Radiosensitizing Effects Of Arsenic Trioxide On Human Breast Cancer MCF-7 Cell Line Exposed To 89 Strontium Chloride

Objectives: To investigate morphological changes, cell proliferation inhibition, cell cycle distribution, cell apoptosis, changes of bcl-2 and bax gene expression of human breast cancer MCF-7 cell line treated with arsenic trioxide (As₂O₃) alone and combined with strontium chloride((89)^SrCl₂) internal irradiation in vitro, to explore the practicability of As₂O₃ used as a useful radiosensitizer, this may provide the experimental reference to the treatment of osseous metastasis foci of breast cancer using As₂O₃ combined with (89)^SrCl₂.Methods: (1) MCF-7 cells were grown in RPMI 1640 medium containing 10% FBS and 100U/ml penicillin and streptomycin(complete medium) at 37℃in 5%CO2. Cells were propagated according to protocol given by the American Type Culture Collection. (2) The MTT method was used to determine the proliferation of MCF-7 cells treated by As₂O₃. The survival curve of cell, the horizontal axis and longitudinal axis representing concentration of As₂O₃ and cell survival ratio respectively, was drew with excel software. According to the curve, the 50% inhibiting concentration (IC50) was calculated, and then the appropriate concentration (20% IC50) of As₂O₃ was selected for subsequent experiments. (3) Experiment was divided into four groups: control group, As₂O₃ group, (89)^SrCl₂ group and As₂O₃ combination with (89)^SrCl₂ group. 24h after treatment, different experiments were conducted. (4) The morphological changes of cells were observed through inverted microscope and the records of pictures were captured by digital camera. (5) Clonogenic assay was carried out to calculate survival fractions (SFs) of cells, dealt with the 20% IC50 of As₂O₃ combined with (89)^SrCl₂, and exceeded 50 cells were considered as one clone. Survival curve was fitted with single-target multi-hit model, X-axis and Y-axis representing absorbed dose and SF respectively. Then the sensitization enhancement ratio(SER) was calculated. (6) The cell cycle and apoptosis rates of cells were detected by flow cytometry (FCM). (7) The expressions of bcl-2 and bax mRNA were examined by semi-quantitative RT-PCR. (8) The activities of Bcl-2 and Bax protein were determined by Western blot. (9) The expression of Bcl-2 and Bax were determined by immunocytochemistry .Results: (1) The inhibition effect of As₂O₃ on MCF-7 breast cancer cells was increased concomitantly with the elevation of concentrations. Its IC50 at 24h was 11.7μM. (2) Typical morphological changes were observed through the optical microscope in (89)^SrCl₂ group and combination group. Cell proliferation of combination group was obviously inhibited.(3) It was showed that, through survival curves and related parameters,both the average lethal dosage(D0) and quasithreshold dose(Dq) of the irradiated MCF-7 cells pretreated with As₂O₃ were less than that of the cells without pretreatment. The SER were 1.26 and 1.43 respectively. (4) The results of cell cycle analysis showed the four groups had different cell cycle distribution.The percentage of G2/M phase cells increased after treated with As₂O₃ or (89)^SrCl₂ or As₂O₃ combining with (89)^SrCl₂. The percentage of G2/M phase cell in combination group was significantly higher than that of (89)^SrCl₂ group(P<0.05). (5)There was the statistical significance, the results of apoptosis analysis showed that the death cells and cells at early stage of apoptosis in combination group were increased compared with in (89)^SrCl₂ group(P<0.05). (6) The results of RT-PCR and Western Blot showed that, compared to those of (89)^SrCl₂ group, expression of bcl-2 gene was decreased in combination group, but it was not so with bax gene. It was therefore clear that the value of bcl-2/bax in combination group was less than that of (89)^SrCl₂ group(P<0.05). (7) The foundings from immunocytochemistry exhibited that Bcl-2 and Bax protein mainly located in the cell cytoplasm as dispersed small particles. The expression of intracellular Bcl-2 was reduced after treated with As₂O₃ or (89)^SrCl₂, the most weak level of Bcl-2 was further observed in combination group. Similar to the results of Western Blot, the expression of intracellular Bax in any group did not showed obvious change. Conclusion: 1²Î¼M of As₂O₃ could increase the radiosensitvity of human breast cancer MCF-7 cell line by inducing G2 phase delay. The lethal effect of (89)^SrCl₂ on MCF-7 cells thus was enhanced concomitantly with As₂O₃ pretreatment. The mechanism could be involved in the rising of the apoptosis rate of cells owing to the reduction of bcl-2/bax rate.