High Catalase Activity: Another Possible Pathway for Imatinib-Resistant CML Patients.

Imatinib (imatinib mesylate: Novartis Pharmaceuticals, Basel, Switzerland) is a well-known selective inhibitor of BCR-ABL tyrosine kinase activity, a hallmark enzyme in the pathogenesis of chronic myeloid leukemia (CML). Resistance to imatinib in CML patients has been associated with different and complex types of mechanisms ranging from nonspecific multidrug resistance protein, preventing the drug to reach its target, to BCR-ABL kinase domain point mutations, reducing the affinity of the enzyme to Imatinib. More recently, it was described that modulation of reduced glutathione (GSH) intracellular levels restores sensitivity to Imatinib in Imatinib-resistant cell line. This suggests that induction of hydrogen peroxide (H₂O₂), a well-known apoptotic reactive oxygen species (ROS), might be another Imatinib anti-leukemic mechanism action. Additionally, it is known that catalase activity is enhanced through phosphorilation by the c-abl tyrosine kinase (TK). In a preliminary work, our group found normal levels of GSH but high levels of antioxidant enzyme catalase in CML patients. The aim of this work is to evaluate the possible role of catalase as another pathway to Imatinib resistance. White blood cells from blood donors, CML patients and patients with reactional leukocytosis caused by infectious bacterian diseases were obtained using Ficoll-Hypaque. Catalase activity was measured by monitoring decomposition of 10mM H₂O₂ at 240nm according to the method described by Aebi. The viability of eritroleukemia cell lineage K562 previously treated or not with 100μM 3-aminotriazole (catalase inhibitor) was evaluated by MTT method with Imatinib 1, 5 and 25μM for 24 and 48 hours. Imatinib induced intracellular ROS generation was monitored by flow cytometry with 40μM 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), a well-known ROS capturing reagent. Catalase activity was increased up to 30x in CML patients but not in reactional leukocytosis. When BCR-ABL positive cell line K562 was treated with Imatinib (50μM), catalase activity levels decreased in 38% compared with untreated cells. Also, we found that 3-aminotriazole can overcome the resistance to Imatinib in K562 cells resulting in 25% enhancement of dead cells. The protection of catalase against Imatinib-ROS generation was confirmed by DCFH-DA/FACs in cells incubated with PEG-catalase (monomethoxypolyethylene glycol covalent attached to catalase). Taken together, these results suggest that catalase might be another attractive target for overcoming resistance to Imatinib in Imatinib-resistant CML cells with high catalase activity.