EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro

Abstract

1 E ects 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 mg kg 71 body weight, EB1089 caused signi®cant inhibition of tumour progression over the 28 day treatment period in the absence of a signi®cant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 mg kg 71 ) 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 mg kg 71 ) for 14 days. 4 E ects 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 e ects 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) 2 D 3 or EB1089 (1610 78 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.

Differential and antagonistic effects of 9-cis-retinoic acid and vitamin D analogues on pancreatic cancer cells in vitro

“…This is an interesting observation as the cells do express VDR (Kawa et al, 1996 and unpublished data) and are clearly responsive to the analogues, which have been shown to induce apoptosis in various other cell types (James et al, 1995(James et al, , 1998Danielsson et al, 1997). Furthermore, EB1089 and CB1093 not only failed to induce apoptosis by themselves, but were also able to completely block Pretreatment with EB1089 for 3 days was shown to be enough to completely block induction of apoptosis by 9cRA.…”

“…(Korsmeyer, 1999). For example, in breast cancer cells which undergo apoptosis in response to vitamin D compounds, downregulation of the anti-apoptotic protein Bcl-2 and an increased Bax/Bcl-2 ratio can be observed (James et al, 1998). This is also observed in HL-60 leukaemia cells, and is thought to be a mechanism whereby vitamin D derivatives potentiate induction of apoptosis by 9cRA (James et al, 1999).…”

1α,25-Dihydroxyvitamin D3 (calcitriol) and its analogue, 19-nor-1α,25(OH)2D2, potentiate the effects of ionising radiation on human prostate cancer cells

“…Observations that support combining 1,25(OH) 2 D 3 with ionising radiation include the following: (a) both IR and 1,25(OH) 2 D 3 induce apoptosis in prostate carcinoma cells by apparently distinct pathways (Welsh et al, 1995;Billis et al, 1998;James et al, 1998;Pirianov et al, 1999;Ding and Fisher, 2001;McGuire et al, 2001;Sheard, 2001;Liu et al, 2002); (b) other agents that induce cellular differentiation, for example, phenylacetate, platelet-derived growth factor and retinoic acid, are known to enhance the cytotoxic effects of IR (Bill et al, 1992b;Miller et al, 1997;Hoffmann et al, 1999); (c) EB 1089 has been shown to enhance the radiation sensitivity of breast carcinoma cells (Sundaram and Gewirtz, 1999). These findings strongly suggest that 1,25(OH) 2 D 3 may enhance the cytotoxic effects of IR on prostate cancer cells.…”

A phase II trial of the vitamin D analogue Seocalcitol (EB1089) in patients with inoperable pancreatic cancer

“…The synthetic analogue seocalcitol is 50 -200 times more potent than vitamin D in inhibiting growth and inducing differentiation of cancer cell lines Kissmeyer et al, 1997). In vivo studies in animal models have shown that seocalcitol can cause regression of established tumours, prevent the development of metastases, and prolong survival time in tumour-bearing animals (Colston et al, 1992(Colston et al, , 1997James et al, 1998;Lokeshwar et al, 1999;Nickerson and Huynh, 1999), with significant inhibition of tumour progression achieved at doses that do not cause significant hypercalcaemia (Colston et al, 1992). Furthermore, seocalcitol inhibits growth of pancreatic cancer cells in vitro (Zugmaier et al, 1996;Pettersson et al, 2000) and inhibits growth in vivo of pancreatic cancer xenografts in immunodeficient mice (Colston et al, 1997).…”