High glucose activates ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic foot ulcers.

Increased matrix metalloproteinase 9 (MMP9) expression is involved in delayed wound healing in diabetic foot ulcers. We created skin wounds in normal SD rats and STZ-induced diabetic SD rats, then we found protein levels of activator protein-1 (AP1), a crucial transcription factor to increase MMP9 transcription, as well as MMP9 was up-regulated in epithelium of diabetic skin tissues. Then, we evaluated the mRNA and protein stability of AP1 subunits C-FOS/C-Jun in HaCaT cells after high glucose treatment. Results showed that high glucose could increase protein stability of C-FOS and C-Jun. Additionally, high glucose also activated extracellular signaling-related kinase 1/2 (ERK1/2). ERK1/2 inhibitor could rescue phosphorylation of C-FOS and C-Jun, increased protein stability of C-Jun, and increased MMP9 expressions. Thus, our study demonstrated that high glucose could activate ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic skin and HaCaT cells.

Transcriptional response of human articular chondrocytes treated with fibronectin fragments: an in vitro model of the osteoarthritis phenotype.

OBJECTIVE: Fibronectin is a matrix protein that is fragmented during cartilage degradation in osteoarthritis (OA). Treatment of chondrocytes with fibronectin fragments (FN-f) has been used to model OA in vitro, but the system has not been fully characterized. This study sought to define the transcriptional response of chondrocytes to FN-f, and directly compare it to responses traditionally observed in OA. DESIGN: Normal human femoral chondrocytes isolated from tissue donors were treated with either FN-f or PBS (control) for 3, 6, or 18 h. RNA-seq libraries were compared between time-matched FN-f and control samples in order to identify changes in gene expression over time. Differentially expressed genes were compared to a published OA gene set and used for pathway, transcription factor motif, and kinome analysis. RESULTS: FN-f treatment resulted in 3,914 differentially expressed genes over the time course. Genes that are up- or downregulated in OA were significantly up- (P < 0.00001) or downregulated (P < 0.0004) in response to FN-f. Early response genes were involved in proinflammatory pathways, whereas many late response genes were involved in ferroptosis. The promoters of upregulated genes were enriched for NF-κB, AP-1, and IRF motifs. Highly upregulated kinases included CAMK1G, IRAK2, and the uncharacterized kinase DYRK3, while growth factor receptors TGFBR2 and FGFR2 were downregulated. CONCLUSIONS: FN-f treatment of normal human articular chondrocytes recapitulated many key aspects of the OA chondrocyte phenotype. This in vitro model is promising for future OA studies, especially considering its compatibility with genomics and genome-editing techniques.

Importin ß1 regulates cell growth and survival during adult T cell leukemia/lymphoma therapy.

There is no cure for adult T cell leukemia/lymphoma (ATLL) associated with human T cell leukemia virus type 1 (HTLV-1), and novel targeted strategies are needed. NF-κB and AP-1 are crucial for ATLL, and both are transported to the nucleus by an importin (IPO)α/ß heterodimeric complex to activate target genes. In this study, we aimed to elucidate the function of IPOß1 in ATLL. The expression of IPOß1 was analyzed by western blotting and RT-PCR. Cell growth, viability, cell cycle, apoptosis and intracellular signaling cascades were examined by the water-soluble tetrazolium-8 assay, flow cytometry and western blotting. Xenograft tumors in severe combined immune deficient mice were used to evaluate the growth of ATLL cells in vivo. IPOß1 was upregulated in HTLV-1-infected T cell lines. Further, IPOß1 knockdown or the IPOß1 inhibitor importazole and the IPOα/ß1 inhibitor ivermectin reduced HTLV-1-infected T cell proliferation. However, the effect of inhibitors on uninfected T cells was less pronounced. Further, in HTLV-1-infected T cell lines, inhibitors suppressed NF-κB and AP-1 nuclear transport and DNA binding, induced apoptosis and poly (ADP-ribose) polymerase cleavage, and activated caspase-3, caspase-8 and caspase-9. Inhibitors also mediated G1 cell cycle arrest. Moreover, the expression of NF-κB- and AP-1-target proteins involved in cell cycle and apoptosis was reduced. In vivo, the IPOα/ß1 inhibitor ivermectin decreased ATLL tumor burden without side effects. IPOß1 mediated NF-κB and AP-1 translocation into ATLL cell nuclei, thereby regulating cell growth and survival, which provides new insights for targeted ATLL therapies. Thus, ivermectin, an anti-strongyloidiasis medication, could be a potent anti-ATLL agent.

TAK1/AP-1-Targeted Anti-Inflammatory Effects of Barringtonia augusta Methanol Extract.

Barringtonia augusta methanol extract (Ba-ME) is a folk medicine found in the wetlands of Thailand that acts through an anti-inflammatory mechanism that is not understood fully. Here, we examine how the methanol extract of Barringtonia augusta (B. augusta) can suppress the activator protein 1 (AP-1) signaling pathway and study the activities of Ba-ME in the lipopolysaccharide (LPS)-treated RAW264.7 macrophage cell line and an LPS-induced peritonitis mouse model. Non-toxic concentrations of Ba-ME downregulated the mRNA expression of cytokines, such as cyclooxygenase and chemokine ligand 12, in LPS-stimulated RAW264.7 cells. Transfection experiments with the AP-1-Luc construct, HEK293T cells, and luciferase assays were used to assess whether Ba-ME suppressed the AP-1 functional activation. A Western blot assay confirmed that C-Jun N-terminal kinase is a direct pharmacological target of Ba-ME action. The anti-inflammatory effect of Ba-ME, which functions by ß-activated kinase 1 (TAK1) inhibition, was confirmed by using an overexpression strategy and a cellular thermal shift assay. In vivo experiments in a mouse model of LPS-induced peritonitis showed the anti-inflammatory effect of Ba-ME on LPS-stimulated macrophages and acute inflammatory mouse models. We conclude that Ba-ME is a promising anti-inflammatory drug targeting TAK1 in the AP-1 pathway.

ß-Mangostin inhibits the metastatic power of cervical cancer cells attributing to suppression of JNK2/AP-1/Snail cascade.

ß-Mangostin is a natural mangostin with potent anticancer activity against various cancers. In this study, we further explored the anticancer activity of ß-mangostin on cervical cancer cells. ß-Mangostin did not affect cell viability and cell cycle distribution in HeLa and SiHa cells; however, it dose-dependently inhibited the migration and invasion of both the human cervical cancer cell lines. In addition, we observed that ß-mangostin suppressed the expression of integrin αV and ß3 and the downstream focal adhesion kinase/Src signaling. We also found that Snail was involved in the ß-mangostin inhibited cell migration and invasion of HeLa cell. Then, our findings showed that ß-mangostin reduced both nuclear translocation and messenger RNA expression of AP-1 and demonstrated that AP-1 could target to the Snail promoter and induce Snail expression. Kinase cascade analysis and reporter assay showed that JNK2 was involved in the inhibition of AP-1/Snail axis by ß-mangostin in HeLa cells. These results indicate that ß-mangostin can inhibit the mobility and invasiveness of cervical cancer cells, which may attribute to the suppression of both integrin/Src signaling and JNK2-mediated AP-1/Snail axis. This suggests that ß-mangostin has potential antimetastatic potential against cervical cancer cells.

Effect of 1,25(OH)2 D3 and 20(OH)D3 on interleukin-1ß-stimulated interleukin-6 and -8 production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Vitamin D-1,25(OH)2 D3 or 1,25D3-maintains healthy osseous tissue, stimulates the production of the antimicrobial peptide cathelicidin and has anti-inflammatory effects, but it can cause hypercalcemia. Evidence links diminished serum levels of 1,25D3 with increased gingival inflammation. Periodontitis progression is associated with increased local production of inflammatory mediators by immune cells and gingival fibroblasts. These include interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8, both regulated by signaling pathways, including NF-κB and MAPK/AP-1. The objectives were to determine the effects of 1,25D3 or a non-calcemic analog, 20-hydroxyvitamin D3 -20(OH)D3 or 20D3-on IL-1ß-stimulated IL-6 and IL-8 production, and NF-κB and MAPK/AP-1 activation, by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were incubated ± IL-1ß, with or without exposure to 1,25D3 or 20D3. IL-6 and IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay. NF-κB (p65) and AP-1 (phospho-cJun) and were measured in nuclear extracts via binding to specific oligonucleotides. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß-stimulated IL-6 and IL-8 levels were both significantly inhibited (40%-60%) (P<.045) by 1,25D3, but not 20D3 (0%-15% inhibition, not statistically significant). Both 1,25D3 and 20D3 significantly and similarly inhibited IL-1ß-stimulated nuclear levels of p65 and phospho-cJun (P<.02). CONCLUSION: Reduction of the activation of NF-κB and AP-1 alone is not able to inhibit strongly the IL-1ß stimulated IL-6 and IL-8 gene expression. 1,25D3 but not 20D3 may affect some of the many other factors/processes/pathways that in turn regulate the expression of these genes. However, the results suggest that topical application of ligands of the vitamin D receptor may be useful in the local treatment of periodontitis while reducing adverse systemic effects.

Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

Magnolol Inhibits RANKL-induced osteoclast differentiation of raw 264.7 macrophages through heme oxygenase-1-dependent inhibition of NFATc1 expression.

Magnolol (1) isolated from Magnolia officinalis exhibits many beneficial effects such as anti-inflammatory and antioxidant activity. The aim of this study was to evaluate the effects of magnolol (1) on RANKL-induced osteoclast differentiation and investigate the underlying molecular mechanisms. Treatment with magnolol (1) significantly inhibited osteoclast differentiation of RAW 264.7 macrophages and bone-resorbing activity of osteoclasts in the RANKL-induced system. Moreover, RANKL-activated JNK/ERK/AP-1 and NF-κB signaling, ROS formation, and NFATc1 activation were attenuated by magnolol (1). A novel finding of this study is that magnolol (1) can increase heme oxygenase-1 (HO-1) expression and Nrf2 activation in RANKL-stimulated cells. Blocking HO-1 activity with tin protoporphyrin IX markedly reversed magnolol (1)-mediated inhibition of osteoclast differentiation, NFATc1 nuclear translocation, and MMP-9 activity, suggesting that HO-1 contributes to the attenuation of NFATc1-mediated osteoclastogenesis by magnolol (1). Therefore, the inhibitory effect of magnolol (1) on osteoclast differentiation is due to inhibition of MAPK/c-fos/AP-1 and NF-κB signaling as well as ROS production and up-regulation of HO-1 expression, which ultimately suppresses NFATc1 induction. These findings indicate that magnolol (1) may have potential to treat bone diseases associated with excessive osteoclastogenesis.

Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 µM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

Inhibition of interleukin 1ß-stimulated interleukin-6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways.

BACKGROUND AND OBJECTIVE: In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1ß. IL-1ß regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1ß-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1ß-stimulated IL-6 production in gingival epithelial cells. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1ß in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1ß-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1ß stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1ß-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). CONCLUSION: In S-G epithelial cells, IL-1ß appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1ß-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.

Meroterpenoids from the alga-derived fungi Penicillium thomii Maire and Penicillium lividum Westling.

Ten new austalide meroterpenoids (1-10) were isolated from the alga-derived fungi Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663. Their structures were elucidated by extensive spectroscopic analysis and by comparison with related known compounds. The absolute configurations of some of the metabolites were assigned by the modified Mosher's method and CD data. Compounds 1, 2, 8, and 9 were able to inhibit AP-1-dependent transcriptional activity in JB6 Cl41 cell lines at noncytotoxic concentrations. Austalides 1-5, 8, and 9 exhibited significant inhibitory activity against endo-1,3-ß-D-glucanase from a crystalline stalk of the marine mollusk Pseudocardium sachalinensis.

Benzyl alcohol derivatives from the mushroom Hericium erinaceum attenuate LPS-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity.

On the search for anti-inflammatory compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the fruiting bodies of Hericium erinaceum resulted in the isolation and identification of five benzyl alcohol derivatives (1-5). In this study, their anti-inflammatory effects on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW 264.7 macrophage cells. The structures of isolates were identified by comparing their spectroscopic data with previously reported values. The analysis of their inhibitory activities on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells showed that erinacerin B (2) and hericenone E (4) decreased the levels of NO and PGE2 production in a concentration-dependent manner. Next, this study was performed to examine their mechanism of action on the regulation of NO and PGE2 production. Compounds 2 and 4 were found to block the LPS-induced phosphorylation of two major inflammatory transcription factors, NF-κB (p65/p50) and AP-1 (c-Jun and c-Fos). Taken together, these results suggest that down-regulation of LPS-induced NO and PGE2 production by compounds 2 and 4 is mediated through the modulation of NF-κB and AP-1 activation in macrophage cells. These results impact the development of potential health products for preventing and treating inflammatory diseases.

Inhibitory effect of reinioside C on vascular smooth muscle cells proliferation induced by angiotensin II via inhibiting NADPH oxidase-ROS-ENK1/2-NF-kappaB-AP-1 pathway.

The proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin II (Ang II) plays a vital role in the pathogenesis of arteriosclerosis and restenosis. In the present study, the effect of reinioside C, a main active ingredient of Polygala fallax Hemsl, on proliferation of VSMCs induced by Ang II was investigated. It was found that Ang II (1 microM) markedly stimulated proliferation of VSMCs. Pretreatment of reinioside C (3, 10 or 30 microM) concentration-dependently inhibited the proliferative effect of Ang II. To determine the possible mechanism, NADPH oxidase subunits (Nox-1, Nox-4) mRNA expression, intracellular ROS level, phosphorylation of ERK1/2, NF-kappaB activity, and mRNA expression of AP-1 subunits (c-fos, c-jun) and c-myc were measured. The results demonstrated that reinioside C attenuated Ang II-induced NADPH oxidase mRNA expression, generation of ROS, ERK1/2 phosphorylation, activation of NF-kappaB, and mRNA expression of AP-1 and c-myc in VSMCs in a concentration-dependent manner. The effects of Ang II were also inhibited by diphenyleneiodonium (DPI, the NADPH oxidase inhibitor), PD98059 (the ERK1/2 inhibitor) and pyrrolidine dithiocarbamate (PDTC, the NF-kappaB inhibitor). These results suggest reinioside C attenuates Ang II-induced proliferation of VSMCs by inhibiting NADPH oxidase-ROS-ERK1/2-NF-kappaB-AP-1 pathway.

BML-111 attenuates hemorrhagic shock-induced acute lung injury through inhibiting activation of mitogen-activated protein kinase pathway in rats.

BACKGROUND: Hemorrhagic shock activates cellular stress signals and can lead to systemic inflammatory response, organ injury, and death. Mitogen-activated protein kinase (MAPK) acts as a sensor of tissue injury in models of ischemia-reperfusion injury. Lipoxins are endogenous lipid mediators with potent anti-inflammatory and pro-resolving actions. We hypothesized that BML-111 (a lipoxin A4-receptor agonist) attenuates hemorrhagic shock-induced acute lung injury (ALI) through inhibiting activation of the MAPK pathway. METHODS: We randomized Sprague-Dawley rats into four groups: sham, hemorrhagic shock-resuscitation (HS), HS plus BML-111 (BML-111), and HS plus BML-111 and BOC-2 (BOC-2). Two hours after resuscitation, we collected samples of lung. We obtained bronchoalveolar lavage fluid for neutrophil count. We performed optical microscopy to examine pathologic changes in lungs. Wet/dry ratios, myeloperoxidase expression, interleukin (IL)-1ß and IL-6 levels in lung were measured. We evaluated MAPK activation and the DNA binding activity of activator protein-1 in lung. RESULTS: Treatment with BML-111 reduced the lung damage and wet/dry ratio, neutrophil count in bronchoalveolar lavage fluid, expression of myeloperoxidase, and production of IL-1ß and IL-6 in lung. Phosphorylation of MAPK was also decreased by BML-111 in lung. Furthermore, the DNA binding activity of activator protein-1 was blocked by BML-111. An antagonist of the lipoxin A4-receptor, BOC-2, reversed the protective effect of BML-111 on ALI induced by hemorrhagic shock. CONCLUSIONS: This study indicates that BML-111 attenuated hemorrhagic shock-induced ALI via the MAPK/activator protein-1 signaling pathway. Therefore, BML-111 may have therapeutic potential for hemorrhagic shock-induced ALI.

DC-81-enediyne induces apoptosis of human melanoma A375 cells: involvement of the ROS, p38 MAPK, and AP-1 signaling pathways.

Melanoma is one of the most chemoresistant cancers in patient care. The remission rate of current therapy remains low. DC-81, an antitumor antibiotic produced by Streptomyces species, belongs to pyrrolo[2,1-c][1,4]benzodiazepine (PBD), which is a potent inhibitor of nucleic acid synthesis. An enediyne contains either DNA intercalating groups or DNA minor groove binding functions and these are potent DNA-damaging agents due to their ability to generate benzenoid diradicals. We have previously reported an efficient synthesis and antitumor activity of a series of novel PBD hybrids linked with enediyne. The purpose of this study was to examine the mechanism of the antiproliferative effect of DC-81-enediyne agent on human melanoma A375 cells. DC-81-enediyne induced an increase in Ca(2+) level and reactive oxygen species (ROS) generation as detected by flow cytometric assay. Western blot analysis showed that DC-81-enediyne induced the phosphorylation of p38 and activating transcription factor 2 (ATF-2). By using the luciferase reporter assay, activating protein-1 (AP-1) activity was further enhanced after A375 cells were treated with graded concentrations of DC-81-enediyne. DC-81-enediyne treatment-induced A375 cell apoptosis was significantly abrogated by the addition of Ca(2+), ROS, and p38 inhibitors. Collectively, our studies indicate that DC-81-enediyne induces A375 cell apoptosis through an increased Ca(2+) and ROS generation, which involves p38 phosphorylation and enhanced ATF-2/AP-1 expressions, leading to caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 CytoDeath staining, and subsequent apoptotic cell death.

Anti-fibrotic effect of 6-bromo-indirubin-3'-oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3ß inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFß-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFß, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFß, and α-SMA increased in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFß treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFß treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

Grassypeptolides F and G, cyanobacterial peptides from Lyngbya majuscula.

Grassypeptolides F (1) and G (2), bis-thiazoline-containing cyclic depsipeptides with a rare ß-amino acid, extensive N-methylation, and a large number of d-amino acids, are reported from an extract of the Palauan cyanobacterium Lyngbya majuscula. Both 1 and 2 were found to have moderate inhibitory activity against the transcription factor AP-1 (IC50 = 5.2 and 6.0 µM, respectively).

Antitumor actinopyranones produced by Streptomyces albus POR-04-15-053 isolated from a marine sediment.

Four new antitumor pyranones, PM050511 (1), PM050463 (2), PM060054 (3), and PM060431 (4), were isolated from the cell extract of the marine-derived Streptomyces albus POR-04-15-053. Their structures were elucidated by a combination of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS. They consist of an α-methoxy-γ-pyrone ring containing a highly substituted tetraene side chain glycosylated at C-10 in the case of 1 and 4. Compounds 1 and 4 displayed strong cytotoxicity against three human tumor cell lines with GI50 values in the submicromolar range, whereas 2 showed subnanomolar activity as an inhibitor of EGFR-MAPK-AP1-mediated mitogenic signaling, causing inhibition of EGF-mediated AP1 trans-activation and EGF-mediated ERK activation and slight inhibition of EGF-mediated JNK activation. Taken together, these results suggest that members of the pyranone family of compounds could be developed as potential antitumor agents.

Comparative evaluation of two structurally related flavonoids, isoliquiritigenin and liquiritigenin, for their oral infection therapeutic potential.

Isoliquiritigenin (1) and liquiritigenin (2) are structurally related flavonoids found in a variety of plants. The purpose of this study was to perform a comparative analysis of biological properties of these compounds in regard to their therapeutic potential for oral infections. Compound 1 demonstrated significant antibacterial activity against three major periodontopathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. In contrast, 2 exerted less pronounced effects on the above bacterial species. Neither compound was effective against cariogenic bacteria (Streptococcus mutans and Streptococcus sobrinus). Furthermore, 1 exhibited a stronger inhibitory activity than 2 toward P. gingivalis collagenase and human matrix metalloproteinase 9. Finally, the capacity of 1 to attenuate the inflammatory response of macrophages induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) was much higher when compared to 2. The activation of transcriptional factors nuclear factor-κB (NF-κB) p65 and activator protein-1 (AP-1) associated with the LPS-induced inflammatory response in macrophages was inhibited strongly by 1, but less affected by 2.

Cancer chemopreventive activity of maslinic acid: suppression of COX-2 expression and inhibition of NF-κB and AP-1 activation in Raji cells.

Chronic inflammation is one of the predisposing factors for neoplastic transformation. Targeting inflammation through suppression of the pro-inflammatory pathway by dietary phytochemicals provides an important strategy for cancer prevention. Maslinic acid is a novel natural triterpenoid known to inhibit proliferation and induce apoptosis in some tumor cell lines. Although maslinic acid has cytotoxic and pro-apoptotic effects on cancer cells, the underlying mechanisms of its effects on the inflammatory pathway have yet to be elucidated. It has been reported that abnormal expression of pro-inflammatory enzyme cyclooxygenase-2 (COX-2) causes promotion of cellular proliferation, suppression of apoptosis, enhancement of angiogenesis and invasiveness. In the present study, the suppressive effect of maslinic acid on COX-2 expression and the binding activity of upstream transcription factors NF- κB and AP-1, which are known to regulate COX-2 transcriptional activation, were assessed using Raji cells. The anti-inflammatory action of maslinic acid was benchmarked against oleanolic acid and other standard drugs. Western blot analysis and electrophoretic mobility shift assay (EMSA) were employed to analyze COX-2 expression as well as NF- κB and AP-1 binding activity. Our results showed that maslinic acid suppresses COX-2 expression in a concentration-dependent manner. Likewise, the constitutive nuclear NF- κB (p65) activity as well as phorbol 12-myristate 13-acetate (PMA)- and sodium N-butyrate (SnB)-induced AP-1 binding activity in Raji cells were significantly reduced following treatment with maslinic acid. Since maslinic acid suppresses COX-2 expression in Raji cells at concentrations that also lowered the NF- κB (p65) and AP-1 binding activity, it is possible that the suppression of COX-2 by this natural triterpenoid might be achieved, at least in part, via the NF- κB and AP-1 signaling pathways.