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

(-)-Epicatechin is a flavanol that selectively stimulates mitochondrial respiration in pancreatic cancer cells.

<p>(A) Oxygen consumption by cytochrome c oxidase (COX) from Panc-1 cells exposed to different (-)-epicatechin concentrations. Turnover is defined as consumed O₂ (nM)/(min · total protein (mg)). Respiration rates are increased by (-)-epicatechin in a concentration-dependent manner. *p<0.05, compared to 0 µM (-)-epicatechin. (B) Oxygen consumption by cytochrome c oxidase (COX) from human normal fibroblast (HNF) cells exposed to 0 or 100 µM (-)-epicatechin. Respiration rates are not increased by 100 µM (-)-epicatechin.</p

(-)-Epicatechin stimulates checkpoint kinase protein 2 phosphorylation at threonine 68 and p21 expression in Panc-1 cells.

<p>(A) (-)-Epicatechin stimulates p21 expression. Protein levels in 20 µM (-)-epicatechin treated samples were normalized to samples treated with 6 Gy. (B) 20 µM (-)-epicatechin stimulates Chk2 phosphorylation. Protein levels in (-)-epicatechin treated samples were normalized to samples that were treated with 6 Gy, and *p<0.05.</p

(-)-Epicatechin does not sensitize normal cells to radiation.

<p>(A) 20 µM (-)-Epicatechin does not sensitize HNF cells to radiation (REF = 0.9). (B) (-)-Epicatechin alone does not inhibit colony formation in HNF (p>0.05).</p

(-)-Epicatechin inhibits p21 expression and does not stimulate checkpoint kinase protein 2 phosphorylation at threonine 68 in HNF cells.

<p>(A) (-)-Epicatechin inhibits p21 expression. Protein levels in 20 µM (-)-epicatechin treated samples were normalized to samples treated with 6 Gy, and *p<0.05. (B) 20 µM (-)-epicatechin does not stimulate Chk2 phosphorylation. Protein levels in (-)-epicatechin treated samples were normalized to samples that were treated with 6 Gy.</p

The effect of (-)-epicatechin on ETC protein expression in combination with radiation.

<p>(A) A representative blot for ETC protein expression in Panc-1 cells that were treated with 0–200 µM (-)-epicatechin and 0 or 6 Gy. (B) (-)-Epicatechin alone does not significantly change ETC protein expression. Protein levels are normalized to the untreated control. (B) (-)-Epicatechin induced minimal changes in ETC protein expression when combined with 6 Gy. Values were normalized to 6 Gy alone, and * p<0.05 relative to 6 Gy/0 µM (-)-epicatechin.</p

(-)-Epicatechin sensitizes cancer cells to radiation.

<p>(A) (-)-Epicatechin sensitizes Panc-1 cells to radiation with an average REF = 1.7 (B) (-)-Epicatechin sensitizes U87 cells to radiation with an average REF of 1.5. (C) (-)-Epicatechin sensitizes MIA PaCa-2 cells to radiation with an average REF = 1.2.</p

(-)-Epicatechin stimulates caspase 3 cleavage and expression in Panc-1 cells.

<p>Panc-1 cells were exposed to 0 or 20 µM (-)-epicatechin for 1 hour, then exposed to 6 Gy of radiation, and incubated for 72 hours. (-)-Epicatechin stimulates pro-caspase 3 expression and cleavage.</p