Abstract 580: Combined treatment of breast cancer cells with epigallocatechin gallate (EGCG) and Tamoxifen delays cell-growth in long-term culture: Implications for recurrent breast cancer
Authors: Aiyer Harini S. , Warri Anni M. , Woode Denzel R. , Hilakivi-Clarke Leena , Clarke Robert 
Published: August 2012

Abstract
Recent health surveys have revealed that green tea is one of the most commonly consumed natural products among breast cancer survivors. Although breast cancer survival has increased during the past two decades due to adjuvant endocrine therapy with antiestrogens (AE) such as Tamoxifen (TAM) and Faslodex (FAS) the risk of acquired endocrine-resistance still persists for many women. This can be mimicked by a prolonged culture of breast cancer cells with sequentially increasing concentrations of 4-hydroxy tamoxifen (4OHT; 1 nM -1 µM), applying a selection pressure for cells to acquire resistance to 4OHT. Such cells maintain their resistant phenotype even after removal of the pressure (Brunner et al., 1993). The aim of this study was to determine whether a green tea polyphenol (EGCG) can affect the development of TAM-resistance in breast cancer. We tested the efficacy of EGCG, in this long-term [LT] cell culture model. Estrogen receptor (ER)α-positive MCF-7 cells (n=2/group) were cultured in media containing: i) Vehicle (0.01% EtOH;, LT-V); ii) LT-4OHT (1 nM to 1 µM; increasing every 3rd passage; LT-4OHT); or iii) 4OHT (1 nM to 1 µM) + EGCG (100 nM;single dose;LT-EGCG). Growth rate was recorded for each passage (P), measured as the time required reach confluence (70%), after which the cells were subcultured. We will present results of this LT study including mechanisms by which EGCG may alter resistance-development, focusing on signaling pathways such as ER, NFkB and unfolded protein response (UPR) known to be involved in endocrine resistance. During the initial passages (P1-P6; [4OHT] ≤ 5 nM) the cells grew at the same rate regardless of the treatment. Starting at P8, time to confluence was delayed for LT-4OHT ([10 nM]; 7d) compared to the LT-V. This delay was even more pronounced at P15 ([100 nM]; ≤ 21d), suggesting that cells were under a growth inhibition/selection pressure. The growth rate of LT-EGCG did not differ from LT-4OHT prior to P15, but in subsequent passages a delay was evident (14 to 18 d). To test whether the cells maintained their (delayed growth) phenotype, they were first cultured in regular media for 24h and tested for growth inhibition by 4OHT (1 nM-1µM) and FAS (100 nM). LT-V cells (P27) were more susceptible to the inhibitory effects of 1 µM 4OHT (73 ± 11% versus 0 µM 4OHT) than LT-4OHT cells (P19; 105 ± 40%), confirming the development of resistance in LT-4OHT cells. In contrast, LT-EGCG cells had retained their sensitivity to 4OHT, similar to LT-V (P19; 75 ± 8%). All treatment groups were equally responsive to FAS (≥30%). These results suggest that EGCG may delay the development of a 4OHT-resistance in breast cancer cells. It will be confirmed whether the effect prevails after removal of selection pressure (4OHT). This data will provide a better understanding of green tea's potential to prevent TAM-resistance in breast cancer patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 580. doi:1538-7445.AM2012-580