Received for publication October 19, 2005.
Revised February 17, 2006.
Accepted for publication February 22, 2006.

Actions of a Histone Deacetylase Inhibitor, NSC3852, Link Reactive Oxygen Species to Cell Differentiation and Apoptosis in MCF-7 Human Mammary Tumor Cells

Abstract

NSC3852 has cell differentiation and anti-proliferative activity in human breast cancer cells in tissue culture and antitumor activity in mice bearing P388 and L1210 leukemic cells. We investigated the mechanism of NSC3852 action in MCF-7 human breast cancer cells using electron spin resonance (ESR). Reactive oxygen species (ROS) were detected in MCF-7 cell suspensions incubated with NSC 3852 using the spin trap, DMPO. Formation of the DMPO-OH adduct was quenched by the addition of superoxide dismutase, but not by catalase, and we concluded that superoxide was generated in the NSC 3852-treated cells. The flavoprotein inhibitor, diphenylene iodonium (DPI) suppressed ROS production providing evidence for the involvement of a flavindependent enzyme system in the ROS response to NSC3852. A biologically significant oxidative response to NSC 3852 occurred in MCF-7 cells. An early marker of oxidative stress was a decrease in the [GSH] / [GSSG] ratio1 h after NSC3852 addition. Oxidative DNA damage, marked by the presence of 8-oxoguanine, and DNA strand breakage occurred in cells exposed to NSC3852 for 24 h. Apoptosis peaked 48 h after exposure to NSC3852. Pre-treatment with the glutathione precursor, N- acetyl cysteine (NAC) prevented DNA strand breakage and apoptosis. Pre- treatment with NAC also reversed NSC3852 decreases in E2F1, Myc and phosphorylated retinoblastoma (Rb) indicative of redox-sensitive pathway(s) in MCF-7 cells during G₁ phase of the cell cycle. We conclude that ROS formation is involved in the apoptotic and cell differentiation responses to NSC3852 in MCF-7 cells.

Key words: apoptosis, breast cancer, cell cycle, cell differentiation, histone deacetylase, reactive oxygen species