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Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.

Therapeutic radiation results in a cascade of cellular events culminating either in repair of DNA damage and cell survival, or failure to repair damage and cell death. Gene expression is partially under epigenetic control in the form of DNA methylation. We investigated whole genome DNA methylation profiles of MDA-MB-231 breast cancer cells following therapeutic radiation to determine genes and pathways that are differentially methylated. MDA-MB-231 cells were treated with a single dose of 0, 2, or 6 Gy and incubated at 1, 2 4, 8, 24, 48, and 72 hrs before obtaining DNA extracts. DNA was subjected to whole genome DNA methylation profile analysis using an Infinium BeadArray which has the ability to determine CpG methylation at 450,000 genomic sites and covering all genes. Compared to unirradiated cells, 2 and 6 Gy irradiated cells showed 419-1661 CpG sites with decreased methylation and 514-2657 CpG sites with increased methylation, depending on time and dose. The number of hypomethylated genes in 2 Gy irradiated cells increased over time while the number of hypermethylated genes decreased in the first 8 hrs and then increased to a maximum at 72 hrs. In 6 Gy irradiated cells, no pattern was detected, although there was overall more DNA methylation changes than in 2 Gy irradiated cells, indicated a dose effect. Gene ontology analysis using Genomatix Genome Analyzer showed significant changes in DNA repair, cell cycle, apoptosis, and cell survival pathway gene methylation. DNA repair genes were differentially methylated as early as 1 hr after irradiation and sustained through 72 hrs. Epigenetic changes persisted after DNA repair, as shown by gamma-H2AX immunofluorescence, had taken place. Cell cycle, cyclin-dependent kinase, and cyclin-dependent kinase inhibitor genes were differentially methylated 4-24 hrs post-irradiation, correlating with a G2 cell cycle arrest as determined by flow cytometry. These data suggest that epigenetic regulation in the form of DNA methylation is integrated with the cellular response to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4349. doi:1538-7445.AM2012-4349

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