Jaya Gautam

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

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 demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT1 receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 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-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin 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-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.

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

BackgroundTriple-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.MethodsQuantitative 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.ResultsTNBC 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 Gβγ-activated kinase signaling during invasion, and Gβγ-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.Conclusions5-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.

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

Abstract 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‐&kgr;B 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 TNF&agr; and IL‐1&bgr;, 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. Graphical abstract Figure. No Caption available. Highlights5‐HT induces adhesive invasion of commensal E. coli to colon epithelial cells.Commensal E. coli induces colon epithelial NOX2 activation via TLR‐2 and TLR‐4.5‐HT amplifies commensal E. coli‐induced up‐regulation of TLR2/TLR4, IL‐8, and ICAM‐1 via NOX2.5‐HT enhances E. coli‐induced down‐regulation of E‐cadherin.Inoculation of commensal E.coli with high 5‐HT levels induces fatal colon inflammation in mice.

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

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.

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, p110β, 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.

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.

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

Abstract The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro‐inflammatory cytokines including TNF‐&agr;. Once generated, TNF‐&agr; 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‐&agr;‐induced and TNBS‐induced colon inflammation. TI‐1‐162 showed inhibitory effect on the TNF‐&agr;‐induced adhesion of U937 monocytic cells to HT‐29 colonic epithelial cells (IC50 = 0.83 ± 0.12 &mgr;M), which is an in vitro model representing the initial step of colitis. In addition, TI‐1‐162 suppressed TNF‐&agr;‐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‐&agr; 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‐&agr;‐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. Graphical abstract Figure. No Caption available.

4‐Hydroxynonenal‐induced GPR109A (HCA2 receptor) activation elicits bipolar responses, Gαi‐mediated anti‐inflammatory effects and Gβγ‐mediated cell death

In this study, we examined the possibility that 4‐hydroxynonenal (4‐HNE) acting as a ligand for the HCA2 receptor (GPR109A) elicits both anti‐inflammatory and cell death responses.

Protective effects of 6-ureido/thioureido-2,4,5-trimethylpyridin-3-ols against 4-hydroxynonenal-induced cell death in adult retinal pigment epithelial-19 cells

Dysfunction or progressive degeneration of retinal pigment epithelium (RPE) contributes in the initial pathogenesis of age-related macular degeneration (AMD) causing irreversible vision loss, which makes RPE the prime target of the disease. The present study aimed to identify compounds to protect 4-hydroxynonenal (4-HNE)-induced RPE cell death by inhibiting NADPH oxidase 4 (NOX4) activity, not just as free radical scavengers, using ARPE-19, a human adult retinal pigment epithelial cell line, as a RPE representative. Novel thirty-two 6-ureido/thioureido-2,4,5-trimethylpyridin-3-ol derivatives 17 were synthesized and tested. We found that there was a strong correlation between level of protective effect of compounds 17 against 4-HNE-induced APRE-19 cell death and that of inhibitory activity against 4-HNE-induced superoxide production, and that most of the compounds 17 showed minimal DPPH radical scavenging activity. Compound 17-28 showed the best protective activity against 4-HNE-induced superoxide production (79.5% inhibition) and cell death (85.1% recovery) at 10 μM concentration, which was better than that of VAS2870, a NOX2/4 inhibitor. In addition, compound 17-28 blocked 4-HNE-induced apoptosis of ARPE-19 cells in a concentration-dependent manner. The results indicate that compound 17-28 may be a lead compound to develop AMD therapeutics.

Up-regulation of cathepsin S expression by HSP90 and 5-HT7 receptor-dependent serotonin signaling correlates with triple negativity of human breast cancer

AbstractPurpose Cathepsin S (CTSS) is expressed in a variety of cancers and stimulates tumor progression. However, the regulatory mechanism and role of CTSS in breast cancer progression are poorly understood. The aim of this study was to examine the relationships between CTSS expression and breast cancer grade and stage, and the signaling molecules involved in CTSS expression.MethodsImmunohistochemical staining was performed in tissue microarray sections of 1451 human invasive breast cancer samples to determine epithelial (E-CTSS) and stromal CTSS (S-CTSS) expression. Gene and protein expression levels in human breast cancer cell lines were measured by polymerase chain reaction and western blotting. Small interfering RNA transfection and a Matrigel transwell invasion assay were used to confirm the signaling pathways regulating CTSS expression.ResultsIn patient tumor tissue blocks, high grade, late stage, and triple negativity were associated with elevated CTSS protein expression, and expression levels were related to the clinical outcomes of patients with invasive breast cancer. CTSS expression was also higher in triple-negative breast cancer (TNBC) cell lines than in hormone-responsive cells, and CTSS expression patterns matched those of tryptophan hydroxylase 1 (TPH1) and 5-hydroxytryptamine receptor 7 (5-HT7). Treatment of TNBC cells (MDA-MB-231 and HCC-1395) with 5-HT significantly enhanced CTSS protein expression, whereas pharmacological inhibition or knockdown of 5-HT7 significantly inhibited its expression. Correspondingly, cancer cell invasion was increased by 5-HT treatment and suppressed by 5-HT7 knockdown. The expression of CTSS was regulated by PI3K/Akt and Ras/Raf/MAPK signaling pathways, and these signaling pathways were stabilized by HSP90 and enhanced by the 5-HT7 receptor-dependent autocrine effect of 5-HT in TNBC cells.ConclusionOur findings suggest CTSS as a candidate target for development of a strategy to inhibit breast cancer invasion, and indicate that HSP90 and 5-HT7 (regulators of CTSS) should be considered as alternative targets for the management of TNBC invasion and metastasis.