Inhibition of Cell Cycle Progression by Hydroxytyrosol Is Associated with Upregulation of Cyclin-Dependent Protein Kinase Inhibitors p21WAF1/Cip1 and p27Kip1 and with Induction of Differentiation in HL60 Cells

Recent evidence indicates that the cancer preventive activity of olive oil can be mediated by the presence of minor components, such as antioxidant phenolic compounds. However, their mechanisms of action remain largely unknown. In this study, we investigated the in vitro effects of one of the main olive oil phenols, hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], on proliferation, cell cycle progression, apoptosis, and differentiation of HL60 human promyelocytic leukemia cells. 3,4-DHPEA showed a potent inhibitory activity on DNA synthesis, as evidenced by a 92% reduction of [ 3 H]-thymidine incorporation at 100 μmol/L, and an induced apoptosis, as evidenced by the release of cytosolic nucleosomes and flow cytometry. This phenol, 3,4-DHPEA, was also able to inhibit the progression of the cell cycle in synchronized HL60 cells, which accumulated in the G 0 /G 1 phase of the cell cycle after 25 h of treatment. Furthermore, 3,4-DHPEA induced differentiation on HL60 cells with a maximum effect (22% of cells) at 100 μmol/L after 72 h of treatment. Among the different proteins involved in the regulation of the cell cycle, 3,4-DHPEA reduced the level of cyclin-dependent kinase (CDK) 6 and increased that of cyclin D3. With regard to the CDK inhibitors, p15 was not altered by 3,4-DHPEA treatment, whereas the expression of p21 WAF1/Cip1 and p27 Kip1 was increased at both protein and mRNA levels. To our knowledge, these results provide the first evidence that 3,4-DHPEA may effect the expression of genes involved in the regulation of tumor cell proliferation and differentiation.