Medicinal Chemistry : RESEARCH ; NOX1 to NOX2 Switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7

( Suhrid Banskota ),( Sushil C. Regmi ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2015 영남대학교 약품개발연구소 연구업적집 Vol.25 No.-

Background:Although matrix metalloproteinase(MMP)-7 expression is correlated with increased metastatic potential in human colon cancer cells, the underlying molecular mechanism of incasive phenotype remains unknown. In the current study, we investigated the regulatory effects of membrane NADPH oxidase (NOX) and AMP activated protein kinase (AMPK) on MMP-7 expression and invasive phenotype change in colon cancer cells. Methods:Production of superoxide anion was measured by lucigenin chemiluminescence assay using whole cells and protein extracts(NADPH oxidase activity), and intracellular rective oxygen species (ROS) by fluorescence microscopy using 2`,4`-dichlorofluorescein diacetate(DCF-DA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. siRNA transfecition was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Luciferase reporter assay was performed to identify transcription factors linked to gene expression.Results:Under basal conditions, less invasive human colon cancer cells (HT29 and Caco-2) showed low MMP-7 expression but high NOX1 expression and AMPK phosphorylation. Treatment of HT29 and Caco-2 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) induced an invasive phenotype response along with corresponding increases basal conditions of highly invasive human colon cancer cells(SW620 and HCT116). In addition, inverse regulation between WMPK phosphorylation and NOX2 and MMP-7 expression was obserced in HT29 cells treated with different by treatment with Vit. E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype. TPA-induced of MMP-7 expression was suppressed by AP-1, NF-kB, and MAPK(ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-KB inhibitors. Conclusions:Molecular switch from NOX1 to NOX2 in colon cancer cells induces ROS production and subsequently enhances MMP-7 expression by deactivating AMPK, which otherwise inhibits stimulus-induced autoregulation of ROS and NOX2 gene expression.

BJ-1108,a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog, Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

( Suhrid Banskota ),( Jaya Gautam ),( Sushil C Regmi ),( Pallavi Gurung ),( Myo Hyeon Park ),( Seung Joo Kim ),( Tae Gyu Nam ),( Byeong Seon Jeong ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demon-strate that 5-HT-induced angiogenesis was mediated through the 5-HT, receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPKIERKlp38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-11 08, a derivative of 6-amino-2,4,5-trimethylpyri-oln-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-11 08 induced a significant reduction in the size and weight of excised tumors in breast cancer ceil-inocu-lated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-11 08 signifi-cantly suppressed 5-HT-induced ROS generation and phosphorylation of P13K1Akt but not of Src. Unlike NOX inhibitors, BJ-11 08, which showed better antioxidant activity than vita-min C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-11 08 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K1NOX signaling but not through Src, ERK, or p38.

Serotonin disturbs colon epithelial tolerance of commensal <i>E. coli</i> by increasing NOX2-derived superoxide

Banskota, Suhrid,Regmi, Sushil Chandra,Gautam, Jaya,Gurung, Pallavi,Lee, Yu-Jeong,Ku, Sae Kwang,Lee, Jin-Hyung,Lee, Jintae,Chang, Hyeun Wook,Park, Sang Joon,Kim, Jung-Ae Elsevier 2017 FREE RADICAL BIOLOGY AND MEDICINE Vol.106 No.-

<P><B>Abstract</B></P> <P>Adherent-invasive <I>E. coli</I> colonization and Toll-like receptor (TLR) expression are increased in the gut of inflammatory bowel disease (IBD) patients. However, the underlying mechanism of such changes has not been determined. In the current study, it was examined whether gut serotonin (5-hydroxytryptamine, 5-HT) can induce adherent-invasive <I>E. coli</I> colonization and increase TLR expression. In a co-culture system, commensal <I>E. coli</I> strain (BW25113, BW) adhered minimally to colon epithelial cells, but this was significantly enhanced by 5-HT to the level of a pathogenic strain (EDL933). Without inducing bacterial virulence, such as, biofilm formation, 5-HT enhanced BW-induced signaling in colon epithelial cells, that is, NADPH oxidase (NOX)-dependent superoxide production, the up-regulations of IL-8, TLR2, TLR4, and ICAM-1, and the down-regulations of E-cadherin and claudin-2. In a manner commensurate with these gene modulations, BW induced an increase in NF-κB and a decrease in GATA reporter signals in colon epithelial cells. However, 5-HT-enhanced BW adhesion and colon epithelial responses were blocked by knock-down of NOX2, TLR2, or TLR4. In normal mice, 5-HT induced the invasion of BW into gut submucosa, and the observed molecular changes were similar to those observed <I>in vitro</I>, except for significant increases in TNFα and IL-1β, and resulted in death. In dextran sulfate sodium-induced colitis mice (an IBD disease model), in which colonic 5-HT levels were markedly elevated, BW administration induced death in along with large amount of BW invasion into colon submucosa, and time to death was negatively related to the amount of BW injected. Taken together, our results demonstrate that 5-HT induces the invasion of commensal <I>E. coli</I> into gut submucosa by amplifying commensal bacteria-induced epithelial signaling (superoxide production and the inductions of NOX2 and TLR2/TLR4). The authors suggest that these changes may constitute the molecular basis for the pathogenesis of IBD.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 5-HT induces adhesive invasion of commensal <I>E. coli</I> to colon epithelial cells. </LI> <LI> Commensal <I>E. coli</I> induces colon epithelial NOX2 activation via TLR-2 and TLR-4. </LI> <LI> 5-HT amplifies commensal <I>E. coli</I>-induced up-regulation of TLR2/TLR4, IL-8, and ICAM-1 via NOX2. </LI> <LI> 5-HT enhances <I>E. coli</I>-induced down-regulation of E-cadherin. </LI> <LI> Inoculation of commensal <I>E.coli</I> with high 5-HT levels induces fatal colon inflammation in mice. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Ameliorating effect of TI-1-162, a hydroxyindenone derivative, against TNBS-induced rat colitis is mediated through suppression of RIP/ASK-1/MAPK signaling

Gurung, Pallavi,Banskota, Suhrid,Katila, Nikita,Gautam, Jaya,Kadayat, Tara Man,Choi, Dong-Young,Lee, Eung Seok,Jeong, Tae Cheon,Kim, Jung-Ae Elsevier 2018 european journal of pharmacology Vol.827 No.-

<P>The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro-inflammatory cytokines including TNF-alpha. Once generated, TNF-alpha stimulates production of various pro-inflammatory cytokines and disrupts mucosal barrier by inducing inflamed mucosal epithelial cell death. In the present study, we investigated inhibitory effects of TI-1-162, a hydroxyindenone derivative, against TNF-alpha-induced and TNBS-induced colon inflammation. TI-1-162 showed inhibitory effect on the TNF-alpha-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells (IC50 = 0.83 +/- 0.12 mu M), which is an in vitro model representing the initial step of colitis. In addition, TI-1-162 suppressed TNF-alpha-stimulated caspase-3 activation and HT-29 cell apoptosis. These in vitro inhibitory activities of TI-1-162 correlated to recovery changes in in vivo colon tissues, such as downregulation of adhesion molecules (ICAM-1, VCAM-1) and chemokines (CCL11, CXCL1, CXCL2, CXCL3, CX3CL1) revealed by gene expression array and Western blot analyses. Such molecular recovery of colon epithelium from TNBS-treated rats corresponded to the recovery in body weight, colon weight/length, and myeloperoxidase level by TI-1-162 (10 and 30 mg/kg/day, orally). In relation to action mechanism, TI-1-162 did not disturb TNF-alpha binding to its receptor, but suppressed phosphorylation of RIP-1, ASK-1, JNK and p38, and nuclear translocation of NF-kB and AP-1, which corresponded to down regulation of inflammatory cytokines in TNF-alpha-treated cells (HT-29 and U937) and TNBS-treated rat colon tissues. Taken together, the results indicate that the protective effects of TI-1-162 against colon inflammation and epithelial cell death are associated with its inhibitory action in RIP/ASK-1/MAPK signaling pathway downstream to TNF receptor 1.</P>

4-Hydroxynonenal-induced GPR109A (HCA₂ receptor) activation elicits bipolar responses, G_αi -mediated anti-inflammatory effects and G_βγ -mediated cell death : Bipolar responses of 4-HNE-activated GPR109A

Gautam, Jaya,Banskota, Suhrid,Shah, Sajita,Jee, Jun-Goo,Kwon, Eunju,Wang, Ying,Kim, Dong Young,Chang, Hyun Wook,Kim, Jung-Ae Wiley (Blackwell Publishing) 2018 British journal of pharmacology Vol.175 No.13

Down-regulation of cathepsin S and matrix metalloproteinase-9 via Src, a non-receptor tyrosine kinase, suppresses triple-negative breast cancer growth and metastasis

Jaya Gautam,Suhrid Banskota,이현지,이유정,전용현,김정애,정병선 생화학분자생물학회 2018 Experimental and molecular medicine Vol.50 No.-

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. In the present study, we demonstrated that Src, a non-receptor tyrosine kinase, might provide an effective therapeutic strategy to overcome TNBC invasion and metastasis, which are mediated via the synergistic action of the lysosomal enzyme cathepsin S (CTSS) and gelatinase MMP-9. Knock-down of MMP-9 and CTSS using siRNAs resulted in a synergistic suppression of MDA-MB-231 cell invasion, which was similarly observed with pharmacological inhibitors. During the screening of new drug candidates that suppress both CTSS and MMP-9, BJ-2302, a novel 7-azaindolin-2-one derivative, was discovered. Src, an upstream activator of both pathways (PI3K/Akt and Ras/Raf/ERK) responsible for the expression of CTSS and MMP-9, was identified as a high-affinity target of BJ-2302 (IC90: 3.23 μM) through a Src kinase assay and a drug affinity responsive target stability (DARTS) assay. BJ-2302 effectively suppressed MDA-MB-231 cell invasion (Matrigel invasion assay) and metastasis (chorioallantoic membrane assay xenografted with MDA-MB-231-luc2-tdTomato cancer cells). Unlike Z-FL-COCHO (potent CTSS inhibitor), BJ-2302 did not induce any cytotoxicity in MCF-10A normal breast epithelial cells. Additionally, BJ-2302 (1 mg/kg) strongly suppressed TNBC cell proliferation in vitro and tumor growth in a xenograft mouse tumor model. The anti-metastatic and anti-tumor effects of BJ-2302 were superior to those of Z-FL-COCHO (1 mg/kg) or batimastat (30 mg/kg), a pan-MMP inhibitor. In summary, inhibition of Src kinase suppressed TNBC tumor growth and metastasis, and Src inhibitors such as BJ-2302 may constitute a novel therapeutic tool to treat breast cancer that expresses high levels of CTSS and MMP-9.

Synthesis and antiangiogenic activity of 6-amido-2,4,5-trimethylpyridin-3-ols

( Hyunji Lee ),( Suhrid Banskota ),( Dong Guk Kim ),( Jae Hui Been ),( You Jin Jin ),( Jaya Gautam ),( Hyeonjin Jang ),( Tae Gyu Nam ),( Jung Ae Kim ),( Byeong Seon Jeong ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

We recently reported that 6-aminoalkyl-2,4,5-trimethylpyridin-3-ols, novel series of 6-aminopyridin-3-ol-based antioxidants, have high antiangiogenic activities. In pursuit of wider variety in the analogues, we here report the synthesis and antiangiogenic activities of 6-amidoalkyl-2,4,5-trimethylpyridin-3-ols, which would not be considered excellent antioxidants because of the poorer electron-donating effect of the C(6)-amido group than the corresponding C(6)-amino group. The selected 6-amido compounds showed up to several fold-higher antiangiogenic activities and up to an order of magnitude better antitumor activities in the chick embryo chorioallantoic membrane (CAM) assay than SU4312, a positive control. We also found that paracetamol, as a direct phenolic analogue of our simplest 6-amidopyridin-3-ol, showed a moderate level of antiangiogenic activity. We propose this study will offer a basis for a scaffold of novel angiogenesis inhibitors that can perturb angiogenesis-related pathologies.ⓒ2014Elsevier Ltd. All rights reserved.

Synthesis and biological evaluation of pyridine-linked indanone derivatives: Potential agents for inflammatory bowel disease

Kadayat, Tara Man,Banskota, Suhrid,Bist, Ganesh,Gurung, Pallavi,Magar, Til Bahadur Thapa,Shrestha, Aarajana,Kim, Jung-Ae,Lee, Eung-Seok Elsevier 2018 Bioorganic & medicinal chemistry letters Vol.28 No.14

<P><B>Abstract</B></P> <P>A series of pyridine-linked indanone derivatives were designed and synthesized to discover new small molecules for the treatment of inflammatory bowel disease (IBD). Compounds <B>5b</B> and <B>5d</B> exhibited strongest inhibitory activity against TNF-α-induced monocyte adhesion to colon epithelial cells (an <I>in vitro</I> model of colitis). In TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced rat colitis model, oral administration of the compounds <B>5b</B> and <B>5d</B> ameliorated colitis with significant recovery in altered expressions of E-cadherin, TNF-α and IL-1β expressions, indicating <B>5b</B> and <B>5d</B> as potential agents for therapeutics development against IBD.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Synthesis of pyridine-linked indanone derivatives <B>2</B>, <B>5a–l</B>, <B>8a–c</B> and <B>11a–c</B>. </LI> <LI> Potent anti-inflammatory effect by compounds <B>2</B>, <B>5a</B>, <B>5b</B>, <B>5d</B>, <B>5f</B>, and <B>5h</B>. </LI> <LI> Hydroxyl group substitution is favorable but alkoxy groups at indanone ring is less favored. </LI> <LI> <B>5b</B> and <B>5d</B> as potential agents for inflammatory bowel disease. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Anti-angiogenic activity of macrolactin A and its succinyl derivative is mediated through inhibition of class I PI3K activity and its signaling

강유라,Sushil Chandra Regmi,김미영,김동희,김정애,Suhrid Banskota,Jaya Gautam 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.2

In the current study, macrolactin compounds,macrolactinA(MA) and 7-O-succinyl macrolactinA(SMA),were investigated for their anti-angiogenic activities andaction mechanism. MA and SMA inhibited in vitro andin vivo angiogenesis induced by three different classes of proangiogenicfactors, VEGF, IL-8, and TNF-a. SMA exhibitedstronger anti-angiogenic activity than MA, and such antiangiogenicactivity of SMA was consistently observed inMDA-MB-231 human breast cancer cell-inoculated CAMassay showing dose-dependent suppression of tumor growthand tumor-induced angiogenesis. In an in vitro PI3K competitiveactivity assay, SMA induced concentration-dependentinhibition of class I PI3K isoforms, p110a, p110b,p110d, and p110c. In addition, non-receptor tyrosine kinasec-Src, which is involved in the activation of PI3K heterodimer,was suppressed byMAand SMA.Correspondingly,MAand SMA significantly inhibited the stimulus-induced phosphorylationof Akt, mTOR, p70S6K, and ribosomal S6 inhuman umbilical vein endothelial cells (HUVECs). At thesame time, the stimulus-induced production of reactiveoxygen species (ROS) and activation of NF-jB were significantlysuppressed by MA and SMA. Moreover, the macrolactinssuppressed NF-jB-regulated HSP90 proteinexpression, which stabilizes phosphorylated Akt andNADPH oxidase. Suppression of NF-jB in macrolactintreatedHUVECs with concurrent inhibition of rS6 indicatesthat MAs effectively block angiogenesis through down-regulationof genes related to angiogenesis at both transcriptionaland translational levels. Taken together, the results demonstratethat anti-angiogenic effect ofMAandSMAis mediatedthrough inhibition of PI3K/Akt andNADPHoxidase-derivedROS/NF-jB signaling pathways. These results further indicatethat MA and SMA may be applicable for treatment ofvarious diseases associated with angiogenesis.

Medicinal Chemistry : RESEARCH ARTICLE ; Anti-angiogenic activity of macrolactin A and its succinyl derivative is mediated through inhibition of class I PI3K activity and its signaling

( Youra Kang ),( Sushil Chandra Regmi ),( Mi Yeong Kim ),( Suhrid Banskota ),( Jaya Gautam ),( Dong Hee Kim ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2015 영남대학교 약품개발연구소 연구업적집 Vol.25 No.-

In the current study, macrolactin compounds, macrolactin A (MA) and 7-0-succinyl macrolactin A (SMA), were investigated for their anti-angiogenic activities and action mechanism. MA and SMA inhibited in vitro and in vivoangiogenesis induced by three differentclasses of pro-angiogenic factors, VEGF, IL-8, and TNF-α. SMA exhibited stronger anti-angiogenic activity than MA, and such anti-MDA-MB-231 human breast cancer cell-inoculated CAM assay showing dose-dependent suppression of tumor growth and tumor-induced angiogenesis. In an in vitro PI3K com-petitive activity assay, SMA induced concentration-depen-dent inhibition of class I PI3K isoforms, p110α, p110β, p110δ, and p110★. In addition, non-receptor tyrosine kinase c-Src, which is involved in the activation of PI3K heterodi-mer, was suppressed by MA and SMA. Correspondingly, MA and SMA significantly inhibited the stimulus-induced phos-phorylation of Akt, mTOR, p70S6K, and ribosomal S6 in human umbilical vein endothelial cells (HUVECs). At the same time, the stimulus-induced production of reactive oxygen species (ROS) and activation of NF-κB were signif-icantly suppressed by MA and SMA. Moreover, the macro-lactins suppressed NF-κB-regulated HSP90 protein expression, which stabilizes phosphorylated Akt and NADPH oxidase. Suppression of NF-κB in macrolactin-treated HUVECs with concurrent inhibition of rS6 indicates that Mas effectively block angiogenesis through down-reg-ulation of genes related to angiogenesis at both transcriptional and translational levels. Taken together, the results demon-strate that anti-angiogenic effect of MA and SMA is mediated through inhibition of PI3K/Akt and NADPH oxidase-derived ROS.NF-ΚB signaling pathways. These results further indi-cate that MA and SMA may be applicable for treatment of various diseases associated with angiogenesis.