Potential mechanisms for the cytotoxicity elicited by the combination of EGCG and raloxifene in MDA-MB-231 cells

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Previously we have shown that epigallocatechin gallate (EGCG) (25 μM) and 4-OH tamoxifen (4-OHT) (1 μM) are synergistically cytotoxic toward MDA-MB-231 ERα negative human breast cancer cells. Furthermore, studies have shown that raloxifene is cytotoxic in a number of cancer cell lines. We aimed to determine if the synergistic effect observed with the combination of EGCG and 4-OHT is observed in combination with raloxifene. The sulforhodamine B assay was used to determine changes in cell number. After 7 days of treatment, EGCG (25 μM) and raloxifene (4 μM) were shown to synergistically decrease cell number by 76 ± 3.8% from control, and time-course studies demonstrated that a significant repression (p<0.05) of cell number occurred after 36 h. FACS analysis was utilized to determine the roles of both apoptosis induction and changes in the cell cycle. Analysis of apoptosis induction via staining with annexin-V/propidium iodide (PI) demonstrated that a significant percentage of cells in the combination treatment were apoptotic (25 ± 3.3%) 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 PI. A maximal increase in the proportion of cells in the G1-phase of the cell cycle (116% of control) occurred following 24 h of combination treatment, 12 h after significant induction of apoptosis, and thus was not considered to be the principal mechanism of enhanced apoptosis. Alterations in the expression and phosphorylation of various signaling proteins were investigated via Western blotting. These results demonstrated that combination treatment significantly reduced the phosphorylation of EGFR, AKT and S6K proteins by 21.2 ± 3.3%, 31.5 ± 1.7% and 39.5 ± 3.4% (p<0.05) from control, respectively. However, the total expression of these proteins was not significantly altered with EGCG+raloxifene treatment. The effect these changes had on the transcriptome was examined via microarray analysis, conducted after 18 h of treatment using 20 K human oligo arrays. A significant result from these arrays showed that both EGCG and EGCG+raloxifene treatments increased the RNA expression of metallothioneins (MT) 1B, 2A and 3. However, raloxifene treatment decreased the RNA expression of MT1B, MT2A and MT3 from control. MT expression is considered as a potential prognostic marker of breast cancer in the clinic, where an increased MT protein expression is associated with a poorer outcome. In conclusion, our results indicate that the combination of EGCG and raloxifene induces apoptosis in a synergistic fashion. While the mechanism responsible for the induction of apoptosis is unlikely to be mediated via alterations in the cell cycle, it may be a result of changes in activity of intracellular signaling proteins such as EGFR, AKT and S6K. Furthermore, these changes may be associated with modulation of MT RNA expression.