Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition ofa human ovarian cancer cell line

Although gonadotropin-releasing hormone agonists (GnRHa) have been used in the therapy of the endocrine-dependent cancers, their biological mechanism remained obscure. We have studied the roles of mitogen-activated protein ki nase family in the antiproliferative effect of GnRHa on the Caov-3 human ov arian cancer cell line. Reverse transcription-PCR assays confirmed mRNA for GnRH receptor in Caov-3 cells. In the presence of 1 mu M GnRHa, the prolif eration of cells was significantly reduced to 76% of controls after 24 h, a nd the effect was sustained up to 4 days. Although GnRHa had no effect on t he activation of the Jun N-terminal kinase (JNK), treatment of Caov-3 cells with GnRHa activated extracellular signal-regulated protein kinase (ERK), and its effect was more than that induced by GnRH. Activation of ERK by GnR Ha occurred within 5 min, with the maximum occurring at 3 h and sustained u ntil 24 h. GnRHa also activated ERK kinase (mitogen-activated protein/ERK k inase) and resulted in an increase in phosphorylation of son of sevenless ( Sos), and Shc. Furthermore, we examined the mechanism by which GnRHa induce d ERK activation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the beta gamma snbunits of G proteins, blocked the GnRHa-induc ed ERK activation. Phorbol 12-myristate 13-acetate (PMA) also induced the E RK activity, but pretreatment of the cultured cells with PMA to down-regula te protein kinase C did not abolish the activation of ERK by GnRHa. Elimina tion of extracellular Ca2+ by EGTA also did not abolish the activation of E RK by GnRHa. To examine the role of ERK cascade in the antiproliferative ef fect of GnRHa, PD98059, an inhibitor of mitogen-activated protein/ERK kinas e, was used. This inhibitor canceled the antiproliferative effect of GnRHa and apparently reversed the GnRH-induced dephosphorylation of the retinobla stoma protein, the hyperphosphorylation of which is a hallmark of G(1)-S tr ansition in the cell cycle. These results provide evidence that GnRHa stimu lation of ERK activity may be mediated by G beta gamma protein, not by PMA- sensitive protein kinase C nor extracellular Ca2+ in the Caov-3 human ovari an cancer cell line, suggesting that this cascade may play an important rol e in the antiproliferative effect of GnRHa.