Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

The association between KLF4 as a tumor suppressor and the prognosis of hepatocellular carcinoma after curative resection.

Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor in klfs family, is known for its crucial role in regulating cell growth, proliferation, and differentiation. This research aimed to explore the prognostic significance of KLF4 in hepatocellular carcinoma's (HCC) patients after curative resection and the role of KLF4 in HCC progression. There were 185 HCC patients who had hepatectomy from July 2010 to July 2011 included in this study. KLF4 expression was detected by microarray immunohistochemical technique, western blot, and qRT-PCR. Then, the correlation between the prognosis of patients and KLF4 expression was evaluated based on patients' follow-up data. The research found KLF4 expression was significantly downregulated in HCC tissues compared to para-tumorous tissues. More importantly, the overall survival rate (OS) and recurrence-free survival rate (RFS) of HCC patients with low KLF4 expression were both significantly decreased compared to those with a high level of KLF4. Further function and mechanism analysis showed that KLF4 could inhibit the proliferation, migration, invasion and epithelial-mesenchymal transition of HCC cells. The study revealed that KLF4 was not only a tumor suppressor in HCC but also can be regarded as a valuable prognostic factor and potential biological target for diagnosis and treatment in HCC patients.

Melatonin inhibits colon cancer RKO cell migration by downregulating Rho­associated protein kinase expression via the p38/MAPK signaling pathway.

Melatonin is predominately produced and secreted by the pineal gland, and inhibits cell growth in various cancer cell lines such as colorectal cancer. However, the precise mechanisms involved have not been fully elucidated. In the present study, the potential molecular mechanism underlying the efficacy of melatonin on migration in RKO colon cancer cells was investigated. The effects of melatonin and H­1152, a selective inhibitor of Rho­associated protein kinase (ROCK), on the migration of RKO cells were analyzed by an in vitro wound healing assay. The localization of zonula occludens­1 (ZO­1) and occludin were observed by immunofluorescence. Reverse transcription­quantitative polymerase chain reaction (qPCR) was performed to analyze the relative mRNA levels of ROCK, ZO­1 and occludin. In addition, western blot analysis was implemented to examine the expression of ROCK, phospho (p)­myosin phosphatase targeting subunit 1 (MYPT1), p­myosin light chains (MLC) and p­p38. The results revealed that the expression levels of ROCK2, p­MYPT1 and p­MLC in RKO cells were decreased, and the membrane protein expression of ZO­1 and occludin increased when the cells were treated with melatonin. qPCR demonstrated that melatonin downregulated ROCK2 gene expression, and upregulated the expression of the ZO­1 and occludin genes. The levels of ZO­1 and occludin localized in the tight junctions were markedly increased in the immunofluorescence assay. In addition, the phosphorylation levels of p38 were reduced when the cells were treated with melatonin, and treatment with H­1152 downregulated p38 phosphorylation. The results indicated that melatonin may inhibit the migration of RKO colon cancer cells by downregulating ROCK expression via the p38/mitogen­activated protein kinase signaling pathway.

All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway.

Ameliorative effect of melatonin against increased intestinal permeability in diabetic rats: possible involvement of MLCK-dependent MLC phosphorylation.

The increased intestinal permeability and functional impairment play an important role in type 2 diabetes (T2D), and melatonin may possess enteroprotection properties. Therefore, we used streptozotocin-induced diabetic rat model to investigate the regulation of intestinal permeability by melatonin. Rats were randomly divided into three groups, including control, diabetes mellitus (DM), and DM rats treated with melatonin. Melatonin was administered (10 mg/kg/day) by gavage for 24 weeks. The DM rats significantly increased the serum fasting blood glucose and lipid levels, which were alleviated by melatonin treatment. Importantly, the intestinal epithelial permeability was significantly increased in DM rats but was ameliorated following treatment with melatonin. These findings also indicated the expression of myosin light chain kinase (MLCK) and phosphorylation of MLC targeting subunit (MYPT) induced myosin light chain (MLC) phosphorylation level was markedly elevated in hyperglycemic and hyperlipidemic status. They were partly associated with down-regulated membrane type 1 and 2 (MT1 and MT2) expression, and up-regulated Rho-associated protein kinase (ROCK) expression and increased extracellular signal-regulated kinase (ERK) phosphorylation. However, the changes in target protein expression were reversed by melatonin. In conclusion, our results show melatonin beneficial effects on impaired intestinal epithelial permeability in T2D by suppressing ERK/MLCK- and ROCK/MCLP-dependent MLC phosphorylation.

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.

Atorvastatin inhibits hyperglycemia-induced expression of osteopontin in the diabetic rat kidney via the p38 MAPK pathway.

Osteopontin (OPN), a large phosphoglycoprotein adhesion molecule, which is up-regulated in the kidneys of humans and mice with diabetes, has emerged as a potentially key pathophysiological contributor in diabetic nephropathy. Here, we investigated the role of OPN in kidney injury caused by diabetic nephropathy and the effect of atorvastatin on the expression of OPN and on diabetic nephropathy. Diabetes was induced with streptozotocin in rats, and atorvastatin (5 mg/kg) was orally administered once a day for 8 weeks. We analyzed the expression and regulation of OPN in the kidneys of streptozotocin-induced diabetic Sprague-Dawley albino rats by immunohistochemistry and western blot analysis. The expression of OPN was increased in diabetic rat kidney, and atorvastatin inhibited this process. Atorvastatin also decreased the expression and phosphorylation of p38. In vitro, atorvastatin inhibited the high glucose-induced OPN expression in Madin-Darby canine kidney epithelial cells through the p38 MAPK signaling pathway. These results suggested that atorvastatin reduced the expression of OPN through inhibition of the p38 MAPK pathway. The expression of OPN was associated with kidney injury. These molecules may represent therapeutic targets for the prevention of acute kidney injury induced by diabetes.

KLF4 Expression Correlates with the Degree of Differentiation in Colorectal Cancer.

BACKGROUND/AIMS: Krüppel-like factor 4 (KLF4) is an epithelial-specific transcription factor primarily expressed in the gastrointestinal tract that mediates growth arrest in the colonic epithelium. We tried to find whether KLF4 expression is associated with the progression and differentiation of colorectal cancer. METHODS: We detected KLF4 expression in 109 colorectal specimens (40 normal appearing mucosa, 7 adenomas, and 62 carcinomas) by immunohistochemistry using a tissue microarray. Western blot and RT-PCR analyses were also performed. RESULTS: The upregulation of KLF4 expression in carcinoma tissue was statistically significant (p<0.05) when compared to normal appearing mucosa. The negative and weak positive staining rates in normal appearing mucosa, adenoma, and carcinoma were 42.5%, 71.4%, and 82.3%, respectively, indicating a decreased degree of KLF4 expression over the course of progressive transformation of normal cells into malignant derivatives. KLF4 protein levels showed no correlation with sex, age, or metastatic state (p>0.05), while KLF4 protein expression correlated with the diagnostic stage (p<0.05). Furthermore, strong KLF4 staining was detected in 22.9% (11/48) and 0% (0/14) of well/moderately and poorly differentiated colorectal cancers, respectively. Our results clearly indicate that KLF4 protein expression significantly correlates with the degree of differentiation in colorectal cancers (p<0.05). KLF4 expression in RKO cells is also upregulated by butyrate, an inducer of differentiation. CONCLUSIONS: Downregulation of KLF4 expression may lead to more poorly differentiated tumors.

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.

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.