Effects of 1,25 dihydroxyvitamin D3 and its analogues on induction of apoptosis in breast cancer cells.
Vitamin D derivatives have been shown both to inhibit the proliferation of cultured breast cancer cells and to cause regression of experimental mammary tumours in vivo. We have investigated the ability of several vitamin D analogues to promote the regression of experimental rat mammary tumours. Our results revealed that one vitamin D compound in particular, EB1089 (1(S),3(R)-dihydroxy-20(R)-5'-ethyl- 5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z ),7(E) ,10(19)-triene), was highly effective at inhibiting tumour progression, without causing a significant rise in serum calcium concentration. Tumour regression occurs when the rate of cell death is greater than the rate of cell proliferation. Apoptosis (programmed or active cell death) is an active, energy-dependent process in which a distinct series of biochemical and molecular events leads to the death of cells by specific signals. We have examined effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2(D)3) and the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells. The effects of the vitamin D compounds on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and p53 were examined by Western analysis. In MCF-7 cell cultures treated for six days with 1,25(OH)2(D)3 or EB1089 (1 x 10(-8) M), bcl-2 protein was reduced in comparison to control levels, whereas p53 protein was increased. In addition, the p21 protein, whose gene WAF-1 is induced by wild type p53, was also increased by both vitamin D compounds. Using Northern analysis, it was observed that 24-h treatment of MCF-7 cells with 1 x 10(-8) M 1,25(OH)2(D)3 or EB1089 resulted in an induction of TRPM-2 (clusterin) mRNA, a gene associated with onset of apoptosis in the involuting prostate. Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal deoxynucleotidyl transferase (TdT) assay, 3'-OH DNA breaks indicative of DNA fragmentation were detected histochemically in MCF-7 cells treated with 1 x 10(-8) M 1,25(OH)2(D)3 or EB1089 for four days prior to fixation and TdT reaction. Further evidence of apoptosis was obtained following six days treatment of MCF-7 cell cultures with 5 x 10(-8) M 1,25(OH)2(D)3 or EB1089, utilizing a cell death ELISA assay, which measures the presence of histone-associated oligonucleosome complexes generated from DNA fragmentation. Taken together our findings indicate that vitamin D derivatives may play a role in regulating the expression of genes and protein products implicated in apoptosis.
Department of Clinical Biochemistry, St. George's Hospital Medical School, London, U.K.
S Y
SY
James
A G
AG
Mackay
K W
KW
Colston
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000737:chemistry / Q000473:pathology*; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000627:therapeutic use; D002277:Carcinoma / Q000737:chemistry / Q000473:pathology*; D051152:Clusterin; D053938:DNA Fragmentation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy; D018832:Molecular Chaperones; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D051381:Rats; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D000970:Antineoplastic Agents; C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016159:Tumor Suppressor Protein p53; D002117:Calcitriol; C078903:seocalcitol