姜黄素调节p53/AMPK信号通路对子宫内膜癌细胞系生物学行为的影响

Effects of curcumin on proliferation, migration, invasion and apoptosis of endometrial carcinoma HEC-1-B cells through p53/AMPK signaling pathway

关键词:  姜黄素  腺苷酸活化蛋白激酶  腺苷酸活化蛋白激酶抑制剂  子宫内膜癌

Key words:  curcumin  AMPK  compound C  endometrial cancer

摘要:

目的 探究姜黄素对人子宫内膜癌细胞系（HEC-1-B）的增殖、迁移、侵袭及凋亡的影响及其作用机制。方法 首先，用不同浓度的姜黄素（0、5、10、20、40 μmol/L）处理HEC-1-B细胞，噻唑蓝（MTT）法检测对细胞的增殖率；将HEC-1-B细胞分为：空白对照组、腺苷酸活化蛋白激酶（AMPK）抑制剂（compound C）组（10 μmol/L）、姜黄素组（40 μmol/L）以及姜黄素（40 μmol/L）+compound C（10 μmol/L）组，药物作用时间均为48 h，通过MTT法检测HEC-1-B细胞的增殖，划痕实验和Transwell实验分别检测HEC-1-B细胞的迁移和侵袭，流式检测HEC-1-B细胞的凋亡，实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测HEC-1-B细胞中Caspase-3、Bcl-2、Bax mRNA相对表达量，蛋白免疫印迹（Western blot）检测HEC-1-B细胞中cleaved-Caspase 3、Bcl-2、Bax以及AMPK信号通路蛋白的表达情况。结果 与空白对照组相比，5 μmol/L组中HEC-1-B细胞增殖率没有显著性改变（P>0.05），10、20和40 μmol/L组细胞增殖率均下降（P < 0.05），并呈时间-剂量依赖性；与对照组相比，姜黄素组细胞增殖、迁移和侵袭能力均降低而凋亡升高（P < 0.05），Caspase-3、Bax表达量升高而Bcl-2表达量降低（P < 0.05），同时AMPK和p53磷酸化水平也升高（P < 0.05）；compound C组中细胞增殖、迁移、侵袭均升高而凋亡降低（P < 0.05），Caspase-3、Bax表达量下降而Bcl-2表达升高（P < 0.05），p-AMPK和p-p53蛋白表达量降低（P < 0.05）；姜黄素+compound C组中无论细胞的生物学性状及各蛋白表达情况无明显变化（P>0.05）。结论 姜黄素可能通过p53/AMPK信号通路影响子宫内膜癌HEC-1-B细胞增殖迁移、侵袭和凋亡等生物学功能。

Abstract:

Objective Exploring the biological effects of curcumin on human endometrial cancer cell line HEC-1-B by affecting the AMPK signaling pathway. Methods Firstly, HEC-1-B cells were treated with different concentrations of curcumin(0, 5, 10, 20, 40 μmol/L), and the inhibition rate of cell proliferation was detected by MTT assay. Divided HEC-1-B cells into control group(untreated) and compound C group(10 μmol/L), Curcumin group(20 μmol/L) and curcumin(20 μ mol/L)+compound C(10 μmol/L group), the drug action time was 48 hours. The proliferation of HEC-1-B cells was detected by MTT method, and the migration and invasion of HEC-1-B cells were detected by scratch assay and transwell, respectively. The apoptosis of HEC-1-B cells was detected by flow cytometry. The expression levels of caspase 3, Bcl-2, Bax mRNA in HEC-1-B cells were detected by RT-PCR, and the expression of caspase 3, Bcl-2, Bax, and AMPK signaling pathway proteins in HEC-1-B cells were detected by Western blot. Results Compared with 0 μmol/L, the proliferation inhibition rate of 5 μmol/L curcumin was not significant(P>0.05). The proliferation inhibition rates of 10 μmol/L, 20 μmol/L, and 40 μmol/L curcumin were significantly increased(P < 0.05) and showed a dose-dependent effect; Compared with the control group, the curcumin group showed a significant decrease in cell proliferation, migration, and invasion(P < 0.05), a significant increase in apoptosis(P < 0.05), an increase in caspase 3 and Bax expression(P < 0.05), and a decrease in Bcl-2 expression(P < 0.05);The expression of p-AMPK and p-p53 proteins increased(P < 0.05);In the compound C group, cell proliferation, migration, and invasion were significantly increased(P < 0.05), apoptosis was significantly reduced(P < 0.05), caspase 3 and Bax expression were reduced(P < 0.05), and Bcl-2 expression was increased(P < 0.05);The expression of p-AMPK and p-p53 proteins decreased(P < 0.05);There was no significant change in various indicators of cells in the curcumin+compound C group(P>0.05). Conclusion Curcumin affects the proliferation, migration, invasion, and apoptosis of endometrial cancer HEC-1-B cells by interfering with the p53/AMPK signaling pathway.