Y.-C. Chang

Chemoinhibitory effect of mulberry anthocyanins on melanoma metastasis involved in the Ras/PI3K pathway.

Anthocyanins richly exist in mulberry plants and have been well characterized to have various bioactive properties. However, the antimetastasis properties of mulberry anthocyanins (MACs) remain unclear. The objectives of this study were to investigate the inhibitory effects of MACs on the metastasis of B16-F1 cells under noncytotoxic concentrations. Further investigation revealed that the antimetastatic effect of MACs was also evident in a C57BL/6 mice model. First, MACs exhibited an inhibitory effect on the migration ability by wound healing assay and Boyden chamber assay. In the cancer cell metastasis process, matrix degrading proteinases are required. B16-F1 cells treated with MACs at various concentrations showed reduced extracellular matrix (ECM) proteinases including matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) by gelatin zymography assay. The results of the Western blotting assay demonstrated that the expression levels of Ras, phosphoinositide 3-kinase (PI3K), phospho-Akt, and nuclear factor kappa B (NF-kappaB) in the MACs-treated B16-F1 cells were reduced. Therefore, it was suggested that MACs could mediate B16-F1 cell metastasis by reduction of MMP-2 and MMP-9 activities involving the suppression of the Ras/PI3K signaling pathway. Besides, B16-F1 melanoma cells were also injected into the right groin of the C57BL/6 mice, and the mice were fed with MACs at the same time. The hematoxylin-eosin stain (H&E stain) and immunohistochemistry stain showed that the MACs inhibited the mtastasis of B16-F1 cells in vivo. Taken together, the findings proved the inhibitory effect of MACs on the growth and metastasis of B16-F1 cells. These results indicated that MACs might be offered for future application as an antimetastatic agent.

Mulberry Anthocyanins Inhibit Oleic Acid Induced Lipid Accumulation by Reduction of Lipogenesis and Promotion of Hepatic Lipid Clearance

Mulberry (Morus alba L.) has been considered to possess different benefits such as protecting liver; improving fever, urine excretion disorder, hypertension, and diabetic syndrome; and preventing cardiovascular diseases. Recently, mounting evidence has shown that mulberry anthocyanin extract (MAE) is beneficial to hyperlipidemia; however, the mechanisms remain unclear. The present study was aimed to investigate the protective effects of MAE on hepatocyte cultured with high fatty acid and the underlying mechanisms. By using human hepatoma cell HepG2 as cell model, the results showed that MAE suppressed fatty acid synthesis and enhanced fatty acid oxidation, contributing to amelioration of lipid accumulation induced by oleic acid (OA). Moreover, MAE also inhibited acetyl coenzyme A carboxylase (ACC) activities by stimulating adenosine monophosphate-activated protein kinase (AMPK). MAE attenuated the expression of sterol regulatory element-binding protein-1 (SREBP-1) and its target molecules, such as fatty acid synthase (FAS). Similar results were also found in the expressions of enzymes involved in triglyceride and cholesterol biosyntheses including glycerol-3-phosphate acyltransferase (GPAT), 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCoR), adipocyte-specific fatty acid binding protein (A-FABP), and SREBP-2. In contrast, the lipolytic enzyme expressions of peroxisome proliferator activated receptor α (PPARα) and carnitinepalmitol- transferase-1 (CPT1) were increased. This study suggests the hypolipidemic effects of MAE occur via phosphorylation of AMPK and inhibition of lipid biosynthesis and stimulation of lipolysis. Therefore, the mulberry anthocyanins may actively prevent nonalcoholic fatty liver disease.

Platelet-rich fibrin increases cell attachment, proliferation and collagen-related protein expression of human osteoblasts

BACKGROUND Platelet-rich fibrin (PRF) prepared by Choukrouns technique is derived from an autogenous preparation of concentrated platelets without any manipulation. PRF was found to increase osteoblast growth and proliferation. However, the underlying mechanisms are not yet completely understood. This study aimed to determine the effects of PRF on cell attachment, proliferation, phosphorylated Akt, heat shock protein 47 (HSP47) and lysyl oxidase (LOX) expression on human osteoblasts. METHODS Blood collection was carried out from 10 healthy volunteers. Cell attachment and proliferation were measured by colorimetric assay with WST-1 and alamar blue in human osteoblast cell line U2OS cells, respectively. Western blot was employed to evaluate the expression of p-Akt, HSP47 and LOX. RESULTS PRF alone was found to stimulate U2OS cell attachment compared with untreated controls (p < 0.05). PRF was found to increase osteoblast proliferation during a 5-day incubation period (p < 0.05). PRF was found to increase Akt phosphorylation in a time-dependent manner (p < 0.05). Collagen-related proteins HSP47 and LOX were significantly elevated by stimulation with PRF compared with untreated controls (p < 0.05). CONCLUSIONS It is suggested that PRF is capable of increasing osteoblast attachment, proliferation and simultaneously upregulating collagen-related protein production. These actions in combination would effectively promote bone regeneration.

Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels

AIM To evaluate the mechanisms of cytotoxicity of chlorhexidine (CHX) in human osteoblastic cells in vitro. METHODOLOGY Cytotoxicity, cell proliferation and collagen synthesis assays were performed to elucidate the toxic effects of CHX on the human osteoblastic cell line U2OS. To determine whether glutathione (GSH) levels were important in the cytotoxicity of CHX, cells were pre-treated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. RESULTS CHX demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P < 0.05). The 50% inhibition concentration of CHX was approximately 0.005%. CHX also inhibited cell proliferation and collagen synthesis (P < 0.05). The addition of OTZ acted as a protective effect on the CHX-induced cytotoxicity (P < 0.05). In contrast, the addition of BSO enhanced the CHX-induced cytotoxicity (P < 0.05). CONCLUSIONS The levels of CHX tested inhibited cell growth, proliferation and collagen synthesis on U2OS cells. CHX has significant potential for periapical toxicity. GSH depletion might be one of the mechanisms underlying CHX cytotoxicity.

The upregulation of cystatin C in human gingival fibroblasts stimulated with cyclosporine A

BACKGROUND AND OBJECTIVE Cystatin C is a 13 kDa non-glycosylated, basic protein belonging to the cystatin family. It is consistently and dramatically upregulated in a variety of fibrotic diseases. However, little is known about the correlation between cystatin C and cyclosporine A-induced gingival overgrowth. The aim of this study was to compare cystatin C expression in normal, healthy gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanism that may result in cystatin C expression. MATERIAL AND METHODS Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Three human gingival fibroblast (HGF) strains were established from crown-lengthening surgery. The reverse transcriptase-polymerase chain reaction was used to investigate the effects on HGFs exposed to cyclosporine A. In addition, predominant periodontal pathogens (Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis) and proinflammatory cytokines (interleukin-1alpha and tumor necrosis factor-alpha) were added to seek the possible regulatory mechanisms of cystatin C expression. RESULTS The cystatin C staining in gingival tissue was stronger in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Intensive staining for cystatin C expression was observed mainly in the cytoplasm of fibroblasts, epithelial cells and inflammatory cells. Moreover, cystatin C expression was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). A concentration of 200 ng/mL cyclosporine A was found to increase cystatin C expression in HGFs in a time-dependent manner (p < 0.05). The addition of periodontal pathogens and proinflammatory cytokines significantly increased the expression of cystatin C compared with cyclosporine A alone (p < 0.05). CONCLUSION The increased ability of protein accumulation by cystatin C is one of several factors mediating cyclosporine A-induced gingival overgrowth. In addition, cyclosporine A may predispose to gingival overgrowth in inflammatory environments.

Up-regulation of osteolytic mediators in human osteosarcoma cells stimulated with nicotine

BACKGROUND AND OBJECTIVE Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-kappaB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine. MATERIAL AND METHODS Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcription-polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively. RESULTS Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells (p < 0.05). The gelatin zymograms revealed that matrix metalloproteinase (MMP)-2 and MMP-9 were secreted by U2OS cells. The secretion of MMP-2 and MMP-9 occurred in a dose-dependent manner that was dependent on the concentration of nicotine (p < 0.05). Casein zymography exhibited a caseinolytic band with a molecular weight of 70 kDa, indicative of the presence of tissue-type plasminogen activator. Tissue-type plasminogen activator was also found to be up-regulated by nicotine in a dose-dependent manner (p < 0.05). CONCLUSION Taken together, the results of the present study indicated that smoking modulation of bone destruction in periodontal disease may involve various osteolytic mediators, such as interleukin-1, interleukin-8, RANKL, MMP-2, MMP-9, and tissue-type plasminogen activator.

The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate

AIM To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7. METHODOLOGY Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferronis t-test for multi-group comparisons test. RESULTS BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P < 0.05). BisGMA was found to induce two modes of cell death. The mode of cell death changed from apoptosis to necrosis as the concentrations of BisGMA elevated. Caspase-3, caspase-8 and caspase-9 activities were significantly induced by BisGMA in a dose-dependent manner (P < 0.05). Moreover, BisGMA exhibited genotoxicity via a dose-related increase in the numbers of micronucleus and DNA strand breaks (P < 0.05). CONCLUSIONS Cytotoxicity and genotoxicity induced by BisGMA are mediated by DNA damage and caspase activation.

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

OBJECTIVES Hypoxia inducible factor (HIF)-1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF-1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF-1α expression in normal human oral epithelium and areca quid chewing-associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF-1α expression. METHODS Twenty-five OSCC from areca quid chewing-associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N-acetyl-l-cysteine (NAC), AP-1 inhibitor curcumin, extracellular signal-regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. RESULTS Hypoxia inducible factor-1α expression was significantly higher in OSCC specimens than normal specimen (P<0.05). Arecoline was found to elevate HIF-1α expression in a dose- and time-dependent manner (P<0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline-induced HIF-1α expression (P<0.05). CONCLUSIONS Hypoxia inducible factor-1α expression is significantly upregulated in areca quid chewing-associated OSCC and HIF-1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.

The upregulation of tumour necrosis factor-α and surface antigens expression on macrophages by bisphenol A-glycidyl-methacrylate.

AIM To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7. METHODOLOGY Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferronis t-test for multigroup comparisons. RESULTS BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P < 0.05) during 2-h incubation period. BisGMA was found to increase TNF-α secretion in a dose-dependent manner (P < 0.05). In addition, CD11, CD14, CD45, CD54, CD40, CD80, and MHC II were significantly stimulated by BisGMA in a dose-dependent manner (P < 0.05). However, MHC I expression was not affected by BisGMA (P > 0.05). CONCLUSIONS Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)(5)-GP, was produced by acetylation of a glycoside, isolated from an extract of Gardenia fructus. Previously, we have reported that C6 glioma cells could be inhibited in culturing as well as in bearing rats by treating with (Ac)(5)-GP. In this study, the effect and action of (Ac)(5)-GP on inducing cell death was examined in rat C6 glioma cells. Treatment of C6 glioma cells with (Ac)(5)-GP caused cell death, chromatin condensation, and internucleosomal DNA ladder. Also, cell cycle arrest at G(0)/G(1) phase revealed that (Ac)(5)-GP-induced cell death appears to be mediated by apoptosis. In addition, the results also showed that p53 and c-Myc increased due to treatment of (Ac)(5)-GP in a dose-response and time-dependent manner. Concomitant with the expression of p53 and c-Myc, decreased level of Bcl-2 and increased level of Bax protein were observed. These results suggest that cell death caused by (Ac)(5)-GP through apoptosis and cell cycle arrest at G(0)/G(1) may be associated with the induction of p53, c-Myc and may be mediated with apoptosis-related Bcl-2 family proteins.