Chen Shao-Xuan, Du Shi-Yu, Wang Yun-Ting, Zhao Hong-Chuan, Zhang Yan-Li, Yao Li
Department of Endoscopy Center, China-Japan Friendship Hospital, No. 2 Yinghua Dong Street, Chaoyang District, Beijing, 100029, China.
Department of Gastroenterology, China-Japan Friendship Hospital, Beijing, 100029, China.
Tumour Biol. 2016 Jan;37(1):323-9. doi: 10.1007/s13277-015-3798-z. Epub 2015 Jul 27.
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.
在肿瘤坏死因子家族成员中，Fas配体与其受体结合后可强烈诱导肿瘤浸润淋巴细胞（TIL）凋亡。因此，FasL充当抗肿瘤免疫反应的抑制剂。本研究表明，视黄酸吗啉酰胺（RAMA）以剂量和时间依赖性方式显著抑制结肠癌细胞中FasL的表达。在CLT85和HT26结肠癌细胞中，48小时后30μM浓度时FasL mRNA和蛋白的抑制作用显著。在CLT85细胞和HT26细胞中，与30μM的RAMA孵育后，FasL mRNA分别下降了约2.6倍和3.2倍。蛋白质印迹结果显示，RNA干扰抑制环氧合酶（COX）-2和COX-1后，CLT85和HT26细胞中FasL mRNA和蛋白表达均下降。然而，当用COX-2特异性沉默RNA（siCOX-2）和siCOX-1处理的CLT85和HT26细胞暴露于RAMA时，FasL表达的抑制作用进一步受到抑制。用siCOX-2处理的CLT85和HT26细胞暴露于RAMA后，FasL mRNA分别降低了约87%和54%。与对照HT26和CLT85细胞中分别为19.5%和37.3%相比，Jurkat T细胞与经RAMA处理的HT26和CLT85细胞共培养后，Jurkat T细胞的活力仅分别降低了2%和4.3%。实时逆转录聚合酶链反应（RT-PCR）和免疫印迹结果表明，在mRNA和蛋白水平上，抑制EP1可阻止RAMA诱导的CLT85细胞中FasL的抑制。因此，RAMA可能是治疗结肠肿瘤的一种有效治疗剂。
