20 research outputs found

    Combinations of Kinase Inhibitors Protecting Myoblasts against Hypoxia

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    <div><p>Cell-based therapies to treat skeletal muscle disease are limited by the poor survival of donor myoblasts, due in part to acute hypoxic stress. After confirming that the microenvironment of transplanted myoblasts is hypoxic, we screened a kinase inhibitor library <i>in vitro</i> and identified five kinase inhibitors that protected myoblasts from cell death or growth arrest in hypoxic conditions. A systematic, combinatorial study of these compounds further improved myoblast viability, showing both synergistic and additive effects. Pathway and target analysis revealed CDK5, CDK2, CDC2, WEE1, and GSK3β as the main target kinases. In particular, CDK5 was the center of the target kinase network. Using our recently developed statistical method based on elastic net regression we computationally validated the key role of CDK5 in cell protection against hypoxia. This method provided a list of potential kinase targets with a quantitative measure of their optimal amount of relative inhibition. A modified version of the method was also able to predict the effect of combinations using single-drug response data. This work is the first step towards a broadly applicable system-level strategy for the pharmacology of hypoxic damage.</p></div

    Top 10 kinases that show a protective response in hypoxia when inhibited.

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    <p>The data were obtained using KIEN on the entire kinase inhibitor library. The coefficient <i>β</i><sub><i>k</i></sub>, a measure of the protective impact of each kinase, was calculated and the kinases were ranked using this parameter in order to estimate the main kinases influencing the protective mechanisms against hypoxia. In bold we show kinases important for the response of both cell lines.</p><p>Top 10 kinases that show a protective response in hypoxia when inhibited.</p

    Fully factorial combination studies.

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    <p>(a) The EMD kinase inhibitors were rank ordered according to their ability to protect myoblasts in hypoxia (n = 4) (b) All possible combinations of five candidate compounds were tested at two different doses (1 μM as high dose and 0.25 μM as low dose) for their ability to protect myoblasts in hypoxia. The cell viability in the compound-treated groups was measured relative to the DMSO-treated control group at 5 days of culture using ATPlite assay (n = 3). The combinations were rank ordered according to their protective capability. (c) The normalised cell viability for each combination was shown. The pie symbols show the relative contribution of five kinase inhibitors.</p

    Primary screening of kinase inhibitor libraries.

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    <p>(a) Myoblast proliferation was observed under hypoxic and normoxic conditions for 6 days. Cellular ATP production was measured by an ATPlite assay and is indicated in Relative Luminescence Units (RLU) (n = 12, p<0.01). (b) The EMD kinase inhibitor library was screened for their capability to protect cells from 0.1% hypoxia at 1 μM concentration. Five kinase inhibitors were selected based on their efficiency in improving cell viability in hypoxic condition. (c) Dose-dependent protective effects of five candidate compounds on primary myoblasts cultured in hypoxic condition for 5 days. The cell viability in the compound-treated groups was normalised against DMSO-treated control group using ATPlite assay (n = 6, p<0.01 for all groups).</p

    Hypoxia-induced cell death in transplanted primary myoblasts.

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    <p>(a) Schematic representation of myoblasts isolated from Luciferase x EGFP mice and transplanted into the tibialis anterior muscle of NOD/SCID recipient mice. (b) Representative images of bioluminescence signaling in recipient mice at the indicated time points post-transplantation (values indicated as photons s<sup>–1</sup> cm<sup>–2</sup>). (c) Quantification of bioluminescence signaling following the transplantation of 500,000, 100,000 or 10,000 cells over 3 d (n = 4, left). Quantification of the percentage of surviving myoblasts 3 d post-transplantation (right). (d) Representative images of GFP+ myoblasts 1 d post-transplantation (green = GFP, blue = nuclei and red = laminin, hypoxyprobe, HIF-1α or cCasp3 as indicated). Scale bar, 200 μm. (e-g) Quantification of mean gray values from hypoxyprobe, HIF-1α or cCasp3 staining in areas occupied by GFP<sup>+</sup> donor myoblasts (or GFP<sup>−</sup>control) (n = 4). Data are represented as average ± SEM. Student’s t test was used for all statistical analyses (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001).</p

    Pathway analysis.

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    <p>Green nodes represent five selected kinase inhibitors whereas red nodes represent direct/indirect targets of those inhibitors. Each edge represents the influence of the inhibitors or upstream kinases on the phosphorylation levels of downstream kinases.</p

    All-trans retinoic acid suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and metastasis in esophageal squamous cell carcinoma

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    <div><p>Esophageal squamous cell carcinoma (ESCC) is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis. Adjuvant therapies, chemotherapy, radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma. All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development, growth, neural function, immune function, reproduction, and vision. It is one of the most potent therapeutic agents used for treating cancers, especially lung adenocarcinomas. ATRA inhibits metastatic potential and angiogenesis in several tumor models. We investigated the effects of ATRA on the expression of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and receptor Tie-2 in EC1 cells in vitro. We also assessed the growth and migration of EC1 cells in vitro. ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours, relative to the control. ATRA plus fluorouracil treatment reduced the viability more strongly than either drug alone, indicating an additive effect. Moreover, ATRA decreased EC1 migration by 87%. Furthermore, ATRA treatment led to a marked decrease of the transcript levels of Ang-1, Ang-2, Tie-2, VEGF, and VEGF receptors, as assessed by real-time RT-PCR. Importantly, the protein levels of Ang-1, Ang-2 and Tie-2 were reduced by ATRA treatment. In vivo, we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice. Importantly, ATRA treatment decreased the expression of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells. Collectively, our findings demonstrate that ATRA exhibits a dose- and temporal-dependent effect on the metastatic behavior, suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and the progression of xenograft tumors of EC1 cells.</p></div

    ATRA treatment reduces cell migration in EC1 cells.

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    <p>EC1 cells were cultured in RPMI-1640 supplemented with 10% FBS and seeded in 6 well plates. Scratches on cell monolayer were made using pipette tips when cells became confluent. Cells were then treated with 3 concentrations of ATRA (0.1, 1, 10 μmol/L), fluorouracil (100 mg/L), or untreated for 24 hours. Images were chosen from 10 random fields to calculate the average distances. Data were presented as average length of cell-free void ± SD. (B) Representative pictures of wound healing assay. *p<0.05; **p<0.01; *** p<0.001. Student t-test.</p

    Schematic illustration of ATRA effect on cell viability and angiogenesis in esophageal carcinoma.

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    <p>ATRA treatment induces cell apoptosis by controlling cell death regulators (survivin and bcl-2) via RAR-β, inhibits VEGF-VEGFR signaling pathway via RAR-α, and regulates the expression of angiogenic factors (Ang-1, Ang-2 and Tie-2) and angiogenesis. In vivo, ATRA suppressed tumor growth and metastasis. Previously reports and the current study demonstrate that ATRA inhibits cell viability, angiogenesis and metastasis of esophageal cancer cells in vitro and in vivo.</p
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