Investigations of the highly efficient processing technique, chemical constituents, and anti-inflammatory effect of N-ethyl-2-pyrrolidinone-substituted flavan-3-ol (EPSF)-enriched white tea.

N-Ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) are a newly discovered compound class in tea with various bioactivities. This study aimed to develop a novel processing technique to enhance EPSF contents in white tea efficiently. Using optimal processing parameters of 125 °C and 30 min in a high-temperature sterilizing oven, total EPSF content significantly increased by 1.42-18.80-fold to 1.57-6.22 mg/g without impacting sensory characteristics. Metabolomics analysis revealed elevated levels of nucleosides, nucleotides, bases, theaflavins, flavonol aglycones, EPSFs, and most flavone-C-glycosides, as well as decreased levels of amino acids, procyanidins, theasinensins, several flavanols, and flavonol-O-glycosides after EPSF-enrichment treatment. Furthermore, the EPSF-enriched white tea exhibited notable anti-inflammatory effects, mitigating xylene-induced ear edema in mice and carrageenan-induced paw edema and cotton ball-induced granulomas in rats. This study developed a new processing technique for highly efficient enhancement of EPSFs in white tea and demonstrated that EPSF-enriched white tea has a potential to serve as effective anti-inflammatory dietary supplement.

The Effect of 4-(Dimethylamino)phenyl-5-oxopyrrolidines on Breast and Pancreatic Cancer Cell Colony Formation, Migration, and Growth of Tumor Spheroids.

A series of hydrazones, azoles, and azines bearing a 4-dimethylaminophenyl-5-oxopyrrolidine scaffold was synthesized. Their cytotoxic effect against human pancreatic carcinoma Panc-1 and triple-negative breast cancer MDA-MB-231 cell lines was established by MTT assay. Pyrrolidinone derivatives 3c and 3d, with incorporated 5-chloro and 5-methylbenzimidazole fragments; hydrazone 5k bearing a 5-nitrothien-2-yl substitution; and hydrazone 5l with a naphth-1-yl fragment in the structure significantly decreased the viability of both cancer cell lines. Compounds 3c and 5k showed the highest selectivity, especially against the MDA-MB-231 cancer cell line. The EC50 values of the most active compound 5k against the MDA-MB231 cell line was 7.3 ± 0.4 µM, which were slightly higher against the Panc-1 cell line (10.2 ± 2.6 µM). Four selected pyrrolidone derivatives showed relatively high activity in a clonogenic assay. Compound 5k was the most active in both cell cultures, and it completely disturbed MDA-MB-231 cell colony growth at 1 and 2 µM and showed a strong effect on Panc-1 cell colony formation, especially at 2 µM. The compounds did not show an inhibitory effect on cell line migration by the 'wound-healing' assay. Compound 3d most efficiently inhibited the growth of Panc-1 spheroids and reduced cell viability in MDA-MB-231 spheroids. Considering these different activities in biological assays, the selected pyrrolidinone derivatives could be further tested to better understand the structure-activity relationship and their mechanism of action.

Synthesis and In Vitro Evaluation as Potential Anticancer and Antioxidant Agents of Diphenylamine-Pyrrolidin-2-one-Hydrazone Derivatives.

The title compounds were synthesized by the reaction of 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide with various aldehydes bearing aromatic and heterocyclic moieties and acetophenones, and their cytotoxicity was tested via MTT assay against human triple-negative breast cancer MDA-MB-231, human melanoma IGR39, human pancreatic carcinoma Panc-1, and prostate cancer cell line PPC-1. Furthermore, the selectivity of compounds towards cancer cells compared to fibroblasts was also investigated. Four compounds were identified as the most promising anticancer agents out of a series of pyrrolidinone-hydrazone derivatives bearing a diphenylamine moiety. These compounds were most selective against the prostate cancer cell line PPC-1 and the melanoma cell lines IGR39, with EC50 values in the range of 2.5-20.2 µM against these cell lines. In general, the compounds were less active against triple-negative breast cancer MDA-MB-231 cell line, and none of them showed an inhibitory effect on the migration of these cells. In the 'wound healing' assay, N'-((5-nitrothiophen-2-yl)methylene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide was identified as the most promising derivative that could be further developed as an antimetastatic agent. N'-(5-chloro- and N'-(3,4-dichlorobenzylidene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazides most efficiently reduced the cell viability in IGR39 cell spheroids, while there was no effect of the investigated pyrrolidinone-hydrazone derivatives on PPC-1 3D cell cultures. Antioxidant activity determined via FRAP assay of N'-(1-(4-aminophenyl)ethylidene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide was 1.2 times higher than that of protocatechuic acid.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction.

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid). To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

Bursatella leachii Purple Ink Secretion Concentrate Exerts Cytotoxic Properties against Human Hepatocarcinoma Cell Line (HepG2): In Vitro and In Silico Studies.

Liver cancer is a leading cause of cancer death globally. Marine mollusc-derived drugs have gained attention as potential natural-based anti-cancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. Using liquid chromatography-mass spectrometry, the main biomolecules in the purple ink secretion released by the sea hare, named Bursatella leachii (B. leachii), were identified as hectochlorin, malyngamide X, malyngolide S, bursatellin and lyngbyatoxin A. The cytotoxic effects of B. leachii ink concentrate against human hepatocarcinoma (HepG2) cells were determined to be dose- and time-dependent, and further exploration of the underlying mechanisms causing the programmed cell death (apoptosis) were performed. The expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to the B. leachii ink concentrate. The gene expression levels of pro-apoptotic BAX, TP53 and Cyclin D1 were increased after treatment with the B. leachii ink concentrate. Applying in silico approaches, the high scores predicted that bioactivities for the five compounds were protease and kinase inhibitors. The ADME and cytochrome profiles for the compounds were also predicted. Altogether, the B. leachii ink concentrate has high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.

The Enhanced Cytotoxic Effects in B-Cell Leukemia and Lymphoma Following Activation of Prostaglandin EP4 Receptor and Targeting of CD20 Antigen by Monoclonal Antibodies.

Anti-CD20 monoclonal antibodies (MAbs) have revolutionized the treatment of B-cell leukemia and lymphoma. However, many patients do not respond to such treatment due to either deficiency of the complementary immune response or resistance to apoptosis. Other currently available treatments are often inadequate or induce major side effects. Therefore, there is a constant need for improved therapies. The prostaglandin E2 receptor 4 (EP4) receptor has been identified as a promising therapeutic target for hematologic B-cell malignancies. Herein, we report that EP4 receptor agonists PgE1-OH and L-902688 have exhibited enhanced cytotoxicity when applied together with anti-CD20 MAbs rituximab, ofatumumab and obinutuzumab in vitro in Burkitt lymphoma cells Ramos, as well as in p53-deficient chronic lymphocytic leukemia (CLL) cells MEC-1. Moreover, the enhanced cytotoxic effects of EP4 receptor agonists and MAbs targeting CD20 have been identified ex vivo on primary lymphocytes B obtained from patients diagnosed with CLL. Incubation of cells with PgE1-OH and L-902688 preserved the expression of CD20 molecules, further confirming the anti-leukemic potential of EP4 receptor agonists in combination with anti-CD20 MAbs. Additionally, we demonstrated that the EP4 receptor agonist PgE-1-OH induced apoptosis and inhibited proliferation via the EP4 receptor triggering in CLL. This work has revealed very important findings leading towards the elucidation of the anticancer potential of PgE1-OH and L-902688, either alone or in combination with MAbs. This may contribute to the development of potential therapeutic alternatives for patients with B-cell malignancies.

Constitutive, Basal, and ß-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling.

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist ß-alanine, we observed a release of IL-6. ß-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for ß-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical, on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration.

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone's ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called "BMP2 enhancers". In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as "osteopromotive substance" in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.

Synthesis and Bioactivity of Ancorinoside B, a Marine Diglycosyl Tetramic Acid.

The sponge metabolite ancorinoside B was prepared for the first time in 16 steps and 4% yield. It features a ß-d-galactopyranosyl-(1→4)-ß-d-glucuronic acid tethered to a d-aspartic acid-derived tetramic acid. Key steps were the synthesis of a fully protected d-lactose derived thioglycoside, its attachment to a C20-aldehyde spacer, functionalization of the latter with a terminal N-(ß-ketoacyl)-d-aspartate, and a basic Dieckmann cyclization to close the pyrrolidin-2,4-dione ring with concomitant global deprotection. Ancorinoside B exhibited multiple biological effects of medicinal interest. It inhibited the secretion of the cancer metastasis-relevant matrix metalloproteinases MMP-2 and MMP-9, and also the growth of Staphylococcus aureus biofilms by ca 87% when applied at concentrations as low as 0.5 µg/mL. This concentration is far below its MIC of ca 67 µg/mL and thus unlikely to induce bacterial resistance. It also led to a 67% dispersion of preformed S. aureus biofilms when applied at a concentration of ca 2 µg/mL. Ancorinoside B might thus be an interesting candidate for the control of the general hospital, catheter, or joint protheses infections.

Synthesis and preliminary biological evaluation of antibacterial and antifungal 5-arylidene tetramic acid-cadmium(II) complexes.

The synthesis and biological evaluation of 5-arylidene-N-acetyl-tetramic acids cadmium(II) complexes are reported. Eleven novel compounds were prepared, characterized by nuclear magnetic resonance experiments and screened for their antimicrobial activity against five bacterial species (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus [MRSA]) and two fungi (Candida albicans and Cryptococcus neoformans). The complexes showed similar or enhanced activities against MRSA in comparison to the corresponding ligands and, additionally, promising antifungal activities against C. neoformans. The most active compounds 3c and 3h showed remarkable activities against MRSA (minimum inhibitory activity [MIC] values of 32 and 4 µg/ml, respectively) and C. neoformans (MIC values of 8 and 16 µg/ml, respectively), accompanied by no human cell toxicity and hemolytic activity within the tested concentration range. The results demonstrate that appropriately functionalized tetramic acids attached with lipophilic alkanoyl chain and after complexation with cadmium(II) ions may act as valuable lead compounds for further investigations toward the development of novel antibacterial and/or antifungal agents.

CXCL13/CXCR5 axis facilitates endothelial progenitor cell homing and angiogenesis during rheumatoid arthritis progression.

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays important roles in several cellular functions such as infiltration, migration, and motility. We report significantly higher levels of CXCL13 expression in collagen-induced arthritis (CIA) mice compared with controls and also in synovial fluid from RA patients compared with human osteoarthritis (OA) samples. RA synovial fluid increased endothelial progenitor cell (EPC) homing and angiogenesis, which was blocked by the CXCL13 antibody. By interacting with the CXCR5 receptor, CXCL13 facilitated vascular endothelial growth factor (VEGF) expression and angiogenesis in EPC through the PLC, MEK, and AP-1 signaling pathways. Importantly, infection with CXCL13 short hairpin RNA (shRNA) mitigated EPC homing and angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with CIA. CXCL13 is therefore a novel therapeutic target for RA.

Design, synthesis and biological evaluation of novel pyrrolidone-based derivatives as potent p53-MDM2 inhibitors.

Inhibition of the interactions of the tumor suppressor protein p53 with its negative regulators MDM2 in vitro and in vivo, representing a valuable therapeutic strategy for cancer treatment. The natural product chalcone exhibited moderate inhibitory activity against MDM2, thus based on the binding mode between chalcone and MDM2, a hit unsaturated pyrrolidone scaffold was obtained through virtual screening. Several unsaturated pyrrolidone derivatives were synthesized and biological evaluated. As a result, because the three critical hydrophobic pockets of MDM2 were occupied by the substituted-phenyl linked at the pyrrolidone fragment, compound 4 h demonstrated good binding affinity with the MDM2. Additionally, compound 4 h also showed excellent antitumor activity and selectivity, and no cytotoxicity against normal cells in vitro. The further antitumor mechanism studies were indicated that compound 4 h could successfully induce the activation of p53 and corresponding downstream p21 proteins, thus successfully causing HCT116 cell cycle arrest in the G1/M phase and apoptosis. Thus, the novel unsaturated pyrrolidone p53-MDM2 inhibitors could be developed as novel antitumor agents.

Dual targeting of MEK and PI3K effectively controls the proliferation of human EGFR-TKI resistant non-small cell lung carcinoma cell lines with different genetic backgrounds.

BACKGROUND: Molecular targeted therapy for non-small cell lung carcinoma (NSCLC) is restricted due to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This study evaluated the effects of dual targeting of MEK and PI3K in human EGFR-TKI resistant NSCLC cell lines. METHODS: EGFR-TKI resistant NSCLC cell lines H1975, H460, and A549, with different mutation and amplification status in EGFR, K-RAS, PIK3CA, and MET genes, were treated with a MEK162 (MEK inhibitor) and BKM120 (PI3K inhibitor) combination or a BIBW2992 (EGFR inhibitor) and ARQ197 (MET inhibitor) combination and assayed for cell proliferation, apoptosis, and cell cycle distribution. RESULTS: Dual targeting of MEK and PI3K efficiently inhibited the cell proliferation, induced apoptosis and the G0/G1 cell cycle, and decreased the phosphorylation of ERK1/2, AKT, S6, and 4E-BP1. H460 cells with K-RAS and PIK3CA mutation were most sensitive to MEK162 and BKM120 combinations. H1975 cells with EGFR and PIK3CA mutation and MET amplification were sensitive to BIBW2992 and ARQ197 combinations. CONCLUSION: Dual targeting regulated the proliferation of EGFR-TKI-resistant NSCLC cells, especially mutants in K-RAS and PIK3CA that are promising for EGFR-TKI-resistant NSCLC therapeutics.

Rhinovirus-induced Human Lung Tissue Responses Mimic Chronic Obstructive Pulmonary Disease and Asthma Gene Signatures.

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.

N,N-Ru(II)-p-cymene-poly(N-vinylpyrrolidone) surface functionalized gold nanoparticles: from organoruthenium complex to nanomaterial for antiproliferative activity.

Organometallic Ru-arene complexes are promising as anticancer agents, but the lack of tumor uptake and poor solubility in the physiological medium impede their development. In order to deal with these challenges, we developed gold nanoparticles coated with Ru(arene)-functionalized PNVP-Py, where PNVP-Py is pyridine end-functionalized poly(N-vinylpyrrolidone). It is demonstrated that these particles exhibit higher anti-proliferative activity than the individual organometallic ruthenium(ii) complex of the type [Ru(η6-p-cymene)(NN)Cl]PF6, where NN is bis(4-methoxyphenylimino)acenaphthene, against colorectal adenocarcinoma cell lines. More specifically, a RuII(η6-p-cymene) complex containing a NN bidentate ligand has been prepared and characterized by spectral studies and X-ray crystallography. To tether the isolated complex onto the surface of the AuNPs, PNVP-Py, which contains a pyridine group at one end to coordinate to the Ru-complex and a suitable functional group at the other end to bind on the surface of the AuNPs, has been prepared and utilized to obtain the macromolecular complex [Ru(η6-p-cymene)(NN)(PNVP-Py)]Cl2. Next, stable Ru(p-cym)(NN)(PNVP-Py)@AuNPs were obtained via a ligand exchange reaction of citrate-stabilized AuNPs with a macromolecular complex by a direct 'grafting to' approach and characterized well. Despite the lower DNA cleavage activity, the nanoconjugate exhibits better cytotoxicity than the individual complex against HT-29 colorectal adenocarcinoma cells on account of its enhanced permeability across the cell membrane. The AO/EB staining assay revealed that the nanoconjugate is able to induce an apoptotic mode of cell death, which was further quantitatively evaluated by Annexin V-FITC/PI double assay. An immunofluorescence assay indicated the higher potency of the nanoconjugate to inhibit cyclin D1 gene expression that is required for cancer cell growth. To the best of our knowledge, this is the first report of the modification of an organometallic Ru(arene) complex into a Ru(arene)metallopolymer-gold nanoconjugate for the development of ruthenium-based nanomedicine for cancer treatment.

Kinomic profile in patient-derived glioma cells during hypoxia reveals c-MET-PI3K dependency for adaptation.

Hypoxic microenvironment is a hallmark of solid tumors, especially glioblastoma. The strong reliance of glioma-propagating cells (GPCs) on hypoxia-induced survival advantages is potentially exploitable for drug development. Methods: To identify key signaling pathways for hypoxia adaptation by patient-derived GPCs, we performed a kinase inhibitor profiling by screening 188 small molecule inhibitors against 130 different kinases in normoxia and hypoxia. Potential kinase candidates were prioritized for in vitro and in vivo investigations using a ranking algorithm that integrated information from the kinome connectivity network and estimated patients' survival based on expression status. Results: Hypoxic drug screen highlighted extensive modifications of kinomic landscape and a crucial functionality of c-MET-PI3K. c-MET inhibitors diminished phosphorylation of c-MET and PI3K in GPCs subjected to hypoxia, suggesting its role in the hypoxic adaptation of GPCs. Mechanistically, the inhibition of c-MET and PI3K impaired antioxidant defense, leading to oxidative catastrophe and apoptosis. Repurposed c-MET inhibitors PF04217903 and tivantinib exhibited hypoxic-dependent drug synergism with temozolomide, resulting in reduced tumor load and growth of GPC xenografts. Detailed analysis of bulk and single-cell glioblastoma transcriptomes associates the cellular subpopulation over-expressing c-MET with inflamed, hypoxic, metastatic, and stem-like phenotypes. Conclusions: Thus, our "bench to bedside (the use of patient-derived GPCs and xenografts for basic research) and back (validation with independent glioblastoma transcriptome databases)" analysis unravels the novel therapeutic indications of c-MET and PI3K/Akt inhibitors for the treatment of glioblastoma, and potentially other cancers, in the hypoxic tumor microenvironment.

The PSMD14 inhibitor Thiolutin as a novel therapeutic approach for esophageal squamous cell carcinoma through facilitating SNAIL degradation.

Metastasis and chemoresistance are major causes of poor prognosis in patients with esophageal squamous cell carcinoma (ESCC), manipulated by multiple factors including deubiquitinating enzyme (DUB). DUB PSMD14 is reported to be a promising therapeutic target in various cancers. Here, we explored the antitumor activity of Thiolutin (THL), the PSMD14 inhibitor, as a new therapy strategy in ESCC. Methods: Through 4-NQO-induced murine ESCC model, we investigated the expression of PSMD14 in esophageal tumorigenesis. Ubiquitin-AMC assay was performed to evaluate DUB activity of PSMD14 with THL treatment. The effect of THL on epithelial-to-mesenchymal transition (EMT), invasion, stemness and chemosensitivity was detected by using in vitro and in vivo experiments. Immunoprecipitation and in vivo ubiquitination assay were conducted to examine whether THL could impair the deubiquitination and stability of SNAIL regulated by PSMD14. Results: Compared with normal esophageal epithelium, PSMD14 was upregulated in 4-NQO-induced murine esophageal epithelium dysplasia and ESCC tissues. THL could significantly weaken DUB activity of PSMD14. Furthermore, the results of in vitro and in vivo assays showed that THL efficiently suppressed motility and stemness and increased sensitivity to cisplatin in ESCC. Mechanically, THL impaired the interaction between PSMD14 and SNAIL, then promoted the ubiquitination and degradation of SNAIL to inhibit EMT which plays a crucial role in ESCC metastasis, stemness and chemosensitivity. TCGA database analysis revealed that high concomitant PSMD14/SNAIL expression predicted shorter overall survival in esophageal cancer. Conclusion: Our findings demonstrate for the first time that suppression of PSMD14/SNAIL axis by THL could be a novel and promising therapeutic approach for ESCC clinical therapy.

Analgesic and Anticancer Activity of Benzoxazole Clubbed 2-Pyrrolidinones as Novel Inhibitors of Monoacylglycerol Lipase.

Ten benzoxazole clubbed 2-pyrrolidinones (11-20) as human monoacylglycerol lipase inhibitors were designed on the criteria fulfilling the structural requirements and on the basis of previously reported inhibitors. The designed, synthesized, and characterized compounds (11-20) were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Compounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative), with an IC50 value of 8.4 and 7.6 nM, were found most active, respectively. Both of them showed micromolar potency (IC50 value above 50 µM) against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of 2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three hydrogen bonds (~2 Å) with Ala51, Met123, and Ser122, the amino acid residues responsible for the catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test, compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They significantly demonstrated the reduction in pain response, having better potency than the positive control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA, for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth inhibition (% GI), respectively.

Fluorochloridone induces autophagy in TM4 Sertoli cells: involvement of ROS-mediated AKT-mTOR signaling pathway.

BACKGROUND: Fluorochloridone (FLC), a selective pyrrolidone herbicide, has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is unclear. The aim of this study was to investigate the mechanism of FLC-induced reproductive toxicity on male mice with particular emphasis on the role of autophagy in mice' TM4 Sertoli cells. METHODS: Adult C57BL/6 mice were divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160 µM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species (ROS), expression of autophagy marker proteins (LC3, Beclin-1 and P62) and AKT-related pathway proteins (AKT, mTOR) were observed. The ROS scavenger N-acetylcysteine (NAC) and AKT agonist (SC79) were used to treat TM4 cells to observe the changes of AKT-mTOR pathway and autophagy. RESULTS: In vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3II/LC3I, Beclin-1 and P62 protein, which is related to the degradation of autophagy. In vitro, FLC triggered TM4 cell autophagy by increasing the formation of autophagosomes and upregulating of LC3II/LC3I, Beclin-1 and P62 levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of NAC. Additionally, decreased autophagy and increased cell viability were observed in TM4 cells treated with SC79 and FLC, compared with FLC alone, indicating that FLC-induced autophagy may be pro-death. CONCLUSION: Taken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.

Brivaracetam prevents astroglial l-glutamate release associated with hemichannel through modulation of synaptic vesicle protein.

The antiepileptic/anticonvulsive action of brivaracetam is considered to occur via modulation of synaptic vesicle protein 2A (SV2A); however, the pharmacological mechanisms of action have not been fully characterised. To explore the antiepileptic/anticonvulsive mechanism of brivaracetam associated with SV2A modulation, this study determined concentration-dependent effects of brivaracetam on astroglial L-glutamate release associated with connexin43 (Cx43), tumour-necrosis factor-α (TNFα) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor of rat primary cultured astrocytes using ultra-high-performance liquid chromatography. Furthermore, interaction among TNFα, elevated extracellular K+ and brivaracetam on expression of SV2A and Cx43 was determined using capillary immunoblotting. TNFα and elevated extracellular K+ predominantly enhanced astroglial L-glutamate release associated with respective AMPA/glutamate receptor and hemichannel. These effects were enhanced by a synergistic effect of TNFα and elevated extracellular K+ in combination. The activation of astroglial L-glutamate release, and expression of SV2A and Cx43 in the plasma membrane was suppressed by subchronic brivaracetam administration but were unaffected by acute administration. These results suggest that migration of SV2A to the astroglial plasma membrane by hyperexcitability activates astroglial glutamatergic transmission, perhaps via hemichannel activation. Subchronic brivaracetam administration suppressed TNFα-induced activation of AMPA/glutamate receptor and hemichannel via inhibition of ectopic SV2A. These findings suggest that combined inhibition of vesicular and ectopic SV2A functions contribute to the antiepileptic/anticonvulsive mechanism of brivaracetam action.