金雀异黄素联合顺铂对SK-OV-3 人卵巢腺癌细胞生物学行为影响的研究

Effect of genistein combined with cisplatin on biological behavior of ovarian cancer SK-OV-3 cells

摘要:

目的 观察金雀异黄素联合顺铂对SK-OV-3人卵巢腺癌细胞生物学行为的影响，探讨其可能的作 用机制。方法 培养SK-OV-3人卵巢腺癌细胞，分别给予金雀异黄素（20 μmol/L、40 μmol/L、80 μmol/L、 160 μmol/L、200 μmol/L）、顺铂（1 μg/ml、2 μg/ml、4 μg/ml、8 μg/ml、16 μg/ml）单独作用24 h、48 h和72 h， 采用偶氮唑蓝（MTT）法检测细胞增殖情况，筛选合适的作用浓度和作用时间；SK-OV-3人卵巢腺癌细胞分为 空白组（含正常生长的SK-OV-3细胞并加入等量培养液）、金雀异黄素组、顺铂组和联合用药组。药物作用48 h 后，细胞划痕实验检测各组SK-OV-3细胞迁移能力；Hoechst33342荧光染色法检测各组SK-OV-3细胞核的形 态；流式细胞术检测各组细胞凋亡率；Western blotting 检测各组SK-OV-3 细胞PI3K、Akt、p-Akt、Bcl-2、 Bax、Caspase-3蛋白相对表达量。结果 MTT法检测结果显示，随着金雀异黄素、顺铂浓度的增加，作用时间 的延长，细胞增殖抑制率上升（P <0.05）。由此选定金雀异黄素160 μmol/L、顺铂4 μg/ml进行后续实验。细胞 划痕实验、Hoechst33342染色法、流式细胞术结果均证明，与空白组比较，金雀异黄素组、顺铂组及联合用药组 均可降低SK-OV-3细胞迁移能力，促进细胞凋亡（P <0.05），且联合用药组效果好于金雀异黄素组和顺铂组 （P <0.05）；Western blotting检测结果表明，与空白组比较，金雀异黄素组、顺铂组及联合用药组SK-OV-3细胞 p-Akt、PI3K及Bcl-2蛋白相对表达量、p-Akt/Akt比值均降低（P <0.05），Bax、Caspase-3蛋白相对表达量升高 （P <0.05）；联合用药组p-Akt、PI3K及Bcl-2蛋白相对表达量、p-Akt/Akt比值较金雀异黄素组、顺铂组均降低， Bax、Caspase-3蛋白相对表达量均升高（P <0.05）。结论 金雀异黄素对SK-OV-3人卵巢腺癌细胞增殖在一定 范围内具有抑制作用；金雀异黄素可协同顺铂促进SK-OV-3人卵巢腺癌细胞的凋亡作用。

Abstract:

Objective To observe the effect of genistein combined with cisplatin on the biological behavior of ovarian cancer SK-OV-3 cells, and to confirm that genistein and cisplatin have synergistic effects, which can promote the apoptosis of ovarian cancer cells and inhibit its proliferation and migration. Methods Ovarian cancer SK-OV-3 cells were cultured and treated with genistein (20, 40, 80, 160, 200 μmol/L) and cisplatin (1, 2, 4, 8, 16 μg/ml) for 24, 48, 72 h, respectively. The cell proliferation was detected by MTT, and the appropriate concentration and duration were screened. Ovarian cancer SK-OV-3 cells were divided into 4 groups: blank group (including the normal growth of SK-OV-3 cells and join the amount of nutrient solution) and the experimental group (genistein group, cisplatin group, genistein combined with cisplatin). After 48 hours of drug treatment, the Cell Scratch Test was conducted to determine the effect of single or combined treatment of genistein and cisplatin on SK-OV-3 cell migration; Hoechst33342 was used to detect the effect of each group of drugs on the morphology of ovarian cancer SK-OV-3 nucleus; Annexin V/PI was used to detect apoptosis; Western blotting analysis was used to detect protein expression levels of PI3K, Akt, p-Akt, Bcl-2, Bax and Caspase-3. Results The results showed that the inhibition rate of cell proliferation increased with the increase of genistein and cisplatin concentration (P < 0.05). The contration was selected as 160 μmol/L for genistein and 4 μg/ml for cisplatin. The results of cell score test, Hoechst33342 and flow cytometry showed that, compared with the control group, genistein alone and combined with cisplatin could significantly reduce SK-OV-3 cell migration ability and promote cell apoptosis (P < 0.05), and the results of combination therapy were significantly superior to those of single therapy (P < 0.05). Western blotting results showed that, compared with the control group, the protein expressions of p-Akt, PI3K and Bcl-2 were downregulated and the protein expressions of Bax and Caspase-3 were upregulated in single treatment and combined treatment group, the ratio of p-Akt protein to Akt protein decreased, and the results of combined treatment were more significant than those of single treatment (P < 0.05). Conclusions Genistein has inhibition on SK-OV-3 cell proliferation in ovarian cancer within limits, which is dose-time dependent. Genistein and cisplatin can promote apoptosis of ovarian cancer cells.