The antiproliferative effect of GnRH agonists and the mechanism of the resistance to cisplatin in human ovarian cancer cell line

促性腺激素释放激素激动剂(GnRHa)已用于内分泌依赖型肿瘤的治疗，但其生物学机制尚不清楚。我们研究了丝裂原活化蛋白激酶(MAPK)家族在GnRHa对人卵巢癌顺铂耐药细胞株Caov-3的抗增殖作用中的作用。逆转录-聚合酶链式反应检测证实CAOV-3细胞有GnRH受体的mRNA表达。1μM GnRHa作用2 4h后，细胞增殖率明显下降至对照组的76%，并持续至4d。GnRHa对Jun氨基末端激酶的激活无影响，但GnRHa激活细胞外信号调节蛋白激酶的作用强于GnRHa。GnRHa对细胞外信号调节蛋白激酶的激活发生在5min内，3h达最大值，并持续至2 4h。为探讨ERK级联通路在GnRHa、…抗增殖作用中的作用更多使用MEK抑制剂PD98059。该抑制物可取消GnRHa的抗增殖作用，并明显逆转GnRH诱导的视网膜母细胞瘤蛋白(Rb)的去磷酸化，该蛋白的过度磷酸化是细胞周期中G1-S转变的标志。这些结果表明，GnRHa对ERK活性的刺激可能在GnRHa抑制人卵巢癌细胞株Caov-3增殖中起重要作用。接下来，我们研究了JNK和ERK级联在顺铂耐药的CAOV-3和敏感的A2780人卵巢癌细胞系中的作用。顺铂作用于Caov-3和A2780细胞后，JNK和ERK均被激活，顺铂激活JNK在30min达到高峰，3h达到高峰，之后下降，而顺铂激活ERK呈现双相模式(30min和3h达到峰值)，表明不同的时间框架。顺铂对JNK的激活在1000μM时最大，ERK的激活在100μM时最大，浓度越高，激活作用越弱，表现出不同的剂量依赖性。在对顺铂高度耐药的CAOV-3细胞(IC_(50)=380±25μM)和对顺铂敏感的A2780细胞(IC_(50)=84±4μM)中，外源性显性负性c-jun(Dnjun)的表达均降低了细胞的存活率：表达顺铂的CAOV-3和A2780细胞对顺铂的IC_(50)分别降低了7.6%和4.2倍。我们进一步研究了ERK级联对使用PD98059顺铂治疗后的生存能力的影响。该化合物诱导了对顺铂的敏感性，但对顺铂没有影响，导致Caov-3和A2780细胞对顺铂的IC50分别降低了9.7倍和1.8倍。我们的发现表明，顺铂诱导的DNA损伤不同地激活了JNK和ERK级联，抑制这两个级联中的任何一个都会使卵巢癌细胞对顺铂敏感。较少

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 kinase (MAPK) family in the antiproliferative effect of GnRHa on the cisplatin resistant Caov-3 human ovarian cancer cell line. Reverse-transcriptase PCR assays confirmed mRNA for GnRH receptor in Caov-3 cells. In the presence of 1 μM GnRHa, the proliferation of cells was significantly reduced to 76% of controls after 24 h and the effect was sustained up to 4 days. Although GnRHa had no effect on the 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 GnRHa occurred within 5 min, with the maximum at 3 h and sustained until 24 h. To examine the role of ERK cascade in the antiproliferative effect of GnRHa, … More PD98059, an inhibitor of MEK, was used. This inhibitor canceled the antiproliferative effect of GnRHa and apparently reversed the GnRH-induced dephosphorylation of the retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of G1-S transition in the cell cycle. These results provide evidence that GnRHa stimulation of ERK activity may play an important role in the antiproliferative effect of GnRHa in the Caov-3 human ovarian cancer cell line. Next, we have studied the roles of JNK and ERK cascade in both the cisplatin resistant Caov-3 and sensitive A2780 human ovarian cancer cell lines. Treatment of both Caov-3 and A2780 cells with cisplatin but not the transplatin isomer activates JNK and ERK.Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern (peaks at 30 min and 3 h), indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 μM, whereas activation of ERK was maximal at 100 μM and was less at higher concentrations, indicating the different dose-dependency. Exogenous expression of dominant negative c-Jun (dnJun) in both Caov-3 cells, which are highly resistant to cisplatin (IC50=380±25 μM), and A2780 cells, which are sensitive to cisplatin (IC50=84±4 μM), decreased viability following treatment with cisplatin : the IC50 for cisplatin was 7.6-and 4.2-fold less in dnJun expressing Caov-3 and A2780 cells, respectively. We further examined the role of ERK cascade on the viability following cisplatin treatment using PD98059. The treatment by this compound induced sensitivity to cisplatin, but not to transplatin, leading to a 9.7-and 1.8-fold less IC50 for cisplatin in Caov-3 and A2780 cells, respectively. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin. Less