Mild overexpression of a novel transcription factor Grainy head like-2 (Grhl2) elicits hybrid phenotype in human astrocytoma cells

The Grainy head like-2 (Grhl2) transcription factor plays a major role in embryonic and cancer development. The role of Grhl2 has been intensively studied in various cancers but not for brain cancer. Hence, in this study, we provide a preliminary understanding on the role of Grhl2 that regulate the transition of astrocytoma cells. The human A172 astrocytoma cell line, a mesenchymal cell characterized by mild overexpression of Grhl2 transcription factor, was used in this study. At first, the Grhl2 stably overexpressing A172 clones into three types i.e., Grhl2+ (mild), Grhl2++ (moderate) and Grhl2+++ (high) based on mRNA and protein expression levels of Grhl2 were characterized. Phenotypic characteristics of vector and Grhl2+ cells were observed using phase contrast microscopy. Quantitative PCR (qPCR), Western blot and immunofluorescence were used to detect the level of mesenchymal markers (N-cadherin/vimentin) and also epithelial markers (E-cadherin/ ?- catenin) in vector and Grhl2+ cells. The migration and invasion characteristics of vector and Grhl2+ cells were determined by scratch assay and Boyden chamber assay. Further, the Grhl2+ cells were characterized to determine the effect of temozolomide chemotherapy drug which were widely used in treating brain cancer. As expected, in phase contrast image, we observed the mesenchymal characteristic of A172 cells becomes hybrid phenotype i.e., mixture of mesenchymal (spindle-like fibroblast morphology) and epithelial (cobblestone like appearance) cells upon Grhl2 mild expression (Grhl2+) when compared to vector cells. Further, we found that there was a significant upregulation of E-cadherin at both mRNA and protein levels in Grhl2+ cells when compared to vector cells. There was a significant upregulation of ?-catenin, N-cadherin and vimentin at mRNA levels, but there was no significant upregulation at the protein levels in Grhl2+ cells compared to the vector cells. The migration and invasion were diminished in Grhl2+ cells when compared to the vector control cells. We observed that the Grhl2+ were sensitive to the temozolomide compared to the vector cells. This infers that the Grhl2+ cells were unable to attain complete transition of mesenchymal to epithelial state, and hence we categorized the Grhl2+ cells as hybrid phenotype. The results provide a better understanding of the largely unknown function of Grhl2 in human astrocytoma cells as tumor progression or suppression.

Effect of Smad7 deficiency on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen I secretion in vitro / 解剖学报

Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen secretion in vitro. Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method. The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts, Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus. The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer. Cell migration was evaluted by cell wound scratch assay. Western blotting was used to detect expression of α-smooth muscle actin（α-SMA） and collagen Ⅰ（Col Ⅰ）. Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods. The multiplicity of infection（MOI） that lentivirus transduction of myocardial fibroblasts was 100. After lentivirus transduction, 88.33% myocardial fibroblasts expressed green fluorescent protein, showed that the lentivirus could significantly reduce the protein expression of Smad7. Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts, increased the protein expression of α-SMA and decreased collagen secretion. The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts, increased α-SMA protein expression and reduced ColⅠ protein expression. Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function, that is related to the pathological mechanism that lead to myocardial fibrosis

Effects of usnicoyinamide on proliferation, apoptosis, cell cycle, and migration of SGC-7901 gastric cancer cells / 中草药

Objective: To investigate the inhibitory effect of usnicoyinamide on the proliferation of gastric cancer cell line SGC-7901 and its mechanism. Methods: SGC-7901 cells were cultured in vitro and divided into two groups: control group and experimental group with different concentrations of usnicoyinamide. The morphology of each group of cells was observed by a microscope; Proliferation of SGC-7901 cells was measured by MTT assay; The mechanism of apoptosis was studied by AnnexinV/PI double staining and DAPI fluorescence staining; Flow cytometry was used to detect the effect of usnicoyinamide on the cell cycle; Effect of usnicoyinamide on invasion and migration of SGC-7901 cells was detected by cell scratch test. Results: After SGC-7901 cells were treated with usnicoyinamide, the cells were wrinkled, deformed and adherent cells fell off; The results of MTT showed that the inhibition of the proliferation of SGC-7901 cells was a significant dose-effect relationship and time-dependent; The results of AnnexinV/PI double staining showed that nicotine increased the late apoptosis rate of SGC-7901 cells, and DAPI staining showed obvious nuclear concentration and nuclear fragmentation of apoptosis. The results of flow cytometry showed that the cell cycle of SGC-7901 cells stagnated in S phase; Scratch test showed that the mobility of SGC-7901 cells was decreased more obviously with the prolongation of time and the increase of concentration. Conclusion: Usnicoyinamide can inhibit the proliferation of gastric cancer cell line SGC-7901, and its mechanism may be achieved by inducing late apoptosis, inducing S phase cell arrest and inhibiting the invasion and migration of SGC-7901 cells.

Fatty acid composition of vegetable oil blend and in vitro effects of pharmacotherapeutical skin care applications

Vegetable oils have been used for a plethora of health benefits by their incorporation in foods, cosmetics, and pharmaceutical products, especially those intended for skin care. This study aimed to investigate the cutaneous benefits of a vegetable oil blend (VOB) formulation and its fatty acid composition. The anti-inflammatory activity was studied in macrophages of RAW 264.7 cells by investigating the release of nitric oxide (NO), superoxide anion generation (O2-), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). ABTS cation radical scavenging capacity assay, ferric reducing antioxidant potential (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and NO free radical scavenging assays were used to evaluate the antioxidant activity. VOB was tested for its ability to stimulate fibroblast proliferation and migration using the scratch assay, and antibacterial activity by the microdilution test. The fatty acid profile of a freshly prepared VOB formulation was determined by gas chromatography before and after accelerated stability testing. Chemical composition of VOB revealed the presence of oleic acid (C18:1n-9; 63.3%), linoleic acid (C18:2n-6; 4.7%), and linolenic acid (C18:3n-6; 5.1%) as major mono- and polyunsaturated fatty acids. No changes in the organoleptic characteristics and fatty acid composition were observed after the accelerated stability test. VOB 100 µg/mL reduced the healing time by increasing the total number of cells in the wounded area by 43.0±5.1% compared to the negative control group. VOB also suppressed the pro-inflammatory TNF-α and IL-6 cytokines, and NO and O2- production in lipopolysaccharide-stimulated macrophage cells. In conclusion, the VOB formulation contributed to the improvement of current therapeutic strategies for cutaneous applications in skin care.

Effect of Knocking Down ski on Migration of Astrocytes in Rats / 中国康复理论与实践

Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfec-tion, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blot-ting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P47.197, P69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.

An automatic measurement method for detecting the migration of A549 cells induced by Cdc42 overexpression / 国际生物医学工程杂志

Objective To explore whether Cdc42 is an independent influence factor in regulating the migration of A549 cells by using quantitative measurement method,and to verify the effectiveness of an automatic measurement and calculation method for in vitro cell migration assay.Methods Cdc42 was overexpressed in human lung adenocarcinoma A549 cell line,and then in vitro scratch assay was applied to evaluate the migration of the cells.Different methods were used to measure the images acquired at different time points for quantitative analysis.Accuracy and repeatability of different measurement methods were analyzed.Results The results showed that overexpression of Cdc42 alone significantly advanced the migration of A549 cells (P＜0.05).The new method is efficient,accurate and reproducible as compared to manual measurement,and has significant advantages in target recognition and noise removal as compared to the existing measurement method in Image Pro Plus 6.0.Conclusions Over expression of Cdc42 significantly increased A549 cell migration ability in vitro.The new method can realize the automatic quantitative analysis of cell migration in vitro.