Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR–ABL positive cells

Abstract

Chronic myeloid leukemia (CML) is characterized by constitutive BCR–ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR–ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%–30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR–ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR–ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L‐NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1 μM) of imatinib. Mechanistically, imatinib or BCR–ABL siRNA following deglutathionylation of NF‐κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase‐3 however associated with increased caspase‐3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

Oxidative and Nitrosative Stress in Major Depressive Disorder: A Case Control Study

“…The neutrophils from healthy volunteers, patients, and their first-degree relatives were isolated as described previously [ 47 ], and the purity was ascertained by a Flow cytometer (FACS Calibur, Becton Dickinson, NJ, USA) using a CD15 antibody and it was always more than 95%. The viability of the isolated PMNs was more than 95%, as assessed by the Trypan blue exclusion test [ 47 ].…”

“…The relative fold differences between the groups were calculated by using the comparative cycle threshold (2 −ΔΔCp ) method. β-Actin was used as an internal standard to calculate the relative mRNA expression [ 47 , 50 ].…”

“…Neutrophils were isolated from the venous blood samples collected in sodium citrate (0.129 M, pH 6.5, 9:1-v/v). Platelet rich plasma was removed by centrifugation at 250× g for 20 min at 20 • C (Sigma centrifuge, Roedermark, Germany) and the buffy coat was subjected to a Percoll density gradient at 700 g for 30 min at 25 • C. The neutrophils from healthy volunteers, patients, and their first-degree relatives were isolated as described previously [47], and the purity was ascertained by a Flow cytometer (FACS Calibur, Becton Dickinson, NJ, USA) using a CD15 antibody and it was always more than 95%. The viability of the isolated PMNs was more than 95%, as assessed by the Trypan blue exclusion test [47].…”