Introduction Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid which has anti-colorectal cancer (CRC) activity. The molecular mechanism (s) underlying the anti-neoplastic activity of EPA are not understood. Trihydroxy-EPA, also known as Resolvin E1 (RvE1), is an oxygenated derivative of EPA, that has been shown to inhibit NK-κB signalling, which is implicated in colorectal carcinogenesis. RvE1 has been shown to bind to two G-protein coupled receptors, ChemR23 and BLT1. We investigated whether ChemR23 and BLT1 receptors are expressed in human CRC.
Methods Seven human CRC cell lines (HCA7, LoVo, T84, HRT18, HT29, Caco2 and HCT116) were characterised for ChemR23 and BLT1 expression by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence. Jurkat and THP-1 cells were used as positive controls for ChemR23 and BLT1, respectively. Membrane protein fraction analysis was carried out using a transmembrane protein extraction kit. Densitometric analysis was performed using BIO-RAD Quantity One Software. Human CRC tissue was examined for ChemR23 expression by immunohistochemistry on formalin-fixed, paraffin-embedded tissue blocks.
Results ChemR23 and BLT1 messenger RNA expression was detected in all seven human CRC cell lines. ChemR23 protein (45kDa) expression was also observed in all human CRC cell lines, with Caco2 cells expressing around two-fold more ChemR23 receptor protein relative to a-tubulin than other CRC cell lines. However, BLT1 receptor protein was not detected in any of the human CRC cell lines, but was confirmed in monocytic THP-1 cells (38kDa). ChemR23 protein was enriched in the membrane protein fraction of Caco2 cells. ChemR23 protein levels increased with confluency in Caco2 cells. There was a three-fold increase in ChemR23 protein expression in 100% confluent Caco2 cells compared with less confluent cell cultures. In contrast, HCA-7 cells did not display confluence-dependent changes in ChemR23 protein expression. Immunofluorescence demonstrated predominant cytoplasmic localisation of ChemR23 with a heterogeneous population of ChemR23-expressing and negative cells. ChemR23 immunohistochemistry on primary CRC tissue demonstrated homogeneous ChemR23 immunoreactivity in CRC cells with some stromal cell staining, including endothelial cells.
Conclusion ChemR23 (but not BLT1) protein is expressed by human CRC cells (particularly Caco2) in vitro and in cancer cells in human primary CRCs. ChemR23 protein expression varies in vitro in a confluence-dependent manner, with heterogeneous expression by Caco2 cells. ChemR23 is localised predominantly in cancer cells in human CRC. Investigation of ChemR23-dependent anti-CRC activity of RvE1 is warranted.
Disclosure of Interest None Declared.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.