The effect and mechanism of ursolic acid on reducing cholesterol abundant and inducing apoptosis in colorectal cancer
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摘要: 目的 基于蛋白组学探讨熊果酸抑制结直肠癌进展的可能生物学机制。方法 以结肠癌HCT-116细胞为研究对象，通过CCK-8法检测不同浓度熊果酸对HCT-116的增殖抑制率。收集熊果酸处理组和对照组样本进行蛋白质提取、蛋白质组学测序并筛选差异蛋白。对差异蛋白进行GO和KEGG富集分析，对目标通路进一步进行GSEA富集分析验证。IPA聚焦关键信号通路，Western blot、细胞内胆固醇含量检测、流式细胞术等实验验证IPA结果。结果 熊果酸在一定范围内呈浓度依赖抑制HCT-116细胞增殖，通过非线性拟合计算出熊果酸对HCT-116细胞的IC50、IC20、IC10分别为7.733、5.926、5.072 μg/mL。选取非毒性剂量1.6 μg/mL和3.2 μg/mL进行后续实验。非毒性剂量下，熊果酸依然呈时间依赖和浓度依赖地抑制HCT-116增殖。蛋白质组学发现对照组与熊果酸处理组之间存在468个差异蛋白，对差异蛋白进行GO和KEGG富集分析显示胆固醇代谢、自噬、铁死亡等生物进程被富集，GSEA进一步发现差异蛋白在胆固醇代谢通路上显著富集(标准化富集评分=1.76，P＜0.001)。IPA显示熊果酸显著激活LXR/RXR信号[Z-score=3.00，-log10(P)=9.49]。进一步实验验证结果显示，熊果酸能够上调LXR/RXR靶蛋白E3泛素连接酶MYLIP、三磷酸腺苷结合匣转运蛋白G1、载脂蛋白E的表达，降低细胞内总胆固醇含量，诱导细胞凋亡，胆固醇回补能够拮抗熊果酸所诱导的细胞凋亡。结论 熊果酸激活LXR/RXR信号增加靶蛋白E3泛素连接酶MYLIP、三磷酸腺苷结合匣转运蛋白G1、载脂蛋白E的表达，导致结直肠癌细胞内胆固醇含量降低，增殖被抑制，凋亡增加。Abstract: Objective To investigate the mechanism of ursolic acid(UA) suppresses colorectal cancer progression based on proteomics.Methods HCT-116 was selected as the study target, and the CCK-8 assay was used to detect the inhibition rates on HCT-116 after the treatment with different concentrations of UA. Extracted proteins from UA-treated and control cells were analyzed by proteomic analysis. Differential expression proteins(DEPs) were screened and then analyzed by GO and KEGG enrichment analysis. The target pathway was further verified by GSEA pathway enrichment analysis. Ingenuity pathway analysis(IPA) focused on key signaling pathways, and the results of IPA were verified by Western blot, measurement of intracellular total cholesterol level, and flow cytometry.Results UA inhibited HCT-116 cell proliferation in a dose-dependent manner. The IC50, IC20and IC10 of UA on HCT-116 were 7.733 μg/mL, 5.926 μg/mL and 5.072 μg/mL, respectively. Next, 1.6 μg/mL and 3.2 μg/mL UA were selected for follow-up experiments. UA inhibited HCT-116 cell proliferation in a time-and dose-dependent manner at non-toxic concentrations. Proteomics identified 468 DEPs between the control group and the UA-treated group. GO and KEGG enrichment analysis showed that the DEPs were associated with many biological processes such as cholesterol metabolism, autophagy, and ferroptosis. GSEA enrichment analysis further confirmed that the cholesterol metabolic pathway was related to the DEPs(NES=1.76, P < 0.001). IPA showed that UA significantly activated the LXR/RXR signaling(Z-score=3.00, -log10(P)=9.49). The verification experiment results showed that UA up-regulated the expression of LXR/RXR target proteins MYLIP, ABCG1, and apolipoprotein E(APOE). In addition, UA decreased the total cholesterol level and induced apoptosis in HCT-116 cells. Furthermore, cholesterol supplementation could antagonize UA-induced apoptosis.Conclusion UA activates LXR/RXR signaling and increases the expression of target proteins MYLIP, ABCG1 and APOE, which results in the decrease of cholesterol level, inhibition of proliferation, and increase of apoptosis in colorectal cancer cells.
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Key words:
- colorectal cancer /
- ursolic acid /
- cholesterol metabolism /
- LXR/RXR signaling
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