The Effect Of Heavy Ion Radiation On Invasion And Migration Of Non-small Cell Lung Cancer And Molecular Mechanism

Lung cancer is a high incidence rate,high death rate and low cure rate of malignant tumor,non-small cell lung cancer（NSCLC）was the main pathological type.NSCLC occupy 85%of the lung cancer and its five year survival rate is only 18%.Radiation therapy is one of the most important treatment methods for lung cancer,Because of the low radiation sensitivity the high rate of local recurrence and distant metastasis is the major failure of treatment,so find new rays to improve the local control rate and distant metastasis was an important direction.Heavy ion which has the unique features of physics and good radiation biology characteristics was become a research focus in the radiation therapy field.Tumor invasion and metastasis are the main cause of treatment failure and the bystander effect induced by ionizing radiation may be involved in tumor invasion and metastasis.In this study,In order to explore the efficacy and safety of the lung cancer treated with heavy ion,evidence-based medical research method was used,using an in vitro cell model（lung adenocarcinoma A549 cells）as the research object different ray(X-ray and heavy ion¹²C⁶⁺)and different doses of irradiation were delivered in following studies:（1）The electronic bibliographic databases,including Cochrane library,Pub Med,EMBASE,China Journal Full-text Database,Chinese Biomedical Database,Wanfang Database to assemble the studies of the heavy ion（carbon ion）radiotherapy for stage I non-small cell lung cancer were searched.The Meta Analyst Beta 3.13 and STATA 12.0 software were used to combine the extracted data.Results 10 studies（7 trials）were included,the results of meta-analysis show that,the overall survival rates of 1,3 and 5 years were 95.1%,68.5%and 45.9%;the local control rates of 1,3 and 5 years were 90.2%,76.7%and 81.2%;the cause specific survival rate of 1,3 and 5years were 82.9%,58.5%and 57.3%;the rate of primary recurrence and total recurrence were45.8%and11%;The rate of total death,death from lung cancer were 52.4%and 28.3%.At the same time there were difference between T1 stage and T2 stage in five year overall survival rate,total recurrence,lung cancer death,grade 1 and grade 2 skin adverse effect.Available data demonstrate that heavy ion radiotherapy in general is a safe and feasible treatment modality to stage I NSCLC.Although current results are promising,more evidence is required before heavy ion radiotherapy can become internationally the standard treatment for lung cancer patients.（2）To observe the biological effects of heavy ion ¹²C⁶⁺and X-ray on the survival,proliferation and apoptosis of A549 cells,and to explore the effects of two kinds of radiation on cell adhesion,migration and invasion and related mechanisms.The logarithmic growth A549 cells were irradiated by carbon heavy ion and X-ray,adhesion ability was tested with the MTS method;cell migration and invasion ability were detected with Transwell method;Real time-PCR and WB（Wester blotting）were performed to detect the expressions of MMP-2 and MMP-9 m RNA and protein.The results showed that both heavy ion ¹²C⁶⁺and X-ray all inhibited cell survival,proliferation and promoted apoptosis with increasing radiation dose,but heavy ion ^12C6+significantly inhibited the survival,proliferation and enhanced apoptosis of A549 cells compared with the same dose of X-rays,The Cell adhesion was enhanced after two rays irradiation,heavy ion ¹²C⁶⁺was more significant than X-rays;The cell migration and invasion were significantly suppressed by heavy ion ¹²C⁶⁺compared with X ray.Fluorescence quantitative RT-PCR and Western Blotting experiments showed that MMP2 and MMP9 expression were down-regulated（P<0.05）after X-ray irradiation but significantly down-regulated（P<0.05）with ¹²C⁶⁺irradiation.Two rays can improve the adhesive ability of A549 cells,compared to X ray,heavy ion ¹²C⁶⁺significantly inhibited A549 cell migration and invasion ability.（3）Ionizing radiation induced by-stander effect may be involved in the process of tumor invasion and metastasis.A549 cell metastasis affected by radiation-induced by-stander effect was detected with metabolomics technology.The exponential phase to use the logarithmic growth WI-38 cells,which were irradiated 24h and 48h after cultured by different doses 0、1、2 and 4Gy carbon heavy ion.Transferred the collected cell culture medium to the well-grown A549 cells,cultured again for 24h.and then detected cell migration and invasion ability with Transwell method.Choose the appropriate radiation dose,analysis differences in change of metabolic factors in transfer medium.Result it is found that transfer medium have the most visible impact to metastasis of tumor cells after 2 Gy irradiation for 24h and 48h culture medium.There is no significant transfer effect after the 4Gy irradiation.With liquid mass spectrometry（LC-MS）analysis of metabolomics,the results show that metabolomics factors in cell culture medium irradiated by two periods of 2Gy have significant differences（p<0.05）.Differential metabolic factors are mainly involved in tryptophan,glycolysis,cyanoacid amino acid,ornithine and citric acid metabolism cycle pathway.Heavy ion radiation-induced by stander effect inhibit metastasis of tumor cells in a certain period of time,which may change metabolites of cells and indirectly caused the growth of micro-environment of cancer cells,transfer of tumor cells will be inhibited.The above evidence-based medical research results provide a basis for clinical research and application of heavy ion treatment of NSCLC;the results obtained from the basic research provide a theoretical basis for the clinical application of heavy ion in the lung cancer treatment.In order to explore the treatment with heavy ion combined with biological targeted therapy and accurate treatment have predicted the direction,and it is expected to break through the bottleneck of lung cancer treatment and improve the diagnosis and treatment of lung cancer patients..