RESUMO
The study was conducted to evaluate the effects of human relaxin on apoptosis in the human trophoblast derived HTR-8/SV neo cell line, which is a possible model of human extravillous trophoblasts (EVTs). HTR-8/SV neo cells, cultured in phenol red free RPMI1640 medium, were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions. RT-PCR was used for evaluating relaxin receptor: RXFP1 and RXFP2 expression in HTR-8/SV neo cells. The cell death was examined by TUNEL assay. Furthermore, we investigated caspase-3, cleaved PARP and Bcl-2 expressions by Western blot analysis to recognize the translational effects of anti-apoptotic and pro-apoptotic proteins. RXFP1 and RXFP2 mRNA expression was observed in HTR-8/SV neo cells. Compared with untreated control cultures, treatment with rH2 relaxin, decreased TUNEL-positive rate in HTR-8/SV neo cells was observed. Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 and cleaved PARP, but in contrast increased Bcl-2 expression in those cells. These results suggest that rH2 relaxin has anti-apoptotic effects on HTR8/SV neo cells by decreasing pro-apoptotic caspase-3 and cleaved PARP expression and up-regulating anti-apoptotic Bcl-2 expression.
Assuntos
Apoptose/efeitos dos fármacos , Relaxina/farmacologia , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Caspase 3/análise , Linhagem Celular , Meios de Cultura Livres de Soro , Feminino , Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Proto-Oncogênicas c-bcl-2/análise , RNA Mensageiro/análise , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Proteínas Recombinantes/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos/químicaRESUMO
OBJECTIVE: To evaluate the effects of human relaxin on proliferation and apoptosis in cultured human uterine leiomyoma cells and normal myometrial cells. DESIGN: In vitro experiment. SETTING: Research laboratory at Kobe University Graduate School of Medicine. PATIENT(S): Nine patients undergoing hysterectomy for uterine leiomyoma. INTERVENTION(S): Cultured leiomyoma cells and normal myometrial cells were treated with human recombinant (rH2) relaxin. MAIN OUTCOME MEASURE(S): Human relaxin receptor LGR7 expressions in cultured leiomyoma cells and myometrial cells were evaluated by immunocytochemical staining. Cell proliferation, proliferating cell nuclear antigen-positive rate, and TUNEL-positive rate were assessed by MTS assay, immunocytochemistry, and TUNEL assay, respectively. Caspase-3 expression was evaluated by Western blot analysis. RESULT(S): LGR7 expression was observed both in cultured human leiomyoma cells and myometrial cells. Compared with untreated control cultures, treatment with rH2 relaxin increased the number of viable cultured leiomyoma cells and the proliferating cell nuclear antigen-positive rate in those cells but not in myometrial cells. Moreover, treatment with rH2 relaxin decreased the TUNEL-positive rate in cultured leiomyoma cells but not in myometrial cells. Similarly, Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 in cultured leiomyoma cells but not in myometrial cells. CONCLUSION(S): These results suggest that rH2 relaxin selectively inhibits apoptosis by down-regulating caspse-3 expression and induces proliferation in cultured human leiomyoma cells without affecting apoptosis or proliferation in normal myometrial cells.