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Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using
Objective:
The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs. Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment.
Methods:
The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined. Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT). In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C.
Results:
Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs. 10% variability after free phosphorothioates). Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs. 22% MRK16 staining). Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed. In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control).
Conclusion:
The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs. A more promising approach seems to be the prophylactic treatment with AS-ODNs.
Insights
Antisense oligodeoxynucleotides (AS-ODNs) encapsulated with cationic lipids enhance cellular uptake and inhibit multidrug resistance (MDR) in leukemia cells. Prophylactic AS-ODN treatment shows promise for managing MDR.
Area of Science:
- Molecular biology
- Cancer research
- Drug delivery systems
Background:
- Multidrug resistance (MDR) in leukemia poses a significant challenge to effective chemotherapy.
- The P-glycoprotein (P170) is a key mediator of MDR, contributing to drug efflux and treatment failure.
- Antisense oligodeoxynucleotides (AS-ODNs) offer a targeted approach to downregulate genes involved in MDR, such as mdr1.
Purpose of the Study:
- To investigate the efficacy of chimeric AS-ODNs encapsulated with cationic lipids in overcoming MDR in human leukemia cells.
- To compare the effectiveness of lipid-conjugated AS-ODNs with traditional free AS-ODNs (unmodified or phosphorothioated).
- To evaluate the potential of AS-ODNs for prophylactic treatment in drug-sensitive leukemia cells.
Main Methods:
- Drug-resistant and drug-sensitive human leukemia cell lines were treated with AS-ODNs.
- Cellular uptake of AS-ODNs was analyzed.
- MDR-related markers, including mdr1-mRNA and P170 expression, were quantified using RT-PCR and flow cytometry (FCM).
- Cellular accumulation and efflux of Rhodamine 123 (R123) were assessed by FCM.
- Sensitivity to vincristine was determined using the MTT assay.
Main Results:
- Cationic lipid encapsulation significantly increased cellular AS-ODN uptake but also introduced variability.
- Both AS-ODN types reduced P170 expression, with chimeric AS-ODNs showing a stronger effect (30% vs. 22% reduction).
- Inhibition of P170 expression correlated with increased sensitivity to vincristine.
- AS-ODN treatment in drug-sensitive cells led to near-complete inhibition of mdr1-mRNA expression (5% of control).
Conclusions:
- Complete reversal of the MDR phenotype in established drug-resistant leukemia cells using AS-ODNs is challenging.
- Prophylactic treatment with AS-ODNs presents a more promising strategy for managing MDR in leukemia.
- Lipid-based delivery systems enhance AS-ODN uptake and may improve therapeutic outcomes in leukemia treatment.