Relationship Between Induction Of Apoptosis By 1,25-dihydroxyvitamin D₃ In Breast Cancer Cell And Apoptosis-related Proteins

Backgroud 1 ,25-Dihydroxyvitamin D3 (1,25D) is the biologically active form of vitamin D3, acts through the nuclear vitamin D3 receptor (VDR).In addition to its calcium regulatory properties, 1,25D inhibits proliferation and induces differentiation of a variety of malignant cells . Although it is clear that 1,25D inhibits the growth of both estrogen receptor-positive and estrogen receptor-negative breast cancer cells and promotes their differentiation in vitro and in vivo, minimizing the tumor's cell number and volume, It is unclear 1,25D exert potent antiproliferative effects by inhibiting growth or by inducing apoptosis .The knowledge of the precise molecular mechanism by which 1,25D and the VDR mediate apoptosis is poorly characterized. Objective To investigate the effect of 1, 25-dihydroxyvitamin D3 on growth and apoptosis and its possible mechanism in breast cancer cell line MCF-7.We observed the morphologyical and biochemical markers of apoptosis in MCF-7 cells to determine if the apoptosis occurred after treatment MCF-7 with 1,25D, and detected the expression levels of apoptosis-related proteins VDR,ER ,bcl-2,bax to determine the relationship between their changes and apoptosis in this study. Methods Estrogen receptor(ER)-positive human breast carcinoma cell line MCF-7 was treated by 1,25-dihydroxyvitamin D3 with concentration of 10-10, 10-9,10-8,10-7 mol/L for the periods of 24,48,72,96h in vitro.Cell proliferation was evaluated by MTT assay,distribution of cell cycle and rateapoptosis were determined by flow cytometry, The morphologic changes of apoptotic cells were examined by means of light and electron microscopy. The number of the apoptotic cells was detected by In Situ Cell Death Detection Kit and HE .The expression of VDR, ER, Bcl-2 and Bax were examined using immunohistochemistry S-P method in different time. Results (1) 1,25D could inhibit the growth after treatment MCF-7 with 10-9 mol/L 1.25D for 72h , 10-8 mol/L 1,25D for 48h and 10-7mol/L for 24h, inhibiting growth in a dose-dependent and time-dependent manner. (2) Apoptosis was detected by HE, microscope, flow cytometry and in situ cell death detection, Fluorescein assay. MCF-7 cells treated with 10-7 mol/L 1,25D for 48h or 10-8 mol/L for 72h exhibites characteristics of apoptosis, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and DNA fragmentation,and increasing of the apoptotic cells is in a time-dependent and concentration-dependent manner. (3) Treatment MCF-7 cells with 1,25-dihydroxyvitamin D3 resulted in time-and concentration-dependent decrease in Bcl-2,ER and increase inVDR, Bax proteins. Conclusions The apoptosis induced by 1, 25-dihydroxyvitamin D3 is related to the increase of VDR protein and the decrease of Bcl-2 and ER protein.