Evidence for role of transforming growth factor‐β in RRR‐a‐tocopheryl succinate‐induced apoptosis of human mda‐mb‐435 breast cancer cells

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Abstract
MDA‐MB‐435 human breast cancer cells treated with 10 μg/ml of RRR‐α‐tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA‐MB‐435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase‐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‐β (TGF‐β), respectively, per 106 cells. Although purified TGF‐β1, is not an effective apoptotic agent for MDA‐MD‐435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 μg/ml) + 10 ng/ml of purified TGF‐β1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF‐β‐signaling pathway by transient transfection of MDA‐MB‐435 cells with antisense oligomers to TGF‐β type II receptor (TGF‐βR‐II) blocked VES‐induced apoptosis. Likewise, addition of neutralizing antibodies to TGF‐β1 or to all three mammalian isoforms of TGF‐β (TGF‐β1 ‐β2, β3) blocked VES‐ and CM‐induced apoptosis. Furthermore, inhibitors of TGF‐β conversion from an inactive latent form to a biologically active form inhibited VES‐induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF‐β or the TGF‐β receptor‐signaling pathway with antisense oligomers or lig‐and‐neutralizing antibodies or prevention of activation of TGF‐β indicates a role for TGF‐β signaling in VES‐induced apoptosis.