Wu Sheng-Tang, Ku Wei-Chi, Huang Chi-Jung, Wang Yen-Chieh, Lin Chih-Ming, Chen Shao-Kuan
Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei, Taiwan.
J Biomed Sci. 2016 Sep 29;23(1):67. doi: 10.1186/s12929-016-0282-z.
Renal cell carcinoma (RCC) is an adult malignancy with 2:1 men-to-women ratio, which implies the possible role of sex hormones in RCC carcinogenesis. One of the predominant sex hormones in women before menopause, 17-β-estradiol (or E2), may regulate RCC growth by cellular mechanisms that are still not fully understood.
The expression levels of E2 receptors (ER1 and ER2) were determined in different RCC cell lines. The DNA damage response induced by E2 was determined by a DNA double-strand break marker γH2AX. To study the possible effect of E2 on oxidative stress response, RCC cells were stained with 2,7-dichlorofluorescein diacetate and analyzed by flow cytometry. Upregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) ser40 phosphorylation in response to oxidative stress was detected by immunoblotting. Finally, annexin V/propidium iodide (PI) double staining assay was used to determine E2-induced cellular apoptosis.
Variable expression of ER1 and ER2 were found in the RCC cell lines studied (786-O, A498, and ACHN), in which ACHN and A498 showed highest and lowest ER expression, respectively. In A498 cells, E2 induced DNA double-strand breaks with positive staining of γH2AX. On the other hand, the level of reactive oxidative species were elevated in ACHN cells after E2 treatment. The E2-induced oxidative stress also induced the Ser40 phosphorylation and nuclear translocation of Nrf2. Finally, we also demonstrated that E2 induced apoptosis as revealed by annexin V/PI double staining.
In this study, we demonstrated the cellular effects of E2 on DNA repair, ROS production as well as Nrf2 activation, and apoptosis in RCC cell lines. Together these cellular alterations may contribute to the reduced viability of RCC cells following E2 treatment.
肾细胞癌（RCC）是一种成人恶性肿瘤，男女比例为2:1，这暗示了性激素在RCC致癌过程中可能发挥的作用。绝经前女性体内主要的性激素之一17-β-雌二醇（或E2），可能通过尚未完全明确的细胞机制调节RCC的生长。
测定不同RCC细胞系中E2受体（ER1和ER2）的表达水平。通过DNA双链断裂标志物γH2AX测定E2诱导的DNA损伤反应。为研究E2对氧化应激反应的可能影响，用二乙酸2,7-二氯荧光素对RCC细胞进行染色，并通过流式细胞术进行分析。通过免疫印迹检测氧化应激反应中核因子（红细胞衍生2）样2（Nrf2）丝氨酸40磷酸化的上调情况。最后，采用膜联蛋白V/碘化丙啶（PI）双染法测定E2诱导的细胞凋亡。
在所研究的RCC细胞系（786-O、A498和ACHN）中发现ER1和ER2的表达存在差异，其中ACHN和A498分别显示出最高和最低的ER表达。在A498细胞中，E2诱导DNA双链断裂，γH2AX染色呈阳性。另一方面，E2处理后ACHN细胞中的活性氧水平升高。E2诱导的氧化应激还诱导了Nrf2的丝氨酸40磷酸化和核转位。最后，我们还通过膜联蛋白V/PI双染法证明E2诱导了细胞凋亡。
在本研究中，我们证明了E2对RCC细胞系中DNA修复、活性氧产生、Nrf2激活以及细胞凋亡的细胞效应。这些细胞改变共同可能导致E2处理后RCC细胞活力降低。
