1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells.
1,25-Dihydroxyvitamin D3 [1,25(OH)2(D)3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. To complement data which documents the anti-proliferative effects of 1,25(OH)2(D)3, we assessed the role of apoptosis in vitamin D-mediated growth arrest of MCF-7 cells. Time course studies indicated that 100 nM 1,25(OH)2(D)3 significantly reduces MCF-7 cell numbers within 48 h of treatment. Morphological assessment demonstrated that MCF-7 cells treated with 1,25(OH)2(D)3 for 48 h exhibit characteristic apoptotic features, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and nuclear matrix re-organization. In situ end labelling with terminal transferase indicated that cells exhibiting apoptotic morphology in 1,25(OH)2(D)3-treated cultures were positive for DNA strand breaks. These morphological features of apoptosis were accompanied by an increase in the cell death rate assessed as soluble DNA-histone complexes indicative of DNA fragmentation. To complement the morphological data, we assessed the temporal expression of two proteins which have been associated with apoptosis in mammary cells and tumors. The steady state mRNA levels for TRPM-2/clusterin and cathepsin B mRNA were significantly up-regulated in MCF-7 cells treated with 1,25(OH)2(D)3 compared to control cells. Time-dependent increases in the expression of TRPM-2/clusterin and cathepsin B proteins were detected by Western blotting in 1,25(OH)2(D)3-treated cells. These findings indicate that, in addition to its anti-proliferative effects, 1,25(OH)2(D)3 activates the apoptotic cell death pathway in MCF-7 breast cancer cells.
Department of Biochemistry, University of Ottawa, Canada.
M
M
Simboli-Campbell
C J
CJ
Narvaez
M
M
Tenniswood
J
J
Welsh
D034961:Antigens, Nuclear; D017209:Apoptosis / Q000187:drug effects*; D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000473:pathology*; D002117:Calcitriol / Q000494:pharmacology*; D002277:Carcinoma / Q000473:pathology*; D002401:Cathepsin B / Q000032:analysis / Q000235:genetics; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D051152:Clusterin; D053938:DNA Fragmentation; D000431:Ethanol / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D008941:Spindle Apparatus; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D014408:Biomarkers, Tumor; C495242:CLU protein, human; D018797:Cell Cycle Proteins; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; C050045:NUMA1 protein, human; D009363:Neoplasm Proteins; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D000431:Ethanol; D002401:Cathepsin B; D002117:Calcitriol