Study On The Function Of Caspase-3Protein In Lung Cancer Cells By The Heavy Ion Irradiation

The lung cancer is one of the highest mortality of malignant tumors, its morbidity and mortality tend to increase year by year. Radiotherapy has become the principal means of its treatment. About85%of lung cancer patients need to receive radiation therapy at some stage in their course of the disease. Studies have shown that ionizing radiation can induce apoptosis through a variety of signal transduction pathway for therapeutic purposes. Caspase-3is the most important protease in Caspase family. It is the common downriver effective parts of multi-apoptosis pathways and the only way to apoptotic enzyme reaction. We have studied12C6+-ray irradiation of human lung cell line H1299. Dynamic changes of the cell cycle and apoptosis were observed, and the expression caspase-3, Caspase-9, Bcl-2and Bax proteins of the cells irradiated by12C6+at different doses and times was detected. This study is to explore the theoretical mechanism and apoptosis pathway of heavy ion radiation therapy of lung cancer.Several important experimental results were acquired as summarized as below:1Heavy ion beam could depress the cancerous cell replicationFCM was used to check the cell cycle distributions of H1299cells after heavy ion beam irradiations. As the dose of the heavy ion radiation increased, the G2/M cells were increased obviously; the level of retardarce was dose-dependent. As the dose was over6GyE, the G1cells were increased and the cells become to die. That shows the heavy ion beam could depress the cancerous cell replication and induce cell death.Assay OD value of the hole with MTT. The result shows that the inhibition is time-and dose-dependent. In the same time, as the dose of12C6+increased, the cell survival rate decreases2Heavy ion beam could induce H1299cells undergone apoptosisThe cellular form through the fluorescence microscope after radiated for24h was observed. It clearly shown cell nuclear morphology changes such as membrane blebbing and formation of apoptotic body which are characteristic of apoptosis. Apoptosis induction increased is time and dose dependent manner.FCM showed that the rate of apoptosis of H1299radiated by heavy ion beam was increased as the dose increased. That indicates the heavy ion can induce the apoptotic of H1299in vitro.3Heavy ion irradiation and related apoptosis gene expressionThrough the Caspase-3activeness analytical reagent box examination, with the dose of irradiation increasing, Caspase-3activity was significantly increased, and the dose-dependen. It clued on that enzyme activity of Caspase-3of H1299would be higher after being irradiated by12C6+and the change of apoptosis rate was positive correlation.The expression of protein Caspase-3was detected with Western Blotting and RT-PCR in irradiated cell H1299. The results shown that:irradiation of12C6+can increase protein expression of Caspase-3, and there is dose-depended.The immunohistochemistry test result demonstrates that the expression of gene Caspase-3,Caspase-9and Bax was increased with the increase of radial dose, but Bcl-2’s was decreased, Bcl-2/Bax decreased as well. That indicates:the heavy ion radiation can activate Caspase-3. The Bax massive expressions have suppressed Bcl-2function to a certain extent. It suggests heavy ion radiation induces the H1299cell apoptosis possibly is weakly through the caspase-3way regulation.4Caspase-3activity inhibition assayWhen H1299cells were pretreated with Caspase-3inhibitor and cells were observed activity and apoptosis rate displayed after24h:Caspase-3inhibitor (Z-DEVD-fmk) that specifically blocks the activation of Caspase-3, while reducing cell apoptosis.This research tests from above may obtain the following conclusion:The heavy ion radiation can suppress H1299cell multiplication in vitro. Radiation-inducing can lead H1299to apoptosis by the non-P53-dependent pathway. The expression of protein Caspase-3and the radio sensitivity are proportional. The heavy ion possibly is through activates Caspase-3in the H1299and reduces the Bcl-2/Bax ratio to induce the apoptosis. The protein Caspase-3participates in the regulation mitotic cycle, gives play to the anti-tumor. By regulating the expression of Caspase-3gene may change the radio sensitivity of tumor cells, which is expected to increase tumor radiation therapy to provide new ideas and new methods.