OBJECTIVETo investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK.

METHODSFifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin β1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats.

RESULTSGFK attenuated the up-regulation of CTGF, integrin α5, and integrin β1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01).

CONCLUSIONSCTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

Animals ; Collagen ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Cyclin D1 ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Gene Expression Regulation ; drug effects ; Integrin alpha5 ; genetics ; metabolism ; Integrin beta1 ; genetics ; metabolism ; Liver ; drug effects ; enzymology ; pathology ; Liver Cirrhosis ; drug therapy ; enzymology ; genetics ; pathology ; Male ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

BACKGROUNDThe initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated.

METHODSA porous ACP/PLLA composite and PLLA scaffold (as a control) were incubated in fetal bovine serum (FBS) containing phosphate-buffered saline (PBS), and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy (SEM). The expression levels of the genes encoding integrin subunits α1, α5, αv, β1, focal adhesion kinase (FAK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using real-time reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits α1, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite.

CONCLUSIONSThe ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits α1, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.

Biocompatible Materials ; chemistry ; Calcium Phosphates ; chemistry ; Cell Adhesion ; physiology ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alpha1 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin alphaV ; metabolism ; Integrin beta1 ; metabolism ; Integrins ; genetics ; metabolism ; Lactic Acid ; chemistry ; Osteoblasts ; cytology ; Porosity ; Tissue Engineering ; methods

OBJECTIVETo observe the expressions of α-SMA, integrin α5 and fibronectin (Fn) in acute paraquat poisoned rats and the effect of PDTC. To investigate the mechanism of paraquat-induced pulmonary fibrosis.

METHODSSprague-Dawley rats were randomly divided into three experimental groups: Control group (6 rats), PQ group (36 rats) and PQ+PDTC group (36 rats). On the 1st, 3rd, the 7th, the 14th, the 28th and the 56th day after exposure, the protein expression of α smooth muscle actin (α-SMA) was evaluated by western blot. The mRNA levels of integrin α5 and fibronectin (Fn) were analyzed with real-time quantitative PCR (RT-PCR). Meanwhile, the lung pathological changes were observed and semi-quantified.

RESULTST With the time passing, the expression of α-SMA in PQ group increased gradually compared with control group (P < 0.05 or P < 0.01). The increasing extent was gently on the 3 rd, the 7 th day. While increasing extent was rapidly from the 28 th to the 56 th day. RT-PCR showed PQ significantly increased Fn mRNA level on all time points and increased integrin α5 mRNA level from the 7 rd to 56 th day compared with control group (P < 0.05 or P < 0.01). PDTC treatment significantly deceased α-SMA, Fn, and integrin α5 levels compared with PQ group in corresponding time points (P < 0.05 or P < 0.01) Noteworthy, in PQ+PDTC group, the occurrence of pathological changes were drastically attenuated and pathologic score significantly decreased (P < 0.05 or P < 0.01).

CONCLUSIONSα-SMA, integrin α5 and fibronectin could play an important role in the development of pulmonary fibrosis caused by paraquat poisoning. PDTC, asa strong NF-κB inhibitor, may inhibit NF-κB activity and further significantly decreased expressions of α-SMA, integrin α5 and fibronectin which were important part of ECM, leading to drastically attenuated pulmonary fibrosis. However, the mechanisms of PDTC intervention still remains to be explored.

Actins ; metabolism ; Animals ; Disease Models, Animal ; Fibronectins ; metabolism ; Integrin alpha5 ; metabolism ; Male ; Paraquat ; poisoning ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Pyrrolidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates ; pharmacology

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.
Animals ; Bone Morphogenetic Proteins/metabolism ; Cartilage/*cytology/metabolism ; *Cell Differentiation ; Chondrocytes/cytology/enzymology ; Extracellular Matrix Proteins/deficiency/*metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Integrin alpha5/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Mice ; Neoplasm Proteins/deficiency/*metabolism ; Osteogenesis ; Protein Binding ; Signal Transduction ; Smad Proteins/metabolism

OBJECTIVETo investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism.

METHODSHuman myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting.

RESULTSThe exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA.

CONCLUSIONRGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Integrin alpha5 ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; PPAR gamma ; agonists ; Thiazolidinediones ; administration & dosage ; pharmacology ; Tretinoin ; pharmacology ; Up-Regulation

The aim of this study was to evaluate the homing capabilities of hematopoietic stem/progenitor cells (HSPCs) derived from human placenta tissues (PT). Single cell suspension of human PT was prepared by mechanical method. The expression levels of homing-related adhesion molecules (HRAM) including CD11a, CD49d, CD44, CD49e, CD62L and CD54 on CD34(+) cells and the percentages of CD34(+) cells and their subpopulations in nucleated cells (NC) from fresh human PT, umbilical cord arterial blood (UCAB) and umbilical cord venous blood (UCVB) were detected by using flow cytometry. The results showed that the percentage of CD34(+) cells and CD34(+)CD38(-) cells in placenta were higher than those in UCAB and UCVB. There were no significant difference in percentage of HSPC between UCAB and UCVB. Placenta-derived CD34(+) cells strongly expressed CD11a, CD49d, CD44, CD49e and CD54, among which expression levels of CD49e and CD54 on placenta-derived CD34(+) cells were significantly higher than those on UCAB and UCVB-derived CD34(+) cells. While the percentage of CD34(+)CD62L(+) cells in placenta was only lower than that in UCVB. It is concluded that human placenta is rich in HSPC. Moreover, the expression levels of most HRAM in CD34(+) cells from PT are higher than those from UCAB and UCVB or are close to them. It suggested that HSPCs derived from PT might have stronger homing capabilities than those from UCB.
Antigens, CD34 ; biosynthesis ; Cell Adhesion Molecules ; biosynthesis ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; metabolism ; Humans ; Hyaluronan Receptors ; biosynthesis ; Integrin alpha5 ; biosynthesis ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Placenta ; cytology

OBJECTIVETo explore the correlation between the expression of integrin subunits alpha5 and beta1 in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage.

METHODSExpressions of integrin subunits alpha5 and beta1 were examined in 30 cases of PCa and 30 cases of normal prostatic tissues by immunohistochemical assay.

RESULTSExpressions of integrin subunits alpha5 and beta1 in PCa were lower than those in normal prostatic tissues (P <0.05) respectively.

CONCLUSIONCompared with normal prostatic tissues, expressions of integrin subunits alpha5 and beta1 in PCa were rather weaker or even faded. Expressions of integrin subunits alpha5 and beta1 revealed an positive correlation with tumor's Gleason grade and negative with clinical TNM stage. The results indicate that integrin subunits alpha5 and beta1 have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Aged ; Aged, 80 and over ; Animals ; Humans ; Integrin alpha5 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Male ; Mice ; Middle Aged ; Neoplasm Staging ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo analyze the drug-sensitivity-related genes to anti-tumor drugs navalbine (NVB) and docetaxel (Doc) in four SCLC and six NSCLC cell lines.

METHODSThe sensitivity of 4 SCLC lines and 6 NSCLC lines to NVB and to Doc was determined with MTT test. The expression of 1291 anti-tumor drug sensitivity-related genes in the 10 cell lines was assayed by cDNA macroarray technique, and cluster analysis was performed to find the relationship between the results obtained by the above mentioned two measurements.

RESULTS(1) The anti-tumor effect of NVB on the 10 cell lines was apparently better than that of Doc. (2) The drug sensitivity-related genes in these 10 cell lines showed a more close positive correlation with Doc than that with NVB, whereas more genes showed negative correlation with NVB than that with Doc. But in 6 NSCLC cell lines, more genes showed the same positive or negative correlation with the two drugs. (3) 51 genes in the 10 cell lines showed correlation with Doc or NVB. 13 of them had negative correlation with Doc, 11 of them showed positive correlation. 24 of them showed negative correlation with NVB, 3 of them showed positive correlation. 67 genes in 6 NSCLC cell lines showed a correlation with sensitivity to Doc or NVB, among them 34 had negative correlation with Doc, 4 had positive correlation. 25 genes had negative correlation with NVB, 4 had positive correlation. (4) Rab 1, Rab 3, Rho B, Rho C, Rac 1, Rac 2, Gho GDI beta, CD44, integrin alpha5, integrin alpha6, integrin beta5, vinculin showed to be cytoskeleton-related genes differently expressing in SCLC or NSCLC cell lines.

CONCLUSIONThere is obvious difference in the drug sensitivity-related genes to NVB or Doc between SCLC and NSCLC cell lines.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cluster Analysis ; Cytoskeletal Proteins ; metabolism ; Cytoskeleton ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Integrin alpha5 ; metabolism ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; Taxoids ; pharmacology ; Vinblastine ; analogs & derivatives ; pharmacology ; rab1 GTP-Binding Proteins ; metabolism ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) on multiple myeloma (MM) cell migration and adhesion and it related signaling pathways.

METHODSExpression of adhesion molecules on MM cells of RPMI8226, XG-1 and XG-7 cells was analysed by flow cytometry, the influence of SDF-1 on CD29 and CD49e distribution by immunofluorescence, the effect of SDF-1 on chemotaxis of MM cells by transwell assay. Activation of phosphoinositide-3 kinase (PI3K) in MM cells treated with SDF-1 and by immunoblotting.

RESULTS3 strains of MM cell line expressed many adhesion molecule. RPMI8226, XG-7 cells were all high level of expression of CD29 (> 70%). XG-1, XG-7 cells were all high level of expression of CD44 (> 80%), and XG-7 cells was of CD49d (> 90%). In all of 3 strains, the levels of expression of CD49e were low (< 30%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of MM cells and the redistribution of CD29 and CD49e. SDF-1 promoted MM cells adhesion to endothelial cells, stimulated phosphorylation of P85 subunit of PI3K in MM cells and induced MM cells migration, which were inhibited by G protein inhibitor PTX and PI3K inhibitor wortmannin.

CONCLUSIONSDF-1 can promote MM cell adhesion to endothelial cells, trigger establishment of a polarized morphology of MM cells and redistribution of adhesion molecules and induce MM cells migration via PI3K signaling pathway.

Blotting, Western ; Cell Adhesion ; drug effects ; physiology ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; physiology ; Chemokine CXCL12 ; pharmacology ; physiology ; Enzyme Activation ; drug effects ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans ; Integrin alpha4 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin beta1 ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; physiopathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; drug effects ; physiology

OBJECTIVETo investigate the differentiation induction effect of 2-methoxyestradiol (2ME2), an estrogen derivative on myeloma cell line CZ-1.

METHODSThe changes of CZ-1 cells in morphology, expression of surface CD49e and quantity of light chain secretion in the supernatant were observed when treated with 0.1 approximately 0.5 micromol/L 2ME2 for 48 h.

RESULTS2ME2 could induce differentiation of CZ-1 cells. The cells appeared decreased in size of nucleus, increased in cytoplasma, decreased in the ratio of nucleus to plasma, decreased in number or disappearance of nucleolus, and thickness and pyknosis of chromatin. The expression of CD49e was increased from (12.20 +/- 1.57)% to (24.80 +/- 1.26)% (P < 0.05). Light chain secretion in the supernatant was increased from (35.97 +/- 2.60) microg/ml to (79.67 +/- 1.88) microg/ml (P < 0.05).

CONCLUSIONLow concentrations of 2ME2 could induce differentiation of myeloma cell line CZ-1.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Estradiol ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Humans ; Integrin alpha5 ; analysis ; Multiple Myeloma ; metabolism ; pathology ; Tubulin Modulators ; pharmacology