Objective: To investigate the effect of MACC1 on RSL3-induced ferroptosis in colorectal cancer cells and explore its molecular mechanism.
Methods: MACC1 expression was detected in SW620, HCT116, LOVO and RKO cells using Western blotting. The effects of different concentrations of RSL3 (an inducer of ferroptosis) or Fer-1 (an inhibitor of ferroptosis) alone, or 10 μmol/L RLS3 combined with 10 μmol/L Fer-1, on viability of SW620 cells were examined using MTT assay. The survival of SW620 cells with mRNA interference of MACC1 was analyzed following treatment with RSL3, and RT-qPCR and Western blotting were performed to detect the changes in MACC1 expressions after RSL3 treatment at different concentrations and the changes in GPX4 expression after MACC1 knockdown. Flow cytometry and laser confocal microscopy were used to analyze the changes in ROS-induced lipid peroxidation in SW620 cells after MACC1 knockdown.
Results: SW620 cells had the highest MACC1 expression among the 4 colorectal cancer cell lines. Treatment with RSL3 significantly inhibited the viability of SW620 cells in a dose-dependent manner, while Fer-1 did not significantly affect the survival of SW620 cells. RSL3 alone reduced SW620 cell survival by 50% (P < 0.01), and the combined treatment with RSL3 and Fer-1 caused no significant changes in cell survival (P > 0.05). Treatment with RSL3 concentration-dependently suppressed MACC1 expressions at both the mRNA and protein levels in SW620 cells (P < 0.01). MACC1 knockdown obviously enhanced the cytotoxic effect of RSL3, inhibited the expression of GPX4, and increased ROS-induced lipid peroxidation in SW620 cells (P < 0.05).
Conclusion: MACC1 knockdown enhances RSL3-induced ferroptosis in cultured colorectal cancer cells by inhibiting the expression of GPX4.
目的: 探讨结肠癌转移相关基因1(MACC1)对RAS选择性致死化合物3(RSL3)诱导的结直肠癌细胞铁死亡的影响及其分子机制。
方法: 体外培养结直肠癌细胞SW620，HCT116，LOVO及RKO细胞，Western blot实验检测细胞中MACC1表达；以SW620细胞为实验材料，MTT法检测不同浓度(0、2.5、5、10、20、40 μmol/L)铁死亡诱导剂RSL3以及不同浓度(0、5、10、20 μmol/L)铁死亡抑制剂Fer-1对SW620细胞存活率的影响，分析单独使用10 μmol/L RLS3以及10 μmol/L RLS3和10 μmol/L Fer-1联合作用对SW620细胞存活率的影响，并检测干扰MACC1后不同浓度RSL3对SW620细胞存活率的影响；实时定量RT-PCR和Western blot法检测不同浓度RSL3(0、2.5、5、10 μmol/L)对MACC1在mRNA和蛋白水平的影响，并检测干扰MACC1后GPX4在mRNA和蛋白水平的表达；流式细胞仪及激光共聚焦实验检测干扰MACC1后，SW620细胞中脂质过氧化Lipid ROS水平的变化。
结果: 4种结直肠癌细胞中SW620细胞MACC1表达量最高；铁死亡诱导剂RSL3抑制SW620细胞的存活率，细胞存活率随RSL3浓度升高而降低，呈剂量依赖性；不同浓度的铁死亡抑制剂Fer-1对SW620细胞的存活率没有影响；与对照Ctrl组细胞存活率相比，单独使用RSL3细胞存活率降低了50%(P < 0.01)，而联合使用RSL3与Fer-1处理SW620细胞，细胞的存活率得到恢复(P > 0.05)；不同浓度的RSL3作用于SW620细胞后，细胞中MACC1基因在mRNA和蛋白水平被显著抑制(P < 0.01)，具有一定的药物浓度依赖性。siRNA干扰MACC1基因表达后，增强RSL3对SW620细胞毒作用，并抑制细胞中GPX4的表达(P < 0.01)，增加细胞中Lipid ROS水平(P < 0.05)。
结论: MACC1通过调控GPX4影响RSL3诱导的结直肠癌细胞铁死亡。
Keywords: RAS-selective lethal 3; ferroptosis; glutathione peroxidase 4; metastasis-associated in colon cancer 1.