Retinoic acid morpholine amide (RAMA) inhibits expression of Fas ligand through EP1 receptor in colon cancer cells
- 175 Downloads
Among the members of tumour necrosis factor family Fas ligand on binding to its receptor strongly induces apoptosis of tumour-infiltrating lymphocytes (TIL). Thus, FasL acts as an inhibitor of anti-tumour immune response. The present study demonstrates that retinoic acid morpholine amide (RAMA) significantly suppresses FasL expression in colon cancer cells in a dose- and time-dependent manner. The suppression of FasL mRNA and proteins was significant at a concentration of 30 μM after 48 h in CLT85 and HT26 colon cancer cells. There was around 2.6- and 3.2-fold decrease in FasL mRNA after incubation with 30 μM of RAMA in CLT85 cells and HT26 cells, respectively. The results from Western blot showed a decrease in FasL mRNA and protein expression in both CLT85 and HT26 cells after suppression of cyclooxygenase (COX)-2 and COX-1 by RNAi. However, when COX-2-specific silencer RNA (siCOX-2)- and siCOX-1-treated CLT85 and HT26 cells were exposed to RAMA, inhibition of FasL expression was further suppressed. The siCOX-2-treated CLT85 and HT26 cells on exposure to RAMA showed ∼87 and ∼54 % reduction in FasL mRNA, respectively. Co-culture of Jurkat T cells with RAMA-treated HT26 and CLT85 cells decreased the viability of Jurkat T cells by only 2 and 4.3 %, respectively, compared to 19.5 and 37.3 % in control HT26 and CLT85 cells. The results from real-time reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting showed that suppression of EP1 prevented RAMA-induced FasL suppression in CLT85 cells at both the mRNA and protein levels. Thus, RAMA can be a potent therapeutic agent for the treatment of colon tumours.
KeywordsTumour necrosis Immune response Cytotoxicity Fas-sensitive
This work was supported by Project (No: 2013DFA31510) and China-Japan Friendship Hospital Youth Science and Technology Excellence Project (No. 2015-QNYC-B-02).
Conflicts of interest
Shao-Xuan Chen, Li Yao and Shi-Yu Du designed the study. Yun-Ting Wang, Hong-Chuan Zhao and Yan-Li Zhang wrote the manuscript. Shi-Yu Du carried out the cell culture and Western blot. All authors approved the final version of the manuscript.
- 7.Bennett MW, O’Connell J, Houston A, Kelly J, O’Sullivan GC, Collins JK, et al. Fas ligand upregulation is an early event in colonic carcinogenesis. J ClinPathol. 2001;54:598–604.Google Scholar
- 11.Barnhart BC, Legembre P, Pietras E, Bubici C, Franzoso G, Peter ME. CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J. 2004;22:22.Google Scholar
- 18.Moon RC, McCormick DL, Mehta RG. Inhibition of carcinogenesis by retinoids. Cancer Res. 1983;43:2469.Google Scholar