Inhibition of Ku70 in a high-glucose environment aggravates bupivacaine-induced dorsal root ganglion neurotoxicity.

BACKGROUND: Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. METHODS: We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. RESULTS: Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. CONCLUSION: These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.

Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4- CD8- unconventional αß T cells (UTCαß). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαß associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαß polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors.

Cimifugin Inhibits Inflammatory Responses of RAW264.7 Cells Induced by Lipopolysaccharide.

BACKGROUND RAW264.7 cells are induced by lipopolysaccharide (LPS) as a rheumatoid arthritis (RA) model. The present study investigated the effect of cimifugin on the proliferation, migration, chemotaxis, and release of inflammation-related factors and inflammation-related signaling pathways of LPS-induced RAW264.7 cells. MATERIAL AND METHODS MTS assay was used to determine the proliferation of RAW264.7 cells. Transwell assay was employed to examine the migration and chemotaxis of the cells. ELISA was performed to measure the contents of chemotactic factors and inflammatory factors in cell culture supernatants. Western blotting was carried out to detect the expression of factors related with MAPKs and NF-κB signaling pathways. RESULTS Cimifugin (0-100 mg/L) had no cytotoxicity for RAW264.7 cells. LPS stimulation induced morphological differentiation of RAW264.7 cells, but intervention by cimifugin inhibited the activation effect by LPS by about 50%. Cimifugin (100 mg/L) decreased the migration and chemotaxis of RAW264.7 cells to 1/3 of that in control cells by decreasing the release of migration- and chemotaxis-associated factors by at least 30%. Cimifugin (100 mg/L) suppressed the release of inflammatory factors from RAW264.7 cells to less than 60% of that in the LPS group. In addition, cimifugin (100 mg/L) inhibited the activities of MAPKs and NF-κB signaling pathways. CONCLUSIONS The present study demonstrates that cimifugin reduces the migration and chemotaxis of RAW264.7 cells and inhibits the release of inflammatory factors and activation of related signaling pathways induced by LPS. Cimifugin may have potential pharmacological effects against RA.

Hepatocellular toxicity of oxalicumone A via oxidative stress injury and mitochondrial dysfunction in healthy human liver cells.

The marine­derived oxalicumone A (POA) has been demonstrated as a potent anti­tumor bioactive agent for a variety of human carcinoma, but to the best of our knowledge, remains to be evaluated in healthy liver cells. As many drugs distribute preferentially in the liver, the present study aimed to investigate the effects of POA on apoptosis, oxidative stress and mitochondrial function in L­02 healthy liver cells. A Cell­Counting kit­8 assay demonstrated that POA inhibits the proliferation of L­02 cells in a dose­ and time­dependent manner. Furthermore, POA induced apoptosis by increasing the percentage of cells in early apoptosis and the sub­G1 cell cycle, along with causing S­phase arrest in L­02 cells. Additionally, POA activated caspase 3, increased the protein expression levels of Fas ligand and B­cell lymphoma X­associated protein, and decreased the expression of the anti­apoptotic protein B­cell lymphoma 2. POA additionally reduced the content of GSH and the activity of superoxide dismutase, elevated malondialdehyde and nitric oxide levels, increased reactive oxygen species production and the levels of alanine aminotransferase and aspartate aminotransferase, which suggested that POA induced lipid peroxidation injury in L­02 cells and that oxidative stress serves an important role. Furthermore, POA caused alternations of mitochondrial function, including an abrupt depletion of adenosine triphosphate synthesis, mitochondrial permeability transition pore opening and depletion of mitochondrial membrane potential in L­02 cells. These data suggested that POA exerts cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial function and oxidative stress by POA may therefore be critical in POA­induced toxicity in L­02 cells.

Synthesis, characterization and antinociceptive activities of Novel 2-(2,4-dichlorophenyl)-4H-chromen-4-one.

A novel flavone derivative has been synthesized in good yield from ketone and aldehyde. The structure has been established by different spectroscopic techniques like 1H NMR, 13C NMR, IR and elemental analysis. The compound was then screened for its acute toxicity and antinociceptive activity studies on animal model. The novel compound was safe upto a maximum dose of 500mg/kg body weight oral dose in mice and showed 65.92 and 82.18% pheriperal analgesic activity at 15 and 30mg/kg body weight doses. Central antinociceptive activity of the compound was 53.13 and 64.44% at 15 and 30mg/kg body weight respectively.

Toxicity study of oxalicumone A, derived from a marine-derived fungus Penicillium oxalicum, in cultured renal epithelial cells.

Oxalicumone A (POA), a novel dihydrothiophene-condensed chromone, was isolated from the marine­derived fungus Penicillium oxalicum. Previous reports demonstrated that POA exhibits strong activity against human carcinoma cells, thus it has been suggested as a bioactive anticancer agent. To research the toxic effect of POA on cultured normal epithelial human kidney­2 (HK­2) cells and evaluate its clinical safety, cell survival was evaluated by the Cell Counting Kit-8 assay and apoptosis was evaluated by Hoechst 33258 staining, flow cytometry, caspase­3 activity assay and western blotting. 2',7'-Dichlorofluorescin diacetate and JC­1 dye staining was used to evaluate reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP), respectively. The results indicated that POA inhibited HK-2 cell growth and promoted apoptosis, by increasing levels of Fas cell surface cell receptor and the B­cell lymphoma 2 associated protein X apoptosis regulator (Bax)/B­cell lymphoma 2 apoptosis regulator (Bcl-2) ratio. POA treatment also induced release of ROS and loss of MMP in HK­2 cells. Compared with untreated control, a significant decrease was also demonstrated in superoxide dismutase activity and glutathione content with POA treatment, accompanied by enhanced release of N­acetyl­ß­D­glucosaminidase, increased leakage of lactate dehydrogenase, increased malondialdehyde formation and increased release of nitric oxide. In conclusion, the present in vitro study revealed that POA exhibits antiproliferation activity on HK­2 cells, through stimulation of apoptosis and oxidative stress injury, which may be relevant to its clinical application. The present study may, therefore, offer valuable new information regarding the use of POA as a candidate novel antitumor drug for clinical use.

A central role for R7bp in the regulation of itch sensation.

Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates Gαi/o -directed GTPase activating protein activity mediated by the Gß5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of Gαo (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.

Pyranochromones from Dictyoloma vandellianum A. Juss and Their Cytotoxic Evaluation.

One new chromone 3,3-dimethylallylspatheliachromene methyl ether (1), as well as five known chromones, 6-(3-methylbut-2-enyl) allopteroxylin methyl ether (2), 6-(3-methylbut-2-enyl) allopteroxylin (3), 3,3-dimethylallylspatheliachromene (4), 5-O-methylcneorumchromone K (5) and spatheliabischromene (6), two alkaloids, 8-methoxy-N-methylflindersine (7) and 8-methoxyflindersine (8), and two limonoids, limonin diosphenol (9) and rutaevin (10), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16-F10, HepG2, K562 and HL60 and non-tumor cells PBMC were evaluated for compounds 1 - 6. Compound 1 was the most active showing IC50 values ranging from 6.26 to 14.82 µg/ml in B16-F10 and K562 cell lines, respectively, and presented IC50 value of 11.65 µg/ml in PBMC cell line.

Effect of iguratimod and methotrexate on RANKL and OPG expression in serum and IL-1ß-induced fibroblast-like synoviocytes from patients with rheumatoid arthritis.

The receptor activator of nuclear factor κB ligand (RANKL)/receptor activator of nuclear factor κB (RANK)/osteoprotegerin (OPG) system plays a key role in rheumatoid arthritis (RA)-associated bone erosion. The upregulation of the RANKL/OPG ratio promotes bone erosion. The objective of this study is to explore the effects of iguratimod, a small-molecule disease-modifying antirheumatic drug (DMARD), alone or in combination with methotrexate (MTX), on RANKL and OPG expression in RA. We performed an enzyme-linked immunosorbent assay (ELISA) to investigate the modulatory effects of iguratimod, MTX, or their combination on serum RANKL and OPG levels of patients with RA before and after treatment for 12 and 24 weeks. Furthermore, fibroblast-like synoviocytes (FLS) from patients with RA were interleukin (IL)-1ß-stimulated and then treated with different concentrations of iguratimod, MTX, or both, and RANKL and OPG expressions were investigated by using ELISA, quantitative real-time polymerase chain reaction (qPCR) and western blot analysis. We found that RANKL levels and the RANKL/OPG ratio significantly decreased in both serum and IL-1ß-induced RA FLS after treatment. Moreover, combination therapy with iguratimod and MTX showed an even stronger inhibition than each drug alone did. Our results suggest that iguratimod and MTX, especially in combination, efficaciously protected against bone erosion by suppressing the production of RANKL.

Natural product pectolinarigenin inhibits osteosarcoma growth and metastasis via SHP-1-mediated STAT3 signaling inhibition.

Signal transducer and activator of transcription 3 (STAT3) has important roles in cancer aggressiveness and has been confirmed as an attractive target for cancer therapy. In this study, we used a dual-luciferase assay to identify that pectolinarigenin inhibited STAT3 activity. Further studies showed pectolinarigenin inhibited constitutive and interleukin-6-induced STAT3 signaling, diminished the accumulation of STAT3 in the nucleus and blocked STAT3 DNA-binding activity in osteosarcoma cells. Mechanism investigations indicated that pectolinarigenin disturbed the STAT3/DNA methyltransferase 1/HDAC1 histone deacetylase 1 complex formation in the promoter region of SHP-1, which reversely mediates STAT3 signaling, leading to the upregulation of SHP-1 expression in osteosarcoma. We also found pectolinarigenin significantly suppressed osteosarcoma cell proliferation, induced apoptosis and reduced the level of STAT3 downstream proteins cyclin D1, Survivin, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma extra-large (Bcl-xl) and myeloid cell leukemia 1 (Mcl-1). In addition, pectolinarigenin inhibited migration, invasion and reserved epithelial-mesenchymal transition (EMT) phenotype in osteosarcoma cells. In spontaneous and patient-derived xenograft models of osteosarcoma, we identified administration (intraperitoneal) of pectolinarigenin (20 mg/kg/2 days and 50 mg/kg/2 days) blocked STAT3 activation and impaired tumor growth and metastasis with superior pharmacodynamic properties. Taken together, our findings demonstrate that pectolinarigenin may be a candidate for osteosarcoma intervention linked to its STAT3 signaling inhibitory activity.

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer, in Sprague Dawley rats and Gottingen minipigs.

PURPOSE: Extensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer (123)I-CMICE-013 to support applications for clinical trials. METHODS: Sprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 µg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues. RESULTS: The acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain. CONCLUSION: The lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 µg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.

NU7441 Enhances the Radiosensitivity of Liver Cancer Cells.

OBJECTIVE: Radiation therapy, one of the major treatments for liver cancer, causes DNA damage and cell death. Since the liver cancer cells have a strong capacity to repair irradiative injury, new medicines to enhance this treatment are urgently required. In this study, we investigated the effect of NU7441, a synthetic small-molecule compound, as a specific inhibitor of DNA-dependent protein kinase (DNA-PK) in radiosensitization of hepatocellular carcinoma HepG2 cells. METHODS: Cell Counting Kit-8 (CCK-8) was first used to evaluate the proliferation of HepG2 cells under NU7441 treatment. SDS-PAGE and Western blot were then performed to study the protein expression leading to the DNA damage repair. Further, neutral single cell gel electrophoresis and immunofluorescence assay were carried out to assess DNA repair. Finally, flow cytometry was implemented to examine the changes in cell cycle. RESULTS: NU7441 reduced the CCK-8 counts in the HepG2 culture, further enhanced 60Cox03B3; radiation injury to HepG2 cells, which was manifested by decreasing the DNA-PKcs (S2056) protein expression, increasing x03B3;H2AX foci number, prolonging the tail moment of the comet cells, and inducing cell cycle arrest at G2/M phase. CONCLUSION: NU7441 inhibited the growth of liver cancer cells, enhanced the radiosensitization of these cancer cells by interfering with the DNA repair and cell cycle checkpoint. These data implicate NU7441 as a potential radiotherapy sensitizer for the treatment of liver cancer.

LY294002 prevents lipopolysaccharide­induced hepatitis in a murine model by suppressing IκB phosphorylation.

Although fulminant hepatitis represents a ubiquitous human health problem, there is a lack of effective therapeutic strategies that have few side­effects and the precise mechanisms underlying fulminant hepatitis are not fully understood. Phosphoinositide 3­kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in hepatic diseases. Although previous research indicates that PI3K is involved in cardiac diseases, including myocardial infarction, it currently remains unclear whether the inhibition of PI3K is essential for ameliorating the severity of lipopolysaccharide (LPS)­induced hepatitis. The aim of the present study was to investigate whether pharmacological blockade of PI3K ameliorates the development of LPS­induced murine acute hepatic injury. A murine model of LPS­induced acute hepatic injury was used to investigate the therapeutic effect of the pan­PI3K inhibitor, LY294002 on murine fulminant hepatitis and to investigate potential underlying mechanisms. The current report presents the in vivo role of LY294002 in protecting the mice from fulminant hepatitis. LY294002 was observed to exert significant protective effects on the liver by reducing the activities of alanine aminotransferase and aspartate aminotransferase, as well as by improving the histological architecture of the liver. In LPS­induced hepatitis, treatment with LY294002 clearly inhibited intrahepatic synthesis of various disease­relevant proinflammatory cytokines, including tumor necrosis factor­α, interleukin (IL)­6, IL­1ß and interferon­Î³. Furthermore, LY294002 was observed to significantly inhibit IκB phosphorylation in LPS­injured mouse liver samples. Therefore, LY294002 may protect the liver from LPS­induced injury by inhibition of the IκB­nuclear factor κ­light­chain­enhancer of activated B cell dependent signaling pathway. Thus, the current report provides evidence that LY294002 exerts potent effects against LPS­induced hepatic injury, indicating its potential therapeutic value for the treatment of acute hepatitis.

Quantitative Structure-Cytotoxicity Relationship of 3-Styryl-2H-chromenes.

BACKGROUND: Sixteen 3-styryl-2H-chromenes were subjected to quantitative structure-activity relationship analysis based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to find their new biological activities. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines, three mesenchymal and two epithelial normal oral cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Tumor-selectivity (TS) was evaluated by the ratio of the mean CC50 (50% cytotoxic concentration) against normal human oral cells to that against OSCC cell lines. Anti-HIV activity was evaluated by the ratio of CC50 to EC50 (50% cytoprotective concentration from HIV infection). Potency-selectivity expression (PSE) was determined by the ratio of TS/CC50 against OSCC. Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method. RESULTS: All 3-styryl-2H-chromene derivatives showed relatively high tumor selectivity. Especially, the compound that has a methoxy group at 7-position of the chromene ring and chlorine at 4'-position of phenyl group in styryl moiety [ 12: ] showed the highest TS and PSE values, exceeding those of resveratrol, doxorubicin and 5-FU. All compounds showed no anti-HIV activity. Among 330 chemical descriptors, 8, 74 and 16 descriptors significantly correlated to the cytotoxicity of normal and tumor cells, and tumor-specificity, respectively. CONCLUSION: Multivariate statistics with chemical descriptors for molecular shape and flatness may be useful for the evaluation of tumor-specificity of 3-styryl-2H-chromenes.

Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis.

Diplodiosis, a neuromycotoxicosis of cattle and sheep grazing on mouldy cobs infected by Stenocarpella maydis, is considered the last major veterinary mycotoxicosis for which the causative mycotoxin is still unknown. The current study was aimed at characterizing the cell death observed in mouse neuroblastoma (Neuro-2a), Chinese hamster ovary (CHO-K1) and Madin-Darby bovine kidney (MDBK) cell lines exposed to the S. maydis metabolites (i.e. diplodiatoxin and dipmatol) by investigating the roles of necrosis and apoptosis. Necrosis was investigated using the lactate dehydrogenase (LDH) leakage and propidium iodide (PI) flow cytometry assays and apoptosis was evaluated using the caspase-3/7 and Annexin V flow cytometry assays. In addition, transmission electron microscopy (TEM) was used to correlate the cell death pathways observed in this study with their typical morphologies. Both diplodiatoxin and dipmatol (750 µM) induced necrosis and caspase-dependent apoptosis in Neuro-2a, CHO-K1 and MDBK cells. Ultrastructurally, the two mycotoxins induced mitochondrial damage, cytoplasmic vacuolation and nuclear fragmentation in the three cell lines. These findings have laid a foundation for future studies aimed at elucidating in detail the mechanism of action of the S. maydis metabolites.

Plant-derived flavones as inhibitors of aurora B kinase and their quantitative structure-activity relationships.

Although several plant-derived flavones inhibit aurora B kinase (aurB), quantitative relationships between the structural properties of plant-derived flavones and their inhibitory effects on aurB remain unclear. In this report, these quantitative structure-activity relationships were obtained. For quercetagetin, found in the Eriocaulon species, showing the best IC50 value among the flavone derivatives tested in this report, further biological tests were performed using cell-based assays, including Western blot analysis, flow cytometry, and immunofluorescence microscopy. In vitro cellular experiments demonstrated that quercetagetin inhibits aurB. The molecular-binding mode between quercetagetin and aurB was elucidated using in silico docking. Quercetagetin binds to aurB, aurA, and aurC and prevents the active phosphorylation of all three aurora kinases. In addition, quercetagetin triggers mitotic arrest and caspase-mediated apoptosis. These observations suggest that quercetagetin is an aurora kinase inhibitor. Induction of mitosis-associated tumor cell death by quercetagetin is a promising strategy for developing novel chemotherapeutic anticancer agents.

3-benzylidene-4-chromanones: a novel cluster of anti-tubercular agents.

In a quest for developing novel anti-tubercular agents, a series of 3-benzylidene-4-chromanones 1a-l were evaluated for growth inhibition of Mycobacterium tuberculosis H37Rv. Three promising compounds 1d, g, j emerged as the lead compounds with the IC50 and IC90 values of less than 1 µg/mL. Evaluation of the potent compounds 1d, g, j and k against Vero monkey kidney cells revealed that these compounds are far more toxic to M. tuberculosis than to Vero cells. Structure-activity relationships demonstrated that 3-benzylidene-4-chromanones are more potent against M. tuberculosis than the related 2-benzylidene cycloalkanones and the meta substituted chromanone derivatives are more active than their ortho- and para-counterparts. Some guidelines for amplifying the project are presented.

Quantitative structure-cytotoxicity relationship of 3-styrylchromones.

BACKGROUND: Fifteen 3-styrylchromones were subjected to quantitative structure-activity relationship (QSAR) analysis based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to explore their biological activities. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and three human oral normal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-selectivity was evaluated by the ratio of the mean CC50 (50% cytotoxic concentration) against normal human oral cells to that against OSCC cell lines. Anti-HIV activity was evaluated by the ratio of CC50 to EC50 (50% cytoprotective concentration from HIV infection). Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method followed by the density functional theory (DFT) method. RESULTS: All 3-styrylchromone derivatives showed moderate-to-high tumor selectivity. Especially, compounds that have a methoxy group at 6-position of the chromone ring and hydroxyl group at 4'-position of phenyl group in styryl moiety [ 11: ] showed the highest tumor-selectivity. On the other hand, their cytotoxicity against normal cells showed good correlation to the descriptors that reflect hydrophobic interaction and molecular shapes. CONCLUSION: Multivariate statistics with chemical descriptors for the location of substituted group, molecular shape and electrostatic interaction may be useful for designing the most favorable compound with higher tumor selectivity.

Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key target for antiviral intervention. The goal of this study was to identify the binding site and unravel the molecular mechanism by which natural flavonoids efficiently inhibit HCV RdRp. Screening identified the flavonol quercetagetin as the most potent inhibitor of HCV RdRp activity. Quercetagetin was found to inhibit RdRp through inhibition of RNA binding to the viral polymerase, a yet unknown antiviral mechanism. X-ray crystallographic structure analysis of the RdRp-quercetagetin complex identified quercetagetin's binding site at the entrance of the RNA template tunnel, confirming its original mode of action. This antiviral mechanism was associated with a high barrier to resistance in both site-directed mutagenesis and long-term selection experiments. In conclusion, we identified a new mechanism for non-nucleoside inhibition of HCV RdRp through inhibition of RNA binding to the enzyme, a mechanism associated with broad genotypic activity and a high barrier to resistance. Our results open the way to new antiviral approaches for HCV and other viruses that use an RdRp based on RNA binding inhibition, that could prove to be useful in human, animal or plant viral infections.

Cytotoxicity of diplodiatoxin, dipmatol and diplonine, metabolites synthesized by Stenocarpella maydis.

The cytotoxicity of three Stenocarpella maydis metabolites (diplodiatoxin, dipmatol and diplonine) was investigated on Neuro-2a, CHO-K1 and MDBK cell lines. Diplodiatoxin was the most cytotoxic followed by dipmatol. Conversely, diplonine was not cytotoxic. Diplodiatoxin and dipmatol affected mitochondrial succinate dehydrogenase (MTT assay) and the overall viability of cells as assessed in real-time (xCELLigence assay). The results obtained so far indicate that diplodiatoxin and dipmatol exert their toxicity possibly via the necrotic cell death pathway.