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Down-regulation of catalase gene expression in the doxorubicin-resistant AML subline AML-2/DX100
1Department of Pharmacology, Chosun University Medical School, 375 Seosuk-dong, Dong-gu, Kwangju, 501-759, South Korea.
Abstract:
A major obstacle to successful cancer chemotherapy is the development of multidrug resistance (MDR). The previous study revealed that a doxorubicin-resistant AML subline (AML-2/DX100) overexpressed an MDR-associated protein (MRP) but not P-glycoprotein. The AML-2/DX100 also showed various levels of resistance to daunorubicin and vincristine but was paradoxically sensitive to hydrogen peroxide (5-fold), t-butyl hydroperoxide (3-fold), and paraquat (2-fold) when compared to the drug-sensitive parental AML-2 cells (AML-2/WT). We compared the activities of antioxidant enzymes to detoxify reactive oxygen species (ROS), including superoxide dismutases, glutathione S-transferase, catalase, glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase in both AML-2/WT and AML-2/DX100. Interestingly, of these antioxidant enzymes, catalase activity of AML-2/DX100 decreased significantly to about one-third that of AML-2/WT (P < 0.000005). The decreased activity of catalase was due to reduced expression of the catalase gene; confirmed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The decreased activity of catalase was maintained even in the absence of doxorubicin for 3 months as well as by the treatment of probenecid, an MRP inhibitor. In addition, there was no difference in catalase activity between HL-60 and another MRP-overexpressing subline HL-60/Adr. Taken together, the paradoxical increase in the sensitivity of an MRP-overexpressing AML-2/DX100 in response to peroxides and paraquat is due to the down-regulation of catalase gene expression, which totally independent of overexpression of MRP. It is therefore possible that decreased catalase activity could be exploited as an Achilles' heel in resistant cells such as this.
Insights
Multidrug resistance (MDR) in cancer chemotherapy is a challenge. This study found that decreased catalase activity, not MRP overexpression, explains why some resistant AML cells are paradoxically sensitive to peroxides.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Multidrug resistance (MDR) is a significant hurdle in cancer chemotherapy, often involving the overexpression of drug efflux pumps like multidrug resistance-associated protein (MRP).
- A doxorubicin-resistant acute myeloid leukemia (AML) subline (AML-2/DX100) overexpressed MRP but exhibited paradoxical sensitivity to reactive oxygen species (ROS) generating agents like peroxides and paraquat.
Purpose of the Study:
- To investigate the underlying mechanisms behind the paradoxical sensitivity of MRP-overexpressing AML cells to ROS-generating agents.
- To compare the activities of key antioxidant enzymes in drug-sensitive and resistant AML cell lines.
Main Methods:
- Comparative analysis of antioxidant enzyme activities (superoxide dismutases, glutathione S-transferase, catalase, glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase) between AML-2/WT and AML-2/DX100 cells.
- Gene expression analysis using Western blot and reverse transcription-polymerase chain reaction (RT-PCR) to confirm catalase down-regulation.
- Assessment of catalase activity stability in the absence of doxorubicin and in the presence of an MRP inhibitor (probenecid).
Main Results:
- Catalase activity was significantly reduced (to about one-third) in the MRP-overexpressing AML-2/DX100 cells compared to the drug-sensitive AML-2/WT cells.
- This reduction in catalase activity was directly attributed to decreased catalase gene expression.
- The diminished catalase activity was independent of MRP overexpression and persisted even after drug withdrawal or MRP inhibition.
Conclusions:
- The paradoxical sensitivity of MRP-overexpressing AML cells to peroxides and paraquat is due to down-regulation of catalase gene expression.
- This down-regulation of catalase is independent of MRP overexpression.
- Reduced catalase activity represents a potential therapeutic vulnerability, or 'Achilles' heel,' in certain resistant cancer cells.