DCC-2036 increases sub-G₁ population in FIP1L1-PDGFRα-positive cells.

<p>EOL-1 cells and BaF3 cells expressing WT or T674I FIP1L1-PDGFRα were treated with indicated concentrations of DCC-2036 (24 hours for EOL-1 cells, 36 hours for BaF3 cells); then cells were collected, washed, stained with propidium iodide and analyzed with flow cytometry. A) Representative graphs of three independent experiments; B) Statistical charts. Columns, mean; error bars, SD.</p

MEK-dependent up-regulation of Bim-EL confers the DCC-2036 induced apoptosis.

<p>A, knockdown of Bim attenuated the apoptosis in EOL-1 cells. EOL-1 cells were transfected with human Bim siRNA or non-specific control pool (Mock siRNA), respectively. Then cells were exposed to the indicated concentrations of DCC-2036. Twenty-four hours after treatments, the levels of indicated proteins were analyzed with immunoblotting (<i>top</i>). Cell death was detected with trypan blue in triplicate (<i>bottom</i>). Columns, mean; error bars, SD. One-way ANOVA with <i>post hoc</i> intergroup comparison with control by Tukey test. ***<i>P</i><0.0001. B, DCC-2036 decelerated the turnover rate of Bim-EL protein. BaF3 cells expressing WT or T674I FIP1L1-PDGFRα were exposed to 400 nM DCC-2036 for 2 hours, and then cycloheximide (CHX) was added (100 μg/mL). Cells were harvested at the indicated time points and underwent the Western blotting. C, DCC-2036 elicited a proteosome-dependent degradation of Bim-EL protein. After a 2-hour pretreatment with MG132, BaF3-T674I cells were also treated with or without 400 nM DCC-2036 for 12 more hours. Then cells were harvested, and the levels of Bim-EL protein were analyzed by Western blotting. D, MEK-dependent upregulation in Bim-EL protein in DCC-2036 treated cells. EOL-1 cells were treated with a MEK inhibitor (U0126, 20 μM), a PI3K inhibitor (LY294002, 25 μM), DCC-2036 (6 nM) or combination of them, respectively, for 24 hours, then cells were harvested and the indicated proteins were analyzed by immunoblotting. E, DCC-2036 decreases Bim-EL polyubiquitination. BaF3-T674I cells were transfected with Bim-EL along with His-ubiquitin (His-Ub). After 24 hours, the cells were treated with or without DCC-2036 at 400 nM for 8 hours, and then collected for <i>in vivo</i> ubiquitination of Bim-EL. The ubiquitinated BimEL was quantified by densitometry using Image J. The scale of ubiquitinated BimEL were normalized to that of relevant total BimEL, and then normalized to control. The ratio was shown. Ni–nitrilotriacetic acid (NTA), nickel bead precipitate; WCE, whole-cell extracts.</p

DCC-2036 abrogates the growth of xenografted T674I PDGFRα tumors transplanted in nude mice.

<p>A, the growth curves of subcutaneous xenografts of BaF3-T674I cells are shown. Nude mice bearing BaF3-T674I xenograft tumors were treated with vehicle or DCC-2036 (200 mg/kg, administered by oral gavage daily) from day 3 to 16 after inoculation of BaF3-T674I cells. The estimated tumor size was plotted versus the days after DCC-2036 treatment. Points, mean; bars, SD. Student's <i>t</i> test. B, After 13 days of DCC-2036 treatment, mice were sacrificed, tumors were dissected, weighed and photographed. Top, representative tumors from the control and experiment group are shown; bottom, comparison of tumor weights in control and experimental group. Columns, mean; bars, SD. Student's <i>t</i> test. C, Immunoblotting of the phosphorylated and total levels of indicated proteins in xenograft tissues inoculated in nude mice with vehicle or DCC-2036 treatment. D, Immunohistochemical analysis of Ki67 in xenograft tissues from mice. Hematoxylin and eosin (H&E)-stained serial sections of the same xenografts are presented.</p

DCC-2036 induces apoptosis of FIP1L1-PDGFRα-expressing cells with flow cytometry assay.

<p>EOL-1 cells and BaF3 cells expressing WT or T674I FIP1L1-PDGFRα were cultured with DCC-2036 for indicated durations at 6 nM (EOL-1 cells) or 400 nM (BaF3 cells), and then cells were collected, washed, fixed and stained with Annexin V-FITC/PI (EOL-1) or Annexin V-PE/7-AAD (BaF3 cells) to detect the cell death with flow cytometry. A, representative of three independent experiments; B, statistical charts, One-way ANOVA with <i>post hoc</i> intergroup comparison with control by Tukey test. *<i>P</i><0.05, ***<i>P</i><0.0001. Data are expressed as mean ± SD.</p

DCC-2036 induces apoptosis of FIP1L1-PDGFRα-expressing cells with Western blotting assay.

<p>A, immunoblotting of PARP cleavage and caspase-3 activation was shown in EOL-1 cells and BaF3 cells in a concentration- and time-dependent manner. B, Impact of DCC-2036 on apoptosis-related proteins. EOL-1 cells, BaF3-WT and BaF3-T674I cells were exposed to a fixed concentration of DCC-2036 (6 nM for EOL-1 cells, 400 nM for BaF3 cells) for indicated durations and then levels of apoptosis-related proteins were detected by Western blotting. C, the cytosolic AIF and cytochrome <i>c</i> were detected in EOL cells (6 nM) and BaF3 cells (400 nM) in a time- dependent manner by immunoblotting.</p

1,3-Substituted β‑Carboline Derivatives as Potent Chemotherapy for the Treatment of Cystic Echinococcosis

Echinococcosis is a global public health issue that generally occurs in areas with developed animal husbandry. In search of safe and effective therapeutic agents against echinococcosis, we designed and synthesized new 1,3-substituted β-carboline derivatives based on harmine. Among them, compounds 1a, 1c, and 1e displayed potent inhibitory activity against Echinococcus granulosus in vitro, significantly better than albendazole and harmine. The morphological detection revealed that 1a, 1c, and 1e significantly changed the ultrastructure of Echinococcus granulosus protoscolices (PSCs). Furthermore, pharmacokinetic studies suggested that 1a possessed a better metabolic property. Encouragingly, 1a exhibited a highest cyst inhibition rate as 76.8% in vivo and did not display neurotoxicity in mice. Further mechanistic research illustrated that 1a has the potential to induce autophagy in PSCs, which may be responsible for the therapeutic effect of the drugs. Together, 1a could be a promising therapeutic agent against echinococcosis, warranting further study

1,3-Substituted β‑Carboline Derivatives as Potent Chemotherapy for the Treatment of Cystic Echinococcosis

Echinococcosis is a global public health issue that generally occurs in areas with developed animal husbandry. In search of safe and effective therapeutic agents against echinococcosis, we designed and synthesized new 1,3-substituted β-carboline derivatives based on harmine. Among them, compounds 1a, 1c, and 1e displayed potent inhibitory activity against Echinococcus granulosus in vitro, significantly better than albendazole and harmine. The morphological detection revealed that 1a, 1c, and 1e significantly changed the ultrastructure of Echinococcus granulosus protoscolices (PSCs). Furthermore, pharmacokinetic studies suggested that 1a possessed a better metabolic property. Encouragingly, 1a exhibited a highest cyst inhibition rate as 76.8% in vivo and did not display neurotoxicity in mice. Further mechanistic research illustrated that 1a has the potential to induce autophagy in PSCs, which may be responsible for the therapeutic effect of the drugs. Together, 1a could be a promising therapeutic agent against echinococcosis, warranting further study

Effects of D9 on histone methylation and transcriptome in AML.

<p>Western blot analysis showing the level of cleaved PARP, EZh2 and a series of histone lysine methylation marks in MOLM-14 (a) and KG-1a cells (b) treated by D9 at indicated concentrations for 48 and 72 hours. (c) Heat map of differential genesets between sensitive and resistant cell lines with D9 treatment. Three sensitive (MOLM-14, MV4-11 and TF-1) and three resistant cell lines (Mono-Mac-1, KG-1a and THP-1) were treated with D9 at 1 or 5 μM for 48 hours. Total RNA was isolated for microarray and SAM analysis. 327 genes were up-regulated and 220 genes were down-regulated upon D9 treatment in sensitive cells relative to resistant cells using 10% false discovery rate (FDR) cut-off. (d) Ingenuity Pathway Analysis (IPA) of differentially up-regulated geneset and down-regulated geneset (dii) showing their strong connections to PI3K/AKT and MEK/ERK signaling pathways.</p

EC50 profile of D9 in human cancer cell lines.

<p>(a) Chemical structure of D9. (b) Bar graphs showing EC50 of D9 measured in a panel of solid (bi) and blood (bii) cancer cell lines using cell viability assay.1 x 10^³ cells of individual cell lines were seeded into 96-well plates in triplicates and D9 at 10 different doses was added 24 hours post cell seeding. Proliferation was measured after 96 hours treatment of D9 using an ATP based cell viability assay. EC50 of D9 was calculated by nonlinear regression (curve fit) using GraphPad PRISM3. Data represent the means of EC50 of D9 measured in three independent experiments, with each experiment run in triplicates.</p

Effects of D9 on AKT and ERK phosphorylation in AML.

<p>(a)Western blot analysis of p-ERK (T202/204), total ERK, p-AKT (S473), total AKT and ACTIN in indicated AML cell lines treated with D9 for 24 hours and 48 hours. (b) Western blot analysis showing the effects of D9 on Bim and Survivin in AML cell lines as in (a). (c) The frozen primary AML patient blasts were recovered for 24 hours and the dead cells were removed using Dead Cell Removal Kit just before D9 treatment. The blasts were treated for 96 hours for cell viability assay and 48 hours for western blot analysis. The diagrams showed the drug response curves of AML patient blasts towards D9 (ci), measurement of EC50 of D9 using cell viability assay (cii) and western blot analysis of PARP, p-ERK (T202/204), total ERK, p-AKT (S473), total AKT and ACTIN in AML patient blasts as well as MOLM-14 with and without D9 treatment (ciii). Each data point in the plots of drug response curves of D9 represents the mean ± SEM of six replicates at each specified concentration of D9, N = 3.</p