Effects of CysLT₁R antagonists on HCT-116 xenograft tumor angiogenesis.

<p>(<b>A</b> and <b>D</b>) Representative CD31 stained images (×100). (<b>B</b> and <b>E</b>) Vessel density was determined with CD31-positive counts in three different fields (hot spots). (<b>C</b> and <b>F</b>) Quantitative analysis of CD31-positive areas using Adobe Photoshop. The quantitative data shown are the mean ± SEM. *<i>P</i><0.05 by Student’s <i>t</i> test.</p

Effects of CysLT₁R antagonists on HCT-116 xenograft tumor growth.

<p>(<b>A</b>) Experimental protocol for the pretreatment groups; BalbC (nu/nu) mice were subcutaneously injected into two flanks with HCT-116 cells pretreated with ZM198,615 or Montelukast (50 µM), and received treatment intraperitoneally from the day of inoculation with DMSO, ZM 198.615, or Montelukast (5 mg/kg/day). (<b>B</b>) Tumor incidence of mice treated with DMSO (DMSO I group), ZM198,615 (Pre-ZM group), or Montelukast (Pre-Montelukast group) and (<b>C</b>) tumor weight compared to the DMSO I group at the end of the experiment (day 21). (<b>D</b>) Representative tumor images from the pretreatment group. (<b>E</b>) Experimental protocol for the treatment study; non-pretreated HCT-116 cells were subcutaneously injected into two flanks of nude mice. DMSO (DMSO II group), ZM198,615 (ZM group), or Montelukast (Montelukast group) treatment began on day 6 after tumor cell inoculation. (<b>F</b>) Tumor volumes over a 21-day period and (<b>G</b>) tumor weight at the end of the experiment (day 21). (<b>H</b>) Representative tumor images from the treatment group. The quantitative data shown are the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Tumor volume analysis was performed by two-way ANOVA and tumor weight analysis was performed by Student’s <i>t</i> test.</p

Effects of CysLT₁R antagonists on HCT-116 xenograft tumor proliferation and apoptosis.

<p>(<b>A</b> and <b>C</b>) Representative Ki-67-stained images from paraffin sections of xenograft tumors (×400). (<b>B</b> and <b>D</b>) One Ki-67-stained hot spot was selected from each tumor and 3 separate areas within these hot spots were analyzed at high power field (×400). Ki-67 positive area fraction was determined as ratio of stained area to total high power field area. (<b>E</b> and <b>G</b>) Representative M30 CytoDEATH-stained images from paraffin sections of xenograft tumors (×200). Black and white arrows indicate positively stained cells. Boxed regions within the main panels shows the positively stained cells indicated by the white arrows at higher magnification (×400). (<b>F</b> and <b>H</b>) Average apoptotic cell number per field was determined by M30- positive counts (black arrows) in median-sized xenograft tumor sections taken from the middle part. The quantitative data shown are the mean ± SEM. *<i>P</i><0.05 by Student’s <i>t</i> test.</p

Effects of CysLT₁R antagonists on apoptosis in HCT-116 cells.

<p>(<b>A</b>) Representative flow cytometry panels display apoptosis of HCT-116 cells treated with ZM198,615 (ZM) or Montelukast (Mo) using Annexin V-PE and 7-AAD-staining. (<b>B</b>) The level of cleaved caspase 3 fragments (19 and 17 kDa) in HCT-116 cells treated with CysLT₁R antagonists as determined by Western blot analysis. Data shown are representative of three separate experiments.</p

Effects of CysLT₁R antagonists on HCT-116 cell proliferation and cell cycle.

<p>Cell proliferation was measured using the WST-1 cell proliferation assay, and absorption of the samples was measured at 440 nm. Cells were treated with (<b>A</b>) ZM198,615 or (<b>B</b>) Montelukast for 24, 48, 72, or 96 h. (<b>C</b>) Cell cycle analysis was carried out with propidium iodide staining. Percentages of the total cell population in different phases of the cell cycle were analyzed by flow cytometry. The quantitative data shown are the mean ± SEM from three separate experiments. <i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001 by paired <i>t</i> test.</p

Effects of the CysLT₁R antagonist Montelukast on HT-29 and SW-480 xenograft tumor growth.

<p>(<b>A</b>) Experimental protocol; untreated SW-480 or HT-29 cells were subcutaneously injected into both flanks of BalbC (nu/nu) mice. These mice received daily intraperitoneal injections with DMSO or Montelukast (5 mg/kg) for 14 days, starting 7 days after tumor cell inoculation. (<b>B</b>) Tumor weight and (<b>C</b>) volume at the experimental endpoint (day 21). (<b>D, E</b>) Tumor diameters over a 21-day period. (<b>F</b>) Representative <i>in situ</i> tumor images. The quantitative data shown are the mean ± SEM. *<i>P</i><0.05. Tumor volume and weight analysis was performed by Student’s <i>t</i> test and tumor diameter analysis was performed by two-way ANOVA.</p

Effects of CysLT₁R antagonists on cell cycle, apoptosis, and angiogenesis in HCT-116 xenograft tumors.

<p>Tumor samples (three or four tumors from each group) were subjected to Western blot analysis. Membranes were probed for (<b>A</b> and <b>D</b>) p21^WAF/Cip1; (<b>B</b> and <b>E</b>) cleaved caspase 3; and (<b>C</b> and <b>D</b>) VEGF. Data were normalized on the basis of β-actin levels<b>.</b> Densitometric analysis of protein expression represents the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 by Student’s <i>t</i> test.</p

Effects of CysLT₁R antagonists on HCT-116 cell adhesion and colony formation.

<p>(<b>A</b>) Briefly, HCT-116 cells were pretreated with ZM198,615 (ZM) or Montelukast (Mo) for 30 min, stained with 0.5% crystal violet and quantified with spectrophotometry at 550 nm. Relative adhesive cell number compared to the DMSO-treated control cells. (<b>B</b>) Cell viability as determined by trypan blue staining after 30 min treatment with or without CysLT₁R antagonists, just prior to the initiation of the adhesion assay. (<b>C</b>) Representative photographs of crystal violet-stained colonies treated with ZM198,615 (ZM) or Montelukast (Mo) in 6-well plates. (<b>D</b>) Relative colony number and (<b>E</b>) relative colony size were measured using ImageJ software. The quantitative data shown are the mean ± SEM from three separate experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by paired <i>t</i> test or one-way ANOVA.</p

Basal expression level of CysLTs in cell culture media of indicated colon cancer cell lines.

<p>The data shown are the mean ± SEM.</p

Additional file 1 of Discovery of a small molecule that inhibits Bcl-3-mediated cyclin D1 expression in melanoma cells

Additional file 1: Supplementary Figure 1