Isolated and combined action of tamoxifen and metformin in wild-type, tamoxifen-resistant, and estrogen-deprived MCF-7 cells

Resistance to tamoxifen (TAM) and aromatase inhibitors represents a major drawback to the treatment of hormone-dependent breast cancer, and strategies to overcome this problem are urgently needed. The anti-diabetic biguanide metformin (MF) exerts pleiotropic effects which could enhance the effectiveness of available hormonal therapies. This study modeled several aspects of hormonal therapy in women and examined the effectiveness of MF under those conditions. For cell growth evaluation, wild-type (wt), TAM-resistant (TAM-R), and long-term estradiol-deprived (LTED) MCF-7 cells, as a model of aromatase inhibitor resistance, were grown in the presence or absence of TAM or MF for 5 days. For immunoblot analysis and aromatase activity measurements, these cells were grown for 48 h. Wild-type and LTED cells were equally sensitive to the growth inhibitory effects of TAM and MF, while TAM-R cells were less sensitive to TAM than to MF. Partial additive effects on cell number of TAM combined with MF were greatest (if compared with isolated TAM action) in TAM-R and LTED cells. In contrast to the decrease in PCNA values in TAM-resistant cells treated with the TAM and MF combination, no other changes were found in the levels of this proliferation marker. These findings suggested a major component of apoptosis in the growth inhibitory effect. This was confirmed with Western blot analysis of PARP and caspase 7 as well as with apoptosis ELISA assay. MF also altered signaling pathways. AMP-kinase was stimulated by MF approximately equally in MCF-7, TAM-R, and LTED cells, while inhibition by biguanide of p-S6K as a downstream target of mTOR was strongest in TAM-R cells. Under the influence of MF, expression of ER-alpha was decreased in wt MCF-7 cells suggesting possible involvement of this compound in estrogen signaling. Metformin interacts additively with TAM to reduce neoplastic cells growth. The cellular context (including loss of sensitivity to TAM and estrogen deprivation) is of importance in influencing breast cancer responses to MF and to a combination of MF and TAM.