Anticancer effects of Daidzein against the human melanoma cell lines involves cell cycle arrest, autophagy and deactivation of PI3K/AKT signalling pathways.

PURPOSE

Melanoma is one of the fatal human malignancies. Its incidence in humans is increasing constantly and therefore there is urgent need to develop effective therapies for its management. This study was therefore undertaken to investigate the anticancer effects of Daidzein on human melanoma cells and also an attempt was made to decipher the underlying mechanisms.

METHODS

MTT assay was used to determine the melanoma A-375 cells viability. Αcridine orange (AO)/ Εthidium bromide (EB) and Annexin V/propidium iodide (PI) assays were used to detect the cell apoptosis. Autophagy was detected by electron microscopy and cell cycle analysis was performed by flow cytometry. The protein expression was determined by western blot analysis.

RESULTS

The results of MTT assay showed that Daidzein causes significant decrease in the proliferation of the melanoma A-375 cells and showed an IC50 of 18 µM. However, the IC50 of Daidzein was very high against the normal HEMn-LP cells, indicative of low cytotoxicity. Flow cytometry showed significant arrest of the A-375 cells at the G0/G1 phase of the cell cycle. Western blot analysis showed that the molecule suppressed the expression cell cycle regulatory proteins such as cyclin D1, CDK4, CDK6 and p27. DAPI and annexin V/PI staining assays showed that Daidzein prompted apoptosis in A-375 melanoma cells which was concomitant with depletion of Bcl-2, increase of Bax and activation of cleavage of caspase-3 and caspase-9. Electron microscopic analysis showed that the molecule led to the development of autophagosomes in A-375 cells, which was also concomitant with increase in the expression of LC3B II and decrease in the expression of p62. Finally, Daidzein also suppressed the phosphorylation of PI3K and AKT, causing deactivation of the PI3K/AKT signalling pathway.

CONCLUSION

Daidzein may prove beneficial in the development of melanoma systemic therapy.