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

Abstract

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 microM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco-2 cells produced three IGFBPs of M(r): 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 microM 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.

Identification of IGFBP-6 as a significantly downregulated gene by β-catenin in desmoid tumors

“…Using an anti-IGFBP-6 antibody, an apporximately 32-kDa band was seen only in the CM of fascia ( Figure 4). The size of the band was consistent with that previously described for IGFBP-6 (Kim et al, 2002). None of the desmoid CM samples demonstrated any detectable bands.…”

Transcriptional activation of insulin-like growth factor binding protein 6 by 17β-estradiol in SaOS-2 cells

“…IGFBP-6 is expressed in many cell types, such as fibroblasts, myoblasts, smooth muscle cells, keratinocytes, and osteoblasts (Yan et al, 2001;Strohbach et al, 2008). Because of its binding preference, IGFBP-6 might regulate IGF-II-induced cell proliferation, differentiation, and survival (Bach et al, 1994;Dake et al, 2002;Kim et al, 2002;Gallicchio et al, 2003;Fu et al, 2007). Generally, IGFBPs play an IGF-dependent role in high affinity IGF binding.…”

Overexpression of mature insulin-like growth factor (IGF)-II leads to growth arrest in Caco-2 human colon cancer cells

“…Caco-2 cells were maintained and subcultured as previously described [9]. To determine the growth characteristics of individual clones, cells were plated in 24 well plates with DMEM/F12 containing 10% FBS.…”

“…Multiple isoforms of pro IGF-II (pIGF-II) resulting from alternative processing and glycosylation have been identified [7,8]. The human colon adenocarcinoma cell line, Caco-2, utilized in the present study, secretes both mature 7500 M r and higher M r forms of IGF-II (pIGF-II) [9,10], and IGF-II acts as an autocrine growth regulator of these cells [11,12]. We have previously observed that compounds which inhibit cancer cell proliferation also inhibit the production of mIGF-II and pIGF-II [10].…”