Study Of Biological Effects And Proteomics On Breast Cancer Cells MDA-MB-231 Induced By X-ray Irradiation

Breast cancer is one of the most common malignant tumors in female tumor. Radiation therapy is an important method for the treatment of breast cancer. Individual or tumor cells have different radiant sensitivity, which leads to the difference of the curative effect of radiotherapy. In radiotherapy, an important method to improve the curative effect of radiation therapy is reducing the radiation resistance of tumor cells and increasing the radiation sensitivity of tumor cells. Therefore, it is necessary to study the effects of X-ray on biological characteristics and changes of related protein expression in breast cancer cells. What’s more, it can help us to better understand the radiosensitivity and the molecular regulation mechanism in tumor cells. 1. Biological effects on MDA-MB-231 cells induced by X ray irradiationObjective: By comparing the effects of different doses of X-ray irradiat ion on the proliferation, cycle and apoptosis of MDA-MB-231 cells, we analysed the relationship between the biological characteristics and cell radiation sensitivity.Methods: the MDA-MB-231 cells in exponential growth phase were used for following experiment. The proliferation of MDA-MB-231 cells was detected by MTT after X-ray irradiation of different doses(0, 1, 2, 4, 8, 10 Gy) 24 h, 48 h and 72 h.And the effect of different doses of X-ray irradiation on MDA-MB-231 cell growth inhibit was analysed. PI single staining assay and annexin V / PI double staining were used to detect the distribution of cell cycle and apoptosis. Flow cytometry were used to calculate and analyze the effect of different doses of X-ray irradiation on MDA-MB-231 cell cycle and apoptosis.Results: The results of MTT showed that the proliferation ability of MDA-MB-231 cells was decreased at 24 h, 48 h and 72 h after X- ray irradiation. The inhibition rate of cells varied with the dose and time. At 48 h and 72 h points, cell inhibition rate increased along with the dose increased. The inhibition rate of each dose point at 72 h was significantly lower than that at 48 h. Compared to control group, each dose point showed a certain inhibitory effect at 24 h. At eac h dose point, cell inhibition rate first increased and then decreased along with prolongation of the time. After different doses of X-ray irradiation treating MDA- MB-231 cells. At 24 h and 48 h, total cell apoptosis rate increased along with increasing dos e, and total apoptosis rate of 24 h was greater than 48 h at the same dose. The early apoptosis rates of each dose point at 24 h and 48 h do not exist signif icant difference. there is no dose dependence at 24 h, but with the dose increase apoptosis gradually increased at 48 h. Late apoptosis rate was significantly different at 24 h and 48 h in all dose points. There were dose dependents at 24 h and 48 h, and late apoptosis rate at 24 h was more than that at 48 h at the same dose. The results of cell cycle analysis show that after X-ray irradiation for 24 h and 48 h, the impact of MDA-MB-231 cell cycle mainly for G2 / M arrest. The percentage of G2/M cells with the dose increase showed an increasing trend at 24 h and 48 h. Low dose(1 and 2 Gy), the G2 / M phase no significant difference(P > 0.05), high dose(4, 8, 10 Gy), G2 / M phase arrest exist signif icant difference(P < 0.05).Conclusion: X-ray irradiation can effectively inhibit the proliferation of MDA-MB-231 cells, so that the cell cycle was arrest in the G2/M phase, and inducing apoptosis. 2. Differential protein expression pattern in MDA-MB-231 breast cancer cells after X-ray irradiationObjective: to analysis of X-ray irradiation on MDA-MB-231 cell protein expression profiles, we compared the protein expression profiles, screened and identif ied differentially expressed proteins to obtain molecular markers related to X-ray irradiation effect on MDA-MB-231 cells. these results offer an useful help for exploring the mechanism of tumor radiosensitiv ity and developing corresponding radiation sensitization product.Methods: The MDA-MB-231 cell in the logar ithmic growth phase were treated with 4 Gy and 0 Gy X-ray. After 48 h irradiation, two groups were analyzed by Two dimensional electrophoresis. 2 DL graph were captured by using Versadoc4000 images system. some obvious difference protein spots were selected to dig, enzymolysis and extract, and analysed by using API 4800 tandem time-of-flight mass spectrometer MALDI-TOF/TOF respectively in gel(Applied Biosystems).Results: By comparing and analyzing, we screened 32 protein spots in this experiment. Compared breast cancer cells lines MDA-MB-231 without X- ray treatment, 17 protein spots were up-regulated, 15 protein spots were down regulated in 4 Gy treatment groups. 32 differentially expressed protein spots were identified by mass spectrometry. The specific information of the 8 differentially expressed proteins were obtained by Protein mass spectrometry. They were: Hsc70, GRP78, impdh2. EIF4 H, GAPDH, vim,TUBA1 B and TUBA8, respectively.Conclusion:There is a great influence on the expression profile of MDA-MB-231 cell protein with treatment by radiation and unradiation.The differentially expressed proteins were identified by mass spectrometry.They were heat shock protein 70 member(HSC70), inosine monophosphate dehydrogenase(IMPDH2), eukaryotic translation initiation factor(EIF4H), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), vimentin(VIM) and microtubule associated proteins(TUBA1B,T UBA8). These proteins are the effectors of X-ray radiation in MDA-MB-231 cells, which may be involved in the regulation of cell radiation sensitivity, and these proteins may be the biological targets for the clinical treatment of breast cancer.