Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species­mediated MMP­9 activation and invasion in MCF­7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Inhibitory effect of Sanguisorba hakusanensis Makino ethanol extract on atopic dermatitis-like responses in NC/Nga mice and human keratinocytes.

Atopic dermatitis (AD) is an allergic, inflammatory skin disease caused by immune dysregulation. In this study, we investigated anti-atopic and anti-inflammatory activities of Sanguisorba hakusanensis ethanol extract (SHE) both in vivo using NC/Nga mice and in vitro using human HaCaT keratinocytes. Oral administration of SHE suppressed several atopic symptoms associated with house dust mites (induced with Dermatophagoides farinae extract) in NC/Nga mice and decreased serum levels of inflammatory mediators such as immunoglobulin E, histamine, and inflammatory chemokines. Additionally, SHE treatment reduced the infiltration of immune cells such as mast cells and macrophages in AD skin lesions. In vitro, interferon-γ- and tumor necrosis factor-α-stimulated HaCaT cells exhibited increased expression of T helper 1 and 2 chemokines; their expression was inhibited by SHE treatment. The anti-inflammatory effects of SHE treatment involved blocking of the mitogen-activated protein kinase and signal transducer and activator of transcription 1 signaling pathways. In conclusion, SHE exerts potent anti-atopic and anti-inflammatory effects and should be considered for the clinical treatment of AD.

Terminalia chebula Retz. extract ameliorates the symptoms of atopic dermatitis by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-ĸB signaling in vitro.

BACKGROUND: Terminalia chebula (TC) is a traditional medicinal plant used for treating various diseases in humans. However, pharmacological mechanisms underlying the effects of TC in atopic treatment remain unelucidated. HYPOTHESIS/PURPOSE: We investigated the therapeutic effects of TC extract in a mouse model of atopic dermatitis (AD) in vivo and the anti-inflammatory mechanism in vitro. STUDY DESIGN/METHODS: For the in vivo study, AD was induced by Dermatophagoides farinae extract (Dfe) in NC/Nga mice. After 14 days of oral administration, the effects of TC concentrations of 30, 100, and 300 mg/kg were analyzed by assessing morphological changes visually; measuring serum levels of inflammatory chemokines/cytokines, IgE, histamine, MDC, TARC, RANTES, and TSLP using ELISA kits; and counting infiltrated mast cells. For in vitro analyses, we used IFNγ/TNF-α-stimulated human keratinocyte cell lines to study the mechanism of action. The production of chemokines/cytokines in the IFNγ/TNF-α-stimulated HaCaT cells was measured using ELISA and a bead array kit. The signaling pathways were analyzed by western blotting and the expression of the transcriptional factors using RT-PCR and luciferase assay. RESULTS: Administration of TC significantly alleviated AD-like symptoms in vivo and decreased the ear thickness, dermatitis score, keratinization, and mast cell infiltration. It also resulted in decreased serum levels of IgE, histamine, and inflammation-related mediators MDC, TARC, RANTES, and TSLP compared with those in the Dfe treatment group. Moreover, TC downregulated the expression of the inflammatory chemokines RANTES and MDC in IFNγ/TNF-α-stimulated HaCaT cells. TC inhibited phosphorylated STAT1/3 and NK-κB subunits and nuclear translocation of NF-κB. It also suppressed the transcription of IFNγ, IL-6, IL-8 and MCP-1 in the IFNγ/TNF-α-stimulated HaCaT cells. TC and its constituents, chebulic acid, gallic acid, corlagin, chebulanin, chbulagic acid, ellagic acid, and chebulinic acid, strongly inhibited the nuclear translocation of NF-κB, STAT1, and STAT3 and decreased the expression of inflammatory cytokines at the mRNA level. CONCLUSIONS: Overall, TC extract alleviated AD-like symptoms by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-κB signaling in vitro. In addition, our results show the in vivo effect of partial improvements in AD, as well as the in vitro effect on inflammatory factors by the constituents of TC. This finding provides that TC extract and its components could be potential therapeutic drugs for AD.

Spatholobus suberectus Dunn Water Extract Ameliorates Atopic Dermatitis-Like Symptoms by Suppressing Proinflammatory Chemokine Production In Vivo and In Vitro.

S. patholobus suberectus Dunn, a traditional Chinese herbal medicine, has various pharmacological activities, such as anti-inflammatory properties. However, to the best of our knowledge, its therapeutic effect on atopic dermatitis (AD) has not been investigated. In this study, we explored the effect of S. suberectus Dunn water extract (SSWex) on AD in vivo and in vitro. In Dermatophagoides farina extract (DfE)-treated NC/Nga mice, the oral administration of SSWex alleviated AD-like symptoms, such as ear thickness, dermatitis score, epidermal thickness, immune cell infiltration, and levels of AD-related serum parameters (immunoglobulin E, histamine, and proinflammatory chemokines). In HaCaT cells, the production of proinflammatory chemokines induced by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was inhibited by SSWex pretreatment. SSWex treatment inhibited the phosphorylation of mitogen-activated protein kinase and activation and translocation of transcriptional factors, such as signal transducer and activator of transcription 1 and nuclear factor kappa B in IFN-γ/TNF-α-stimulated HaCaT cells. These results indicate that SSWex may be developed as an efficient therapeutic agent for AD.

Alpinia officinarum water extract inhibits the atopic dermatitis-like responses in NC/Nga mice by regulation of inflammatory chemokine production.

Alpinia officinarum (AO) has been traditionally used in Asia as an herbal medicine to treat inflammatory and internal diseases. However, the therapeutic effect of AO on atopic dermatitis (AD) is unclear. Therefore, we examined whether Alpinia officinarum water extract (AOWex) affects AD in vivo and in vitro. Oral administration of AOWex to NC/Nga mice with Dermatophagoies farina extract (DfE)-induced AD-like symptoms significantly reduced the severity of clinical dermatitis, epidermal thickness, and mast cell infiltration into the skin and ear tissue. Decreased total serum IgE, macrophage-derived chemokine (MDC), and regulated on activation, normal T-cell expressed and secreted (RANTES) levels were observed in DfE-induced NC/Nga mice in the AOWex-treated group. These effects were confirmed in vitro using HaCaT cells. Treatment with AOWex inhibited the expression of proinflammatory chemokines such as MDC, RANTES, IP-10 and I-TAC in interferon-γ and tumor necrosis factor-α-stimulated HaCaT cells. The anti-inflammatory effects of AOWex were due to its inhibitory action on MAPK phosphorylation (ERK and JNK), NF-κB, and STAT1. Furthermore, galangin, protocatechuic acid, and epicatechin from AOWex were identified as candidate anti-AD compounds. These results suggest that AOWex exerts therapeutic effects against AD by alleviating AD-like skin lesions, suppressing inflammatory mediators, and inhibiting major signaling molecules.

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.

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.

Laminaria japonica Suppresses the Atopic Dermatitis-Like Responses in NC/Nga Mice and Inflamed HaCaT Keratinocytes via the Downregulation of STAT1.

Atopic dermatitis (AD) is a skin allergy accompanied by acute and chronic dermal inflammation. In traditional oriental medicine, Laminaria japonica has been used to treat various diseases, including inflammatory diseases. Therefore, to determine the therapeutic potential of L. japonica against AD, we investigated the inhibitory effects of L. japonica water extract (LJWE) on the inflammatory mediators and AD-like skin lesions. We determined the cell viability of LJWE-treated HaCaT cells using the cell counting kit-8 assay and the levels of inflammatory cytokines using cytometric bead array kits. Additionally, we analyzed the modulatory effects of LJWE on the signaling pathways in tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells via Western blotting. Furthermore, we determined the in vivo effect of LJWE on NC/Nga mice and found that LJWE remarkably improved the skin moisture, reduced dermatitis severity, and inhibited the overproduction of inflammatory mediators in 2,4-dinitrochlorobenzene-sensitized NC/Nga mice. We also observed that LJWE inhibits the expression of inflammatory chemokines in human keratinocytes by downregulating the p38 mitogen-activated protein kinase signaling pathway and activating the signal transducer and activator of transcription 1. In conclusion, LJWE has the therapeutic potential against AD by healing AD-like skin lesions, and suppressing inflammatory mediators and major signaling molecules.

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.

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.

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.

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.

Sophorae Flos extract inhibits RANKL-induced osteoclast differentiation by suppressing the NF-κB/NFATc1 pathway in mouse bone marrow cells.

BACKGROUND: Sophorae Flos (SF) is a composite of flowers and buds of Styphnolobium japonicum (L.) Schott and has been used in traditional Korean and Chinese medicine for the treatment of hemostasis and inflammation. Previous studies reported that SF possesses anti-obesity properties, as well as anti-allergic, anti-proliferative, and anti-inflammatory activities. However, the effect of SF in bone resorption has not been studies. In this study, we examined the potential of SF extract (SFE) to inhibit receptor activator of NF-κB ligand (RANKL) -induced osteoclast differentiation in cultured mouse-derived bone marrow macrophages (BMMs). METHODS: BMMs, that act as osteoclast precursors, were cultured with M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 4 days to generate osteoclasts. Osteoclast differentiation was measured by tartrate-resistant acidic phosphatase (TRAP) staining and the TRAP solution assay. Osteoclast differentiation marker genes were analyzed by the quantitative real-time polymerase chain reaction analysis. RANKLs signaling pathways were confirmed through western blotting. RESULTS: SFE significantly decreased osteoclast differentiation in a dose-dependent manner. SFE inhibited RANKL-induced osteoclastogenesis by suppressing NF-κB activation. By contrast, SFE did not affect phospholipase C gamma 2 or subsequent cAMP response element binding activation. SFE inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). CONCLUSIONS: SFE attenuated the RANKL-mediated induction of NF-κB through inhibition of IκBα phosphorylation, which contributed to inhibiting of RANKL-induced osteoclast differentiation through downregulation of NFATc1.

3D-printed phantom study for investigating stent abutment during gastroduodenal stent placement for gastric outlet obstruction.

BACKGROUND: Placing a self-expandable metallic stent (SEMS) is safe and effective for the palliative treatment of malignant gastroduodenal (GD) strictures. SEMS abutment in the duodenal wall is associated with increased food impaction, resulting in higher stent malfunction and shorter stent patency. The desire to evaluate the mechanism and significance of stent abutment led us to design an in vitro experiment using a flexible anthropomorphic three-dimensional (3D)-printed GD phantom model. RESULTS: A GD phantom was fabricated using 3D printer data after performing computed tomography gastrography. A partially covered (PC) or fully covered (FC) stent was placed so that its distal end abutted onto the duodenal wall in groups PC-1 and FC-1 or its distal end was sufficiently directed caudally in groups PC-2 and FC-2. The elapsed times of the inflowing of three diets (liquid, soft, and solid) were measured in the GD phantom under fluoroscopic guidance. There was no significant difference in the mean elapsed times for the liquid diet among the four groups. For the soft diet, the mean elapsed times in groups PC-1 and FC-1 were longer than those in groups PC-2 and FC-2 (P = 0.018 and P < 0.001, respectively). For the solid diet, the mean elapsed time in group PC-1 was longer than that in group PC-2 (P < 0.001). The solid diet could not pass in group FC-1 due to food impaction. The mean elapsed times were significantly longer in groups FC-1 and FC-2 than in groups PC-1 and PC-2 for soft and solid diets (all P < 0.001). CONCLUSIONS: This flexible anthropomorphic 3D-printed GD phantom study revealed that stent abutment can cause prolonged passage of soft and solid diets through the stent as well as impaction of solid diets into the stent.

Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts.

Reactive oxygen species (ROS) play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3) metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC) or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

Suppression of TPA-induced cancer cell invasion by Peucedanum japonicum Thunb. extract through the inhibition of PKCα/NF-κB-dependent MMP-9 expression in MCF-7 cells.

Metastatic cancers spread from their site of origin (the primary site) to other parts of the body. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is important in metastatic cancers as it plays a major role in cancer cell invasion. The present study examined the inhibitory effect of an ethanol extract of Peucedanum japonicum Thunb. (PJT) on MMP-9 expression and the invasion of MCF-7 breast cancer cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis, gelatin zymography, and reverse transcription-quantitative PCR revealed that PJT significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, PJT attenuated TPA-induced nuclear translocation and the transcriptional activation of nuclear factor (NF)-κB. The results indicated that the PJT-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involved the suppression of the PKCα/NF-κB pathway in MCF-7 cells. Thus, the inhibition of MMP-9 expression by PJT may have potential value as a therapy for restricting the invasiveness of breast cancer.

Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways.

Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3',4',7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis.

Differential in vitro and cellular effects of iron chelators for hypoxia inducible factor hydroxylases.

Hypoxia inducible factor 1α (HIF-1α), an essential transcriptional factor, is negatively regulated by two different types of oxygen and Fe(2+) -dependent HIF hydroxylases, proline hydroxylase (PHD) and factor inhibiting HIF (FIH), under normoxia. Iron chelators have therefore been used for inducing HIF-1α expression by inhibiting the hydroxylases. In this study, the iron chelators displayed differential effects for PHD and FIH in cells depending on their iron specificity and membrane permeability rather than their in vitro potencies. The membrane permeability of the strict Fe(2+) -chelator potentially inhibited both hydroxylases, whereas the membrane impermeable one showed no inhibitory effect in cells. In contrast, the depletion of the extracellular Fe(3+) ion was mainly correlated to PHD inhibition, and the membrane permeable one elicited low efficacy for both enzymes in cells. The 3'-hydroxyl group of quercetin, a natural flavonoid, was critical for inhibition of intracellular hydroxylases. Since the 3'-methylation of quercetin is induced by catechol-O-methyl transferase, the enzyme may regulate the intracellular activity of quercetin. These data suggest that the multiple factors of iron-chelators may be responsible for regulating the intracellular activity HIF hydroxylases.