阿帕替尼下调NRF2/HO-1通路抑制卵巢癌CAOV-3细胞增殖与凋亡

Apatinib inhibits proliferation and apoptosis of ovarian cancer CAOV-3 cells by suppressing NRF2/HO-1 pathway

目的观察阿帕替尼对卵巢癌CAOV-3细胞增殖和凋亡的影响及可能作用机制。方法取对数生长期卵巢癌CAOV-3细胞,随机分为对照组和阿帕替尼组,分别给予0(对照)、5、10、20、40μmol·L^(-1)阿帕替尼处理。CCK-8法观察细胞增殖,平板克隆实验观察细胞克隆形成能力,Annexin V-FITC/PI双染法检测细胞凋亡,Western blot法检测凋亡相关蛋白Bcl-2、Bax、cleaved caspase-3蛋白及NRF2通路蛋白表达情况,并比较细胞内活性氧(ROS)和总谷胱甘肽(GSH)水平。结果与对照组相比,5~40μmol·L^(-1)阿帕替尼组细胞增殖抑制率升高(P<0.05),呈时间-浓度依赖性;1、5、10μmol·L^(-1)阿帕替尼组CAOV-3细胞克隆形成率显著降低(P<0.05)。与对照组相比,10、20μmol·L^(-1)阿帕替尼组凋亡率增加,促凋亡蛋白Bax和cleaved caspase-3蛋白表达升高、抗凋亡蛋白Bcl-2表达降低,细胞内GSH水平降低、ROS水平升高,核因子相关因子2(NRF2)及下游蛋白血红素加氧酶1(HO-1)的表达随阿帕替尼浓度增加而下降(均P<0.05),且阿帕替尼组间比较差异显著(P<0.05)。结论阿帕替尼可能通过下调NRF2/HO-1通路抑制CAOV-3细胞内GSH水平,促进ROS产生,并调控线粒体凋亡蛋白,发挥抑制增殖和诱导凋亡的作用。

AIM To observe the effects of apatinib on the proliferation and apoptosis of ovarian cancer CAOV-3 cells, and its possible mechanism. METHODS The ovarian cancer CAOV-3 cells in logarithmic growth phase were randomly divided into control group and apatinib group, and were treated with 0(control), 5, 10, 20, 40 μmol·L^(-1) apatinib, respectively. The CCK-8 method was used to detect the cell proliferation. The clone experiment was performed to observe the cloning ability of cells. The Annexin V-FITC/PI double staining method was performed to detect cell apoptosis and Western blot was used to observe the expression of apoptosis-related proteins Bcl-2, Bax, cleaved caspase-3 and proteins in NRF2 pathway. The intracellular reactive oxygen species(ROS) and total glutathione(GSH) levels were detected and compared. RESULTS Compared with the control group, the inhibition rates of cell proliferation in 5-40 μmol·L^(-1) apatinib group were increased(P<0.05), which were obviously time-concentration dependent. Compared with the control group, the CAOV-3 cell clone formation rate in the 10 and 20 μmol·L^(-1) apatinib group were decreased(P<0.05), the apoptosis rate was increased, and the expression levels of pro-apoptotic proteins Bax and cleaved caspase-3 protein were higher, while the expression level of anti-apoptotic protein Bcl-2 was lower(P<0.05). The intracellular GSH level was decreased and the level of ROS increased after apatinib treatment(P<0.05). The expressions of nuclear factor erythroid-derived 2-like 2(NRF2) and its downstream protein heme oxygenase 1(HO-1) were decreased with apatinib concentration increasing(P<0.05), and there were significant differences between the two apatinib groups(P<0.05). CONCLUSION Apatinib could inhibit proliferation of CAOV-3 cells by downregulating NRF2/HO-1 pathway, suppressing the level of GSH, increasing the level of ROS, and regulating mitochondrial apoptotic proteins to promote cell apoptosis.