CXC195通过抑制PI3K-AKT-mTOR信号通路和激活内质网应激调控人肝癌细胞增殖及凋亡

发布时间：2018-06-28 12:32

本文选题：CXC195 + 肝癌细胞　；参考：《南昌大学》2016年博士论文

【摘要】：目的:研究CXC195在体外诱导HepG2细胞凋亡并抑制细胞增殖。探讨PI3K-AKT-mTOR信号通路和内质网应激共同诱导HepG2细胞凋亡。为CXC195治疗肝癌提供帮助。材料和方法:体外培养HepG2细胞株作为实验组,对照组使用L-02细胞株。化学反应合成CXC195。联合台盼蓝拒染法和MTT比色法检测CXC195分别作用HepG2细胞和L-02细胞后细胞的存活情况,流式细胞术分析细胞凋亡,检测浓度为10μM,25μM,50μM,100μM,150μM,200μM的CXC195作用于HepG2细胞和L-02细胞的平均生长存活率。用Western blotting检测150μM CXC195作用HepG2细胞后,PI3K-AKT-mTOR信号通路PI3K、AKT、mTOR及磷酸化蛋白和内质网应激相关蛋白表达变化。结果:(1)CXC195抑制HepG2细胞生长:台盼蓝拒染法和MMT比色法检测细胞存活结果显示,加入CXC195后,人肝癌细胞(HepG2)和正常肝细胞(L-02)的生长存活率均明显降低。并且,随着CXC195剂量从10μM逐渐增加到200μM,HepG2细胞存活率从97.3%逐渐降到42.9%,L-02细胞存活率从98.5%逐渐降到65.1%。不同剂量CXC195作用相同细胞生长存活率下降有显著差异(P0.01),相同剂量CXC195作用于两组细胞比较,存活的肝癌细明显少于正常肝细胞L-02。MMT比色法检测结果表明,用150μMCXC195处理HepG2细胞12、24、48、72小时,细胞存活率也明显依次下降(P0.01)。流式细胞术检测发现,150μM CXC195作用HepG2细胞24小时后,细胞存活于G0/G1期较多(71.62%vs45.17%,P0.01),S期(17.53%vs41.11,P0.01)和G2/M期(10.85%vs13.72%,P0.05)细胞比例明显减少。结果表明:与正常肝细胞(L-02)相比,CXC195能更多抑制肝癌细胞(HepG2)生长,CXC195对肝癌细胞生长的抑制作用与药物浓度和药物作用时间呈正相关。并且CXC195主要作用于S期和G2/M期肝癌细胞HepG2。(2)CXC195诱导HepG2细胞凋亡:AnnexinV/PI双染色法标记后,通过流式细胞仪检测发现,各浓度CXC195诱导人肝癌细胞(HepG2)凋亡率明显高于正常肝细胞(L-02)(P0.01)。随着CXC195剂量从10μM逐渐增加到150μM,HepG2细胞凋亡率从5.04%逐渐上升到35.21%,而L-02细胞凋亡率从4.03%逐渐上升到19.30%。随着CXC195剂量增加,HepG2细胞凋亡数量明显增多(P0.01),相同剂量CXC195作用于两组细胞比较,HepG2细胞的凋亡率明显高于对照组L-02细胞。结果表明:CXC195诱导肝癌细胞(HepG2)凋亡作用显著强于正常肝细胞(L-02),且随着药物浓度增加,诱导凋亡作用逐渐增强。(3)CXC195对HepG2肝癌细胞PI3K-AKT-mTOR信号通路的影响:采用Western blotting分析,浓度为150μΜ的CXC195作用HepG2细胞,Bcl-2/Bax比值、p-PI3K和PI3K、p-AKT和AKT及p-mTOR和mTOR的表达量均非常明显少于未加CXC195对照组(P0.01)。联合使用通路上PI3K、AKT、mTOR对应的抑制剂LY294002,SH-6和雷帕霉素处理5μmol/L后,细胞凋亡率超过50%,同时通路相关蛋白p-PI3K和PI3K、p-AKT和AKT及p-mTOR和mTOR的表达量较单用150uM CXC195均非常明显减少(P0.01)。结果表明:CXC195通过抑制PI3K-AKT-mTOR信号传导通路相关蛋白诱导HepG2细胞凋亡,降低HepG2细胞增殖。(4)采用Western blotting检测发现,在经过浓度为150μΜCXC195作用后,HepG2细胞中GRP78,GRP94、CHOP、p-PERK,PERK,p-eIF2α,eIF2α、ATF4、IRE1α,p-ASK,ASK,p-p38,p38,p-JNK、JNK、Cleaved ATF6和ATF6表达量明显高于对照组。说明了内质网应激参与了CXC195诱导的HepG2细胞凋亡过程。结论:CXC195能显著抑制HepG2肝癌细胞增殖,诱导细胞凋亡,主要针对S期和G2/M期细胞。而对正常肝细胞(L-02)抑制生长和诱导凋亡的作用则明显较弱。CXC195的这一作用可能是通过抑制PI3K-AKT-mTOR信号通路蛋白表达和激活内质网应激来实现的。这些研究结果提示CXC195在体外对肝癌细胞有一定治疗效果。
[Abstract]:Objective: To study the apoptosis of HepG2 cells induced by CXC195 in vitro and to inhibit cell proliferation in vitro. To explore the PI3K-AKT-mTOR signaling pathway and endoplasmic reticulum stress to induce apoptosis of HepG2 cells. To provide help for CXC195 in the treatment of liver cancer. Materials and methods: in vitro culture, HepG2 cell lines were used as experimental group, and L-02 cell lines were used to synthesize CXC195. by chemical reaction. The survival of HepG2 cells and L-02 cells was detected by CXC195 and MTT colorimetric assay, and the cell apoptosis was analyzed by flow cytometry. The detection concentration was 10 mu M, 25 mu M, 50 mu M, 100 mu M, 150 micron, and 200 micron M CXC195 acted on the average growth survival rate of HepG2 cells and cells. 150 mu The expression of PI3K, AKT, mTOR and phosphorylated protein and endoplasmic reticulum stress related protein expression in PI3K-AKT-mTOR signaling pathway after the action of XC195 on HepG2 cells. Results: (1) CXC195 inhibits HepG2 cell growth: trypan blue stain method and MMT colorimetric assay show cell survival results, after adding CXC195, human hepatoma cells (HepG2) and normal hepatocyte (L-02) birth The long survival rate decreased significantly, and as the dose of CXC195 increased from 10 to 200 M, the survival rate of HepG2 cells decreased from 97.3% to 42.9%. The survival rate of L-02 cells decreased from 98.5% to 65.1%. at different doses of CXC195, and there was a significant difference in the survival rate of the same cells (P0.01). The same dose of CXC195 was used in the two groups of cells. The survival rate of liver cancer was less than that of normal hepatocyte L-02.MMT colorimetric assay. The results showed that the survival rate of HepG2 cells was decreased in 12,24,48,72 hours with 150 mu MCXC195 (P0.01). Flow cytometry found that the cells survived more G0/G1 (71.62%vs45.17%, P0.01) after 24 hours of HepG2 cells with 150 M CXC195. The proportion of phase (17.53%vs41.11, P0.01) and G2/M (10.85%vs13.72%, P0.05) cells decreased significantly. The results showed that CXC195 could inhibit the growth of hepatoma cells (HepG2) more than normal hepatocytes (L-02). The inhibitory effect of CXC195 on the growth of hepatoma cells was positively correlated with the drug concentration and the time of drug action. And CXC195 was mainly used in S phase and G2. HepG2. (2) CXC195 induced apoptosis of HepG2 cells in phase /M: after AnnexinV/PI double staining, the apoptosis rate of human hepatoma cells (HepG2) induced by CXC195 was significantly higher than that of normal liver cells (L-02) (P0.01). The apoptosis rate of CXC195 was gradually increased from 5.04% to 150 micron. Up to 35.21%, while the apoptosis rate of L-02 cells increased from 4.03% to 19.30%., with the increase of CXC195 dose, the number of apoptotic HepG2 cells increased significantly (P0.01). The same dose of CXC195 acted on the two groups of cells, and the apoptosis rate of HepG2 cells was significantly higher than that of the control group L-02 cells. The results showed that the apoptosis effect of CXC195 induced hepatoma cells (HepG2) was significant. Stronger than normal hepatocytes (L-02), and with the increase of drug concentration, the induction of apoptosis gradually increased. (3) the effect of CXC195 on the PI3K-AKT-mTOR signaling pathway of HepG2 hepatoma cells: Western blotting analysis, the CXC195 action of HepG2 cells with a concentration of 150 micron, Bcl-2/Bax ratio, p-PI3K and PI3K. The apoptosis rate of PI3K, AKT, mTOR corresponding to LY294002, SH-6 and rapamycin treated with 5 u mol/L was less than that of the control group (P0.01). The results show that CXC195 can induce HepG2 cell apoptosis by inhibiting PI3K-AKT-mTOR signal transduction pathway related proteins and reduce the proliferation of HepG2 cells. (4) Western blotting detection was used to detect GRP78, GRP94, CHOP, HepG2 cells in HepG2 cells. The expression of Cleaved ATF6 and ATF6 was significantly higher than that in the control group. It showed that endoplasmic reticulum stress participated in the apoptosis process of HepG2 cells induced by CXC195. Conclusion: CXC195 can significantly inhibit the proliferation of HepG2 hepatoma cells and induce apoptosis, which is mainly aimed at S phase and G2/M phase cells, but the inhibitory effect of normal liver cells (L-02) on growth and induction of apoptosis is obvious. This effect of weak.CXC195 may be achieved by inhibiting the expression of PI3K-AKT-mTOR signaling pathway protein and activation of endoplasmic reticulum stress. These results suggest that CXC195 has a certain therapeutic effect on liver cancer cells in vitro.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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