Inhibition of NADPH Oxidase (NOX) 2 Mitigates Colitis in Mice with Impaired Macrophage AMPK Function.

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKß1-deficient (AMPKß1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKß1LysM mice, whereas sodium salicylate (SS; AMPK ß1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKß1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKß1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Antitumor effect of 3-(quinolin-2-ylmethylene)-4,6-dimethyl-5-hydroxy-7-azaoxindole down-regulating the Gas6-Axl axis.

In this study, a new series of 3-arylidene-4,6-dimethyl-5-hydroxy-7-azaoxindole compounds with a wide range of functional groups were designed, synthesized, and evaluated for their antitumor activity. Among the 35 compounds, compound 6-15, with a quinoline moiety, showed cytotoxic IC50 values superior to those of sunitinib against the seven cancer cell lines (MCF-7, MDA-MB-231, HT-29, DU145, U937, A549, and PANC-1). However, its inhibitory activity against receptor tyrosine kinases (VEGFR2, PDGFRß, c-KIT, FGFR1, FLT3, CSF1R, EGFR, Axl, and Axl mutant) was 100 -3000-fold weaker than that of sunitinib. Interestingly, compound 6-15 exerted a 3.6-fold stronger cytotoxicity than sunitinib in the gemcitabine-resistant PANC-1 cell line and significantly inhibited Axl, which was in contrast with the effect of sunitinib. Nonetheless, both compounds suppressed the expression of growth arrest-specific 6 (Gas6), the ligand of Axl. The inhibitory effect of compound 6-15 on the Gas6-Axl axis was similar to that of Gas6 knockdown by siRNA in PANC-1 cells in terms of apoptosis induction, increase in Bax/Bcl-2 ratio, Axl down-regulation, and PI3K/Akt inhibition. The inhibitory effect of compound 6-15 on tumor growth in mouse tumor models with A549 and PANC-1 xenografts was much greater than that of cisplatin or gemcitabine. Taken together, the current findings demonstrate that compound 6-15 is a promising anticancer drug candidate that acts by inhibiting the Gas6-Axl axis.

Metformin-induced reductions in tumor growth involves modulation of the gut microbiome.

BACKGROUND/PURPOSE: Type 2 diabetes and obesity increase the risk of developing colorectal cancer. Metformin may reduce colorectal cancer but the mechanisms mediating this effect remain unclear. In mice and humans, a high-fat diet (HFD), obesity and metformin are known to alter the gut microbiome but whether this is important for influencing tumor growth is not known. METHODS: Mice with syngeneic MC38 colon adenocarcinomas were treated with metformin or feces obtained from control or metformin treated mice. RESULTS: We find that compared to chow-fed controls, tumor growth is increased when mice are fed a HFD and that this acceleration of tumor growth can be partially recapitulated through transfer of the fecal microbiome or in vitro treatment of cells with fecal filtrates from HFD-fed animals. Treatment of HFD-fed mice with orally ingested, but not intraperitoneally injected, metformin suppresses tumor growth and increases the expression of short-chain fatty acid (SCFA)-producing microbes Alistipes, Lachnospiraceae and Ruminococcaceae. The transfer of the gut microbiome from mice treated orally with metformin to drug naïve, conventionalized HFD-fed mice increases circulating propionate and butyrate, reduces tumor proliferation, and suppresses the expression of sterol response element binding protein (SREBP) gene targets in the tumor. CONCLUSION: These data indicate that in obese mice fed a HFD, metformin reduces tumor burden through changes in the gut microbiome.

Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK.

BACKGROUND: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation. METHODS: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKß1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot. RESULTS: Genetic deletion of AMPKß1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the ß1 subunit. CONCLUSIONS: Together, these data suggest that the development of therapeutic agents targeting AMPKß1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease.

Inhibition of tumor growth and angiogenesis of tamoxifen-resistant breast cancer cells by ruxolitinib, a selective JAK2 inhibitor.

Tamoxifen (TAM) is the most widely used treatment for estrogen receptor-positive breast cancer patients. Unfortunately, the majority of these patients exhibit TAM resistance following treatment. We previously reported that proliferation and migration were greater in TAM-resistant MCF-7 (TAMR-MCF-7) cells than in parental MCF-7 cells. Janus kinases (JAKs) are cytosolic tyrosine kinases that transduce signals from plasma membrane cytokines and growth factor receptors. JAK2 selectively phosphorylates signal transducer and activator of transcription (STAT)-3, and the JAK2-STAT3 signaling pathway is known as a crucial signaling pathway for the regulation of cancer progression and metastasis. In the present study, basal phosphorylation of STAT3 was revealed to be greater in TAMR-MCF-7 cells than in control MCF-7 cells. Ruxolitinib, a potent JAK2 inhibitor, was demonstrated to attenuate STAT3 phosphorylation and the proliferation of TAMR-MCF-7 cells. Ruxolitinib also suppressed the enhanced cell migration of TAMR-MCF-7 cells through the inhibition of epithelial mesenchymal transition. Vascular endothelial growth factor (VEGF), a representative target gene of the JAK2-STAT3 pathway, functions as a key regulator of invasion and angiogenesis. Ruxolitinib significantly inhibited VEGF mRNA expression and transcriptional activity. The present study also performed a chick embryo chorioallantoic membrane assay to assess tumor growth and angiogenesis in TAMR-MCF-7 cells. Ruxolitinib reduced tumor weight and the number of blood vessels produced by TAMR-MCF-7 cells in a concentration-dependent manner. These results indicated that JAK2 could be a new therapeutic target for TAM-resistant breast cancer.

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

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.

Antitumor activity of BJ-1207, a 6-amino-2,4,5-trimethylpyridin-3-ol derivative, in human lung cancer.

Enhanced expression of NADPH oxidase (NOX) and the subsequent production of reactive oxygen species (ROS) are associated with lung cancer. In the present study, fifty 6-amino-2,4,5-trimethylpyridin-3-ol derivatives were screened for anticancer activity by targeting NOX2-derived ROS. The compounds suppressed ROS production and decreased cancer cell viability (R2 = 0.79). Among the derivatives, the compound coded BJ-1207, which contained a 4-(hydroxydiphenylmethyl)piperidine moiety, exhibited the most effective anticancer activity against A549 lung cancer cell line and eight other cancer cell lines, including H1299, MCF-7, MDA-MB-231, HT-29, SW620, Mia PaCa-2, PANC-1, and U937. BJ-1207 also showed significantly lower inhibitory effects on kinase insert domain receptor (KDR) and c-KIT tyrosine kinase but higher inhibitory activity on NOX than those of sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor. In addition, BJ-1207-induced inhibition of RTK-downstream signaling pathways, such as ROS production, and expression of target genes, such as stem cell factor and transforming growth factor-α, were similar to those induced by sunitinib. In the xenograft chick tumor model, BJ-1207 inhibited lung tumor growth to a similar or much greater extent than that of sunitinib or cisplatin, respectively. Overall, the present study showed that BJ-1207, a vitamin B6-derived 2,4,5-trimethylpyridin-3-ol compound with azacyclonol moiety at C (6)-position of the pyridine ring, inhibited NOX activity and that it is a promising lead compound for developing anticancer drugs against lung cancer.

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.

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

PURPOSE: 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. METHODS: Immunohistochemical 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. RESULTS: In 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. CONCLUSION: Our 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.

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.

5-Hydroxy-7-azaindolin-2-one, a novel hybrid of pyridinol and sunitinib: design, synthesis and cytotoxicity against cancer cells.

Angiogenesis plays important roles in tumor growth and metastasis. Sunitinib (Sutent®) is an antitumor agent targeting receptor tyrosine kinases which are involved in angiogenesis as well as cancer cell growth and survival. Using the pyridin-3-ol scaffold, which was previously reported as an excellent antioxidant and antiangiogenic platform, we have synthesized sunitinib mimics 6 by hybridizing bicyclic pyridinol 4 as a key scaffold and pyrrole-2-carbaldehydes 7 as side chains. Cytotoxicity assays showed that compounds 6 have comparable to better anticancer activity than sunitinib against five different cancer cell lines. In addition, compounds 6 showed even lower levels of cytotoxicity against normal cells, resulting in up to 26-fold better safety windows, than sunitinib. Signaling pathway-associated transcription factor reporter assay and western blot analyses revealed that apoptosis induction in MDA-MB-231 human breast cancer cells by 6F is mainly mediated through the p53 increase and down-regulation of phospho-signal transducer and activator of transcription 3 (STAT3) and its target gene products, cyclin D, Bcl-2, and survivin. The data strongly suggest that our hybrid compounds can provide a novel anticancer scaffold with improved and safer cytotoxicity profiles than sunitinib.

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 Gßγ/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.

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.

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.