Li Jian-jun, Li Hong-yu, Chen Yan-zhi, Li Guang, Xin Yan
Department of Radiotherapy, The first Affiliated Hospital, China Medical University, Shenyang 110001, China.
Zhonghua Zhong Liu Za Zhi. 2006 May;28(5):345-8.
To investigate the effect of exogenous phosphatase and tensin homologue 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.
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.
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 implantation (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 G(2)/M blockage in ASPC-1-pEAK8-PTEN cells were remarkably higher than those in ASPC-1 cells.
Exogenous PTEN can block ASPC-1 cell cycle at the G(2)/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.
研究外源性10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）对人胰腺癌细胞系（ASPC-1）在常氧或1%低氧条件下暴露24小时后的细胞周期、血管内皮生长因子（VEGF）和表皮生长因子受体（EGFR）蛋白表达以及细胞增殖能力的影响。
采用脂质体法将含PTEN的真核表达质粒（pEAK8）或不含PTEN的质粒体外转染ASPC-1细胞。筛选、扩增阳性细胞克隆，命名为ASPC-1-pEAK8-PTEN或ASPC-1-pEAK8细胞。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测目的基因表达。通过蛋白质印迹分析评估PTEN、VEGF和EGFR蛋白。采用流式细胞术检测细胞周期和凋亡诱导情况。在裸鼠体内评估肿瘤生长能力，并在常氧或低氧条件下检测克隆形成存活能力。
ASPC-1-pEAK8-PTEN细胞中PTEN mRNA和蛋白表达显著高于ASPC-1-pEAK8或ASPC-1细胞。ASPC-1-pEAK8-PTEN细胞中VEGF蛋白表达下降23.4%，但EGFR无变化。与ASPC-1细胞相比，常氧条件下ASPC-1-pEAK8-PTEN细胞的接种效率下降28.0%（F = 4.283，P < 0.05）。植入后5周，接种ASPC-1-pEAK8-PTEN的裸鼠肿瘤体积与接种ASPC-1的裸鼠相比有显著差异（t = 4.834，P < 0.01）。ASPC-1-pEAK8-PTEN组的肿瘤抑制率为42.4%。VEGF和EGFR表达分别下降31.4%和25.0%。与ASPC-1细胞相比，低氧条件下ASPC-1-pEAK8-PTEN细胞的接种效率下降33.2%（F = 9.152，P < 0.01）。低氧8小时后，ASPC-1-pEAK8-PTEN细胞的细胞凋亡和G(2)/M期阻滞明显高于ASPC-1细胞。
外源性PTEN可使ASPC-1细胞周期阻滞于G(2)/M期，增强低氧诱导的细胞凋亡，在低氧条件下抑制VEGF和EGFR蛋白表达，抑制ASPC-1细胞的增殖和生长。
