Synthesis and activity of N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)acetamide analogues as anticolitis agents via dual inhibition of TNF-α- and IL-6-induced cell adhesions.

Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are the critical pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Inhibition of these cytokines and related signaling pathways has been a target for the development of IBD therapeutics. In the current study, 6-acetamido-2,4,5-trimethylpyridin-3-ol (1) and various analogues with the amido scaffold were synthesized and examined for their inhibitory activities in in vitro and in vivo IBD models. The parent compound 1 (1 µM) showed an inhibitory activity against TNF-α- and IL-6-induced adhesion of monocytes to colon epithelial cells, which was similar to tofacitinib (1 µM), a JAK inhibitor, but much better than mesalazine (1,000 µM). All the analogues showed a positive relationship (R2 = 0.8943 in a linear regression model) between the inhibitory activities against TNF-α-induced and those against IL-6-induced adhesion. Compound 2-19 turned out to be the best analogue and showed much better inhibitory activity against TNF-α- and IL-6-induced adhesion of the cells than tofacitinib. In addition, oral administration of compound 1 and 2-19 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 in colon tissues. More importantly, compound 2-19 (1 mg/kg) was more efficacious in ameliorating colitis than compound 1 and sulfasalazine (300 mg/kg), the commonly prescribed oral IBD drug. Taken together, the results suggest that compound 2-19 can be a novel platform for dual-acting IBD drug discovery targeting both TNF-α and IL-6 signaling.

Dual Inhibition of NOX2 and Receptor Tyrosine Kinase by BJ-1301 Enhances Anticancer Therapy Efficacy via Suppression of Autocrine-Stimulatory Factors in Lung Cancer.

NADPH oxidase-derived reactive oxygen species (ROS) potentiate receptor tyrosine kinase (RTK) signaling, resulting in enhanced angiogenesis and tumor growth. In this study, we report that BJ-1301, a hybrid of pyridinol and alpha-tocopherol, exerts anticancer effects by dual inhibition of NADPH oxidase and RTK activities in endothelial and lung cancer cells. BJ-1301 suppresses ROS production by blocking translocation of NADPH oxidase cytosolic subunits to the cell membrane, thereby inhibiting activation. The potency of RTK inhibition by BJ-1301 was lower than that of sunitinib (a multi-RTK inhibitor), but the inhibition of downstream signaling pathways (e.g., ROS generation) and subsequent biological changes (e.g., NOX2 induction) by BJ-1301 was superior. Consistently, BJ-1301 inhibited cisplatin-resistant lung cancer cell proliferation more than sunitinib did. In xenograft chick or mouse tumor models, BJ-1301 inhibited lung tumor growth, to an extent greater than that of sunitinib or cisplatin. Treatments with BJ-1301 induced regression of tumor growth, potentially due to downregulation of autocrine-stimulatory ligands for RTKs, such as TGFα and stem cell factor, in tumor tissues. Taken together, the current study demonstrates that BJ-1301 is a promising anticancer drug for the treatment of lung cancer. Mol Cancer Ther; 16(10); 2144-56. ©2017 AACR.

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

Adherent-invasive E. coli 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 E. coli colonization and increase TLR expression. In a co-culture system, commensal E. coli 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 in vitro, except for significant increases in TNFα and IL-1ß, 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 E. coli 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.

Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) has a high risk of relapse and there are few chemotherapy options. Although 5-hydroxytryptamine (5-HT, serotonin) signaling pathways have been suggested as potential targets for anti-cancer drug development, the mechanism responsible for the action of 5-HT in TNBC remains unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. Cell proliferation was measured using CellTiter 96 Aqueous One Solution. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Levels of 5-HT and vascular endothelial growth factor (VEGF) were measured using ELISA kits. Chick chorioallantoic membrane (CAM) assay and mouse tumor model were used to investigate the in vivo effects of SB269970, a 5-HT7 receptor antagonist, and BJ-1113, a novel synthetic compound. RESULTS: TNBC cell lines (MDA-MB-231, HCC-1395, and Hs578T) expressed higher levels of tryptophan hydroxylase 1 (TPH1) than hormone-responsive breast cancer cell lines (MCF-7 and T47D). In MDA-MB-231 cells, 5-HT promoted invasion and proliferation via 5-HT7 receptor, and interestingly, the stimulatory effect of 5-HT on MDA-MB-231 cell invasion was stronger than its effect on proliferation. Likewise, downstream signaling pathways of 5-HT7 differed during invasion and proliferation, that is, Gα-activated cAMP and Gßγ-activated kinase signaling during invasion, and Gßγ-activated PI3K/Akt signaling during proliferation. Also, 5-HT increased the protein expressions of TPH1 and VEGF in MDA-MB-231 cells. These results provide insight of the stimulatory effect of 5-HT on breast cancer progression; 5-HT was found to act more strongly during the first stage of metastasis (during invasion and migration) than during the later proliferative phase after local invasion. Interestingly, these actions of 5-HT were inhibited by BJ-1113, a 6-amino-2,4,5-trimethylpyridin-3-ol analog. BJ-1113 blocked intracellular signaling pathways initiated by 5-HT7 receptor activation, and exhibited anti-proliferative and anti-invasive activities against MDA-MB-231 cells. Furthermore, the inhibitory effect of BJ-1113 against MDA-MB-231 tumor growth was greater than that of SB269970, a 5-HT7 receptor antagonist. CONCLUSIONS: 5-HT7 receptor which mediates 5-HT-induced cancer progression is a potential therapeutic target in TNBC, and BJ-1113 offers a novel scaffold for the development of anti-cancer agents against TNBC.

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

In the current study, macrolactin compounds, macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), were investigated for their anti-angiogenic activities and action mechanism. MA and SMA inhibited in vitro and in vivo angiogenesis induced by three different classes of pro-angiogenic factors, VEGF, IL-8, and TNF-α. SMA exhibited stronger anti-angiogenic activity than MA, and such anti-angiogenic activity of SMA was consistently observed in 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 competitive activity assay, SMA induced concentration-dependent inhibition of class I PI3K isoforms, p110α, p110ß, p110δ, and p110γ. In addition, non-receptor tyrosine kinase c-Src, which is involved in the activation of PI3K heterodimer, was suppressed by MA and SMA. Correspondingly, MA and SMA significantly inhibited the stimulus-induced phosphorylation 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 significantly suppressed by MA and SMA. Moreover, the macrolactins 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-regulation of genes related to angiogenesis at both transcriptional and translational levels. Taken together, the results demonstrate 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 indicate that MA and SMA may be applicable for treatment of various diseases associated with angiogenesis.

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

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.

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

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.

Apple flavonoid phloretin inhibits Escherichia coli O157:H7 biofilm formation and ameliorates colon inflammation in rats.

Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagic Escherichia coli O157:H7. The antioxidant phloretin, which is abundant in apples, markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx(2)), autoinducer-2 importer genes (lsrACDBF), curli genes (csgA and csgB), and dozens of prophage genes in E. coli O157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production in E. coli O157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor of E. coli O157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensal E. coli biofilms.