Search

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (Hordeum vulgare var. hexastichon) grass (HVA) significantly inhibited RANKL-induced osteoclast formation and protected mice from LPS-induced bone loss. A phytochemical investigation of HVA afforded nine indole alkaloids, including one new compound [hordeumin A (1)] and eight known analogues (2-9). Of them, four (1, 2, 4, and 5) were anti-osteoclastogenic compounds. Of these four, compound 5 significantly suppressed RANKL-induced osteoclast formation, actin ring formation, and bone resorption in a concentration-dependent manner. It also suppressed the RANKL-induced NF-κB and MAPK signaling pathways and the activation of c-Fos and NFATc1. Compound 5 also reduced the expression levels of osteoclast-specific marker genes, including TRAP, CtsK, DC-STAMP, OSCAR, and MMP9. Our findings suggest that HVA and its alkaloid constituents could be valuable candidates for the prevention and treatment of osteolytic bone diseases.

One new oleanane triterpenoid, 2-oxo-3β,23-dihydroxyolean-12-en-28-oic acid (1), one new megastigmane, (1S,3S,9R)-megastigman-5-en-3,9,11-triol 11-O-β-D-glucopyranoside (6) and nine known compounds, 23-hydroxyursolic acid (2), 6α-hydroxybetulic acid (3), alphitolic acid (4), 3-oxo-6α-hydroxylup-20(29)-en-28-oic acid (5), (6R,9R)-3-oxo-α-ionol 9-O-β-D-glucopyranoside (7), byzantionoside B (8), dihydrovomifoliol O-β-D-glucopyranoside (9), 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-rhamnopyranoxypropyl)-methoxyphenoxy]-1,3-propanediol (10), and sweroside (11) were isolated from the methanol extract of the Viburnum lutescens Blume leaves. Their chemical structures were elucidated by 1D- and 2D-NMR and MS data and comparing their NMR data with those reported in the literature. In addition, the anti-osteoclastogenic activities of all compounds were also evaluated. The TRAP staining on RAW 264.7 cells indicated that compounds 1, 2, and 5 potently inhibited the osteoclastogenesis in RAW 264.7 cells in a concentration dependent manner.

Low-dose metronomic chemotherapy (LDMC) inhibits tumor angiogenesis and growth by targeting tumor-associated endothelial cells, but the molecular mechanism has not been fully elucidated. Here, we examined the functional role of regulated in development and DNA damage responses 1 (REDD1), an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in LDMC-mediated endothelial cell dysfunction. Low-dose doxorubicin (DOX) treatment induced REDD1 expression in cultured vascular and lymphatic endothelial cells and subsequently repressed the mRNA expression of mTORC1-dependent translation of vascular endothelial growth factor receptor (Vegfr)-2/3, resulting in the inhibition of VEGF-mediated angiogenesis and lymphangiogenesis. These regulatory effects of DOX-induced REDD1 expression were additionally confirmed by loss- and gain-of-function studies. Furthermore, LDMC with DOX significantly suppressed tumor angiogenesis, lymphangiogenesis, vascular permeability, growth, and metastasis in B16 melanoma-bearing wild-type but not Redd1-deficient mice. Altogether, our findings indicate that REDD1 is a crucial determinant of LDMC-mediated functional dysregulation of tumor vascular and lymphatic endothelial cells by translational repression of Vegfr-2/3 transcripts, supporting the potential therapeutic properties of REDD1 in highly progressive or metastatic tumors., Cancer: Stress protein implicated in effects of low-dose metronomic chemotherapy Sustained low-dose metronomic chemotherapy impairs the function of tumor-associated blood vessels and lymphatic tissues due to the action of a stress response protein called REDD1. A research team from South Korea led by Young-Myeong Kim of Kangwon National University School of Medicine in Chuncheon showed that low doses of doxorubicin, a widely used anti-cancer drug, induces the expression of REDD1. This in turn inhibits the activity of vascular endothelial growth factor receptors-2/-3 that are needed for the formation of blood and lymphatic vessels, both of which can promote tumor growth. Cancer-bearing mice that lacked a working version of REDD1 did not respond to continuous low-dose treatment with doxorubicin, demonstrating the critical nature of this protein in mediating the drug regimen’s effects. The authors propose that pharmacologically enhancing REDD1 activity could help combat highly aggressive or metastatic tumors.

Low-dose metronomic chemotherapy has been introduced as a less toxic and effective strategy to inhibit tumor angiogenesis, but its anti-angiogenic mechanism on endothelial progenitor cells (EPCs) has not been fully elucidated. Here, we investigated the functional role of regulated in development and DNA damage response 1 (REDD1), an endogenous inhibitor of mTORC1, in low-dose doxorubicin (DOX)-mediated dysregulation of EPC functions. DOX treatment induced REDD1 expression in bone marrow mononuclear cells (BMMNCs) and subsequently reduced mTORC1-dependent translation of endothelial growth factor (VEGF) receptor (Vegfr)-2 mRNA, but not that of the mRNA transcripts for Vegfr-1, epidermal growth factor receptor, and insulin-like growth factor-1 receptor. This selective event was a risk factor for the inhibition of BMMNC differentiation into EPCs and their angiogenic responses to VEGF-A, but was not observed in Redd1-deficient BMMNCs. Low-dose metronomic DOX treatment reduced the mobilization of circulating EPCs in B16 melanoma-bearing wild-type but not Redd1-deficient mice. However, REDD1 overexpression inhibited the differentiation and mobilization of EPCs in both wild-type and Redd1-deficient mice. These data suggest that REDD1 is crucial for metronomic DOX-mediated EPC dysfunction through the translational repression of Vegfr-2 transcript, providing REDD1 as a potential therapeutic target for the inhibition of tumor angiogenesis and tumor progression. [BMB Reports 2021; 54(9): 470-475].

Syntenin acts as an adaptor and scaffold protein through its two PSD-95, Dlg, and ZO-1 (PDZ) domains, participating in multiple signaling pathways and modulating cellular physiology. It has been identified as an oncogene, promoting cancer development, metastasis, and angiogenesis in various carcinomas. Syntenin-1 is also associated with the production and release of exosomes, small extracellular vesicles that play a significant role in intercellular communication by containing bioactive molecules such as proteins, lipids, and nucleic acids. The trafficking of exosomes involves a complex interplay of various regulatory proteins, including syntenin-1, which interacts with its binding partners, syndecan and activated leukocyte cell adhesion molecule (ALIX). Exosomal transfer of microRNAs, a key cargo, can regulate the expression of various cancer-related genes, including syntenin-1. Targeting the mechanism involving the regulation of exosomes by syntenin-1 and microRNAs may provide a novel treatment strategy for cancer. This review highlights the current understanding of syntenin-1’s role in regulating exosome trafficking and its associated cellular signaling pathways.

The white-flowered leaves of Eclipta prostrata L. together with leaves of Scoparia dulcis and Cynodon dactylon are mixedly boiled in water and given to diabetic patients resulting in the significant improvement in the management of diabetes. However, the active constituents from this plant for antidiabetic and anti-obesity properties are remaining unclear. Thus, this study was to discover anti-diabetes and anti-obesity activities through protein tyrosine phosphatases (PTP)1B inhibitory effects. We found that the fatty acids (23, 24) showed potent PTP1B inhibition with IC50 values of 2.14 and 3.21 µM, respectively. Triterpenoid-glycosides (12-15) also exhibited strong to moderate PTP1B inhibitory effects, with IC50 values ranging from 10.88 to 53.35 µM. Additionally, active compounds were investigated for their PTP1B inhibitory mechanism and docking analysis. On the other hand, the anti-inflammatory activity from our study revealed that compounds (1-4, 7, 8, 10) displayed the significant inhibition nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Especially, compound 9 showed the potent inhibitory effects in LPS-induced NO production on RAW264.7 cell. Therefore, further Western blot analysis was performed to identify the inhibitory expression including heme oxygenase-1 (HO-1) and inhibitor of kappaB (IκB) phosphorylation.

One new 1,4-bis-phenyl-1,4-butanedione glycoside (14), one new eudesmane-type sesquiterpenoid (16), and 16 known compounds were isolated from the leaves and stems of Nelumbo nucifera Gaertn. The structures of the isolated compounds were elucidated by interpretation of their 1D and 2D NMR spectroscopic and HRESIMS data. Time-dependent density functional theory calculations and Electronic Circular Dichroism (ECD) spectroscopy was used to determine absolute configurations of the new eudesmane-type sesquiterpenoid (16). All the isolated compounds were examined for their antiosteoclastogenic activity. Preliminarily results of the TRAP staining on RAW 264.7 cells indicated that compounds 1 and 11 possess potential inhibitory effects on RANKL-induced osteoclast formation. Further bioassay investigation was carried out to reveal that compounds 1 and 11 suppressed RANKL-induced osteoclast formation in a concentration-dependent manner with the inhibition up to 55% and 78% at the concentration of 10 μM, respectively. In addition, the structure-activity relationship analysis showed that the 1,3-dioxole substitute and the double bond at C-6a/C-7 in the aporphine skeleton may be responsible for the antiosteoclastogenic activity. The findings provided valuable insights for the discovery and structural modification of aporphine alkaloids as the antiosteoclastogenic lead compounds.

PURPOSE Thermal ablation is a novel treatment alternative for benign thyroid nodules, and one of the most promising thermal ablation techniques is radiofrequency ablation (RFA). Considering the increasing use of thyroid RFA, some scientific societies have proposed clinical practice guidelines. We systemically reviewed and compared these guidelines for thyroid RFA to identify a standard treatment strategy that represents the positions of most societies. METHODS We searched the MEDLINE and EMBASE databases for studies with human participants that were published in English between January 1, 2000 and August 2, 2019. Studies containing clinical practice guidelines for the RFA of benign thyroid nodules were included. We extracted data regarding indications, pre- and post-procedural evaluations, treatment techniques, and the need to obtain informed consent. RESULTS Of the 83 studies found, four studies were included, and one study was added after searching the bibliographies of those articles. The five included studies were guidelines developed by the Korean Society of Thyroid Radiology, a group of experts from Italian scientific societies, the Italian Working Group on Minimally Invasive Treatments of the Thyroid, the United Kingdom's National Institute for Health and Clinical Excellence, and a group of four professional Austrian thyroid associations. Indications, pre- and post-procedural evaluations, and techniques were similar across studies; however, differences in each of these categories were found. CONCLUSION While the reviewed guidelines are similar with regard to major categories, international guidelines for the RFA of benign thyroid nodules should be established in the future.

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (

Regulated in development and DNA damage response 1 (REDD1) expression is upregulated in response to metabolic imbalance and obesity. However, its role in obesity-associated complications is unclear. Here, we demonstrate that the REDD1–NF-κB axis is crucial for metabolic inflammation and dysregulation. Mice lacking Redd1 in the whole body or adipocytes exhibited restrained diet-induced obesity, inflammation, insulin resistance, and hepatic steatosis. Myeloid Redd1-deficient mice showed similar results, without restrained obesity and hepatic steatosis. Redd1-deficient adipose-derived stem cells lost their potential to differentiate into adipocytes; however, REDD1 overexpression stimulated preadipocyte differentiation and proinflammatory cytokine expression through atypical IKK-independent NF-κB activation by sequestering IκBα from the NF-κB/IκBα complex. REDD1 with mutated Lys219/220Ala, key amino acid residues for IκBα binding, could not stimulate NF-κB activation, adipogenesis, and inflammation in vitro and prevented obesity-related phenotypes in knock-in mice. The REDD1-atypical NF-κB activation axis is a therapeutic target for obesity, meta-inflammation, and metabolic complications.

Bone homeostasis is maintained by a combination of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Excessive osteoclast activity is linked to several bone-related disorders, including osteoporosis and rheumatoid arthritis. Pharmacological therapy might have a number of adverse effects. Therefore, the development of natural anti-osteoclastogenic drugs with greater efficacy and fewer adverse effects is desirable. In this study, the anti-osteoclastogenic effects of 23-hydroxyursolic acid (HUA), a triterpene isolated from Viburnum lutescens, were investigated in vitro and in vivo. HUA significantly inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced mature osteoclast differentiation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and F-actin ring formation. It also inhibited the expression of osteoclast-specific marker genes such OSCAR, MMP-9, TRAP, DC-STAMP, and CtsK, as well as transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. Mice orally administered with HUA (25 and 50 mg/kg) exhibited significant protection against bone loss and osteoclast formation induced by lipopolysaccharide (LPS). HUA suppressed RANKL-induced nuclear factor kappa B (NF-κB) activation and phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). These results suggest that HUA attenuates osteoclast formation in vitro and in vivo by suppressing the RANKL-mediated AP1, NF-κB, and NFATc1 pathways. Therefore, HUA may be a lead compound for the prevention or treatment of osteolytic bone disorders.

Elevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored.This study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model.The content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining.Mussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP.Mussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.

Small extracellular vesicles (sEVs) play a pivotal role in tumor progression by mediating intercellular communication in the tumor microenvironment (TME). Syntenin-1 induces malignant tumor progression in various types of human cancers, including human lung cancer and regulates biogenesis of sEVs. However, the function of syntenin-1-regulated sEVs and miRNAs in sEVs remains to be elucidated. In the present study, we aimed to demonstrate the role of oncogenic Ras/syntenin-1 axis in the release of sEVs and elucidate the function of syntenin-1-mediated miRNAs in sEVs in lung cancer progression. The results revealed that oncogenic Ras promoted the release of sEVs by inducing syntenin-1 expression; disruption of syntenin-1 expression impaired the release of sEVs as well as sEV-mediated cancer cell migration and angiogenesis. Moreover, we identified three miRNAs, namely miR-181a, miR-425-5p, and miR-494-3p, as onco-miRNAs loaded into syntenin-1-dependent sEVs. Remarkably, miR-494-3p was highly abundant in sEVs and its release was triggered by syntenin-1 expression and oncogenic Ras. Ectopic expression of the miR-494-3p mimic enhanced the migration and proliferation of lung cancer cells as well as tube formation in endothelial cells; however, the miR-494-3p inhibitor blocked sEV-mediated effects by targeting tyrosine-protein phosphatase nonreceptor type 12 (PTPN12), a tumor suppressor. sEVs promoted tumor growth and angiogenesis by downregulating PTPN12 expression; however, the miR-494-3p inhibitor significantly suppressed these effects in vivo, confirming that miR-494-3p acts as a major onco-miRNA loaded into lung cancer cell-derived sEVs. Eventually, the oncogenic Ras/syntenin-1 axis may induce cancer progression by increasing miR-494-3p loading into sEVs in lung cancer cells in the TME.

Of the 32 Trichosanthes species in China, T. kirilowii Maxim. is the most renowned species used in traditional Chinese medicine and has diverse pharmacological properties. However, most of the phytochemical studies of T. kirilowii have focused on the fruits and seeds. In our investigation of the chemical constituents of T. kirilowii roots, two previously undescribed sterols [trichosanhemiketal A and B (1 and 2)], together with 13 known compounds, were isolated and their structures were elucidated. To the best of our knowledge, this represents the first isolation of compounds with a 13,14-seco-13,14-epoxyporiferastane (1–2) skeleton from the Cucurbitaceae family. The anti-inflammatory activity of the isolated compounds was determined through an analysis of their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophage RAW264.7 cells. Of the compounds, 4, 5, 6, and 8 showed significant inhibitory activities, with IC50 values of 8.5, 15.1, 25.4, and 28.5 µM, respectively. In addition, compound 4 inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression in a concentration-dependent manner.

As part of our ongoing program to develop anti-inflammatory agents, an extract derived from Saururus chinensis collected in Korea was found to inhibit nitric oxide (NO) production in RAW264.7 cells. Bioassay-guided fractionation led to the isolation two new (1 and 2) and six known dineolignans (3–8). To the best of our knowledge, manassatin B1 (3) was isolated from S. chinensis for the first time. All structures were elucidated using extensive spectroscopic analysis. Of these compounds, 2 and 8 inhibited lipopolysaccharide (LPS)-induced production of NO and showed IC50 values of 5.80 and 1.52 µM, respectively. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was also significantly suppressed by the administration of 2 and 8. In addition, these lignans induced the expression of heme oxygenase-1 (HO-1) in a concentration-dependent manner. Nuclear translocation of nuclear-E2-related factor 2 (Nrf2), a key regulator of HO-1 protein expression, was also induced in RAW264.7 cells treated with 2 and 8. These findings suggested that these lignans exerted anti-inflammatory effects in RAW264.7 cells through modulation of the Nrf2/HO-1 pathway and that they were potential HO-1 inducers for preventing or treating inflammation.

Objective: To examine the in vitro and in vivo anti-inflammatory effects of the alkaloid enriched extract (ELA) from the roots of Eurycoma longifolia. Methods: The in vitro antiinflammatory effects of ELA were evaluated by examining its inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The level of NO produced in the culture media was determined by Griess method. The iNOS and COX-2 protein expressions were analyzed by Western blot. The in vivo effect of ELA was evaluated on LPS-induced septic shock in mice model. Mice mortality was monitored for 5 days after injection of LPS. The chemical contents of the ELA were determined by using various chromatographic and spectroscopic techniques. Results: The ELA was found to exhibit a significant anti-inflammatory effect in both in vitro and in vivo models. The results demonstrated that ELA dose-dependently inhibited LPS-induced NO production as well as the protein iNOS and COX-2 expressions. In the septic shock model, ELA dose-dependently protected mice from LPS-induced mortality. Further study on the isolated components of ELA indicated that 9,10-dimethoxycanthin-6-one may contribute significantly to the anti-inflammatory effects of the extract. Conclusions: These results suggest that ELA exhibits the anti-inflammatory activity via suppression of pro-inflammatory mediators such as NO, iNOS, and COX-2 and protects mice from LPS-induced mortality in septic shock model.

Transforming growth factor-β (TGF-β) signaling pathways are well-recognized for their role in proliferation and epithelial–mesenchymal transition (EMT) of cancer cells, but much less is understood about their contribution to interactions with other signaling events. Recent studies have indicated that crosstalk between TGF-β and Ras signaling makes a contribution to TGF-β-mediated EMT. Here, we demonstrate that Jumonji domain containing-3 (JMJD3 also called KDM6B) promotes TGF-β-mediated Smad activation and EMT in Ras-activated lung cancer cells. JMJD3 in lung cancer patients was significantly increased and JMJD3 expression in lung tumor tissues was correlated with expression of K-Ras or H-Ras in particular, and its expression was regulated by Ras activity in lung cancer cells. JMJD3 promotes TGF-β-induced Smad activation and EMT in Ras-activated lung cancer cells through the induction of syntenin, a protein that regulates TGF-β receptor activation upon ligand binding. Tissue array and ChIP analysis revealed that JMJD3 epigenetically induces syntenin expression by directly regulating H3K27 methylation levels. Mechanical exploration identified a physical and functional association of JMJD3 with syntenin presiding over the TGF-β in Ras-activated lung cancer cells. Taken together, these findings provide new insight into the mechanisms by which JMJD3 promotes syntenin expression resulting in oncogenic Ras cooperation with TGF-β to promote EMT.

The white-flowered leaves of Eclipta prostrata L. together with leaves of Scoparia dulcis and Cynodon dactylon are mixedly boiled in water and given to diabetic patients resulting in the significant improvement in the management of diabetes. However, the active constituents from this plant for antidiabetic and anti-obesity properties are remaining unclear. Thus, this study was to discover anti-diabetes and anti-obesity activities through protein tyrosine phosphatases (PTP)1B inhibitory effects. We found that the fatty acids (23, 24) showed potent PTP1B inhibition with IC

Albanol B (ABN-B), an arylbenzofuran derivative isolated from mulberries, has been shown to have anti-Alzheimer&rsquo, s disease, anti-bacterial and antioxidant activities. The aim of this study was to investigate the anti-cancer effect of this compound against lung cancer cells. The results show that ABN-B inhibited the proliferation of four human lung cancer cell lines (A549, BZR, H1975, and H226) and induced apoptosis, based on the cleavage of caspase-7 and PARP (poly (ADP-ribose) polymerase), as well as the downregulation of Bcl-2. ABN-B also induced cell cycle arrest at G2/M by down-regulating the expression of CKD1 (cyclin-dependent kinase 1) and cyclin B1, but up-regulating p21 (cyclin-dependent kinase inhibitor 1) expression. Notably, ABN-B increased the production of mitochondrial reactive oxygen species (ROS), however, treatment with mito-TEMPO (a specific mitochondrial antioxidant) blocked ABN-B-induced cell cycle arrest at G2/M and apoptosis, as well as the up-regulation of p21 and down-regulation of CDK1 and cyclin B1 induced by ABN-B. At the molecular level, ABN-B-induced mitochondrial ROS production increased the phosphorylation levels of AKT (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2), while the inhibition of these kinases blocked the ABN-B-induced up-regulation of p21 and down-regulation of CDK1 and cyclin B1. Moreover, ABN-B significantly suppressed tumor growth in Ex-3LL (Lewis lung carcinoma) tumor-bearing mice. Taken together, these results suggest that ABN-B can exert an anti-cancer effect by inducing apoptosis and cell cycle arrest at G2/M through mitochondrial ROS production in lung cancer cells.

Phytochemical investigation of Citrus unshiu peels led to the isolation of eight new flavonols (7–9, 11–15) and sixteen known compounds (1–6, 10, 16–24). Their structures were elucidated using spectroscopic analysis (1D, 2D NMR, and HR-MS). Besides, all isolated compounds (1–24) were evaluated for their inhibitory effects on receptor activator of RANKL-induced osteoclastogenesis in BMMs. Among them, dimethylmikanin (1), quercetogetin (2), 3,3′,4′,5,7,8-hexamethoxyflavone (3), 3-methoxynobiletin (4) showed a significant inhibitory effect on RANKL-induced osteoclast differentiation at a concentration of 10 μM. Moreover, 3-methoxynobiletin (4) suppressed RANKL-induced osteoclastogenesis by decreasing the number of osteoclasts and osteoclast actin-ring formation in a dose-dependent manner without causing any cytotoxic effects on BMMs. At the molecular level, 3-methoxynobiletin (4) inhibited RANKL-induced c-Fos expression and subsequently NFATc1 activation, as well as the expression of osteoclastogenesis-related marker genes c-Src and CtsK. These findings suggested that 3-methoxynobiletin (4) attenuated osteoclast differentiation by inhibiting RANKL-mediated c-Fos signaling and that it may have therapeutic potential for treating or preventing bone resorption-related diseases, such as osteoporosis.

Fourteen triterpenes, lup-20(29)-ene-3β,6β-diol (1), betulin (2), lupeol caffeate (3), 3β-caffeoyloxylup-20(29)-en-6α-ol (4), betulin-3β-yl-caffeate (5), 3β-trans-feruloylbetulin (6), betulinaldehyde 3-caffeate (7), 3-O-trans-caffeoylbetulinic acid (8), dammarenediol II 3-caffeate (9), 12-oleanene-3β,6α-diol (10), 11α-hydroxy-3β-amyrin (11), nivadiol (12), 29-hydroxyfriedelin (13), and celastrusin A (14) were isolated from Celastrus orbiculatus Thunb. and evaluated for their activity on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs). Compounds betulin (2), betulin-3β-yl-caffeate (5), 3β-trans-feruloylbetulin (6), and 3-O-trans-caffeoylbetulinic acid (8) significantly inhibited osteoclast formation in a dose-dependent manner. Among these, betulin-3β-yl-caffeate (5) exhibited the most potent inhibitory activity. We demonstrated that betulin-3β-yl-caffeate (5) suppressed F-actin-ring formation and bone resorption activity. At the molecular level, betulin-3β-yl-caffeate (5) inhibited RANK-induced expression of c-Fos and the induction of nuclear factor of activated T cells 1 (NFATc1), a key transcription factor for osteoclast formation, and it also downregulated mRNA expression of osteogenesis-associated marker genes including tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and matrix metalloprotein (MMP). These results indicate that betulin-3β-yl-caffeate (5) may be a promising candidate for the treatment of osteoclast-related diseases such as osteoporosis.

Apoptosis pathways in cells are classified into two pathways: the extrinsic pathway, mediated by binding of the ligand to a death receptor and the intrinsic pathway, mediated by mitochondria. Apoptosis is regulated by various proteins such as Bcl-2 (B-cell lymphoma 2) family and cellular FLICE (Fas-associated Death Domain Protein Interleukin-1&beta, converting enzyme)-inhibitory protein (c-FLIP), which have been reported to inhibit caspase-8 activity. In this study, it was found that C5 (3&beta, Acetyl-nor-erythrophlamide), a compound of cassaine diterpene amine from Erythrophleum fordii, induced cell apoptosis in a variety of types of cancer cells. Induction of apoptosis in cancer cells by C5 was inversely related to the level of Bcl-2 expression. Overexpression of Bcl-2 into cancer cells significantly decreased C5-induced apoptosis. It was also found that treatment of cancer cells with a caspase-8 inhibitor significantly suppressed C5-induced apoptosis, however, treatment with caspase-9 inhibitors did not affect C5-induced apoptosis, suggesting that C5 may induce apoptosis via the extrinsic pathway by activating caspase-8. It was confirmed that treatment with C5 alone induced an association of FADD with procaspase-8, however, overexpression of c-FLIP decreased C5-induced caspase-8 activation. In conclusion, C5 could be utilized as a new useful lead compound for the development of an anti-cancer agent that has the goal of apoptosis.

Degalactotigonin (1) and three other steroidal compounds solasodine (2), O-acetyl solasodine (3), and soladulcoside A (4) were isolated from the methanolic extract of Solanum nigrum, and their chemical structures were elucidated by spectroscopic analyses. The isolated compounds were evaluated for cytotoxic activity against human pancreatic cancer cell lines (PANC1 and MIA-PaCa2) and lung cancer cell lines (A549, NCI-H1975, and NCI-H1299). Only degalactotigonin (1) showed potent cytotoxicity against these cancer cell lines. Compound 1 induced apoptosis in PANC1 and A549 cells. Further study on its mechanism of action in PANC1 cells demonstrated that 1 significantly inhibited EGF-induced proliferation and migration in a concentration-dependent manner. Treatment of PANC1 cells with degalactotigonin induced cell cycle arrest at G0/G1 phase. Compound 1 induced downregulation of cyclin D1 and upregulation of p21 in a time- and concentration-dependent manner and inhibited EGF-induced phosphorylation of EGFR, as well as activation of EGFR downstream signaling molecules such as Akt and ERK.

Five new lactones, litsenolide F1 (1), lisealactone H1 (10), lisealactone H2 (11), akolactone D (13), and akolactone E (14), along with thirteen known compounds were isolated from the pericarps of Litsea japonica (Thunb.) Jussieu. Their chemical structures were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR, HRMS, and chemical methods. The isolated compounds were evaluated for their inhibitory effects on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among them, 2-alkylidene-3-hydroxy-4-methylbutanolide derivatives (compounds 1–9) exhibited the most potent activity, with IC50 values in the range of 2.9–12.8 μM. In additon, compounds 1, 3, 4, and 6 showed inhibition of iNOS and COX-2 expression in concentration-dependent manner. Compound 3 suppresses mRNA expression of iNOS, COX-2, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Based on these evidence, the isolated lactones from L. japonica could be promissing candidates for the development of new anti-inflammatory agents.

An undescribed 1,3-diphenylpropane derivative, kazinol V and six undescribed prenylated flavonoids, broussonols F–H and broussonols K–M were isolated from the roots of Broussonetia kazinoki Siebold, together with 12 known compounds. This is the first report of the isolation and structure determination of broussonol I from a natural source. The chemical structure of the undescribed compounds was determined using conventional NMR and HRMS data. Absolute configurations were assigned using time-dependent density functional theory calculations and Electronic Circular Dichroism (ECD) spectroscopy. The isolated compounds were screened for their effects on RANKL-induced osteoclast formation using RAW264.7 cells. Among them, broussonols F, G, and K showed strong, dose-dependent antiosteoclastogenic activities. Broussonol K exhibited the most potent inhibitory activity and possessed bone resorption suppressive activity.

Two new phloroglucinols (7 and 10), along with 12 known derivatives (1-6, 8-9, and 11-14), were isolated from the roots of Dryopteris crassirhizoma (Aspiadaceae). Their chemical structures were elucidated by various spectroscopic methods (1H NMR, 13C NMR, COSY, HMQC, and HMBC) and high-resolution mass spectrometry. All isolates (1-14) were tested for their inhibitory effects on melanin production in B16F10 murine melanoma cells. Norflavaspidic acid AB (8), norflavaspidic acid PB (9), and flavaspidic acid PB (12) inhibited melanin production with IC50 values of 181.3, 35.7, and 38.9 μM, respectively. Compound 9 was also shown to inhibit tyrosinase activity in a dose-dependent manner and melanogenesis with an IC50 value of 5.9 μM in B16F10 cells stimulated with 3-isobutyl-1-methylxanthine (IBMX).

Five new compounds, 10-oxomornigrol F (1), (7″R)-(-)-6-(7″-hydroxy-3″,8″-dimethyl-2″,8″-octadien-1″-yl)apigenin (2), ramumorin A (3), ramumorin B (4), and (4S,7S,8R)-trihydroxyoctadeca-5Z-enoic acid (5), together with 31 known compounds (6-36), were isolated from the twigs of Morus alba (Moraceae). The chemical structures of these compounds were established using spectroscopic analyses, 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and Mosher's methods. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophage RAW 264.7 cells. Compounds 1, 2, 13, 17, 19, 25-28, and 32 showed inhibitory effects with IC50 values ranging from 2.2 to 5.3μg/mL. Compounds 1, 2, 17, 25, and 32 reduced LPS-induced inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner. In addition, pretreating the cells with compound 1, 17, and 32 significantly suppressed LPS-induced expression of cyclooxygenase-2 (COX-2) protein.

A phytochemical investigation into the bark of Erythrophleum fordii yielded four new compounds, two new cassaine diterpenoids (erythrofordin T and U, 1 and 2) and two new cassaine diterpenoid amines (erythroformine A and B, 6 and 7), as well as nine known compounds. We report for the first time the isolation of erythrofordin V (3) from a natural source and that of the remaining eight known diterpenoids (4-5, 8-13) from E. fordii. All structures were elucidated using spectroscopic analysis. Cytotoxic activity of the isolated compounds (1-13) was examined in vitro against three non-small cell lung cancer cell lines (A549, NCI-H1975, and NCI-H1229) using the MTT assay. Cassaine diterpene amines (6-10, 12, 13) exhibited potent cytotoxic activity against all three cell lines with IC50 values between 0.4μM and 5.9μM. Erythroformine B (7) significantly induced apoptosis in all three cancer cells in a concentration-dependent manner.

// Nara Tae 1 , Suhyun Lee 1 , Okwha Kim 1 , Juhee Park 1 , Sunghun Na 2 and Jeong-Hyung Lee 1 1 Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-Do 243-41, Republic of Korea 2 Department of Obstetrics and Gynecology, Kangwon National University Hospital, school of Medicine, Kangwon National University, Chuncheon, Gangwon-Do 243-41, Republic of Korea Correspondence to: Jeong-Hyung Lee, email: jhlee36@kangwon.ac.kr Keywords: syntenin, VEGFR2 endocytosis, ephrin-B2, endothelial cell, angiogenesis Received: July 04, 2016 Accepted: February 27, 2017 Published: March 22, 2017 ABSTRACT Syntenin, a tandem PDZ-domain-containing scaffold protein, is involved in the regulation of diverse biological functions, including protein trafficking, exosome biogenesis, and cancer metastasis. Here, we present the first study to explore the significance of syntenin in endothelial cells. Syntenin knockdown in human umbilical vein endothelial cells (HUVECs) impaired vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, vascular permeability, and nitric oxide (NO) production. Syntenin knockdown also suppressed expression of the VEGFR2 target genes VEGF, MMP2 , and Nurr77 as well as VEGF-induced angiogenesis in vitro and in vivo . And it decreased cell-surface levels of ephrin-B2. Biochemical analyses revealed that syntenin exists in complex with VEGFR2 and ephrin-B2. Syntenin knockdown abolished the association between VEGFR2 and ephrin-B2, suggesting syntenin functions as a scaffold protein facilitating their association in HUVECs. Consistent with these observations, knocking down syntenin or ephrin-B2 abolished VEGF-induced endocytosis and VEGFR2 phosphorylation and activation of its downstream signaling molecules. Treatment with MG132, a proteasome inhibitor, rescued the downregulation of ephrin-B2 and VEGFR2 signaling induced by syntenin knockdown. These findings demonstrate that syntenin promotes VEGF signaling and, through its PDZ-dependent interaction with ephrin-B2, enhances VEGF-mediated VEGFR2 endocytosis and subsequent downstream signaling and angiogenesis in endothelial cells.

In the present study, ten alkaloids, namely chabamide (1), pellitorine (2), retrofractamide A (3), pyrroperine (4), isopiperolein B (5), piperamide C9:1 (8E) (6), 6,7­dehydrobrachyamide B (7), 4,5-dihydropiperine (8), dehydropipernonaline (9), and piperine (10), were isolated from the fruits of Piper nigrum. Among these, chabamide (1), pellitorine (2), retrofractamide A (3), isopiperolein B (5), and 6,7­dehydrobrachyamide B (7) exhibited significant inhibitory activity on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells, with IC50 values of 6.8, 14.5, 30.2, 23.7, and 38.5 μM, respectively. Furthermore, compound 1 inhibited lipopolysaccharide-induced NO production in bone marrow-derived macrophages with IC50 value of 9.5 μM. Consistent with NO inhibition, treatment of RAW264.7 cells with chabamide (1), pellitorine (2), and 6,7­dehydrobrachyamide B (7) suppressed expression of inducible NO synthase and cyclooxygenase-2. Chabamide (1), pellitorine (2), and 6,7­dehydrobrachyamide B (7) induced heme-oxygenase-1 expression at the transcriptional level. In addition, compound 1 induced the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and upregulated the expression of Nrf2 target genes, NAD(P)H:quinone oxidoreductase 1 and γ-glutamyl cysteine synthetase catalytic subunit, in a concentration-dependent manner in RAW264.7 cells. These findings suggest that chabamide (1) from P. nigrum exert antiinflammatory effects via the activation of the Nrf2/heme-oxygenase-1 pathway; hence, it might be a promising candidate for the treatment of inflammatory diseases. Copyright © 2017 John Wiley & Sons, Ltd.

The chemical characterization of Selaginella tamariscina leaves resulted in the isolation of five lignanoside derivatives (1-4 and 6) and one neolignan (5). These compounds include three new lignanosides, tamariscinosides D-F (1-3), and one liriodendrin (4) that were isolated for the first time from this plant, together with two known compounds, (2R,3S)-dihydro-2-(3,5-dimethoxy-4-hydroxyphenyl)-7-methoxy-5-acetyl-benzofuran (5) and moellenoside B (6). The chemical structures of these isolated compounds were determined using 1D and 2D NMR, MS, and CD spectroscopic data, and the results were compared to data previously reported in the literatures. These compounds were also evaluated in terms of their inhibition of NO production in lipopolysaccharide (LPS)-stimulated activity in the macrophage cell line RAW 264.7. Among them, compounds 1, 2, 5, and 6 exhibited a significant inhibition with IC50 values ranging from 32.3 to 55.8μM.

Four new diterpene-type compounds normiltirone (

Four new diterpene-type compounds normiltirone (3) and isosalviamides F-H (14–16) together with twelve known compounds (1, 2, 4–13) were isolated from the roots of Salvia miltiorrhiza. Their structures were mainly elucidated from detailed spectroscopic data. All isolated compounds were evaluated for their ability to inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages. Compound 11 showed a strong inhibitory effect, with an IC50 value of 3.4 ± 1.2 μM.

Targeting the vascular endothelial growth factor (VEGF)/its receptor-2 (VEGFR-2) system has become a mainstay of treatment for many human diseases, including retinal diseases. We examined the therapeutic effect of recently developed N-acetylated Arg-Leu-Tyr-Glu (Ac-RLYE), a human plasminogen kringle-5 domain-derived VEGFR-2 antagonists, on the pathogenesis of diabetic retinopathy. Ac-RLYE inhibited VEGF-A-mediated VEGFR-2 activation and endothelial nitric oxide synthase (eNOS)-derived NO production in the retinas of diabetic mice. In addition, Ac-RLYE prevented the disruption of adherens and tight junctions and vascular leakage by inhibiting S-nitrosylation of β-catenin and tyrosine nitration of p190RhoGAP in the retinal vasculature of diabetic mice. Peptide treatment preserved the pericyte coverage of retinal capillaries by upregulating angiopoietin-2. These results suggest that Ac-RLYE potentially prevents blood-retinal barrier breakdown and vascular leakage by antagonizing VEGFR-2; Ac-RLYE can be used as a potential therapeutic drug for the treatment of diabetic retinopathy.

Three polyacetylenes, one novel and two known. were isolated from the root of Angelica tenuissima. U ing 1H- and13C_NMR, COSY. HMBC, and HMQC, their truciure were found to be (3R,8S)-heptadeca-1-en-4.6-diyne-3,8-diol (1), falcarindiol (2). and oplopandiol (3). Absolute configuration of compound 1 were established using Mosher`s esterification. In addition. the polyacetylenes (1 - 3) were evaluated for their anti-inflammatory activity. Compounds 1 and 3 showed potent inhibitory activity against lipopolysaccharide-induced nitric oxide (N0) production in RAW267.7 macrophage cell with IC50 value of 4.31 and 5.06 μM. respectively. Compound 1. strongly inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in a concentration-dependent manner.

Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3&alpha, 23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3&alpha, hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-&kappa, B ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F&ndash, actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.

The flower buds of Cleistocalyx operculatus are used as an important ingredient in herbal tea and herbal products in several tropical countries. However, their protective effects and underlying mechanisms on lipopolysaccharide (LPS)-induced endotoxic shock remain unclear. The aim of this study was to investigate the anti-inflammatory effects of ethanol extract of C. operculatus flower buds (ECO) and its major constituent 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) in macrophages and in an experimental LPS-induced sepsis mouse model. ECO inhibited the LPS-induced production and expression of pro-inflammatory mediators in macrophages. In an endotoxic shock mouse model, the oral administration of ECO rescued LPS-induced mortality, and attenuated LPS-induced increases in the serum levels of pro-inflammatory mediators, and damage of the lung and liver tissues. ECO increased the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2), as well as the expression of Nrf2 target genes, including heme oxygenase-1 (HO-1), in macrophages. Similar to the effects of ECO, DMC also inhibited the LPS-induced inflammatory response in macrophages and endotoxic shock in mice, and activated the Nrf2/HO-1 pathway. In conclusion, our findings suggested that ECO and its major constituent, DMC, attenuated LPS-induced endotoxic shock by activating the Nrf2/HO-1 pathway.

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4'-Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c-Jun-N-terminal kinase signaling pathway without affecting extracellular signal-regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c-Fos, nuclear factor-activated T cells cytoplasm 1, cathepsin K, and c-src by RANKL. We used a lipopolysaccharide (LPS)-induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF-treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL-induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS-induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption.