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 adenocarcinoma 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 addition of 1 μM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco‐2 cells produced three IGFBPs of Mr: 34,000 (IGFBP‐2), 24,000 (IGFBP‐4), and 32,000 (IGFBP‐6). The concentrations of IGFBP‐2 and IGFBP‐4 decreased by 48 ± 6 and 70 ± 13%, respectively, whereas that of IGFBP‐6 increased by 698 ± 20% with 1 μM tRA. tRA decreased mRNA levels of 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 mRNA or peptide. To examine if endogenous IGFBP‐6 inhibits cell proliferation, Caco‐2 cells were transfected with an IGFBP‐6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP‐6 grew more slowly than vector controls and achieved final densities 30–55% lower than those of vector controls. Accumulation of IGFBP‐6 mRNA and concentrations of IGFBP‐6 peptide in conditioned media were increased by 200–250 and 220–250%, respectively, in the IGFBP‐6 clones compared with controls. Increased expression of IGFBP‐6, which has a high binding affinity for IGF‐II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP‐6‐mediated disruption of the IGF‐II autocrine loop in these colon cancer cells. J. Cell. Physiol. 190: 92–100, 2002. © 2002 Wiley‐Liss, Inc.