RESUMO
Over-expression of cleaved cyclin E in breast tumors is closely associated with tumor progression and resistance to antiestrogens. 17ß-Estradiol (E2) has been recently shown to induce cyclin E processing in breast cancer cells. Tamoxifen has been used in patients with estrogen-sensitive breast cancer, yet resistance to antiestrogens and recurrence will appear in some of the patients after its continued use. We therefore addressed possible effects of tamoxifen on the generation of cleaved cyclin E and its signal mechanism(s) in estrogen-responsive MCF-7 breast cancer cells that express both G protein-coupled protein (GPR) 30 and estrogen receptor α (ERα). 4-Hydroxytamoxifen (OHT, tamoxifen's active form) failed to prevent E2-induced proteolysis of cyclin E and migration, but rather triggered cyclin E cleavage coincident with augmented migration. OHT-induced cyclin E truncation also occurred in SK-BR-3 cells that express GPR30 and lack ERα, but not in MDA-MB-231 cells that express neither GPR30 nor ERα. G1, a specific GPR 30 agonist, caused dramatic proteolysis of cyclin E and enhanced migration. Furthermore, OHT-stimulated cleavage of cyclin E and migration were tremendously attenuated by G15, a GPR30 antagonist, or siRNA against GPR30. In addition, inhibitors for EGFR or ERK1/2 remarkably suppressed OHT-induced truncation of cyclin E, suggesting involvement of EGFR signaling. Collectively, our data indicate that OHT contributes to the production of proteolyzed cyclin E via GPR30 with augmented migration in MCF-7 cells.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Ciclina E/biossíntese , Ciclina E/genética , Processamento de Proteína Pós-Traducional , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Tamoxifeno/análogos & derivados , Movimento Celular/fisiologia , Ciclina E/metabolismo , Feminino , Humanos , Células MCF-7 , Tamoxifeno/toxicidade , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To study effects of Rhaponticum uniflorum on the activities and mRNA expression of cytochrome P4503A1 in rats hepatocytes. METHODS: Rat hepatocytes sandwich cultures were treated by Rhaponticum uniflorum in different concentrations (15.6, 31.2, 62.4 mg/ml) for 48 hours. The enzymic activities of CYP3A1 were reflected by the activities of liver microsomal erythromyeine N-demethylase (ERD). The level change in the mRNA expression were tested by RT-PCR. RESULTS: After treatment with the medicine, there was concentration-dependent inhibition in both enzymic activity and mRNA level of CYP3A1 compared with the control. The rates of the inhibitory effect to CYP3A1 activity were 26.4%, 44.2% and 65.4% respectively, while those of the inhibitory effect to mRNA level were 29.4%, 49.8% and 56.0%. CONCLUSION: Rhaponticum uniflorum inhibits the enzymic activity of CYP3A1 and lowers CYP3A1 expression at the transcriptive levels.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Medicamentos de Ervas Chinesas/farmacologia , Hepatócitos/efeitos dos fármacos , Leuzea/química , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Células Cultivadas , Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/metabolismo , Masculino , Plantas Medicinais/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721. METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent, and cells were screened by G418. RESULTS: Overexpression of alpha5beta1 or beta1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21(cip1) and p27(kip1). The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved. When phosphorylation of PKB was solely blocked by wortmannin, p27(kip1) protein level was increased. Moreover, S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of beta1-7721 or alpha5beta1-7721 cells. CONCLUSION: S-phase delay induced by overexpression of integrin beta1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21(cip1) and p27(kip1) proteins, and may be involved in the unoccupied alpha5beta1 because of lack of its ligands.
