The Role Of Akt-FoxO1Pathway In Curcumin Induced Apoptosis Of MDA-MB-468Human Breast Cancer Cells

Curcumin is a chemical composition in rhizomes of Zingiberaceae and Araceae. Recent research suggested that curcumin had multiple compelling biological activities in vitro and in animal models, which include antitumor, anti-inflammatory, antioxidant, anti-bacteria and anti-viral. But the molecular mechanisms by which curcumin induces apoptosis of cancer cells are not completely understood.Akt (protein kinase B (PKB)) becomes activated by phosphorylation at Thr308and Ser473. FoxO family transcription factors localized in the nucleus are downstream targets of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which regulate the transcription of genes involved in cell cycle arrest and apoptosis such as p27and Bim. But FoxOs can be phosphorylated by phospho-Akt, and then exported from the nucleus, thereby losing the activities of regulatory transcription.In this study, we used the MDA-MB-468cells to characterize the role of Akt-FoxO1pathway in curcumin induced apoptosis of breast cancer cells. The results should help understanding the molecular mechanisms of apoptosis induced by curcumin in cancer cells.1. The effect of curcumin on viability of MDA-MB-468cellsWe determined the viability of MDA-MB-468cells treated with curcumin by MTT assay. The results showed that curcumin reduced the cell viability, and the IC5o value was41.6μM. MDA-MB-468cells were treated with curcumin at0,10,20,30and40μM for24hours, and apoptosis was determined by Annexin-V and PI double staining. Flow cytometry revealed a dose-dependent increase of curcumin-induced apoptosis of MDA-MB-468cells. These results showed that curcumin had an inhibitory effect on viability and induced apoptosis of MDA-MB-468cells.2. The effect of curcumin on the expression of FoxO1, Akt, P-Akt and p27in MDA-MB-468cellsFoxO1is localized in the nucleus as a transcription factor to regulate transcription of various genes involved in cell cycle arrest and apoptosis such as p27kipl and Bim. But it could be phosphorylated by P-Akt, and then exported from the nucleus, thereby inhibiting FoxO1-dependent transcription of genes.To understand the role of Akt-FoxO1pathway in the curcumin-induced apoptosis of MDA-MB-468cells, we measured the expression of FoxO1at mRNA and protein levels by Q-PCR and Western blot following curcumin treatment. The levels of Akt,P-Akt and p27in the curcumin treated cells were determined. We found that curcumin could induced FoxO1expression at mRNA and protein levels, decreased the levels of P-Akt and increased the levels of FoxO targeted p27kip1.3. The effect of curcumin on the nuclear translocation of FoxO1in the MDA-MB-468cellsAs the downstream target of Akt, FoxO1could be phosphorylated and then exported from the nucleus. To determine the change of nuclear translocation of FoxO1after curcumin treatment, we measured the relative amounts of FoxO1in nucleus and cytoplasm. The results showed that the amounts of P-FoxO1were decreased, and moreover the levels of FoxO1in nucleus were increased by curcumin.4. The effect of P-Akt inhibitor and curcumin co-treatment on the levels of P-Akt, P-FoxO1and FoxO1To confirm the decline of P-FoxO1levels is mediated by P-Akt, we treated MDA-MB-468cells with a combination of curcumin and Wortmannin, which is the inhibitor of P-Akt. We found that the levels of P-Akt and P-FoxO1declined after curcumin and Wortmannin co-treatment, which were more significant than curcumin treatment alone, and the results of Wortmannin treatment alone were similar with curcumin treatment. The Western blot results showed that Wortmannin had no effect on the expression of FoxO1.These results showed that curcumin not only decreased the levels of P-Akt, but also increased the expression of FoxO1to increased the amounts of FoxO1in nucleus.