Zou Duo-Bing, Wei Xiao, Hu Ruo-Lei, Yang Xiao-Ping, Zuo Li, Zhang Su-Mei, Zhu Hua-Qing, Zhou Qing, Gui Shu-Yu, Wang Yuan
Laboratory of Molecular Biology and Department of Biochemistry, the First Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, Anhui, China E-mail :
Asian Pac J Cancer Prev. 2015;16(14):5835-42. doi: 10.7314/apjcp.2015.16.14.5835.
Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms.
The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38).
The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38.
Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.
褪黑素主要由松果体产生，对多种癌症类型的细胞生长具有良好的抑制作用。然而，褪黑素对结肠癌抗肿瘤活性的潜在分子机制尚未完全阐明。在本研究中，我们研究了褪黑素对人结肠癌RKO细胞迁移的影响及其潜在的分子机制。
用褪黑素、SB203580（p38抑制剂）和佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA，丝裂原活化蛋白激酶激活剂）单独或联合处理RKO细胞48小时后，通过MTT法检测细胞活力。通过体外划痕试验分析褪黑素、肌球蛋白轻链激酶（MLCK）的选择性抑制剂ML-7、SB203580和PMA对RKO细胞迁移的影响。通过实时定量逆转录-聚合酶链反应评估MLCK的相对mRNA水平。进行蛋白质印迹分析以检测MLCK的表达、肌球蛋白轻链（pMLC）和p38（pp38）的磷酸化。
褪黑素处理后，人结肠癌RKO细胞的增殖和迁移受到显著抑制。RKO细胞的MLCK表达水平和MLC磷酸化水平降低，实时定量逆转录-聚合酶链反应显示褪黑素对抑制MLCK表达有显著作用。此外，当细胞用褪黑素处理时，呈现相同趋势的p38磷酸化水平也降低。此外，ML-7（25μmol/l）可下调p38的磷酸化。
褪黑素可抑制RKO细胞的增殖和迁移，进一步实验证实p38丝裂原活化蛋白激酶通过下调MLCK的表达和活性在调节褪黑素诱导的迁移抑制中起重要作用。
