Ribisins and Certain Analogues Exert Neuroprotective Effects through Activation of the Keap1-Nrf2-ARE Pathway.

Oxidative stress contributes to the pathogenesis of various neurodegenerative diseases and induction of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is a validated neuroprotective strategy. Synthetically-derived samples of members of the ribisin class of natural product together with a range of analogues were evaluated for their neuroprotective capacities. Four of the twenty-four compounds tested were found to strongly stimulate antioxidant response element-dependent transcriptional activity in human-derived SH-SY5Y cells. Further, in rat pheochromocytoma PC12 cells and mouse brain cortical cultures these compounds upregulated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target gene products, namely heme oxygenase (HO-1) and NAD(P)H quinone reductase 1 (NQO1). Functionally speaking, the compounds conferred protection in these cell models challenged with H2 O2 . In silico molecular modeling suggests that certain of the ribisins can dock in the Nrf2-binding Kelch domain in Keap1, while cysteine labeling by biotinylated iodoacetamide suggest that cysteine residues within Keap1 react with the ribisins. It is thus proposed that the most active compounds exert their neuroprotective activities by targeting Keap1, thereby activating Nrf2 and so increasing transactivation of Nrf2-responsive genes that encode for detoxifying and antioxidant enzymes.

A systematic review of the association of obesity with the outcomes of inflammatory rheumatic diseases.

This was a systematic review of the literature on the association between obesity and the outcome of inflammatory rheumatic diseases. We conducted a literature search using PubMed®, Embase and PsycINFO®. Articles were classified into three categories based on the effects of obesity on the outcomes of inflammatory rheumatic diseases. The subject population, country, type of studies, number of patients, measurement of obesity and outcomes assessed were presented. Quality was appraised using Kmet et al's criteria. 4,331 articles were screened and 60 were relevant to the objective. Obesity had a negative, positive and neutral association with outcomes of inflammatory rheumatic diseases in 38 (63.3%) studies with 57,612 subjects, 11 (18.3%) studies with 3,866 subjects, and 11 (18.3%) studies with 3,834 subjects, respectively. In most studies, the disease population had been diagnosed with rheumatoid arthritis (RA). Tumour necrosis factor-α inhibitors were mostly associated with negative outcomes. More studies examining subjects outside Europe and North America and diseases other than RA are warranted.

Novel dual-targeting anti-proliferative dihydrotriazine-chalcone derivatives display suppression of cancer cell invasion and inflammation by inhibiting the NF-κB signaling pathway.

Chalcones present in edible plants possess anti-cancer and anti-inflammatory properties, with the Michael acceptor moiety reported to be responsible for their biological activities. In this study, two novel dihydrotriazine-chalcone compounds previously identified to exert anti-proliferative effects through dual-targeting of dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR), were evaluated for their anti-invasive and anti-inflammatory abilities. At non-lethal concentrations, the compounds suppressed in vitro migration of MDA-MB-231 breast carcinoma cells, which was correlated with a dose-dependent downregulation of phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) expression and secretion. At similar concentrations, these chalcone-based compounds suppressed expression of inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated murine macrophage-like RAW 264.7 cells, as well as tumor necrosis factor alpha (TNF-α) in LPS-stimulated human monocytes isolated from healthy donors. Mechanistically, inhibition of cancer cell invasion and inflammation by the compounds were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway, which corroborated with the reported mechanism of action of chalcones. Their abilities to target multiple biological mediators relevant to multi-step carcinogenesis and with bioactivities stronger than those of the parent chalcone scaffold have warranted dihydrotriazine-chalcone compounds as promising candidates for use in pharmacological intervention of aggressive cancers.

Measurement properties of patient reported outcome measures for spondyloarthritis: A systematic review.

OBJECTIVES: This systematic review aimed to identify studies investigating measurement properties of patient reported outcome measures (PROMs) for spondyloarthritis (SpA), and to evaluate their methodological quality and level of evidence relating to the measurement properties of PROMs. METHODS: This systematic review was guided by the preferred reporting items for systematic review and meta-analysis (PRISMA). Articles published before 30 June 2017 were retrieved from PubMed®, Embase®, and PsychINFO® (Ovid). Methodological quality and level of evidence were evaluated according to recommendations from the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN). RESULTS: We identified 60 unique PROMs from 125 studies in 39 countries. Twenty-one PROMs were validated for two or more SpA subtypes. The literature examined hypothesis testing (82.4%) most frequently followed by reliability (60.0%). A percentage of 77.7% and 42.7% of studies that assessed PROMs for hypothesis testing and reliability, respectively had "fair" or better methodological quality. Among the PROMs identified, 41.7% were studied in ankylosing spondylitis (AS) only and 23.3% were studied in psoriatic arthritis (PsA) only. The more extensively assessed PROMs included the ankylosing spondylitis quality of life (ASQoL) and bath ankylosing spondylitis functional index (BASFI) for ankylosing spondylitis, and the psoriatic arthritis quality of life questionnaire (VITACORA-19) for psoriatic arthritis. CONCLUSION: This study identified 60 unique PROMs through a systematic review and synthesized evidence of the measurement properties of the PROMs. There is a lack of validation of PROMs for use across SpA subtypes. Future studies may consider validating PROMs for use across different SpA subtypes.

Design, Synthesis, and Biological Evaluation of Coupled Bioactive Scaffolds as Potential Anticancer Agents for Dual Targeting of Dihydrofolate Reductase and Thioredoxin Reductase.

The dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) enzymes are involved in the process of tumor cell growth and survival. The 4,6-diamino-1,2-dihydro-1,3,5-triazine scaffold is well-established as a useful scaffold for DHFR inhibition, while chalcones have been reported to be inhibitors of TrxR. In this study, 15 novel compounds designed by the structural combination of the 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds via a diether linker were successfully synthesized and characterized. All of the compounds demonstrated dual inhibition against DHFR and TrxR when they were assessed by in vitro enzyme assays. The compounds also exhibited antiproliferative activity against the MCF-7 and HCT116 cells. The more potent analogs 14 and 15 were found to inhibit cellular DHFR and TrxR activities in HCT116 cells. Therefore, this study provided compelling evidence that 14 and 15 could exert their anticancer property via multitarget inhibition at the cellular level.

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads.

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,ß-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

Applying the designed multiple ligands approach to inhibit dihydrofolate reductase and thioredoxin reductase for anti-proliferative activity.

The development of multi-targeting drugs is currently being explored as an attractive alternative to combination therapy, especially for the treatment of complex diseases such as cancer. Dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) are enzymes belonging to two unrelated cellular pathways that are known to contribute towards cancer cell growth and survival. In order to evaluate whether simultaneous inhibition of DHFR and TrxR by dihydrotriazines (DHFR-targeting) and chalcones (TrxR-targeting) may be beneficial, breast MCF-7 and colorectal HCT116 carcinoma cells were treated with combinations of selected dihydrotriazines and chalcones at a 1:1 M ratio. Two combinations demonstrated synergy at low-to-moderate concentrations. Based on this result, four merged dihydrotriazine-chalcone compounds were designed and synthesized. Two compounds, 11a [DHFR IC50 = 56.4 µM, TrxR IC50 (60 min) = 12.6 µM] and 11b [DHFR IC50 = 2.4 µM, TrxR IC50 (60 min) = 10.1 µM], demonstrated in vitro inhibition of DHFR and TrxR. The compounds showed growth inhibitory activity against MCF-7 and HCT116 cells, but lacked cytotoxicity. Molecular docking experiments showed 11b to possess rational binding modes to both the enzymes. In conclusion, this study has not only identified the dihydrotriazine and chalcone scaffolds as good starting points for the development of dual inhibitors of DHFR and TrxR, but also demonstrated the synthetic feasibility of producing a chemical entity that could result in simultaneous inhibition of DHFR and TrxR. Future efforts to improve the antiproliferative profiles of such compounds will look at alternative ways of integrating the two pharmacophoric scaffolds.

Thioredoxin-dependent regulation of AIF-mediated DNA damage.

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.

Sulforaphane and its methylcarbonyl analogs inhibit the LPS-stimulated inflammatory response in human monocytes through modulating cytokine production, suppressing chemotactic migration and phagocytosis in a NF-κB- and MAPK-dependent manner.

Sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)-butane], an aliphatic isothiocyanate (ITC) naturally derived from cruciferous vegetables and largely known for its chemopreventive potential also appears to possess anti-inflammatory potential. In this study, structural analogs of SF {compound 1 [1-isothiocyanato-4-(methylcarbonyl)-butane] and 2 [1-isothiocyanato-3-(methylcarbonyl)-propane]} containing a carbonyl group in place of the sulfinyl group in SF, were evaluated for their anti-inflammatory activities. In RAW 264.7 cells, the ITCs at non-toxic concentrations caused an inhibition of NO and prostaglandin E2 (PGE2) release through suppressing expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as a reduction in matrix metalloproteinase-9 (MMP-9) expression, secretion and gelatinolytic activity. Further work performed on human monocytes isolated from blood of healthy donors revealed that the ITCs not only suppressed the expression and release of pro-inflammatory mediators IL-1ß, IL-6, TNF-α and MMP-9, but also suppressed their antibody-independent phagocytic and chemotactic migratory abilities. These anti-inflammatory activities were mediated through suppression of the NF-κB and MAPK signaling pathways. In addition, the ITCs were revealed to interact with the cysteines in inhibitor of nuclear factor-κB kinase ß subunit (IKKß), which could contribute at least partly to the suppression of NF-κB signaling. In conclusion, results obtained in this study provide deeper insights into the anti-inflammatory properties of SF and its methylcarbonyl analogs and the underlying mechanisms. These compounds thus serve as promising candidates for clinical applications in controlling inflammatory conditions.

3-(2-oxoethylidene)indolin-2-one derivatives activate Nrf2 and inhibit NF-κB: potential candidates for chemoprevention.

Induction of cytoprotective phase 2 enzymes through inhibition of Keap1, a repressor of transcription factor Nrf2, is a cancer-prevention strategy. Compounds that elicit antiinflammatory and cytoprotective effects are promising candidates for chemoprevention. Novel analogues of 1-methyl-3-(2-oxopropylidene)indolin-2-one ('supercinnamaldehyde'; SCA) were synthesized, and their abilities to induce cytoprotective responses through Nrf2 induction and to suppress inflammatory responses were examined. 1-Methyl-3-(2-oxo-2-phenylethylidene)indolin-2-one (6) was identified as the lead compound. The compounds showed induction of Nrf2-dependent phase 2 enzymes in Keap1+/+ mouse embryonic fibroblasts (MEFs), which was abrogated in Keap1-/- MEFs. The compounds also displayed a suppressive effect on NF-κB signaling that was at least partly responsible for inhibition of lipopolysaccharideinduced inflammatory responses. These SCA analogues exhibited cytoprotective and anti-inflammatory activities and may be developed further as chemopreventive agents.

A novel shogaol analog suppresses cancer cell invasion and inflammation, and displays cytoprotective effects through modulation of NF-κB and Nrf2-Keap1 signaling pathways.

Natural compounds containing vanilloid and Michael acceptor moieties appear to possess anti-cancer and chemopreventive properties. The ginger constituent shogaol represents one such compound. In this study, the anti-cancer potential of a synthetic novel shogaol analog 3-phenyl-3-shogaol (3-Ph-3-SG) was assessed by evaluating its effects on signaling pathways. At non-toxic concentrations, 3-Ph-3-SG suppressed cancer cell invasion in MDA-MB-231 and MCF-7 breast carcinoma cells through inhibition of PMA-activated MMP-9 expression. At similar concentrations, 3-Ph-3-SG reduced expression of the inflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and prostanglandin-E2 (PGE2) in RAW 264.7 macrophage-like cells. Inhibition of cancer cell invasion and inflammation by 3-Ph-3-SG were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway. The 3-Ph-3-SG also demonstrated cytoprotective effects by inducing the antioxidant response element (ARE)-driven genes NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1). Cytoprotection by 3-Ph-3-SG was achieved at least partly through modification of cysteine residues in the E3 ubiquitin ligase substrate adaptor Kelch-like ECH-associated protein 1 (Keap1), which resulted in accumulation of transcription factor NF-E2 p45-related factor 2 (Nrf2). The activities of 3-Ph-3-SG were comparable to those of 6-shogaol, the most abundant naturally-occurring shogaol, and stronger than those of 4-hydroxyl-null deshydroxy-3-phenyl-3-shogaol, which attested the importance of the 4-hydroxy substituent in the vanilloid moiety for bioactivity. In summary, 3-Ph-3-SG is shown to possess activities that modulate stress-associated pathways relevant to multiple steps in carcinogenesis. Therefore, it warrants further investigation of this compound as a promising candidate for use in chemotherapeutic and chemopreventive strategies.

Antioxidant and Nrf2 inducing activities of luteolin, a flavonoid constituent in Ixeris sonchifolia Hance, provide neuroprotective effects against ischemia-induced cellular injury.

Ixeris sonchifolia Hance is an herb distributed in northeastern part of China and has been used by natives to invigorate circulation. In the present study, bioactivity-guided fractionation of I. sonchifolia Hance extract was performed with the aim to isolate and identify the compounds underlying the potential protective effects against ischemia brain injury. Among the four fractions isolated from the herb extract, the ethyl acetate fraction was found to scavenge DPPH radicals, induce ARE-dependent transcriptional activity and upregulate Nrf2 protein levels. The isolation work focused on this fraction revealed the presence of two categories of compounds: flavonoids and sesquiterpene lactones. Among the five isolated flavonoids, luteolin was evaluated to possess direct and indirect antioxidant activities by scavenging free radicals and inducing the upregulation of ARE-dependent phase II enzymes. Concomitant with the findings from the cell-based assays, in the middle cerebral artery occlusion-induced ischemia rat model, administration of luteolin at 4 mg/kg displayed neuroprotective effects by reducing infarct area and inhibiting neuronal cell death. In summary, the obtained results suggest that flavonoids in I. sonchifolia Hance, in particular luteolin, contribute at least partly to the neuroprotective effects against ischemia-induced cellular injury and can be potentially developed for treatment of ischemia-reperfusion induced diseases.

Identification of Michael acceptor-centric pharmacophores with substituents that yield strong thioredoxin reductase inhibitory character correlated to antiproliferative activity.

AIMS: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target. A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which include the Michael reaction acceptors containing an α,ß-unsaturated carbonyl moiety. We aimed to identify features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR inhibitory character. RESULTS: Structurally dissimilar Michael acceptor-based natural compounds such as isobutylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2'-diOH-5,5'-diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 µM, respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to be targeted. INNOVATION: Identification of Michael acceptor-centric pharmacophores among diversified compounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhibitors is feasible. CONCLUSION: A strong TrxR inhibitory character correlated to the antiproliferative potency is attributed to structural features that include an α,ß-unsaturated carbonyl moiety centered in a DPPen or DPPro pharmacophore bearing hydroxyl and fluorine substitutions.

Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.

Shogaols at proapoptotic concentrations induce G(2)/M arrest and aberrant mitotic cell death associated with tubulin aggregation.

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G(2)/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G(2)/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G(2)/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G(2) checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.

Pachymic acid impairs breast cancer cell invasion by suppressing nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

Pachymic acid (PA), a lanostane-type triterpenoid derived from Poria cocos, possesses demonstrated anti-inflammatory and anti-cancer activities. Nonetheless, the biological properties and mechanism/s of action of PA remain largely undefined. In this study, the activity of PA against breast cancer cell invasion was evaluated. Invasiveness of human-derived MDA-MB-231 and MCF-7 breast carcinoma cells was suppressed by PA at non-lethal concentrations, which was associated with a decrease in matrix metalloproteinase-9 (MMP-9) secretion as a result of PA-mediated down-regulation of MMP-9 mRNA expression. In order to elucidate the underlying anti-invasive mechanism, the effect of PA on transcription factors activator protein-1 (AP-1) and nuclear factor kappaB (NF-κB) was examined using luciferase-based reporter gene assays. PA was found to bring about a reduction in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activity of NF-κB, but not that of AP-1. In accord with the luciferase activity data, western blot analysis showed that PA inhibited NF-κB signaling pathway, but did not alter the phosphorylation states of mitogen-activated protein kinases including ERK, JNK, and p38 kinase. The inhibition of PA on NF-κB signaling pathway was further attributed to PA-mediated diminution in PMA-induced degradation of inhibitor of kappaBα (IκBα) through preventing phosphorylation of the upstream signal IκB kinase (IKK). A decrease in p65 nuclear translocation was achieved, which led to attenuation of NF-κB transactivation. Taken together, it was concluded that by targeting NF-κB signaling, PA inhibited breast cancer cell invasion through decreasing MMP-9 expression. PA may thus be potentially exploited for use in tumor metastasis intervention.

Inhibition of hypoxia-inducible factor-1alpha (HIF-1alpha) protein synthesis by DNA damage inducing agents.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that is composed of a hypoxia-inducible alpha subunit (HIF-1alpha and HIF-2alpha) and a constitutively expressed beta subunit (HIF-1beta). HIF mediates the adaptation of cells and tissues to low oxygen concentrations. It also plays an important role in tumorigenesis and constitutes an important therapeutic target in anti-tumor therapy. We have screened a number of reported HIF inhibitors for their effects on HIF-transcriptional activity and found that the DNA damage inducing agents camptothecin and mitomycin C produced the most robust effects. Camptothecin is a reported inhibitor of HIF-1alpha translation, while mitomycin C has been reported to induce p53-dependent HIF-1alpha degradation. In this study we demonstrate that the inhibitory effect of mitomycin C on HIF-1alpha protein expression is not dependent on p53 and protein degradation, but also involves HIF-1alpha translational regulation. Initiation of a DNA damage response with the small molecule p53 activator NSC-652287 (RITA) has been reported to inhibit HIF-1alpha protein synthesis by increasing the phosphorylation of eIF2alpha. However, we show here that even when eIF2alpha phosphorylation is prevented, the DNA damage inducing drugs mitomycin C, camptothecin and NSC-652287 still inhibit HIF-1alpha protein synthesis to the same extent. The inhibitory effects of camptothecin on HIF-1alpha expression but not that of mitomycin C and NSC-652287 were dependent on cyclin-dependent kinase activity. In conclusion, specific types of DNA damage can bring about selective inhibition of HIF-1alpha protein synthesis. Further characterization of the involved mechanisms may reveal important novel therapeutic targets.

Functionalized aurones as inducers of NAD(P)H:quinone oxidoreductase 1 that activate AhR/XRE and Nrf2/ARE signaling pathways: synthesis, evaluation and SAR.

The chemopreventive potential of functionalized aurones and related compounds as inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) are described. Several 4,6-dimethoxy and 5-hydroxyaurones induced NQO1 activity of Hepa1c1c7 cells by 2-fold at submicromolar concentrations, making these the most potent inducers to be identified from this class. Mechanistically, induction of NQO1 was mediated by the activation of AhR/XRE and Nrf2/ARE pathways, indicating that aurones may be mixed activators of NQO1 induction or agents capable of exploiting the proposed cross-talk between the AhR and Nrf2 gene batteries. QSAR analysis by partial least squares projection to latent structures (PLS) identified size parameters, in particular those associated with non-polar surface areas, as an important determinant of induction activity. These were largely determined by the substitution on rings A and B. A stereoelectronic role for the exocyclic double bond as reflected in the E(LUMO) term was also identified. The electrophilicity of the double bond or its effect on the conformation of the target compound are possible key features for induction activity.

Cinnamaldehydes inhibit thioredoxin reductase and induce Nrf2: potential candidates for cancer therapy and chemoprevention.

Trans-cinnamaldehyde (CA) and its analogs 2-hydroxycinnamaldehyde and 2-benzoyloxycinnamaldehyde have been reported to possess antitumor activity. CA is also a known Nrf2 activator. In this study, a series of ortho-substituted cinnamaldehyde analogs was synthesized and screened for antiproliferative and thioredoxin reductase (TrxR)-inhibitory activities. Whereas CA was weakly cytotoxic and TrxR inhibiting, hydroxy and benzoyloxy substitutions resulted in analogs with enhanced antiproliferative activity paralleling increased potency in TrxR inactivation. A novel analog, 5-fluoro-2-hydroxycinnamaldehyde, was identified as exhibiting the strongest antitumor effect (GI(50) 1.6 microM in HCT 116 cells) and TrxR inhibition (IC(50) 7 microM, 1 h incubation with recombinant TrxR). CA and its 2-hydroxy- and 2-benzoyloxy-substituted analogs possessed dual TrxR-inhibitory and Nrf2-inducing effects, both attributed to an active Michael acceptor pharmacophore. At lethal concentrations, TrxR-inhibitory potencies correlated with the compounds' antiproliferative activities. The penultimate C-terminal selenocysteine residue was shown to be a possible target. Conversely, at sublethal concentrations, these agents induced an adaptive antioxidant response through Nrf2-mediated upregulation of phase II enzymes, including TrxR induction. We conclude from the results obtained that TrxR inactivation contributes at least partly to cinnamaldehyde cytotoxicity. These Michael acceptor molecules can potentially be exploited for use in different concentrations in chemotherapeutic and chemopreventive strategies.