外源性PTEN增强乏氧诱导胰腺癌细胞系ASPC-1的凋亡

Exogenous PTEN enhances apoptosis in pancreas cancer cell line ASPC-1 induced by hypoxia

目的研究外源性PTEN对乏氧胰腺癌细胞系ASPC-1细胞周期、血管内皮生长因子 (VEGF)和表皮生长因子受体(EGFR)蛋白表达及细胞增殖能力的影响。方法将质粒pEAK8和 pEAK8-PTEN分别转染ASPC-1细胞,获得ASPC-1-pEAK8(A-pE)细胞和ASPC-1-pEAK8-P-FEN(A-pE-P)细胞,给予乏氧(1％O2)处理,并应用RT-PCR、Western印迹杂交、流式细胞术、成克隆分析及观察裸鼠移植瘤生长等方法进行检测。结果A-pE-P细胞PTEN mRNA及其蛋白表达明显高于ASPC-1细胞和A-pE细胞。与ASPC-1细胞相比,常氧时,A-pE-P细胞VEGF蛋白表达量下降了23．4％,EGFR蛋白表达量无明显变化,细胞克隆形成率降低了28．0％(F=4．283,P<0．05),荷瘤裸鼠5周时,A-pE-P 细胞组和ASPC-1细胞组瘤结节体积比较,差异有统计学意义(t=4．834,P<0．01),A-pE-P细胞组的抑瘤率为42．4％。乏氧时,A-pE-P细胞VEGF蛋白表达量下降了31．4％,EGFR蛋白表达量降低了 25．0％,细胞克隆形成率降低了33．2％(F=9．152,P<0．01)。A-pE-P细胞较ASPC-1细胞G2／M期阻滞明显,乏氧培养8 h时可引起细胞大量凋亡。结论外源性PTEN使ASPC-1细胞阻滞在G2／M 期,增强乏氧诱导细胞的凋亡,抑制乏氧诱导VEGF、EGFR表达的增加,抑制肿瘤细胞的增殖。

Objective To investigate the effect of exogenous phosphatase and tensin homologne deleted on chromosome ten （ PTEN ） on cell cycle, the expression of vascular endothelial growth factor （VEGF） and epidermal growth factor receptor （EGFR） proteins, and cellular proliferation ability in human pancreas cancer cell line （ ASPC-1 ） exposed to normal oxygen or hypoxia 1% for 24 h were determined. Methods ASPC-1 cells were transfected in vitro with an eukaryotic expression plasmid （pEAK8） containing PTEN or not by lipofectin. Positive cell clones were selected, amplified and named ASPC-1- pEAK8-PTEN or ASPC-1-pEAK8 cells. RT-PCR and Western blot were used to determine the target gene expression. PTEN, VEGF and EGFR proteins were assessed by Western blot assay. Cell cycle and the induction of apoptosis were detected by flow cytometry. The tumor growth ability in vivo was assessed in nude mice, and cologenic survival ability was assayed under normal oxygen or hypoxia condition. Results The expression of PTEN mRNA and protein in ASPC-1-pEAK8-PTEN cells were significantly higher than that in ASPC-1-pEAK8 or ASPC-1 cells. The expression of VEGF protein in ASPC-1-pEAK8-PTEN cells decreased by 23.4%, but EGFR showed no change. The plating efficiency was decreased by 28.0% （ F =4.283, P 〈 0.05） under normal oxygen condition, compared with those in ASPC-1 cells. The tumor volume in nude mice with ASPC-1-pEAK8-PTEN were significantly different compared to those with ASPC-1 5 weeks after implantion （t =4.834,P 〈0.01 ）. The tumor inhibitory rate was 42.4% in ASPC-1-pEAK8-PTEN group. The expressions of VEGF and EGFR were decreased by 31.4% and 25.0%, respectively. In comparison with ASPC-1 cells, the plating efficiency of ASPC-1-pEAK8-PTEN cells was decreased by 33.2% （ F = 9. 152, P 〈0.01 ） under hypoxic condition. The cellular apoptosis 8 h after hypoxia and G2/M blockage in ASPC-1-pEAK8-PTEN cells were remarkably higher than those in ASPC-1 cells. Conclusion Exogenous PTEN can block ASPC-1 cell cycle at the G2/M phase, enhance the cell apoptosis induced by hypoxia, inhibit the expression of VEGF and EGFR proteins under hypoxic condition, and inhibit the proliferation and growth of ASPC-1 cells.