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

虽然促性腺激素释放激素激动剂（GnRHa）已被用于治疗内分泌依赖性癌症，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对顺铂耐药人卵巢癌cav -3细胞系的抗增殖作用中的作用。逆转录酶PCR检测证实了Caov-3细胞中GnRH受体的mRNA表达。在1 μM GnRHa的作用下，24 h后细胞的增殖率明显降低，为对照组的76%，且效果持续4天。虽然GnRHa对Jun n -末端激酶（JNK）的激活没有影响，但用GnRHa处理Caov-3细胞激活细胞外信号调节蛋白激酶（ERK），其作用大于GnRH诱导的作用。GnRHa在5分钟内激活ERK，最大激活时间为3小时，持续至24小时。为了研究ERK级联在GnRHa的抗增殖作用中的作用，我们使用了MEK抑制剂PD98059。该抑制剂取消了GnRHa的抗增殖作用，并明显逆转了gnrh诱导的视网膜母细胞瘤蛋白（pRb）的去磷酸化，pRb的过度磷酸化是细胞周期G1-S转变的标志。这些结果证明GnRHa刺激ERK活性可能在GnRHa对Caov-3人卵巢癌细胞系的抗增殖作用中起重要作用。接下来，我们研究了JNK和ERK级联在顺铂耐药Caov-3和敏感的A2780人卵巢癌细胞系中的作用。用顺铂治疗Caov-3和A2780细胞，但不使用移植异构体，可激活JNK和ERK。顺铂对JNK的激活发生在30分钟，在3小时达到平台，此后下降，而顺铂对ERK的激活呈现双相模式（在30分钟和3小时达到峰值），表明不同的时间框架。顺铂对JNK的激活作用在1000 μM时达到最大，而对ERK的激活作用在100 μM时达到最大，且浓度越高，ERK的激活作用越弱，显示出不同的剂量依赖性。外源表达显性阴性c-Jun （dnJun）在高顺铂耐药的cav -3细胞（IC50=380±25 μM）和对顺铂敏感的A2780细胞（IC50=84±4 μM）中，顺铂治疗后生存能力下降：表达cav -3和A2780的dnJun细胞对顺铂的IC50分别降低7.6和4.2倍。我们进一步研究了使用PD98059顺铂治疗后ERK级联对细胞存活率的作用。该化合物诱导对顺铂的敏感性，但对移植不敏感，导致顺铂在Caov-3和A2780细胞中的IC50分别降低9.7和1.8倍。我们的研究结果表明，顺铂诱导的DNA损伤不同程度地激活了JNK和ERK级联反应，抑制这两种级联反应中的任何一种都会使卵巢癌细胞对顺铂敏感。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