Tumor Multidrug Resistance Reversal

Multidrug resistance (MDR) is defined as resistance of tumor cells to multiple, structurally dissimilar and functionally divergent drugs commonly used in cancer chemotherapy. Since MDR is one of the main obstacles to successful chemotherapy of cancer, extensive investigation has been made to understand the resistance mechanism and to develop strategies to reverse the resistance.The product of human MDR1 gene, P-glycoprotein (P-gp) is a prototypical MDR protein, was originally identified as a 170 kDa glycoprotein abundantly expressed in MDR cells, which effluxes intercellular d(?)g to reduce effective drug concertrations. To decrease P-gp expression is a main approach to reverse MDR phenotypes in the present stage.Using RNA interference to suppress MDR1 gene expression has been a strategy to reverse multidrug resistance. In the first chapter, we descripted endonuclease-prepared small interfering RNA (esiRNA) decreased MDR1 expression and increased chemosensitivity in the resistant MCF-7/R cells. In the esiMDR1-transfected MCF-7/R cells the MDR1 mRNA was reduced by about 50%, drug accumulation increased about 30%, and IC₅₀ value for daunorubicin was reduced from 4.5 μM to 1.2 μM. These results provide evidence that esiRNA of MDR1 could be an alternative to P-gp inhibitors.In the second chapter, we investigate whether antioxidant (catalase or N-acetylcysteine) might participate in the regulation of intrinsic MDR1 overexpression in HepG2 cells cultures. Addition of catalase and NAC to the culture led to a significant increase of the expression of MDR1 mRNA (by reverse transcription polymerase chain reaction) and drug transporter activity (Rhodamine123 drug efflux). The reduced intracellular reactive oxygen species (ROS) after catalase or N-acetylcysteine treatment was observed. Furthermore, the JNK inhibitor SP600125 abolished the positive effects of catalase on drug transporter activity in a dose-dependent manner. These data suggest that catalase can upregulate P-gp expression via reducing intracellular ROS, and JNK may mediate this process.In the third chapter we investigated the modification of the expression and function of P-gp by various concentration of TNFα. In HepG2 cells, 300 U/ml TNFα changed the expression of MDR1 faintly, but it cannot sensitize chemotherapy.Nevertheless 30000 U/ml TNFα up-regulated the expression of MDR1 significantly. In Caco-2 cells, 30000 U/ml TNFα down-regulated the activity of the MDR1 promoter by 50% and sensitized chemotherapy. In MCF-7/R cells, 300 U/ml TNFα did not change the expression of P-gp dramatically, but they decreased the value of IC₅₀ under adriamycin. All these apparent discrepancies in the regulation of MDR1 mRNA expression, protein expression, and P-gp activity might result from the gene contexts of various cell lines.In summary, in this work we investigated the influence of expression and function of P-gp by esiMDR1, antioxidant and TNFα. The effects of these reagents on MDR1 expression will provide experimental evidence for comprehensive cancer therapies.