The hormonally active form of
vitamin D3,
1,25-dihydroxyvitamin D3, and its two analogues,
EB 1089 and
CB 1093, are novel putative
anticancer agents with an interesting profile of induction of growth inhibition, differentiation, and apoptosis in
tumor cells. To study the signaling pathways mediating these events, we used two human
breast cancer cell lines: MCF-7 cells, expressing a wild-type
p53 tumor suppressor protein, and T47D cells, lacking a functional p53.
Vitamin D compounds induced a growth arrest followed by apoptosis in both cell lines at concentrations ranging from 1 to 100 nM, indicating that p53 is not necessary for growth-inhibitory effects induced by
vitamin D compounds. Surprisingly, apoptosis induced by these compounds occurred also independently of known
caspases. Inhibition of
caspase activation by overexpression of a
cowpox-derived
caspase inhibitor CrmA or by addition of inhibitory
peptides acetyl-
Asp-Glu-Val-Asp-
aldehyde (200 microM),
acetyl-Ile-Glu-Thr-Asp-aldehyde (50 microM), and Z-Val-Ala-D,L-Asp-fluoromethylketone (1 microM) showed no effect on the induction of growth arrest or apoptosis by
vitamin D compounds under assay conditions in which apoptosis induced by TNF or
staurosporine was effectively inhibited. Moreover, overexpression of
caspase-3 in MCF-7 cells had no sensitizing effect to
vitamin D compounds, and neither caspase-3-like
protease activity nor cleavage of a
caspase substrate
poly(ADP)ribose polymerase was detected in lysates from apoptotic cells following the treatment with these compounds. Contrary to CrmA, overexpression of an antiapoptotic
protein Bcl-2 in MCF-7 cells conferred a nearly complete protection from apoptosis induced by
vitamin D compounds. Taken together, these data indicate that
vitamin D compounds induce apoptosis via a novel
caspase- and p53-independent pathway that can be inhibited by Bcl-2. This may prove useful in the treatment of
tumors that are resistant to therapeutic agents that are dependent on the activation of p53 and/or
caspases.