Spectroscopic Characterization of the 3+ and 2+ Oxidation States of Europium in a Macrocyclic Tetraglycinate Complex

The 3+ and 2+ oxidation states of europium have drastically different magnetic and spectroscopic properties. Electrochemical measurements are often used to probe Eu^III/II oxidation state changes, but a full suite of spectroscopic characterization is necessary to demonstrate conversion between these two oxidation states in solution. Here, we report the facile conversion of an europium­(III) tetraglycinate complex into its Eu^II analogue. We present electrochemical, luminescence, electron paramagnetic resonance, UV–visible, and NMR spectroscopic data demonstrating complete reversibility from the reduction and oxidation of the 3+ and 2+ oxidation states, respectively. The Eu^II-containing analogue has kinetic stability within the range of clinically approved Gd^III-containing complexes using an acid-catalyzed dissociation experiment. Additionally, we demonstrate that the 3+ and 2+ oxidation states provide redox-responsive behavior through chemical-exchange saturation transfer or proton relaxation, respectively. These results will be applicable to a wide range of redox-responsive contrast agents and Eu-containing complexes

WA inhibited tumor growth in murine MPM allograft.

<p>Female BALB/c mice bearing AB 12 tumors were treated with either vehicle (V) or WA as described in methods. Animals were monitored every day for tumor volumes (growth) as well as any activity alteration. Tumor volumes were measured every other day for the control and treated groups (A). <i>Points</i>, mean tumor volume in each experimental group containing 4 mice. <i>Bars</i>, SD. Tumors were collected after 17 days of treatment, and the tumor biopsies were analyzed by the proteasomal chymotrypsin-like activity assay (<i>B</i>) and Western blotting for ubiquitinated proteins (Ub-prs) and Bax (<i>C</i>) essentially as detailed in methods. D, The tumor tissues from the control and treated groups were immuno-stained for apoptosis (by TUNEL assay) and levels of CDKI p27, oncogene c-myc and CARP-1 proteins as noted in methods. Magnifications are 400×.</p

Withaferin A Inhibits the Proteasome Activity in Mesothelioma <em>In Vitro</em> and <em>In Vivo</em>

<div><p>The medicinal plant <em>Withania somnifera</em> has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors <em>in vivo</em> in part by inhibiting proteasome activity and stimulating apoptosis. Together our <em>in vitro</em> and <em>in vivo</em> studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.</p> </div

Antiproliferative effect of WA on human MPM cells.

<p>Cells were treated with vehicle (Control, denoted as 0) or indicated doses of WA for 72 h. Determination of viable/live cells was carried out by MTT assay. The data in the histograms represent means of three independent experiments; bars, S.E.</p

CARP-1 is required for MPM cell growth inhibition by WA.

<p>Knockdown of CARP-1 blocks WA effects. Cells were transfected with 100 nM each of scrambled or CARP-1 siRNAs for 72 h and then were either untreated (Control, DMSO) or treated with 10 µM WA for further 24 h. Cell lysates were subjected to western blotting as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041214#pone-0041214-g004" target="_blank">Fig. 4</a> for levels of CARP-1 and actin proteins or subjected to MTT assay for determination of cell viabilities essentially as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041214#pone-0041214-g001" target="_blank">Fig. 1</a>. Columns in B represent means of three independent experiments; bars, S.E. * and #, <i>p</i> = ,0.01 relative to WA-treated, scrambled siRNA-transfected cells.</p

CARP-1 Functional Mimetics: A Novel Class of Small Molecule Inhibitors of Medulloblastoma Cell Growth

<div><p>Medulloblastomas (MBs) constitute an aggressive class of intracranial pediatric tumors. Current multimodality treatments for MBs include surgery, ionizing radiation, and chemotherapy. Toxic side effects of therapies coupled with high incidence of recurrence and the metastatic spread warrant development of more effective, less toxic therapies for this disease. CARP-1/CCAR1 is a peri-nuclear phospho-protein that is a co-activator of the cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase. CARP-1 functional mimetics (CFMs) are a novel class of small molecule compounds that interfere with CARP-1 binding with APC/C subunit APC-2, and suppress growth of a variety of cancer cells in part by promoting apoptosis. Here we investigated MB growth inhibitory potential of the CFMs and found that CFM-4 inhibits growth of MB cells in part by inducing CARP-1 expression, promoting PARP cleavage, activating pro-apoptotic stress-activated protein kinases (SAPK) p38 and JNK, and apoptosis. Gene-array-based analysis of the CFM-4-treated Daoy MB cells indicated down-regulation of a number of key cell growth and metastasis-promoting genes including cell motility regulating small GTP binding protein p21Rac1, and extracellular matrix metallopeptidase (MMP)-10. Moreover, CFM-4 treatment stimulated expression of a number of molecules such as neurotrophin (NTF)3, and NF-κB signaling inhibitors ABIN1 and 2 proteins. Overexpression of NTF3 resulted in reduced MB cell viability while knock-down of NTF3 interfered with CFM-4-dependent loss of viability. CFMs also attenuated biological properties of the MB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Together our data support anti-MB properties of CFM-4, and provide a proof-of-concept basis for further development of CFMs as potential anti-cancer agents for MBs.</p></div

WA inhibits MPM cell proteasome activity.

<p>(A, B) Kinetic effects of WA on proteasome inhibition and apoptosis induction in H2595 human MPM cells. Cells were treated with 10 µM WA for various times. Chymotryptic activity of the proteasome was determined as in methods (A) and accumulation of ubiquitinated proteins, levels of p21, PARP, cleaved PARP, and actin proteins were determined by Western blotting (B). (C, D) WA inhibits proteasomal chymotrypsin-like activity in AB12 murine MPM cells and induces apoptosis. Cells were treated with 10 µM WA for indicated time periods, followed by measurement of proteasomal CT-like activity (C), Western blotting analysis for accumulation of ubiquitinated proteins, and levels of Bax, PARP, cleaved PARP, and actin proteins (D) as described in methods. <i>Bars</i>, SD; Ub prs, Ubiquitinated proteins.</p

WA suppresses MPM survival and metastasis promoting genes while enhances expression/activation of pro-apoptotic genes.

<p>MPM cells were either untreated (DMSO; denoted as −) or treated with 10 µM WA (denoted as +) and cell lysates were analyzed by western blotting as in methods. Levels of c-myc, c-Jun (A), and CARP-1 (B) proteins were determined in the cells treated with WA for 12 h. In addition, MPM cells were either untreated (DMSO, denoted as 0) or treated with 10 µM WA for the indicated time periods, and the levels of CARP-1, vimentin (C), and phospho-p38 (D) were determined by western blotting. The membranes in panels A–C were subsequently analyzed for levels of actin to assess protein loading, while the membrane in panel D was probed for expression of total p38.</p

Dosage and kinetic effects of WA on apoptosis induction in human MPM cells.

<p>H2373 cells were treated with different concentrations of WA for 16 h (A, B), or 10 µM WA for various times (C–E), followed by Western blotting analysis for levels of Bax, IκB-α, PARP, and actin proteins as detailed in methods (A and C). B, D, Caspase 3/7 activation was determined from by WA treated cell lysates as in methods. <i>Bars</i>, SD. E, Photomicrographs showing apoptosis-associated morphological changes in WA-treated MPM cells for indicated times. 0, Control, DMSO treatment.</p

CFMs reduce viabilities of the human MB cells.

<p>Cells were treated with vehicle (Control) or indicated doses of CFMs for 12 h (A) or 24 h (B). In panel C and D, the cells were treated with respective CFM for noted dose and time. Determination of viable/live cells was carried out by MTT assay. The data in the histograms represent means of three independent experiments; bars, S.E. ∗, p = <0.05 relative to Control (A, B) or 0.00 (C, D).</p