| BACKGROUND AND OBJECTBreast cancer is the most commmon cancer in women in the world. More than one million new cases were noted erery year. Radiation therapy is a key strategy for the treatment of many epithelial carcinomas. In breast cancer, post-operative radiotherapy (RT) is one of the most commonly used and elective strategies for local control. But there are many limitations in radiation therapy, such as tumor recurrence alter radiotherapy, radiation resistance, and side effects.Therefore, enhancing radiosensitivity of tumors and reducing radiation side effect is one of the major medical challenges.Berberine, an isoquinoline derivative alkaloid. It has been reported that berberine can be used as an anti-diarrhea, anti-arrhythmia and anti-inllammatory agent. Additionally, berberine has also been shown to have antitumor effects on many cancer cell lines. Mainly the mechanism includes inhibiting the proliferation, invasion, and metastasis of tumor cells, and inducing the cell apoptosis. It has been reported that berberine significantly reduces the incidence of radiation-induced lung injury and intestinal injury. Berherine has been shown to radiosensiti/.e lung cancer cells by inducing autophagy. and esophageal cancer cells by the downregulation of the homologous recombination repair protein, RAD51. The present study aimed to investigate the effects of the concomitant administration of berberine and radiation on breast cancer.METHODSThe effects of berberine on the radiosensitivity of MCF-7and MDA-MB-468cells were evaluated by using cell clonogenic assays. The cells were treated with the vehicle control (DMSO) or15μiM berberine for24h. The cells were then irradiated using a Faxitron Cabinet X-ray System (Faxitron X-ray Corp., Wheeling, IL, USA) to deliver the indicated doses (0,1,2,3and4Gy) at room temperature. Cells pre-treated with berberine or dimethyl sulfoxide (DMSO) for24h were irradiated using a Faxitron Cabinet X-ray System to deliver the indicated doses (0,1,2,3and4Gy). After incubation for14-21days to allow the formation of macroscopic colonies, the plates were fixed with methanol and stained with Giemsa. Colonies containing at least50cells in size were counted. The fraction surviving a given X-ray dose was calculated based on the survival of non-irradiated cells treated with the vehicle or berberine. Survival (S) data after a radiation dose (D) were fit by a weighted, stratified, linear regression.Changes in cell cycle distribution were determined by flow cytometry. Cell cycle analysis by flow cytometry. Cells were harvested with trypsin, washed with phosphate-buffered saline (PBS) and then stained with buffer including50μg/ml propidium iodide (Sigma-Aldrich) for30min at room temperature. For fluorescence-activated cell sorting (FACS) analysis, data were collected using a FACSCalibur (BD Bioscience, San Jose, CA, USA) flow cytometer and analyzed by ModFit (Verity, Topsham, ME, USA). The cell-cycle distribution was evaluated by counting>20,000cells for each sample.y-H2AX foci were detected by immunofluorescence staining. Immunofluo-rescence staining for y-H2AX. Cells were grown on coverslips in6-well plates and treated with X-ray and/or berberine151μM. At specific times, cells were treated, The slides were then examined on a Leica fluorescent microscope. Images were captured by a charge coupled device camera. For each treatment condition, y-H2AX foci were counted in at least100cells from randomly captured images.The levels of Ku70, Ku86and RAD51proteins were evaluated by western blot analysis, in the MCF-7and MDA-MB-468cells treated with15μM erberine for24-48h. In the MCF-7cells treated with15μM berberine for24h prior to6Gy irradiation, the levels of Ku70and Ku86and RAD51protein were evaluated at the indicatedtime-points (0,2,6and24h), in comparision with the cells treated with irradiation alone.RESULTS We observed that berberine increased the MCF-7and MDA-MB-468cell radiosensitivity with cell clonogenic assays. Berberine sensitizes breast cancer cells to IR. In the MCF-7cell line, clonogenic assay revealed that berberine pre-treatment (15μM,24h) reduced the surviving fraction at2Gy (SF2) of the irradiated cells from31.2±0.8to20.5±3.9%in the cells treated with radiation alone. The combinationtreatment caused a reduction of approximately30%in the SF2(P=0.002). The data were further analyzed according to the linear quadratic model;the cells treated with the combination treatment, respectively,leading to survival curves which were significantly different(P<0.01) as tested with the linear regression analysis. A similar response was observed in the other human breastcanccr cell line, MDA-MB-468, with the SF2being reducedto20.1±0.6%when the irradiated cells were pre-treated withberberine at15μM for24h, in comparison with33.1±3.1%for the cells treated with radiation alone (P<0.01). According to the linear regression analysis, a statistical difference between2groups was obtained (P<0.001).The radiation-induced G2/M cell cycle delay was reduced in the MCF-7cells pre-teated with berberine. Berberine treatment causes cell cycle arrest. We performed flow cytometry analysis of the cells treated with15μM berberine or DMSO for24h by propidium iodide staining to evaluate the effect of berberine treatment on the cell cycle progression of human breast cancer cells. Berberine treatment induced an increase in the proportion of cells in the G1phaseand a marked decrease in the proportion of cells in the S phase in comparison with the control MCF-7and MDA-MB-468cells. When the MCF-7cells were exposed to6Gy irradiation alone for8-24h, a significant cell cycle arrest in the G2/M phase was observed, with a decrease in the percentage of cells in the C0/G1phase and S phase. Following pre-treatment with berberine. the radiation-induced G2/M phase arrest did not occur within24h alter irradiation, with an increase in the percentage of cells in the G0/G1phase compared with irradiation alone.Berberine pre-treatment prolonged the persistence of DNA double-strand breaks in the MCF-7cell line. In the MCF-7cells not pre-treated with berberine. the majority of the γ-H2AX foci cleared4h following exposure to1.0Gy of X-ray radiation. By contrast, a delayed clearance of the X-ray-induced γ-H2AX foci was observed in the MCF-7cells pre-treated with berberine (15μM). These results indicate that berberine pre-treatment radiosensitizes the cancer cells via the impairment of the repair of X-ray-induced DSBs.In comparison with the control cells, the protein levels of RAD51were decreased in the MCF-7and MDA-MB-468cells treated with berberine, and in the cells pre-treated with15μM berberine for24h, the level of RAD51protein decreased significantly at the indicated time-points (0,2,6and24h) following X-ray exposure. However, these changes were not observed in the cells treated with irradiation alone.CONCLUSIONSIn conclusion, berberine sensitizes human breast cancer cells to ionizing radiation by inducing cell cycle arrest and the downregulation of the homologous recombination repair protein, RAD51. Berberine may be a promising radiosensitizer for the treatment of breast cancer. |
PDF Full Text Request