EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro.
1. Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea-induced rat mammary tumours in vivo were investigated. 2. At a dose of 0.5 microg kg(-1) body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 microg kg(-1)) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death. 3. Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3' DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 microg kg(-1)) for 14 days. 4. Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF-7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089. 5. The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and bax were examined by Western analysis. In MCF-7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1 x 10(-8) M), bcl-2 protein levels were decreased in a time-dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl-2/bax ratio favouring increased susceptibility of MCF-7 cells to undergo apoptosis. 6. These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.
James
S Y
SY
Division of Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School London.
Mercer
E
E
Brady
M
M
Binderup
L
L
Colston
K W
KW
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D053938:DNA Fragmentation; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051381:Rats; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000031:analogs & derivatives / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D014807:Vitamin D; D002117:Calcitriol; C078903:seocalcitol