| Objective: The incidence of breast cancer is increasing yearly all over the world,Now it ranks first in female malignant tumor in our country and it is an important factor that threatens women's health. From the seventies of the 20 th century, American oncologist Fisher proposed that breast cancer is a systemic disease, Systemic comprehensive treatment of breast cancer has begun to receive attention, then formed a comprehensive treatment mode that based on local treatment and combined with radiotherapy, chemotherapy and endocrine therapy as adjuvant therapy. In recent years, with the deepening of the research on the biological characteristics of tumor, biological therapy has been promoted to an important position, including targeted therapy, gene therapy, immune cell therapy and so on. Interferon alpha(IFN-?) as an earlier application of immune regulator, also earlier applied to the clinical treatment of tumor, now it has been used in the treatment of over 14 types of cancer and has received a good effect and less toxic side effects, but it is still not being used in breast cancer. In this paper, we studied the effect of IFN-? combined with chemotherapy on breast cancer cell line to explore whether there is a synergistic anti proliferation effect between them. Then we studied the changes of the rrelated gene expression to explore the mechanism of anti tumor proliferation. This study provides a theoretical basis to IFN-? as an immune regulatory factor in the comprehensive treatment of breast cancer.Methods: Firstly, we used different concentrations of IFN-? and anti-cancer agents adriamycin(ADM), cis-diamine dichloroplatinum(CDDP) in breast cancer cell lines of MDA-MB-453, MDA-MB-231, MCF-7, BR3 and HCC-1937. And the optical density of each hole under 450 nm were measured by the WST-8 using enzyme linked immunoassay instrument, then we draw the cell growth curve to calculate the cell proliferation of each group and compare the sensitivity of different breast cancer cell lines to IFN-?. In the next step, we selected the breast cancer cell line which is sensitive to the effect of IFN-? on anti-proliferation, then we cultured the cells in the solution combined with IFN-? and anti-cancer agents for 96 h, and the optical density of each hole under 450 nm were measured by the WST-8 using enzyme linked immunoassay instrument, then we calculated whether there is a synergistic effect on breast cancer cells with IFN-? and anti-cancer agents by Calcu Syn, Combination index(CI) values at respective fractions affected(Fa), which showed relative suppression levels of cell viability, were calculated based on the WST assay. CI<1, CI=1 and CI>1 indicate synergistic, additive and antagonistic actions, respectively. The group which existed a synergistic effect were selected. Then we detected the cell cycle distribution of cell proliferation by flow cytometry. Finally, we detected the changes of TRAIL, Cyt-c and Bax in cell expression after the use IFN-? and anti-cancer agents alone and combined by q RT-PCR.Results: 1 The anti-proliferation effect of IFN-?, ADM and CDDP in breast cancer cell lines The breast cancer cell lines of MDA-MB-453, MDA-MB-231, MCF-7, BR3, HCC-1937 were seeded in 96 hole plate and were cultured with different concentrations of IFN-?(0, 3ng/ml, 10ng/ml, 30ng/ml, 100ng/ml, 300ng/ml, 1000ng/ml,) different concentrations of ADM(0, 0.01?M, 0.03?M, 0.1?M, 0.3?M, 1?M, 3?M), different concentrations of CDDP(0, 0.1?M, 0.3?M, 1?M, 3?M, 10?M, 30?M) respectively. Cell viabilities were assessed with a WST-8 kit after the effect of each drugs. The results showed that the anti proliferative effect induced by the different concentrations of IFN-? observed on the breast cancer cell lines of MCF-7, MDA-MB-453 and MDA-MB-231 is obviously, meanwhile, dose dependent effect exist in a given range of concentration 0-1000ng/ml, the difference has statistical significance(P<0.05) Moreover, the anti proliferation effect of IFN-? on breast cancer cell line MCF-7 was more sensitive than that of MDA-MB-231 and MDA-MB-453, and the difference was statistically significant(P < 0.05). The anti proliferative effect of different concentrations of IFN-? on breast cancer cell lines HCC-1937 and BR3 was not obvious, and the difference was not statistically significant(P > 0.05). Different concentrations of ADM and CDDP have anti proliferation effect on different breast cancer cell lines, and the anti proliferation effect was enhanced with the increase of drug concentration, the difference was statistically significant(P<0.05). 2 The anti proliferative effect of IFN-? combined with ADM or CDDP on breast cancer cell line MCF-7 was synergistic The proliferative activity of breast cancer cell line MCF-7 was set to 100% in the control group, Cells were divided into horizontal and vertical concentration gradients. Horizontal concentration gradients was set according to IFN-? concentration gradient(0, 10ng/ml, 30ng/ml, 100ng/ml, 300ng/ml) and the vertical concentration gradients was set according to ADM or CDDP concentration gradient(ADM: 0, 0.003?M, 0.01?M, 0.03?M, 0.1?M, 0.3?M, 1?M. CDDP: 0?0.03?M, 0.1?M, 0.3?M, 1?M, 3?M, 10?M, 30?M). After 96 h of culturing, the optical density of each hole under 450 nm were measured by the WST-8 kit, then we calculate the cell proliferation of each group and draw the cell growth curve when cells were treated with IFN-? combined with ADM, and cultured for 96 hours. Then calculated by Calcu Syn, when Fa is between 0.2-0.8, the CI value is less than 1. It shows that there is a synergistic effect. There is also a synergistic effect when IFN-? and CDDP were simultaneously applied to the cell, when Fa is between 0.2-0.8, the CI value is less than 1. It shows that there is a synergistic effect between IFN-? and CDDP. 3 The distribution of cell cycle in breast cancer cell line MCF-7 will change when apoptosis induced by IFN-? combined with ADM or CDDP After the treatment of IFN-?(300ng/ml), ADM(0.03?M) and CDDP(0.3?M) in breast cancer cell line MCF-7 for 96 h, cell cycle analyzed by flow cytometry. The proportion of sub G1 in the three groups was higher than that in the control group(2.85% in the control group, 13.83% in the IFN-? group, 7.98% in the ADM group and 8.53% in the CDDP group), the difference was statistically significant(P<0.05). The results showed that IFN-??ADM and CDDP had the effect of inducing apoptosis on tumor cells at the designed concentration. After the treatment of IFN-? combination with ADM or CDDP in breast cancer cell line MCF-7 for 96 h the proportion of sub G1 in the combined treatment group was higher than that in the single treatment group.(29.49% in the IFN-? combined with ADM group, 26.79% in the IFN-? precedes ADM group, 25.17% in the IFN-? combined with CDDP group, 26.11% in the IFN-? precedes CDDP group). Compared with the control group and single drug group, the difference was statistically significant(P<0.05). 4 The expression of Bax,Cyt-C and TRAIL can be regulated at the transcriptional level in human breast cancer cell line MCF-7 that was terated by IFN-? combined with CDDP The breast cancer cell line MCF-7 was treated with IFN-?, CDDP and their combination for 96 hours. Set the dosing sequence for secondary variables(12h interval). Then we extracted RNA and reverse transcribed it to c DNA. Bax, Cyt-C and TRAIL expression level was detected by q RT-PCR. Results showed that the expression of Bax, Cyt-C and TRAIL were significant up regulated in the group of IFN-? combined with CDDP and IFN-? precedes CDDP compared with single drug group and blank control group. The difference was statistically significant(P < 0.05). It indicated that the combination of IFN-? and CDDP could increase the release of Cyt-C by up regulating the expression of Bax, thus enhancing the mitochondrial apoptotic pathway. The expression of TRAIL in the combined group also up regulated, the difference was statistically significant(P<0.05). It indicated that the combination of IFN-? and CDDP could increase death receptor mediated apoptosis pathway(Fas-Fas L) by up regulating the expression of TRAIL.Conclusion: 1 The anti proliferative activity of IFN-? was different in different breast cancer cell lines, and the MCF-7 is more obvious, and it has dose dependent in a certain range. 2 IFN-? combined with ADM or CDDP can significantly enhance the anti proliferation effect of anti-cancer agents on MCF-7 cell line, and there is a synergistic effect between the two. 3 IFN-? combined with ADM or CDDP increase sub-G1-phase population in MCF-7. 4 IFN-? can induce apoptosis and enhance the effect of anti-cancer agents by up regulate the express of Bax, Cyt-C, and TRAIL in MCF-7 cell line, and can activate mitochondrial apoptosis pathway and death receptor mediated apoptosis pathway. |
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