RESUMEN
Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.
Asunto(s)
Anticarcinógenos/farmacología , Quinasas CDC2-CDC28 , Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Aceites Volátiles/farmacología , Western Blotting , Manzanilla , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Genes de Retinoblastoma , Humanos , Fosforilación , Plantas Medicinales , Proteínas Serina-Treonina Quinasas/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
Apigenin is a plant flavonoid which has been shown to significantly inhibit UV-induced mouse skin tumorigenesis when applied topically, and may represent an alternative sunscreen agent in humans. We have investigated the molecular mechanism(s) by which apigenin inhibits skin tumorigenesis. Initial studies examined the effects of apigenin on the cell cycle. DNA flow cytometric analysis indicated that culturing cells for 24 h in medium containing apigenin induced a G2/M arrest in two mouse skin derived cell lines, C50 and 308, as well as in human HL-60 cells. The G2/M arrest was fully reversible after an additional 24 h in medium without apigenin. We investigated the effects of apigenin on cyclin B1 and p34cdc2, since cyclin B1/p34cdc2 complexes regulate G2/M progression. Western blot and immune complex kinase assays using whole cell lysates from 308 and C50 cells treated for 24 h with 0-70 microM doses of apigenin demonstrated that apigenin treatment did not change the steady-state level of p34cdc2 protein, but did inhibit p34cdc2 H1 kinase activity in 308 cells. Western blot analysis showed that apigenin treatment of C50 cells and 308 cells inhibited the accumulation of cyclin B1 protein in a dose-dependent manner. The apigenin levels detected in cultured keratinocytes were relevant to those detected in epidermal cells of Sencar mice treated with tumor inhibitory doses of apigenin. In conclusion, we present evidence that apigenin induces a reversible G2/M arrest in cultured keratinocytes, the mechanism of which is in part due to inhibition of the mitotic kinase activity of p34cd2, and perturbation of cyclin B1 levels.
Asunto(s)
Anticarcinógenos/farmacología , Ciclina B , Quinasas Ciclina-Dependientes , Flavonoides/farmacología , Fase G2/efectos de los fármacos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Mitosis/efectos de los fármacos , Aceites Volátiles/farmacología , Animales , Anticarcinógenos/farmacocinética , Línea Celular , Manzanilla , Ciclina B1 , Ciclinas/efectos de los fármacos , Ciclinas/metabolismo , Flavonoides/farmacocinética , Células HL-60 , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Desnudos , Aceites Volátiles/farmacocinética , Plantas Medicinales , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Previous studies in our laboratory have shown that promotion of two-stage skin carcinogenesis in the SENCAR mouse model was inhibited in mice fed energy-restricted/low-fat diets, and elevated in mice fed high-fat diets. Studies reported here describe the influence of dietary energy restriction from fat and carbohydrate (ER) or high-fat (HF) diet on early promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and on late promotion by mezerein (MEZ). Female SENCAR mice were initiated topically with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone at 9 weeks of age. For the following 2 weeks they received 3.2 nmol TPA in 0.2 ml acetone twice weekly, and for the next 16 weeks they received 10 nmol MEZ in 0.2 ml acetone twice weekly. All mice were fed control diet before TPA began and following the final MEZ treatment. Control mice received the control diet (c) throughout TPA and MEZ (C/C). The six experimental groups received: (1) ER diet throughout TPA and MEZ treatment (ER/ER); (2) HF diet throughout TPA and MEZ treatment (HF/HF); (3) ER during TPA (ER/C); (4) ER during MEZ (C/ER); (5) HF diet during TPA (HF/C); or (6) HF diet during MEZ (C/HF). Papilloma incidence and multiplicity, and carcinoma incidence were similarly reduced in the mice fed ER diet during MEZ (ER/ER and C/ER groups). In comparing the HF groups, papilloma multiplicity was highest in the HF/C group, intermediate in the C/C and lowest in the C/HF groups, but papilloma and carcinoma incidences were not modified by the HF diet protocols. Papilloma regression was greater in the C/HF group (27%, 4 regressions/15 tumor-bearing mice) than in the controls (0/18) during weeks 21-23, immediately following the end of MEZ treatment (P < 0.05).
Asunto(s)
Carcinógenos/toxicidad , Cocarcinogénesis , Dieta con Restricción de Grasas , Carbohidratos de la Dieta/farmacología , Grasas de la Dieta/farmacología , Diterpenos , Ingestión de Energía , Papiloma/inducido químicamente , Papiloma/prevención & control , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/prevención & control , Terpenos/toxicidad , 9,10-Dimetil-1,2-benzantraceno , Animales , Peso Corporal/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos SENCAR , Papiloma/etiología , Neoplasias Cutáneas/etiología , Acetato de TetradecanoilforbolRESUMEN
Studies were conducted to evaluate the ability of dietary dried cabbage supplements to inhibit pancreatic carcinogenesis in hamsters and skin tumorigenesis in mice. Pancreatic cancer was induced by treatment with 40 mg/kg body wt N-nitrosobis-(2-oxopropyl)amine (BOP). Cabbage was fed from before carcinogen treatment in low fat diet and, beginning 1 week after BOP treatment, cabbage was given in low fat and high fat diets in comparison with the respective non-cabbage-containing diets. Dried cabbage was incorporated at 9 and 11% levels into the low and high fat diets. Feeding cabbage in the high fat diet elevated the yield of BOP-induced pancreatic ductular carcinoma (1.6 carcinomas/effective animal) in comparison with that observed in hamsters fed cabbage in a low fat diet or in those given a high fat diet without cabbage, 0.6-0.8 carcinomas/effective animal (P less than 0.05). Furthermore, the incidence of BOP-induced gall bladder adenocarcinoma was elevated in cabbage-fed hamsters irrespective of dietary fat intake. Effects of dietary fat and cabbage on food consumption, body weight, and serum T3 and T4 values are described. Skin tumorigenesis was induced in SENCAR mice by 10 nmol 7,12 dimethylbenz[a]anthracene (DMBA) and promoted beginning 1 week later with twice weekly applications of 2 micrograms 12-O-tetradecanoyl-13-phorbol acetate (TPA). Dried cabbage was incorporated into AIN semi-purified diets from before DMBA treatment and throughout TPA treatment. Skin papilloma yield was elevated in DMBA-initiated TPA-promoted mice that were fed diets containing 10% cabbage. Mice fed cabbage developed an average of 8.45 papillomas per mouse following 22 weeks of promotion while mice given control diet developed 7.25 papillomas per mouse (P less than 0.001). Cabbage feeding did not influence survival, food consumption or body weight of the mice. These results suggest the need for further research on the use of cabbage as a chemopreventive measure.