Triterpenoids Display Single Agent Anti-tumor Activity in a Transgenic Mouse Model of Chronic Lymphocytic Leukemia and Small B Cell Lymphoma

The synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and derivatives display anti-tumor activity against a variety of cultured tumor cell lines and in mouse xenografts. In this report, we have studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on chronic lymphocytic leukemia (CLL), using patients' CLL cells and a mouse model of CLL and small B cell lymphoma (SBL).CDDO and CDDO-Im efficiently induced apoptosis of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating mice with CLL/SBL with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung. CDDO-Im was shown to be more potent than CDDO, while treatment with empty liposomes had no impact on disease. CDDO-Im treatment initially resulted in an increase of circulating B cells, which correlates with a reduction in resident lymphocytes in spleen, and lungs, suggesting that CDDO-Im induces mobilization of tumor cells from lymphoid organs and infiltrated tissues into the circulation. Analysis of blood cells recovered from treated mice also showed that CDDO-Im is a potent inducer of tumor cells death in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL/SBL cells but had little effect on the viability of normal B and T cells in vivo.The presented data demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL, and support the clinical testing of CDDO-based synthetic triterpenoids in patients with CLL

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009

Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

<div><p>Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the <i>in vitro</i> and <i>in vivo</i> consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.</p></div

<i>In vivo</i>, Plk1 inhibition leads to mitotic arrest, DNA damage and senescence.

<p>A) HCT116 tumor bearing mice were dosed orally once a day with 23.3 mg/kg of MLN0905. Tumor tissues were harvested and stained for pHisH3 (green staining) and γH2AX (brown staining). Representative tumor sections are shown with either vehicle or MLN0905 treatment 24 hours after the first dose. pHisH3 and γH2AX signals were quantified (see graph, shown is mean ±SD) and arrows indicate when doses were delivered. B) Tumor tissues were harvested at the indicated times and stained for β-galactosidase activity. Images shown indicate β-galactosidase staining in the MLN0905 treated tumors but not in the time-matched vehicle controls. Staining was quantified and is represented as % tumor area positive (see graph, shown is mean ±SD; *p≤0.04, two-tailed paired t-test).</p

Plk1 inhibition leads to prolonged mitotic arrest followed by detection of DNA damage in HT-29 cells.

<p>Cells were treated with increasing concentrations of MLN0905 and mitotic arrest (via pHisH3) and DNA damage (via γH2AX) were analyzed at indicated times. A) Immunoblotting was used to demonstrate prolonged mitotic arrest precedes DNA damage (representative blot shown from two independent experiments). B) The same samples used in A) were quantified for DNA damage (γH2AX) using FACS analysis. C) In a separate experiment immunofluorescent chemistry was also used to demonstrate mitotic arrest (pHisH3) precedes DNA damage (γH2AX) (representative images shown from two independent experiments; 200 nM MLN0905 used).</p

Plk1 inhibition leads to mitotic arrest and DNA damage in a wide range of human cancer cell lines.

<p>Cells were treated with MLN0905 (Calu-6, 10 nM; DLD-1, 90 nM; A549, 30 nM; SW480, 20 nM) for the indicated times and immunoblotting was used to monitor mitotic arrest (pHisH3) and DNA damage (γH2AX).</p

Plk1 inhibition leads to transient mitotic arrest followed by DNA damage in HCT116 cells.

<p>Cells were treated with increasing concentrations of MLN0905 and at indicated times analyzed for mitotic arrest (via pHisH3) and DNA damage (via γH2AX). A) Immunoblotting indicates transient mitotic arrest leads to DNA damage (representative blot shown from two independent experiments). B) The same samples used in A) were quantified for DNA damage (γH2AX staining) using FACS analysis. C) In a separate experiment immunofluorescent chemistry was also used to demonstrate mitotic arrest precedes DNA damage (representative images shown from two independent experiments; 50 nM MLN0905 used).</p