Protein Kinase C-βii-Dependent Up-Regulation Of Heme Oxygenase-1 In Chronic Lymphocytic Leukemia Cells Is Crucial For Resistance Against Imatinib

Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors(TKI) remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1) inhibition plus TKI were found to produce growth inhibition in imatinib-resistant K562/IM cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In this study, we aimed to explore the molecular mechanism of HO-1 explosive expression and relationship between HO-1 and Protein Kinase C-βII(PKC-βII) in imatinib-resistant CML cells.

We explored that mRNA expression of various drug-resistant-relative gene between K562 cells and K562/IM cells via high throughout screening assay detected by real-time PCR. According to the results mentioned above, relative protein expression and phosphorylation examined by western blot. And protein translocation was observed and recorded by fluorescence microscope. Cells apoptosis, reactive oxygen species(ROS) and intracellular calcium were analyzed by flow cytometry. Cells proliferation inhibition was detected by CCK-8 assay. The peripheral blood mononuclear cells(PBMNCs) were purified from 45 patients with freshly diagnosed CML or imatinib-resistant CML(including all kinds of drug-resistance involved in various of reasons). Compared the differences of mRNA expression between HO-1 and PKC-βII in those patients.

Our results indicated that PKC-βII contributed to activation of HO-1 expression through PI3K/AKT pathway. PKC-βII phosphorylation localized to membrane from cytoplasim to activate PI3K/AKT pathway, meanwhile, the 7.8 folder higher (p<0.01) expression of HO-1 following the nucleus translocation of NF-E2–related factor 2(Nrf-2) was observed after pAKT up-regulation. The accumulation of ROS and intracellular calcium overload were reduced by HO-1 to protect cells against cytotoxicity, less apoptotic imatinib-resistant K562 cells were detected at the same concentration of imatinib(the total apoptotic rate of K562/IM:42.4%±3.3% vs. K562: 72.1%±4.1%, at the concentration of 1 μM, p<0.01). In addition, the results of HO-1 and PKC-βII mRNA expression in PBMNCs from 45 patients indicated that HO-1 activation kept positively correspondent with expression of PKC-βII in primary leukemic cells obtained from those patients. In addition, blockage of PI3K/AKT, silence of PKC-βII or HO-1 by small interfere RNA( or inhibited by targeted inhibitor) could significantly increase apoptotic rate of imatinib-resistant CML cells(the apoptotic rate of regulated expression/ natural expression: when blockage of PI3K/AKT: 1.54±0.17; silence of PKC-βII: 2.09±0.23; inhibition of HO-1: 1.87±0.12, p<0.05).

Selectively targeted-PKC-βII plus imatinib produced growth inhibition in primary leukemic cells. In summary, these data showed that HO-1 expression involved in PKC-βII activation in imatinib-treated CML cells, and targeted- PKC-βII may be the same promising novel strategy as HO-1 inhibition in imatinib resistant CML.

No relevant conflicts of interest to declare.