ANX2T inhibitor sensitizes primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine.

<p><b>A)</b> Graph shows the percentage of NTPL-20 cells that bound to Saos-2 monolayer in the presence or absence of ANX2T inhibitor or anti-ANX2 antibody with respect to the input. Error bars indicate SD of the Mean from three independent experiments. Asterisks denote P < 0.01. <b>B)</b> Primary ALL cells (NTPL-20) were either plated on tissue culture plastic or Saos-2 monolayers and treated with 1 μM dexamethasone or 10 nM vincristine. Twenty-four hours post incubation, leukemic cells were collected by vigorous pipetting and stained with propidium iodide. The percentage of viable cells in the population was determined by flow cytometry based on exclusion of propidium iodide. Graph represents the percentage of viable cells in each sample normalized to the control (cells plated on osteoblasts in the presence of DMSO considered as 100%). Asterisks indicate statistical significance (*P < 0.05, **P < 0.01). <b>C</b>) Representative images of histological sections of femur from a mouse transplanted with NTPL-20 cells. Dual immunohistochemistry staining was performed showing osteocalcin (osteoblast specific marker, in brown) and CD45 (marker for ALL cells, in red). The black square in the upper panel marks the region of the slide, which is magnified in the lower panel. Scale bars = 100 μm. Similar data was obtained in three different mice.</p

Disruption of interaction between ANX2 and p11 suppresses short-term homing and engraftment of primary ALL cells in immune-compromised mice.

<p><b>A)</b> NSG-B2m mice were injected with either anti-ANX2 antibody or an isotype-matched control antibody (IgG) before transplantation of 10 million NTPL-20 cells. Twenty-four hours post cell injection, mice were sacrificed and peripheral blood, spleen, liver, and femurs were harvested. Femurs were flushed to collect bone marrow. The percentage of human cells was detected by flow cytometry. The error bars denote SE of the Mean (n = 6 each). Asterisks indicate statistical significance with P < 0.05. <b>B)</b> NSG-B2m mice were injected with either anti-p11 antibody or an isotype-matched control antibody (IgG) before transplantation of 10 million NTPL-20 cells. The percentage of human cells in mouse organs was detected by flow cytometry twenty four hours post transplantation as described in A. The error bars denote SE of the Mean (n = 5 each). Asterisks indicate statistical significance with P < 0.05. <b>C</b>) Mice were pre-treated with either ANX2T inhibitor or vehicle before NTPL-20 cell transplantation. The graph shows the percentage of human cells in indicated organs determined twenty-four hours post cell injection. The error bars represent SE of the mean (n = 5). Asterisk denotes P < 0.05. <b>D</b>) Mice were pre-treated with either ANX2T inhibitor or vehicle before NTPL-90 cell transplantation. The graph shows the percentage of human cells in indicated organs determined twenty-four hours post cell injection. The error bars represent SE of the mean (n = 5). Asterisk denotes p < 0.05. <b>E)</b> The graph shows the increase in the percentage of human cells in mouse blood over time. Error bars denote SE of the mean from five mice each. *P < 0.05. <b>F)</b> Percentage of engraftment in bone marrow and spleen in mice at two weeks post transplantation with NTPL-20 cells is shown in the graph. Error bars represent SE of the mean from four mice each. *P < 0.01.</p

p11 is upregulated at mRNA and protein levels and is localized to the cell surface in ALL cells.

<p><b>A)</b> Analysis of microarray data from GSE7440 showing fold-increase in transcript levels of ANX2 and p11. CCR = patients under continuous complete remission for more than four years (n = 28). Relapse = patients who relapsed within three years of initial diagnosis (n = 31). Error bars denote Standard Deviation (SD) of the Mean. Asterisk indicates statistical significance with a confidence limit of 95%. <b>B)</b> Quantitative RT-PCR data showing mRNA levels of ANX2 and p11 in pediatric normal immortalized B-cells (AG09390 and AG15007) and pediatric (Nalm6, REH) or adult (RS4;11) pre B-ALL cell lines. Error bars denote SD of the Mean from four independent experiments. Asterisks indicate statistical significance with a confidence limit of 95%. <b>C)</b> Immunoblots showing ANX2 and p11 levels in the indicated cell lines. GAPDH was used as a loading control. Representative blots from three independent experiments are shown. <b>D)</b> Representative immunoblots showing ANX2 and p11 levels in lysates from mononuclear cells isolated from healthy volunteers (Normal 1 and 2), or from leukemic cells from pediatric patients. Samples were collected using IRB approved protocols. <b>E)</b> Flow cytometry profiles of fixed, non-permeabilized RS4;11 cells incubated with (red trace) or without (blue trace) addition of the indicated primary antibodies. <b>F)</b> Mean fluorescence intensity was plotted from three independent experiments. Error bars denote SD of the Mean. Asterisks indicate statistical significance (P < 0.01).</p

Knockdown of p11 reduced ALL cell binding to osteoblasts.

<p><b>A)</b> Nalm6 cells were transfected with either scrambled or p11-specific shRNA (sequence 1 or sequence 2). Forty-eight hours post transfection, cells were plated on poly-lysine coated slides, fixed, stained with anti-p11 antibody followed by Alexa Flour 488-conjugated anti-mouse secondary antibody, and visualized by confocal microscopy. Scale bar = 10 μm. Representative images in gray scale are shown. <b>B)</b> The mean fluorescence intensity corresponding to p11 protein levels was determined from 100 cells per condition. The error bars denote SD of the Mean (*P < 0.01). <b>C)</b> Transfected cells were subjected to adhesion assay as described in Materials and Methods. Error bars denote SD of the Mean from two independent experiments in quadruplicates. Asterisks indicate statistical significance (*P < 0.001).</p

ANX2/p11 interaction mediates binding of ALL cells to osteoblasts.

<p><b>A)</b> Cell adhesion assay showing the percentage of RS4;11 cells bound to Saos-2 monolayer with respect to the input, which was considered 100%. Error bars denote SD of the Mean from three independent experiments. Asterisk indicates statistical significance, P < 0.01. <b>B)</b> Immunoprecipitates using control IgG or anti-p11 antibody from lysates of Nalm6 cells treated with or without ANX2T inhibitor (50 μM) were blotted using ANX2 or p11 antibodies. Cell lysate corresponding to 10% of the total protein input in the co-immunoprecipitation assay was loaded in the lane denoted as “Lysate”. <b>C)</b> Graph shows the percentage of cells that bound to Saos-2 monolayer in the presence or absence of ANX2T inhibitor with respect to the input. Error bars indicate SD of the Mean from three to four independent experiments. Note the dose dependent decrease in the percentage of bound Nalm6 and RS4;11 cells post treatment with ANX2T inhibitor (*P < 0.05, **P < 0.01).</p

Dexamethasone-Loaded Block Copolymer Nanoparticles Induce Leukemia Cell Death and Enhance Therapeutic Efficacy: A Novel Application in Pediatric Nanomedicine

Nanotechnology approaches have tremendous potential for enhancing treatment efficacy with lower doses of chemotherapeutics. Nanoparticle (NP)-based drug delivery approaches are poorly developed for childhood leukemia. Dexamethasone (Dex) is one of the most common chemotherapeutic drugs used in the treatment of childhood leukemia. In this study, we encapsulated Dex in polymeric NPs and validated their antileukemic potential in vitro and in vivo. NPs with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly­(ethylene glycol) (PEG) and poly­(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The blank NPs were nontoxic to cultured cells in vitro and to mice in vivo. Encapsulation of Dex into the NPs (Dex-NP) did not compromise the bioactivity of the drug. Dex-NPs induced glucocorticoid phosphorylation and showed cytotoxicity similar to the free Dex in leukemic cells. Studies using NPs labeled with fluorescent dyes revealed leukemic cell surface binding and internalization. In vivo biodistribution studies showed NP accumulation in the liver and spleen with subsequent clearance of the particles with time. In a preclinical model of leukemia, Dex-NPs significantly improved the quality of life and survival of mice as compared to the free drug. To our knowledge, this is the first report showing the efficacy of polymeric NPs to deliver Dex to potentially treat childhood leukemia and reveals that low doses of Dex should be sufficient for inducing cell death and improving survival

GCs enhanced cell-cell aggregation and reduced the motility and invasiveness of kidney cancer cells.

<p>(A) Phase contrast images of a cell-cell aggregation assay showing the ability of GCs to enhance cohesion of cells. The graph represents the mean of three independent experiments performed in quadruplicate wells ±SE. (B) The distance migrated by cells pre-treated with DMSO or GCs in a wound healing assay after 24 hr. The data represent the mean±SE of three independent experiments. Asterisk indicates p-value <0.05 by Student’s <i>t</i>-test comparing DMSO versus drug treatment. (C) The reduction of Caki-1 migration by drug treatment was not a result of changes in cell proliferation as shown by an MTT assay. This was performed in three independent experiments with 12 wells per condition. The graph is from a single representative experiment with bars representing the mean±SE. (D) A Transwell invasion assay showing the invasive capacity of Caki-1 cells through collagen coated inserts at 24 hr. Data represent means±SE of three independent experiments performed in duplicate wells. Asterisk indicates significant p-value < 0.05 by Student’s <i>t</i>-test.</p

GC compounds elevate NaK-β₁ expression in kidney cancer cells.

<p>(A) NaK-β₁ transcript levels were determined by Q-PCR and expressed as a fold change over DMSO-treated cells. Drugs induced a dose-dependent increase in NaK-β₁ transcripts. Data represents the mean±SE of 3 independent experiments performed in quadruplicates. (B) Immunoblot analysis showing NaK-β₁ levels in Caki-1 cells treated with DMSO or GCs. Actin confirmed equal loading of protein. (C) All three drugs induced increased NaK-β₁ at the plasma membrane at 100 nM and 10 μM in a cell-surface biotinylation assay. (D) Immunostaining of NaK-β₁ (green) in Caki-1 drug treated cells imaged by confocal microscopy. TRIAM (panel B), DEX (panel C), and FLUOR (panel D) exhibited more intense NaK-β₁ staining both intracellularly and at the cell surface compared to DMSO (panel A) treated cells. Note the thicker staining along the cell-cell contacts. Scale bar represents 20 μm.</p

Characterization of shRNA-β clones.

<p>(A) Immunoblots showing the levels of NaK-β₁ and NaK-α₁ in Caki-1 cells stably transfected with scrambled shRNA (SCRAM) or shRNA against NaK-β₁ (shRNA-β). Two clones (cl#1 and cl#2) were further studied. Actin was used as a loading control. (B) Rubidium uptake assay showing Na,K-ATPase pump activity was comparable in SCRAM and shRNA-β cells. (C) Cells were treated with TRIAM, DEX, or FLUOR, and NaK-β₁ and NaK-α₁ expression was evaluated by immunoblotting.</p

Glucocorticoids Suppress Renal Cell Carcinoma Progression by Enhancing Na,K-ATPase Beta-1 Subunit Expression

<div><p>Glucocorticoids are commonly used as palliative or chemotherapeutic clinical agents for treatment of a variety of cancers. Although steroid treatment is beneficial, the mechanisms by which steroids improve outcome in cancer patients are not well understood. Na,K-ATPase beta-subunit isoform 1 (NaK-β₁) is a cell-cell adhesion molecule, and its expression is down-regulated in cancer cells undergoing epithelial-to mesenchymal-transition (EMT), a key event associated with cancer progression to metastatic disease. In this study, we performed high-throughput screening to identify small molecules that could up-regulate NaK-β₁ expression in cancer cells. Compounds related to the glucocorticoids were identified as drug candidates enhancing NaK-β₁ expression. Of these compounds, triamcinolone, dexamethasone, and fluorometholone were validated to increase NaK-β₁ expression at the cell surface, enhance cell-cell adhesion, attenuate motility and invasiveness and induce mesenchymal to epithelial like transition of renal cell carcinoma (RCC) cells <i>in vitro</i>. Treatment of NaK-β₁ knockdown cells with these drug candidates confirmed that these compounds mediate their effects through up-regulating NaK-β₁. Furthermore, we demonstrated that these compounds attenuate tumor growth in subcutaneous RCC xenografts and reduce local invasiveness in orthotopically-implanted tumors. Our results strongly indicate that the addition of glucocorticoids in the treatment of RCC may improve outcome for RCC patients by augmenting NaK-β₁ cell-cell adhesion function.</p></div