Study On The In Vitro And In Vivo Mechanisms Of The Reversal Multidrug Resistance On B-MD-C1 (ADR^+/+) By Quercetin

Objective: To study on the in vitro and in vivo effects and mechanisms of the reversal multidrug resistance on B-MD-C1(ADR+/+) cells by quercetin at the molecule level and systemic level,respectively,in order to find a potent but harmfulless drug for improving the effect of chemotherapy and ameliorating the prognosis of patients.Methods:1.The cytotoxicity of quercetin on B-MD-C1 cells and its chemosensibility on B-MD-C1(ADR+/+) cells were evaluated by means of MTT.2. The apoptosis of B-MD-C1 cells after treatment with various concentrations of quercetin (10, 20, 40, 80μmol/L) for 72h was studied by fluorescence microscope after AO/EB staining.3. Expression of mdr1 and TopoⅡαgene after treatment with various concentrations of quercetin (10, 20, 40μmol/L) for 48h were examined by RT-PCR.4. Changes of subcellular distribution of ADM in B-MD-C1 cells were observed by confocal scanning laser microscopy (CSLM).5. Expression of P-glycoprotein after treatment with various concentrations of quercetin (10, 20, 40μmol/L) for 72h was detected by means of immunohistochemisty.6. Expression of TopoⅡαprotein after treatment with various concentrations of quercetin (10, 20, 40μmol/L) for 48h was studied by FCM.7. B-MD-C1 cells were inoculated subcutaneouly into right shoulder of nude mice to establish endometrial cancer model. Then 17 BALB/c nude mice with xenograft tumor were randomized into control group, Quercetin group, ADM group, Quercetin+ADM group, B-MD-C1(wt) control group, with treatment for 10 days.The potency and side effect of quercetin and/or ADM were observed,the histopathological character was ovserved by HE staining,and the expression of P-glycoprotein, TopoⅡα, bcl-2, bax, Fas and FasL were detected by immunohistochemistry.Results:1. Quercetin can significantly inhibit the growth of B-MD-C1 cells in vitro(p<0.05). The growth inhibition is in dose-independent and time-independent manner. The IC50 value of B-MD-C1(ADR+/+) cells to ADM for 24h, 48h, 72h were 152.729±2.710,122.819±2.794, 108.730±1.994μmol/L. The IC50 value of B-MD-C1(wt) cells to ADM for 24h, 48h, 72h were 165.679±2.219, 148.344±2.171, 123.683±2. 157μmol/L, respectively. 2. No significant apoptosis was observed after treatment by quercetin (equal to or less than 40μmol/L) on B-MD-C1 cells.3.The reversing fold of quercetin with the concentrations of 10,20,40μmol/L for B-MD-C1 (ADR+/+) cells to ADM are 9.12±0.32,10.29±0.60,11.46±0.18,respectively.4. After treatment with different concentrations of quercetin (10, 20, 40μmol/L) for 48h, the expression level of mdr1 gene and P-glycoprotein in B-MD-C1 (ADR+/+) cells were decreased significantly (p<0.05).5. Quercetin can restore the abnormal distribution of ADM in B-MD-C1(ADR+/+) cells and increase their chemosensibilitity to ADM.6. The expression of TopoⅡαgene and its encoding protein were down-regulated after treatment with various concentrations quercetin, and showed significant difference compare to the control group(p<0.05).7. Injection at the side of tumor of quercetin and ADM combination resulted in a significant inhibition on the growth of B-MD-C1 (ADR+/+) cells in vivo (p<0.05). HE staining showed that transplantation tumors were poorly differentiated adenocarcinoma. Immunohistochemical staining of P-gp, Fas, FasL and Bax showed no significant difference in any group (p>0.05). Immunohistochemical staining of bcl-2 showed that group B (Que), D (Que+ADM) were significant lower than group A (control), C (ADM) (p<0.05). Immunohistochemical staining of TopoⅡαshowed that group B (Que), D (Que+ADM) were significant more than group A (control), C (ADM) (p<0.05).Conclusion:1. Quercetin can reverse the multidrug resistance in vitro by down-regulating the expression of mdr1 gene and P-glycoprotein and restoring the distribution of ADM in B-MD-C1 (ADR+/+) cells.2. Quercetin can reverse the atypical multidrug resistance in vitro by up-regulating the expression of TopoⅡαgene and TopoⅡαprotein in B-MD-C1 (ADR+/+) cells.3. Quercetin can reverse multidrug resistance in vivo by up-regulating the expression of TopoⅡαand down-regulating the expression of bcl-2 protein.