丁酸钠通过自噬途径降解HIF-1α抑制结直肠癌细胞生长  

Sodium butyrate inhibits the growth of colorectal cancer cells by promoting autophagic degradation of HIF-1α

作　　者：张秋玉 卞中博 孙小蝶 秦勇 刘华缓 毛联智 孙素霞 ZHANG Qiuyu;BIAN Zhongbo;SUN Xiaodie;QIN Yong;LIU Huahuan;MAO Lianzhi;SUN Suxia(Department of Nutrition and Food Hygiene,School of Public Health,Southern Medical University,Guangdong Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院营养与食品卫生学系,广东广州510515

出　　处：《现代肿瘤医学》2022年第5期762-767,共6页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:81773429)。

摘　　要：目的:研究丁酸钠(sodium butyrate,NaB)对结直肠癌(colorectal cancer,CRC)细胞生长的影响及可能机制。方法:不同浓度NaB(0、1、2、5、10 mmol/L)处理结直肠癌HCT116细胞24 h后,MTT和平板克隆实验检测细胞生长情况,实时荧光定量PCR和Western blot检测缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和乳酸脱氢酶A(lactate dehydrogenase-A,LDHA)的表达。3-甲基腺嘌呤(3-methyladenine,3-MA)或氯喹(chloroquine,CQ)抑制自噬后NaB处理HCT116细胞,Western blot检测LC3Ⅱ蛋白表达。环己酰亚胺(cycloheximide,CHX)抑制蛋白质合成后NaB处理HCT116细胞,Western blot检测HIF-1α蛋白表达。蛋白酶体抑制剂MG132、氯化钴(cobalt chloride,CoCl2)或CQ抑制泛素蛋白酶体途径和自噬后NaB处理HCT116细胞,Western blot检测HIF-1α和LDHA的表达,MTT检测细胞活力。结果:2 mmol/L NaB即可显著抑制HCT116细胞活力和克隆形成能力(P<0.01)、下调HIF-1α和LDHA的表达(P<0.01)。5 mmol/L3-MA可阻断LC3Ⅱ生成(P<0.05),抑制NaB对LC3Ⅱ的上调(P<0.05);50μmol/L CQ可促进LC3Ⅱ在细胞内的蓄积(P<0.01),上调NaB促发的LC3Ⅱ蛋白水平的表达(P<0.05)。200μmol/L CHX处理HCT116细胞5 h可下调HIF-1α的表达(P<0.05),联用NaB将下调HIF-1α的时间提前至1 h(P<0.01)。CQ逆转了NaB对HIF-1α、LDHA和细胞活力的抑制作用(P<0.05),MG132与CoCl2无法逆转(P>0.05)。结论:NaB通过自噬途径下调HIF-1α蛋白稳定性,进而抑制其下游信号LDHA表达来达到抑制结直肠癌细胞生长的目的。Objective:The purpose of this study was to explore the effect of sodium butyrate(NaB)on the growth of colorectal cancer cells and its possible mechanisms.Methods:HCT116 cells were treated with NaB(0,1,2,5,10 mmol/L)for 24 h.Cell viability and clonogenic ability were assessed with MTT assay and plate cloning experiment,respectively.Real-time PCR and Western blot were used to detect the expression of hypoxia inducible factor-1α(HIF-1α)and lactate dehydrogenase-A(LDHA).HCT116 cells were treated with 3-methyl adenine(3-MA),chloroquine(CQ),protein synthesis inhibitor(CHX),proteasome inhibitor(MG132)or cobalt chloride(CoCl;)and then expoured to 5 mmol/L NaB.The expression of LC3Ⅱand HIF-1αwere detected by Western blot.Cell viability were assessed with MTT assay.Results:In comparison with the control cells,2 mmol/L NaB significantly inhibited the viability and clonogenic ability of HCT116 cells(P<0.01),and down-regulated the expressions of HIF-1αand LDHA(P<0.01).The production of LC3Ⅱwas blocked by 5 mmol/L 3-MA(P<0.05),which inhibited the effect of NaB on LC3Ⅱ(P<0.05).Treatment with 50μmol/L CQ can promote the accumulation of LC3Ⅱ(P<0.01)and up-regulate the level of LC3Ⅱprotein induced by NaB(P<0.05).Treatment with 200μmol/L CHX for 5 h repressed HIF-1α(P<0.05),and NaB advanced the time of decreasing HIF-1αprotein level to 1 h(P<0.01).Treatment of HCT116 cells with CQ alleviated the inhibitory effects of NaB on HIF-1α,LDHA and cell viability(P<0.05),while MG132 and CoCl;did not affect the inhibitory effects of NaB on HCT116 cells(P>0.05).Conclusion:NaB down-regulates the stability of HIF-1αprotein through autophagy,and then inhibits the expression of its downstream protein LDHA,so as to inhibit the growth of colorectal cancer cells.

关 键 词：结直肠癌 丁酸钠 自噬 HIF-1Α 

分 类 号：R735.3[医药卫生—肿瘤]