The Affective Factors Of The Radiosensitivity In MGC803, ASPC-1 Cell Lines

In solid tumors, hypoxia can promote malignant progression and confer resistance to irradiation and chemotherapy by altering gene expression. The aim of this study was to investigate the effect of hypoxia on cell cycles, expression of mitochondrion ATP6 (mt ATP6) , mitochondrion mtCyt-b(mtCyt-b) , and PTEN mRNA, expression of vascular endothelial growth factor( VEGF) and epidermal growth factor receptor(EGFR) proteins, and radiosensitivity in a human gastric cancer cell line ( MGC803 ). In addition, we studied the effect of heteroenous PTEN, LY294002 and PD98059, inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) , on human pancreas cancer cell line (ASPC-1) under normal oxygen and hypoxia conditions, probed into the mechanism that PTEN, LY294002 and PD98059 affect the proliferation and radiosensitivity of ASPC-1 cell under normal oxygen and hypoxia conditions.MethodsWe measured the MGC803 cell cycles by flow cytometry, expression of mtATP6, mtCyt-b and PTEN mRNA by RT-PCR, and expression of VEGF and EGFR proteins by western blot assay in different time after hypoxia exposure. The survival rates of the cells irradiated (4Gy) or not were assessed by cologenic survival assay after 14days culture. And ASPC-1 cell was transfected with a eu-karyotic expression plasmid(Peak8) containing PTEN or not in vitro by a lipo-some-mediated lipofectin method, and then positive cell clones were selected and amplified. We measured hypoxic ASPC-1 cells irradiated (4Gy) or not by flow cytometry, western blot and cologenic survival assay, which were treated with heteroenous PTEN, LY294002 and PD98059.ResultsThe expression of PTEN was evaluated in MGC803 cell after hypoxia exposure in vitro showing maximal expression at 24hr hypoxia. Semiquantitative RT-PCR analysis showed that the relative expression of mtATP6 in MGC803 cell line after 24hr hypoxia was significantly reduced comparing with the expression under normal oxygen condition, and mtCyt-b was transiently reduced after 8hr hypoxi-a. The expression of VEGF and EGFR proteins was significantly increased after 24hr hypoxia. The MGC803 Gj phase cell and apoptosis cell transiently increased after hypoxia comparing with normal oxygen condition. After 24hr hy-poxic exposure, cell cycles distribution was similar to that of Ohr hypoxia. The surviving fraction of MGC803 cell without radiation under hypoxia condition gradually decreased, whereas, that with radiation gradually increased. A good correlation was observed between hypoxia time and surviving fraction in MGC803 cell irradiated or not. But there were no association between hypoxia time and the changes of cell cycles. After exposure in either LY294002 (20 M) or PD98059 (25uM) , VEGF and EGFR expression remarkably decreased under hypoxia conditions. Cell cycles did not change significantly after hypoxia comparing with that under normal oxygen condition except for G, phase. The surviving fractions in hypoxia radiation group were higher than that in normal oxygen radiation group (P <0.01). Hypoxia cells treated with LY294002, with irradiation or not, had lower surviving fractions than those with PD98059. The surviving fraction of ASPC-1 cell under the cooperation of LY294002 and PD98059 had a remarkably effect than that under either one alone ( P < 0. 05 ). We transfected plasmid pEAK8 contained PTEN or not into ASPC-1 cell and found that the PTEN mRNA and protein could be efficiently expressed in transfected ASPC-1 cell contained plasmid pEAK8-PTEN, which could greatly enhance radiation-induced G2/M arrest, apoptosis, growth inhibition and radiosensitivity. ASPC-1 cell transfected with the plasmid pEAKS contained PTEN was preferentially killed through apoptosis under hypoxia conditions. The apoptotic peak of ASPC-1 cell transfected with PTEN was found much higher than that without transfectionby FCM after 8hr hypoxia.ConclusionOur findings suggested that hypoxia played an important role not only in modulating cell cycles of MGC803 and ASPC-1 cells but also in regulating the radi...