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

Caco-2 cells produce both mature 7500 Mr and higher Mr forms of IGF-II (pro IGF-II - pIGF-II) and pIGF-II production is much higher than that of mature IGF-II (mIGF-II). The present study was performed to determine whether overexpression of mIGF-II or pIGF-II stimulates Caco-2 cell growth. A pIGF-II cDNA construct that expresses IGF-II including the E-domain was prepared by cloning a 1250 bp BamH I-Apa I human prepro IGF-II cDNA fragment downstream of the CMV promoter in pcDNA3. To create a mIGF-II cDNA construct which does not express the E-domain, two stop codons were inserted right after the glutamine residue of the D-peptide by site directed mutagenesis utilizing the pIGF-II cDNA expression construct as the template. Caco-2 cells were stably transfected with the mIGF-II or pIGF-II construct. Secretion of the mature and higher Mr forms of IGF-II into serum-free medium was higher in clones transfected with the mIGF-II or pIGF-II expression constructs compared to vector controls. Both IGF-II clonal cell lines grew faster than the control Caco-2 cells until six days of culture. However, at day 12 the final cell density of the pIGF-II expressing cells was higher than that of the mature IGF-II clones. Western blot analysis of cell lysates at day 8 through day 12 with anti-IGF-I receptor (IGF-IR) β subunit antibody revealed that the mature IGF-IR levels were lower in both IGF-II overexpressing cell lines compared to the vector control clone. Furthermore, it was shown that at day 12 the IGF-IR levels were significantly lower in mIGF-II clones than in pIGF-II clones. These results indicate that mIGF-II is more effective in down-regulating the IGF-IR than pIGF-II. We propose that overexpression of mIGF-II causes down-regulation of the IGF-IR, leading to growth arrest of Caco-2 cells.