Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR1 ribozymes

Abstract

Human cancers, including hepatocellular carcinoma (HCC), are characterized by a high degree of drug resistance. The multidrug resistance (MDR) transporters MDR1-P-glycoprotein and MRP2 (multidrug-associated protein 2) are expressed in almost 50% of human cancers, including HCCs. In this study, we analyzed the effect of anti-MDR1 ribozymes, especially AFP promoter-driven anti-MDR1 ribozymes, to specifically chemosensitize HCC cells. Epirubicin-selected HB8065/R cells were used as MDR1-P-glycoprotein-overexpressing cells. Adenoviral vectors were constructed to allow an efficient gene transfer of anti-MDR1 ribozyme constructs. AFP promoter-driven anti-MDR1 ribozymes reduced the IC(50) 30-fold for epirubicin in HCC cells, whereas human colorectal cancer cells were unaffected. Target sequences were either the translational start site or codon 196 of the human MDR1 gene. Adenoviral delivery of CMV promoter-driven anti-MDR1 ribozymes resulted in a reduced IC(50) for epirubicin and doxorubicin (60- and 20-fold, respectively). They completely restored chemosensitivity in stably transfected anti-MDR1 ribozyme-expressing HCC cells as well as in HCC cells transduced with adenoviruses expressing wild-type anti-MDR1 ribozymes. Adenoviral delivery of ribozymes was so efficient that chemosensitization of HCC cells could be demonstrated in cell cultures without further selection of transduced cells for single anti-MDR1 ribozyme-expressing HCC cell clones. Northern blots showed a decreased MDR1 mRNA expression, and fluorescence-activated cell sorting (FACS) analysis revealed a significantly reduced expression of MDR1-P-glycoprotein on the cell surface of HB8065/R cells after transduction with the anti-MDR1 ribozymes. In conclusion, our data demonstrate that adenoviral delivery of ribozymes can chemosensitize HCC cells and that chemosensitization can be specifically achieved by ribozymes driven by an AFP promoter directed against human MDR1.