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 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 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 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