Abstract
BCR-ABL1-STAT5 is an oncogenic signaling pathway in human chronic myelogenous leukemia (CML) and it represents a valid target for anti-CML drug design. Resistance to direct BCR-ABL1 inhibitors is a common clinical issue, so STAT5 inhibition has become an interesting alternative target. In this study, the effects of NPQ-C6, a novel naphtoquinone-coumarin conjugate, were evaluated on human CML-derived K562 cells. Live-Cell Imaging analysis revealed that NPQ-C6 inhibited 2D (IC50AUC = 1.4 ± 0.6 μM) growth of CML cells. NPQ-C6 increased sub-G1 and reduced G0/G1 cell cycle phases in a dose- and time-dependent manner. This effect on cell cycle was related to increased levels of apoptotic nuclei, cleavage of caspase-3, -9, and PARP and annexin V-positive cells. NPQ-C6 increased γH2AX, a double-strand DNA break marker. NPQ-C6 showed a wide range of modulatory effects on cell signaling through an early increased phosphorylation of JNK, P38-MAPK and AKT, and decreased phosphorylation of ERK1/2, BCR-ABL1, and STAT5. NPQ-C6 inhibited expression of c-MYC and PYM-1, two target gene products of BCR-ABL1/STAT5 signaling pathway. Cytokine-induced activation of STAT5/STAT3-dependent transcriptional and DNA binding activities were also inhibited by NPQ-C6. Notably, NPQ-C6 maintained its activity on BCR-ABL1/STAT5/c-MYC/PIM-1 oncogenic pathway in imatinib-resistant cells. Molecular modeling suggested BCR-ABL1 and JAK2 proteins as NPQ-C6 targets. In summary, our data show a novel multikinase modulator that might be therapeutically effective in BCR-ABL1-STAT5-related malignancies.
Highlights
Human chronic myelogenous leukemia (CML) is a hematological stem cell disorder characterized by excessive proliferation of cells of the myelogenous lineage (Ren, 2005)
We report the NPQcoumarin hybrid compound 7-(3,4-dimethoxyphenyl)-6H,7Hbenzo[h]chromeno[4,3-b]chromene-6,8,9-trione (NPQ-C6) as a unique inhibitor BCR-ABL1-STAT5 oncogenic pathway that was effective against Imanitib mesylate (IM)-resistant CML cells
Monoclonal and polyclonal antibodies used in the Western blotting analyzes were as follows: pTyr694-STAT5, pTyr705-STAT3, pTyr1007/1008JAK2, pTyr177-BCR, pThr183/Tyr185-JNK, pSer473-AKT, pThr308-AKT, pThr202/pTyr204ERK1/2, BCR, PIM-1, AKT, ERK1/2, JAK2, and STAT3 were obtained from Cell Signaling Technology (Leiden, Netherlands)
Summary
Human chronic myelogenous leukemia (CML) is a hematological stem cell disorder characterized by excessive proliferation of cells of the myelogenous lineage (Ren, 2005). The main mark of CML is the Philadelphia chromosome, result of a genetic translocation that originates a fusion gene whose protein product is the chimerical BCR-ABL1 tyrosine kinase which is constitutively active. This oncoprotein is found in 95% of patients with CML and in approximately 5–10% of adults with acute leukemia without evidence of antecedent CML (Ren, 2005). BCR-ABL1 modulates intracellular signal pathways involved in proliferation (activation), apoptosis (inhibition) and cellular adhesion mechanisms (debilitation) (Ren, 2005; McCubrey et al, 2008; Sinclair et al, 2013). STAT5 activity is associated with poor prognosis in CML and its deletion in BCR-ABL1++ cells induces apoptosis, even in cells which have developed resistance to tyrosine kinase inhibitors (TKI) (Ren, 2005)
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