Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition.

In this study, a series of 2- and/or 3-substituted juglone derivatives were designed and synthesized. Among them, 9, 18, 22, 30, and 31 showed stronger inhibition activity against cell surface PDI or recombinant PDI and higher inhibitory effects on U46619- and/or collagen-induced platelet aggregation than juglone. The glycosylated derivatives 18 and 22 showed improved selectivity for inhibiting the proliferation of multiple myeloma RPMI 8226 cells, and the IC50 values reached 61 and 48 nM, respectively, in a 72 h cell viability test. In addition, 18 and 22 were able to prevent tumor cell-induced platelet aggregation and platelet-enhanced tumor cell proliferation. The molecular docking showed the amino acid residues Gln243, Phe440, and Leu443 are important for the compound-protein interaction. Our results reveal the potential of juglone derivatives to serve as novel antiplatelet and anticancer dual agents, which are available to interrupt platelet-cancer interplay through covalent binding to PDI catalytic active site.

Preparation and Evaluation of 6-Gingerol Derivatives as Novel Antioxidants and Antiplatelet Agents.

Ginger (Zingiber officinale) is widely used as a spice and a traditional medicine. Many bioactivities have been reported for its extracts and the isolated compounds, including cardiovascular protective effects. Different pathways were suggested to contribute to these effects, like the inhibition of platelet aggregation. In this study, we synthesised fourteen 6-gingerol derivatives, including eight new compounds, and studied their antiplatelet, COX-1 inhibitor, and antioxidant activities. In silico docking of selected compounds to h-COX-1 enzyme revealed favourable interactions. The investigated 6-gingerol derivatives were also characterised by in silico and experimental physicochemical and blood-brain barrier-related parameters for lead and preclinical candidate selection. 6-Shogaol (2) was identified as the best overall antiplatelet lead, along with compounds 3 and 11 and the new compound 17, which require formulation to optimize their water solubility. Compound 5 was identified as the most potent antioxidant that is also promising for use in the central nervous system (CNS).

Chemical Constituents and Bioactive Principles from the Mexican Truffle and Fermented Products of the Derived Fungus Ustilago maydis MZ496986.

To look in-depth into the traditional Mexican truffle, this study investigated the phytochemical and pharmacological properties of field-collected corn galls and the fermentate of its pathogen Ustilago maydis MZ496986. Here, we established the chemical profiles of both materials via the gradient HPLC-UV method and successfully identified six previously unreported chemical entities, ustilagols A-F (1-6), and 17 known components. Compounds 3, 5, and 9 exhibited potent nitric oxide production inhibitory activities in murine brain microglial BV-2 cells (IC50 = 6.7 ± 0.5, 5.8 ± 0.9, and 3.9 ± 0.1 µM) without cytotoxic effects. DIMBOA (9) also attenuates lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW 264.7 macrophages (IC50 = 58.1 ± 7.2 µM). Ustilagol G (7) showed potent antiplatelet aggregation in U46619-stimulated human platelets (IC50 = 16.5 ± 5.3 µM). These findings highlighted the potential of corn galls and U. maydis MZ496986 fermentate as functional foods for improving inflammation-related discomforts and vascular obstruction.

Discovery of 7, 4'-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice.

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

3,5,2',4'-Tetramethoxystilbene, a fully methylated resveratrol analog, prevents platelet aggregation and thrombus formation by targeting the protease-activated receptor 4 pathway.

Thrombin is a potent platelet activator and a key mediator of blood coagulation, thereby playing a crucial role in cardiovascular disease. Recently, protease-activated receptor 4 (PAR4), one of thrombin receptors in human platelets, is emerging as a promising target for antiplatelet therapy. 3,5,2',4'-Tetramethoxystilbene (TMS), a resveratrol analog, have demonstrated promising effects on preventing atherosclerosis and hypertension, whereas its antiplatelet effect has never been investigated. Herein we show that TMS at concentrations of a few micromolar selectively inhibits PAR4-mediated human platelet aggregation, ATP secretion, integrin αIIbß3 activation, and signaling pathways. In a whole-blood model of arterial flow, TMS also significantly reduced in vitro thrombus formation. Analysis of the structure-activity relationships of TMS and a panel of stilbene analogs reveal that full methylation of hydroxy groups of the stilbenes is the critical structural determinant for the anti-PAR4 activity. Our results suggest that fully methylated resveratrol analogs with anti-PAR4 activity are potential candidates for development of novel antiplatelet agents.

The Impact of Urate-Lowering Therapy in Post-Myocardial Infarction Patients: Insights From a Population-Based, Propensity Score-Matched Analysis.

The role of urate-lowering therapy (ULT) for the primary prevention of cardiovascular (CV) events has been widely discussed, but its evidence for the secondary prevention of myocardial infarction (MI) is limited. Therefore, we conduct a population-based, propensity score-matched cohort study to investigate the CV outcomes among patients with post-MI with and without ULT. A total of 19,042 newly diagnosed in-hospital patients with MI were selected using the Taiwan National Health Insurance Database between January 1, 2005, and December 31, 2016. After 1:1 propensity score matching with covariates, patients with MI with (n = 963) and without (n = 963) ULT were selected for further analysis. The primary outcome was the all-cause mortality and the secondary outcomes were composite CV outcomes, including hospitalization for recurrent MI, stroke, heart failure, and cardiac arrhythmias. ULT users were associated with lower all-cause mortality (adjusted hazard ratio (adjHR), 0.67; 95% confidence interval (CI), 0.51-0.87) compared to the ULT nonusers. In addition, ULT users had a significantly lower risk of recurrent MI, which needed revascularization by percutaneous coronary intervention or coronary artery bypass grafting (adjHR, 0.67; 95% CI, 0.53-0.86) than the ULT nonusers. The primary and secondary outcomes were not different between patients with post-MI who received uricosuric agents and xanthine oxidase inhibitors. The anti-inflammatory effect of ULT plays an essential role in MI management. From a real-world setting, this study shows that ULT is associated with the lower risk of all-cause mortality in patients with post-MI. In addition, the result shows the possible lower incidence of repeat revascularization procedures in the ULT users.

Decoding Multiple Biofunctions of Maca on Its Anti-allergic, Anti-inflammatory, Anti-thrombotic, and Pro-angiogenic Activities.

Four active partition layers and ten isolates, including (5R)- and (5S)-macapyrrolidone A (1a, 1b), and four new alkaloids, (5R)- and (5S)-macapyrrolidone B (2a, 2b) and macapyrrolins D, E (3, 4), were isolated from maca (Lepidium meyenii Walp.), an indigenous food plant from Peru. Derived from the n-hexane layer, the macamide-rich fraction exhibited pro-angiogenic activity on EPC and HUVEC cells. Anti-thrombotic activity was displayed by the polar part of maca extracts (n-butanol and water layers). Both 75% methanol aq. (midlower polar part) and n-hexane (low polar part) layers, which showed signs of fatty acid content, markedly inhibited superoxide and elastase release in an anti-inflammatory assay. The 75% methanol aq. layer showed strong anti-allergic activity, and macapyrrolin A (5) was found active based on ß-hexosaminidase release inhibition assays and a ChemGPS-NP experiment. These valuable bioactivity results suggest that maca is a food plant with good benefits for human health.

Bioactive polyketides from the pathogenic fungus of Epicoccum sorghinum.

MAIN CONCLUSION: We discovered and identified a series of characteristic substances, including one new polyketide, epicorepoxydon B, of the important pathogenic fungus, Epicoccum sorghinum, of sorghum. The fungal extract and some isolated polyketides are sensitive to a malignant triple-negative breast cancer cell line, MDA-MB-231. Sorghum (Kaoliang) grain is an important crop with high economic value and several applications. In Taiwan, sorghum has been used in the wine industry, and "Kinmen Kaoliang Liquor" is a well-known Asian brand. Fungal contamination is one of the major threats affecting the production of sorghum grain resulting in economic losses as well as human and animal health problems. Several fungal species can infect sorghum grain and generate some toxic secondary metabolites. Epicoccum sorghinum is one of the major fungal contaminants of sorghum grains and a potent producer of mycotoxins such as tenuazonic acid (TeA). However, except for TeA, few studies focused on chemical compounds produced by this fungus. To explore the potential biological and toxic effects of E. sorghinum, a chemical investigation was carried out on the ethyl acetate extract of the fungus because it showed cytotoxic activity against a triple-negative breast cancer cell line, MDA-MB-231 (54.82% inhibition at 20 µg/mL). One new polyketide, epicorepoxydon B (1), along with six known compounds including 4,5-dihydroxy-6-(6'-methylsalicyloxy)-2-hydroxymethyl-2-cyclohexenl-one (2), epicorepoxydon A (3), 3-hydroxybenzyl alcohol (4), 6-methylsalicylic acid (5), gentisyl alcohol (6), and 6-(hydroxymethyl)benzene-1,2,4-triol (7) were obtained, and their structures were established by the interpretation of their MS and NMR spectroscopic data. The cytotoxic activity of all isolated polyketides 1-7 was evaluated, and compounds 2, 6, and 7 exhibited potent activities against A549, HepG2, and MDA-MB-231 human cancer cell lines with IC50 value ranging from 1.86 to 18.31 µM. The structure-activity relationship of the isolated compounds was proposed.

Golden berry 4ß-hydroxywithanolide E prevents tumor necrosis factor α-induced procoagulant activity with enhanced cytotoxicity against human lung cancer cells.

Inflammation in the tumor microenvironment is positively correlated with cancer progression and metastasis as well as the risk of thromboembolism in lung cancer patients. Here we show, in human non-small cell lung cancer (NSCLC) cell lines, the master inflammatory cytokine tumor necrosis factor (TNF-α) induced tissue factor expression and procoagulant activity, and these effects were potently inhibited by 4ß-hydroxywithanolide E (4HW), a natural compound isolated from Physalis peruviana. Furthermore, combination of 4HW and TNF-α caused synergistic cytotoxicity against NSCLC cells by inducing caspase-dependent apoptosis. The underlying mechanism by which 4HW reverses the procoagulant effect of TNF-α but enhances its cytotoxic effect appears to be due to inhibition of NF-κB, which is a key switch for both inflammation-induced coagulation and cell survival. Our results suggest that 4HW may have a potential application for treating inflammation-derived cancer progression and cancer-associated hypercoagulable state.

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase.

BACKGROUND/PURPOSE: Juglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time. STUDY DESIGN/METHODS: Human platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay. RESULTS: Juglone (1 - 5 µM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca2+ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation. CONCLUSION: Juglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway.

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

Identifying new liver X receptor alpha modulators and distinguishing between agonists and antagonists by crystal ligand pocket screening.

Background: Modulators of LXRα are of high pharmacological interest as LXRα regulates fatty acid metabolism, inflammatory processes and cancer. We aim to identify new LXRα modulators and to recognize a distinguishable feature of agonists. Results&methodology: The ligand self-dock and largest-cavity-size searching purposely located two appropriate ligand-binding sites to reach the two aims. One is identifying the new modulators from Maybridge library. 20 new compounds are confirmed by the in vitro reporter gene assay. The other is denoting an agonist by at least one best docking pose having one hydrogen bond to LXRα Helix12 His421. Conclusion: Based on the quality x-ray binding pocket, we can identify new LXRα modulators and distinguish between agonists and antagonists by molecular docking.

Anti-inflammatory, Antiplatelet Aggregation, and Antiangiogenesis Polyketides from Epicoccum sorghinum: Toward an Understating of Its Biological Activities and Potential Applications.

The ethyl acetate extract of an endophyte Epicoccum sorghinum exhibited anti-inflammatory activity at a concentration of <10 µg/mL. By bioassay-guided fractionation, one new compound, named epicorepoxydon A (1), and one unusual bioactive compound, 6-(hydroxymethyl)benzene-1,2,4-triol (6), together with six known compounds, were isolated from E. sorghinum. The structures of all isolates were established by spectroscopic analyses. The relative configuration of 1 was deduced by the NOESY spectrum and its absolute configuration was determined by X-ray single-crystal analysis. The biological activities of all isolates were evaluated using four types of bioassays including cytotoxicity, anti-inflammatory, antiplatelet aggregation, and antiangiogenesis activities. Compounds 4 and 6 showed potent anti-inflammatory activity, compound 2 possessed potent antiplatelet aggregation and antiangiogenesis activities, and compound 6 demonstrated antiangiogenesis activity. This fungal species can cause a human hemorrhagic disorder known as onyalai. In this study, we identified the active components with antiplatelet aggregation and antiangiogenesis activities, which may be related to the hemorrhagic disorder caused by this fungus. Moreover, we proposed a biosynthetic pathway of the isolated polyketide secondary metabolites and investigated their structure-activity relationship (SAR). Our results suggested that E. sorghinum is a potent source of biologically active compounds that can be developed as antiplatelet aggregation and anti-inflammatory agents.

Cardiac Rhythm and Molecular Docking Studies of Ion Channel Ligands with Cardiotoxicity in Zebrafish.

Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.

Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.

Different effects of 4ß-hydroxywithanolide E and withaferin A, two withanolides from Solanaceae plants, on the Akt signaling pathway in human breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) represents a clinical challenge because it lacks sensitivity to hormone therapy or other available molecule-targeted agents. In addition, TNBC frequently exhibits over-activation of the PI3K/Akt survival pathway that can contribute to chemotherapy resistance. 4ß-Hydroxywithanolide E (4-HW) and withaferin A (WA) are two withanolides from Solanaceae plants that exhibit promising anticancer activity in vitro and in vivo. PURPOSE: The aim of this study is to investigate and compare the effects of 4-HW and WA on TNBC cells and underling mechanisms. STUDY DESIGN/METHODS: The anticancer effects of 4-HW and WA were evaluated by cell viability, cell cycle arrest, and apoptosis assays. PI3K/Akt signaling and the expression of survivin, Bcl-2 family proteins and cyclin-dependent kinase inhibitors were evaluated by Western blot. The role of PI3K/Akt signaling in the withanolides-induced anticancer effects was examined by using a PI3K inhibitor and overexpression of a constitutively active form of Akt. RESULTS: In TNBC MDA-MB-231 cells, 4-HW and WA displayed different kinetic effect on cell availability. Cell cycle analysis revealed that 4-HW induced the G1-phase arrest while WA caused the G2/M-phase block. Both withanolides induced apoptosis, but WA also caused necrosis. 4-HW inhibited the PI3K/Akt pathway and survivin expression as well as up-regulated the cyclin-dependent kinase inhibitors p21 and p27. In contrast, WA is a more potent inhibitor of Hsp90 and elicited Akt activation at low doses but inhibited Akt signaling at higher doses by depleting the Akt protein. The PI3K inhibitor LY294002 mimicked the effects of 4-HW and potentiated the cytotoxic activity of WA. In contrast, overexpressing a constitutively active form of myristoylated Akt rescue cancer cells from 4-HW-induced cell death. CONCLUSION: The withanolides 4-HW and WA potently inhibit the viability of TNBC cells through induction of cell cycle arrest and apoptosis/necrosis. The PI3K/Akt pathway plays distinct roles in cancer cells respond to 4-HW and WA. These results suggest the potential applications of the withanolides for the treatment of TNBC.

YC-1 Prevents Tumor-Associated Tissue Factor Expression and Procoagulant Activity in Hypoxic Conditions by Inhibiting p38/NF-κB Signaling Pathway.

Tissue factor (TF) expressed in cancer cells has been linked to tumor-associated thrombosis, a major cause of mortality in malignancy. Hypoxia is a common feature of solid tumors and can upregulate TF. In this study, the effect of YC-1, a putative inhibitor of hypoxia-inducible factor-1α (HIF-1α), on hypoxia-induced TF expression was investigated in human lung cancer A549 cells. YC-1 selectively prevented hypoxia-induced TF expression and procoagulant activity without affecting the basal TF levels. Surprisingly, knockdown or pharmacological inhibition of HIF-1α failed to mimic YC-1's effect on TF expression, suggesting other mechanisms are involved. NF-κB, a transcription factor for TF, and its upstream regulator p38, were activated by hypoxia exposure. Treatment of hypoxic A549 cells with YC-1 prevented the activation of both NF-κB and p38. Inhibition of p38 suppressed hypoxia-activated NF-κB, and inhibited TF expression and activity to similar levels as treatment with an NF-κB inhibitor. Furthermore, stimulation of p38 by anisomycin reversed the effects of YC-1. Taken together, our results suggest that YC-1 prevents hypoxia-induced TF in cancer cells by inhibiting the p38/NF-κB pathway, this is distinct from the conventional anticoagulants that systemically inhibit blood coagulation and may shed new light on approaches to treat tumor-associated thrombosis.

The chemopreventive isothiocyanate sulforaphane reduces anoikis resistance and anchorage-independent growth in non-small cell human lung cancer cells.

The capacity of cancer cells to resist detachment-induced apoptosis, i.e. anoikis, as well as anchorage-independent growth are crucial prerequisites for tumor metastasis. Therefore, agents interfering these properties may provide novel anti-metastatic strategies. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is known as a potent chemopreventive agent, but its effect on anoikis resistance has not been investigated. In this study, two non-small cell lung cancer (NSCLC) cell lines, A549 and CL1-5 cells, were treated with SFN under either suspension or adhesion conditions. SFN exhibited more potent cytotoxicity against suspending rather than adherent cancer cells. The selective cytotoxicity was due to the induction of anoikis, as evident by chromatin condensation, Annexin V binding, and activation of the mitochondrial apoptotic pathway. SFN also inhibited NSCLC cell to form spherical colonies, suggesting that anchorage-independent growth was prevented by SFN. Consistently, SFN treatment led to inactivation of FAK and Akt, down-regulation of ß-catenin, and up-regulation of the cyclin-dependent kinase inhibitor p21. Because A549 cells with wild-type p53 are more sensitive to SFN than p53-mutant CL1-5 cells, p53 dependency of SFN responses were determined in p53-knockdown A549 cells. Knockdown of p53 attenuated the ability of SNF to inhibit anoikis resistance and sphere formation in A549 cancer cells, suggesting that the presence of p53 in NSCLC cancer cells is involved in the sensitivity to SFN. These results provide new insight into mechanisms underlying the chemopreventive ability of SFN and suggest a potential benefit of SFN to interfere with tumor metastasis.

ß-Nitrostyrene derivatives attenuate LPS-mediated acute lung injury via the inhibition of neutrophil-platelet interactions and NET release.

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are high-mortality and life-threatening diseases that are associated with neutrophil activation and accumulation within lung tissue. Emerging evidence indicates that neutrophil-platelet aggregates (NPAs) at sites of injury increase acute inflammation and contribute to the development of ALI. Although numerous studies have increased our understanding of the pathophysiology of ALI, there is still a lack of innovative and useful treatments that reduce mortality, emphasizing that there is an urgent need for novel treatment strategies. In this study, a new series of small compounds of ß-nitrostyrene derivatives (BNSDs) were synthesized, and their anti-inflammatory bioactivities on neutrophils and platelets were evaluated. The new small compound C7 modulates neutrophil function by inhibiting superoxide generation and elastase release. Compound C7 elicits protective effects on LPS-induced paw edema and acute lung injury via the inhibition of neutrophil accumulation, proinflammatory mediator release, platelet aggregation, myeloperoxidase activity, and neutrophil extracellular trap (NET) release. NET formation was identified as the bridge for the critical interactions between neutrophils and platelets by confocal microscopy and flow cytometry. This research provides new insights for elucidating the complicated regulation of neutrophils and platelets in ALI and sheds further light on future drug development strategies for ALI/ARDS and acute inflammatory diseases.

HPW-RX40 prevents human platelet activation by attenuating cell surface protein disulfide isomerases.

Protein disulfide isomerase (PDI) present at platelet surfaces has been considered to play an important role in the conformational change and activation of the integrin glycoprotein IIb/IIIa (GPIIb/IIIa) and thus enhances platelet aggregation. Growing evidences indicated that platelet surface PDI may serve as a potential target for developing of a new class of antithrombotic agents. In the present study, we investigated the effects of HPW-RX40, a chemical derivative of ß-nitrostyrene, on platelet activation and PDI activity. HPW-RX40 inhibited platelet aggregation, GPIIb/IIIa activation, and P-selectin expression in human platelets. Moreover, HPW-RX40 reduced thrombus formation in human whole blood under flow conditions, and protects mice from FeCl3-induced carotid artery occlusion. HPW-RX40 inhibited the activity of recombinant PDI family proteins (PDI, ERp57, and ERp5) as well as suppressed cell surface PDI activity of platelets in a reversible manner. Exogenous addition of PDI attenuated the inhibitory effect of HPW-RX40 on GPIIb/IIIa activation. Structure-based molecular docking simulations indicated that HPW-RX40 binds to the active site of PDI by forming hydrogen bonds. In addition, HPW-RX40 neither affected the cell viability nor induced endoplasmic reticulum stress in human cancer A549 and MDA-MB-231 cells. Taken together, our results suggest that HPW-RX40 is a reversible and non-cytotoxic PDI inhibitor with antiplatelet effects, and it may have a potential for development of novel antithrombotic agents.