Carcinogenesis vol.12 no.4 pp.671-678, 1991
Diverse caroteimoids protect against
John S.Bertram, Ao Pung, Melissa Churley, T.Joseph Kappock IV, Lynne R.Wilkins1 and Robert V.Cooney
The ability of diverse carotenoids to inhibit methylcholanthrene-induced transformation of 10T1/2 cells has been investigated. When delivered using tetrahydrofuran as a novel solvent, all carotenoids were absorbed by cultured cells. When continuously administered to methylcholanthrene-treated cultures 7 days after removal of the carcinogen, canthaxanthin, /3-carotene, a-carotene and lycopene inhibited the production of transformed foci in a dose-dependent manner in the above order of potency. This activity was not associated with drug toxkity or antiproliferative effects. Renierapurpurin and bixin did not inhibit transformation at concentrations S 10~5 M. Lutein was inhibitory at 10~5 M, but was inactive at lower concentrations. Because of differences in stability in culture medium (a-carotene < /3carotene < canthaxanthin < lycopene < lutein) and structure, cellular levels of drug differed up to 8-fold after administration of identical concentrations of compounds. Carotenoids with polar groups achieved highest cellular levels, however cellular uptake did not correlate with activity. For example, lutein, the most polar and most stable, reached the highest concentration in cells yet required a concentration of 10~5 M for activity in the transformation assay, while acarotene, the least stable and least concentrated by cells, was comparably active at 3 x 10~6 M. a-Tocopherol, a potent lipid-phase antioxidant, was as active as lycopene in the transformation assay but at a 10-fold higher concentration did not approach the activity of /3-carotene or canthaxanthin. Because the most potent of the carotenoids tested (i.e. /3carotene, a-carotene, canthaxanthin) all have the potential for conversion to retinoids (though this has never been demonstrated in mammals for canthaxanthin), it is suggested that these compounds have two components to their action; one related to their antioxidant properties, the other to their pro-vitamin A activities.
Introduction Consumption of carotenoids, plant pigments which play a crucial role as electron transport agents in photosynthesis and additionally protect plants from oxidative damage (1), has frequently been correlated with a decreased risk of cancer at several sites. In case control studies, patients with cancers of the larynx, lung, esophagus, stomach, bladder, cervix and ovary reported dietary •Abbreviations: DMBA, 7,12-dimethylbenz[ajanthracene; CTX, canthaxanthin; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; MCA, methylcholanthrene; BUT, butylated hydroxytoluene; BME, basal Eagle's medium; TF, transformation frequency; RAR, retinoic acid receptors. © Oxford University Press
:nc
histories significantly lower in carotenoid and/or retinoid content than did matched controls. Similar associations have also been noted in prospective studies of diet and cancer (2). Most epidemiologic studies have focused on those carotenoids with provitamin A activity, principally /5-carotene. Based on these results, and those demonstrating that vitamin A itself, or its analogs the retinoids, have significant cancer chemopreventive properties (3), a number of interventional trials with carotenoids are underway in humans. Positive results have so far been reported for regression of oral leukoplakia (4). In experimental animals, /3carotene has been reported to reduce the incidence of UV-induced skin tumors (5), 7,12-dimethylbenz[a]anthracene (DMBA*) induced buccal pouch carcinomas (6) and to cause regression of established transplanted tumors (7). A major unanswered question in cancer epidemiology and chemoprevention is: does /3-carotene, the carotenoid with highest potential pro-vitamin A activity also have the highest chemopreventive potential? If so, this would imply that carotenoids function via metabolic conversion to retinoids. Resolution of this question would help focus epidemiologic studies since many hundreds of carotenoids are found in plants (8) and human serum contains up to 20 different carotenoids (9). Tomatoes for example contain lycopene, a non-provitamin A carotenoid, more active in quenching singlet oxygen (10), and often present in human serum at higher concentrations than /3carotene (11). Furthermore, two recent epidemiologic studies have shown an association between serum lycopene (12) or tomato consumption (13) and decreased risk for cancer. The relationship between carotenoid structure and function is also of mechanistic importance since its resolution might lead to synthesis of improved chemopreventive agents. We have approached these questions by using a defined in vitro system of C3H/10T1/2 mouse embryo fibroblasts. These cells undergo neoplastic transformation in response to many chemical and physical carcinogens in a dose-dependent manner (14) and represent one of the most widely characterized in vitro models for carcinogenesis research (15). Of importance in the present context is their response to retinoids: when added after chemical initiation, retinoids reversibly suppress neoplastic transformation (16). This ability of retinoids mirrors their known activity in animals and structure-activity studies have shown excellent correlations between in vitro and in vivo properties of diverse retinoids (17). Recently, we have demonstrated that two carotenoids, /3-carotene and canthaxanthin,- also reversibly suppress chemically and physically induced neoplastic transformation in 10T1/2 cells (18). No evidence for conversion of /3-carotene to retinoids could be detected in these studies (19). Research in animals has the drawback that the mouse and the rat are poor models for absorption and tissue distribution of carotenoids in humans (20). Moreover research using cell culture systems has previously been hindered by difficulties in formulating these highly lipophilic compounds into a bioavailable form. In the cases of/3-carotene and canthaxanthin (CTX) (18), this difficulty was overcome by incorporating the carotenoid into 671
Downloaded from http://carcin.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 22, 2015
Molecular Oncology Program and 'Epidemiology Program, Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA
TITTTli
J.S.Bertram el al.
Materials and methods Chemicals THF 99.9% [with and without 0.025% butylated hydroxytoluene (BHT)] was purchased from Aldrich Chemical Co., Milwaukee, Wl. All HPLC solvents were Optima grade from Fisher Scientific, Kent, WA. Deuterated THF and water were from Sigma Chemical Co., St Louis, MO. /3-Carotene, lycopene, lutein and a-tocopherol were also obtained from Sigma. a-Carotene, CTX, bixin and renierapurpurin were donated by Hoffmann-LaRoche, Basel, Switzerland. The purity of carotenoids was determined by HPLC analysis after storage and prior to use. Samples found to be
Diverse caroteimoids protect against
John S.Bertram, Ao Pung, Melissa Churley, T.Joseph Kappock IV, Lynne R.Wilkins1 and Robert V.Cooney
The ability of diverse carotenoids to inhibit methylcholanthrene-induced transformation of 10T1/2 cells has been investigated. When delivered using tetrahydrofuran as a novel solvent, all carotenoids were absorbed by cultured cells. When continuously administered to methylcholanthrene-treated cultures 7 days after removal of the carcinogen, canthaxanthin, /3-carotene, a-carotene and lycopene inhibited the production of transformed foci in a dose-dependent manner in the above order of potency. This activity was not associated with drug toxkity or antiproliferative effects. Renierapurpurin and bixin did not inhibit transformation at concentrations S 10~5 M. Lutein was inhibitory at 10~5 M, but was inactive at lower concentrations. Because of differences in stability in culture medium (a-carotene < /3carotene < canthaxanthin < lycopene < lutein) and structure, cellular levels of drug differed up to 8-fold after administration of identical concentrations of compounds. Carotenoids with polar groups achieved highest cellular levels, however cellular uptake did not correlate with activity. For example, lutein, the most polar and most stable, reached the highest concentration in cells yet required a concentration of 10~5 M for activity in the transformation assay, while acarotene, the least stable and least concentrated by cells, was comparably active at 3 x 10~6 M. a-Tocopherol, a potent lipid-phase antioxidant, was as active as lycopene in the transformation assay but at a 10-fold higher concentration did not approach the activity of /3-carotene or canthaxanthin. Because the most potent of the carotenoids tested (i.e. /3carotene, a-carotene, canthaxanthin) all have the potential for conversion to retinoids (though this has never been demonstrated in mammals for canthaxanthin), it is suggested that these compounds have two components to their action; one related to their antioxidant properties, the other to their pro-vitamin A activities.
Introduction Consumption of carotenoids, plant pigments which play a crucial role as electron transport agents in photosynthesis and additionally protect plants from oxidative damage (1), has frequently been correlated with a decreased risk of cancer at several sites. In case control studies, patients with cancers of the larynx, lung, esophagus, stomach, bladder, cervix and ovary reported dietary •Abbreviations: DMBA, 7,12-dimethylbenz[ajanthracene; CTX, canthaxanthin; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; MCA, methylcholanthrene; BUT, butylated hydroxytoluene; BME, basal Eagle's medium; TF, transformation frequency; RAR, retinoic acid receptors. © Oxford University Press
:nc
histories significantly lower in carotenoid and/or retinoid content than did matched controls. Similar associations have also been noted in prospective studies of diet and cancer (2). Most epidemiologic studies have focused on those carotenoids with provitamin A activity, principally /5-carotene. Based on these results, and those demonstrating that vitamin A itself, or its analogs the retinoids, have significant cancer chemopreventive properties (3), a number of interventional trials with carotenoids are underway in humans. Positive results have so far been reported for regression of oral leukoplakia (4). In experimental animals, /3carotene has been reported to reduce the incidence of UV-induced skin tumors (5), 7,12-dimethylbenz[a]anthracene (DMBA*) induced buccal pouch carcinomas (6) and to cause regression of established transplanted tumors (7). A major unanswered question in cancer epidemiology and chemoprevention is: does /3-carotene, the carotenoid with highest potential pro-vitamin A activity also have the highest chemopreventive potential? If so, this would imply that carotenoids function via metabolic conversion to retinoids. Resolution of this question would help focus epidemiologic studies since many hundreds of carotenoids are found in plants (8) and human serum contains up to 20 different carotenoids (9). Tomatoes for example contain lycopene, a non-provitamin A carotenoid, more active in quenching singlet oxygen (10), and often present in human serum at higher concentrations than /3carotene (11). Furthermore, two recent epidemiologic studies have shown an association between serum lycopene (12) or tomato consumption (13) and decreased risk for cancer. The relationship between carotenoid structure and function is also of mechanistic importance since its resolution might lead to synthesis of improved chemopreventive agents. We have approached these questions by using a defined in vitro system of C3H/10T1/2 mouse embryo fibroblasts. These cells undergo neoplastic transformation in response to many chemical and physical carcinogens in a dose-dependent manner (14) and represent one of the most widely characterized in vitro models for carcinogenesis research (15). Of importance in the present context is their response to retinoids: when added after chemical initiation, retinoids reversibly suppress neoplastic transformation (16). This ability of retinoids mirrors their known activity in animals and structure-activity studies have shown excellent correlations between in vitro and in vivo properties of diverse retinoids (17). Recently, we have demonstrated that two carotenoids, /3-carotene and canthaxanthin,- also reversibly suppress chemically and physically induced neoplastic transformation in 10T1/2 cells (18). No evidence for conversion of /3-carotene to retinoids could be detected in these studies (19). Research in animals has the drawback that the mouse and the rat are poor models for absorption and tissue distribution of carotenoids in humans (20). Moreover research using cell culture systems has previously been hindered by difficulties in formulating these highly lipophilic compounds into a bioavailable form. In the cases of/3-carotene and canthaxanthin (CTX) (18), this difficulty was overcome by incorporating the carotenoid into 671
Downloaded from http://carcin.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 22, 2015
Molecular Oncology Program and 'Epidemiology Program, Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA
TITTTli
J.S.Bertram el al.
Materials and methods Chemicals THF 99.9% [with and without 0.025% butylated hydroxytoluene (BHT)] was purchased from Aldrich Chemical Co., Milwaukee, Wl. All HPLC solvents were Optima grade from Fisher Scientific, Kent, WA. Deuterated THF and water were from Sigma Chemical Co., St Louis, MO. /3-Carotene, lycopene, lutein and a-tocopherol were also obtained from Sigma. a-Carotene, CTX, bixin and renierapurpurin were donated by Hoffmann-LaRoche, Basel, Switzerland. The purity of carotenoids was determined by HPLC analysis after storage and prior to use. Samples found to be
