EVIDENCE FOR ROLE OF TRANSFORMING GROWTH-FACTOR-BETA IN RRR-ALPHA-TOCOPHERYL SUCCINATE-INDNCED APOPTOSIS OF HUMAN MDA-MB-435 BREAST-CANCER CELLS

MDA-MB-435 human breast cancer cells treated with 10 mu g/ml of RRR-al pha-tocopheryl succinate (vitamin E succinate, VES) for one, two, thre e, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respec tively. Likewise, cells cultured for one, two, and three days with con ditioned media (CM) obtained from MDA-MB-435 cells treated with VES ex hibit 10%, 36%, and 74% apoptosis, respectively. A quantitative lucife rase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) ce lls. Although purified TGF-beta(1) is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with su boptimal levels of VES (2.5 and 5 mu g/ml) + 10 ng/ml of purified TGF- beta(1) enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB- 435 cells with antisense oligomers to TGF-beta type II receptor (TGF-b eta R-II) blocked VES-induced apoptosis. Likewise, addition of neutral izing antibodies to TGF-beta(1) or to all three mammalian isoforms of TGF-beta (TGF-beta(1), -beta(2), -beta(3)) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an ina ctive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF -beta or the TGF-beta receptor-signaling pathway with antisense oligom ers or ligand-neutralizing antibodies or prevention of activation of T GF-beta indicates a role for TGF-beta signaling in VES-induced apoptos is.