CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia

Abstract

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

A Novel Naphthoquinone-Coumarin Hybrid That Inhibits BCR-ABL1-STAT5 Oncogenic Pathway and Reduces Survival in Imatinib-Resistant Chronic Myelogenous Leukemia Cells

“…The human cell lines K562 (CML), HEL (erythroleukemia), HL60 (acute myeloid leukemia), MOLM.13 (acute myeloid leukemia), MV4.11 (acute monocytic leukemia), PC3 (prostate cancer), HCT-15 (colorectal adenocarcinoma), BT-549 (breast cancer), MCF7 (breast cancer), MRC.5 cells (non-malignant lung fibroblasts) and non-human cell lines NCTC3749 (mouse lymphoma) were grown in RPMI-1640 medium. The K562-R cell line, a CML resistant to IM obtained by subculturing K562 in a steplike arrangement of increasing concentration of IM (Guerra et al, 2017), was maintained in RPMI-1640. The human cell lines HeLa (epithelial cervix cancer), MDA-MB-231 (breast cancer), HS-578T (breast cancer) and T47D (breast cancer adenocarcinoma) and non-human cell lines L1210 (mouse lymphocytic leukemia cells), Vero (monkey non-malignant kidney cells) and Raw (mouse macrophage), were grown in DMEM.…”

“…The transfection medium was removed and the cells were treated with vehicle (0.05% DMSO) or compound (0.1–10 μM) in the absence of serum and for 1 h. Following pre-incubation with the drug, rhGH (50 nM) (Maamra et al, 1999) was added and cells were incubated for an additional 6 h. The stable reporter cell line HeLa/STAT3-luc (Panomics, United States), was used to determine the effects of compounds on Oncostatin M (OSM, Humanzyme, United States)-regulated STAT3 transcriptional activity (Stephens et al, 1998). Cells were seeded at a density of 240000 cells per well in 6-well culture plates for 24 h and they were serum deprived (0.5% FBS) for 16 h. Then, cells were treated with vehicle (0.05% DMSO) or compound (0.1 to 10 μM) during 1 h before oncostatin M (50 ng/ml) were added for additional 6 h (Guerra et al, 2017). Cells were lysed in Passive Lysis Buffer (Promega, United States) and luciferase activity was determined by the luciferase assay system (Thermo Scientific, United States).…”

“…Nuclei to the left of the 2N peak containing hypodiploid DNA were considered as apoptotic. Fluorescent microscopy and flow cytometric analysis of PI-stained nuclei were performed to evaluate cell cycle and viability by using a FACSCalibur cytometer and CellQuest software (BD Biosciences, Belgium) (Guerra et al, 2017). Apoptosis was also determined by translocation of phosphatidylserine to the cell surface using the annexin V-FITC apoptosis detection kit (BD Pharmingen, Belgium) according to the manufacturer’s protocol.…”

“…The naphthoquinone (NPQ)-based derivatives are privileged chemical structures commonly used to generate antitumor agents (Hsu et al, 2006; Zhao et al, 2015; Guerra et al, 2017). Some quinone-antitumor agents have been used to treat solid tumors (e.g., doxorubicin) or acute lymphoblastic and myeloblastic leukemias (e.g., daunorubicin).…”

“…Some quinone-antitumor agents have been used to treat solid tumors (e.g., doxorubicin) or acute lymphoblastic and myeloblastic leukemias (e.g., daunorubicin). NPQ-based derivatives exhibit pharmacological activities by a number of mechanisms, including oxidative stress, Michael-type arylation of thiol groups in biomolecules, DNA intercalation and bioreductive alkylation via quinone methide formation, autophagy, inhibition of topoisomerases, cell cycle arrest, apoptosis, or inhibition of c-MYC and BCR-ABL1/STAT5 pathway (Hsu et al, 2006; Zhao et al, 2015; Guerra et al, 2017; Hueso-Falcon et al, 2017). Coumarins are also considered as privileged chemical structures which exhibit a wide range of biological effects, including anticancer activities, generally associated with low toxicity (Medina et al, 2015).…”