Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.
Highlights
Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) resulting from reciprocal translocation between chromosome 9 and chromosome 22 [t(9; 22) (q34; q11)], eventually forming the breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) oncogene, which encodes a constitutively activated tyrosine kinase [1]
K562/G01 cells demonstrated significantly higher expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) protein than the parental K562 cells (p 0.005) (Figures 1(b) and 1(c)). erefore, Nrf2 is more expressed in the imatinib-resistant cell line K562/G01 at both transcription and protein levels, which supports our hypothesis that Nrf2 plays a role in imatinib resistance in K562/G01 cells
Our in vitro preliminary study confirms that Nrf2 expression is a key factor of resistance to imatinib in K562/G01 cell lineage with independence of BCR-ABL mutation
Summary
Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) resulting from reciprocal translocation between chromosome 9 and chromosome 22 [t(9; 22) (q34; q11)], eventually forming the breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) oncogene, which encodes a constitutively activated tyrosine kinase [1]. Imatinib mesylate (IM), as the first-generation tyrosine kinase inhibitors (TKIs), targeted represses the tyrosine kinase activity of BCR/ABL fusion protein [2]. Either administered alone or combined with other therapies, it has become one of the first-line drugs for the targeted treatment of CML [3]. There are still 15% to 25% of patients having primary or secondary drug resistance due to T315I mutation, clonal evolution, overexpression or hyperactivation of some members of the SRC family of kinases, activation of additional pro-oncogenic pathways, leukemia stem cell intrinsic resistance, and mutations in epigenetic regulators [3,4,5]. Nrf was persistently overexpressed in CML and acute myeloid leukemia (AML) patients [10].
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