Study On Autophagy Radiation -induced And Its Relationship With Apoptosis In MCF-7 Cells

Breast cancer is one of the common malignant tumors for women. There are about 120 million women suffering from breast cancer and 50 million cases dying of the disease every year in the global. In recent years, the breast cancer incidence of women shows an ascendant trend in China. The treatments of breast cancer mainly include surgery, radiation and chemotherapy. But these treatments have certain limitations, and still cannot reach to the purpose of curing the breast cancer. Especially during the process of breast cancer cell apoptosis induced by radiotherapy, the tolerance of the cells to ionizing radiation often appears and they escape the apoptosis. However, autophagy is considered to be another programmed cell death path to distinguish from cell apoptosis, some tumor cells which escape the apoptosis may develop the autophagy death. So, the autophagy death of breast cancer cells induced by ionizing radiation is becoming a new focus of breast cancer treatment.The methods in biochemistry and molecular biology were applied, the inhibitors and inducers of autophagy or apoptosis were used, and gene expression and interference model were established in the study. The cells proliferation, apoptosis, the cells cycle procession, gene and protein expression of MCF-7 cells were observated to study autophagy and apoptosis process induced by ionizing radiation. The purpose of the study is to explore the mechanism of autophagy and apoptosis in tumor cells induced by ionizing radiation, and provide theoretical and experimental basis for improving breast cancer radiotherapy in clinical application.1 Detections of autophagy and apoptosis in MCF-7 cells induced by ionizing radiation1.1 Detection of autophagy in MCF-7 cells induced by ionizing radiationThe MCF-7 cells were irradiated with 4 Gy X-rays and dyed with MDC 12 h after culture. The pointlike distribution of autophagy body were observed by laser confocal microscopy. The result shows that ionizing radiation can induce the autophagy of tumor cells.1.2 Detection of apoptosis in MCF-7 cells induced by ionizing radiation The MCF-7 cells were irradiated with 4 and 8 Gy X-rays, DNA gel electrophoresis present a typical ladder distribution. The result expresses ionizing radiation can induce the apoptosis of tumor cells.2 Effects of inducers or inhibitors for autophagy and apoptosis on MCF-7 cells2.1 proliferation of 4-Gy irradiation combinated with autophagy/apoptosis inducers or inhibitors on MCF-7 cellsAfter the MCF-7 cells treated with 4-Gy X-irradiation in cmbination with autophagy inducer (rapamycin). autophagy inhibitor (3-MA) or apoptosis inhibitor (z-VAD-fmk), respectively, the cell absorbency value reduced significantly (P< 0.05). and the inhibit of cell proliferation significantly increased (P< 0.05). as compared with that in the control. The cell proliferation rates 24,48 and 72 h after irradiation were significantly lower than those at 0 h (P< 0.05 or P< 0.01). The results indicate that ionizing radiation combinated with autophagy inducer can inhibit the MCF-7 cell proliferation, and it combinated with autophagy inhibitor can promote the cell proliferation.2.2 Apoptotic effects of 4-Gy X-irradiation combinated with autophagy/apoptosis inducers or inhibitors on MCF-7 cellsThe apoptoses of MCF-7 cells induced with different treatments were detected by flow cytometry (FCM). As compared with that in the controls, the apoptosis percentage of cells treated by using autophagy or apoptosis inhibitors decreased significantly (P< 0.05). The apoptosis percentage of MCF-7 cells after 4-Gy X-irradiation combinated with autophagy inducers increased significantly (P< 0.01). The apoptosis percentage of the cells treated by using 4-Gy X-irradiation combinated with autophagy inhibitor and inducer or apoptosis inhibitor increased significantly as compared with that in the controls (P< 0.05 or P< 0.01). The apoptosis percentage of the cells in the 4-Gy+rapamycin group increased significantly as compared with that in the 4-Gy+3-MA and 4-Gy+z-VAD-FMK groups (P< 0.05). These results suggest that ionizing radiation in combination with autophagy inducer may promote the MCF-7 cell apoptosis. and the inducing autophagy inducer can promote the apoptosis of MCF 7-cells.2.3 Gene expression of MCF-7 cells induced with ionizing radiation jointing with inducers or inhibitors of autophagy and apoptosis The relative expressions of LC3B, beclin-1, p53 and Bax mRNA increased in MCF-7 cells treated by ionizing radiation jointing with autophagy inducer, while those of Aktl and Bcl-2 mRNA decreased. The relative expressions of beclin-1, LC3B, p53 and Bax mRNA decreased in the cells treated by ionizing radiation jointing with autophagy inhibitor, while those of Aktl and Bcl-2 mRNA rose. The relative expressions of LC3B, Bax and p53 mRNA decreased in the cells treated by ionizing radiation jointing with apoptosis inhibitor, while those of Aktl and Bcl-2 mRNA increased, while that of beclin-1 mRNA did not change. The results show that although the cell autophagy and apoptosis are two different programmed cell death ways, their death ways have overlap. Ionizing radiation jointing with autophagy inducer affects not only the autophagy pathway in the cells but also the apoptosis pathway.3 Role of beclin-1 gene in autophagy and apoptosis in MCF-7 cells induced by ionizing radiation3.1 Constructions of beclin-1 over-expression and interference modelsThe purpose gene fragments of beclin-1 from the MCF-7 cells were extracted with RT-PCR method, which inserted into pcDNA3.1(-) eukaryotic expression vector through carrying liposome method and constructed pcDNA3.1(-)-beclin-1 recombinate vector. The protein expression of beclin-1 was detected with Western blot. The beclin-1 protein expression carried with plasmid was significantly higher than that in the control, suggesting the beclin-1 over expression model had been built successfully. The psuper-beclinlRi vector was transfected into the MCF-7 cells by using phosphate calcium transfection method. Through the Western blot detection, interferenced beclin-1 protein expression was significantly lower than that in the control, suggesting the interferenced model was built successfully.3.2 Biological effects of 4-Gy X-irradiation on beclin-1 gene over expression and interference models in MCF-7 cells3.2.1 Effects of 4-Gy X-irradiation on the growth of MCF-7, MCF-7-beclinl and MCF-7-beclinlRi cellsThe growing speed of MCF-7 cells in the MCF-7-beclinl+4 Gy group cells decreased significantly as compared with the MCF-7+0 Gy, MCF-7+4 Gy and MCF-7-beclinlRi+4 Gy groups (P< 0.05-P< 0.001). more significantly in the MCF-7-beclinl+4 Gy group:the cell growth in the MCF-7+0 Gy group was the fastest, suggesting the over expression of beclin-1 gene restrains the growth of MCF-7 cells.3.2.2 Effects of 4-Gy X-irradiation on apoptosis in MCF-7, MCF-7-beclinl and MCF-7-beclinlRi cellsThe apoptosis percentages of MCF-7, MCF-7-beclinl and MCF-7-beclinlRi cells 8 h after 4-Gy X-irradiation as compared with MCF-7+0 Gy group rose significantly (P< 0.01-P< 0.001). There was the statistical difference in the the apoptosis percentages between the MCF-7-beclinl Ri+4 Gy, MCF-7-beclinl+4 Gy and MCF-7+4 Gy groups (P< 0.05), and there was also in between the MCF-7-beclinl Ri+4 Gy and MCF-7-beclinl+4 Gy groups (P< 0.05). The cell apoptosis percentage in the MCF-7-beclinl group was the highest. The results show that the beclin-1 gene expression promotes the apoptosis of MCF-7cells.3.2.3 Protein expressions of beclin-1 and p53 in MCF-7, MCF-7-beclinl and MCF-7-beclinl Ri cells after 4-Gy X-irradiationThe expression of beclin-1 protein in the MCF-7 cells increased 4 h after 4-Gy X-irradiation, which reached to the greatest 8 h later, and decreased 16 and 32 h after irradiation than that 4 and 8 h later, but still higher than that in the control. The expression of p53 protein began to rise 8 h after irradiation, which increased gradually with the extension of time, and increased and maintained in the higher level 32 h later. The beclin-1 protein expression in the MCF-7-beclinl Ri cells were the lowest 4 h after irradiation, which began to rise 8 h later, reached to maximum 16 h later and decreased 32 h later, but remained at higher level. The expression of p53 protein began to rise 4 h after irradiation, which reached the peak 16 h later, and reduced 32 h later, but still higher than that 4 and 8 h later. The expression of beclin-1 protein in the MCF-7-beclinl cells increased gradually with the extension of time, which reached to the peak 32 h later. The expression of p53 protein did not change significantly with time prolongation. The expression of beclin-1 in MCF-7 cells gene induced by ionizing radiation promoted the increase of p53 protein expression, suggesting that the cell autophagy induced by ionizing radiation can promote cell apoptosis.4 Role of Aktl gene in autophagy and apoptosis in MCF-7 cells induced by ionizing radiation4.1 Establishment of Aktl gene over expression and interferenced models in MCF-7 cells The Aktl siRNA gene interference expression vector was constructed, and packed into 293T cells by using calcium phosphate precipitation. The virus particles after collection infected directly the target MCF-7 cells. The Aktl gene expression in the model group reduced significantly with Western blot method, suggesting that the Aktl gene interference model was successfully built.The over expression model of MCF-7-Aktl in the MCF-7 cells was established by using liposome transfected method. The Aktl gene expression increased significantly with Western blot method, showing that the expression model of Aktl expression was built successfully.4.2 Effects of 4-Gy X-irradiation on growth in the MCF-7, MCF-7-Aktl and MCF-7-AktlRi cellsThe effects of Aktl gene over expression and interferenced models on the MCF-7 cell proliferation were detected with MTT method. As compared with that in the MCF-7 cells of the MCF-7+4 Gy and MCF-7-Aktl+4 Gy groups, the A490 absorbency value of the cells in MCF-7-AktlRi+4 Gy group decreased significantly (P< 0.001), and the cell growth was the slowest, while that in MCF-7+0 Gy group was the fastest, suggesting that Aktl gene controlled negatively the growth and reproduction in the MCF-7 cells.4.3 Effects of 4-Gy X-irradiation on the apoptosis in the MCF-7, MCF-7-Aktl and MCF-7-AktlRi cellsThe apoptosis of MCF-7 cells in the MCF-7+0 Gy, MCF-7+4 Gy, MCF-7-Aktl+4 Gy and MCF-7-ktlRi+4 Gy groups after 4-Gy X-irradiation was detected by using flow cytometry. As compared with that in the MCF-7+0 Gy group, the apoptosis percentage in other groups all rose significantly (P< 0.001); there was statistical difference in the apoptosis percentage of MCF-7 cells between the MCF-7-AktlRi+4 Gy, MCF-7-Aktl+4 Gy and MCF-7+4 Gy groups (P< 0.01), and there was also significant differences between the MCF-7-AktlRi+4 Gy and MCF-7-Aktl+4 Gy groups (P< 0.001). The cell apoptosis percentage in the Aktl interference model group was the largest, suggesting that Aktl genes controlled negatively the apoptosis of MCF-7 cells.4.4 Gene expressions related with autophagy and apoptosis in the MCF-7, MCF-7-Aktl and MCF-7-AktlRi cells after 4-Gy X-irradiationThe relative expressions of MAP1LC2B/GAPDH. Bcl-2/GAPDH and p53/GAPDH genes 4,8,16.24,48 h after 4-Gy X-irradiation were detected with real time PCR. The relative expressions of MAP1LC2B/GAPDH gene of MCF-7 cells in all groups 16 h after irradiation were the highest. There was significant difference in the relative expression of MAP1LC2B/GAPDH gene between the MCF-7+4 Gy, MCF-7-ktl+4 Gy, MCF-7-AktlRi+4 Gy and MCF-7+0 Gy groups 8 and 16 h (P < 0.05-P< 0.001). In addition to 4 h after irradiation, there was significant difference between in the relative expression of MAP1LC2B mRNA between the MCF-7-AktlRi, MCF-7+0 Gy, MCF-7+4 Gy and MCF-7-ktl+4 Gy groups (P< 0.001).With the extension of time, in addition to the MCF-7+0 Gy group, the relative expressions of Bcl-2 gene of MCF-7 cells in other three groups presented a increasing tendency. The relative expression of Bcl-2/GAPDH gene in the MCF-7+4 Gy, MCF-7-Aktl+4 Gy and MCF-7-AktlRi+4 Gy groups at different time points after irradiation was significantly lower than that in the MCF-7+0 Gy group (P< 0.05-P < 0.001). There was significant difference in the relative expression of Bcl-2 mRNA between the MCF-7-ktlRi+4 Gy, MCF-7+0 Gy, MCF-7+4 Gy and MCF-7-Aktl+ 4 Gy groups (P< 0.001).The relative expression of p53/GAPDH mRNA in the MCF-7+4 Gy, MCF-7-Aktl+4 Gy and MCF-7-AktlRi+4 Gy groups at different time points after irradiation all was significantly lower than that in the MCF-7+0 Gy group (P< 0.05-P< 0.001). There was significant difference in the relative expression of p53 mRNA between the MCF-7-AktlRi+4 Gy, MCF-7+0 Gy, MCF-7+4 Gy and MCF-7-Aktl+4 Gy groups (P< 0.001).The results show that after Aktl gene was interfered, the relative expression of MAP1LC2B mRNA related with autophagy increased, that of Bcl-2 mRNA related with apoptosis also increased, while that of p53 mRNA declined tendenciously, suggesting that Aktl gene in the MCF-7 cells negatively regulated the cell autophagy and apoptosis.