新型PI3K小分子抑制剂抗胰腺癌的作用与机制研究

发布时间：2019-04-19 01:41

【摘要】：目的观察新型PI3K小分子抑制剂BYL-93对人胰腺癌细胞株CFPAC-1和Capan-2的作用,从细胞水平上验证BYL-93的抗胰腺癌活性。为拓展PI3K抑制剂的应用提供实验依据,同时也为开发新的小分子靶向治疗药物提供科学依据。方法培养CFPAC-1和Capan-2细胞,采用CCK-8法检测细胞增殖;DAPI染色观察细胞核;流式细胞术测定细胞凋亡及其周期;JC-1法检测线粒体跨膜电位变化;Western blot法检测PI3K-AKT信号通路的蛋白表达和磷酸化水平。对CFPAC-1细胞进行划痕实验、Transwell小室体外侵袭实验,实时荧光定量PCR法测定E-cadherin、Snail基因转录水平。结果BYL-93对CFPAC-1和Capan-2的增殖都产生了抑制效应,CFPAC-1和Capan-2第 24、48 小时的 IC50 值分别为 22.7μM、5.3μM 和 9.0μM、2.4μM;5、10、20μM BYL-93均能诱导CFPAC-1细胞凋亡,2.5、5、1OμM BYL-93均能诱导Capan-2细胞凋亡;不同浓度BYL-93作用后,可将CFPAC-1和Capan-2明显阻滞于细胞G1期;BYL-93在CFPAC-1和Capan-2两株细胞上呈现显著下降线粒体膜电位的效果(p0 001);BYL-93作用于CFPAC-1和Capan-2后,AKT、mTOR、70S6、S6蛋白的磷酸化水平降低,PARP发生裂解,且BYL-93对CFPAC-1细胞的作用略强;随着BYL-93浓度的升高,CFPAC-1细胞的迁移能力逐渐降低,其抑制迁移能力呈浓度梯度依赖性;BYL-93能明显抑制胰腺癌细胞的侵袭转移,上调E-cadherin mRNA表达,下调Snail mRNA表达。结论BYL-93具有抑制CFPAC-1和Capan-2肿瘤细胞的增殖、转移的作用,验证BYL-93的抗胰腺癌活性。
[Abstract]:Aim to observe the effect of BYL-93, a novel small molecule inhibitor of PI3K, on human pancreatic cancer cell lines CFPAC-1 and Capan-2, and to verify the anti-pancreatic cancer activity of BYL-93 at the cell level. It can provide experimental basis for expanding the application of PI3K inhibitors and provide scientific basis for the development of new small molecular targeted therapeutic drugs. Methods CFPAC-1 and Capan-2 cells were cultured, cell proliferation was detected by CCK-8 method, cell nucleus was observed by DAPI staining, apoptosis and cell cycle were measured by flow cytometry, mitochondrial transmembrane potential (mtDNA) was measured by JC-1 method. The protein expression and phosphorylation level of PI3K-AKT signaling pathway were detected by Western blot. Scratches of CFPAC-1 cells, invasion of Transwell cells in vitro, and transcription of E-cadherin and snail genes were measured by real-time fluorescence quantitative PCR. Results BYL-93 inhibited the proliferation of CFPAC-1 and Capan-2. The IC50 values of CFPAC-1 and Capan- 224h were 22.7 渭 M, 5.3 渭 M and 9.0 渭 M, 2.4 渭 M, respectively. 5, 10 and 20 渭 M BYL-93 could induce apoptosis of CFPAC-1 cells, and 2.5, 5 and 1 渭 M BYL-93 could induce apoptosis of Capan-2 cells. After different concentrations of BYL-93, CFPAC-1 and Capan-2 could be blocked obviously in G1 phase of cells. BYL-93 significantly decreased the mitochondrial membrane potential in CFPAC-1 and Capan-2 cell lines (p0.001). When CFPAC-1 and Capan-2 were treated with BYL-93, the phosphorylation level of AKT,mTOR,70S6,S6 protein was decreased and PARP was lysed, and the effect of BYL-93 on CFPAC-1 cells was slightly stronger. With the increase of BYL-93 concentration, the migration ability of CFPAC-1 cells decreased gradually, and its ability to inhibit migration showed a concentration-gradient dependent manner. BYL-93 could significantly inhibit the invasion and metastasis of pancreatic cancer cells, upregulate the expression of E-cadherin mRNA and down-regulate the expression of Snail mRNA. Conclusion BYL-93 can inhibit the proliferation and metastasis of CFPAC-1 and Capan-2 tumor cells, and verify the anti-pancreatic cancer activity of BYL-93.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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