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

尽管促性腺激素释放激素激动剂（GnRHa）已用于治疗内分泌依赖性癌症，但其生物学机制仍不清楚。我们研究了丝裂原激活蛋白激酶 (MAPK) 家族在 GnRHa 对顺铂耐药的 Caov-3 人卵巢癌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。在 1 μM GnRHa 存在下，24 小时后细胞增殖显着降低至对照的 76%，且效果可持续长达 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细胞会激活细胞外信号调节蛋白激酶（ERK），其作用比GnRH诱导的激活作用更大。GnRHa对ERK的激活发生在5 min内，3 h时达到最大值，并持续至24 h。为了检查 ERK 级联在 GnRHa 抗增殖作用中的作用，使用了 MEK 抑制剂 PD98059。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，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发挥重要作用。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。用顺铂而不是转铂异构体处理Caov-3和A2780细胞都会激活JNK和ERK。顺铂对JNK的激活发生在30分钟，在3小时达到平台，然后下降，而顺铂对ERK的激活显示出双相模式（在30分钟和3小时处达到峰值），表明不同的时间范围。顺铂对 JNK 的激活在 1000 μM 时达到最大，而 ERK 的激活在 100 μM 时达到最大，并且在较高浓度时激活较少，表明不同的剂量依赖性。对顺铂高度耐药的 Caov-3 细胞 (IC50=380±25 μM) 和对顺铂敏感的 A2780 细胞 (IC50=84±4 μM) 中显性阴性 c-Jun (dnJun) 的外源表达，经顺铂处理后活力下降：顺铂的 IC50 分别降低了 7.6 倍和 4.2 倍。 dnJun 分别表达 Caov-3 和 A2780 细胞。我们进一步研究了 ERK 级联对使用 PD98059 顺铂治疗后生存能力的作用。该化合物的处理诱导对顺铂的敏感性，但不诱导对转铂的敏感性，导致Caov-3和A2780细胞中顺铂的IC50分别降低9.7倍和1.8倍。我们的研究结果表明，顺铂诱导的 DNA 损伤会不同程度地激活 JNK 和 ERK 级联，并且抑制这些级联中的任何一个都会使卵巢癌细胞对顺铂敏感。较少的

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。