α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway.

Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) induces apoptosis in MCF-7 and HCC-1954 human breast cancer cells in a dose- and time-dependent manner. α-TEA induces increased levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor-5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE-like inhibitory protein (c-FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c-FLIP (L), caspase-8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase-9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α-TEA to enhance DR5 protein levels, downregulate c-FLIP(L) protein levels and induce apoptosis. Combination of α-TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c-FLIP (L) protein levels. siRNA knockdown of c-FLIP produces a low level of spontaneous apoptosis and enhances α-TEA- and TRAIL-induced apoptosis. Taken together, these studies show that α-TEA induces TRAIL/DR5 mitochondria-dependent apoptosis in human breast cancer cells, and that TRAIL/DR5-dependent increases in DR5 and decreases in c-FLIP expression are triggered by TRAIL or α-TEA treatments.

RRR-gamma-tocopherol induces human breast cancer cells to undergo apoptosis via death receptor 5 (DR5)-mediated apoptotic signaling.

Goal of this study was to investigate the pro-apoptotic properties of RRR-gamma-tocopherol (gammaT) in human breast cancer cells. gammaT was shown to induce cancer cells but not normal cells to undergo apoptosis, sensitize cancer cells to Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced apoptosis, and increase death receptor 5 (DR5) mRNA, protein and cell surface expression. Knockdown of DR5 attenuated gammaT-induced apoptosis. Investigations of post-receptor signaling showed: caspase-8, Bid and Bax activation, increases in mitochondria permeability, cytochrome c release and caspase-9 activation. Thus, gammaT is a potent pro-apoptotic agent for human breast cancer cells inducing apoptosis via activation of DR5-mediated apoptotic pathway.

Differential response of human ovarian cancer cells to induction of apoptosis by vitamin E Succinate and vitamin E analogue, alpha-TEA.

A vitamin E derivative, vitamin E succinate (VES; RRR-alpha-tocopheryl succinate), and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy acetic acid (alpha-TEA), induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells in culture to undergo apoptosis but not normal human mammary epithelial cells, immortalized, nontumorigenic breast cells, or normal human prostate epithelial cells. Human ovarian and cervical cancer cell lines are exceptions, with alpha-TEA exhibiting greater proapoptotic effects. Although both VES and alpha-TEA can induce A2780 and subline A2780/cp70 ovarian cancer cells to undergo DNA synthesis arrest within 24 h of treatment, only alpha-TEA is an effective inducer of apoptosis. VES or alpha-TEA treatment of cp70 cells with 5, 10, or 20 microg/ml for 3 days induced 5, 6, and 19% versus 9, 36, and 71% apoptosis, respectively. Colony formation data provide additional evidence that cp70 cells are more sensitive to growth inhibition by alpha-TEA than VES. Differences in stability of the ester-linked succinate moiety of VES versus the ether-linked acetic acid moiety of alpha-TEA were demonstrated by high-performance liquid chromatography analyses that showed alpha-TEA to remain intact, whereas VES was hydrolyzed to the free phenol, RRR-alpha-tocopherol. Pretreatment of cp70 cells with bis-(p-nitrophenyl) phosphate, an esterase inhibitor, before VES treatment, resulted in increased levels of intact VES and apoptosis. Taken together, these data show alpha-TEA to be a potent and stable proapoptotic agent for human ovarian tumor cells and suggest that endogenous ovarian esterases can hydrolyze the succinate moiety of VES, yielding RRR-alpha-tocopherol, an ineffective apoptotic-inducing agent.

Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis.

A nonhydrolyzable ether analogue of RRR-alpha-tocopherol, 2,5,7,8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid, called RRR-alpha-tocopheryloxyacetic acid or RRR-alpha-tocopherol ether-linked acetic acid analogue (alpha-TEA), exhibits antitumor activity in vitro and in vivo using a syngeneic BALB/c mouse mammary tumor model (line 66 clone 4 stably transfected with green fluorescent protein). Treatment of cells with 5, 10, and 20 micro g/ml alpha-TEA for 3 days produced 6, 34, and 50% apoptosis, respectively, and treatment of cells with 10 micro g/ml for 2, 3, 4, and 5 days produced 20, 35, 47, and 58% apoptosis, respectively. A liposomal formulation of alpha-TEA administered by aerosol reduced s.c. tumor growth and lung metastasis. Alpha-TEA-treated animals showed a significant decrease in tumor volumes over 17 days of aerosol treatment (P < 0.001). Forty percent of aerosol as well as untreated control mice had visible, macroscopic lung metastases versus none (0%) of the alpha-TEA-treated mice. On the basis of fluorescence microscopic examination of the surface (top and bottom) of flattened whole left lung lobes, an average of 60 +/- 15 and 102 +/- 17 versus 11 +/- 4 fluorescent microscopic metastases was observed in aerosol control and untreated control versus alpha-TEA-treated animals, respectively. Alpha-TEA formulated in ethanol + peanut oil (5 mg/mouse/day) delivered by gavage did not reduce s.c. primary tumor burden; however, fluorescent microscopic lung metastases were significantly reduced (P < 0.0021). In summary, alpha-TEA formulated in liposomes and delivered by aerosol is a potent antitumor agent and reduces lung metastasis.

Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis.

PURPOSE: To test the anticancer properties of a nonhydrolyzable ether-linked acetic acid analogue of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (alpha-TEA), and a derivative of camptothecin, 9-nitrocamptothecin (9-NC)singly and in combination against mouse mammary tumor cells (line 66 clone 4 stably transfected with green fluorescent protein; 66cl-4-GFP) cultured in vitro or transplanted subcutaneously into the inguinal region of female BALB/c mice to form established tumors. METHODS: Following in vitro treatment of 66cl-4-GFP cells with alpha-TEA and suboptimal concentrations of 9-NC, singly or in combination, apoptosis was measured by morphological evaluation of nuclei stained with 4',6-diamidino-2-phenylindole (DAPI), and DNA synthesis arrest was measured by tritiated thymidine uptake. For in vivo analyses alpha-TEA and 9-NC, both water-insoluble compounds, were formulated into liposomes using dil-auroylphosphatidylcholine and administered by aerosol to deliver doses calculated to be 36 and 0.4 microg/mouse per day, respectively, (singly or each separately for combined treatments) 7 days per week. RESULTS: Treatment of 66cl-4-GFP cells in culture for 3 days with a combination of alpha-TEA (10 microg/ml; singly produces 38% apoptosis), and suboptimal concentrations of 9-NC(15.6, 31.3, 62.5, or 125 ng/ml; singly produce 2-7% apoptosis), produced 47%, 58%, 64%, and 69% apoptosis. Likewise, combinations of alpha-TEA 9-NC inhibited DNA synthesis more than either agent administered singly. A significant reduction (P< 0.001)in growth of subcutaneous transplanted tumors was observed with liposome-formulated and aerosolized delivery of alpha-TEA + 9-NC to BALB/c mice. The incidence of macroscopic lung metastasis was 83% in control vs 8 % in alpha-TEA-, 9-NC-, or combination-treated mice. Fluorescence microscopic examination of lungs and axillary and brachial lymph nodes showed a statistically significant decrease in metastasis observed in alpha-TEA-,9-NC-, and combination- vs control-treated animals. Analyses of primary tumor tissue for proliferation and apoptosis showed treatment groups to have lower Ki-67 and higher terminal deoxynucleotidyl transferase-mediated nick end labeling, respectively. Treatments showed no measurable effects on two angiogenesis parameters,namely intratumoral blood volume as assessed by hemoglobin content and intratumoral blood vessel density as assessed with CD31 staining. CONCLUSIONS: Combination treatments enhanced antiproliferative and proapoptotic activities in cell culture, and when formulated in liposomes and delivered via aerosolization to treat an aggressive and metastatic syngeneic murine mammary tumor, the combination treatment showed a significant reduction in tumor volume in comparison to either treatment alone. Mechanistically, it appears that neither enhanced apoptosis, reduced cell proliferation,nor reduced blood vessel density can fully account for the enhanced effects of the combination treatment.

Alpha-TEA plus cisplatin reduces human cisplatin-resistant ovarian cancer cell tumor burden and metastasis.

A novel nonhydrolyzable ether-linked acetic acid analog of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (alpha-TEA) in combination with cisplatin, reduces tumor burden of A2780/cp70 (cp70) cisplatin-resistant human ovarian cancer cells xenografted into immune compromised nude mice. Two xenograft studies were conducted using cp70 cells stably expressing green fluorescent protein (cp70-GFP) subcutaneously transplanted into NU/NU mice. For studies 1 and 2, alpha-TEA was formulated in liposomes and delivered by aerosol such that approximately 36 microg and 72 microg of alpha-TEA were deposited in the respiratory tract of each mouse each day, respectively. Cisplatin at 5 mg/kg was administered by intraperitoneal injections once weekly for the first 3 weeks in Study 1 and on the third and 10th days following treatment initiation in Study 2. The combination alpha-TEA + cisplatin treatment reduced tumor burden and metastasis of cp70-GFP cells in comparison to control mice or mice treated with alpha-TEA or cisplatin singly. A significant reduction (P < 0.001) in growth of subcutaneous transplanted tumors was obtained with alpha-TEA + cisplatin for both studies. Visible metastases were observed in the lungs of animals from control and cisplatin-treated groups but not in animals from the alpha-TEA- or alpha-TEA + cisplatin-treated groups. The alpha-TEA + cisplatin significantly reduced the total number of lung and axillary lymph node micrometastasis (P < 0.03 and P < 0.0001, respectively). Analyses of tumor sections showed the alpha-TEA + cisplatin treatment group, in comparison to control, to have a significantly lower level of cell proliferation (Ki-67 staining; P < 0.0001) and a significantly higher level of apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling [TUNEL]; P < 0.0001). In summary, combinations of alpha-TEA + cisplatin significantly reduced tumor burden and metastases in a xenograft model of cisplatin-resistant human ovarian cancer cells. These data show promise for combination alpha-TEA + cisplatin chemotherapy for ovarian cancer.

Comparison of vitamin E derivatives alpha-TEA and VES in reduction of mouse mammary tumor burden and metastasis.

A novel nonhydrolyzable ether derivative of RRR-alpha-tocopherol, RRR-alpha-tocopherol ether acetic acid analog [2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (alpha-TEA)], and a hydrolyzable ester derivative RRR-alpha-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with alpha-TEA or VES induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of alpha-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike alpha-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that alpha-TEA delivered by either method significantly reduced tumor cell proliferation as measured by Ki67, and increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative alpha-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.

Aerosol delivery of liposomal formulated paclitaxel and vitamin E analog reduces murine mammary tumor burden and metastases.

Paclitaxel is a chemotherapeutic agent used for the treatment of metastatic breast cancer. 2,5,7,8-Tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA) is an analog of vitamin E that inhibits primary tumor growth and the incidence of lymphatic and pulmonary metastases in preclinical animal models. Here, the efficacy of sequential treatment with paclitaxel and alpha-TEA was tested in the BALB/c syngeneic 66cl-4-GFP mammary cancer model. Both agents were formulated into liposomes and delivered by inhalation in an effort to increase anti-tumor efficacy and minimize paclitaxel toxicity. Combination treatment consisting of twelve days of every-other-day treatment with aerosolized paclitaxel (approximately 0.46 microg/mouse/treatment) followed by a daily regimen of aerosolized alpha-TEA (36 microg/mouse/treatment) significantly decreased primary tumor burden when compared to untreated or liposome control groups and was significantly better than individual treatments (P < 0.05). Importantly, combination treatment was significantly better at reducing lung and lymph node micrometastatic foci when compared to control and individual treatment groups (P < 0.05). Immunohistochemical analyses of tumor sections showed combination treatment when compared to liposome control or individual treatments to significantly decrease total number of cells staining positive for the endothelial cell marker CD31 or for the Ki67 marker of cellular proliferation and increase the number of apoptotic (TUNEL positive) tumor cells (P < 0.001). Studies addressing the toxicity of alpha-TEA demonstrated that alpha-TEA formulated in liposomes and delivered by aerosol (72 microg/mouse/day) or gavage (5 mg/mouse/day) for 25 days did not cause blood, liver, or kidney toxicity. In conclusion, sequential inhalation delivery of liposomal-formulated paclitaxel and alpha-TEA produces significantly better anti-tumor outcomes than single treatments.

Anticancer actions of natural and synthetic vitamin E forms: RRR-alpha-tocopherol blocks the anticancer actions of gamma-tocopherol.

Two naturally occurring dietary sources of vitamin E (i.e. RRR-alpha-tocopherol (alphaT) and RRR-gamma-tocopherol (gammaT)), the manufactured synthetic form of vitamin E, all-racemic-alpha-tocopherol (all-rac-alphaT), as well as a potent antitumor analog of vitamin E, RRR-alpha-tocopherol ether-linked acetic acid analog (alpha-TEA), were assessed for anticancer actions. Data showed that gammaT, all-rac-alphaT, and alpha-TEA but not alphaT or alphaT+gammaT significantly inhibited tumor burden of human MDA-MB-231 cells in nude mice. Immunohistochemical analyses of tumor tissue showed that all-rac-alphaT and alpha-TEA increased apoptosis and decreased proliferation in tumor cells while gammaT was associated with increased tumor cell apoptosis only. In vitro data showed alpha-TEA and gammaT but not all-rac-alphaT or alphaT to inhibit colony formation and induce apoptosis. Anticancer actions of alpha-TEA and gammaT involved death receptor 5 protein upregulation, Survivin protein downregulation and poly (ADP-ribose) polymerase cleavage, all of which were blocked by co-treatment with alphaT. In summary, both gammaT and alpha-TEA exhibited promising anticancer properties in vivo and in vitro, whereas all-rac-alphaT exhibited promising anticancer properties in vivo only. Importantly, alphaT not only failed to exhibit anticancer properties but it also reduced anticancer actions of gammaT in vivo and gammaT and alpha-TEA in vitro.

In vitro and in vivo evaluation of anticancer actions of natural and synthetic vitamin E forms.

The goal of these studies was to investigate the potential anticancer properties of two naturally occurring plant sources and two manufactured synthetic forms of vitamin E, i. e., RRR-alpha-tocopherol (alphaT), RRR-gamma-tocopherol (gammaT), all-rac-alpha-tocopherol (all-rac-alphaT), and all-rac-alpha-tocopheryl acetate (all-rac-alphaTAc) in breast cancer models. Vitamin E compounds were evaluated in vitro for inhibition of colony formation and induction of apoptosis in human MDA-MB-435 and MCF-7 breast cancer cells and murine 66cl-4 mammary cancer cells and in vivo for ability to reduce tumor growth and lung and lymph node metastases using the transplantable syngeneic BALB/c mouse 66cl-4-GFP mammary cancer model. gammaT inhibited colony formation and induced apoptosis in all three cancer cell lines. alphaT and all-rac-alphaT were less effective and all-rac-alphaTAc was ineffective. gammaT-induced apoptosis was correlated with activation of caspases-8 and -9 and down-regulation of protein expression of c-FLIP and survivin. In vivo study 1 analyses showed that all-rac-alphaT and all-rac-alphaTAc significantly inhibited tumor growth and inhibited both visible and microscopic size lung metastases. In vivo study 2 analyses showed that alphaT and gammaT reduced tumor growth, but only gammaT reduced tumor growth significantly in comparison to control. In conclusion, synthetic, but not natural, vitamin E exhibits promising anti-cancer properties in vivo.

Vitamin E analog alpha-TEA and celecoxib alone and together reduce human MDA-MB-435-FL-GFP breast cancer burden and metastasis in nude mice.

Alpha-TEA, a nonhydrolyzable ether analog of vitamin E (RRR-alpha-tocopherol), and celecoxib, a specific COX-2 inhibitor, were delivered separately or in combination to investigate their anticancer properties, using MDA-MB-435-FL-GFP human breast cancer xenografts in nude mice. Liposomal formulated alpha-TEA administered as an aerosol and celecoxib fed at 500 or 1250 mg/kg diet for 31 days separately or in combination significantly reduced tumor volume in comparison to control (p < 0.001 for all treatment groups). Of special note, the combinations of alpha-TEA + celecoxib (1250) inhibited tumor volume significantly better than either single treatment (p < 0.001 and p < 0.001). Average number of macroscopic lung metastases were significantly reduced in all treatment groups in comparison to control, with the exception of celecoxib (500). Mean numbers of microscopic lung and lymph node metastases in all treatment groups were significantly lower than control. Furthermore, the mean number of microscopic lung metastases in the alpha-TEA+celecoxib (1250) group were significantly lower than either separate treatment. Analyses of 5 microm tumor sections showed that all treatments, with the exception of celecoxib (500) alone, significantly enhanced apoptosis (TUNEL) and significantly decreased cell proliferation (Ki-67). alpha-TEA and alpha-TEA + celecoxib (1250) treatments significantly reduced blood vessel density (CD-31) in comparison to control. These data show promise for combination alpha-TEA + celecoxib chemotherapy for breast cancer.