Therapeutic Effect of Enzymatically Hydrolyzed Cervi Cornu Collagen NP-2007 and Potential for Application in Osteoarthritis Treatment.

Cervi cornu extracts have been used in traditional medicine for the treatment of various disorders, including osteoporosis. However, since it is not easy to separate the active ingredients, limited research has been conducted on their functional properties. In this study, we extracted the low-molecular-weight (843 Da) collagen NP-2007 from cervi cornu by enzyme hydrolyzation to enhance absorption and evaluated the therapeutic effect in monosodium iodoacetate-induced rat osteoarthritis (OA) model. NP-2007 was orally administered at 50, 100, and 200 mg/kg for 21 days. We showed that the production of matrix metalloproteinase-2, -3, and -9, decreased after NP-2007 treatment. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and prostaglandin E2 were also reduced after treatment of NP-2007. Furthermore, the administration of NP-2007 resulted in effective preservation of both the synovial membrane and knee cartilage and significantly decreased the transformation of fibrous tissue. We verified that the treatment of NP-2007 significantly reduced the production of nitric oxide and pro-inflammatory cytokines including TNF-α, IL-1ß, and IL-6 in lipopolysaccharides-stimulated RAW 264.7 cells by regulation of the NF-kB and MAPK signaling pathways. This study indicates that NP-2007 can alleviate symptoms of osteoarthritis and can be applied as a novel treatment for OA treatment.

Anti-Inflammatory Effect and Signaling Mechanism of Glycine max Hydrolyzed with Enzymes from Bacillus velezensis KMU01 in a Dextran-Sulfate-Sodium-Induced Colitis Mouse Model.

The purpose of this study was to investigate the effect that Glycine max hydrolyzed with enzymes from Bacillus velezensis KMU01 has on dextran-sulfate-sodium (DSS)-induced colitis in mice. Hydrolysis improves functional health through the bioconversion of raw materials and increase in intestinal absorption rate and antioxidants. Therefore, G. max was hydrolyzed in this study using a food-derived microorganism, and its anti-inflammatory effect was observed. Enzymatically hydrolyzed G. max (EHG) was orally administered once daily for four weeks before DSS treatment. Colitis was induced in mice through the consumption of 5% (w/v) DSS in drinking water for eight days. The results showed that EHG treatment significantly alleviated DSS-induced body weight loss and decreased the disease activity index and colon length. In addition, EHG markedly reduced tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 production, and increased that of IL-10. EHG improved DSS-induced histological changes and intestinal epithelial barrier integrity in mice. Moreover, we found that the abundance of 15 microorganisms changed significantly; that of Proteobacteria and Escherichia coli, which are upregulated in patients with Crohn's disease and ulcerative colitis, decreased after EHG treatment. These results suggest that EHG has a protective effect against DSS-induced colitis and is a potential candidate for colitis treatment.

Fermented Aloe arborescens Miller Leaf Extract Suppresses Acute Alcoholic Liver Injury via Antioxidant and Anti-Inflammatory Effects in C57BL/6J Mice.

This study confirmed the change in functional composition and alcohol-induced acute liver injury in Aloe arborescens after fermentation. An acute liver injury was induced by administration of ethanol (3 g/kg/day) to C57BL/6J mice for 5 days. A fermented A. arborescens Miller leaf (FAAL) extract was orally administered 30 minutes before ethanol treatment. After fermentation, the emodin content was approximately 13 times higher than that of the raw material. FAAL extract significantly attenuated ethanol-induced aspartate aminotransferase, alanine aminotransferase, and triglyceride increases in serum and liver tissue. Histological analysis revealed that FAAL extract inhibits inflammatory cell infiltration and fat accumulation in liver tissues. The cytochrome P450 2E1, superoxide dismutase, and glutathione (GSH), which involved in alcohol-induced oxidative stress, were effectively regulated by FAAL extract in serum and liver tissues, except for GSH. FAAL also maintained the antioxidant defense system by upregulating heme oxygenase 1 and nuclear factor erythroid 2-related factor 2 protein expression. In addition, FAAL extract inhibited the decrease in alcohol dehydrogenase and aldehyde dehydrogenase activity, which promoted alcohol metabolism and prevented the activation of inflammatory response. Our results suggest that FAAL could be used as a potential therapeutic agent for ethanol-induced acute liver injury.

Anti-inflammatory effect and signaling mechanism of 8-shogaol and 10-shogaol in a dextran sodium sulfate-induced colitis mouse model.

Ethnopharmacological relevance: Ginger (Zingiber officinale Roscoe) has been used for food and applied in Ayurvedic medicine in India for thousands of years. With a reputation for strong anti-inflammatory properties, it has been used for to treat colds, migraines, nausea, arthritis, and high blood pressure in China and Southeast Asia. The physiological activity of ginger is attributed to its functional components, including gingerol and shogaol, and their derivatives. Aim of the study: We aimed to investigate the effects of 8- and 10-shogaol and their bioactive signaling mechanisms in a dextran sodium sulfate (DSS)-induced colitis mouse model. The anti-colitis efficacy of 6-, 8-, and 10-derivatives of gingerol and shogaol was comparatively analyzed. Materials and methods: Colitis was induced by providing mice with drinking water containing 5% DSS (w/v) for 8 days. The 6-, 8-, and 10-derivatives of gingerol and shogaol were orally administered for two weeks at a dose of 30 mg/kg. Changes in body weight and disease activity index were measured. The levels of pro-inflammatory cytokines, iNOS and COX-2, as well as the phosphorylation of NF-κB were analyzed using ELISA, PCR, or western blotting. Mucin expression and mRNA levels were measured using alcian blue staining and PCR, respectively. The tight-junction-associated proteins occludin and ZO-1 were assessed using immunohistological staining. Results: The 6-, 8-, and 10-derivatives of gingerol and shogaol exhibited anti-inflammatory effects by regulating NF-κB signaling. Among the compounds administered, 10-shogaol was the most effective against DSS-induced inflammation. Comparative analysis of the chemical structure showed that shogaol, a dehydrated analog of gingerol, was more effective. 6- and 10-shogaol showed similar effects on DSS-induced morphological changes in the colonic mucus layer, mucin expression, and tight junction proteins. Conclusions: 6-, 8-, and 10-Gingerol and 6-, 8-, and 10-shogaol significantly improved the clinical symptoms and intestinal epithelial barrier damage in DSS-induced colitis in mice. The derivatives effectively inhibited DSS-induced inflammation through the regulation of NF-κB signaling. Moreover, 10-shogaol showed the most potent anti-inflammatory effect among the six compounds used in this study. The results indicate that 8- and 10-shogaol, both main ingredients in ginger, may serve as therapeutic candidates for the treatment of colitis.

Downregulation of Matriptase Inhibits Porphyromonas gingivalis Lipopolysaccharide-Induced Matrix Metalloproteinase-1 and Proinflammatory Cytokines by Suppressing the TLR4/NF-κB Signaling Pathways in Human Gingival Fibroblasts.

Matriptases are cell surface proteolytic enzymes belonging to the type II transmembrane serine protease family that mediate inflammatory skin disorders and cancer progression. Matriptases may affect the development of periodontitis via protease-activated receptor-2 activity. However, the cellular mechanism by which matriptases are involved in periodontitis is unknown. In this study, we examined the antiperiodontitis effects of matriptase on Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS)-stimulated human gingival fibroblasts (HGFs). Matriptase small interfering RNA-transfected HGFs were treated with PG-LPS. The mRNA and protein levels of proinflammatory cytokines and matrix metalloproteinase 1 (MMP-1) were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA), respectively. Western blot analyses were performed to measure the levels of Toll-like receptor 4 (TLR4)/interleukin-1 (IL-1) receptor-associated kinase (IRAK)/transforming growth factor ß-activated kinase 1 (TAK1), p65, and p50 in PG-LPS-stimulated HGFs. Matriptase downregulation inhibited LPS-induced proinflammatory cytokine expression, including the expression of IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-Iß. Moreover, matriptase downregulation inhibited PG-LPS-stimulated MMP-1 expression. Additionally, we confirmed that the mechanism underlying the effects of matriptase downregulation involves the suppression of PG-LPS-induced IRAK1/TAK1 and NF-κB. These results suggest that downregulation of matriptase PG-LPS-induced MMP-1 and proinflammatory cytokine expression via TLR4-mediated IRAK1/TAK1 and NF-κB signaling pathways in HGFs.

Aurora kinase A induces migration and invasion by inducing epithelial-to-mesenchymal transition in colon cancer cells.

Aurora kinase is a family of serine/threonine kinases intimately associated with mitotic progression and the development of human cancers. Studies have shown that aurora kinases are important for the protein kinase C (PKC)-induced invasion of colon cancer cells. Recent studies have shown that aurora kinase A promotes distant metastasis by inducing epithelial-to-mesenchymal transition (EMT) in colon cancer cells. However, the role of aurora kinase A in colon cancer metastasis remains unclear. In this study, we investigated the effects of aurora kinase A on PKC-induced cell invasion, migration, and EMT in human SW480 colon cancer cells. Treatment with 12-O-tetradecanoylphorbol- 13-acetate (TPA) changed the expression levels of EMT markers, increasing α-SMA, vimentin, and MMP-9 expression and decreasing E-cadherin expression, with changes in cell morphology. TPA treatment induced EMT in a PKC-dependent manner. Moreover, the inhibition of aurora kinase A by siRNAs and inhibitors (reversine and VX-680) suppressed TPA-induced cell invasion, migration, and EMT in SW480 human colon cells. Inhibition of aurora kinase A blocked TPA-induced vimentin and MMP-9 expression, and decreased E-cadherin expression. Furthermore, the knockdown of aurora kinase A decreased the transcriptional activity of NF-κB and AP-1 in PKC-stimulated SW480 cells. These findings indicate that aurora kinase A induces migration and invasion by inducing EMT in SW480 colon cancer cells. To the best of our knowledge, this is the first study that showed aurora kinase A is a key molecule in PKC-induced metastasis in colon cancer cells. [BMB Reports 2022;55(2): 87-91].

Downregulation of matriptase suppresses the PAR­2/PLCγ2/PKC­mediated invasion and migration abilities of MCF­7 breast cancer cells.

Matriptases, members of the type II transmembrane serine protease family, are cell surface proteolytic enzymes that mediate tumor invasion and metastasis. Matriptase is highly expressed in breast cancer and is associated with poor patient outcome. However, the cellular mechanism by which matriptase mediates breast cancer invasion remains unknown. The present study aimed to determine the role of matriptase in the protein kinase C (PKC)­mediated metastasis of MCF­7 human breast cancer cells. Matriptase small interfering RNA­mediated knockdown significantly attenuated the 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced invasiveness and migration of MCF­7 cells, and inhibited the activation of phospholipase C γ2 (PLCγ2)/PKC/MAPK signaling pathways. Matriptase­knockdown also suppressed the expression of MMP­9 and inhibited the activation of NF­κB/activator protein­1 in MCF­7 cells. Additionally, GB83 [an inhibitor of protease­activated receptor­2 (PAR­2)] inhibited PKC­mediated MMP­9 expression and metastatic ability in MCF­7 cells. Furthermore, downregulation of matriptase suppressed TPA­induced MMP­9 expression and invasiveness via PAR­2/PLCγ2/PKC/MAPK activation. These findings shed light on the mechanism underlying the role of matriptase in MCF­7 cell invasion and migration ability, and suggest that matriptase modulation could be a promising therapeutic strategy for preventing breast cancer metastasis.

Evodiae fructus Extract Inhibits Interleukin-1ß-Induced MMP-1, MMP-3, and Inflammatory Cytokine Expression by Suppressing the Activation of MAPK and STAT-3 in Human Gingival Fibroblasts In Vitro.

Periodontitis is a Gram-negative bacterial infectious disease. Numerous inflammatory cytokines, including interleukin-1ß (IL-1ß), regulate periodontitis pathophysiology and cause periodontal tissue destruction. In human gingival fibroblasts (HGFs), IL-1ß stimulates the production of matrix metalloproteinases (MMPs) and proinflammatory cytokines via various mechanisms. Several transcription factors, such as signal transducer and activator of transcription 3 (STAT-3), activator protein 1 (AP-1), and nuclear factor-κB (NF-κB), regulate gene expression. Mitogen-activated protein kinases (MAPKs) regulate these transcription factors. However, the MAPK/STAT-3 activation signal in HGFs is unknown. We investigated the potential inhibitory effects of the extract of Evodiae fructus (EFE), the dried, ripe fruit of Evodia rutaecarpa, on MMP and proinflammatory cytokine expression in IL-1ß-stimulated HGFs. EFE inhibited the expression of MMP-1, MMP-3, and proinflammatory cytokines (TNF-α, IL-6, and IL-8) in IL-1ß-stimulated HGFs through the inhibition of IL-1ß-induced MAPK/STAT-3 activation. Also, these results suggest that the EFE may be a useful for the bioactive material for oral care.

Bruton's agammaglobulinemia tyrosine kinase (Btk) regulates TPA­induced breast cancer cell invasion via PLCγ2/PKCß/NF­κB/AP­1­dependent matrix metalloproteinase­9 activation.

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B­lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen­activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor­κB (NF­κB) activation, suggesting an anti­metastatic effect of BTK inhibitors on MCF­7 cells that leads to the downregulation of matrix metalloproteinase (MMP)­9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti­metastatic activity of BTK inhibitors was examined in MCF­7 cells focusing on MMP­9 expression in 12­O­tetradecanoylphorbol­13­acetate (TPA)­stimulated MCF­7 cells. The expression and activity of MMP­9 in MCF­7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX­774 (10 µM)] significantly attenuated TPA­induced cell invasion and migration in MCF­7 cells and inhibited the activation of the phospholipase Cγ2/PKCß signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP­9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA­induced MMP­9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF­κB/activator protein­1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP­9 expression in MCF­7 cells.

Aqueous extract of Chrysanthemum morifolium Ramat. inhibits RANKL-induced osteoclast differentiation by suppressing the c-fos/NFATc1 pathway.

OBJECTIVE: The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN: Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. RESULTS: CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. CONCLUSION: Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.

Immuno-enhancement effects of Platycodon grandiflorum extracts in splenocytes and a cyclophosphamide-induced immunosuppressed rat model.

BACKGROUND: Platycodon grandiflorum is a flowering plant that is used in traditional medicine for treating pulmonary and respiratory disorders. It exerts various pharmacological effects, including immunomodulatory and anti-cancer activities. The purpose of this study was to confirm the in vitro and in vivo immune-enhancing effects of P. grandiflorum extract (PGE) on splenocytes isolated from cyclophosphamide (CP)-induced immunosuppressed rats. METHODS: For in vitro analysis, splenocytes were treated with PGE at various doses along with CP. Cell viability was measured by a WST-1 assay, and NK cell activity and cytotoxic T lymphocyte (CTL) activity was also examined. In addition, immunoglobulin A (IgA), IgG, and cytokine levels were measured. For in vivo analysis, Sprague Dawley rats were treated with various doses of PGE along with CP. Complete blood count (CBC) was performed, and plasma levels of IgA, IgG, TNF-α, IFN-γ, IL-2, and IL-12 were quantified. Additionally, tissue damage was assessed through histological analyses of the thymus and spleen. RESULTS: PGE treatment enhanced cell viability and natural killer cell and cytotoxic T lymphocyte activity, and increased the production of CP-induced inflammatory cytokines (TNF-α, IFN-γ, IL-2, and IL-12) and immunoglobulins (IgG and IgA) in splenocytes. In addition, in CP-treated rats, PGE treatment induced the recovery of white blood cell, neutrophil, and lymphocyte counts, along with mid-range absolute counts, and increased the serum levels of inflammatory cytokines (TNF-α, IFN-γ, IL-2, and IL-12) and immunoglobulins (IgG and IgA). Moreover, PGE attenuated CP-induced spleen and thymic damage. CONCLUSIONS: Our results confirmed that PGE exerts an immune-enhancing effect both in vitro and in vivo, suggesting that PGE may have applications as a component of immunostimulatory agents or as an ingredient in functional foods.

Reversine inhibits MMP-3, IL-6 and IL-8 expression through suppression of ROS and JNK/AP-1 activation in interleukin-1ß-stimulated human gingival fibroblasts.

OBJECTIVE: Periodontitis is an inflammatory disease of the supporting tissue around teeth commonly caused by gram-negative bacterial infections. Interleukin (IL)-1ß, a cytokine involved in host immune and inflammatory responses, is known to induce the activation of various intracellular signaling pathways. One of these signaling mechanisms involves the regulation of gene expression by activation of transcription factors (AP-1 and NF-κB). These transcription factors are controlled by mitogen-activated protein kinases (MAPKs), which increase cytokine and matrix metalloproteinase (MMP) expression. We examined the preventive effects of reversine, a 2,6-disubstituted purine derivative, on cytokine and MMP-3 expression in human gingival fibroblasts (HGFs) stimulated with IL-lß. STUDY DESIGN: Western blot analyses were performed to verify the activities of MAPK, p65, p50, and c-Jun and the expression of MMPs in IL-1ß-stimulated HGFs. Cytokine and MMP-3 expression in IL-1ß-stimulated HGFs was measured by real-time quantitative polymerase chain reaction. RESULTS: Reversine decreased the IL-1ß-induced expression of proinflammatory cytokines (IL-6 and IL-8) and MMP-3 in HGFs. Furthermore, the mechanism underlying the effects of reversine involved the suppression of IL-1ß-stimulated MAPK activation and AP-1 activation. CONCLUSION: Reversine inhibits IL-1ß-induced MMP and cytokine expression via inhibition of MAPK/AP-1 activation and ROS generation. Therefore, we suggest that reversine may be an effective therapeutic candidate for preventing periodontitis.

Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism.

PURPOSE: Peroxisome proliferator-activated receptor γ (PPARγ) is involved in the pathology of numerous diseases including atherosclerosis, diabetes, obesity, and cancer. Matrix metalloproteinases (MMPs) play a significant role in tissue remodeling related to various processes such as morphogenesis, angiogenesis, tissue repair, invasion, and metastasis. We investigated the effects of PPARγ on MMP expression and invasion in breast cancer cells. METHODS: MCF-7 cells were cultured and then cell viability was monitored in an MTT assay. Western blotting, gelatin zymography, real-time polymerase chain reaction, and luciferase assays were performed to investigate the effect of the synthetic PPARγ ligand troglitazone on MMP expression. Transcription factor DNA binding was analyzed by electrophoretic mobility shift assay. A Matrigel invasion assay was used to assess the effects of troglitazone on MCF-7 cells. RESULTS: Troglitazone did not affect MCF-7 cell viability. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced MMP-9 expression and invasion in MCF-7 cell. However, these effects were decreased by troglitazone. TPA increased nuclear factor κB and activator protein-1 DNA binding, while troglitazone inhibited these effects. The selective PPARγ antagonist GW9662 reversed MMP-9 inhibition by troglitazone in TPA-treated MCF-7 cells. CONCLUSION: Troglitazone inhibited nuclear factor κB and activator protein-1-mediated MMP-9 expression and invasion of MCF-7 cells through a PPARγ-dependent mechanism.

Silencing of casein kinase 2 inhibits PKC­induced cell invasion by targeting MMP­9 in MCF­7 cells.

Casein kinase 2 (CK2) is a serine/threonine protein kinase that has been considered to represent an important factor in mammary tumorigenesis. Increased expression of matrix metalloproteinase­9 (MMP­9) via nuclear factor­κB (NF­κB) activation has been demonstrated to promote breast cancer cell invasion. In the present study, the involvement of CK2 in protein kinase C (PKC) induced cell invasion in MCF­7 breast cancer cells was investigated as well as the underlying molecular mechanisms. The mRNA and protein levels of MMP­9 in MCF­7 cells were investigated using reverse transcription­quantitative polymerase chain reaction, western blot analyses and a zymography assay. Cell invasiveness was investigated using a Matrigel invasion assay, and it was revealed that small interfering RNA specific for CK2 suppressed PKC induced cell invasion by regulating MMP­9 expression via activation of the p38 kinase/c­Jun N­terminal kinase/NF­κB pathway. In addition, it was demonstrated that CK2 inhibitors [apigenin (20 µM), emodin (20 µM) or 2­dimethylamino­4,5,6,7­tetrabromo­1H­benzimidazole (2 µM)] suppressed PKC induced cell invasion and MMP­9 expression. The results of the present study suggested that CK2 is an important factor involved in the induction of MCF­7 breast cancer cell invasion by PKC. Therefore, CK2 may represent novel candidates for therapy intended to inhibit invasion in breast cancer.

Reversine inhibits MMP-1 and MMP-3 expressions by suppressing of ROS/MAPK/AP-1 activation in UV-stimulated human keratinocytes and dermal fibroblasts.

UVB has been shown to stimulate the generation of reactive oxygen species (ROS), which subsequently results in the activation of various intracellular signalling pathways and transcription factors (AP-1, NF-κB). These transcription factors are regulated by MAPKs, which increase cytokine and MMP expression. We examined the preventive effects of reversine on MMP-1 and MMP-3 expressions in NHEKs and NHDFs exposed to UVB irradiation. Also, we confirmed that reversine decreased pro-inflammatory cytokine expression in NHEKs. The mechanism underlying the MMP inhibitory effects of reversine occurred via the suppression of UVB-induced ROS generation and MAPK/AP-1 activation. Therefore, reversine is an effective therapeutic candidate for preventing skin photoageing.

Crotonis Fructus Extract Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Expression of Matrix Metalloproteinase-9 via the Activator Protein-1 Pathway in MCF-7 Cells.

PURPOSE: Metastatic cancers spread from the primary site of origin to other parts of the body. Matrix metalloproteinase-9 (MMP-9) is essential in metastatic cancers owing to its major role in cancer cell invasion. Crotonis fructus (CF), the mature fruits of Croton tiglium L., have been used for the treatment of gastrointestinal disturbance in Asia. In this study, the effect of the ethanol extract of CF (CFE) on MMP-9 activity and the invasion of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells was examined. METHODS: The cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The expression of MMP-9 was examined by Western blotting, zymography, and real-time polymerase chain reaction. An electrophoretic mobility gel shift assay was performed to detect activator protein-1 (AP-1) DNA binding activity and cell invasiveness was measured by an in vitro Matrigel invasion assay. RESULTS: CFE significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, CFE attenuated the TPA-induced activation of AP-1. CONCLUSION: The results indicated that the inhibitory effects of CFE against TPA-induced MMP-9 expression and MCF-7 cell invasion were dependent on the protein kinase C δ/p38/c-Jun N-terminal kinase/AP-1 pathway. Therefore, CFE could restrict breast cancer invasiveness owing to its ability to inhibit MMP-9 activity.

Salvia miltiorrhiza extract inhibits TPA-induced MMP-9 expression and invasion through the MAPK/AP-1 signaling pathway in human breast cancer MCF-7 cells.

Cancer cell invasion is crucial for metastasis. A major factor in the capacity of cancer cell invasion is the activation of matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix. Salvia miltiorrhiza has been used as a promotion for blood circulation to remove blood stasis. Numerous previous studies have demonstrated that S. miltiorrhiza extracts (SME) decrease lipid levels and inhibit inflammation. However, the mechanism behind the effect of SME on breast cancer invasion has not been identified. The inhibitory effects of SME on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression were assessed using western blotting, reverse transcription-quantitative polymerase chain reaction and zymography assays. MMP-9 upstream signal proteins, including mitogen-activated protein kinases and activator protein 1 (AP-1) were also investigated. Cell invasion was assessed using a matrigel invasion assay. The present study demonstrated the inhibitory effects of the SME ethanol solution on MMP-9 expression and cell invasion in TPA-treated MCF-7 breast cancer cells. SME suppressed TPA-induced MMP-9 expression and MCF-7 cell invasion by blocking the transcriptional activation of AP-1. SME may possess therapeutic potential for inhibiting breast cancer cell invasiveness.

Epigallocatechin gallate inhibits the growth of MDA-MB-231 breast cancer cells via inactivation of the ß-catenin signaling pathway.

Epigallocatechin gallate (EGCG), a major constituent of green tea, has potential as a treatment for a variety of diseases, including cancer. EGCG induces apoptosis and inhibits tumorigenesis through multiple signaling pathways in breast cancer cells. ß-catenin signaling modulators could be useful in the prevention and therapy of breast cancer. However, the precise anticancer effect of EGCG through the ß-catenin signaling pathway in breast cancer is unclear. The present study investigated the association between ß-catenin expression and clinicopathological factors of breast cancer patients, and the effect of EGCG on ß-catenin expression in breast cancer cells. ß-catenin expression was analyzed according to the clinicopathological factors of 74 patients with breast cancer. All patients were females diagnosed with invasive ductal carcinoma. Western blot analysis revealed that ß-catenin was expressed at higher levels in breast cancer tissue than in normal tissue. ß-catenin expression was associated with lymph node metastasis (P=0.04), tumor-node-metastasis stage (P=0.03) and estrogen receptor status (P<0.01). EGCG decreased MDA-MB-231 cell viability and significantly downregulated the expression of ß-catenin, phosphorylated Akt and cyclin D1. Remarkably, additive effects of LY294002 and wortmannin, two phosphatidylinositol-3 kinase inhibitors, were observed. The present results suggest that EGCG inhibits the growth of MDA-MB-231 cells through the inactivation of the ß-catenin signaling pathway. Based on these promising results, EGCG may be a potential treatment for triple negative breast cancer patients.

Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis.

The constituents of Peucedanum japonicum Thunb. (PJ) exhibit biological and pharmacological activities, including anti-obesity, anti-oxidant and anti-allergic activities. The aim of the present study was to examine in vitro effects of PJ in RANKL-induced signaling pathways, which determine osteoclast differentiation. PJ ethanol extract (PEE) exhibited anti-osteoporotic activity by disrupting the phospholipase C (PLC)-Ca2+-c-Fos/cAMP response element-binding protein (CREB)-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway during osteoclastogenesis. Murine bone marrow-derived macrophages (BMMs) were cultured and used to determine the effects of PJ in the receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis. The effects of PEE in the RANKL-mediated signaling cascade were evaluated using a standard in vitro osteoclastogenesis system. PEE treatment of BMMs significantly reduced the number of RANKL-mediated tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (P<0.05 for 5 and 10 µg/ml PEE, P<0.01 for 25 and 50 µg/ml PEE), without cytotoxic effects. Furthermore, the expression of differentiation-related marker genes, including TRAP, Oscar, Cathepsin K, dendrocyte expressed seven transmembrane protein, ATPase H+ Transporting V0 Subunit D2 and NFATc1, were markedly suppressed. PEE induced a transient increase in free cytoplasmic Ca2+ ([Ca2+]i) mobilization via voltage-gated Ca2+ channels and PLC-sensitive pathways. Transient [Ca2+]i increase consequently resulted in the suppression of c-Fos, CREB and NFATc1 activities. These findings highlight the potential use of PJ in treating bone disorders caused by osteoclast overgrowth.

PTEN inhibits replicative senescence-induced MMP-1 expression by regulating NOX4-mediated ROS in human dermal fibroblasts.

The biological function of NADPH oxidase (NOX) is the generation of reactive oxygen species (ROS). ROS, primarily arising from oxidative cell metabolism, play a major role in both chronological ageing and photoageing. ROS in extrinsic and intrinsic skin ageing may be assumed to induce the expression of matrix metalloproteinases. NADPH oxidase is closely linked with phosphatidylinositol 3-OH kinase (PI3K) signalling. Protein kinase C (PKC), a downstream molecule of PI3K, is essential for superoxide generation by NADPH oxidase. However, the effect of PTEN and NOX4 in replicative-aged MMPs expression has not been determined. In this study, we confirmed that inhibition of the PI3K signalling pathway by PTEN gene transfer abolished the NOX-4 and MMP-1 expression. Also, NOX-4 down-expression of replicative-aged skin cells abolished the MMP-1 expression and ROS generation. These results suggest that increase of MMP-1 expression by replicative-induced ROS is related to the change in the PTEN and NOX expression.