Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jul 8:13:336.
doi: 10.1186/1471-2407-13-336.

The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells

Astrid M Bengtsson  1 , Gunilla Jönsson, Cecilia Magnusson, Tavga Salim, Cecilia Axelsson, Anita Sjölander
Affiliations

The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells

Astrid M Bengtsson et al. BMC Cancer. .

Abstract

Background: Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT(1)R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT(2)R) is lost. Further, our previous data indicate that patients with high CysLT(1)R and low CysLT(2)R expression have a poor prognosis. In this study, we examined whether the balance between CysLT(1)R and CysLT(2)R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA).

Methods: To determine the effect of ATRA on CysLT(2)R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions.

Results: ATRA treatment induces CysLT(2)R mRNA and protein expression without affecting CysLT(1)R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT(2)R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT(2)R-specific inhibitor.

Conclusions: This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CysLT2R expression in ATRA-treated colon cancer cell lines. Cells were incubated for 3, 12, or 24 h in the absence or presence of 1 μM ATRA as indicated. (A and C) qPCR for CysLT2R expression. Data are normalized to HPRT-1 in SW480 and Caco-2 cells. (B and D) Protein expression of CysLT2R normalized to β-actin in SW480 and Caco-2 cells as determined by Western blot. (E) qPCR for CysLT1R expression in SW480 cells. Data are normalized to HPRT-1. (F) Protein expression of CysLT1R normalized to β-actin in SW480 cells as determined by Western blot. Changes in CysLT2R expression relative to basal levels in unstimulated cells are shown as mean ± standard error of the mean (SEM; *P < 0.05, **P < 0.01; n.s. = not significant).
Figure 2
Figure 2
Effect of RARα or RARβ inhibition on CysLT2R expression and promoter activity. SW480 cells were transfected with control siRNA (non-target siRNA), RARα siRNA, or/and RARβ siRNA, allowed to rest for 24 h, serum-starved overnight, and subsequently stimulated with 10 μM ATRA. (A and B) qPCR for CysLT2R and RARα expression. Data are normalized to HPRT-1 with and without ATRA stimulation for 3 h. (C) Western blots without and with ATRA stimulation for 3 h. (D) Western blot analyzes of CysLT2R protein expression in membrane fractions from SW480 cells stimulated or not with ATRA for 3 h normalized to unstimulated control. (E) CysLT2R luciferase promoter activity in cells stimulated with or without ATRA for 48 h. Data are normalized to unstimulated controls. (F) CysLT2R luciferase promoter activity in siRNA-treated cells stimulated with or without ATRA for 48 h. Data are normalized to unstimulated control. Values are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 3
Figure 3
Effect of ATRA on cell growth, apoptosis, LTC4S and LTC4 expression. SW480 cells were treated with the CysLT2R inhibitor AP 100984 (AP) and/or 40 nM LTC4 and were or were not stimulated with 1 μM ATRA. (A) 3H-thymidine incorporation measured after 72 h of ATRA treatment, normalized to unstimulated control. (B) Caspase-3 activity after 48 h of ATRA treatment normalized to unstimulated control. (C) qPCR for LTC4S expression after 3–24 h of ATRA stimulation. Data are normalized to HPRT-1. (D) Release of LTC4 into the media form SW480 cells after 24 h incubation in the absence or presence of ATRA. Values are shown as mean ± SEM (*P < 0.05, **P < 0.01).
Figure 4
Figure 4
ATRA does not affect CysLT2R in HCT-116 cells. RAR-mutated HCT-116 colon cancer cells were incubated for 3, 12, or 24 h in the presence or absence of 1 μM ATRA. (A) qPCR for CysLT2R expression in HCT-116 cells. Data are normalized to HPRT-1. (B) Protein expression of CysLT2R normalized to β-actin in HCT-116 cells as determined by Western blot. Data show changes in expression relative to basal levels in unstimulated cells. (n.s. = not significant).
Figure 5
Figure 5
Effect of ATRA on the differentiation markers MUC-2 and alkaline phosphatase activity. SW480 cells were either 1) treated with 1 μM of the CysLT2R inhibitor AP 100984 (AP) and were or were not stimulated with 1 μM ATRA or 2) treated with RAR siRNA and stimulated with 10 μM ATRA. (A and B) qPCR for MUC-2. Data are normalized to HPRT-1 and changes in MUC-2 expression are relative to basal levels in untreated cells. (C) Confocal immunofluorescence microscopy showing ATRA-induced expression of Mucin-2 in SW480 colon cancer cells. After stimulation with 10 μM ATRA for 24 h the cells were fixed, permeabilized and stained with a Mucin-2 antibody. Left panels show Mucin-2 (MUC-2) staining, middle panels nuclear staining with DAPI and right panels merged, the graphs were taken with 40x objective. (D) Alkaline phosphatase activity in Caco-2 cells. Bars show % of ATRA-induces alkaline phosphatase activity and values are shown as mean ± SEM (*P < 0.05, **P < 0.01).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Eaden J. Review article: colorectal carcinoma and inflammatory bowel disease. Aliment Pharmacol Ther. 2004;20(Suppl 4):24–30. - PubMed
    1. Ekbom A, Helmick C, Zack M, Adami HO. Ulcerative colitis and colorectal cancer. A population-based study. N Engl J Med. 1990;323(18):1228–1233. doi: 10.1056/NEJM199011013231802. - DOI - PubMed
    1. Nakamura M, Shimizu T. Leukotriene receptors. Chem Rev. 2011;111(10):6231–6298. doi: 10.1021/cr100392s. - DOI - PubMed
    1. Singh RK, Gupta S, Dastidar S, Ray A. Cysteinyl leukotrienes and their receptors: molecular and functional characteristics. Pharmacology. 2010;85(6):336–349. doi: 10.1159/000312669. - DOI - PubMed
    1. Sharon P, Stenson WF. Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology. 1984;86(3):453–460. - PubMed

Publication types

MeSH terms

LinkOut - more resources