The Study Of Machanism On The Effect Of X-Irradiation On The Adhesion And Apoposis Of Human Lung Adenocarinoma Cell Line

ObjectivesTo investigate the effect of X-irradiation on adhesion molecule E-cadherin/β-catenin and Wnt /β-catenin signaing pathway,then explore the mechanism of X-irradiation inhibiting the metastasis of tumour cell and treating Lung Cancer.Methods1. A549 cells were regularlly subcultured in vitro and were divided into four groups ,then irradiated with 0Gy,2Gy,5Gy and 10Gy doses of X-rays.After irradiating,the cells were seeded for 24h.2. The transwell chambers were employed to evaluate the invasion of A549 cells. The protein and mRNA expression levels of E-cadherin andβ-catenin were analyzed by immunocytochemical method ,Western Blot and RT-PCR.3. MTT assay was employed to evaluate the effects of X-irradiation on the proliferative inhibition of A549 cells.The apopotosis was examines by AO/EB fluorescent and TUNEL labeling examination techniques. Cell cycle was examines by flow cytometry analysis.The nuclear protein and mRNA expression levels ofβ-catenin and CyclinD1 were analyzed by Western Blot and RT-PCR.Results1. The transwell chambers showed that after irradiation the cell invasions were lower than that in control group(P<0.05).The change was in a dose-dependent manner.2. The result of immunocytochemical method ,Western Blot and RT-PCR indicated that before irradiation the protein expression ofβ-catenin in the membrane of A549 was extremely weak and the part of cytolymph was very high. After irradiation the former increased and the latter decreased. (P<0.05) .The total protein and mRNA expression levels of E-cadherin in A549 cells were higher than those in control group(P<0.05). But there were no significant difference in the total protein and mRNA expression levels ofβ-catenin among the four groups(P>0.05).3. MTT assay showed X-irradiation could inhibit the proliferation of A549 cells in a dose-dependent manner. AO/EB fluorescent and TUNEL labeling examination techniques showed that the control cells appeared to be mostly round ,intact and bright green while a little with bright green or jiacinth nuclei,Pyknosis and fragment appearance which characteristic apoptosis.At 24h,the percentage of apoptosis cells was higher.X-irradiation increased the ratio of apoptosis in a dose-dependent manner(P<0.05).4. The flow cytometry analysis showed that the cell cycle was no significant difference after 2Gy irradiation(P>0.05). The cells were blocked in G0/G1 phase in 5Gy and 10Gy X-rays,the change was in a dose-dependent manner(P<0.05).5. The nuclear protein expression levels ofβ-catenin was lower than that in control group(P < 0.05).The expression of CyclinD1 was no significant difference in 2 Gy and decreased from 5Gy.Conclusions1. Up-regulating the expression of E-cadherin andβ-catenin on A549 cells ,which enhanced the adhesion between cells and depressed the invasion. That may be the mechanism of X-irradiation inhibiting the metastasis of Human Lung Adenocarcinoma Cell line .2. X-irradiation down-regulated the expression ofβ-catenin and CyclinD1 on Wnt /β-catenin signaling pathway of A549 cells ,which blocked in G0/G1 phase and induced the apoptosis. That may be the mechanism of X-irradiation killing Human Lung Adenocarcinoma Cell line3. X-irradiation accommodated the disposition ofβ-catenin in the cells. It made the part which was bound to E-cadherin increase ,while the nuclear part decrease .So that X-irradiation might have an anticancer function by enhancing the adhesion, inducing A549 cell line into apoptosis.