Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug-res

来源 :World Journal of Gastroenterology | 被引量 : 0次 | 上传用户:linxl151
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AIM:To investigate the correlation between subcellulardaunorubicin distribution and the multidrug resistancephenotype in drug-resistant cell line SMMC-7721/R.METHODS:The multidrug resistant cell line SMMC-7721/R,a human hepatocellular carcinoma cell line,was established.Antisense oligonucleotides(AS-ODN)were used to obtain different multidrug resistancephenotypes by inhibiting the expression of mdr1 geneand/or muitidrug resistance-related protein gene(mrp)using Lipofectamine as delivery agent.Expression ofmdrl and mrp genes was evaluated by RT-PCR andWestern blotting.Zntracellular daunorubicin(DNR)concentration was measured by flow cytometry.Subcellular DNR distribution was analyzed by confocellaser scanning microscopy.Adriamycin(ADM)and DNRsensitivity was examined by MTr method.RESULTS:Low level expression of mdrl and mrp mRNAsand no expression of P-Glycoprotein(P-gp)andmultidrug resistance-related protein(P_(190))weredetected in parental sensitive cells SMMC-7721/S,butover-expression of these two genes was observed indrug-resistant cell SMMC-7721/R.The expression ofmdrl and mrp genes in SMMC-7721/R cells was down-regulated to the level in the SMMC-7721/S cells by AS-ODN.Intracellular DNR concentration in SMMC-7721/S cells was 10 times higher than that in SMMC-7721/Rcells.In SMMC7721/S cells intracellular DNRdistributed evenly in the nucleus and cytoplasm,whilein SMMC-7721/R cells DNR distributed in a punctatepattern in the cytoplasm and was reduced in thenucleus.DNR concentration in SMMC-7721/R cells co-transfected with AS-ODNs targeting to mdrl and mrpmRNAs recovered to 25 percent of that in SMMC7721/Scells.Intracellular DNR distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cell,and the cells resistance index(RI)to DNRand ADM decreased at most from 88.0 and 116.0 to 4.0 and 2.3,respectively.Co-Transfection of two AS-ODNsshowed a stronger synergistic effect than separatetransfection.CONCLUSIONS:P-gp and P_(190)are two membersmediating MDR in cell line SMMC7721/R.Intracellulardrug concentration increase and subcellular distributionchange are two important factors in multidrugresistance(MDR)formation.The second factor,drugstransport by P-gp and P_(190)from cell nucleus to organellin cytoplasm,may play a more important role. AIM: To investigate the correlation between subcellulardaunorubicin distribution and the multidrug resistancephenotype in drug-resistant cell line SMMC-7721 / R.METHODS: The multidrug resistant cell line SMMC-7721 / R, a human hepatocellular carcinoma cell line, was established. Antisense oligonucleotides (AS-ODN) were used to obtain different multidrug resistance phenotypes by inhibiting the expression of mdr1 gene and / or muitidrug resistance-related protein gene (mrp) using Lipofectamine as delivery agent. Expression of mdrl and mrp genes was evaluated by RT-PCR and Western blotting. Subcellular DNR distribution was analyzed by confocellaser scanning microscopy. Adriamycin (ADM) and DNR sensitivity was examined by MTr method. RESULTS: Low level expression of mdrl and mrp mRNAs and no expression of P-Glycoprotein (P-gp) and multidrug resistance-related protein (P_ (190)) weredetected in parental sensitive cells SMMC-7721 / S, butover- of these two genes was observed indrug-resistant cell SMMC-7721 / R.The expression of mdrl and mrp genes in SMMC-7721 / R cells was down-regulated to the level in the SMMC-7721 / S cells by AS-ODN. Intraracellular DNR concentration in SMMC-7721 / S cells was 10 times higher than that in SMMC-7721 / R cellslls. SMMC7721 / S cells intracellular DNRdistributed evenly in the nucleus and cytoplasm, whilein SMMC-7721 / R cells DNR distributed in a punctate pattern in the cytoplasm and was reduced in thenucleus. DNR concentration in SMMC-7721 / R cells co-transfected with AS-ODNs targeting to mdrl and mrpmRNAs recovered to 25 percent of that in SMMC7721 / Scells. Intracellular DNR distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cells, and the cells resistance index (RI) to DNR and ADM decreased at most from 88.0 and 116.0 to 4.0 and 2.3, respectively. Co-Transfection of two AS-ODNsshowed a stronger synergistic effect than separatetransfection .CONCLUSIONS: P-gp and P_ (190) are two membersmediating MDR in cell line SMMC7721 / R. Intracellulardrug concentration increase and subcellular distributionchange are two important factors in multidrug resistance (MDR) formation. The second factor, drugstransport by P-gp and P_ (190) from cell nucleus to organellin cytoplasm, may play a more important role.
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