EGCG and 4-hydroxytamoxifen synergistically induce cytotoxicity in MDA-MB-231 human breast cancer cells: The role of apoptosis and signaling proteins Free

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Previously we have demonstrated that the combination of epigallocatechin-gallate (EGCG) (25 uM) and 4-OH tamoxifen (4-OHT) (1 uM) are synergistically cytotoxic toward MDA-MB-231 ER alpha negative human breast cancer cells. These treatments were employed to further investigate the mechanisms responsible for this cytotoxic synergism, FACS analysis was employed to determine the roles of both apoptosis induction and changes in the cell cycle. Analysis of apoptosis induction through staining with annexin-V/propidium iodide demonstrated that a significant percentage of cells in the combination treatment were apoptotic (22.8%+3.5) compared to all other treatment groups after 24 h (p<0.05). Changes in the cell cycle were investigated through the analysis of DNA content via staining with propidium iodide. A significant increase in the number of cells in the G1-phase of the cell cycle was observed after 36 h of combination treatment (8.0%+1.3) compared to all other treatments (p<0.05). However, the enhanced apoptosis following combination treatment was unlikely to be caused by an increase in G1-arrest as this occurred after the significant increase in apoptosis. Additionally, the overall increase in cells arrested in G1 was less than 10%. Therefore, alterations in the cell cycle were deemed to be an unlikely mechanism for the cytotoxic synergy between EGCG and 4-OHT. Changes in important cell signaling proteins were then analyzed for their role in the synergistic cytotoxicity. Changes in EGFR, phosphoEGFR, AKT and phosphoAKT protein levels were determined via Western blotting. After 12 h of treatment, there were no significant changes in the protein levels of EGFR, while after 24 h of treatment both EGCG and 4-OHT increased the protein levels of EGFR (19.7%+1 and 17.5%+7.2%, respectively, p<0.05) from the control. After 12 h of treatment both the combination and 4-OHT decreased the protein levels of phosphoEGFR from the control (25%+12 and 32%+13, respectively, p<0.05). However, after 24 h only the combination treatment significantly decreased the protein levels of phosphoEGFR, (24%+8) compared to all other treatments (p<0.05). No changes were observed in the protein levels of AKT after 12 and 24 h of treatment. However, after 24 h the combination treatment significantly decreased the protein levels of phosphoAKT by 36%+10 compared to all other treatments (p<0.05). In conclusion, our results indicate that the combination of EGCG and 4-OHT induce apoptosis in a synergistic fashion. While the mechanism responsible for the induction of apoptosis is unlikely to be via G1-arrest, it may be a result changes in important cell signaling proteins such as phosphoEGFR and phosphoAKT.