The aims of the present study were to compare the ability of various synthetic analogues of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25-(
OH)(2)D(3)] to inhibit proliferation of HT-29 cells, a human
colon adenocarcinoma cell line. HT-29 cells were incubated for 144 h with various concentrations (0-100 nM) of 1 alpha,25-(OH)(2)D(3), or the analogues
EB1089, CB1093 or 1 beta,25-(OH)(2)D(3). All these analogues except 1 beta,25-(OH)(2)D(3) inhibited cell proliferation, but relative potencies and efficacies of
EB1089 and CB1093 were much greater than that of the native
vitamin. Cells grew in serum-free medium, reaching a plateau density at day 10 of culture, and addition of 10 nM 1 alpha,25-(OH)(2)D(3) or 1 beta,25-(OH)(2)D(3) did not alter the long-term growth characteristics of HT-29 cells. However, cells treated with 10 nM
EB1089 or CB1093 grew at a rate slower than control and reached final densities that were 53+/-1 and 36+/-2% lower than control, respectively. Immunoblot analysis of serum-free
conditioned medium using a monoclonal anti-
insulin-like growth factor-(
IGF)-II antibody showed that both 10 nM
EB1089 and CB1093 markedly inhibited secretion of both mature 7500 M(r) and higher M(r) forms of
IGF-II.
Ligand blot and immunoblot analyses of
conditioned media revealed the presence of IGFBPs of M(r) 24,000 (IGFBP-4), 30,000 (glycosylated IGFBP-4), 35,000 (IGFBP-2) and 32,000-34,000 (IGFBP-6). The level of
IGFBP-2 was decreased by 42+/-8 and 49+/-7% by 10 nM
EB 1089 and CB1093, respectively, compared to controls.
IGFBP-6 was increased approximately twofold by
EB1089 and CB1093, and exogenously added
IGFBP-6 inhibited HT-29 cell proliferation. These results suggest that inhibition of HT-29 cell proliferation by
EB1089 and CB1093 may be attributed, at least in part, to the decreased secretion of
IGF-II. The increase in
IGFBP-6 concentration coupled with its high affinity for
IGF-II may also contribute to decreased cellular proliferation by an indirect mechanism involving sequestration of endogenously produced
IGF-II.