Study On Biological Functions Of Tamoxifen And Relevant Mechanism In Breast Cancer Cells

Purpose: The current study was designed to investigate the effect of Tamoxifen oncell growth, apoptosis, migration and invasion as well as radiosensitivity in human breastcancer cells via vitro cell model. These findings will provide experimental evidence forTamoxifen as a new method in radiotherapy of breast cancer.Methods: Human breast cancer cell lines, MDA-MB-435and MCF-7were choosenas the study obeject.1) We used CCK8assay to test the influence of Tamoxifen on cell viability atdifferent concentrations.2) Scratch healing assay and the transwell experiment were used to study theinfluence of Tamoxifen on the cell migration and invasion.3) Flow cytometry was applied to analyze cell cycle progression and AnnexinV-FITC cell apoptosis kit was used detect apoptosis and necrosis. JC-1staining was usedto test the change of mitochondrial membrane potential.4) ROS content and ATP content were also detected by related kits.5) Western blot was used to detect the changes of protein expression.6) Colony formation method was employed to study the effects of Tamoxifen onradiosensitivity of MCF-7cells.7) After irradiated with X-ray, flow cytometric analysis was performed to assess thealteration of cell cycle distribution and apoptosis caused by radiation. Western blot assaywas used to detect the expression of cell cycle and apoptosis-relevent proteins.8) Comet assay was used to analyze DNA damage and repair. Immune fluorescencein situ hybridization was employed to detect γ H2AX foci. Fluorescent probe wasperformed to assess ROS.9) The recombinant plasmid pcDNA3-BTG1was transfected into the breast cancercell lines, MCF-7, via Lipofectamine transfection assay. 10) After irradiated, the expression of BTG1mRNA and protein were monitored byRT-PCR and Western blot assay, respectively.Results:1) Tamoxifen inhibited the growth and proliferation of MDA-MB-435and MCF-7cells in dose-and time-dependent way, the IC50s were2.3μmol/L and1.7μmol/L forMDA-MB-435cells at24h,48h repectively and1.2μmol/L for MCF-7cells at24h.2) Tamoxifen induced G2/M cell cycle arrest via down-regulation ofCDK1/cyclinB1pathway.3) Tamoxifen promoted apoptosis through enhancing mitochondrial ROS generation,4) We also identified that Tamoxifen increased sensitivity to irradiation. The SER forMCF-7was1.54.5) After irradiated with X-rays, Tamoxifen increased the radiosensitivity of MCF-7cells, along with a G2/M arrest, induction of apoptosis.6) After irradiated with X-rays, the "comet" tail length and tail moment significantlyincreased in Tamoxifen group cells. The number of γ H2AX foci also increased inTamoxifen group cells. The ability of DNA damage repair was decreased in Tamoxifengroup cells.7) After irradiated with X-rays, ROS was increased linearly with doses ofTamoxifen, along Tamoxifen group cells were improved the radiosensitivity.8) After irradiated with X-rays, BTG1mRNA was increased along Tamoxifen groupcells were improved the radiosensitivity.Conclusion:Tamoxifen increases cell sensitivity to X-rays, which may be related with a change ofcell cycle progression and apoptosis induction. BTG1may participate in the regulationDNA damage repair, ROS generation and scavenging free radicals. In conclusion, thisstudy provides evidence for Tamoxifen in breast cancer treatment alone or combined withirradiation, and indicates that targeting BTG1may be an effective strategy for breastcancer therapy.