Investigation of the molecular mechanisms of apoptosis induced by a novel vitamin E derivative ([alpha]-TEA) in human breast and ovarian cancer using cell culture

Previous studies from our lab have shown that the vitamin E derivative, RRR-α-tocopheryl succinate (vitamin E succinate, VES) induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo DNA synthesis arrest, cellular differentiation, and apoptosis. Several studies have demonstrated VES to be a potent pro-apoptotic agent inducing apoptosis by restoring both transforming growth factor-β (TGF-β) and Fas (CD95) apoptotic signaling pathways that contribute to the activation of c-Jun N-terminal kinase (JNK)-mediated apoptosis. In an effort to develop a more clinically useful vitamin E-based chemotherapeutic agent, a non-hydrolyzable ether analog of RRR-α-tocopherol; namely, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (called RRR-α-tocopherol ether acetic acid analog; and abbreviated α-TEA) has been produced. Specific aim I studies investigated the individual effects of α-TEA and a naturally occurring from of vitamin E, δ-tocotrienol, as anticancer agents against breast cancer in vitro, and characterized signaling events involved in the pro-apoptotic and differentiation actions of α-TEA in human MDA-MB-435 and MCF-7 breast cancer cell lines. Data reported here showed that both α-TEA and δ-tocotrienol, like VES, induced estrogen-nonresponsive MDA-MB-435 and estrogen-responsive MCF-7 human breast cancer cells to undergo high levels of apoptosis in a concentration- and time-dependent fashion. Like VES, the two compounds induced either no or lower levels of apoptosis in normal human mammary epithelial cells (HMECs) and immortalized but nontumorigenic human MCF-10A cells. The pro-apoptotic mechanisms triggered by the structurally distinct α-TEA and δ-tocotrienol were identical to those previously reported for VES, that is α-TEA- and δ-tocotrienol-induced apoptosis involved sensitizing both cell lines to TGF-β and Fas apoptotic signaling, involving up-regulation of TGF-β receptor II protein expression and signaling apoptosis by TGF-β, and Fas converging on JNK signaling pathway. Specific aim II characterized the apoptotic effects of α-TEA on components of the Fas/CD95 apoptotic pathway in cisplatin-sensitive, A2780S, and cisplatin-resistant, A2780/cp70R human ovarian cancer cells. Both ovarian cancer cells were shown to undergo apoptosis in a dose-dependent manner following α-TEA treatment. α-TEA-induced apoptosis involved downregulation of Akt activation and c-FLIP and survivin expression. Data showed α-TEA to be an efficient inducer of cell death by decreasing constitutive active Akt in ovarian cancer cells. Downregulation of Flip and survivin expression were identified as key proapoptotic events regulated by Akt in α-TEA-induced apoptosis. Specific aim III studies showed α-TEA to induce MDA-MB-435 human breast cancer cells to undergo cellular differentiation. We use four markers to determine α-TEA induces differentiation including Oil red O staining of neutral lipids, downregulation of Her2/neu protein, upregulation of cytokeratin 18 and upregulation of p21. Studies showed α-TEA to induce human MDA-MB-435 cells to undergo cellular differentiation in a manner similar to, if not identical, to VES induced differentiation. Specific aim IV studies showed α-TEA to modulate ErbB family members and signaling. Studies showed α-TEA inhibited cell growth of both A2780S and A2780/CP70R cells and heregulin partially rescued both cell lines from α-TEA-induced apoptosis. α-TEA downregulated ErbB1, ErbB2, and ErbB3 message and protein levels, but not ErbB4, and reduced phosphorylated Akt and ERK1/2 in a time and dose dependent manner. Taken together, these findings demonstrated that α-TEA-induced apoptosis is by downregulation of c-FLIP and survivin through the activity of Akt and elimination of tumor cells through Fas-mediated apoptosis, and showed α-TEA to be a potent inducer of apoptosis in both human breast and ovarian cancer cells in culture.