The Anti-Tumor Activity of Succinyl Macrolactin A Is Mediated through the β-Catenin Destruction Comples via the Suppression of Tankyrase and PI3K/Akt

( Sushil C Regmi ),( Su Young Park ),( Seung Joo Kim ),( Suhrid Banskota ),( Sajita Shah ),( Dong Hee Kim ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

Accumulated gene mutations in cancer suggest that multi-targeted suppression of affected signaling networks is a promising strategy for cancer treatment. In the present study, we report that 7 -O-succinyl macrolactin A (SMA) suppresses tumor growth by stabilizing the β-eaten in destruction complex, which was achieved through inhibition of regulatory compo-nents associated with the complex. SMA significantly reduced the activities of PI3K/Akt, which corresponded with a decrease in GSK3β phosphorylation, an increase in β-catenin phosphorylation, and a reduction in nuclear β-catenin content in HT29 human colon cancer cells. At the same time, the activity of tankyrase, which inhibits the β-catenin destruction complex by destabilizing the axin level, was suppressed by SMA. Despite the low potency of SMA against tankyrase activity (IC₅₀ of 50.1 IJM and 15.51JM for tankyrase 1 and 2, respectively) compared to XAV939 (IC₅₀ of 11 nM for tankyrase 1), a selective and potent tankyrase inhibitor, SMA had strong inhibitory effects on β-catenin-dependent TGF/LEF1 transcriptional activity (IC₅₀ of 39.8 nM), which were similar to that of XAV939 (IC₅₀ of 28.1 nM). In addition to suppressing the colony forming ability of colon cancer cells in vitro, SMA significantly inhibited tumor growth in GT26 syngenic and HT29 xenograft mouse tumor models. Furthennore, treating mice with SMA in combination with 5-FU in a colon cancer xenograft model or with cisplatin in an A549 lung cancer xenograft model resulted in greater anti-tumor activity than did treatment with the drugs alone. In the xenograft tumor tissues, SMA dose-dependently inhibited nuclear β-catenin along with reductions in GSK3β phos-phorylation and increases in axin levels. These results suggest that SMA is a possible can-didate as an effective anti-cancer agent alone or in combination with cytotoxic chemotherapeutic drugs, such as 5-FU and cisplatin, and that the mode of action for SMA involves stabilization of the β-catenin destruction complex through inhibition of tankyrase and the PI3K/Akt signaling pathway.

Original Contribution : Serotonin regulates innate immune responses of colon epithelial cells through Nox2-derived reactive oxygen species

( Sushil Chandra Regmi ),( Su Yong Park ),( Sae Kwang Ku ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Changes in serotonin (5-hydroxytryptamine, 5-HT) content in the gut of patients with inflammatory bowel disease (IBD) and animal models of colitis suggest an important role of 5-HT in the pathogenesis of IBD. In this study, we examined the role and mechanism of action of 5-HT in the inflammatory response of colon epithelial cells in vitro and in vivo. In colon epithelial cells (CCD 841, HT-29, Caco-2), direct application of 5-HT induced production of reactive oxygen species (ROS) and monocyte-epithelial adhesion, an initial event of inflammation, which were blocked not only by 5-HT receptor antagonists (tropisetron, RS39604, and SB269970), antioxidants (ascorbic acid, apocynin), and various inhibitors of NADPH oxidase (DPI), CREB (KG-501), and NF-κB (PDTC), but also by transfection with Nox2 siRNA. Nox2-derived production of ROS corresponded with the rapid and brief activation of Rac. In addition, 5-HT induced Nox2, p67(phox), and Duox2 without altering the level of Nox1 or Duox1 in colon epithelial cells, and silencing of Nox2 suppressed 5-HT-induced Duox2 increase. 5-HT also induced an increase in the expression of MCP-1, IL-8, and ICAM-1 and a decrease in E-cadherin expression. Exogenous application of 5-HT to rat colon through the rectum caused a minimal level of inflammation, which was demonstrated by histological examination, MPO activity, and inflammatory cytokine induction. However, 5-HT combined with a low dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS), the level of which caused a minimal level of colitis, exaggerated colon inflammation accompanied by much more enhanced induction of inflammatory cytokines, IL-6, IL-8, and MCP-1, indicating that colon epithelial cells directly exposed to 5-HT are primed toward inflammation. In the colon at the lesion site, treatment with 5-HT resulted in an increase in the level of epithelial Nox2 but not of constitutively expressed Nox1, which is the opposite result of TNBS treatment. Furthermore, 5-HT treatment of Nox2-knockout mice did not induce colon inflammation, in contrast to 5-HT-treated wild-type mice. The results demonstrate that colon epithelial cells directly exposed to 5-HT are primed for inflammatory reactions, which is an important innate immune response, and the underlying mechanism for the priming is associated with Nox2-activated signaling pathways, including ERK/p38 MAPK, NF-κB, and CREB.ⓒ2014 Elsevier Inc. All rights reserved.

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.

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>

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.

Protective effect of 7-O-succinyl macrolactin A against intestinal inflammation is mediated through PI3-kinase/Akt/mTOR and NF-kB signaling pathways

( Sumin Park ),( Sushil Chandra Regmi ),( Su Young Park ),( Eun Kyong Lee ),( Jae Hoon Chang ),( Sae Kwang Ku ),( Dong Hee Kim ),( Jung Ae Kim ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, are pivotal for the development of inflammatory bowel disease (IBD), and down-regulation of the cytokines and cytokine-induced inflammatory responses therefore constitute pharmacological targets for the development of therapeutic strategies in IBD. In the current study, we found that 7-O-succinyl macrolactin A (SMA), a macrolide, potently inhibited TNF-α-induced adhesion of monocytes to colonic epithelial cells in a concentration-dependent manner, similar to rapamycin, a mTOR inhibitor. In addition, oral administration of SMA resulted in a significant suppression of clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase activity, a marker for inflammatory cell infiltration, as well as microscopic damage score in a histomorphological examination of HE-stained colon tissue. More importantly, SMA was more efficacious in inhibition of intestinal inflammation than 5-aminosalicylic acid (5-ASA), an active metabolite of sulfasalazine, the most commonly prescribed agent for the treatment of IBD. Such anti-inflammatory activity showed correlation with significant suppression of adhesion molecules (ICAM-1 and VCAM-1), T-helper 1-type cytokines (TNF-α, IL-6), and chemokines (MCP-1, IL-8). In addition to inhibition of NF-κB nuclear translocation, SMA also caused significant suppression of TNF-α-induced phosphorylation of PI3K, Akt, mTOR and p70S6 kinase, similar to the effect of rapamycin, an immunosuppressant macrolide. Taken together, the current results suggest that managing both mTOR and NF-κB activation pathways using SMA may be a good therapeutic intervention for the treatment of IBD.ⓒ2014 Elsevier B.V.All rights reserved.

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.

Diverse plant extracts and trans-resveratrol inhibit biofilm formation and swarming of Escherichia coli O157:H7

( Jin Hyung Lee ),( Hyun Scob Cho ),( Sang Woo Joo ),( Sushil Chandra Regmi ),( Jung Ae Kim ),( Choong Min Ryu ),( Shi Yong Ryu ),( Moo Hwan Cho ),( Jintae Lee ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Infection with enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem. Of the 498 plant extracts screened against EHEC, 16 inhibited the formation of biofilm of EHEC by >85% without inhibiting the growth of planktonic cells, and 14 plant extracts reduced the swarming motility of EHEC. The most active extract, Carex dimorpholepis, decreased swimming and swarming motilities and curli formation. Transcriptional analyses showed that the extract of C. dimorpholepis repressed curli genes, various motility genes, and AI-2 quorum sensing genes, which was corroborated by reduction in the production of fimbria, motility, and biofilm by EHEC. Trans-resveratrol at 10μgml(-1) in the extract of C. dimorpholepis was found to be a new anti-biofilm compound against EHEC, but importantly, the extract of C. dimorpholepis and trans-resveratrol did not inhibit the fomation of biofilm in four commensal E. coli strains. Furthermore, the extract of C. dimorpholepis decreased the adhesion of EHEC cells to human epithelial cells without affecting the viability of these cells.

Diverse plant extracts and trans-resveratrol inhibit biofilm formation and swarming of Escherichia coli O157:H7

Lee, Jin-Hyung,Cho, Hyun Seob,Joo, Sang Woo,Chandra Regmi, Sushil,Kim, Jung-Ae,Ryu, Choong-Min,Ryu, Shi Yong,Cho, Moo Hwan,Lee, Jintae Taylor Francis 2013 Biofouling Vol.29 No.10