Inhibition of angiogenesis by LLL12 correlates with inhibition of STAT3 phosphorylation.

<p>A) Suppression of STAT3 phosphorylation by LLL12 in HUVEC cells after VEGF (10 ng/ml) stimulation. HUVEC cells were grown and stimulated with VEGF (10 ng/ml) for 10 mins and treated with LLL12 for 18–20 hrs. Total cellular protein was extracted and both total STAT3 and pSTAT3(Tyr705) expression was determined. GAPDH was used as a loading control. B) LLL12 inhibits HUVEC tube formation. HUVECs were grown in M200, and incubated with PBS or VEGF (10 ng/ml) for 18–20 hr in the absence or presence of LLL12 (100 nM). Tube formation was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035513#s4" target="_blank">Materials and Methods</a>.</p

The STAT3 inhibitor, LLL12, induces cytoskeletal changes in cultured HUVEC cells.

<p>HUVEC cells cultured in 4-well chamber slides were treated with PBS, VEGF (10 ng/mL) alone, VEGF with DMSO or LLL12 (100 nM) for 18 hrs. The cultures were then probed using anti-β-tubulin primary antibodies (green), and F-actin was stained using phalloidin (red). White arrows highlight F-actin localization at the leading edge, while white arrowheads indicate the curling of microtubules at the cell periphery. 200× magnification. Slice depth = 1 µm. Scale bar = 20 µm. Inset 400× magnification.</p

Sensitivity of sarcoma cell lines to LLL12.

<p>Sensitivity of sarcoma cell lines to LLL12.</p

LLL12 inhibits angiogenesis in OS-1 xenografts.

<p>Analysis of vascularity and tumor cell proliferation: Tumors were harvested at completion of the study and examined by hematoxylin and eosin staining. Vascularity of osteosarcoma tumor xenografts in mice was evaluated by CD34 related antigen staining (brown) for endothelial cells, and Ki 67 for proliferation. Apoptotic cells were identified by TUNEL staining. There is a significant effect of LLL12 as compared to vehicle control on tumor vasculature as well as cell proliferation (quantified by staining with anti–Ki-67) in osteosarcoma xenografts, but no significant change in apoptosis in drug treated tumors. All values are expressed as mean plus or minus SEM. *<i>P</i><.01 by Student <i>t</i> test.</p

LLL12 downregulates several angiogenic factors.

<p>LLL12 induced changes in angiogenic factors in OS-1 xenografts were determined using a Proteome profiler antibody array as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035513#s4" target="_blank">Materials and Methods</a>. The effect of LLL12 treatment is quantified in the histogram, untreated tumors (−), tumors from mice receiving LLL12 (+), 5 mg/kg).</p

LLL12 inhibits angiogenesis in vitro.

<p>HUVECs were grown under serum-deficient conditions and stimulated with VEGF (10 ng/ml) in the absence or presence of LLL12 (100 nM). A. Proliferation/viability was determined after 2 days by Calcein AB staining. Migration was determined using the crystal violet assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035513#s4" target="_blank">Materials and Methods</a> as well as scratch assay/wound-healing. Invasion was determined using Matrigel coated membranes (photomicrographs show representative fields) and is quantified (bottom right panel). Each data set represents the mean ± SE for at least 3 independent experiments. B. To study the effect of LLL12 (100 nM) on the cell migration in HUVEC cells, wound-healing assays were carried out by allowing the cells to move to the scar region for 24 hours using VEGF (10 ng/ml) as a positive control in the presence and absence of LLL12 (original magnification ×40).</p

LLL12 inhibits tumor growth <i>in vivo</i> by inhibition of STAT3. A, LLL12 inhibits tumor growth in osteosarcoma xenograft mice.

<p>OS-1 tumors were transplanted into 6-week-old CB17SC <i>scid−/−</i> female mice. After tumors grew to ∼130 mm³, mice were randomized to receive no treatment (control), DMSO or LLL12 (5 mg/kg/d) for a planned six weeks. LLL12 inhibited tumor growth as measured by tumor volume. Representative tumors at the termination of each group are shown. B. Western blot showing STAT3, and p-STAT3 level in six independent tumors from each treatment group. LLL12 completely blocks pSTAT3 levels with compassion to control and DMSO control group.</p

LLL12 inhibits angiogenesis in mice.

<p>Matrigel plugs containing PBS (Control), or VEGF (100 ng/ml) were implanted subcutaneously in mice that were or were not treated with LLL12 (5 mg/kg daily). The Matrigel plugs were excised on day 7, fixed with formalin and 5-µm sections were stained for CD34 staining. The numbers of CD34 positive vessels per high power field (HPF, magnification, 200×) were counted for each experimental condition. Results are mean (n = 4) ± SE. *P<0.05; versus VEGF alone.</p

MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines

<div><p>Background</p><p>Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease.</p><p>Methodology and principal findings</p><p>RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes.</p><p>Conclusions</p><p>These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA.</p></div

Expression of miR-34a in canine OSA cell lines suppresses cell invasion and migration.

<p>(A) Canine OSA8 and Abrams cells expressing control or pre-miR-34a lentiviral constructs were plated in serum free media in the upper wells of plates for Matrigel invasion assays. The lower chamber of each well contained complete media supplemented with 10% fetal bovine serum. Cells were allowed to invade for 24 hours through a layer of Matrigel basement membrane before removal of Matrigel and media from the upper chambers. Cells that had migrated to the lower surface of the insert membrane were stained with crystal violet and the number of invaded cells were counted in ten random fields in triplicate replicates. Experiments were performed in triplicate and data are represented as the mean ± standard deviation (*<i>p</i> ≤ 0.05, Student’s <i>t</i>-test). (B) Migration behavior was assessed in OSA8 and Abrams cell lines expressing empty vector or pre-miR-34a lentiviral constructs using standard wound-healing assays. Cells were seeded in 6-well plates in complete growth medium and allowed to grow to 70% confluency. A scratch was introduced using a P200 pipet tip and after 24 hours, cells were fixed, stained with crystal violet and evaluated by digital photography.</p