PURPOSE: To investigate the role of nitric oxide (NO) on the proliferative and migratory effects of triamcinolone acetonide (TA) in retinal pigment epithelial cells. METHODS: After exposure to 10 nM, 1 micrometer, or 100 micrometer TA for four days, with or without co-exposure of antioxidant N-acetylcyteine, the proliferation and nitrite production of ARPE19 cells were assessed with MTT and Griess assays, respectively. Additionally, a cell migration assay was performed. RESULTS: Cellular survival increased after exposure to TA at low concentration but decreased at high concentration. TA decreased the production of NO and cellular migration significantly, and these effects were abolished by N-acetylcysteine. CONCLUSIONS: TA showed a biphasic response on the proliferation and decreased cellular migration in ARPE19 cells, which may be mediated by nitric oxide.
Cell Migration Assays ; Nitric Oxide ; Retinaldehyde ; Triamcinolone ; Triamcinolone Acetonide

PURPOSE: A composite of aluminum and vanadium (Ti-6Al-4V) is one of the most common compositions of titanium-based alloys. Unfortunately, vanadium has been found to cause adverse reactions. We evaluated the effects of vanadium containing titanium alloy (Ti-6Al-4V) on an osteoblast-like cell line (SaOS-2). MATERIALS AND METHODS: We studied the biologic and morphologic responses of SaOS-2 cell to Ti alloy with grit blasting and Ti coated Ti alloy with grit blasting. We performed energy-dispersive x-ray spectroscopy (EDS) and scanning electron microscopy (SEM) investigations and performed a cell proliferation assay, ALP activity, and cell migration assay of SaOS-2 cells. RESULTS: The morphologic assessment of cells through SEM showed that the two surfaces were covered with similar amounts of small slender osteoblast like cells. The amount of proliferation, ALP activity and the migration extent of SaOS-2 cells on the surfaces of each group were not statistically different. CONCLUSION: We used a grit-blasted Ti-coated Ti alloy, coated using electron beam deposition, and a grit-blasted Ti alloy to evaluate the toxicity of Ti-6Al-4V on SaOS-2 cell. Compared with pure titanium, the vanadium-containing Ti-alloy did not show an adverse effect on SaOS-2 cells.
Alloys ; Aluminum ; Cell Line ; Cell Migration Assays ; Cell Proliferation ; Electrons ; Microscopy, Electron, Scanning ; Osteoblasts ; Spectrum Analysis ; Titanium ; Vanadium

OBJECTIVE: Notch is known as a transmembranous receptor family with four homologous forms - Notch 1, Notch 2, Notch 3, and Notch 4 and related to cell fate regulation and angiogenesis. The purpose is to investigate the effect of follicular stimulating hormone (FSH) on the Notch 1 expression and proliferation in ovarian cancer cells. METHODS: Human ovarian cancer cell line, SK-OV-3 and FSH were used. XTT cell proliferation and cell migration assay were carried out with FSH 100 mIU/mL and Notch 1 siRNA. Western blots and reverse transcriptase-polymerase chain reactions (RT-PCR) were carried out to determine the expression level of the Notch 1 protein and mRNA with FSH treatment in 0, 1, 5, 10, 100, 200, 300 mIU/mL concentrations. Immunofluorescent (IF) stains were performed in SK-OV-3 cell cultures with FSH 100 mIU/mL. Student-t tests were used in statistical analyses. RESULTS: The SK-OV-3 have Notch 1 receptors in their natural status. FSH stimulated SK-OV-3 cells in XTT cell proliferation and cell migration assays and notch 1 siRNA inhibited. The expression level of Notch 1 protein and mRNA were increased in a dose dependent pattern according to FSH concentrations compared to untreated cells. IF stains also showed brighter Notch1 expressions in the FSH treated cells compared to the control cells. CONCLUSION: FSH enhances proliferation & migration and Notch 1 signaling in SK-OV-3 cells. The Notch signaling probably supports one of the cell proliferating mechanisms of FSH in ovarian cancer cells.
Blotting, Western ; Cell Culture Techniques ; Cell Line ; Cell Migration Assays ; Cell Proliferation ; Coloring Agents ; Humans ; Ovarian Neoplasms ; RNA, Messenger ; RNA, Small Interfering

BACKGROUND: We investigate the influence of cell surface adhesion receptor integrin alphavbeta3, alpha5beta1 contributes to proliferation and migration of tumor cell in osteosarcoma for carves out a new treatment model by regulation of integrin roles in human osteosarcoma. MATERIALS AND METHODS: We performed proliferation assay, total 11 cell lines including 7 osteosarcoma cell lines established from patients and 4 osteosarcoma standard cell lines. Murine monoclonal anti-alpha5beta1 and anti-alphavbeta3 (Chemicon International Inc. Temecula, CA) were used for growth inhibition assays. We also performed cell motility assay by using the Boyden chamber to evaluate the effect of integrin mediated cell migration. We used the HOS standard osteosarcoma cell lines and each separates contained serum free media with mouse IgG1 negative control antibody, anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. RESULTS: Proliferation of cells decreased significantly in 10 out of 11 cell lines when blocking with alphavbeta3 or alpha5beta1 respectively. Blocking with anti-alphavbeta3 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 7 cell lines showed significantly more decrease of proliferation with anti-alphavbeta3 antibody than with anti-alpha5beta1antibody. Blocking with anti-alpha5beta1 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 3 cell lines showed significantly more decrease of proliferation with anti-alpha5beta1 antibody than with anti-alphavbeta3 antibody. Including statistically not significant 2 cell lines the growth inhibition of osteosarcoma cell lines was more obvious (10 out of 11) in blocking with anti-alphavbeta3 antibody. The migration of cells was significantly decreased when blocked with anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. CONCLUSION: Under the based on the integrin alphavbeta3, alpha5 beta1 are central role on proliferation and migration of osteosarcoma cells, we could be more approach to new therapeutic endeavors with antibody to integrin alphavbeta3, alpha5beta1 molecular target of osteosarcoma.
Animals ; Cell Line ; Cell Migration Assays ; Cell Movement ; Cell Proliferation* ; Culture Media, Serum-Free ; Humans* ; Immunoglobulin G ; Integrin alphaVbeta3* ; Mice ; Osteosarcoma*

PURPOSE: Burn injury cause pruritis, pain, psychological and functional sequelae. The one of burn injury sequelae is the hypertrophic scar. It is difficult to control devastating fibrotic condition for hypertrophic scar. The objective of this study was to investigated the therapeutic effect on burn hypertrophic scar and wound healing for sequelae of burn injury by Porcine placenta extract (PPE). METHODS: To investigate the effect of PPE, we performed in vitro cell cytotoxity test (MTT assay), antioxidant activity assay (SOD like activity), melanin content assay, cell migration asssay and RT-PCR. RESULTS: As a result of cell cytotoxity test (MTT assay), PPE showed above 80% cell viability. From Antioxidant activity assay (SOD like activity), this effect was similar to vitamin C. In the melanin content assay, melanin synthesis was inhibited 23% on PPE treatment than control. PPE enhanced cell migration on human fibroblast and decreased the expression of hypertropic scar related gene (a-SMA and P311). CONCLUSION: Our data showed anti-oxidant effect, diminution of melanin and decrease of the expression of hypertropic scar related gene on the treatment of PPE. These results may provide the insight into the potential use of porcine placenta extract as support to control skin fibrosis related to burn hypertrophic scar and alternative medicine for burn sequelae.
Antioxidants ; Ascorbic Acid ; Burns ; Cell Migration Assays ; Cell Movement ; Cell Survival ; Cicatrix ; Cicatrix, Hypertrophic ; Complementary Therapies ; Fibroblasts ; Fibrosis ; Humans ; Melanins ; Placenta ; Pruritus ; Skin ; Wound Healing

BACKGROUND: Corticotropin-Releasing Hormone (CRH), an important regulator of stress response, has a potent immunoregulatory effect with the ability to promote the growth of various cancer through CRH receptor type 1 under stress. Although the metastasized cancers through cell migration are more aggressive than the primary cancers, little is known about the effect of CRH on cell migration. Gastric cancer is prone to metastasize to other tissues and it is reported that gastric cancer is response to various stresses such as oxidative stress. Herein, we studied the relationship between CRH and gastric cancer cell migration. METHODS: We used gastric cancer cell line, MKN-28 and tested the CRH receptor type 1 expression on MKN-28 by RT-PCR. To examine the change in the ability of migration by CRH in MKN-28, cells were incubated with CRH and then migration ability was measured using a cell migration assay. RESULTS: We confirmed that CRH receptor type 1 was expressed in MKN-28 and HaCaT cells. The migration ability of MKN-28 cells was increased by CRH in a time-, dose- dependent manner. CONCLUSION: These data suggest that CRH increases migration ability in gastric cancer cell line and that CRH may be a critical regulator in the metastasis of gastric cancer cell.
Cell Line* ; Cell Migration Assays ; Cell Movement* ; Corticotropin-Releasing Hormone* ; Humans* ; Neoplasm Metastasis ; Oxidative Stress ; Receptors, Corticotropin-Releasing Hormone ; Stomach Neoplasms*

PURPOSE: Synuclein-gamma (SNCG), which was initially identified as breast cancer specific gene 1, is highly expressed in advanced breast cancers, but not in normal or benign breast tissue. This study aimed to evaluate the effects of SNCG siRNA-treatment on breast cancer cells and elucidate the associated mechanisms. METHODS: Vectors containing SNCG and negative control (NC) siRNAs were transfected into MDA-MB-231 cells; mRNA levels were determined by real-time polymerase chain reaction. Cell proliferation was evaluated using the MTT assay, cell migration was assessed by the Transwell assay, apoptosis and cell cycle analyses were conducted with the flow cytometer, and Western blot analysis was performed to determine the relative levels of AKT, ERK, p-AKT, and p-ERK expression. RESULTS: SNCG mRNA levels were significantly reduced in MDA-MB-231 cells transfected with SNCG siRNA. Our results indicate that in SNCG siRNA-treated cells, cell migration and proliferation decreased significantly, apoptosis was induced, and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA-treated groups, than in the control and NC groups. CONCLUSION: SNCG siRNA could decrease the migration and proliferation of breast cancer cells by downregulating the phosphorylation of AKT and ERK.
Apoptosis ; Blotting, Western ; Breast ; Breast Neoplasms ; Cell Cycle ; Cell Migration Assays ; Cell Movement* ; Cell Proliferation ; Extracellular Signal-Regulated MAP Kinases ; Phosphorylation* ; Proto-Oncogene Proteins c-akt ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering ; Synucleins*

BACKGROUND: Low temperature plasma (LTP) was recently shown to be potentially useful for biomedical applications such as bleeding cessation, cancer treatment, and wound healing, among others. Keratinocytes are a major cell type that migrates directionally into the wound bed, and their proliferation leads to complete wound closure during the cutaneous repair/regeneration process. However, the beneficial effects of LTP on human keratinocytes have not been well studied. Therefore, we investigated migration, growth factor production, and cytokine secretion in primary human keratinocytes after LTP treatment.METHODS: Primary cultured keratinocytes were obtained from human skin biopsies. Cell viability was measured with the EZ-Cytox cell viability assay, cell migration was evaluated by an in vitro wound healing assay, gene expression was analyzed by quantitative real-time polymerase chain reaction, and protein expression was measured by enzyme-linked immunosorbent assays and western blotting after LTP treatment.RESULTS: Cell migration, the secretion of several cytokines, and gene and protein levels of angiogenic growth factors increased in LTP-treated human keratinocytes without associated cell toxicity. LTP treatment also significantly induced the expression of hypoxia inducible factor-1α (HIF-1α), an upstream regulator of angiogenesis. Further, the inhibition of HIF-1α expression blocked the production of angiogenic growth factors induced by LTP in human keratinocytes.CONCLUSION: Our results suggest that LTP treatment is an effective approach to modulate wound healing-related molecules in epidermal keratinocytes and might promote angiogenesis, leading to improved wound healing.
Anoxia ; Biopsy ; Blotting, Western ; Cell Migration Assays ; Cell Movement ; Cell Survival ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hemorrhage ; Humans ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Keratinocytes ; Plasma ; Real-Time Polymerase Chain Reaction ; Skin ; Wound Healing ; Wounds and Injuries

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Antineoplastic Agents ; pharmacokinetics ; Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; metabolism ; Calcium-Binding Proteins ; genetics ; Cell Line, Tumor ; Cell Migration Assays ; Cell Migration Inhibition ; drug effects ; Cell Proliferation ; drug effects ; Computational Biology ; methods ; Cytoskeleton ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Flow Cytometry ; Gene Expression ; Humans ; Keratin-18 ; genetics ; Oxidation-Reduction ; Protein Biosynthesis ; drug effects ; Protein Transport ; Proteomics ; methods ; Transcription, Genetic ; drug effects ; Ubiquitin-Specific Proteases ; pharmacokinetics ; Vimentin ; genetics ; Xanthones ; pharmacokinetics

PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1–14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41–62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.
Animals ; Carrier Proteins/genetics/*metabolism ; Cell Line, Tumor ; Cell Migration Assays ; *CpG Islands ; *DNA Methylation ; Disease Progression ; Epigenesis, Genetic ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Ovarian Neoplasms/genetics/*metabolism/mortality/pathology ; Polymerase Chain Reaction ; Prognosis ; *Promoter Regions, Genetic ; RNA, Messenger/*metabolism ; Up-Regulation