Inhibition of caco-2 cell proliferation by all-trans retinoic acid: Role of insulin-like growth factor binding protein-6

The present study examined the effects of all-trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco-2 human colon adenoc arcinoma cells. tRA inhibited Caco-2 cell proliferation in a dose-dependent manner, with a 40 +/- 2% decrease in cell number observed 48 h after the a ddition of 1 muM tRA. Ligand blot analysis of IGFBPs in conditioned media r evealed that Caco-2 cells produced three IGFBPs of M-r: 34,000 (IGFBP-2), 2 4,000 (IGFBP-4), and 32,000 (IGFBP-6). The concentrations of IGFBP-2 and IG FBP-4 decreased by 48 +/- 6 and 70 +/- 13%, respectively, whereas that of I GFBP-6 increased by 698 +/- 20% with 1 muM tRA. tRA decreased mRNA levels o f IGFBP-2 and IGFBP-4 by 20 +/- 3 and 50 +/- 8%, respectively, whereas tRA increased IGFBP-6 mRNA by 660 +/- 20%. tRA did not alter levels of IGF-II m RNA or peptide. To examine if endogenous IGFBP-6 inhibits cell proliferatio n, Caco-2 cells were transfected with an IGFBP-6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressin g IGFBP-6 grew more slowly than vector controls and achieved final densitie s 30-55% lower than those of vector controls. Accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide in conditioned media were increased b y 200-250 and 220-250%, respectively, in the IGFBP-6 clones compared with c ontrols. Increased expression of IGFBP-6, which has a high binding affinity for IGF-II, following tRA treatment suggests that the decreased proliferat ion caused by tRA may result, at least in part, from IGFBP-6-mediated disru ption of the IGF-II autocrine loop in these colon cancer cells. (C) 2002 Wi ley-Liss, Inc.