Identification and Verification of Two Novel Differentially Expressed Proteins from Non-neoplastic Mucosa and Colorectal Carcinoma Via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.

Recurrence or metastasis of colorectal cancer (CRC) is common following surgery and/or adjuvant therapy, particularly in patients with an advanced stage of the cancer. Identifying key molecular markers of CRC is beneficial for early diagnosis and early treatment, which may eventually improve the prognosis of patients with CRC. Isobaric mass tags for relative and absolute quantification (iTRAQ) in combination with multidimensional liquid chromatography and tandem mass spectrometry (LC-MS/MS) were used to identify differentially expressed proteins between CRC tissues and paired adjacent normal mucosa. Among the 105 patients, adenocarcinoma was the most common CRC subtype, stage III was the most common Tumor-Node-Metastasis stage and high levels of Ki-67 indicated the rapid proliferation of tumor cells in the samples. The LC-MS/MS-based iTRAQ technology identified 271 differentially expressed proteins, with 130 upregulated proteins and 141 downregulated proteins. Bioinformatics analysis revealed that golgin subfamily A member 2 (GOLGA2) and heterogeneous nuclear ribonucleoprotein D0 (hnRNPD) were located in the center of the upregulated protein network, and were closely associated with the development of CRC. The upregulation of GOLGA2 and hnRNPD was further verified in human tissues using western blotting and immunohistochemistry. GOLGA2 and hnRNPD were identified as two novels differentially expressed proteins in human CRC. Furthermore, the LC-MS/MS-based iTRAQ proteomic approach is a useful tool for searching and identifying differentially expressed proteins, and may be used to provide a comprehensive understanding of the processes that mediate the development of CRC.

Identification and Verification of the Main Differentially Expressed Proteins in Gastric Cancer via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.

BACKGROUND: To find the potential intersections between the differentially expressed proteins and abnormally expressed genes in gastric cancer (GC) patients. METHODS: Gastric cancer tissue and adjacent normal mucosa tissue were used for iTRAQ analysis. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) analysis were used to evaluate gene function. Western blotting and immunohistochemistry (IHC) were applied to verify the protein expression. RESULTS: A total of 2770 proteins were identified, of which 147 proteins were upregulated and 159 proteins were downregulated. GO analysis revealed that the differentially expressed genes were mainly enriched for the terms "cellular process," "binding," and "cell." The results of the KEGG analysis showed that the most abundantly enriched proteins were involved in the "focal adhesion" pathway. The results of the PPI analysis showed that VCAM1 was located at the center of the PPI network. Western blotting and IHC analysis demonstrated that VCAM1, FLNA, VASP, CAV1, PICK1, and COL4A2 were differentially expressed in GC and adjacent normal tissues, which was consistent with the results of the iTRAQ analysis. CONCLUSION: In conclusion, 6 highly differentially expressed proteins were identified as novel differentially expressed proteins in human GC. This exploratory research may provide useful information for the treatment of gastric cancer in the clinic.

Cinobufagin Promotes Cell Cycle Arrest and Apoptosis to Block Human Esophageal Squamous Cell Carcinoma Cells Growth via the p73 Signalling Pathway.

Cinobufagin isolated from traditional Chinese herbs has antitumour, anaesthetic, analgesic and anti-inflammatory effects. Recently, the antitumour activity of cinobufagin has attracted increasing attention from researchers. However, the anticancer activity of this drug on esophageal cancer cells and the precise mechanism are unclear. In this study, we determined the inhibitory effect of cinobufagin on the growth of three esophageal squamous cell carcinoma cell lines and explored its underlying mechanism. EC-109, Kyse-150, and Kyse-520 cells were treated with different concentrations of cinobufagin. The results of the Cell Counting Kit-8 (CCK-8) and clone formation assays showed that cinobufagin significantly reduced cell proliferation in a dose- and time-dependent manner. Also, flow cytometry and Hoechst 33342 staining indicated that the inhibition of growth induced by cinobufagin was mediated by G2/M cell cycle arrest and apoptosis. In addition, the expression of proteins related to cell cycle arrest and apoptosis was assessed by real-time quantitative (q)RT-PCR and Western blot. The results showed that cinobufagin caused G2/M arrest via upregulation of p21 and Wee1 and downregulation of cyclin B1 and Cdc2 at the mRNA and protein levels and induced apoptosis via upregulation of cleaved caspase-3, Puma and Noxa expression and an increased Bax/Bcl-2 ratio. Other data further showed that cinobufagin increased p73 expression and decreased Mdm2 expression, whereas p53 expression was not significantly changed. Taken together, these results suggest that growth inhibition of cinobufagin in esophageal cancer cells might act through the p73 pathway and its downstream molecules.

The anticancer effects of cinobufagin on hepatocellular carcinoma Huh­7 cells are associated with activation of the p73 signaling pathway.

The Na+/K+­ATPase inhibitor cinobufagin exhibits numerous anticancer effects on hepatocellular carcinoma (HCC) cells expressing wild­type p53 via inhibition of aurora kinase A (AURKA) and activation of p53 signaling. However, the effects of cinobufagin on HCC cells expressing mutant p53 remain unclear. In the present study, the anticancer effects of cinobufagin were investigated on HCC Huh­7 cells with mutant p53, and the effects of AURKA overexpression or inhibition on the anticancer effects of cinobufagin were analyzed. Viability, cell cycle progression and apoptosis of cells were determined using an MTT assay, flow cytometry and Hoechst 33342 staining, respectively. The expression levels of p53 and p73 signaling­associated proteins were investigated via western blot analysis. The results demonstrated that the expression levels of AURKA, B­cell lymphoma 2 (Bcl­2), cyclin­dependent kinase 1, cyclin B1, proliferating cell nuclear antigen and heterogeneous nuclear ribonucleoprotein K, as well as the phosphorylation of p53 and mouse double minute 2 homolog, were significantly decreased in Huh­7 cells treated with 5 µmol/l cinobufagin for 24 h. Conversely, the expression levels of Bcl­2­associated X protein, p21, p53 upregulated modulator of apoptosis and phorbol­12­myristate­13­acetate­induced protein 1, were significantly increased by cinobufagin treatment. Overexpression or inhibition of AURKA suppressed or promoted the anticancer effects of cinobufagin on Huh­7 cells, respectively. These results indicated that cinobufagin may induce anticancer effects on Huh­7 cells via the inhibition of AURKA and p53 signaling, and via the activation of p73 signaling, in an AURKA­dependent manner.

PAR-2 promotes invasion and migration of esophageal cancer cells by activating MEK/ERK and PI3K/Akt signaling pathway.

Protease-activated receptor 2 (PAR-2) has been demonstrated to promote invasion and metastasis of certain cancer cells. This study aimed to investigate the mechanism by which PAR-2 regulated invasion and migration of esophageal cancer (EC) cells EC109. A successfully constructed PAR-2 shRNA lentiviral vector (Lenti-PAR-2 shRNA) was stably transfected into EC109 cells, and the expression of PAR-2 in infected cells was detected by quantitative real-time PCR (qRT-PCR) and western blotting. Specific inhibitors, PD98059 (for MEK/ERK) and LY294002 (for PI3K/Akt), were used to confirm the role of MEK/ERK and PI3K/Akt signaling pathways, respectively, in PAR-2-regulated invasion and migration of EC109 cells. A significant decrease in PAR-2 mRNA and protein expression was detected in EC109 cells stably transfected with Lenti-PAR-2 shRNA. The PAR-2 agonist could dramatically promote cell invasion and migration, up-regulate the expression of MMP-9 and TM4SF3, and activate MEK/ERK and PI3K/Akt signaling pathways. However, PAR-2 gene silencing attenuated PAR-2-mediated enhancement of invasion and migration of EC109 cells, significantly down-regulated the mRNA and protein expression of MMP-9 and TM4SF3, and inhibited ERK (Try202/204) and Akt (Ser473) phosphorylation. An effect similar to PAR-2 silencing could be achieved with the two specific MEK/ERK and PI3K/Akt pathway inhibitors. Consequently, these results demonstrated that PAR-2 promoted invasion and migration of esophageal cancer cells EC109 by activating MEK/ERK and PI3K/Akt signaling pathways, which was accompanied by up-regulation of MMP-9 and TM4SF3 expression. Hence, PAR-2 may be a potential candidate for anti-metastasis treatment of EC.

Synthesis and Performances of Phase Change Microcapsules with a Polymer/Diatomite Hybrid Shell for Thermal Energy Storage.

The mechanical behavior of phase-change microcapsules (microPCMs) is of vital significance for practical applications in thermal energy storage. Hence, a new type of microPCMs based on an n-octadecane (C18) core and a melamine-urea-formaldehyde (MUF)/diatomite hybrid shell was developed through in situ polymerization. Based on SEM micrographs, most microPCMs exhibited a nearly spherical and smooth microstructure, with broadened particle size distributions. It was confirmed by Fourier transform infrared (FTIR) that successful polymerization of diatomite into the microPCMs occurred, and that additional diatomite had no effect on the core coated by the shell. In addition, the results of the differential scanning calorimeter (DSC) and Atomic Force Microscopy (AFM) demonstrated that the mechanical properties of the microPCMs were remarkably improved by the addition of a moderate amount of diatomite, but that the heat enthalpy and encapsulated efficiency (η) decreased slightly. The incorporation of 2 wt % diatomite resulted in the average Young's modulus of microPCMs, which was 1.64 times greater than those of microPCMs without diatomite. Furthermore, the melting and crystallization enthalpies and the encapsulated efficiency of the microPCMs were as high as 237.6 J/g, 234.4 J/g and 77.90%, respectively. The microPCMs with a polymer/diatomite hybrid shell may become the potential materials in the application of thermal energy storage.

Effect of yiqi huoxue recipe on cardiac function and ultrastructure in regression of pressure overload-induced myocardial hypertrophy in rats.

OBJECTIVE: To investigate the effect of Yiqi Huoxue Recipe (YHR) on the cardiac function and ultrastructure during the regression of myocardial hypertrophy induced by pressure overload in rats. METHODS: The model of myocardial hypertrophy was established by abdominal aortic banding. Eighty male Wistar rats were divided into six groups, the normal control group I (n=20), the normal control group II (n=12), the hypertension model group I (n=12), the hypertension model group II (n=12), the YHR group (n=12) and the Captopril group (n=12). The observation was carried out in the normal control group I and the hypertension model group I after 4 weeks of modeling, and the other four groups were observed after 16 weeks of modeling (12 weeks of administration). The cardiac function was measured with a multichannel biological signal analysis system, and the myocardium ultrastructure was observed by a transmission electron microscope. RESULTS: (1) Compared with the normal control group I, the systolic blood pressure and cardiac coefficient (left ventricular weight/body weight) in the model I group was higher (P<0.05, P<0.01). (2) In the YHR group, cardiac coefficient and -dp/dt(max) were lower, left ventricular systolic pressure and +dp/dt(min) were higher when compared with the model group II and the Captopril group (P<0.05 or P<0.01). In the Captopril group, only cardiac coefficient was lower when compared with the mode group II (P<0.05). (3) Compared with the normal control group II, +dp/dt(max) was higher (P<0.01) -dp/dt(max) and isovolumetric contraction time (ICT) was lower (P<0.05, P<0.01) in both the YHR group and the Captopril group. (4) Results of the myocardium ultrastructure showed edema under myocardium plasmalemma, enlarged sarcoplasmic reticulum and T tube, and significantly enlarged intercalated disc of the cardiac muscle in the model groups. In the Captopril group, the extension of sarcoplasmic reticulum and T tube as well as the pathological changes of intercalated disc were lighter, with slight edema under the myocardium plasmalemma. In the YHR group, the expansion of the sarcoplasmic reticulum was less than in the Captopril group, part of the pathological changes of intercalated discs was slightly more severe than that in the Captopril group, the dissolution of nuclear chromatin was not found, which was similar to that of the Captopril group, and no injury of the nucleus was found, either. CONCLUSION: YHR could reverse myocardial hypertrophy in rats with abdominal aortic banding and improve the systolic and diastolic function of the left ventricle. The ultrastructure of the myocardium such as arcoplasmic reticulum, intercalated disc, and cell nucleus in abdominal aortic banding rats could be partly reversed by the recipe.