Steven P. Zielske

Molecular Mechanisms and Therapeutic Effects of (−)-Epicatechin and Other Polyphenols in Cancer, Inflammation, Diabetes, and Neurodegeneration

With recent insight into the mechanisms involved in diseases, such as cardiovascular disease, cancer, stroke, neurodegenerative diseases, and diabetes, more efficient modes of treatment are now being assessed. Traditional medicine including the use of natural products is widely practiced around the world, assuming that certain natural products contain the healing properties that may in fact have a preventative role in many of the diseases plaguing the human population. This paper reviews the biological effects of a group of natural compounds called polyphenols, including apigenin, epigallocatechin gallate, genistein, and (−)-epicatechin, with a focus on the latter. (−)-Epicatechin has several unique features responsible for a variety of its effects. One of these is its ability to interact with and neutralize reactive oxygen species (ROS) in the cell. (−)-Epicatechin also modulates cell signaling including the MAP kinase pathway, which is involved in cell proliferation. Mutations in this pathway are often associated with malignancies, and the use of (−)-epicatechin holds promise as a preventative agent and as an adjunct for chemotherapy and radiation therapy to improve outcome. This paper discusses the potential of some phenolic compounds to maintain, protect, and possibly reinstate health.

Radiation-induced epigenetic DNA methylation modification of radiation-response pathways

DNA methylation can regulate gene expression and has been shown to modulate cancer cell biology and chemotherapy resistance. Therapeutic radiation results in a biological response to counter the subsequent DNA damage and genomic stress in order to avoid cell death. In this study, we analyzed DNA methylation changes at >450,000 loci to determine a potential epigenetic response to ionizing radiation in MDA-MB-231 cells. Cells were irradiated at 2 and 6 Gy and analyzed at 7 time points from 1–72 h. Significantly differentially methylated genes were enriched in gene ontology categories relating to cell cycle, DNA repair, and apoptosis pathways. The degree of differential methylation of these pathways varied with radiation dose and time post-irradiation in a manner consistent with classical biological responses to radiation. A cell cycle arrest was observed 24 h post-irradiation and DNA damage, as measured by γH2AX, resolved at 24 h. In addition, cells showed low levels of apoptosis 2–48 h post-6 Gy and cellular senescence became significant at 72 h post-irradiation. These DNA methylation changes suggest an epigenetic role in the cellular response to radiation.

Radiosensitization of Pancreatic Cancer Cells by Metformin through the AMPK Pathway

Pancreatic cancer is relatively radioresistant, however, radiotherapy has been shown to provide efficacy in the treatment of local disease. To increase the effectiveness of radiotherapy in pancreatic cancer, radiosensitizing drugs are under development. In this study, we investigated the radiosensitizing activity of the anti-diabetic drug metformin on pancreatic cancer cells in vitro. We demonstrated that metformin radiosensitized MiaPaCa-2 and Panc1 cells with radiation enhancement ratios (ER) ranging from 1.33–1.45 with metformin concentrations of 30–100 μM, and in addition, we showed that metformin sensitized cells to gemcitabine alone or in combination with radiation treatment. In addition, we found that pancreatic cancer stem cell-like cells showed enhanced radiosensitization in a tumorsphere assay with a REF of 1.66. At these radiosensitizing doses, metformin alone had low toxicity (as shown by >75% clonogenic survival) and did not affect cell cycle. The combination of metformin and radiation yielded greater numbers of γ-H2AX foci after 1 h compared to radiation alone, suggesting increased DNA damage signaling. Examination of the AMPK pathway showed that pharmacological inhibition of AMPK signaling or RNAi of AMPKα1 reversed metformin-mediated radiosensitization. These studies show that metformin radiosensitization of pancreatic cancer cells at micromolar concentration acts through AMPK and may affect DNA damage signaling. The data indicate that metformin may increase the efficacy of radiation therapy for pancreatic cancer.

Epigenetic DNA Methylation in Radiation Biology: On the Field or on the Sidelines?

DNA methylation has been studied with regard to chemotherapeutics for a number of years. The radiation field has just begun to look at this in the context of radiotherapy or radiation exposure. So far, the data suggest that radiation induces epigenetic reprogramming which indicates a purposeful response that influences the cell fate or alters the response to future exposure. Further studies may result in discovery of biomarkers for radiotherapy outcome or prediction of the degree of radiation resistance. Past and ongoing development of DNMT1 inhibitors that lead to DNA hypomethylation appear to sensitize many tumor types to radiation and may be an area with long term clinical implications. J. Cell. Biochem. 116: 212–217, 2015.

Epicatechin Stimulates Mitochondrial Activity and Selectively Sensitizes Cancer Cells to Radiation

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.

The Role of Epigenetics in Radiation Therapy and the DNA Damage Response

The impact of epigenetics in the field of radiation oncology and the DNA damage response is an emerging area of research. Epigenetic mechanisms may potentially play a role in inherent or acquired radioresistance of tumors. In this section, we will discuss what is known about epigenetics, specifically DNA methylation and miRNAs, with regards to the DNA damage response and the exploitation of epigenetics therapeutically. Very little is known about histone modifications and the DNA damage response. Current research in radiation oncology and epigenetics is now at the level of basic science, but is beginning to move to the level of pre-clinical and translational research. The speed of research is accelerating since there are currently epigenetic therapies approved for treatment of certain cancers outside of the radiation oncology clinic.

Abstract 4349: Therapeutic radiation induces an epigenetic DNA methylation response

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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