Study On The Prevention Effect Of Cyclooxygenase-2 Inhibitor On Multidrug Resistance In Breast Cancer Cells

Objective:1.We applied doxerubicin to induce chemoresistance development in human breast cancer MCF-7 cells,and the model of drug resistance was established.Based on this model,MCF-7 cells were applied to prevent doxorubicin-induced chemoresistance by treated with celecoxib, a selective cyclooxygenase-2 inhibitor.MDR1,MRP1,topoâ…¡and GST were detected at mRNA level,and the chemoresistance prevention effect of celecoxib was explored.2.In order to evaluate celecoxib enhancing the cytostatic efficacy of doxorubicin,MCF-7 cells were treated with increasing doses of doxorubicin in absence or presence of celecoxib.3. Setting about the transcriptional control of MDR1 gene,the mechanisms of the interventional effect of celecoxib on MDR1 overexpression induced by doxorubicin were explored from the mRNA expression level, nuclear protein expression level and the DNA binding activity of transcription factors.Methods:1.MTF assay was applied to assess the development of chemoresistance in MCF-7 cells induced by doxorubicin and the cytostatic efficacy of doxorubicin sensitized by celecoxib.MCF-7 cells were treated with increasing doses of doxorubicin in absence or presence of celecoxib,the cytostatic efficacy of doxorubicin on MCF-7 cells was evaluated,and the proper dose of doxorubicin for inducing drug resistance of MCF-7 cell was determined to carry out the following experiments.MCF-7 cells were treated with various doses of doxorubicin in absence or presence of 10uM celecoxib,IC50 values were calculated and applied to evaluate the cytostatic efficacy of doxorubicin sensitized by celecoxib.2.Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of MDR1 MRP1,topoâ…¡,GST,NF-κB,c-Jun and YB-1.3.Flow cytometry(FCM) was complied to measure the expression level of P-gp.4.Intracellular Rhodamine 123(Rho123) retention assay was applied to test the function of P-gp.5.Western blotting was used to investigate the expressin level of proteins,including MRP1,NF-κB,c-Jun and YB-1.6.Electrophoretic gel mobility shift assay(EMSA) was adopted to detect the DNA binding activity of transcription factors NF-κB and AP-1.7.FCM was used to analyze the cell cycle of MCF-7 cells treated with celecoxib and doxorubicin.Results:1.After treated with 0.05ug/ml doxorubicin for 3 days,the growth inhibiting effect of MCF-7 cells was increased,while it was significantly decreased for 7 days,the growth inhibiting rates were 23.7%and 9.03% respectively(P＜0.01);When the concentration of doxorubicin was increased to 0.5ug/ml for 3 days,the growth inhibiting effect of MCF-7 cells was significantly increased,and it was further increased after 7 days, the growth inhibiting rates were 60%and 72.90%;respectively.These date showed MCF-7 cells could develop chemoresistance after treated with 0.05ug/ml doxorubicin for 7 days.2.Compared with sensitive MCF-7 cells,MDR1 and MRPI mRNA were upregulated in MCF-7 cells treated with 0.05ug/ml doxorubicin for 7days(P＜0.01),but Topoâ…¡and GST mRNA were no,different(P＞0.05);MRP1 protein was consisted with mRNA;expression of p-glycoprotein was significantly upregulated (P＜0.01).3.Compared with doxorubicin alone,genes includingMDR1,MRP1,NF-κB,c-jun and YB-1 were significantly downregulated in MCF-7 cells treated with both celecoxib and doxorubicin,and proteins expression were similar to genes;Fluorescence intensity of P-gp in MCF-7 cells treated with both doxorubicin and celecoxib(10uM,20uM) were markedly lower(6.41±0.71 and 5.55±0.50,respectively) than doxorubicin alone(32.66±2.49)(P＜0.01).4.After treated with both celecoxib(10uM,20uM) and 0.05ug/ml doxorubicin for 7 days,Rh123 accumulation in MCF-7 cells was significantly increased,compared with doxorubicin alone(P＜0.01),Fluorescence intensity of Rh123 in three groups cells was 373.03+15.41,341.44±11.75and 72.77±8.88, respectively.5.Compared to untreated MCF-7 cells,the expression of YB-1,NF-κB and AP-1 were upregulated in cells treated with 0.05μg/ml doxorubicin for 7 days;Contrasted with cells treated with 0.05μg/ml doxorubicin,the expression of these transcription factors were significantly decreased in cells treated with both 0.05μg/ml doxorubicin and 10uM celecoxib.6.Compared to untreated MCF-7 cells,the DNA binding ativity of NF-κB and AP-1 in the cells treated with 0.05μg/ml doxorubicin markedly increased;while contrasted with cells treated with 0.05μg/ml doxorubicin,the DNA binding ativity of these transcription factors were significantly decreased in cells treated with both 0.05μg/ml doxorubicin and 10uM celecoxib.It was indicated that celecoxib could significantly inhibit the DNA binding ativity of transcription factors NF-κB and AP-1.7.MCF-7 cells were treated with increasing doses of doxorubicin in presence of 10uM celecoxib for 48 hours,the proliferation of cells was markedly inhibited,IC50 value was(0.384±0.04) ug/ml; while cells were treated with increasing doses of doxorubicin in absence of celecoxib,IC50 value was(0.67±0.03) ug/ml(P＜0.01).8.Compared to untreated MCF-7 cells,the rate of the cells at G₁+G₀ stage increased (P＜0.01),and at S stage decreased markedly(P＜0.01) in cells treated with 0.05ug/ml doxorubicin;this effect was further enhanced when cells were treated with both 0.05ug/ml doxorubicin and 10uM celecoxib (P＜0.01);While the rates of G₁+G₀ stage and S stage were not affected in cells treated with 10uM celecoxib alone.The rates of the cells at G₂+M stage were no different in all groups(P＞0.05) Coclusions:1.After treated with 0.05ug/ml doxorubicin alone,MCF-7 cells could develop chemoresistance,MDR1 were significantly upregulated at the level of mRNA and protein,as well as MRP1.2.Combined celecoxib with doxorubicin could effectively prevent the development of chemoresistance,which partly associated with inhibiting doxorubicin-induced overexpression of MDR1 and MRP1.3.Celecoxib downregulating doxorubicin-induced overexpression of MDR1 was associated with inhibiting the expression and fuction of transcription factors,including YB-1,NF-κB and AP-1 in MCF-7 cells.4.Celecoxib markedly increased the chemotherapeutic sensitive effect of doxorubicin on MCF-7 cells and enhanced the arrestment of cells at G₁+G₀ phase.In conclusion,celecoxib could effectively interfere with the development of chemoresistance in breast cancer cell line MCF-7 by many mechanisms.This study provided a new strategy for clinical tumor chemotherapy.