Quercetin Potentiates Cancer Cells To Doxorubicin-induced Apoptosis Through Mitochondrial Pathway

The chemotherapeutic drug doxorubicin is a cytotoxic anthracycline antibiotic isolated from culture of Streptomyces peuxetus, and has been widely used in the clinical treatment of a broad spectrum of cancers. There has been demonstrated that some flavonoids exhibited a synergistic anti-tumor effect with chemotherapeutics. we demonstrated that sub-toxic doses of quercetin significantly sensitized doxorubicin-induced apoptosis of hepatoma cells, SMMC7721 and QGY7701 cell lines.Therefore, we decided to examine the effect of quercetin on the doxorubicin-induced apoptosis of hepatoma cells.Methods1. Cytotoxicity Assay3The cells were plated in 96-well plates at a density of 5×10~3 cells/well overnight. Then the cells were incubated with quercetin for 48 h in complete RPMI-1640 medium. After drug treatment, the medium was replaced, and the cells were incubated with 0.5 mg/ml of MTT in complete RPMI-1640 medium for 4 h. the surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in DMSO. The intensity was measured at 570 run using a model ELX800 Micro: late Reader. The relative percentage of cell survival was calculated by dividing the absorbance of treated cells by that of the control in each experiment.2. Preparation of cytosol fractionsCells were washed twice with ice-cold PBS, pH 7.2 and resuspended in extractionbuffer. Nuclei were removed by centrifugation at 1,000g for 10 minutes at 4°C. Supernatant was additionally centrifuged for 1 hour at 100,000g and the resulting supernatant (cytosolic fraction) were collected separately and preserved in -20°C and used for Western blotting. 3. Western blot analysisCells were harvested as described above after drug treatment. Cells were then lysed in a sample buffer containing 625 mM Tris-HCl (pH 6.8), 10% SDS, 25% glycerol, 5% b-mercaptoethanol, and 0.015% bromphenol blue, followed by sonication and heat denaturation. The protein (40 mg) was applied to a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and then detected by the proper primary and secondary antibodies before visualization by chemiluminescence. Visualization in 1 min, 3min, 5min, 10min was performed with a Molecular Imager FX using Kodak ID imaging densitometry analysis software on a Macintosh personal computer.Results1. Quercetin potentiates doxorubicin -induced apoptosis in QGY7701 and SMMC7721 cells.To investigate the effect of quercetin on doxorubicin -induced apoptosis, SMMC7721 and QGY7701 cells were treated with doxorubicin alone, or plus quercetin (20μM). 1μM of doxorubicin had slightly inhibitive effect on cell growth (the growth inhibitive rate was 10% around), and this concentration is accessible to clinical treatment, but the inhibitive effect of co-treatment with quercetin (20 μM) and doxorubicin (1 μM) on the growth of these two cell lines was significantly different (P < 0.05). The growth inhibitive rate was about 40% and 30% for SMMC7721 and QGY7701 cells,respectively.2. Quercetin enhances doxorubicin -induced caspase activationTreatment of SMMC7721 cells with quercetin alone did not induce any cleaved caspase-3. In response to doxorubicin, there was a 20-kDa cleavage form, but further cleavage into the active pl7 subunit was not detected. However, co-treatment with quercetin and doxorubicin induced the cleavage of procaspase-3 into the p20 intermediate form and its subsequent cleavage into the active p17 subunit. There were no detectable cleaved caspase-9 or caspase-8 in the control as well as the group treated with quercetin alone. Analyzing caspase-9 p37/p35 cleavage products in SMMC7721 cells demonstrated that doxorubicin in combination with quercetin strongly enhanced caspase-9 activation, which was less pronounced in SMMC7721 cells treated with doxorubicin alone. However, the cleavage of caspase-8 was comparable between treatment with doxorubicin alone and combination treatment.3. Quercetin potentiates doxorubicin -induced apoptosis in hepatoma cells through Bcl-xl/Bax-mediated mitochondrial pathwayCo-treatment with doxorubicin and quercetin induced the higher expression of PUMA protein than the treatment with doxorubicin alone did, whereas quercetin alone did not increase the PUMA protein level. Although the expression level of Bcl-2 protein did not change during different treatments in these cell lines, co-treatment with doxorubicin and quercetin caused a sharp reduction in Bcl-xl level, while treatment with doxorubicin alone did not affect Bcl-xl protein level.Conclusion Quercetin can significantly sensitize doxorubicin -induced apoptosis of hepatomacells through mitochondrial pathway.