Triphenylphosphonium Derivative-Based Combination Therapy for Metastatic Melanoma

302 Crosstalk among ROS, Epigenetics and TPA-Induced EMT in Human Breast Cancer MCF7 Cells Tetsuro Kamiya1, Aki Goto1, Hirokazu Hara1, and Tetsuo Adachi1 1 Gifu Pharmaceutical University, Japan Epithelial mesenchymal transition (EMT) plays a pivotal role in cancer progression, and some transcription factors including Slug and Snail are known to be involved in EMT processes. On the other hand, there is a bunch of findings about the contributions of ROS and epigenetics such as DNA methylation and histone modification to carcinogenesis. However, the crosstalk among ROS, epigenetics and EMT has not been fully elucidated. Here, we investigated above crosstalk mechanisms in human breast cancer MCF7 cells treated with TPA, a PKC activator. Treatment of MCF7 cells with TPA induced cell migration after wound scratch, which was accompanied by the induction of Slug and some other EMT-related genes, but not Snail. PKC or NADPH oxidase (NOX) inhibitor as well as actinomycin D significantly suppressed TPA-induced Slug mRNA expression. Interestingly, TPA treatment markedly induced histone acetylation. Moreover, ChIP assay indicated the significant enrichment of acetylated histone H3, but not H4, within Slug promoter region. Overall, EMT processes in TPA-treated MCF7 cells are regulated through PKC, NOX-derived ROS and histone H3 acetylation within Slug promoter region. doi: 10.1016/j.freeradbiomed.2014.10.199

303 Triphenylphosphonium Derivative-Based Combination Therapy for Metastatic Melanoma Kyle C. Kloepping1, Alora S. Kraus1, Devin K. Hedlund1, Colette M. Gnade1, Melissa A. Fath1, Mitchell C. Coleman1, Brett A. Wagner1, Kranti A. Mapuskar1, Douglas R. Spitz1, and Michael K. Schultz1,2 1 Department of Radiation Oncology (Free Radical and Radiation Biology Program), The University of Iowa, USA, 2Department of Radiology, The University of Iowa, USA Melanoma incidence is increasing faster that any other cancer worldwide and metastatic melanoma is almost uniformly fatal due to the development of resistance to all approved therapies. Evidence suggests that aberrant mitochondria metabolism in melanoma cells results in hyperpolarized mitochondria membranes and chronic-elevated levels of reactive oxygen species (ROS). Our preliminary data show that triphenylphosphonium (TPP) compounds with linear aliphaticcarbon side chains accumulate in melanoma cell mitochondria. Our results suggest that these compounds embed in the mitochondria membrane and disrupt oxidative metabolism, which promotes further increases ROS levels and melanoma-cell cytotoxicity. In this study, we evaluated the effect of TPP sidechain length (5-16 carbon atoms) on mitochondria metabolism and melanoma cell cytotoxicity and the potential for TPP-based combination therapies with inhibitors of ROS metabolism. In vitro clonogenic survival assays and measurements of ROS levels and oxygen consumption were performed with TPP variants alone and in combination with ROS metabolism inhibitors. In vivo mouse studies were performed to determine if TPP treatment reduces melanoma tumor growth. Results suggest that TPP derivatives can increase ROS levels; decrease oxygen consumption; and decrease clonogenic survival of melanoma cells. Further, there is a structure-activity relationship between side-chain length and increased ROS levels and cytotoxicity. In addition, TPP in combination with ROS metabolism inhibitors increase melanoma clonogenic cell death. Mice treated with TPP exhibited decreased melanoma tumor growth rates compared to untreated mice and

S128

tumor lysates showed increases of oxidative stress markers. Our study suggests that modifications to the side chain length of TPPbased drugs promote melanoma cell death via a mechanism involving increased ROS. Further, ROS metabolism inhibitors enhance the sensitivity of melanoma cells to TPP treatments ± highlighting the potential of TPP combination therapies for metastatic melanoma. doi: 10.1016/j.freeradbiomed.2014.10.200

304 Increased Level of Cellular Aging and Inflammatory Markers in Paternal Sperm: Cause of Concern for Sporadic Retinoblastoma in Children Shiv Basant Kumar1, Bhavna Chawla2, and Rima Dada1 1 Lab. for Molecular Reproduction and Genetics, Dept. of Anatomy, AIIMS, New Delhi, India, India, 2Ocular Oncology & Pediatric Ophthalmology Service, Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India Objective: Human live in a sea of free radicals and these are susceptible to early aging. This study was planned to evaluate effects of life style interventions (yoga and meditation) on markers of cellular aging and free radical levels at (0 day) Vs 10 days and 0 day Vs 90 days. Methods: 50 healthy volunteers were enrolled in Integral Health Clinic for certain type of yoga and meditation. Information was obtained about their lifestyle using a questionnaire about their life style habits and socioeconomic status. Venous blood samples were collected after each term (0, 10 and 90 days). Stress and aging markers such as Plasma Cortisol, ȕ ± Endorphin, IL-6, 8Oxo-2'-deoxyguanosine (8-OHdG, mutagenic base), blood Reactive Oxygen Species (ROS) and telomerase levels were measured. Results: The mean Cortisol levels were significantly lower (P = 0.0072) in the subjects (pre yoga) (118.83 ± 30.58) ng/mL compared to 10days after practicing yoga (96.32 ± 36.06) ng/mL, while ROS level decreased from baseline to day 10 (1215.069 ± 0.88, 1020.81 ± 0.79 RLU/min/104 Neutrophils; p=0.024). Although 8-OHdG levels were reduced (10268.23± 3349.71 vs. 9367.57 ± 2709.58pg/mL) after yoga intervention, the difference was not statistically significant (p=0.459). Telomerase levels were elevated post intervention [(0.59 (0.114 - 2.043) IU/Cell Vs 2.40 (0.568 - 5.448) IU/Cell] but telomere length did not show any change. Conclusions: Thus yoga-based lifestyle intervention reduced the markers of stress even within 10 days of practice. Telomerase level upregulation is key factor in maintenance of Telomere length which maintains genomic integrity. Thus yoga based life style interventions may be recommended as therapeutic in reducing oxidative stress and oxidative DNA damage. doi: 10.1016/j.freeradbiomed.2014.10.201

305 Silencing FXYD3 Protein Enhances Cytotoxicity Effect of Doxorubicin and Gamma Irradiation in Human Breast Cancer Cells Chia-Chi Liu1, Janusz M Gebicki2, and Helge H Rasmussen1,3 1 Kolling Medical Research Institute, Sydney Medical School, University of Sydney, Australia, 2Dept of Biological Sciences,

SFRBM 2014