Additional file 1: of Discordant lymphoma consisting of mediastinal large B-cell lymphoma and nodular sclerosis Hodgkin lymphoma in the right supraclavicular lymph nodes: a case report

Gene rearrangements and clonality analysis of immunoglobulin heavy chain gene, Kappa light chain gene, and Lambda light chain gene were identified in mediastinal large B-cell lymphoma and in nodular sclerosis Hodgkin lymphoma in the right supraclavicular lymph nodes using the IdentiCloneTM IGH/IGK/IGL Gene Clonality Assay (InVivoScribe Technologies, CA, USA). (TIF 1129 kb

Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

<div><p>Background</p><p>Glioblastoma multiforme (GBM), the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ) is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ. </p> <p>Methodology/Principal Findings</p><p>Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ.</p> <p>Conclusions/Significance</p><p>These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.</p> </div

Effect of co-treatment with TMZ and eEF-2 kinase inhibitors on growth of glioma cells.

<p>(<b>A</b>) Human glioma cell lines LN229 and U251 with or without silencing of eEF-2 kinase expression were plated in 96-well plates (5×10³/well) and treated with TMZ (100 μM) at 37°C for 24 h; (<b>B</b>) LN229 and U251 cells were plated in 96-well plates (5×10³/well) and treated with TMZ (100 μM) in the presence or absence of 0.5μM of NH125 at 37°C for 24 h. Cell viability was measured by CCK-8 assay. (<b>C</b>) LN229 and U251 cells were transfected with an eEF-2 kinase-targeted siRNA or a non-targeting control siRNA (NT control). At different time points, expression of eEF-2 kinase was analyzed by Western blot,. β-actin was used as a loading control. (<b>D</b>) LN229 and U251 cells were treated with NH125 (0.5μM) or vehicle for various periods of time. At the end of treatment, the level of phospho-eEF2 (Thr56) was examined by western blot. β-actin was used as a loading control. Each bar represents mean ± SD of triplicate determinations; results shown are the representative of three identical experiments; data are expressed as the percentage of the controls. ** <i>p</i> < 0.01.</p

Effect of the combinatorial treatment with TMZ and NH125 on glioma growth mouse in an intracranial xenograft model.

<p>The human glioma LN229 cells were implanted into the anesthetized nude mice (5×10⁶ cells/per site) through a burr hole on the skulls. Three days later, the inoculated mice were divided into four groups (10 mice per group) and subjected to different treatments: (1) Control groups receive appropriate vehicles (0.5% DMSO in PBS); (2) TMZ (8 mg/kg) on days 1- 10, i.p.; (3) NH125 (1 mg/kg) on days 1, 3, 5, 7, 9 and 11, i.p.; (4) TMZ plus NH125. (<b>A</b>) HE staining of the paraffin sections of the tumor xenografts. (<b>B</b>) The survival curves of the tumor-bearing mice. Median survival (days): vehicle: 21days; NH125: 25 days; TMZ: 28 days; TMZ+NH125: 31 days. The pooled-variance two-tailed <i>t</i>-test showed <i>p</i> <0.05 for TMZ+NH125 vs TMZ. (<b>C</b>) TUNEL staining of the paraffin sections of the tumor xenografts. </p

Effect of inhibiting eEF-2 kinase on the TMZ-induced apoptosis in glioma cells.

<p>LN229 and U251 cells were treated with the indicated concentration of TMZ for 24 h in the presence or absence of silencing of eEF-2 kinase expression or in the presence or absence of NH125. At the end of treatment, apoptosis was determined by Western blot of cleaved PARP and caspase-3 (<b>A</b> and <b>B</b>) and by cytometric analysis of Annexin V staining (<b>C</b> and <b>D</b>). β-actin was used as a loading control. **<i>p</i> < 0.01, <i>t</i>-test. Each bar represents mean ± SD of triplicate determinations; results shown are the representative of three identical experiments.</p

Effect of co-treatment with TMZ and eEF-2 kinase inhibitors on migration of glioma cells.

<p>(<b>A</b> and <b>B</b>) LN229 and U251 cells were transfected with an eEF-2 kinase-targeted siRNA or a non-targeting control siRNA (NT control), and then plated in 6-well tissue culture dishes (5×10⁵/well). An injury line was made on the confluent monolayer of cells and then treated with the indicated concentration of TMZ at 37°C for 24 h. (<b>C</b> and <b>D</b>) LN229 and U251 were plated in 6-well tissue culture plates (5×10⁵ /well). When cells were confluent, an injury line was made on the monolayer of cells and treated with the indicated concentration of TMZ in the presence or absence of NH125(0.5μM) at 37°C for 24h. Cell migration was observed with a light microscope and imaged at 0 and 24 h. Width of the lines was measured and the mean ± SD from three independent experiments were shown. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, vs Control/ NT control; ^#<i>p</i> < 0.05, ^##<i>p</i> <0.01, vs NH125 or eEF-2 kinase siRNA; ^&<i>p</i> <0.05, ^&&<i>p</i> <0.01, vs TMZ.</p

Protective Effects of Astaxanthin on ConA-Induced Autoimmune Hepatitis by the JNK/p-JNK Pathway-Mediated Inhibition of Autophagy and Apoptosis

<div><p>Objective</p><p>Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.</p><p>Materials and Methods</p><p>Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.</p><p>Results</p><p>Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.</p><p>Conclusion</p><p>This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.</p></div

Nucleotide sequences of primers used for qRT-PCR.

<p>Nucleotide sequences of primers used for qRT-PCR.</p

Effects of olive oil and astaxanthin on the liver function and pathology of healthy mice.

<p>(A) The levels of serum ALT and AST in the four groups did not differ. Data are given as means ± SD (n = 6, P > 0.05). (B) The percentage of different immune cell subsets, serum levels of TNF-α, IL-6, IL-1β, and IFN-γ of four groups were evaluated in each group with ELISAs or flow cytometry (n = 6, P > 0.05). (C) Representative hematoxylin-and-eosin-stained sections of the liver. Original magnification, ×200.</p

Effects of astaxanthin on liver function and pathology of mice with ConA-induced acute hepatitis.

<p>(A) The levels of serum ALT and AST changed depending on the astaxanthin dose, 20 mg/kg or 40 mg/kg. Data are given as means ± SD (n = 8, *P < 0.05 for Oil versus ConA, ^#P < 0.05 for ConA+Astaxanthin (20) versus ConA, ⁺P < 0.05 for ConA+Astaxanthin (40) versus ConA). (B) The necrotic and edematous area stained with hematoxylin and eosin and used for the liver sections was analyzed with Image-Pro Plus 6.0 (magnification, ×200). The results show statistically significant differences among the different groups (n = 8, *P < 0.05 for ConA+Astaxanthin (20) versus ConA, ^#P < 0.05 for ConA+Astaxanthin (40) versus ConA).</p