Improving function of cytotoxic T-lymphocytes by transforming growth factor-ß inhibitor in oral squamous cell carcinoma.

Immunotherapy with immune-checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor-ß (TGF-ß) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T-cells (Tregs) and cancer-associated fibroblasts and inhibiting the function of cytotoxic T-lymphocytes (CTLs) and natural killer cells. TGF-ß may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF-ß on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T-cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF-ß suppressed the function of antigen-specific CTLs in the priming and effector phases in vitro. Additionally, TGF-ß inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T-cell/Treg ratio and between TGFB1 mRNA expression and the Ki-67 expression in CD8+ T-cells, indicating that TGF-ß also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF-ß function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune-checkpoint inhibitors and TGF-ß inhibitors, for OSCCs.

GINS2 was regulated by lncRNA XIST/miR-23a-3p to mediate proliferation and apoptosis in A375 cells.

Melanoma ranks second in aggressive tumors, and the occurrence of metastasis in melanoma results in a persistent drop in the survival rate of patients. Therefore, it is very necessary to find a novel therapeutic method for treating melanoma. It has been reported that lncRNA XIST could promote the tumorigenesis of melanoma. However, the mechanism by which lncRNA XIST regulates the progression of melanoma remains unclear. The proliferation of A375 cells was measured by clonal formation. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis and cycle. The level of GINS2, miR-23a-3p, and lncRNA XIST was investigated by qRT-PCR. Protein level was detected by Western blot, and the correctness of prediction results was confirmed by Dual luciferase. In present study, GINS2 and lncRNA XIST were overexpressed in melanoma, while miR-23a-3p was downregulated. Silencing of GINS2 or overexpression of miR-23a-3p reversed cell growth and promoted apoptosis in A375 cells. Mechanically, miR-23a-3p directly targeted GINS2, and XIST regulated GINS2 level though mediated miR-23a-3p. Moreover, XIST exerted its function on cell proliferation, cell viability, and promoted the cell apoptosis of A375 cells though miR-23a-3p/GINS2 axis. LncRNA XIST significantly promoted the tumorigenesis of melanoma via sponging miR-23a-3p and indirectly targeting GINS2, which can be a potential new target for treating melanoma.

A novel strategy of nanosized herbal Plectranthus amboinicus, Phyllanthus niruri and Euphorbia hirta treated TiO2 nanoparticles for antibacterial and anticancer activities.

Titanium dioxide nanoparticles exhibit good anticancer and antibacterial activities. They are known to be environmentally friendly, stable, less toxic, and have excellent biocompatibility nature. Due to these properties, they are well suited for biological applications particularly in biomedical applications such as drug delivery and cancer therapy. In this research article, three medicinal herbs namely, Plectranthus amboinicus (Karpooravalli), Phyllanthus niruri (Keezhanelli), and Euphorbia hirta (Amman Pacharisi), were used to modify the surface of the TiO2 nanoparticles. The synthesized nanoparticles were subjected to various characterization techniques. The samples are then subjected to MTT assay to determine cell viability. KB oral cancer cells are used for the determination of the anticancer nature of the pure and bio modified nanoparticles. It is observed that Plectranthus amboinicus-Phyllanthus niruri modified TiO2 nanoparticles exhibit excellent anticancer activities among other bio modified and pure samples. The samples are then examined for antibacterial activities against three Gram-negative bacterial strains namely, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Gram-positive bacterial strains namely, Staphylococcus aureus and Streptococcus mutans, respectively. Among the modified and pure samples, Plectranthus amboinicus showed good antibacterial activity against Gram-positive and Gram-negative bacteria. In the Flow cytometry analysis, the generation of p53 protein expression from Plectranthus amboinicus-Phyllanthus niruri modified TiO2 nano herbal particles shows the anti-cancerous nature of the sample. Then to determine the toxic nature of the Plectranthus amboinicus-Phyllanthus niruri modified TiO2 nano herbal particles against normal cells, the NPs were subjected to MTT assay against normal L929 cells, and it was found to be safer and less toxic towards the normal cells.

Biotransformation of the Phenolic Constituents from Licorice and Cytotoxicity Evaluation of Their Metabolites.

Biotransformation of four bioactive phenolic constituents from licorice, namely licoisoflavanone (1), glycyrrhisoflavone (2), echinatin (3), and isobavachalcone (4), was performed by the selected fungal strain Aspergillus niger KCCM 60332, leading to the isolation of seventeen metabolites (5-21). Structures of the isolated compounds were determined on the basis of extensive spectroscopic methods, twelve of which (5-7, 10-17 and 19) have been previously undescribed. A series of reactions including hydroxylation, hydrogenation, epoxidation, hydrolysis, reduction, cyclization, and alkylation was observed in the biotransformation process. All compounds were tested for their cytotoxic activities against three different human cancer cell lines including A375P, MCF-7, and HT-29. Compounds 1 and 12 exhibited most considerable cytotoxic activities against all the cell lines investigated, while compounds 2 and 4 were moderately cytotoxic. These findings will contribute to expanding the chemical diversity of phenolic compounds, and compounds 1 and 12 may serve as leads for the development of potential cancer chemopreventive agents.

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells.

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.

Tetrazolium reduction assays under-report cell death provoked by clinically relevant concentrations of proteasome inhibitors.

High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.

3-Hydroxyhexanoate-based polycationic nanoparticle system for delivering reprogramming factors.

Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell.Methods: We have prepared (3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR.Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.

Copper Coordination Compounds as Biologically Active Agents.

Copper-containing coordination compounds attract wide attention due to the redox activity and biogenicity of copper ions, providing multiple pathways of biological activity. The pharmacological properties of metal complexes can be fine-tuned by varying the nature of the ligand and donor atoms. Copper-containing coordination compounds are effective antitumor agents, constituting a less expensive and safer alternative to classical platinum-containing chemotherapy, and are also effective as antimicrobial, antituberculosis, antimalarial, antifugal, and anti-inflammatory drugs. 64Сu-labeled coordination compounds are promising PET imaging agents for diagnosing malignant pathologies, including head and neck cancer, as well as the hallmark of Alzheimer's disease amyloid-ß (Aß). In this review article, we summarize different strategies for possible use of coordination compounds in the treatment and diagnosis of various diseases, and also various studies of the mechanisms of antitumor and antimicrobial action.

Inhibition of didscoidin domain receptor 1 reduces epithelial-mesenchymal transition and induce cell-cycle arrest and apoptosis in prostate cancer cell lines.

Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.

The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications.

Hydropersulfides and related polysulfides have recently become topics of significant interest due to their physiological prevalence and proposed biological functions. Currently, examination of the effects of hydropersulfide treatment on cells is difficult due to their lack of inherent stability with respect to disproportionation. Herein, it is reported that the treatment of a variety of cell types with cysteine trisulfide (also known as thiocystine; Cys-SSS-Cys), results in an increase in intracellular hydropersulfide levels (e.g., cysteine hydropersulfide; Cys-SSH, and glutathione hydropersulfide; GSSH). Thus, Cys-SSS-Cys represents a possible pharmacological agent for examining the effects of hydropersulfides on cell function/viability. It has also been found that cells with increased intracellular hydropersulfide levels can export Cys-SSH into the extracellular media. Interestingly, the Cys-SSH is the major hydropersulfide exported by cells, although GSSH is the predominant intracellular species. The possible implications of cellular export are discussed.

Focal Cerebral Ischemia and Reperfusion Induce Brain Injury Through α2δ-1-Bound NMDA Receptors.

Background and Purpose- Glutamate NMDARs (N-methyl-D-aspartate receptors) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. α2δ-1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of α2δ-1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods- Mice were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results- Middle cerebral artery occlusion increased α2δ-1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the α2δ-1-NMDAR physical interaction in the mouse brain tissue. Inhibiting α2δ-1 with gabapentin, uncoupling the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or α2δ-1 C-terminus-interfering peptide or Cacna2d1 genetic knock-out reduced middle cerebral artery occlusion-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions- α2δ-1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and α2δ-1-bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting α2δ-1-bound NMDARs, without impairing physiological α2δ-1-free NMDARs, may be a promising strategy for treating ischemic stroke.

Disruption of Functional Activity of Mitochondria during MTT Assay of Viability of Cultured Neurons.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

Enhanced antitumor activity in A431 cells via encapsulation of 20(R)-ginsenoside Rg3 in PLGA nanoparticles.

OBJECTIVE: The objective of this study is to investigate the encapsulation of 20(R)-ginsenoside Rg3 (20(R)-Rg3) using polylactic-co-glycolic acid (PLGA) and promotion for its antitumor activity. SIGNIFICANCE: Preparation and evaluation of the antitumor efficacy of 20(R)-Rg3-loaded PLGA nanoparticles were the first reported. The data will be helpful to apply 20(R)-Rg3 efficiently and broadly in new drug form development and clinical cancer treatment. METHODS: The nanoparticles were prepared using emulsion and solvent evaporation methods. The uniform particle size and good dispersion were further confirmed by scanning electron microscopy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was applied to detect cell proliferation after 20(R)-Rg3-loaded PLGA nanoparticles treatment. Western blotting and immunofluorescent staining were used for observation of key proteins related with proliferation and apoptosis. Cell cycle and apoptosis were analyzed by flow cytometer technology. RESULTS AND DISCUSSION: The results showed that the size of 20(R)-Rg3-loaded PLGA was 97.5 nm in diameter, and zeta potential was -28 mV detected by Malvern particle size analyzer. The encapsulation efficiency was 97.5%, and drug loading was 70.2% measured by high-performance liquid chromatography. The in vitro study showed that the encapsulated 20(R)-Rg3 was consecutively released and the release ratio reached to the highest value (19.36%) at the time point of 96 h. The encapsulated 20(R)-Rg3 significantly inhibited the proliferation and induced apoptosis in A431 cancer cells compared with the unencapsulated 20(R)-Rg3, control and PLGA alone. CONCLUSION: 20(R)-Rg3-loaded PLGA nanoparticles was well prepared and characterized. The antitumor activity was increased after PLGA encapsulation. The data will be beneficial to the development of new dosage forms of 20(R)-Rg3 and extensive application.

Effects of xylazine and dexmedetomidine on equine articular chondrocytes in vitro.

OBJECTIVE: To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. STUDY DESIGN: Prospective, experimental study. STUDY MATERIAL: Equine articular chondrocytes from five male horses. METHODS: Chondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL-1) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL-1) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xylazine (1, 4, 25 mg mL-1) or dexmedetomidine (0.05, 0.175, 0.25 mg mL-1) for 15 minutes to further evaluate: cell viability by neutral red uptake; cell membrane integrity by lactate dehydrogenase release and by fluorescence microscopy with Hoechst 33342 and propidium iodide (PI), and apoptosis by flow cytometry using double staining with annexin V-fluorescein isothiocyanate/PI and by cell morphology. RESULTS: Both drugs reduced cell viability in a dose-dependent manner. Specifically, all xylazine concentrations, except 0.5 mg mL-1 and 1 mg mL-1, significantly reduced cell viability, whereas the effects of dexmedetomidine were evident only at 0.175 mg mL-1 and 0.25 mg mL-1. The highest concentrations of xylazine (25 mg mL-1) and dexmedetomidine (0.25 mg mL-1) caused loss of membrane integrity. Cell morphology and flow cytometry analyses demonstrated signs of late apoptosis in xylazine-treated cells, and signs of late apoptosis and necrosis in dexmedetomidine-treated cells. CONCLUSIONS AND CLINICAL RELEVANCE: This study offers new insights into the potential chondrotoxicity induced by dexmedetomidine and xylazine. Therefore, the intra-articular administration of α2-agonists should be conducted with care, especially for doses of ≥ 4 mg mL-1 of xylazine and 0.175 mg mL-1 and 0.25 mg mL-1 of dexmedetomidine.

Inhibition of protein translation by the DISC1-Boymaw fusion gene from a Scottish family with major psychiatric disorders.

The t(1; 11) translocation appears to be the causal genetic lesion with 70% penetrance for schizophrenia, major depression and other psychiatric disorders in a Scottish family. Molecular studies identified the disruption of the disrupted-in-schizophrenia 1 (DISC1) gene by chromosome translocation at chromosome 1q42. Our previous studies, however, revealed that the translocation also disrupted another gene, Boymaw (also termed DISC1FP1), on chromosome 11. After translocation, two fusion genes [the DISC1-Boymaw (DB7) and the Boymaw-DISC1 (BD13)] are generated between the DISC1 and Boymaw genes. In the present study, we report that expression of the DB7 fusion gene inhibits both intracellular NADH oxidoreductase activities and protein translation. We generated humanized DISC1-Boymaw mice with gene targeting to examine the in vivo functions of the fusion genes. Consistent with the in vitro studies on the DB7 fusion gene, protein translation activity is decreased in the hippocampus and in cultured primary neurons from the brains of the humanized mice. Expression of Gad67, Nmdar1 and Psd95 proteins are also reduced. The humanized mice display prolonged and increased responses to the NMDA receptor antagonist, ketamine, on various mouse genetic backgrounds. Abnormal information processing of acoustic startle and depressive-like behaviors are also observed. In addition, the humanized mice display abnormal erythropoiesis, which was reported to associate with depression in humans. Expression of the DB7 fusion gene may reduce protein translation to impair brain functions and thereby contribute to the pathogenesis of major psychiatric disorders.

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells.

Two new sesquiterpenoids from cultures of the basidiomycete Tremella foliacea.

Two new sesquiterpenoids, trefoliol B (1) and trefoliol C (2), together with known echinocidin A (3), were isolated from cultures of the basidiomycetes Tremella foliacea. The new structures were elucidated on the basis of extensive spectroscopic methods. At the same time, trefoliol B (1) and echinocidin A (3) were tested for their cytotoxicities against five human cancer cell lines and for their inhibitory activities against isozymes of 11ß-hydroxysteroid dehydrogenases (11ß-HSD). No compound showed significant activity (IC50 > 40 µM). Compound 1 showed moderate inhibitory activities against 11ß-HSD1 (human IC50 = 13.1 µM; mouse IC50 = 91.8 µM).

Cytotoxicity, interaction with dentine and efficacy on multispecies biofilms of a modified salt solution intended for endodontic disinfection in a new in vitro biofilm model.

AIM: To investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. METHODOLOGY: In a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P < 0.05 was considered significant. RESULTS: The PDL fibroblasts remained metabolically active after challenges with MSS. Dentine powder did not alter the efficacy of MSS (P > 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine (CHX) or 2% sodium hypochlorite (NaOCl) (P > 0.05). CONCLUSIONS: Modified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and NaOCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered.

A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line.

CONTEXT: Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG). OBJECTIVE: The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. MATERIALS AND METHODS: The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. RESULTS: The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. CONCLUSION: Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.