The Anti-cancer Effect Of Fenofibrate In Triple-negative Breast Cancer And Involved Mechanism

Objective:To investigate the anti-cancer effect of Fenofibrate in triple-negative breast cancer in vitro and in vivo and to explore its possible mechanism.Methods:Utilization of cell count kit-8(CCK-8) method was to study the anti-proliferation effect of Fenofibrate and invert microscope was to observe the morphological changes. 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. The changes of genes and pathways were detected using the gene expression profiling.The inhibition of tumor growth in vivo was assessed by MDA-MB-231xenograft mouse model. Terminal deoxytransferase-catalyzed DNA nick-end labeling assay (TUNEL assay) was used to estimate the proportion of apoptotic cells in tumor tissue. In order to evaluate the safety of Fenofibrate, blood was sampled from rat eyes and counts of leukocyte, erythrocyte, hemoglobin, platelet, AST, ALT and BUN in peripheral blood were detected.Results:1.Fenofibrate had the anti-proliferation effect of breast cancer cell lines in a dose-dependent manner, to which the first five most sensitive ones were MDA-MB-231,MDA-MB-453,BT549,MDA-MB-436,MDA-MB-231-LU (referring to the lung metastasis potency sub-cell line), and their50%inhibitory concentration (IC50) were (16.07±4.44)μM,(26.72±10.04)μM,(34.47±13.88)μM,(74.46±17.75) μM and (82.09±21.21)±M respectively. Observed under the inverted microscope, we found that the cell morphology changed obviously and the number of viable cells decreased24hours after treatment of Fenofibrate.2. Fenofibrate induced MDA-MB-231cell line apoptosis in a both time-and dose-dependent manner accompanied by up-regulation of Bad, down-regulation of Bcl-xl, Survivin and activation of caspase-3.The percentage of apoptotic cells increased from4.8%to26.0%in the24h treatment group, and5.8%to48%in the48h treatment group.3. Treatment with Fenofibrate led to cell cycle arrest of MDA-MB-231cell line at G1phase accompanied by down-regulation of Cyclin D1, Cdk4and up-regulation of p21, p27/Kipl.The percentage of cells in G1phase increased from49.2%to61.3%in the24h treatment group, and47.9%to61.5%in the36h treatment group.4. GW6471,a PPAR-a(The peroxisome proliferator-activated receptor-alpha, PPAR-a specific inhibitor, could not reverse the effect of anti-proliferation and apoptosis in MDA-MB-231cell line induced by Fenofibrate. As for the anti-proliferation effect, the growth ratio of Fenofibrate alone (0,12.5,25,50and100μM) vs. a combination of Fenofibrate (0,12.5,25,50and100μM) and5μM GW6471in72h were (100±9.14)%vs.(99.90±9.23)%,(55.74±5.43)%vs.(58.60±4.10)%,(48.76±5.16)%vs.(41.43±3.66)%,(34.97±.82)%vs.(28.92±2.94)%,(31.69±3.43)%vs.(25.71±2.84)%, p>0.05, there was no significant difference. As for apoptosis, the proportional apoptotic cells of50μM Fenofibrate alone vs. a combination of50μM Fenofibrate and5μM GW6471in24h was (21.55±2.47)%vs.(20.15±1.34)%, p>0.05, there was no significant difference.5. The anti-cancer effect of Fenofibrate may be mainly associated with the activation of NF-κB pathway. In addition, the suppression of Aktl and Erkl/2pathways may also be involved. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, could reverse the effect of apoptosis induced by Fenofibrate. The proportional apoptotic cells of50μM Fenofibrate alone vs. a combination of50μM Fenofibrate and10nM PDTC in24h was (20.45±0.92)%vs.(6.5±0.85)%, p<0.004, there was a significant difference.6. The gene expression profiling analysis showed that in the first ten most obvious changes in gene ontology (GO) biological process classification, seven out of ten were related to cell death, four related to cell apoptosis. In the first ten of the most significant down-regulated pathways, cell cycle ranked first and pathway in cancer ranked fourth.7. Fenofibrate could inhibit the tumor growth in the MDA-MB-231xengograft mouse model and induce apoptosis in tumor tissue with a good safety profile. After32days of Fenofibrate treatment, the tumor volume in Fenofibrate treatment group vs. control group was (1786.76±384.39)mm3vs.(2788.35±759.88)mm3, p<0.001, there was a significant difference. The inhibitory ratio was35.92%. The proportion of apoptotic cells in tumor tissue of Fenofibrate treatment group vs.control group was (36.22±0.94)%vs.(17.84±6.60)%, p<0.0001, there was a significant difference. Fenofibrate had no effects on white blood cell(WBC), red blood cell(RBC), hemoglobin(HGB), platelet(PLT), alanine aminotransferase(ALT), aspartate aminotr-ansferase (AST) and blood urea nitrogen (BUN)(p>0.05), which showed a good safety profile.Conclusions:Fenofibrate exerted anti-cancer effect over triple-negative breast cancer in vivo and in vitro. The possible mechanism may the activation of NF-κB pathway and suppression of Akt1or Erk1/2pathways in a PPAR-a independent way, which lead to apoptosis and/or cell cycle arrest.The effective concentration of Fenofibrate for its anti-cancer effect is easy to achieve in vivo and has a good safety.