Endocrine Abstracts

The insulin-like growth factors together with the IGF receptors, IGFBPs and IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation in vivo and in vitro. Our previous studies demonstrated that overexpression of IGFBP-2 in colon cancer cells (Caco-2) was associated with increased cell proliferation and tumorigenesis. The wild-type Caco-2 cells express and secrete IGFBP-2, IGFBP-4, IGF1R and growth hormone receptors but not IGFBP-3. Therefore, the aim of the present study was to investigate the effects of IGFBP-3 on colony formation and apoptosis in colon cancer cell lines. Caco-2 cells were stably transfected at day 4 with expression vector (pcDNA3) containing an IGFBP-3 cDNA in the sense orientation and vector alone (mock). Clones of transfected Caco-2 cells were selected by detecting IGFBP-3 gene expression using RT-PCR, the 367bp transcript corresponding to IGFBP-3 was identified. The IGFBP-3 concentrations in the conditioned media were also detected by immunoblot analysis. Anchorage independent growth was examined in a methylcellulose based clonogenic assay in the presence of exogenous rhIGF-I. Colonies were then counted under inverted microscopy. Apoptosis was analysed by nuclear DNA fragmentation (TUNEL). The results showed that colony formation was significantly reduced (50%) in the IGFBP-3 transfected cells compared to mock and wild-type cells. In the presence of exogenous rhIGF-I (100ng/ml) colony formation was increased in transfected and wild-type cells. Apoptosis studies showed that expression of IGFBP-3 in the sense transfected Caco-2 cells increased apoptosis compared to the mock and wild-type cells. Exogenous rhIGF-I (100ng/ml) decreased apoptosis both in the transfected and wild-type cells. In conclusion, endogenous IGFBP-3 expression in Caco-2 cells inhibits colony formation and increases apoptosis. IGFBP-3 appears to decrease the tumourigenic potential of colon cancer cells in vitro.