Combinational treatment of gap junctional activator and tamoxifen in breast cancer cells.

Tamoxifen is a drug of choice for endocrine-responsive breast tumor patients. However, tamoxifen resistance has become a major concern for the treatment of breast cancer. Combinational therapies of tamoxifen and different drugs are being frequently studied. In this study, we tested the efficacy of substituted quinolines (code name=PQ1; a gap junctional activator) in combination with tamoxifen in T47D cells. Colony growth assay was performed using soft agar to measure the colony growth, whereas cell proliferation was measured by the MTT assay in T47D cells. The level of Ki67, survivin, and BAX was measured using confocal microscopy along with western blot analysis. Apoptosis-bromodeoxyuridine triphosphate labeling was also examined in the induced treatment of T47D cells. We observed a 55% decrease in the colony growth in the presence of combination of PQ1 and tamoxifen, whereas tamoxifen alone had little effect. A combination of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1 resulted in only 16% cell viability compared with controls at 48 h in T47D cells by the MTT assay. We found a significant increase in BAX protein at 1 h in the presence of 500 nmol/l PQ1 alone, 10 micromol/l tamoxifen alone, and the combination of PQ1 and tamoxifen. A two-fold increase was observed in active caspase 3 in the presence of combinational treatment of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1. In addition, flow cytometric analysis showed a 50% increase in the number of apoptotic cells in the presence of the combination of tamoxifen and PQ1 compared with the control. Furthermore, the results show that combinational treatment of tamoxifen and PQ1 significantly reduces the expression of survivin in T47D cells. Gap junction inhibitor studies with carbenexolone were also performed confirming the role of gap junctions in cell proliferation and cell death. The combinational treatment of PQ1 and tamoxifen has a significant increase in BAX expression, caspase 3 activation, and DNA fragmentation. Tamoxifen alone and in combination with PQ1 showed a decrease in the expression of survivin, whereas PQ1 alone was shown to be independent of the survivin-mediated pathway. This suggests that an increase in gap junction activity can potentiate the effect of tamoxifen. The combinational treatment of tamoxifen and PQ1 also showed a significant decrease in cell viability compared with tamoxifen treatment alone. The gap junction inhibitor carbenexolone was shown to increase cell proliferation by increased cyclin D1 expression, MTT assay, and Ki67 expression. It further decreased cell death. This study shows for the first time that combinational treatment of tamoxifen and PQ1 (a gap junctional activator) can be used to potentiate apoptosis of T47D human breast cancer cells. Thus, a gap junctional activator, PQ1, could potentially alter either the length or dose of tamoxifen clinically used for breast cancer patients.