OBJECTIVETo investigate the effect of exogenous nitric oxide (NO) on the migration of HaCaT cell and its possible mechanism.

METHODSSodium nitroprusside (SNP) was used as the donor of NO. Different concentrations of SNP (0.1, 1.0, 10.0, 100.0, 1000.0 micromol/L) were added into nutrient culture medium of HaCaT cells. Cell migration rate was observed and calculated at post scratching hour (PSH) 0 (immediately after scratching), 6, 12, 24, 48. The most suitable concentration of SNP and culture duration were selected as stimulation condition. Cytoskeletons of HaCaT cells were observed under confocal laser scanning microscope. The expressions of integrin beta 1, RhoA, Rac1 and Cdc42 of cells in experiment group (cultured with 10.0 micromol/L SNP for 24 hours) and negative control group were determined at mRNA and protein levels with RT-PCR and Western blot respectively. Data were processed with one-way analysis of variance (ANOVA) and repeated measure ANOVA.

RESULTSMigration rate of HaCaT cells in each group increased gradually as time after scratching went on. There were significant differences between PSH 6-48 and PSH 0 in cells cultured with 10.0 micromol/L SNP (F = 31.002, P values all below 0.05). Pili were rarely observed in negative control group with slender stress fibers in cells. In comparison, the amount of pili amount increased obviously in experiment group with thickened stress fibers. Compared with those of cells in control group (RhoA protein expression = 0.64 +/- 0.04), integrin beta 1 expression decreased obviously (F = 8.25, P = 0.015), RhoA (0.92 +/- 0.04), Cdc42 and Rac1 were up-regulated at both protein (with F value respectively 7.25, 14.10, 6.50, P values all below 0.05) and mRNA levels (with F value respectively 23.67, 10.39, 9.52, P values all below 0.05).

CONCLUSIONSExogenous NO in suitable concentration can promote the proliferation and migration of HaCaT cell, suggesting it exerts significant effect in wound repair. The changed cytoskeletons and the down-regulated integrin beta 1 expression may be involved in this process.

Cell Line ; Cell Movement ; drug effects ; Cytoskeleton ; drug effects ; metabolism ; Humans ; Nitric Oxide ; pharmacology ; RNA, Messenger ; genetics ; rhoA GTP-Binding Protein ; genetics ; metabolism

In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection, positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product, electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore, the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5-L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5 L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study, we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction, the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.
Cell Movement ; drug effects ; Epithelial Cells ; cytology ; drug effects ; Fermentation ; Humans ; Intestine, Small ; cytology ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Trefoil Factor-2

OBJECTIVETo study the responses of different pancreatic cancer cells to stimulations by nerve growth factor (NGF) and explore the role of Trk-A in such responses.

METHODSFive pancreatic cancer cell lines (MIA-PaCa-2, PANC-1, SW-1990, AsPC-1, and BxPC-3) were exposed to different concentrations of NGF (0, 4, 20, 100, and 500 ng/ml). MTT and Matrigel invasion method were used to observe the changes in the cell proliferation and invasion ability. Trk-A expression in these cells was detected by PCR and Western blotting, and the relations of Trk-A expression to the cell proliferative and invasive abilities following NGF treatment were analyzed.

RESULTSNGF at 100 ng/ml most obviously stimulated the cell proliferation, and PANC-1 cells showed the highest while AsPC-1 cells showed the least sensitivity to 100 ng/ml NGF stimulation. Matrigel invasion test showed that NGF enhanced the invasiveness of PANC-1 and MIA-PaCa-2 cells but produced only limited effect on AsPC-1 cells; the effect of NGF was completely inhibited by the Trk-A inhibitor CEP701. The expression levels of Trk-A mRNA and protein were the highest in PANC-1 cells and the lowest in AsPC-1 cells.

CONCLUSIONNGF can enhance the proliferation and invasiveness of pancreatic cancer cells, and this effect is possibly mediated by Trk-A protein.

Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Neoplasm Invasiveness ; Nerve Growth Factor ; pharmacology ; Pancreatic Neoplasms ; metabolism ; pathology ; Receptor, trkA ; genetics ; metabolism

AIMThe six copies of RGD sequences present in osteopontin were expressed in the E. coli, and the biological activity of the purified products was studied.

METHODScDNA fragments containing six copies of RGD sequences were subcloned into prokaryotic expression vector pGEX-3X including GST coding sequence to construct pGEX-3X-RGD plasmids. E. coli DH5alpha transformed by pGEX-3X-RGD(6) plasmid was induced by different IPTG concentrations for different times to identify the optimal induction condition. Induced GST-RGD fusion protein was purified via GST-Sepharose 4B affinity resin.

RESULTSGST-RGD fusion proteins containing six copies of RGD sequences could be successfully induced and were mainly located in inclusion bodies. After being denatured and dialyzed, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. GST-RGD(6) fusion protein could specifically inhibit adhesion and migration of VSMC stimulated by osteopontin, which could be considered as a basis on producing small peptides containing RGD sequences for inhibition of VSMC adhesion and migration.

CONCLUSIONThe six copies of osteopontin RGD peptide were successfully expressed in E. coli DH5alpha. The purified GST-RGD fusion protein could inhibit the adhesion and migration of VSMC stimulated by osteopontin.

Animals ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Oligopeptides ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology

BACKGROUNDHuman sulfatase-1 (Hsulf-1) is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans (HSPGs), altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation, cell motility, and apoptosis. We investigated the role of combined cancer gene therapy with Hsulf-1 and cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene on a hepatocellular carcinoma (HCC) cell line, HepG2, in vitro and in vivo.

METHODSReverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC. Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy. We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.

RESULTSA significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system. A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed. Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene. In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone, and the combined treatment resulted in a significant increase in survival.

CONCLUSIONSHsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.

Animals ; Apoptosis ; drug effects ; genetics ; Carcinoma, Hepatocellular ; enzymology ; metabolism ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; genetics ; Cytosine Deaminase ; genetics ; metabolism ; Flucytosine ; pharmacology ; Genetic Therapy ; Hep G2 Cells ; Humans ; Liver Neoplasms ; enzymology ; metabolism ; Sulfatases ; genetics ; metabolism

BACKGROUNDTopical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain. The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells. The expression of c-KIT mRNA and protein of human A375 cells were also investigated.

METHODSThe cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups (10, 10(2), 10(3) and 10(4) nmol/L). The cell proliferation was measured with Cell Counting Kit-8 assays. Melanin content was measured with NaOH method. Transwell migration assay was used to measure cell migration. The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.

RESULTSThe cell proliferation of the 10(3) and 10(4) nmol/L tacrolimus groups were significantly lower (0.666 ± 0.062 and 0.496 ± 0.038) as compared with the control (0.841 ± 0.110, P < 0.05). The mean melanin content in all groups treated with different concentration of tacrolimus (10, 10(2), 10(3), 10(4) nmol/L) increased compared with the control group (P < 0.05). Dose-dependent increase in cell migration were seen in all tacrolimus-treated groups (P < 0.01). The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus (10(3) and 10(4) nmol/L) had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control (P < 0.05).

CONCLUSIONSAlthough tacrolimus had no effects on cell proliferation on A375 human melanoma cells, it could increase the melanin content and cell migration. The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control. Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.

Cell Line, Tumor ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Humans ; Immunohistochemistry ; Melanins ; metabolism ; Melanocytes ; cytology ; drug effects ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics ; Tacrolimus ; pharmacology

To investigate the expression of microRNA (miRNA, miR) let-7e-3p in different cervical lesions and its clinical significance.The expression of miR-let-7e-3p in the tissues of normal cervix (=26), high-grade squamous intraepithelial lesion (HSIL) (=37), and cervix carcinoma (=101) were detected by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The correlation of miR-let-7e-3p expression with the clinicopathological parameters of patients with cervical cancer was analyzed. miR-let-7e-3p mimic was transfected into cervical carcinoma Siha cells. The cell cycle and apoptosis were determined by flow cytometry; cell proliferation was determined by CCK-8 kit; and the migration and invasion of cells were determined by Transwell assay.The relative expression levels of miR-let-7e-3p in normal cervix, HSIL, and cervical carcinoma were 1.45±0.24, 0.79±0.05 and 0.46±0.04, respectively (all<0.05). After transfection with miR-let-7e-3p mimic, the S-phase fraction and apoptosis rate of Siha cells were increased significantly compared with control group[(29.76±6.6)% vs (13.38±1.3)%,<0.05; (5.98±1.38)% vs (3.53±0.79)%,<0.05, respectively]. OD of transfected Siha cells at 48, 72 and 96 h were 0.57±0.11,0.65±0.04 and 0.84±0.14, which were significantly lower than those of untransfected Siha cells (0.74±0.05, 0.93±0.10 and 1.47±0.14, all<0.05). The migration and invasion abilities of transfected Siha cells were not significantly changed (all>0.05).The expression of miR-let-7e-3p is down-regulated in cervical neoplasms, which is associated with cell cycle arrest and proliferation inhibition of cervical cancer cells.
Apoptosis ; drug effects ; genetics ; Carcinoma ; chemistry ; genetics ; Cell Cycle ; drug effects ; genetics ; Cell Line, Tumor ; chemistry ; drug effects ; physiology ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; genetics ; Cervical Intraepithelial Neoplasia ; chemistry ; genetics ; physiopathology ; Down-Regulation ; physiology ; Female ; Humans ; MicroRNAs ; analysis ; pharmacology ; Neoplasm Invasiveness ; genetics ; physiopathology ; Neoplastic Processes ; Real-Time Polymerase Chain Reaction ; Transfection ; Uterine Cervical Neoplasms ; chemistry ; genetics ; physiopathology

OBJECTIVETo investigate the effects of 8-methoxypsoralen on human melanocytes [Ca(2+)]i and cytoskeleton actin organization in vitro.

METHODSHuman melanocytes were obtained from normal foreskins. Laser confocal microscope was employed to measure [Ca(2+)]i and rhodamine-conjugated phalloidin was used to visualize the cytoskeleton actin.

RESULTS8-methoxypsoralen increased [Ca(2+)]i and induced organization of actin stress fiber cytoskeleton.

CONCLUSION8-methoxypsoralen might influence the migration of melanocytes by increasing the intracellular free Ca(2+) concentration and cytoskeleton actin reorganization.

Actins ; biosynthesis ; genetics ; Calcium ; metabolism ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeletal Proteins ; biosynthesis ; genetics ; Humans ; Melanocytes ; cytology ; drug effects ; metabolism ; Methoxsalen ; pharmacology ; Skin ; cytology

Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; Neoplasms ; Phosphorylation

OBJECTIVETo study the angiogenesis modulation mechanism of Xuefu Zhuyu Decoction () on the endothelial cell line ECV304.

METHODSECV304 cells were treated with 2.5% Xuefu Zhuyu Decoction-containing serum (XFZYD-CS) for 24 h, 48 h or 72 h. Thiazolyl blue tetrazolium bromide (MTT), fluorescence activating cell sorter (FACS), migration, adhesion and in vitro tube formation assays were conducted to confirm an angiogenesis effect of XFZYD at 3 time points. An analysis of angiogenesis regulator profiles was performed at 3 times with real-time polymerase chain reaction (RT-PCR) superarray.

RESULTSAt 48 h, XFZYD-CS induced ECV304 significantly improved cell viability, number in S phase, migration, adhesion and tube formation. At 24 h and 72 h, only cell migration was elevated. Microarray results showed that the expression of 27 angiogenesis-related genes was changed.

CONCLUSIONXFZYD-CS treatment induced angiogenesis on ECV304 cells with significant cellcular changes occurring at 48 h and genetic changes as early as 24 h.

Angiogenesis Inducing Agents ; pharmacology ; Cell Adhesion ; drug effects ; genetics ; Cell Line ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Neovascularization, Physiologic ; drug effects ; genetics ; Oligonucleotide Array Sequence Analysis ; methods