Induction of Na+-H+ Ion Exchanger 1 Protein By Heme Oxygenase-1 Plays a Crucial Role in Imatinib-Resistant Chronic Myeloid Leukemia Cells Via p38/MAPK Signaling Pathway

Background: Resistance toward imatinib (IM) 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), as a Bcr/Abl-dependent survival molecule in CML cells, is important to resist against TKI in patients with freshly diagnosed CML or IM-resistant CML. However, the resistant mechanism on HO-1 up-regulation was still unclear.

Objective: In this study, our findings indicated that over-expression of Na⁺-H⁺ion exchanger 1 protein (NHE1) companied with HO-1 up-regulation in CML patients’ mononuclear cells who resisted with IM, while not in whom was sensitive to IM. Therefore, we explored the difference in the signaling pathway related to HO-1 and NHE1 in IM-resistant or sensitive CML cell lines.

Method: CCK-8 kits was used to determine the proliferation inhibition of cells. Flow cytometry was used for analyzing cell proliferation rate and apoptosis. Real-time PCR Array was used to search the relative pathway. Apoptosis relative factors expression were detected by real-time transcription and Western blot.

Result: As a result, we found that up-regulation of NHE1 after transfection of K562 cells line with lentivirus vector, lenti-TOPO-EGFP-HO-1. However, this effect disappeared when HO-1 expression was inhibited by transfection with lenti-U6.2-EGFP-siHO-1. Then, the relationship between HO-1 and NHE1 was detected by qPCR array, activation of p38/MAPK pathway mediated by protein kinase C-β II(PKC-β II) was found. NHE1 wasn’t up-regulated as PKC-β was blocked as well as p38/MAPK pathway was inhibited by SB203580 in K562/IM cells. Meanwhile, the apoptotic rate of the IM-resistant cells line K562 was increased by silencing HO-1, or treatment with SB203580, Enzastaurin, and Cariporide alone.

Conclusion: In this study, we demonstrated that HO-1 relative IM-resistance in CML was involved in the regulation of NHE-1 phosphorylation, which mediated by p38/MAPK signaling pathway activated by PKC-β.