丙戊酸钠诱导人结肠癌细胞HT-29自噬及其机制研究

Valproate acid sodium-induced autophagy of human colon cancer cell line HT-29 and its possible mechanism

目的对丙戊酸钠诱导人结肠癌细胞HT-29发生自噬性死亡的过程进行观察,并初步探讨其可能的机制。方法用不同浓度、不同作用时间的丙戊酸钠处理人结肠癌细胞系HT-29,用细胞计数试剂盒CCK8法观察其对细胞增殖的抑制作用,丹酰戊二胺染色法观察细胞自噬,荧光测试仪检测自噬水平,荧光定量PCR法检测以下基因表达水平的变化:微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)、自噬相关基因Beclin-1、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化蛋白激酶B(p-Akt)和磷酸化核糖体蛋白S6激酶(p-p70S6K)。结果随着丙戊酸钠处理浓度的递增及作用时间的延长,其对人结肠癌细胞HT-29的增殖抑制率逐渐提高,呈剂量依赖性及时间依赖性(P均﹤0.01);同时,随着药物浓度的增加,细胞中自噬泡数量增多,荧光强度增强,与对照组比较,差异有统计学意义(P均<0.05)。经丙戊酸钠处理后,自2 mmol/L浓度始,人结肠癌细胞系HT-29自噬相关基因LC3-Ⅱ和Beclin-1表达水平明显升高(P均<0.01),而mTOR、p-Akt和p-p70S6K表达水平明显降低(P均<0.01)。结论丙戊酸钠可诱导结肠癌细胞发生自噬,其诱导结肠癌细胞发生自噬的机制可能与阻断mTOR-Akt信号转导通路及激活Beclin-1信号转导通路有关。

Objective To observe the process of autophagic death of human colon cancer cell line HT-29 induced by valproate acid sodium （VPA） and explore its potential mechanism. Methods Human colon cancer cell line HT-29 was treated with different concentrations of VPA and different time. The inhibiting effect of VPA on cell proliferation was assessed by cell counting kit-8 （CCK-8） ; the cell autophagy was observed by monodansylcadaver- ine （MDC） staining; the level of autophagy was detected by fluorescence tester;The fluorogenic quantitative poly- merase chain reaction （PCR） were used to measure the expression levels of genes [including：microtubule-associated protein light chain 3 Ⅱ （ LC3-Ⅱ ）, autophagy-related gene Beclin-1, mammalian target of rapamycin （ roTOR）, phos- phorylated protein kinase B （p-Akt） and phosphorylation of 1）70 ribosomal protein S6 kinase（p-p70S6K） ]. Results Along with the ascending increase of VPA concentrations and lengthen of action time, the inhibition rates of VAP on HT-29 proliferation increased gradually with dose-dependent manner and time-dependent manner （all P 〈 0.01 ） ; along with the increase of VPA concentrations, the number of autophagie vacuoles increased and fluorescence inten- sity enhanced compared with control group （ all P 〈 0.05 ）. After treated with VPA, the expression levels of HT-29 autophagy-related genes LC3- Ⅱ and Beelin-1 elevated significantly （ all P 〈 0. 01 ）, while the expression levels of mTOR, p-Akt and p-p70S6K declined significantly （ all P 〈 0.01 ）. Conclusions VPA may induce autophagy in colon cancer cell line HT-29, and its mechanism might be related to the disruption of mTOR-Akt signaling pathway and the activation of beclin-1 signaling pathway.