Involvement of activator protein-1 (AP-1) in induction of apoptosis by vitamin E succinate in human breast cancer cells

Abstract

The purpose of this study was to document induction of apoptosis by vitamin E succinate (VES; RRR-alpha-tocopheryl succinate) in human breast cancer cells in culture and to characterize potential c-jun involvement. VES at 18.8 microM (10 micrograms/mL) induced DNA synthesis arrest, reduced total cell numbers, and induced apoptosis in estrogen receptor-positive and estrogen-responsive MCF-7 human breast cancer cells. VES at 10 micrograms/mL induced apoptosis in greater than 60% of cells within 3 d of treatment. Apoptosis was documented by detection of fragmented or condensed nuclei in 4',6-diamindino-2-phenylindole-stained cells, detection of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeled DNA, and DNA laddering. Analyses of mRNA and protein levels of candidate molecules involved in apoptosis showed that MCF-7 cells treated with VES exhibited elevated and persistent expression of c-jun. MCF-7 cells stably transfected with a dominant-negative interfering mutant c-jun, TAM-67, and expressing high levels of mutant jun exhibited approximately 50% blockage of VES-mediated apoptosis. In addition to increased c-jun expression after VES treatment, VES-treated MCF-7 cells exhibited elevated activator protein-1 (AP-1) binding activity. Comparisons of AP-1 binding factors by super-shift analyses with jun-specific antibodies in cells sensitive to VES-induced apoptosis (empty-vector control 7-1 cells) and cells resistant to VES-induced apoptosis (TAM-67-containing TAM-9 cells) showed that the sensitive cells expressed c-jun and jun D and the resistant cells TAM-67 AP-1 binding proteins after VES treatment. These studies suggested that c-jun may be involved in the apoptotic process initiated by VES treatment of human MCF-7 breast cancer cells.