Fenofibrate Enhances Sensitivity Of Anti-estrogen Treatment And Involved Mechanism In Breast Cancer

Objective:To investigate the synergistic effect of fenofibrate in combination with anti-estrogen agents in breast cancer and to explore its possible mechanism in vitro. Methods:Utilization of cell count kit-8 (CCK-8) method was to study the anti-proliferation effect of fenofibrate, anti-estrogen agents and fenofibrate+anti-estrogen agents, meanwhile clone formation assay was to observe the synergistic effect directly. The percentage of apoptotic cells and distribution ratio of cell cycle were evaluated by flow cytometric analysis. The proteins related were measured with Western Blot methods.Results:1. The combination of fenofibrate with anti-estrogen agents (tamoxifen and fulvestrant) could generate synergistic anti-proliferation effects in estrogen receptor (ER) positive breast cancer cell lines. For T47D and MCF-7 cells, fenofibrate combination with anti-estrogen agents showed much lower cell viabilities compared with either agent alone. CI (Combination index) of different concentration, which was calculated by Compusyn, was<0.9. Meanwhile clone formation assay showed the synergistic effect directly.2. The synergistic anti-proliferation effects of fenofibrate+anti-estrogen agents was independent of PPAR-α (the peroxisome proliferator-activated receptor-alpha, PPAR-α). GW6471, a PPAR-α specific inhibitor, was added to control, fenofibrate, anti-estrogen agents and combination group in MCF-7 cell line. GW6471 could not reverse the synergistic effect of anti-proliferation, and CI was still<0.9.3. MCF-7 cells were arrested in G0/G1 phase by the combination of fenofibrate with anti-estrogen agents. The percentage of cells in G0/G1 phase was obviously higher in comparison with that of either agent alone. The percentage of cells in G0/G1 phase increased from 43.2%、49.8% to 62.2% in the combination of tamoxifen and fenofibrate, and it was increased from 25.2%、20.8% to 31.9% in the combination of fulvestrant and fenofibrate. Cyclin D1, CDK4 and CDK6 were down-regulated and p21, p27/Kip1 were up-regulated.4. The combination of fenofibrate with anti-estrogen agents increased the percentage of apoptotic cells accompanied by up-regulation of Bad and cleaved PARP, down-regulation of Survivin. The percentage of apoptotic cells increased from 7.1%、 10.7% to 18.3% in the combination of tamoxifen and fenofibrate, and it was increased from 12.6%、14.7% to 24.3% in the combination of fulvestrant and fenofibrate.5. The synergistic anti-proliferation effects of the combination of fenofibrate with anti-estrogen agents may be associated with autophagy. pmTOR and pAkt were down-regulated and LC3B II/LC3B I were up-regulated. Chloroquine, an inhibitor of autophagy, could reverse the synergistic effect induced by the combination of Fenofibrate with anti-estrogen agents.Conclusions:Fenofibrate could enhance the sensitivity of anti-estrogen agents (tamoxifen and fulvestrant) in ER positive breast cancer, and the effect was PPAR-a independent. The mechanism may be associated with G0/G1 cell cycle arrest, apoptosis and autophagy...