Gründker C, Schulz K, Günthert A R, Emons G
Department of Gynecology and Obstetrics, Georg-August University, Göttingen, Germany.
J Clin Endocrinol Metab. 2000 Oct;85(10):3815-20. doi: 10.1210/jcem.85.10.6859.
More than 80% of human ovarian cancers express LHRH and its receptor as part of a negative autocrine mechanism of growth control. This study was conducted to investigate whether LHRH affects apoptosis in ovarian cancer. EFO-21 and EFO-27 ovarian cancer cells were treated with LHRH agonist Triptorelin or with cytotoxic agent Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow cytometry. Expression of nuclear factor kappa B (NFkappaB) was assessed by RT-PCR and immunoblotting. For determination of Triptorelin-induced NFkappaB activation, cells were transfected with a NFkappaB-secreted alkaline phosphatase reporter gene plasmid (pNFkappaB-SEAP) and cultured for 96 h with or without Triptorelin. The causal relation between Triptorelin-induced NFkappaB activation and Triptorelin-induced protection against apoptosis was investigated using SN50, an inhibitor for nuclear translocation of activated NFkappaB. Apoptosis induction by Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L) for 72 h increased the percentage of apoptotic cells in EFO-21 and EFO-27 ovarian cancer cell lines to 31% or 34%, respectively. In cultures treated simultaneously with Triptorelin (100 nmol/L), the percentage of apoptotic cells was reduced significantly, to 17% or 18%, respectively (P < 0.001). RT-PCR and immunoblotting experiments showed that NFkappaB subunits p50 and p65 were expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21 or EFO-27 cells were transfected with pNFkappaB-SEAP and subsequently treated with Triptorelin (100 nmol/L), NFkappaB-induced SEAP expression increased 5.3-fold or 4.7-fold, respectively (P < 0.001). Triptorelin-induced reduction of Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition of NFkappaB translocation into the nucleus. We conclude that LHRH induces activation of NFkappaB and thus reduces Doxorubicin-induced apoptosis in human ovarian cancer cells. This possibility to protect ovarian cancer cells from programmed cell death is an important feature in LHRH signaling in ovarian tumors, apart from the inhibitory interference with the mitogenic pathway.
超过80%的人类卵巢癌表达促黄体生成素释放激素（LHRH）及其受体，作为生长控制的负自分泌机制的一部分。本研究旨在调查LHRH是否影响卵巢癌中的细胞凋亡。在不存在或存在曲普瑞林的情况下，用LHRH激动剂曲普瑞林或细胞毒性药物阿霉素处理EFO - 21和EFO - 27卵巢癌细胞。通过流式细胞术对凋亡细胞进行定量。通过逆转录聚合酶链反应（RT-PCR）和免疫印迹法评估核因子κB（NFκB）的表达。为了确定曲普瑞林诱导的NFκB激活，用NFκB分泌型碱性磷酸酶报告基因质粒（pNFκB-SEAP）转染细胞，并在有或无曲普瑞林的情况下培养96小时。使用SN50（一种活化NFκB核转位的抑制剂）研究曲普瑞林诱导的NFκB激活与曲普瑞林诱导的抗凋亡保护之间的因果关系。从未观察到曲普瑞林诱导的细胞凋亡。用阿霉素（1 nmol/L）处理72小时后，EFO - 21和EFO - 27卵巢癌细胞系中凋亡细胞的百分比分别增加到31%或34%。在同时用曲普瑞林（100 nmol/L）处理的培养物中，凋亡细胞的百分比显著降低，分别降至17%或18%（P < 0.001）。RT-PCR和免疫印迹实验表明，卵巢癌细胞系EFO - 21和EFO - 27表达NFκB亚基p50和p65。当用pNFκB-SEAP转染EFO - 21或EFO - 27细胞，随后用曲普瑞林（100 nmol/L）处理时，NFκB诱导的SEAP表达分别增加5.3倍或4.7倍（P < 0.001）。曲普瑞林诱导的阿霉素诱导的细胞凋亡减少被SN50介导的NFκB核转位抑制所阻断。我们得出结论，LHRH诱导NFκB激活，从而减少阿霉素诱导的人卵巢癌细胞凋亡。除了对有丝分裂途径的抑制干扰外，这种保护卵巢癌细胞免于程序性细胞死亡的可能性是卵巢肿瘤中LHRH信号传导的一个重要特征。
