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

GnRH激动剂对人卵巢癌细胞增殖的抑制作用及顺铂耐药机制

• 批准号: 10557147
• 负责人: OHMICHI Masahide
• 金额: $ 7.42万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557147/
• 关键词: GnRH agonists; Cisplatin; Resistance; Ovarian cancer; MAP kinase; ERK; JNK; G1 arrest; DNA損傷; ヒト卵巣癌; トランスプラチン; c-Jun; 耐性化; 燐酸化; GnRH; 増殖抑制; GnRH受容体; MEK inhibitor; SOS

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

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肿瘤，但其生物学机制尚不清楚。我们研究了促分裂原活化蛋白激酶（MAPK）家族在GnRHa抑制人卵巢癌顺铂耐药细胞系Caov-3增殖中的作用。逆转录酶PCR检测证实了Caov-3细胞中GnRH受体的mRNA。在存在1 μM GnRHa的情况下，24 h后细胞增殖显著降低至对照的76%，并且效果持续长达4天。GnRHa对Caov-3细胞JNK的激活无明显影响，但对ERK的激活作用强于GnRH，激活时间在5 min内，3 h达高峰，24 h后持续激活。为了检测ERK级联在GnRHa抗增殖作用中的作用， ...更多信息 使用MEK抑制剂PD 98059。这种抑制剂取消了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，其过度磷酸化是细胞周期中G1-S转换的标志。这些结果提供了证据，GnRHa刺激ERK活性可能在GnRHa的抗增殖作用中发挥重要作用的Caov-3人卵巢癌细胞系。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。顺铂处理Caov-3和A2780细胞激活JNK和ERK，但反铂异构体不激活JNK和ERK。顺铂激活JNK发生在30 min，3 h达到平台，此后下降，而顺铂激活ERK显示出双相模式（峰值在30 min和3 h），表明不同的时间框架。顺铂对JNK的激活在1000 μM时最大，而ERK的激活在100 μM时最大，在较高浓度时较小，表明不同的剂量依赖性。在对顺铂高度耐药的Caov-3细胞中显性阴性c-Jun（dnJun）的外源性表达（IC 50 =380±25 μM）和对顺铂敏感的A2780细胞（IC 50 =84±4 μM），顺铂处理后活力降低：顺铂的IC 50在表达dnJun的Caov-3和A2780细胞中分别低7.6倍和4.2倍。我们使用PD 98059进一步检查了ERK级联对顺铂治疗后的活力的作用。该化合物的治疗诱导了对顺铂的敏感性，但对反铂不敏感，导致顺铂在Caov-3和A2780细胞中的IC 50分别降低了9.7倍和1.8倍。我们的研究结果表明，顺铂诱导的DNA损伤不同地激活JNK和ERK级联反应，并且抑制这些级联反应中的任何一个都会使卵巢癌细胞对顺铂敏感。少

项目成果

Hayakawa, J., et al.: "Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell-line."J Biol Chem. 274. 31648-31654 (1999)

Hayakawa, J. 等人：“顺铂差异性激活的细胞外信号调节蛋白激酶或 c-Jun N 端蛋白激酶级联的抑制，使人卵巢癌细胞系变得敏感。”J Biol Chem。

Kimura,A., et al.: "The role of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade in gonadotropin-releasing hormone induced growth inhibition of human ovarian cancer cell line."Cancer Res. 59. 5133-5142 (1999)

Kimura,A. 等人：“丝裂原激活蛋白激酶 (MAPK)/细胞外信号调节激酶 (ERK) 级联在促性腺激素释放激素诱导的人卵巢癌细胞系生长抑制中的作用。”Cancer Res。

Hayakawa,J., et al.: "Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell-line."J Biol Chem. 274. 31648-31654 (1999)

Hayakawa, J. 等人：“顺铂不同程度地激活细胞外信号调节蛋白激酶或 c-Jun N 端蛋白激酶级联的抑制作用，使人卵巢癌细胞系变得敏感。”J Biol Chem。