| Objective:(1) To study the sensitivity of human breast cancer cell line MCF-7andADM-resistant human breast cancer cell line MCF-7/ADM to chemotherapeutics.(2) Toinvestigate the mechanism and reversal effect of MDR (multidrug resistance) by bFGF mAb(basic fibroblast growth factor monoclonal antibody) in MCF-7/ADM.Methods:(1) The ascites containing the bFGF mAb were extracted and then purified throughthe Protein G-Sepharose affinity column.The purity of bFGF mAb was determined bySDS-PAGE electrophoresis,the concentration of purified bFGF mAb stock solution was assayedby BSA standard assay kit and the titration of bFGF mAb after purification was detected byindirect method ELISA.(2) Breast cancer line MCF-7and MCF-7/ADM were selected as experimental cell.Cellsmorphologies were observed by binocular inverted microscope and cell counting method wasused to plot the associated growth curve and calculate cell doubling time.The expression rate ofP-gp protein in cell surface was evaluated by flow cytometry.CCK-8assay was used to detect thesensitive degree of MCF-7and MCF-7/ADM to different antineoplastic agents(ADM, GEM,OXA, VP-16, PTX, DOC, CTX, S-1, MMC),then IC50value and resistance factor of multipleantineoplastic drugs was calculated.(3) The impacts of bFGF mAb on the proliferatin of MCF-7and MCF-7/ADM cells and thereversal effects of bFGF mAb to chemotherapeutics were detected by CCK-8method.The cellcycle distribution,the expression of P-gp and the intracellular fluorescence intensity of Rho123inMCF-7/ADM after bFGF mAb intervention were detected by flow cytometry,and theexpressions of MDR1,bFGF,FGFR,VEGF and EGFR mRNA in MCF-7/ADM were analysed byreal-time fluorescence quantitative PCR assay.Result:(1)13.5ml ascites containing bFGF mAb were extracted. The result of SDS-PAGEelectrophoresis indicated the high purfity of purified bFGF mAb solution,with the concentrationof5.366mg/ml and the titration of purified bFGF mAb solution was1:2560000after purification by the Protein G-Sepharose affinity column.The total weight of bFGF mAb was21.350mg.(2) The MCF-7cells and MCF-7/ADM cells were all presented adherently single-layerepithelioid.The MCF-7cells boundary was clear and easy to gather into a mass, while theMCF-7/ADM cells boundary was blur,with swelling nuclei.The MCF-7/ADM cells could beeasily digested,with large and irregular nuclei,less cytoplasm and stronger refractivity.Thecontact inhibition phase,logarithmic phase and plateau phase were all presented in the cellgrowth curve of MCF-7and MCF-7/ADM. The doubling time of MCF-7/ADM was significantlyhigher than that of MCF-7,with the value of26.77h and22.91h,respectively.The expression rateof P-gp protein in MCF-7/ADM was greatly increased compared with that of MCF-7,with theexpression rate of （97.90±1.16）%（,0.24±0.11）%, respectively.The CCK-8assay indicated thatthe sensitivity of MCF-7and MCF-7/ADM to multiple antitumor drugs variedgreatly.MCF-7/ADM showed higher resistance to GEM,ADM,OXA,VP-16,with resistancefactor of44.3,14.8,8.6,8.2,respectively,while MCF-7/ADM showed general resistance toPTX,DOC,DDP and obviously lowest drug resistance to CTX,S-1,MMC.(3) The inhibitory rates of MCF-7and MCF-7/ADM cells after treatment with1μg/mlbFGF mAb were (19.87±1.05)%,(27.34±2.79)%(P﹤0.01),respectivly.bFGF mAb couldefficiently reverse drug resistance of MCF-7/ADM cells on ADM,GEM and OXA,with thecorresponding reversal index of4.46,4.25and2.18, respectively.Compared with the untreatmentgroup,the cell cycle was arrested at G0/G1phase,the expression level of P-gp wasdown-regulated,the intracellular Rho123fluorescence intensity was increased,and the expressionlevels of MDR1,bFGF,FGFR and EGFR mRNA were all decreased in MCF-7/ADM cells aftertreatment with bFGF mAb (P﹤0.01).There was no significantly difference in the expression ofVEGF mRNA between MCF-7/ADM cells and bFGF mAb treated MCF-7/ADM cells (P＞0.05).Conclusion:(1) The MCF-7/ADM cell line has typical characteristics of MDR and thesensitivity of MCF-7and MCF-7/ADM to different chemotherapeutics,includingADM,GEM,OXA,VP-16,PTX,DOC,DDP,CTX, and S-1were greatly different.(2) bFGF mAb could inhibit the proliferation of adriamycin-resistant human breast cancercell MCF-7/ADM cells and obviously enhance the sensitivity of MCF-7/ADM tochemotherapeutic drug in vitro,leading to reverse MDR of MCF-7/ADM cells.The mechanism was possiblely related with stagnation of cell cycle at G0/G1phase,down-regulation of theMDR1/P-gp expression,inhibition of the P-gp function and an increasion of the concentration ofthe intracellular chemotherapeutic drugs. |
PDF Full Text Request