Phlorofucofuroeckol A from Edible Brown Alga Ecklonia Cava Enhances Osteoblastogenesis in Bone Marrow-Derived Human Mesenchymal Stem Cells.

The deterioration of bone formation is a leading cause of age-related bone disorders. Lack of bone formation is induced by decreased osteoblastogenesis. In this study, osteoblastogenesis promoting effects of algal phlorotannin, phlorofucofuroeckol A (PFF-A), were evaluated. PFF-A was isolated from brown alga Ecklonia cava. The ability of PFF-A to enhance osteoblast differentiation was observed in murine pre-osteoblast cell line MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (huBM-MSCs). Proliferation and alkaline phosphatase (ALP) activity of osteoblasts during differentiation was assayed following PFF-A treatment along extracellular mineralization. In addition, effect of PFF-A on osteoblast maturation pathways such as Runx2 and Smads was analyzed. Treatment of PFF-A was able to enhance the proliferation of differentiating osteoblasts. Also, ALP activity was observed to be increased. Osteoblasts showed increased extracellular mineralization, observed by Alizarin Red staining, following PFF-A treatment. In addition, expression levels of critical proteins in osteoblastogenesis such as ALP, bone morphogenetic protein-2 (BMP-2), osteocalcin and ß-catenin were stimulated after the introduction of PFF-A. In conclusion, PFF-A was suggested to be a potential natural product with osteoblastogenesis enhancing effects which can be utilized against bone-remodeling imbalances and osteoporosis-related complications.

High Glucose with Insulin Induces Cell Cycle Progression and Activation of Oncogenic Signaling of Bladder Epithelial Cells Cotreated with Metformin and Pioglitazone.

Metformin and pioglitazone are two commonly prescribed oral hypoglycemic agents for diabetes. Recent evidence suggests that these drugs may contribute to bladder cancer. This study investigated molecular mechanism underlying effects of metformin and pioglitazone in bladder epithelial carcinogenesis in type 2 diabetes. The cells derived from human bladder epithelial cells (HBlEpCs) were treated with metformin or pioglitazone with high glucose and insulin. Cell viability and proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and a bromodeoxyuridine incorporation assay, respectively, while cell cycle regulatory factors and oncogene expression were analyzed using western blotting. Metformin or pioglitazone suppressed cell viability concentration and time dependently, which was reversed by exposure to high glucose with or without insulin. Prolonged exposure to high glucose and insulin enhanced cyclin D, cyclin-dependent kinase 4 (Cdk4), and Cdk2 expression and suppressed cyclin-dependent kinase inhibitors p21 and p15/16 in HBlEpC cotreated with pioglitazone and metformin. Levels of tumor suppressor proteins p53 and cav-1 were downregulated while those of the oncogenic protein as c-Myc were upregulated under high glucose and insulin supplementation in HBlEpC cotreated with pioglitazone and metformin. Prolonged exposure to high glucose with or without insulin downregulated B cell lymphoma 2-associated X (Bax) and failed to enhance the expression of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) in drug-treated cells. These results suggest that hyperglycemic and insulinemic conditions promote cell cycle progression and oncogenic signaling in drug-treated bladder epithelial cells and uncontrolled hyperglycemia and hyperinsulinemia are probably greater cancer risk factors than diabetes drugs.

Anti-inflammatory effect of hydroxyproline-GQDGLAGPK in desiccation stress-induced experimental dry eye mouse.

The purpose of this study has been the investigation of the effect of novel peptide hydroxyproline-GQDGLAGPK (Hyp-GQDGLAGPK) in desiccation stress-induced dry eye mouse model and compared medicines for dry eye disease including cyclosporine, diquafosol and sodium hyaluronate. Seventy eight NOD.B10.H2 b mice were injected with scopolamine and exposed to an air draft for 10 days, and then the mice were treated with normal saline (n = 13), 1% Hyp-GQDGLAGPK (n = 13), 0.05% cyclosporine (n = 13), 3% diquafosol (n = 13), and 0.1% hyaluronate (n = 13) for 10 days. Thirteen mice were used for histopathologic analysis at DS 10d. The desiccation stress significantly decreased tear production, but the topical treatment of Hyp-GQDGLAGPK recovered to the baseline levels, which was similar to cyclosporine and diquafosol. In addition, Hyp-GQDGLAGPK improved facilitating epithelium stabilization including the corneal irregularity score, fluorescein score and detachment of the corneal epithelium. These improvements in stabilization of the corneal epithelium was superior to that in the cyclosporine and sodium hyaluronate groups. Furthermore, desiccation stress markedly induced expression of autoimmune inflammation-related factors in the lacrimal glands, but it was significantly suppressed by Hyp-GQDGLAGPK treatment. Overall, we found that novel peptide Hyp-GQDGLAGPK has multi-functional effects such as stabilizing the tear film and inhibiting inflammation.

Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway.

OBJECTIVES: Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. MATERIALS AND METHODS: In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. After treatment with either single or combination, cell viability and cell death were determined with WST-1 assay, trypan blue exclusion method, colony forming assay, annexin-V staining assay and western blot assay. The content of ROS was evaluated by FACS analysis using H2DCF-staining method. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. RESULTS: Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. CONCLUSION: Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.

Dioxinodehydroeckol protects human keratinocyte cells from UVB-induced apoptosis modulated by related genes Bax/Bcl-2 and caspase pathway.

Although ultraviolet B (UVB) has a low level of skin penetration, it readily results in epidermal sunburn of keratinocytes that are destined to apoptosis after sun expose, and leads to DNA damage. Dioxinodehydroeckol (DHE), a phlorotannin from Ecklonia cava has been explored for its preventive activity against UVB-induced apoptosis in human keratinocyte (HaCaT) cells; however, the protective effects of treatment with low doses of DHE on UVB-damaged cells post-UVB exposure and their underlying mechanisms still remain unclear. The HaCaT cells were exposed to 20 mJcm(-2) of UVB irradiation which is the minimal erythema dose (MED) for individuals to be able to tan, and the expression levels of Bax/Bcl-2 and caspase-3,-8, -9 which are associated genes with apoptosis were investigated when we either treated cells with DHE doses after UVB irradiation or exposed them to UVB only. Our results suggest insight into proposed mechanistic pathway of protective activity of DHE on the HaCaT cells from UVB-induced apoptosis, indicating the benefit of DHE as a repair agent for skin damage against UVB.

Evaluation of Effective MMP Inhibitors from Eight Different Brown Algae in Human Fibrosarcoma HT1080 Cells.

Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that have important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Marine plants are on the rise for their potential to provide natural products that exhibit remarkable health benefits. In this context, brown algae species have been of much interest in the pharmaceutical field with reported instances of isolation of bioactive compounds against tumor growth and MMP activity. In this study, eight different brown algae species were harvested, and their extracts were compared in regard to their anti-MMP effects. According to gelatin zymography results, Ecklonia cava, Ecklonia bicyclis, and Ishige okamurae showed higher inhibitory effects than the other samples on MMP-2 and -9 activity at the concentrations of 10, 50, and 100 µg/mL. However, only I. okamurae was able to regulate the MMP activity through the expression of MMP and tissue inhibitor of MMP observed by mRNA levels. Overall, brown algae species showed to be good sources for anti-MMP agents, while I. okamurae needs to be further studied for its potential to yield pharmaceutical molecules that can regulate MMP-activity through cellular pathways as well as enzymatic inhibition.

Radical scavenging and anti-inflammatory effects of the halophyte Spergularia marina Griseb.

As a part of ongoing research to develop antioxidant and anti-inflammatory nutraceuticals, an ethanolic extract of Spergularia marina Griseb. was tested for its ability to scavenge radicals and suppress inflammation. The extract was able to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide radicals, respectively, in cell-free environments as well as intracellular radicals in H2O2-stimulated mouse macrophages. Inflammation in mouse macrophages induced by lipopolysaccharides was suppressed by S. marina according to the measurement of nitric oxide generation and expression of inflammatory cytokines, i.e. tumour necrosis factor-α (TNFα), interleukin (IL)-1ß, and IL-6. The anti-inflammatory effect of S. marina was substantiated by the finding that the expression of the inflammatory modulators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was significantly decreased. The chemical composition of S. marina was evaluated by FT-IR analysis of the extract indicating the presence of polyphenols and flavonoids. In conclusion, S. marina is suggested as a novel source for antioxidant and anti-inflammatory agents.

Photoprotective effect of libanoridin isolated from Corydalis heterocarpa on UVB stressed human keratinocyte cells.

Ultraviolet-B (UVB) irradiation acts primarily on the epidermal basal cell layer of the skin, inducing harmful biological effects. In this study, we have investigated the effect of libanoridin isolated from Corydalis heterocarpa against UVB-induced damage in human keratinocyte (HaCaT) cells and the molecular mechanism underlying those effects. Treatment with libanoridin inhibited the cell cytotoxicity and LDH induced by UVB exposure at 40 mJ/cm(2). Additionally, expression levels of type IV collagenases (MMP-2, MMP-9) were decreased by libanoridin. Furthermore, MMP tissue inhibitors were enhanced followed by treatment with libanoridin. Moreover, UVB-induced activation of phosphorylation of three MAPKs such as JNK, ERK, p38 and AP-1 transcription factor were decreased by treatment with libanoridin. Our present study demonstrates that libanoridin has the abilities to inhibit UVB-induced cellular damage via ASK1-MAPK and AP-1 signalling pathways. Therefore, libanoridin may be used as an effective natural compound to prevent skin damage due to UVB exposure.

Anti-inflammatory action of sulfated glucosamine on cytokine regulation in LPS-activated PMA-differentiated THP-1 macrophages.

OBJECTIVE: The aim of this study was to evaluate the effect of sulfated glucosamine (SGlc) on the regulation of inflammatory cytokines and profiles involved in immunological activities. Changes in the inflammatory profiles of lipopolysaccharide (LPS)-activated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage models were investigated following SGlc treatment. METHODS: Human THP-1 macrophages were used to evaluate anti-inflammatory profiles. The cytokine secretion levels were measured by enzyme-linked immunosorbent assay (ELISA). Effects of SGlc on the regulation of mRNA and protein levels were determined using RT-PCR and Western blot analysis. The effect of SGlc on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB protein was also determined by Western blot analysis. RESULTS: Treatment of THP-1 cells with SGlc inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6. In addition, SGlc suppressed the mRNA and protein expression levels of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, IL-1ß, IL-6, 5-lipoxygenase and cytoplasmic phospholipase A(2) in LPS-activated THP-1 macrophages. Furthermore, we confirmed that the LPS-activated transcriptions of MAPKs and NF-κB were inhibited by SGlc treatment in PMA-differentiated THP-1 macrophages. CONCLUSION: These results suggest that SGlc can be considered as a potential anti-inflammatory supplement.

1-(3',5'-dihydroxyphenoxy)-7-(2'',4'',6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin inhibits adipocyte differentiation of 3T3-L1 fibroblasts.

In this study, we isolated the phloroglucinol derivative, 1-(3',5'-dihydroxyphenoxy)-7-(2'',4'',6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (1), from Ecklonia cava and evaluated its potential inhibition on adipocyte differentiation in 3T3-L1 cells. Lipid accumulation along with the expression of several genes associated with adipogenesis and lipolysis was examined at the end of differentiation. Lipid accumulation level was examined by measuring triglyceride content and Oil-Red O staining. The expression levels of several genes and proteins were examined using reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot analysis. Compound 1 significantly reduced lipid accumulation and downregulated peroxisome proliferator-activated receptor-gamma, sterol regulatory element-binding protein 1c, and CCAAT/enhancer-binding proteins alpha in a dose-dependent manner. Moreover, the presence of compound 1 induced downregulation of adipogenic target genes such as fatty acid binding protein 4, fatty acid transport protein 1, fatty acid synthase, acyl-CoA synthetase 1, lipoprotein lipase, and leptin. According to the lipolytic response, compound 1 downregulated perilipin and hormone-sensitive lipase while upregulating tumor necrosis factor alpha. Therefore, these results suggest that compound 1 might decrease lipid accumulation during adipocyte differentiation by modulating adipogenesis and lipogenesis. Furthermore, compound 1 could be developed as a functional agent effective in improving obesity.