Dipeptide species regulate p38MAPK–Smad3 signalling to maintain chronic myelogenous leukaemia stem cells

Naka, Kazuhito; Jomen, Yoshie; Ishihara, Kaori; Kim, Junil; Ishimoto, Takahiro; Bae, Eun Ju; Mohney, Robert P.; Stirdivant, Steven M.; Oshima, Hiroko; Oshima, Masanobu; Kim, Dong-Wook; Nakauchi, Hiromitsu; Takihara, Yoshihiro; Kato, Yukio; Ooshima, Akira; Kim, Seong‐Jin

doi:10.1038/ncomms9039

Cited by 55 publications

(57 citation statements)

References 56 publications

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“…EW‐7197 (2.5 mg/kg) was given to tetracycline‐inducible tg‐CML‐affected mice by oral gavage. Then we isolated a cell fraction containing primitive LT‐CML stem cells (CD150 + CD135 − CD48 − KLS cells) from tg‐CML‐affected mice by flow cytometry using a FACSAria III instrument (BD Biosciences) . Phospho‐Smad3 was detected in cells using anti‐Smad3 (ab75512; Abcam, Cambridge, UK) and rabbit anti‐phospho‐Ser423/425 Smad3 (ab51451; Abcam) antibodies by the highly sensitive Duolink ® in situ PLA technology approach …”

Section: Methodsmentioning

confidence: 99%

“…(27) Phospho-Smad3 was detected in cells using anti-Smad3 (ab75512; Abcam, Cambridge, UK) and rabbit anti-phospho-Ser423 ⁄ 425 Smad3 (ab51451; Abcam) antibodies by the highly sensitive Duolink â in situ PLA technology approach. (27) Determination of WBC numbers in PB. The CML-affected mice received dasatinib (5 mg ⁄ kg ⁄ day) plus vehicle or EW-7197 (2.5 mg ⁄ kg every third day) by oral gavage for 30 days.…”

Section: Methodsmentioning

confidence: 99%

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Novel oral transforming growth factor‐β signaling inhibitor EW‐7197 eradicates CML‐initiating cells

et al. 2016

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Recent strategies for treating CML patients have focused on investigating new combinations of tyrosine kinase inhibitors ( TKI s) as well as identifying novel translational research agents that can eradicate CML leukemia‐initiating cells ( CML ‐ LIC s). However, little is known about the therapeutic benefits such CML ‐ LIC targeting therapies might bring to CML patients. In this study, we investigated the therapeutic potential of EW ‐7197, an orally bioavailable transforming growth factor‐β signaling inhibitor which has recently been approved as an Investigational New Drug ( NIH , USA ), to suppress CML ‐ LIC s in vivo . Compared to TKI treatment alone, administration of TKI plus EW ‐7197 to CML ‐affected mice significantly delayed disease relapse and prolonged survival. Notably, combined treatment with EW ‐7197 plus TKI was effective in eliminating CML ‐ LIC s even if they expressed the TKI ‐resistant T315I mutant BCR ‐ ABL 1 oncogene. Collectively, these results indicate that EW ‐7197 may be a promising candidate for a new therapeutic that can greatly benefit CML patients by working in combination with TKI s to eradicate CML ‐ LIC s.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Novel oral transforming growth factor‐β signaling inhibitor EW‐7197 eradicates CML‐initiating cells

et al. 2016

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“…cDNA was synthesized with oligo(dT) [12][13][14][15][16][17][18] primer, deoxynucleotide triphosphate mix, RT buffer and MultiScribe Reverse Transcriptase, and amplified on a Mx3005P (Agilent Technologies, Santa Clara, CA, U.S.A.) in a reaction mixture containing cDNA with relevant sense and antisense primers (Table 1), and THUNDERBIRD SYBR qPCR Mix. PCR reactions were initiated by template denaturation at 95°C for 15 min, followed by 40 cycles of amplification (denaturation at 95°C for 10 s, and primer annealing and extension at 60°C for 30 s).…”

Section: Methodsmentioning

confidence: 99%

“…14,15) Oligopeptide transporter (PEPT) 2 is highly expressed in chronic myelogenous leukemia stem cells, and pharmacological inhibition of this transporter resulted in a reduction of cellular activity. 16) Transporters generally regulate intracellular-to-extracellular concentration ratios of various nutrients and/or metabolites, and some of them play fundamental roles in cell homeostasis. Thus, they could be promising targets for cancer treatment, and their selective expression in cancer cells may mean that undesired cytotoxicity to normal cells would be minimal.…”

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confidence: 99%

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

Nakamichi

Ishimoto

Yamauchi

et al. 2016

Biological & Pharmaceutical Bulletin

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The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.

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“…The chromosomal abnormality persistently activates tyrosine kinase, resulting in the activation of a downstream signaling pathway that inhibits cell apoptosis [2–4]. In 2002, the US Food and Drug Administration (FDA) approved the tyrosine kinase inhibitor (TKI) imatinib (IM) for CML treatment [5].…”

Section: Introductionmentioning

confidence: 99%

Homoharringtonine suppresses imatinib resistance via the Bcl-6/p53 pathway in chronic myeloid leukemia cell lines

Wang

Ding

et al. 2017

Oncotarget

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BackgroundThe anti-leukemic mechanism of homoharringtonine (HHT) differs from that of IM, and HHT is one of the most useful agents for use in patients with IM resistance or intolerance. The Bcl-6/p53 pathway has been shown to regulate the sensitivity of tumor cells to antitumor drugs. We tested whether HHT blocked the Bcl-6/p53 pathway in order to promote the apoptosis of IM-resistant cells in vitro and in vivo.ResultsPh+ acute lymphoblastic leukemia (ALL) cells and IM-resistant chronic myeloid leukemia (CML) cells showed high expression of Bcl-6 protein. Bcl-6 mediated the upregulation of p53, and and Bcl-6 induced growth inhibition of IM-resistant cells as well as its apoptosis by targeting p53. In addition, Bcl-6 was downregulated moderately after HHT treatment in different cells. The Bcl-6 expression was significantly increased in patients with CML when compared with healthy subjects. Furthermore, the expression of Bcl-6 was higher in patients with CML-blastic phase (CML-BP) than in those with CML-chronic phase (CML-CP).MethodsThe inhibitory effect of drugs on cell growth was detected by Cell Counting Kit-8 (CCK-8), The apoptosis rate and the cell cycle were investigated by flow cytometry. The expression of Bcl-6, p53, Bcl-2, caspase9, and caspase3 proteins was assayed by western blot, Real- Time PCR (qPCR) detect Bcl-6 and p53 mRNA.ConclusionsHHT can suppress the growth and induce apoptosis of IM-resistant cells, the mechanism of which is associated with blocking of the Bcl-6/p53 pathway. Our results could offer a theoretical explanation for HHT use in patients with IM resistance or intolerance.

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Dipeptide species regulate p38MAPK–Smad3 signalling to maintain chronic myelogenous leukaemia stem cells

Cited by 55 publications

References 56 publications

Novel oral transforming growth factor‐β signaling inhibitor EW‐7197 eradicates CML‐initiating cells

Novel oral transforming growth factor‐β signaling inhibitor EW‐7197 eradicates CML‐initiating cells

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

Homoharringtonine suppresses imatinib resistance via the Bcl-6/p53 pathway in chronic myeloid leukemia cell lines

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