Insulin-like Growth Factor Binding Protein-3 Induces G1 Cell Cycle Arrest with Inhibition of Cyclin-dependent Kinase 2 and 4 in MCF-7 Human Breast Cancer Cells

Insulin-like growth factor binding protein (IGFBP)-3 has been shown to potently inhibit proliferation of various cell types in an insulin-like growth factor (IGF)-independent manner. We have previously shown that IGFBP-3 induces apoptosis in an IGF-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. In the present study, we present further evidence that IGFBP-3 inhibits cell proliferation through the induction of cell cycle arrest in the same cell line. Induction of IGFBP-3 in MCF-7 cells inhibited cell proliferation whereas presence of small interfering RNA against IGFBP-3 abolished cell inhibitory effect of IGFBP-3, suggesting that the observed growth inhibition is specific. Flow cytometry analysis showed that induced expression of IGFBP-3 led to an arrest of the cell cycle in G1-S phase. Western immunoblot analysis showed a significant decrease in the levels of the cell cycle-regulated proteins such as cyclin D1, cyclin D3, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2, CDK4, retinoblastoma protein (pRB), and phosph-pRB, suggesting a possible mechanism for cell cycle arrest by IGFBP-3. Northern blot analysis and real-time quantitative PCR demonstrated a significant decrease in gene expression of cyclin D1. Additional phosphorylation assay showed that IGFBP-3 decreased the phosphorylation activity of CDK2 and CDK4. These results show that cellular production of IGFBP-3 leads to G1 cell cycle arrest with inhibition of CDK2 and CDK4. Taken together, IGFBP-3 exerts its growth inhibitory action through not only induction of apoptosis but also the G1 cell cycle arrest in human breast cancer cells.