Related Experiment Videos
Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells
M Kiehntopf1, M A Brach, T Licht
1Department of Medical Oncology and Applied Molecular Biology, Freie Universität Berlin, Universitätsklinikum Rudolf Virchow, Robert-Rössle-Cancer Center, Germany.
Abstract:
How cancer cells become resistant to chemotherapy is not completely understood, but it is believed that resistance is usually associated with overexpression of drug resistance genes. Drug resistance mediated by the MDR-1 gene is the first well characterized form of drug resistance in human cancer. MDR-1 encodes a phosphoglycoprotein, P-GP, that serves as an energy-dependent drug efflux pump, reducing intracellular drug accumulation and thereby cytotoxicity. We have used ribozymes to reverse the multiple drug resistance phenotype. A hammerhead ribozyme recognizing the GUC sequence at position -6 to -4 close to the translation start site of the 4.5 kb MDR-1 mRNA was prepared by in vitro transcription (MDR-1-RZiv) or chemical synthesis (MDR-1-RZs). Both MDR-1-RZiv and MDR-1-RZs specifically cleaved the MDR-1 mRNA into two parts of the expected size under physiological conditions in an extracellular system with MDR-1-RZiv being more effective. Site-specific cleavage was dependent on time, temperature and [MgCl2]. To examine the in vivo potential of MDR-1-RZ, MDR-1-RZiv and MDR-1-RZs were transfected into a human pleural mesothelioma cell line and into one adriamycin-resistant and one vindesine-resistant subline thereof by liposome-mediated transfer. Incorporation of ribozymes resulted in significantly reduced expression of the MDR-1 gene, with MDR-1-RZs being more potent than MDR-1-RZiv in vitro. MDR-1-RZ reduces P-GP overexpression at the protein level. Liposome-mediated transfer of MDR-1-RZiv or MDR-1-RZs reversed the multiple drug resistance phenotype and restored sensitivity towards chemotherapeutic drugs.
Insights
Ribozymes targeting the MDR-1 gene reverse chemotherapy resistance by reducing P-glycoprotein expression. This restores cancer cell sensitivity to drugs, offering a potential strategy against multidrug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Multidrug resistance (MDR) in cancer is a significant challenge, often linked to the overexpression of drug resistance genes.
- The MDR-1 gene encodes P-glycoprotein (P-GP), an efflux pump that reduces intracellular chemotherapy drug concentration and efficacy.
- Understanding and overcoming MDR is crucial for improving cancer treatment outcomes.
Purpose of the Study:
- To investigate the potential of ribozymes to reverse the multidrug resistance phenotype in cancer cells.
- To evaluate the efficacy of MDR-1 specific ribozymes in reducing MDR-1 gene and P-GP expression.
- To assess the restoration of sensitivity to chemotherapeutic agents in resistant cancer cells.
Main Methods:
- Design and synthesis of hammerhead ribozymes (MDR-1-RZiv and MDR-1-RZs) targeting the MDR-1 mRNA.
- In vitro cleavage assays to assess ribozyme specificity and efficiency under physiological conditions.
- Liposome-mediated transfection of ribozymes into human mesothelioma cell lines, including drug-resistant sublines.
- Analysis of MDR-1 gene and P-GP protein expression levels post-transfection.
- Assessment of restored sensitivity to chemotherapeutic drugs.
Main Results:
- Both MDR-1-RZiv and MDR-1-RZs specifically cleaved MDR-1 mRNA, with MDR-1-RZiv showing higher efficacy in vitro.
- Transfection of ribozymes into resistant cell lines significantly reduced MDR-1 gene expression, with MDR-1-RZs being more potent.
- Ribozyme treatment decreased P-GP overexpression at the protein level.
- Liposome-mediated delivery of ribozymes reversed the multidrug resistance phenotype and restored drug sensitivity.
Conclusions:
- Ribozymes targeting the MDR-1 gene can effectively reduce its expression and P-GP protein levels.
- This approach successfully reversed the multidrug resistance phenotype in cancer cells.
- Ribozyme-based strategies hold promise for overcoming chemotherapy resistance and enhancing treatment efficacy.