CysLT1R Antagonists Inhibit Tumor Growth in a Xenograft Model of Colon Cancer.

The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model

Background: Colorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth. Methods: In this study we used human HCT-116 colon cancer ALDH+ cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA. Results: We observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated β-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206+). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1β, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1β, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2. Conclusion: Our data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer

The role of CysLT1R in animal models of colorectal cancer

Cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are potent pro-inflammatory lipids derived from arachidonic acid and mediate their effect through CysLT1R and CysLT2R. There is a strong correlation between long-standing inflammatory bowel disease where these pro-inflammatory mediators are abundant and colorectal cancer. We have shown that LTD4 via its receptor CysLT1 induces expression of proteins associated with colorectal cancer and promotes proliferation, survival and migration in intestinal epithelial cells. In addition, we have demonstrated that that high expression of CysLT1R in colorectal adenocarcinomas predicts poor prognosis in patients. In the presented papers in this thesis we investigated the role of CysLT1R in different mouse models of colorectal cancer. In the mouse xenograft model of colon cancer, we were able to observe a reduced tumor growth in response to CysLT1R antagonist treatment. The inhibition of the tumor growth was accompanied with changes in proliferation and apoptosis as determined by reduced Ki-67 expression, increased expression of p21WAF/Cip1, cleaved caspase 3 and caspase-cleaved keratin 18. An impaired tumor angiogenesis was also demonstrated by detection of increased levels of VEGF and reduced vessel size. We also investigated the role of CysLT1R in 1) FAP/sporadic colorectal cancer by crossing ApcMin/+ mice with mice lacking CysLT1R expression and in 2) colitis-associated colorectal cancer by employing the AOM/DSS-model on mice lacking CysLT1R expression. Interestingly, a reduced polyp formation in a gender-specific manner could be observed in both models. CysLT1R knockout female mice, but not male mice exhibited a reduced polyp formation in the small intestine and colon, respectively. Also, a decreased nuclear expression of β-catenin within the epithelial tumor compartment was determined for CysLT1R mutant female mice in both models. However, the mechanism of tumor progression in FAP/sporadic colorectal cancer and in colitis-associated colorectal cancer might differ as indicated by reduced tumor expression of COX-2 and reduced serum levels of PGE2 in the female double mutant (CysLT1R−/− ApcMin/+) mice, whereas AOM/DSS-treated female single mutant (CysLT1R−/−) mice demonstrated increased serum levels of PGE2. In conclusion, the presented mouse models of colorectal cancer further strengthen our previous in vitro findings and highlight the prospect of CysLT1R as an alternative therapeutic approach

Cysteinyl leukotrienes and their receptors: Bridging inflammation and colorectal cancer.

Long-standing inflammation has emerged as a hallmark of neoplastic transformation of epithelial cells and may be a limiting factor of successful conventional tumor therapies. A complex milieu composed of distinct stromal and immune cells, soluble factors and inflammatory mediators plays a crucial role in supporting and promoting various types of cancers. An augmented inflammatory response can predispose a patient to colorectal cancer (CRC). Common risk factors associated with CRC development include diet and lifestyle, altered intestinal microbiota and commensals, and chronic inflammatory bowel diseases. Cysteinyl leukotrienes are potent inflammatory metabolites synthesized from arachidonic acid and have a broad range of functions involved in the etiology of various pathologies. This review discusses the important role of cysteinyl leukotriene signaling in linking inflammation and CRC

Cysteinyl leukotriene receptor 1 facilitates tumorigenesis in a mouse model of colitis-associated colon cancer

Cysteinyl leukotriene receptor 1 (CysLT1R) has been shown to be up-regulated in the adenocarcinomas of colorectal cancer patients, which is associated with a poor prognosis. In a spontaneous model of colon cancer, CysLT1R disruption was associated with a reduced tumor burden in double-mutant female mice (ApcMin/+/Cysltr1-/-) compared to ApcMin/+ littermates. In the current study, we utilized a genetic approach to investigate the effect of CysLT1R in the induced azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis-associated colon cancer. We found that AOM/DSS female mice with a global disruption of the Cysltr1 gene (Cysltr1-/-) had a higher relative body weight, a more normal weight/length colon ratio and smaller-sized colonic polyps compared to AOM/DSS wild-type counterparts. The Cysltr1-/- colonic polyps exhibited low-grade dysplasia, while wild-type polyps had an adenoma-like phenotype. The Cysltr1-/- colonic polyps exhibited significant decreases in nuclear β-catenin and COX-2 protein expression, while the normal crypts surrounding the polyps exhibited increased Mucin 2 expression. Furthermore, Cysltr1-/- mice exhibited an overall reduction in inflammation, with a significant decrease in proinflammatory cytokines, polyp 5-LOX expression and infiltration of CD45 leukocytes and F4/80 macrophages. In conclusion, the present genetic approach in an AOM/DSS model further supports an important role for CysLT1R in colon tumorigenesis

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

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