Butyrate-Induced Cell Death and Differentiation Are Associated with Distinct Patterns of ROS in HT29-Derived Human Colon Cancer Cells

To investigate the role of reactive oxygen species (ROS) induced by butyrate in tumor cells, we compared HT29R, an HT29-derived human colon cancer cell line refractory to butyrate-induced cell differentiation but highly sensitive to cell death, with the differentiation-positive HT29-12 and HT29-21 cell lines (exhibiting low sensitivity to butyrate-induced cell death), with respect to levels of butyrate-induced free radicals (FRs), ROS, and H2O2. Dose-dependent increase of FRs (as determined by electron spin resonance spectroscopy) and ROS (dichlorofluorescein assay) was induced in HT29R, but not in HT29-12 and HT29-21 cells, where, in contrast to HT29R, a dose-dependent increase of H2O2 release (phenol red assay) was induced by butyrate. The mode of butyrate-induced cell death in HT29R cells was of a mixed type with necrosis predominating, which, however, switched to apoptosis as the major type of cell death in the presence of the drugs 1,5-dihydroxyisoquinoline, resveratrol, or cyclosporine A. The results suggest that FRs and ROS induced by butyrate in HT29R cells are products of cell death, while H2O2 induced in HT29-12 and HT29-21 cells is functionally related to cell differentiation.