Establishment Of Human Breast Carcinoma Drug-resistant Cell Lines And The Study Of Drug-resistant Mechanism

ObjectiveTo establish human breast cancer docetaxel (Doc) and adriamycin (Adr)-resistantcell line models (named MCF-7/Doc and MCF-7/Adr), describe their biologicalcharacteritics and explore the potential molecular mechanisms of acquired drugresistance; To screen the differerntially expressed miRNA among MCF-7/Doc,MCF-7/Adr and MCF-7/S by microRNA Microarray, and confirm the microarrayresult through real-time fluorescent quantitative PCR (RT-qPCR), so as to lay afundation for further study of the regulatory role of miRNA in drug-resistance.Methods1. MCF-7/Doc and MCF-7/Adr were developed from human breast cancer parentalcell line MCF-7/S, by exposing MCF-7/S to gradually increasing concentrations ofDoc or Adr in vitro. Cells morphology changes during the process of acquired drugresistance were observated and recorded under the binocular inverted microscope;Cell counting method by trypan blue staining was used to establish cell growth curvesand get cell doubling time; MTT method was used to detect the sensitivity extent ofsensitive or resistant cells to various anti-cancer drugs; The cycle distribution ofsensitive or resistant cells was described by Flow cytometry (FCM); The expressionof cell surface symbol estrogen receptor (ER) was evaluated by western blot;RT-qPCR and western blot were respectively adopted to analyze mRNA and proteinexpressions of series multidrug-resistance related genes, such as drug-transportersMDR1/P-gp, MRP1and BCRP, metabolism enzymes CYP3A4and GST pi, repairgene Topo IIα, and apoptosis genes Bcl-2and Bax.2. The differentially expressed miRNA among sensitive, docetaxel-resistant andadriamycin-resistant cell lines were screened by microRNA Microarray. Fold change ≥2or≤0.5were thought to be statistically significant. Real time quantitative PCR(RT-qPCR) was applied to verify the reliability of microarray data for the15miRNAs,that were consistently found to be up-regulated or down-regulated in bothMCF-7/Doc and MCF-7/Adr comparing with MCF-7/S.Results1. After16months inducement, the established MCF-7/Doc could grow stably inthe medium containing200nM Doc. Under light microscope, the cells becamerounder, smaller and bulged with less mitotic figures after Doc handling. MTTshowed that MCF-7/Doc was of37-fold resistance to Doc and cross-resistance tomany other anticancer agents in various degrees. Comparing to MCF-7/S, thedoubling time of MCF-7/Doc was prolonged (41.6h vs30.6h, P<0.01) and the cellproportion in G1, G2phase increased while that in S-phase decreased. The mRNA andprotein levels of MDR1/P-gp, MRP1, BCRP, CYP3A4, Bcl-2were up-regulated, andthose of Topo IIα and Bax were down-regulated. At the same time, the ER expressionin MCF-7/Doc was lost.2. After16months inducement, the established MCF-7/Adr could grow stably inthe medium containing1500nM Adr. Under light microscope, the cells becamebigger with nuclei swelling, strong granular sensation and less mitotic figures afterAdr handling. MTT showed that MCF-7/Adr was of274-fold resistance to MCF-7/S,and cross-resistance to many other anticancer agents in various degrees. Comparingto MCF-7/S, the doubling time of MCF-7/Adr was prolonged (33.8h vs30.6h, P<0.05)and the cell proportion in G1, G2phase increased while that in S-phase decreased. ThemRNA and protein levels of MDR1/P-gp, MRP1, BCRP, GSTpi, Bcl-2wereup-regulated, and those of Topo IIα and Bax were down-regulated. At the same time,the ER expression in MCF-7/Adr was lost.3. MicroRNA microarray results showed that, comparing to MCF-7/S, there were33up-regulated and53down-regulated miRNAs in MCF-7/Doc and84up-regulatedand100down-regulated miRNAs in MCF-7/Adr. There were also165differentlyexpressed miRNAs between the two resistant cell lines. The RT-qPCR verificationresults of the15selected miRNAs that changed consistently in both two resistant cell lines accorded with the microarray results.Conclusion1. The established MCF-7/Doc and MCF-7/Adr has the typical multidrug resistantcharacteristics, which can be used as the models for resistance mechanism study.2. The acquired process of MCF-7/S resistance to Doc or Adr is very complicated,which is involved in the various pathways. There may be some common mechanismsas well as drug-special mechanisms between MCF-7/Doc and MCF-7/Adr.3. There are obvious differences in the miRNA expression among MCF-7/S,MCF-7/Doc and MCF-7/Adr cells, suggesting that miRNA may have an importantregulatory role in the acquiring of drug-resistance, which is worthy of further study.