Isoorientin Inhibits Amyloid ß25-35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway.

Alzheimer's disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aß) peptides. Aß induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aß-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3',4',5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aß-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aß-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aß25-35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aß25-35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aß25-35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.

Characterization and Optimization of the Tyrosinase Inhibitory Activity of Vitis amurensis Root Using LC-Q-TOF-MS Coupled with a Bioassay and Response Surface Methodology.

Vitis amurensis roots have been reported to have the potential for skin whitening through the evaluation of melanogenesis and tyrosinase inhibitory activities. In this study, V. amurensis roots were utilized to quickly select whitening ingredients using LC-Q-TOF-MS coupled with tyrosinase inhibitory assay, and to optimize the extraction process for use as a skin whitening functional material by response surface methodology. Results showed that V. amurensis roots exhibited tyrosinase inhibitory effects by two stilbene oligomers, ε-viniferin (1) and vitisin B (2), as predicted by LC-Q-TOF-MS coupled with bioassay. The optimal extraction conditions (methanol concentration 66%, solvent volume 140 mL, and extraction time 100 min) for skin whitening ingredients were established with the yields 6.20%, and tyrosinase inhibitory activity was 87.27%. The relationship between each factor and its corresponding response was confirmed by Pearson's correlation analysis. The solvent volume showed clear linear relationship with yields, and methanol concentration had a strong linear relationship with tyrosinase inhibitory activity for compounds 1 and 2, as well as their combination. Overall, LC-Q-TOF-MS coupled with bioassay was proved to have the potential to effectively find new active constituents, as well as known active constituents; vitisin B can be proposed as a new natural potential whitening agent.

Minecoside Modulates Cell Invasion via Regulation of CXCR4 Expression in Breast and Colon Cancer Cells.

Metastasis, which is closely linked to cancer-related deaths, is a highly complex process. It is an organ-specific process and involves interactions between the host and cancer cells. CXC chemokine receptor 4 is known to be expressed in various tumors and the binding with CXC ligand 12 induces signaling in cancer cell survival, migration, and proliferation. Particularly, the CXC chemokine receptor 4/CXC ligand 12 axis is known to promote the metastasis of breast cancer. Thus, agents that can downregulate CXC chemokine receptor 4 expression have potential against cancer metastasis. Minecoside is an active compound extracted from Veronica peregrina L. It is widely distributed in Korea and has been used as a traditional drug for the treatment of various chronic diseases. However, the anticancer and anti-inflammatory effects of minecoside have yet to be clarified. In this study, we found that minecoside downregulates constitutive CXC chemokine receptor 4 expression in MDA-MB-231 breast cancer cells. This downregulation also occurred at the transcriptional level. Minecoside-mediated suppression of CXC chemokine receptor 4 expression inhibited CXC ligand 12-induced invasion of breast and colorectal cancer cells. Overall, our results suggest that minecoside can be a novel anticancer agent that can inhibit cancer metastasis through inhibition of CXC chemokine receptor 4 expression.

Characterization of Anti-Inflammatory and Antioxidant Constituents from Scutellaria baicalensis Using LC-MS Coupled with a Bioassay Method.

An effective and previously demonstrated screening method for active constituents in natural products using LC-MS coupled with a bioassay was reported in our earlier studies. With this, the current investigation attempted to identify bioactive constituents of Scutellaria baicalensis through LC-MS coupled with a bioassay. Peaks at broadly 17-20 and 24-25 min on the MS chromatogram displayed an inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. Similarly, peaks at roughly 17-19 and 22 min showed antioxidant activity with an 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/2,2-diphenyl-1- picrylhydrazyl (DPPH) assay. For confirmation of LC-MS coupled with a bioassay, nine compounds (1-9) were isolated from an MeOH extract of S. baicalensis. As we predicted, compounds 1, 8, and 9 significantly reduced lipopolysaccharide (LPS)-induced NO production in BV2 cells. Likewise, compounds 5, 6, and 8 exhibited free radical-scavenging activities with the ABTS/DPPH assay. In addition, the structural similarity of the main components was confirmed by analyzing the total extract and EtOAc fractions through molecular networking. Overall, the results suggest that the method comprised of LC-MS coupled with a bioassay can effectively predict active compounds without an isolation process, and the results of molecular networking predicted that other components around the active compound node may also be active.

Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction.

Thiazolidinedione is a five-membered heterocycle that is widely used in drug discovery endeavors. In this study, we report the design, synthesis, and biological evaluation of a series of thiazolidinedione-based HDAC6 inhibitors. In particular, compound 6b exerts an excellent inhibitory activity against HDAC6 with an IC50 value of 21 nM, displaying a good HDAC6 selectivity over HDAC1. Compound 6b dose-dependently induces the acetylation level of α-tubulin via inhibition of HDAC6 in human neuroblastoma SH-SY5Y cell line. Moreover, compound 6b efficiently reverses methamphetamine-induced morphology changes of SH-SY5Y cells via regulating acetylation landscape of α-tubulin. Collectively, compound 6b represents a novel HDAC6-isoform selective inhibitor and demonstrates promising therapeutic potential for the treatment of methamphetamine addiction.

Hair Metabolomics in Animal Studies and Clinical Settings.

Metabolomics is a powerful tool used to understand comprehensive changes in the metabolic response and to study the phenotype of an organism by instrumental analysis. It most commonly involves mass spectrometry followed by data mining and metabolite assignment. For the last few decades, hair has been used as a valuable analytical sample to investigate retrospective xenobiotic exposure as it provides a wider window of detection than other biological samples such as saliva, plasma, and urine. Hair contains functional metabolomes such as amino acids and lipids. Moreover, segmental analysis of hair based on its growth rate can provide information on metabolic changes over time. Therefore, it has great potential as a metabolomics sample to monitor chronic diseases, including drug addiction or abnormal conditions. In the current review, the latest applications of hair metabolomics in animal studies and clinical settings are highlighted. For this purpose, we review and discuss the characteristics of hair as a metabolomics sample, the analytical techniques employed in hair metabolomics and the consequence of hair metabolome alterations in recent studies. Through this, the value of hair as an alternative biological sample in metabolomics is highlighted.

Ascochlorin Suppresses MMP-2-Mediated Migration and Invasion by Targeting FAK and JAK-STAT Signaling Cascades.

Human glioblastomas express higher levels of matrix metalloprotease-2 (MMP-2) than low-grade brain tumors and normal brain tissues. Ascochlorin (ASC) has anti-metastatic, anti-angiogenic, and synergistic effect in various types of cancer cells. However, it remains unknown whether ASC can affect cell migration and invasion in malignant human glioma cells. In this study, we found that ASC indeed inhibits cell migration and invasion in U373MG and A172. ASC significantly suppresses the MMP-2 gelatinolytic activity and expression in U373MG and A172. To determine the molecular mechanism by which ASC suppressed cell migration and invasion, we investigated whether ASC could modulate metastasis via focal adhesion kinase (FAK) and janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, a potential drug target. ASC strongly inhibits the phosphorylation of FAK, and treatment with a FAK inhibitor significantly suppresses cancer cell migration in the presence of ASC. In addition, ASC significantly decreased phosphorylation of JAK2/STAT3, cancer cell migration and nuclear translocation of STAT3. Taken together, these results suggest that ASC inhibits cell migration and invasion by blocking FAK and JAK/STAT signaling, resulting in reduced MMP-2 activity. J. Cell. Biochem. 119: 300-313, 2018. © 2017 Wiley Periodicals, Inc.

Characterization of inhibitory constituents of NO production from Catalpa ovata using LC-MS coupled with a cell-based assay.

An effective screening method for inhibitors of NO production in natural products using LC-QTOF MS/MS coupled with a cell-based assay was proposed. The ethyl acetate fraction of Catalpa ovata exhibited a strong inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. We attempted to identify the active constituents of C. ovata by using LC-QTOF MS/MS coupled with a cell-based assay. Peaks at approximately 14-15 min on the MS chromatogram were estimated to be the bioactive constituents. A new iridoid compound, 6-O-trans-feruloyl-3ß-hydroxy-7-deoxyrehamaglutin A (4), and nine known compounds (1-3, 5-10) were isolated from the ethyl acetate fraction of C. ovata by repeated column chromatography. Compounds 3, 4, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells. Our results indicate that LC-QTOF MS/MS coupled with a cell-based NO production inhibitory assay successfully predicted active compounds without a time-consuming isolation process.

Pomolic Acid Ameliorates Fibroblast Activation and Renal Interstitial Fibrosis through Inhibition of SMAD-STAT Signaling Pathways.

Fibrosis is a common pathological feature in most kinds of chronic kidney disease. Transforming growth factor ß1 (TGF-ß1) signaling is the master pathway regulating kidney fibrosis pathogenesis, in which mothers against decapentaplegic homolog 3 (SMAD3) with signal transducer and activator of transcription 3 (STAT3) act as the integrator of various pro-fibrosis signals. We examine the effects of pomolic acid (PA) on mice with unilateral ureteral obstruction (UUO) and TGF-ß1 stimulated kidney fibroblast cells. UUO mice were observed severe tubular atrophy, and tubulointerstitial fibrosis and extracellular matrix (ECM) deposition at seven days postoperatively. However, PA-treated UUO mice demonstrated only moderate injury, minimal fibrosis, and larger reductions in the expression of ECM protein and epithelial-mesenchymal transition (EMT) progress. PA inhibited the SMAD-STAT phosphorylation in UUO mice. PA effects were also confirmed in TGF-ß1 stimulated kidney fibroblast cells. In this study, we first demonstrated that PA ameliorates fibroblast activation and renal interstitial fibrosis. Our results indicate that PA may be useful as a potential candidate in the prevention of chronic kidney disease.

Crocin Suppresses Constitutively Active STAT3 Through Induction of Protein Tyrosine Phosphatase SHP-1.

The aim of the present study is to investigate the effect of a natural compound crocin, one of the active components of saffron, on human multiple myeloma cells. Crocin effectively suppressed constitutive STAT3 activation, translocation of STAT3 to the nucleus, and its target gene expression. The suppression of STAT3 was mediated through the inhibition of activation of protein tyrosine kinases JAK1, JAK2, and c-Src. We found that crocin induced the expression of SHP-1, a tyrosine protein phosphatase, and pervanadate treatment reversed the crocin-induced downregulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Moreover, suppression of SHP-1 by its inhibitor overturned the effect of crocin on induction of SHP-1 and the inhibition of STAT3 activation. Finally, crocin downregulated the expression of STAT3-mediated gene products including anti-apoptotic (Bcl-2), pro-apoptotic (BAX), invasive (CXCR4), angiogenic (VEGF), and cell cycle regulator (cyclin D1), which are correlated with suppression of proliferation, the accumulation of cells in sub-G1 phase of cell cycle, and induction of apoptosis. Overall, our results suggested that crocin is a novel inhibitor of STAT3 activation pathway and thus may have potential in prevention and treatment of human multiple myeloma. J. Cell. Biochem. 118: 3290-3298, 2017. © 2017 Wiley Periodicals, Inc.

Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway.

BACKGROUND: Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway. METHODS: We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways. RESULTS: METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on mitochondrial membrane integrity was also confirmed by flow cytometry and immunofluorescence staining. CONCLUSIONS: Based on the literatures and our findings, AA is a promising candidate for an anti-neurotoxic agent, and it can potentially be used for the prevention and treatment of various neurological disorders.

Pomolic Acid Inhibits Invasion of Breast Cancer Cells Through the Suppression of CXC Chemokine Receptor Type 4 Expression.

High mortality of cancer-mediated deaths is due to metastasis. CXC chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in migration of breast cancer. Thus, identification of CXCR4 inhibitor has been challenged constantly as an anticancer drug. This study is aimed to investigate the CXCR4 inhibitor that could inhibit tumor metastasis from natural products. We demonstrated that pomolic acid (PA), a component of Euscaphis japonica, could downregulate CXCR4 expression in breast cancer cells. Treatment with proteasomal and lysosomal inhibitors did not show significant effects on PA's ability. When we further explored the molecular mechanism, suppression of CXCR4 occurred at transcriptional level and was correlated with inhibition of nuclear factor-kappaB (NF-κB) activation. Downregulation of CXCR4 by PA was accompanied by the inhibition of CXC motif chemokine 12 (CXCL12)-induced invasion of breast cancer cells. Overall, our results indicate that PA, as a novel inhibitor of CXCR4, can be a promising therapeutic agent for treatment of cancer metastasis.

Phosphorylation of Smac by Akt promotes the caspase-3 activation during etoposide-induced apoptosis in HeLa cells.

The Akt, family of serine/threonine protein kinases functions as key regulators of multiple aspects of cell behavior, such as survival, proliferation, migration, and carcinogenesis. Notably, Akt exerts its anti-apoptotic effects through the phosphorylation of numerous substrates related with cell cycle, genome stability, and cancer development. In this report, nevertheless, we focused our view on the novel role of Akt which involves in a pro-apoptotic action by phosphorylating second mitochondria derived activator of caspases (Smac) protein during etoposide-induced apoptotic processes. Our data reveals that Akt could bind to and phosphorylate Smac at serine residue 67, which enhances the ability of Smac to interact with the cytosolic X-chromosome linked IAP (XIAP) protein. The cellular interaction of wild-type Smac with XIAP was enhanced with similar activation kinetics of Akt activity, while this interaction was markedly attenuated in cells expressing the phosphorylation-defective mutant S67A-Smac during etoposide-induced apoptosis. Moreover, we provide the evidence indicating that the phosphorylation of Smac at ser-67 markedly upregulates the caspase-3 activity by promoting the interaction of Smac with XIAP. Taken together, we propose that the phosphorylation of Smac by Akt might be a novel mechanism that involves in amplification of caspase cascade pathway during etoposide-induced apoptosis in HeLa cells.

Baohuoside I suppresses invasion of cervical and breast cancer cells through the downregulation of CXCR4 chemokine receptor expression.

More than 90 percent of cancer-mediated deaths are due to metastasis, but the mechanisms that control metastasis remain poorly understood. Thus, the therapy targeting this process has been challenged constantly, but no therapy has yet been approved. CXC chemokine receptor 4 (CXCR4), a Gi protein-coupled receptor for the CXC chemokine ligand (CXCL) 12/stromal cell derived factor (SDF) 1α, is known to be expressed in various tumors. Recently, the CXCL12/CXCR4 axis has emerged as a key mediator of tumor metastasis; therefore, the possibility that identification of CXCR4 inhibitors can be a promising strategy for abrogating metastasis has been considered. In this report, we investigate baohuoside I, a component of Epimedium koreanum, as a regulator of CXCR4 expression as well as function in cervical cancer and breast cancer cells. We observed that baohuoside I downregulated CXCR4 expression in a dose- and time-dependent manner in HeLa cells. Treatment with a pharmacological proteasome and lysosomal inhibitors did not have a substantial effect on baohuoside I's ability to suppress CXCR4 expression. When we investigated the molecular mechanism of action, it was observed that the suppression of CXCR4 expression occurred at the level of mRNA. The decrease in the level of CXCR4 expression caused by baohuoside I was correlated with inhibition of the CXCL12-induced invasion of both cervical and breast cancer cells. Overall, our results show that baohuoside I exerts its antimetastatic effect through the downregulation of CXCR4 expression and, thus, has the potential to play a role in the suppression of cancer metastasis.

[Corrigendum] Saffron carotenoids inhibit STAT3 activation and promote apoptotic progression in IL­6­stimulated liver cancer cells.

Following the publication of the above article, an interested reader drew to the authors' attention that, in Fig. 1E on p. 1885, the STAT3 blots shown for the A549 and A2780 cell lines were strikingly similar, such that these data were possibly derived from the same original source where the panels were intended to show the results from differently performed experiments. Upon examining their original data, the authors have realized that an inadvertent error was made in assembling the data in the figure, and the STAT3 data shown correctly for the A549 cell line were erroneously copied across for the A2780 cell line. The corrected version of Fig. 1, showing the correct STAT3 blot for the A2780 cell line in Fig. 1E, is shown on the next page. Note that this error did not affect the overall conclusions reported in the paper. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this. They also apologize to the readership for any inconvenience caused. [Oncology Reports 39: 1883­1891, 2018; DOI: 10.3892/or.2018.6232].

Retraction notice to "Effects of geniposide on hepatocytes undergoing epithelial-mesenchymal transition in hepatic fibrosis by targeting TGFß/Smad and ERK-MAPK signaling pathways" [Biochimie 113 (2015) 26-34].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The corresponding author, Dr Byoungduck Park, requested publication of a corrigendum to correct Fig. 2B which reused control data from a different publication (doi: 10.1016/j.intimp.2015.02.014). Upon further inspection, the Biochimie editorial team noticed that: Comparison of Fig. 2B with Fig. 4C of a previous publication in International Immunopharmacology by two co-authors (doi: 10.1016/j.intimp.2015.02.014) reveals that western blot ß-actin control data from the earlier paper were re-used in a different experiment shown in Fig. 2B of the article in Biochimie, after adjustment of the brightness/contrast. Furthermore, the same bands, after more image manipulation were presented as Smad3 data in Fig. 4C of the Biochimie article. Here the image manipulation involved notably the rotation of the set of bands by 180° and some adjustment of the height/width ratio. The authors apologise for any confusion that may have arisen from their article.

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax.

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.

Minecoside promotes apoptotic progression through STAT3 inactivation in breast cancer cells.

Breast cancer is one of the most common malignant tumors in women worldwide, and is a major cause of mortality and morbidity in cancer patients. Constitutive activation of STAT3 has been found in a variety of malignant tumors, including breast cancer. Since STAT3 activation is capable of regulating various important features of tumor cells, identification of a novel STAT3 inhibitor is considered a potential strategy for treating breast cancer. The aim of the present study was to examine whether minecoside (MIN), an active compound extracted from Veronica peregrina L., exerts an antitumor effect by inhibiting STAT3 signaling pathway in MDA-MB-231 cells. The results revealed that MIN inhibited the constitutive STAT3 activation in a dose- and time-dependent manner. MIN also blocked the nuclear translocation of STAT3 and suppressed STAT3-DNA binding. In addition, MIN downregulated the STAT3-mediated expression of proteins such as Bcl-xL, Bcl-2, CXCR4, VEGF, and cyclin D1. Subsequently, MIN promoted the caspase-dependent apoptosis in MDA-MB-231 cells. Overall, results of the present study provide evidence that MIN exerted anticancer activity via inhibition of the STAT3 signaling pathway. Further studies using animal models are required to determine the potential of this molecule as an anticancer drug.