Breast cancer is associated with a high rate of recurrence, resistance therapy and mortality worldwide. We aimed at investigating the inhibitory effects of Sulindac and vitamin D3 (VD) on MCF-7 human breast cancer cells. MCF-7 cells were cultured with different concentrations of Sulindac and VD over a period of 24, 48 and 72 hours for cell viability and IC50 experiments. Hochst staining was used to evaluate apoptosis, whereas quantitative PCR (qPCR) was performed to measure mRNA levels of BCL-2 and BAX genes. Immunofluorescence staining was used to monitor intracellular β-catenin expression. The protein levels of AKT, AMPK and P65 were measured by western blotting. The result showed that cell viability decreased in treated cells dose/time dependently (P < .05). Hochst staining showed an increase in fragmented nuclei in treated cells. The expression of BCL-2 and BAX genes decreased and increased in treated cells, respectively (P < .05). Immunofluorescence staining indicated that the expression of β-catenin significantly reduced in treated cells. The AKT-1/p-Akt-1 and AMPK/p-AMPK ratio increased in treated cells (P < .05), but the P65/p-P65 ratio did not change significantly (P > .05). Our results indicated that the combination of Sulindac and VD has a growth-inhibiting effect on MCF-7 cells through AMPK/Akt/β-catenin axis.

中文翻译：

---

舒林酸和维生素D3体外通过AMPK/Akt/β-catenin轴协同抑制MCF-7乳腺癌细胞增殖

乳腺癌在全球范围内与高复发率、耐药性治疗和死亡率有关。我们旨在研究舒林酸和维生素 D3 (VD) 对 MCF-7 人乳腺癌细胞的抑制作用。MCF-7 细胞与不同浓度的舒林酸和 VD 一起培养 24、48 和 72 小时，用于细胞活力和 IC50 实验。Hochst 染色用于评估细胞凋亡，而定量 PCR (qPCR) 用于测量 BCL-2 和 BAX 基因的 mRNA 水平。免疫荧光染色用于监测细胞内β-连环蛋白的表达。通过蛋白质印迹测量AKT、AMPK和P65的蛋白质​​水平。结果表明，处理细胞的细胞活力呈剂量/时间依赖性降低（P < .05）。Hochst 染色显示处理过的细胞中破碎的细胞核增加。BCL-2 和 BAX 基因的表达分别在处理过的细胞中减少和增加（P  < .05）。免疫荧光染色表明β-catenin的表达在处理细胞中显着降低。AKT-1/p-Akt-1 和 AMPK/p-AMPK 比值在处理细胞中增加（P  < .05），但 P65/p-P65 比值没有显着变化（P  > .05）。我们的结果表明，舒林酸和 VD 的组合通过 AMPK/Akt/β-catenin 轴对 MCF-7 细胞具有生长抑制作用。