Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R.

Abstract

To investigate the correlation between subcellular daunorubicin distribution and the multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R. 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 multidrug resistance-related protein gene(mrp) using Lipofectamine as delivery agent. Expression of mdr1 and mrp genes was evaluated by RT-PCR and Western blotting. Intracellular daunorubicin (DNR) concentration was measured by flow cytometry. Subcellular DNR distribution was analyzed by confocal laser scanning microscopy. Adriamycin (ADM) and DNR sensitivity was examined by MTT method. Low level expression of mdr1 and mrp mRNAs and no expression of P-Glycoprotein(P-gp) and multidrug resistance-related protein (P(190)) were detected in parental sensitive cells SMMC-7721/S, but over-expression of these two genes was observed in drug-resistant cell SMMC-7721/R. The expression of mdr1 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/R cells. In SMMC7721/S cells intracellular DNR distributed evenly in the nucleus and cytoplasm, while in SMMC-7721/R cells DNR distributed in a punctate pattern in the cytoplasm and was reduced in the nucleus. DNR concentration in SMMC-7721/R cells co-transfected with AS-ODNs targeting to mdr1 and mrp mRNAs 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 DNR and ADM decreased at most from 88.0 and 116.0 to 4.0 and 2.3, respectively. Co-Transfection of two AS-ODNs showed a stronger synergistic effect than separate transfection. P-gp and P(190) are two members mediating MDR in cell line SMMC7721/R. Intracellular drug concentration increase and subcellular distribution change are two important factors in multidrug resistance (MDR) formation. The second factor, drugs transport by P-gp and P(190) from cell nucleus to organell in cytoplasm, may play a more important role.