8 research outputs found

    Imatinib and avasimibe show a significant synergy in inhibiting viability of K562R cells.

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    <p>(a) Representative SRS images of K562 and K562R cells. (b) Quantification of CE% in LDs of K562 and K562R cells. The results are shown as means + SEM, n = 6. Two-way student t test was used for statistical analysis; n.s. indicates no significance. (c) 3D contour plot with colormap (d) linear plot of K562R cells treated with imatinib, avasimibe, or combination of imatinib and avasimibe at a molar concentration ratio of 1: 10 (IM: Ava) for 72 hours. (e) 3D contour plot with colormap (f) linear plot of K562 cells treated with imatinib, avasimibe, or combination of imatinib and avasimibe at a molar concentration ratio of 1: 10 (IM: Ava) for 72 hours.Viability was measured using the Cell Titer Glo assay, with all viabilities normalized to no inhibitor treatment group. The results are shown as means + SEM, n = 3.</p

    Imatinib and avasimibe synergistically suppress K562R xenograft tumor growth in athymic nude mice.

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    <p>(a) Tumor volume (mm<sup>3</sup>) measured by a caliper over the course of treatment for the four treatment groups. (b) Body weight (g) of the mice throughout the course of treatment. The results are shown as means + SEM, n = 8. One-way student t test was used for statistical analysis, * <i>p</i> < 0.05, *** <i>p</i> < 0.001</p

    CE accumulation in CML.

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    <p>(a) Raman Spectra acquired from LDs of four leukemia cell lines, including RCH-ACV (ALL), K562 (CML), Kasumi-2 (ALL), and MOLM14 (AML).(b) Quantification of CE% out of total lipids in LDs in four leukemia cell lines. (c) Representative SRS images of Ba/F3 Cells overexpressing empty vector treated with or without IL-3, BCR-ABL<sup>WT</sup>, or BCR-ABL<sup>T315I</sup> cells. (d) Quantification of LD amount by area fraction analysis from SRS images. (e) Raman spectra of LDs in 32D cells overexpressing empty vector, BCR-ABL, BCR-ABL<sup>T315I</sup>, or BCR-ABL<sup>kinase-dead</sup>. (f) Quantification of CE% in LDs from 32D cells. For quantitative analysis, all the results are shown as means + SEM, n = 4~6. Two-way student t test was used for statistical analysis, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001.</p

    Avasimibe downregulates the MAPK pathway.

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    <p>(a) Contour biaxials of pS6 (y-axis) and pCREB (x-axis) gated on pCRKL+ cells collected by CyTOF in K562R cells. Cells were treated for 0 or 4 hours with10μM avasimibe. (b) Effect of 30 minute 5μM imatinib treatment on sensitive and resistant patients normalized to the basal condition on the pooled MAPK pathway proteins together (All) and individually. Error bars represent standard deviation of fold change in each group of patients. T-tests were conducted comparing fold change in resistant patients to sensitive patients (p-values shown) and for general reduction in phosphorylation (*- p<0.05) (c-d) Bar graphs showing fold change of median protein expression after 10μM avasimibe and combination therapy normalized to 5μM imatinib in resistant and sensitive CML patients (n = 4 for all groups except SCML3 was omitted in the pERK group because zero pERK signal was observed). Imatinib treatment was for thirty minutes while avasimibe treatment was for four hours. (e) Heatmaps of CyTOF screens of non-lymphoid CD34<sup>+</sup> CD38<sup>−</sup> cells from cryopreserved bone marrow from a normal patient (top), cryopreserved bulk PBMCs from an imatinib-sensitive patient (middle), and cryopreserved bulk PBMCs from an imatinib-resistant patient without a BCR-ABL kinase domain mutation (bottom). Cells were treated with no inhibitor, 1μM imatinib, 10μM avasimibe, or imatinib plus avasimibe at the same concentrations. Imatinib stimulation was done for 30 minutes, while avasimibe stimulation was done for four hours. Heatmap tile color represents arcsinh ratio of medians normalized to the basal condition for each patient, see Bendall et al. 2011[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179558#pone.0179558.ref023" target="_blank">23</a>] for details. (f) Biaxials of p-p65/NFκB on the x-axis versus p-p38/MAPK on the y-axis in Lin<sup>-</sup> CD34<sup>+</sup> CD38<sup>−</sup> collected by CyTOF from the resistant patient. Each plot represents one of the four stimulation conditions: basal (top left), imatinib (top right), avasimibe (bottom left), and imatinib + avasimibe (bottom right). The contour represents cell density.</p

    Additive effects of 1R-Chl and imatinib treatments.

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    <p>(A) Murine BM cells transduced with unmutated BCR-ABL were treated with 500 nM imatinib, 500 nM 1R-Chl, and 500 nM 1S-Chl individually, or in combination of either 500 nM 1R-Chl or 500 nM 1S-Chl with 500 nM imatinib. (B) Same treatment routines were used on CML patient MNCs. The CML patient MNCs were treated 500 nM imatinib, 500 nM 1R-Chl, and 500 nM 1S-Chl individually, or 500 nM 1R(S)-Chl in combination with 500 nM imatinib. (C) Same experiments were done on normal blood donor MNCs.</p

    Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.

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    <p>(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC<sub>50</sub> = 260 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>, and dasatinib (10 nM and 100 nM; IC<sub>50</sub> = 0.8 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.</p

    Effects of 1R-Chl on colony formation of CML patient MNCs and normal human MNCs.

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    <p>(A) MNCs from CML patients were cultured with rh stem cell factor, rh IL-3, rh GM-CSF with or without erythropoietin which lead to CFU-GM or BFU-E cell lineages. The experiments were done in duplicate with 1R-Chl ranging from 125 to 1000 nM, 1S-Chl at 500 and 1000 nM, and imatinib at 500 and 5000 nM. The colonies were counted and results were calculated as the percentage of the control plates (without treatment) after 2 weeks. (B) MNCs from normal donors were cultured and plated under the same condition as CML patient cells. The cells were exposed to 500 and 1000 nM 1R-Chl, 500 and 1000 nM 1S-Chl, and 500 and 5000 nM imatinib. The colonies were counted and percent growth inhibition was calculated after 2 weeks.</p

    Cytotoxicity (MTS) assays on resting murine BM cells and human MNCs.

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    <p>(A) Ficoll-Hypaque-separated murine BM cells were plated in triplicate with 1R-Chl (10, 100, 500, and 1000 nM) without any growth factors or mitogens. After 72 and 96 hours, 20 µL of Celltiter 96 Aqueous One solution (Promega, WI) was added. The absorbances of the MTS metabolites were read corresponding to the numbers of metabolically active cells. (B) Ficoll-Hypaque-separated human MNCs were treated as above, and the viabilities of the cells were assessed after 72 and 96 hours.</p
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