Interactions between EGCG and SERMs in a xenograft model of ERα- breast cancer.

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Estrogen receptor negative (ERα-) breast cancers represent a highly aggressive subset of cancers for which there is currently no effective treatment. The green tea polyphenol epigallocatechin gallate (EGCG) is known to inhibit the growth of ERα- MDA-MB-231 breast cancer cells when combined with the selective estrogen receptor modulators (SERM) tamoxifen or raloxifene. The current study was designed to assess the efficacy of EGCG-tamoxifen and EGCG-raloxifene combinations in a xenograft model of ERα- breast cancer and to elucidate the underlying mechanism of tumor suppression. Athymic nude female mice were implanted with MDA-MB-231 breast cancer cells into the flank. After 2 weeks, to allow palpable tumors to form, mice were administered tamoxifen (50 or 75 µg/kg/d, po), raloxifene (225, 500 or 850 µg/kg, po), EGCG (25 mg/kg, ip), EGCG and tamoxifen, EGCG and raloxifene or vehicle control (5 ml/kg/d, po) for 10 or 8 weeks (for studies involving tamoxifen and raloxifene, respectively). Tumor volume measurements were conducted weekly using electronic callipers and protein extraction was performed on tumors upon necropsy. Organ weights recorded upon study completion confirmed good animal health and plasma alanine aminotransferase activity was normal, indicating a lack of hepatotoxicity. The combination of EGCG and tamoxifen synergistically inhibited tumor growth with a 66% reduction in tumor volume and a 50% reduction in tumor weight in the EGCG and tamoxifen (75 µg/kg/d) treated mice compared to control mice (p<0.05). Western blotting of protein extracts from tumors was conducted to identify potential mechanisms underlying tumor suppression. Several signalling pathways downstream of the EGFR were investigated, including the PI₃K-Akt, MAPK and mTOR cascades. Results revealed that combination treatment significantly lowered the levels of EGFR, p-EGFR, mTOR, S6K, p-MEK and CYP1B1 (15, 22, 22, 14, 23 and 25% of control, respectively) compared to all other treatments. Akt, p-Akt, NFκB, 4EBP1, p-4EBP1, b-Raf, pMEK, VEGF and VEGFR were also decreased from control by the combination treatment, but not different from the corresponding individual treatments of EGCG or tamoxifen. MEK, ERK, p-ERK, TGF-α, phospholipase D (1/2) and p-S6K were not altered following any treatment. Raloxifene (850 µg/kg, po) significantly inhibited tumor growth 82% (p<0.05) and tumor weight was reduced 71%, however, combination with EGCG abolished this effect. Furthermore, signalling pathways examined were not modulated to the same degree by EGCG and raloxifene. These results demonstrate that the combination of EGCG and tamoxifen is efficacious as a treatment in a xenograft model of ERα- breast cancer and that decreases in the protein expression of EGFR, mTOR and CYP1B1 represent key pathways that are targeted by the combination treatment and may dominate the mechanism underlying this effect.