, which is largely or completely reversible, inhibits kinase activity towards the substrate Elk1 based on experiments with both endogenous and recombinant Erk.

 doi: 10.1016/j.freeradbiomed.2013.10.799

   Overexpression of AMPKĮ1 and HSP70 and Effects on Cell Bioenergetics in Response to 4-Hydroxy 2Nonenal (4-HNE) in Quail Muscle (QM7) Cells Kentu Lassiter1, Byung-Whi Kong1, Alissa Piekarski1, Sami Dridi1, Nazrul Hacque2, and Walter Bottje1 1 University of Arkansas, Fayetteville, United States, 2National Research Centre on Mithun, Jharnapani, India The purpose of this study was to assess the effects of increased expression of AMPKD1 and HSP70 proteins on cell bioenergetics in an avian muscle cell line (quail muscle 7, QM7) with and without oxidant challenge. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), used to assess oxidative phosphorylation and glycolysis activity, respectively, were obtained using the XF24 Flux Analyzer (Seahorse Biosciences, Billerica, MA). Bioenergetic profiling of the cells was conducted with a µ%2)$¶ DQDO\VLV to monitor OCR during basal (B) conditions, and in response to oligomycin (O), FCCP (F), and antimycin a (A), enabling determination oxygen consumption linked to ATP synthesis, proton leak, oxygen reserve capacity, and non-cytochrome c oxidase activity. BOFA analysis showed that the percentage of O2 consumption devoted to ATP synthesis increased from 26% in the untransfected (control) QM7 cells, to 59% in the cells overexpressing AMPKD1, whereas reserve capacity, proton leak, and non-cytochrome c oxidase OCR were all lower for the AMPKD1 transfected cells. in response to ȝ0 4-HNE (2 h), both the AMPKD1 and HSP70 transfected cell lines were able to maintain some reserve capacity (4%) when compared to control QM7 cells (0%). in comparison to untransfected QM7 cells, both transfected cell lines exhibited increased OCR due to proton leak when compared to the control

6

cells, and a decrease in OCR due to non-cytochrome c oxidase activity. These results suggest that overexpression of AMPKD1 and HSP70 enhanced proton leak that could attenuate mitochondrial reactive oxygen species (ROS) production. Funding provided in part by Arkansas Biosciences Institute grant (WB) and Fellowship from Dept. of Biotechnology, India (NH)

 doi: 10.1016/j.freeradbiomed.2013.10.800

   Zinc Depletion by TPEN induces Apoptosis Involving P38 Protein and Lysosomal Disruption in Neuroblastoma Cell Andreza Candido Matias1, Tânia Maria Manieri1, and Giselle Cerchiaro1 1 Universidade Federal do ABC, São Paulo, Brazil Zinc is an essential trace element for all living organisms and it is important for functional and structural integrity of cells, contributing to a number of important biological processes. the zinc concentration at the cellular level is essential for cell proliferation, differentiation and also apoptosis regulation. Based on this information, the present study investigated the molecular effects and mode of action of an intracellular zinc specific quelator, tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) in neuroblastoma cell line SH-SY5Y. Cells were treated with TPEN 5 or 25 μM. It was observed a decrease both in zinc intracellular level and cell viability. by flow cytometry we observed an increase in lysosomal pH and cell death. Westtern blot showed the activation of p38, caspase 3 and Bax proteins. When the p38 protein was inhibited, the activation of caspase 3 was suppressed. However it was not observed ROS generation suggesting that apoptotic cell death is associated with zinc depletion, requiring p38 protein ativation. Consequently, zinc depletion is responsible for the cell toxicity caused by TPEN in neuroblastoma cells. Support: FAPESP, CNPq

 doi: 10.1016/j.freeradbiomed.2013.10.801

   Assessment of Cell Bioenergetics in HSP90Transfected Male Leghorn Hepatocytes Alissa Piekarski1, Kentu Lassiter1, Byung-Whi Kong1, Sami Dridi1, Nazrul Hacque2, and Walter Bottje1 1 University of Arkansas, Fayetteville, United States, 2National Research Centre on Mithun, Jharnapani, India, India The objective of the present study was to assess bioenergetics in Leghorn Male Hepatocytes (LMH) cells with increased ectopic expression of AMPK and HSP90. an XF24 Flux Analyzer (Seahorse Biosciences, Billerica, MA) was used to assess oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) representing oxidative phosphorylation and glycolysis activity, respectively. Bioenergetic profiling of the cells was conducted with a µ%2)$¶ DQDO\VLV to monitor OCR during basal (B) conditions, and in response to oligomycin (O), FCCP (F), and antimycin a (A), enabling determination oxygen consumption linked to ATP synthesis, proton leak, oxygen reserve capacity, and non-cytochrome c oxidase activity. Initial studies indicate that compared to non-transfected (Control) LMH cells, HSP90

6)5%0