Correlation between expression and differentiation of endocan in colorectal cancer.

AIM: To investigate the expression frequency of endocan in colorectal cancer and analyze the relationship between endocan expression and clinical parameters and to study the role of endocan in colorectal carcinogenesis. METHODS: Expression of endocan in 72 tumor tissue samples of colorectal cancer as well as in 27 normal mucous membrane tissue samples was analyzed using in situ hybridization, immunohistochemistry on tissue microarray, Western blot and reverse-transcript polymerase chain reaction (RT-PCR). RESULTS: The expression of endocan was higher in normal colon and rectum tissue samples than in cancerous tissue samples (mRNA = 92.6%, protein = 36%), and was lower in colorectal cancer tissue samples (mRNA = 70.4%, protein = 36.1%). No correlation was found between staining intensity and clinical parameters such as sex, age, tumor size and TNM stage. However, the expression of endocan was positively correlated with the tissue differentiation in colorectal cancer. CONCLUSION: The expression of endocan is down-regulated in colorectal cancer and is positively correlated with the tissue differentiation in colorectal cancer, suggesting that the expression of endocan is associated with development and differentiation of colorectal cancer.

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK.

BACKGROUND: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Distribution and expression of non-muscle myosin light chain kinase in rabbit livers.

AIM: To study the distribution and expression of non-muscle myosin light chain kinase (nmMLCK) in rabbit livers. METHODS: Human nmMLCK N-terminal cDNA was amplified by polymerase chain reaction (PCR) and was inserted into pBKcmv to construct expression vectors. The recombinant plasmid was transformed into XL1-blue. Expression protein was induced by IPTG and then purified by SDS-PAGE and electroelution, which was used to prepare the polycolonal antibody to detect the distribution and expression of nmMLCK in rabbit livers with immunofluorescene techniques. RESULTS: The polyclonal antibody was prepared, by which nmMLCK expression was detected and distributed mainly in peripheral hepatocytes. CONCLUSION: nmMLCK can express in hepatocytes peripherally, and may play certain roles in the regulation of hepatic functions.

[Effect of vitamin E on myosin light chain kinase activity and endothelial permeability of the artery in atherosclerotic rabbit].

OBJECTIVE: To study the effect of vitamin E (Vit E) on the myosin light chain kinase(MLCK) activity and the endothelial permeability of the artery in atherosclerotic rabbits. METHODS: The MLCK activity of rabbit artery was measured by incorporation of gamma-(32)P. The endothelial permeability was accessed by immunofluorescence. RESULTS: The model of atherosclerosis was established after rabbits were fed with cholesterol for four weeks. The activity of MLCK increased markedly, and there was significantly statistical difference compared with the normal control (P<0.05). When the rabbits were fed with cholesterol for twelve weeks or with cholesterol and Vit E for twelve weeks, the activity of MLCK did not change markedly, and there was no statistical difference compared with the normal control, respectively (P>0.05). The permeability of arterial wall was increased after the rabbits were fed with cholesterol for four weeks, and the permeability increased even more obviously after the rabbits were fed with cholesterol for twelve weeks. The permeability appeared to be decreased when Vit E was added into the cholesterol feeding. CONCLUSION: The change in integrity of arterial wall may be associated with the increase of the activity of MLCK. Vit E may decrease the MLCK activity. Vit E may decrease the endothelial permeability of atherosclerotic rabbits.