IKKϵ negatively regulates RIG-I via direct phosphorylation.

Inhibitor of nuclear factor kappa-B kinase Epsilon (IKKϵ) is an IKK-related kinase. Despite it was originally discovered as a kinase functionally related to TBK-1, studies entailing gene knockout mouse demonstrated that IKKϵ is dispensable for interferon induction by viral infection. In this study, we report that IKKϵ directly phosphorylates a key serine residue within the RNA-binding domain of RIG-I (retinoic acid-inducible gene 1) to inhibit RIG-I-mediate innate immune signaling. Using IKKϵ-deficient MEFs, we found that loss of IKKϵ resulted in increased cytokine production in response to the activation of cytosolic sensors. Biochemical analyses indicated that IKKϵ physically associated with and phosphorylated RIG-I. Mass spectrometry analysis identified that IKKϵ phosphorylated the serine 855 of the RNA-binding pocket of RIG-I carboxyl terminal domain, a residues known to impinge on RNA-binding via phosphorylation. Our findings collectively support the conclusion that IKKϵ modulates innate immune signaling cascades via phosphorylating the RIG-I cytosolic sensor, providing a feedback regulatory mechanism.

Bisphenol A regulates Snail-mediated epithelial-mesenchymal transition in hemangioma cells.

Hemangioma (HA) can be exposed to bisphenol A (BPA) through direct skin absorption. Although numerous studies indicated that BPA can trigger the progression of cancers, there is no study concerning the effects of BPA on development of HA. Our present study revealed that nanomolar BPA can significantly increase the in vitro migration and invasion of HA cells via induction of epithelial-mesenchymal transition (EMT), which was evidenced by the upregulation of vimentin and downregulation of E-cadherin. The BPA treatment also significantly increased the expression and nuclear localization of Snail and the key transcription factor of EMT, while it had no effect on the expression of other transcription factors such as Slug, Twist, or ZEB1. Silencing of Snail by small interfering RNAs attenuated BPA-induced downregulation of cadherin and upregulation of vimentin, suggesting that Snail is essential for BPA-induced EMT. Both estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) were expressed in HA cells; furthermore, BPA treatment can increase the expression of both ERα and GPER. However, only the inhibitor of ERα (ICI 182, 780), and not GPER (G15), can abolish BPA-induced upregulation of Snail. It suggested that ERα is involved in BPA-induced EMT of HA cells. Collectively, our data suggested that BPA can trigger the EMT of HA cells via ERα/Snail signals. It indicated that more attention should be paid to the skin exposure to BPA for HA patients.

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial-mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells.

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial-mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulated miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.

[Preparation and property evaluation of graphene oxide based silver nanoparticles composite materials].

We prepared silver nanoparticles/polyethyleneimine-reduction graphene oxide (AgNP/rGO-PEI) composite materials, and evaluated their quality performance in our center. Firstly, we prepared AgNP/rGO-PEI, and then analysed its stability, antibacterial activity, and cellular toxicity by comparing the AgNP/rGO-PEI with the silver nanoparticles (PVP/AgNP) modified by polyvinylpyrrolidone. We found in the study that silver nanoparticles (AgNP) distributed relatively uniformly in AgNP/rGO-PEI surface, silver nanoparticles mass fraction was 4.5%, and particle size was 6-13 nm. In dark or in low illumination light intensity of 3 000 lx meter environment (lux) for 10 days, PVP/AgNP aggregation was more obvious, but the AgNP/rGO-PEI had good dispersibility and its aggregation was not obvious; AgNP/rGO-PEI had a more excellent antibacterial activity, biological compatibility and relatively low biological toxicity. It was concluded that AgNP/rGO-PEI composite materials had reliable quality and good performance, and would have broad application prospects in the future.

[Determination of content and entrapment efficiency of clindamycin phosphate liposome].

This study was conducted to prepare and determine the content and entrapment efficiency of Clindamycin Phosphate liposome. Evaporating and Ultrasound method was used for preparing Clindamycin Phosphate liposome. HPLC was used for determining the concentration and the entrapment efficiency of Clindamycin Phosphate liposome. The results indicated that Clindamycin Phosphate had a good linear relation in a range of 5.0-50.0 microg/ml. The entrapment efficiency of Clindamycin Phosphate liposome in three groups reached 52.26%, 50.13%, 53.75% respectively. Accordingly, the technique of preparing Clindamycin Phosphate liposome was noted to be feasible, and the method of quality control was shown simple and accurate.

Swertiamarin decreases lipid accumulation dependent on 3-ketoacyl-coA thiolase.

Natural compounds are important resources for drug discovery. Using Caenorhabditis elegans (C. elegans) models, we screened active natural compounds with lipid lowering effects. Swertiamarin was found as a potent candidate to reduce lipid content in C. elegans. Using RNAi screening, we were able to demonstrate that kat-1 (ketoacyl thiolase-1) is necessary for the lipid lowering effect of swertiamarin. Furthermore, the activity of swertiamarin was verified in high fat diet induced obese mice. Consistent with the results in C. elegans, swertiamarin ameliorated high fat diet induced lipid deposition and hyperlipidemia. These results indicate that swertiamarin exerts lipid-lowering effects through kat-1 regulation and could serve as a possible therapeutic option to improve hyperlipidemia induced comorbidities.

Cancer cell membrane cloaking nanoparticles for targeted co-delivery of doxorubicin and PD-L1 siRNA.

Nanoparticles coated with cell membranes have been garnering growing attention due to their homologous binding capability of membrane molecules and consequent self-recognition by their source cells. In the present study, we report on the construction of doxorubicin and PD-L1 siRNA-loaded PLGA nanoparticles and their biological functionalization by cancer cell-derived membrane cloaking. The resulting cancer cell membrane-coated nanoparticles (CCMNPs) presented a core-shell nanostructure with highly specific self-recognition affinity to the homotypic cells, which can be attributed to the transference of cell adhesion molecules with homotypic binding properties. These findings facilitate the application of this bioinspired strategy for effective delivery of siRNA and precise tumour therapy.

Studies of macrophage immuno-modulating activity of polysaccharides isolated from Paecilomyces tenuipes.

The objective of this study was to evaluate the immunomodulatory effects of the Paecilomyces sinensis polysaccharides (PtP) on the activity of macrophages and human monocytes. A water-soluble polysaccharide, with estimated molecular weight of 2.04x10(4) Da, was isolated from P. sinensis. The results indicate that PtP can increase the activity of LDH and ACP in AMphi and PMphi of rats and human mononuclear cells, and enhance the pinocytic activity of macrophages and TNF-alpha production by human peripheral blood mononuclear cells (PBMC), suggesting that PtP had potent immunomodulatory properties and could be explored as a novel potential immunostimulants for the food and pharmaceutical purpose.

Regulatory Roles of Osteopontin in Production of Monocyte-Origin MCP-1.

Osteopontin (OPN), expressed by various immune cells, plays a critical role in leukocyte migration. Although OPN was found to selectively induce the expression of proinflammatory chemokines, the molecular mechanisms that control OPN gene expression and its underlying mechanism for migration and recruitment of inflammatory cells remain largely unknown. In this study, real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine OPN and monocyte chemoattractant protein 1 (MCP-1) expression. Signaling and molecular events between OPN and MCP-1 were analyzed by Western blot. Leukocyte migration in the presence of OPN was measured by chemotaxis assay. Our data indicated that phosphoinositide 3-kinase (PI3K), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) that are activated upon stimulation with lipopolysaccharide were shown to upregulate OPN expression. Endogenous production of OPN was attributable to increased production of MCP-1, and this effect could be blocked by an anti-ß1 integrin antibody and JNK and p38 kinase inhibitors. Furthermore, we found that the effect of OPN on inflammatory cell migration was mediated through inducing the expression of MCP-1 in monocytes. These results support a role of OPN in monocyte migration via MCP-1, which may represent an additional mechanism for innate and adaptive immune responses.

Hyper activation of ß-catenin signalling induced by IKKε inhibition thwarts colorectal cancer cell proliferation.

OBJECTIVE: Aberrant activation of Wnt/ß-catenin signalling contributes significantly to the development of human colorectal cancers and ß-catenin is the key signalling molecule transducing canonical Wnt/ß-catenin signalling. Therefore, ß-catenin is a promising therapeutic target for cancer treatment. This study demonstrates that the oncogenic IKKε kinase phosphorylates ß-catenin to restrain its hyper activation, therefore promoting colorectal cancer (CRC) cell proliferation. MATERIALS AND METHODS: IKKε and ß-catenin expression levels in human colorectal cancer tissues and cell lines were analysed by immunohistochemical staining and Western blotting. The regulation of IKKε on Wnt/ß-catenin signalling pathway was studied by reporter assay and real-time PCR analysis in the context of IKKε stably knocking down. Co-immunoprecipitation was conducted to monitor the interaction between IKKε and ß-catenin. Kinase assay was performed to measure ß-catenin post-translational modifications induced by IKKε. RESULTS: Oncogenic IKKε kinase is required for the proliferation of colorectal cancer cells. Mechanistically, inhibition of IKKε results in ß-catenin hyper activation and thwarts CRC cell proliferation. Furthermore, IKKε phosphorylates ß-catenin and inhibits the activation of ß-catenin signalling. CONCLUSION: Our study suggests that IKKε is a potential target to combat CRC induced by aberrant Wnt/ß-catenin signalling.

MiR-503 inhibits hepatocellular carcinoma cell growth via inhibition of insulin-like growth factor 1 receptor.

MicroRNAs (miRs) have been demonstrated to play key roles in the development and progression of hepatocellular carcinoma (HCC). However, the regulatory mechanism of miR-503 in HCC has not been fully uncovered. In this study, we found that miR-503 was significantly downregulated in HCC tissues compared to nontumorous liver tissues. Moreover, lower miR-503 levels were associated with the malignant progression of HCC, and the expression of miR-503 was also decreased in several common HCC cell lines compared to normal human liver cell line THLE-3. Overexpression of miR-503 inhibited proliferation but induced apoptosis of LM3 and HepG2 cells. Bioinformatical analysis and luciferase reporter assay further identified insulin-like growth factor 1 receptor (IGF-1R) as a novel target of miR-503 in 293T cells. Moreover, overexpression of miR-503 led to a significant decrease in the protein levels of IGF-1R, while knockdown of miR-503 enhanced its protein levels in LM3 and HepG2 cells. Besides, overexpression of IGF-1R reversed the effects of miR-503-mediated HCC cell proliferation and apoptosis, indicating that IGF-1R acts as a downstream effector of miR-503 in HCC cells. Furthermore, IGF-1R was found to be significantly upregulated in HCC tissues compared to nontumorous liver tissues. In addition, the mRNA levels of IGF-1R were inversely correlated to the miR-503 levels in the HCC tissues. Thus, we demonstrate that miR-503 inhibits the proliferation and induces the apoptosis of HCC cells, partly at least, by directly targeting IGF-1R, and suggest that IGF-1R may serve as a promising target for the treatment of HCC.

MicroRNA-22 expression in hepatocellular carcinoma and its correlation with ezrin protein.

OBJECTIVE: Expression of microRNA-22 (miR-22) and ezrin protein (a membrane-cytoskeleton linking protein) in hepatocellular carcinoma (HCC) was investigated. METHODS: Specimens of HCC and paracancerous tissue (control; ~5 cm away from tumour tissue) were collected from 192 patients. miR-22 expression was detected by real-time polymerase chain reaction; ezrin protein expression in tumour tissue was assessed immunohistochemically. Associations between miR-22 expression and clinicopathological features of HCC and ezrin expression were analysed. RESULTS: miR-22 expression was lower in HCC tissue than in paracancerous tissue samples (median relative expression 0.676 versus 1.000 for control tissue). Expression of miR-22 was significantly associated with histological differentiation (relative expression 0.431 for lower grades of differentiation versus 0.918 for higher grades), and was associated with lymphatic metastasis (relative expression 0.518 if metastasis was present, 0.919 if absent). Survival time was shorter in patients with low miR-22 expression than in those with high expression (31.0 ± 2.6 versus 52.2 ± 5.1 months). There was a significant negative correlation between the expression of miR-22 and that of ezrin. CONCLUSIONS: miR-22 is downregulated in HCC and its expression is associated with the differentiation, metastasis and prognosis of the carcinoma. Ezrin is a potential regulatory protein of miR-22.

Study on optimisation of extraction process of tanshinone IIA and its mechanism of induction of gastric cancer SGC7901 cell apoptosis.

The objective of this paper was to investigate the extraction process of tanshinone IIA and its mechanism of induction of gastric cancer SGC7901 cell apoptosis. Extraction process of tanshinone IIA was optimised by orthogonal experimental method, and its effect on gastric cancer SGC7901 cell apoptosis was observed using MTT assay and electron microscopy. The optimum extraction process of tanshinone IIA was as follows: addition of a 10-fold amount of 80% ethanol, one-time extraction, and extraction time of 45 minutes. The study concluded that tanshinone IIA can induce apoptosis of gastric cancer SGC7901 cells.

[In vitro cytotox icity effects of cocultured DC-C IK cells combined with sorafenib against hepa to cellular carcinoma].

AIM: To investigate the in vitro inhibitory effects of DC(dendritic cell)-CIK (cytokine-induced killer cell) cocultured cells combined with sorafenib against hepatocellular carcinoma cell line BEL27402. METHODS: DC and CIK cells were generated in vitro by stimulating human peripheral blood mononuclear cells with different cytokines, and then they were cocultured. The cytotoxicity of DC-CIK cocultured cells (DC-CIK) combined with sorafenib against BEL-7402 cells was determined by CCK8 kit. The apoptosis of BEL27402 cells was measured by Annexin V-FITC Kit. RESULTS: The cytotoxicity rate of BEL27402 cells in DC-CIK +sorafenib group was significantly higher than those in the other there groups, with cytotoxicity rate in DC-CIK + sorafenib group being (72.24 ± 2.42)% , which was 1.8 times that in DC-CIK group, 2.1 times that in sorafenib group , and 1.6 times that in CIK group(P < 0.01). The apoptosis rate of BEL-7402 cells in DC-CIK + sorafenib group was significantly higher than those in the sorafenib and DC-CIK groups, with the apop tosis rate in DC-CIK + sorafenib group being (77.36 ± 1.92)% (P < 0.05). CONCLUSION: DC-CIK cocultured cells combined with sorafenib can inhibit the growth of hepatocellular carcinoma cell line BEL-7402 in vitro. Molecular targeting therapy combined with immunotherapy may be a new way for the comprehensive treatment of hepatocellular carcinoma.

[Study of encapsulation of suicide gene in PEG-PEI/Fe3O4 nano-magnetic fluid and its characteristics in vitro].

AIM: To prepare PEG-PEI Fe(3)O(4) nano-magnetic fluid and invest its characteristics. METHODS: PEG-PEI Fe(3)O(4) nano-magnetic fluid was prepared by chemical co-deposition as gene delivery vector. Then DNA of plasmid pAFP-TK was wrapped into PEG-PEI Fe(3)O(4) nano-magnetic fluid. At the same time, we invest its characteristics with MTT assay. RESULTS: PEG-PEI Fe(3)O(4) nano-magnetic fluid-DNA complexes could protect the DNA from degradation of DNaseI, serum and sonication. AFP positive cells transfected by pAFP-TK were highly sensitive to the GCV treatment, and the survival of cells declined obviously. HSV-TK/GCV suicide gene therapy system had direct cell toxicity. CONCLUSION: This new target strategy of pAFP-TK/GCV suicide gene therapy system in which PEG-PEI Fe(3)O(4) nano-magnetic fluid are used as gene delivery vectors explores a promising area for AFP positive HCC and associated carcinoma therapy.

[Phage display random peptide library for screening the peptides that specifically bind to hepatocellular carcinoma cells].

OBJECTIVE: To screen the peptides that bind specifically to the hepatoma cells using phage display random peptides library. METHODS: Three rounds of panning were conducted in vitro targeting HepG2 cell lines. In nude mice bearing HepG2 tumor, one round of panning was conducted, and 30 phage clones were randomly selected for sequence analysis to identify the consensus sequence. Immunocytochemistry and immunohistochemistry were performed to determine the specificity of the phages to the hepatoma cells. RESULTS: After three rounds of panning in vitro and one round of panning in vivo, the phages binding to HepG2 cells were enriched. Sequence analysis of the randomly selected clones identified the peptide sequence VRKRSECLGAHD as the most frequent sequence. Immunocytochemistry and immunohistochemistry confirmed the specificity of the phage binding to the hepatoma cells. CONCLUSIONS: Specific peptides against hepatoma cells can be obtained from a phage- display peptide library, which provides an experimental basis for developing therapeutic agents targeting hepatoma cells.

Overexpression of EphA2, MMP-9, and MVD-CD34 in hepatocellular carcinoma: Implications for tumor progression and prognosis.

AIM: To investigate the expression of erythropoietin-producing hepatocellular (Eph)A2 receptor, matrix metalloproteinase (MMP)-9, and angiogenesis in hepatocellular carcinoma (HCC), in order to reveal their expression correlations with tumor invasion, metastasis, and prognosis. METHODS: From January 2000 to June 2003, 129 specimens of resected tumors from the patients with HCC were obtained. Corresponding pericarcinomatous liver tissues were also obtained and selected as a control group. Expressions of EphA2, MMP-9, and CD34 were detected with immunohistochemical staining. Microvascular density (MVD) was calculated with counting of CD34-positive vascular endothelial cells. RESULTS: The expressions of EphA2, MMP-9, and MVD in the HCC tissues were significantly higher than those in the pericarcinomatous liver tissues (P < 0.01). Statistical analysis showed there were significant correlations between the expressions of EphA2, MMP-9 and MVD in some classicclinicopathological parameters (i.e. tumor nodule, vein invasion, tumor, node, metastasis stages, extrahepatic metastasis; P < 0.05). The correlation between EphA2 and MMP-9 expression was positive (r = 0.625, P = 0.011). Tumor MVD was closely associated with EphA2 (r = 0.281, P = 0.01) and MMP-9 (r = 0.319, P < 0.01) expressions. In particular, EphA2, MMP-9, and MVD expressions levels were found to be independent prognostic factors after HCC resection. CONCLUSIONS: Overexpressions of EphA2 and MMP-9 relate to tumor progression, metastasis, and prognosis in HCC. The present study suggests that EphA2 is associated with key mediators of angiogenesis and invasion.

[Expression of MMP-2, MMP-9 and collagen type IV and their relationship in colorectal carcinomas].

AIM: To investigate the expression of MMP-2, MMP-9 and collagen type IV and their relationship in colorectal carcinomas. METHODS: The expressions of collagen type IV, MMP-2 and MMP-9 was in normal and colorectal cancer tissues of 30 patients whose clinical stages were Dukes'B and C was detected with immunohistochennical staining. And the relationship among collagen type IV, MMP-2 and MMP-9 was analysed. RESULTS: The expression of collagen type IV was significantly decreased in colorectal cancer tissues. The expression of MMP-2 and MMP-9 was not detected in normal colorectal tissues but it was significantly increased in colorectal cancer tissues. There was a negative correlation between the expression of collagen type IV and MMP-9. CONCLUSION: The expression of collagen type IV in colorectal cancer tissues is lower than that in normal tissues. MMP-9 may play an important role in the degradation of collagen type IV in colorectal cancer.