Wang Shunde, Wang Shuhong, Li Hang, Li Xiaoxia, Xie Menglin, Wen Jiayu, Li Meicai, Long Tengbo
Department of Andrology, Chongqing Three Gorges Central Hospital, Chongqing 404000, P.R. China.
Exp Ther Med. 2018 Jun;15(6):5269-5274. doi: 10.3892/etm.2018.6081. Epub 2018 Apr 20.
The molecular mechanism of the aromatase inhibitor letrozole was investigated. It promotes the proliferation of spermatogonia by regulating the mitogen-activated protein kinase (MAPK) pathway. Six different concentrations were selected for letrozole in order to incubate mouse spermatogonia [GC-1 spermatogonia (spg)] for 24, 48 and 72 h, respectively. Cell Counting Kit-8 (CCK-8) was used to observe the effect of letrozole on the proliferation of GC-1 spg cells, and the effect was further verified by cell plate clone formation assay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the effects of letrozole on MAPK signaling pathways [Ras/extracellular signal-regulated kinase 1 (ERK1)/c-Myc], proliferation indexes [Ki-67 and proliferating cell nuclear antigen (PCNA)]. Bromodeoxyuridine (BrdU) staining was used to study the effects of letrozole and MAPK signaling pathways on cell proliferation. The results of CCK-8 showed that the proliferation rate of GC-1 spg cells was improved. Study results also revealed a significant increase in letrozole concentration along with the time of action. The results of plate clone formation assay further indicated that letrozole could significantly promote the proliferation capacity of GC-1 spg cells (p<0.05). The results of RT-PCR and western blot analysis confirmed letrozole significantly activated the expression of Ras/ERK1/c-Myc in the classical MAPK pathway. A significant increase was noted in the protein levels of Ki-67 and PCNA (p<0.05). By contrast, inhibition of the MAPK pathway resulted in a significant decrease in the levels of the above indexes (p<0.05). The number of BrdU cells in the letrozole group was also higher than that of the control group, while the number of BrdU-stained cells in the letrozole + MAPK inhibition group showed a significant decrease in comparison to the letrozole group. In conclusion, letrozole activated the MAPK signaling pathway and promoted the proliferation of mouse spermatogonia GC-1 spg cells. The present study provides a theoretical basis for the clinical application of letrozole.
研究了芳香化酶抑制剂来曲唑的分子机制。它通过调节丝裂原活化蛋白激酶（MAPK）途径促进精原细胞的增殖。选择六种不同浓度的来曲唑分别孵育小鼠精原细胞[GC-1精原细胞（spg）]24、48和72小时。使用细胞计数试剂盒-8（CCK-8）观察来曲唑对GC-1 spg细胞增殖的影响，并通过细胞平板克隆形成试验进一步验证该效果。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析来检测来曲唑对MAPK信号通路[Ras/细胞外信号调节激酶1（ERK1）/c-Myc]、增殖指标[Ki-67和增殖细胞核抗原（PCNA）]的影响。使用溴脱氧尿苷（BrdU）染色来研究来曲唑和MAPK信号通路对细胞增殖的影响。CCK-8结果显示GC-1 spg细胞的增殖率提高。研究结果还显示来曲唑浓度随作用时间显著增加。平板克隆形成试验结果进一步表明来曲唑可显著促进GC-1 spg细胞的增殖能力（p<0.05）。RT-PCR和蛋白质印迹分析结果证实来曲唑显著激活经典MAPK途径中Ras/ERK1/c-Myc的表达。Ki-67和PCNA的蛋白水平显著升高（p<0.05）。相比之下，抑制MAPK途径导致上述指标水平显著降低（p<0.05）。来曲唑组的BrdU细胞数量也高于对照组，而来曲唑+MAPK抑制组的BrdU染色细胞数量与来曲唑组相比显著减少。总之，来曲唑激活MAPK信号通路并促进小鼠精原细胞GC-1 spg细胞的增殖。本研究为来曲唑的临床应用提供了理论依据。
