Investigating the Anti-tumor and Apoptosis-inducing Effects of Coumarin Derivatives as Potent 15-Lipoxygenase Inhibitors on PC-3 Prostate Cancer Cells.

INTRODUCTION: Prostate cancer is the second most prevalent cancer among men. Despite different treatments, including surgery, chemotherapy, radiation therapy, hormone therapy and immunotherapy for this disease, patients ultimately progress to advanced states. Thus, there is a need for new treatment options targeting cell growth and apoptosis to better control the proliferation and metastasis of these cells. There are many reports indicating overexpression of the 15-lipoxygenase-1 (15-LOX-1) enzyme in prostate tumors. Studies have also shown that inhibition of this enzyme prevents the progression of prostate cancer. OBJECTIVE: This study was conducted to assess the anti-cancer properties of some coumarin derivatives as possible 15- LOX-1 inhibitors, on PC-3 prostate cancer cells. METHODS: In this study, the activity of 15-LOX-1 was evaluated in PC-3 cells by a spectrophotometric assay. In addition, due to high similarity between the 15-LOX-1 and soybean 15-lipoxygenase (SLO) (L1; EC 1, 13, 11, 12) active sites, the soybean SLO was used to investigate inhibitory effects of synthetic coumarin compounds 8- isopentenyloxycoumarin (8-IC), 8-isopentenyloxy-3-carboxycoumarin (8-ICC), 8-geranyloxycoumarin (8-GC), 8- geranyloxy-3-carboxycoumarin (8-GCC), and 8-farnesyloxy-3-carboxycoumarin (8-FCC) on this enzyme. Moreover, the cytotoxic and anticancer effects of the coumarin compounds were examined on PC-3 (Prostate Cancer) and HDF-1 (Human Dermal Fibroblast) cells by alamarBlue assay. Finally, apoptosis-inducing effects of all synthetic compounds were determined by flow cytometry. RESULTS: The IC50 values obtained by the alamarBlue test revealed that 8-IC, 8-GC and 8-GCC had cytotoxic effects on PC-3 cells. Treating both PC-3 and HDF-1 cells with 8-ICC and 8-FCC did not significantly reduce cell number. Furthermore, the IC50 values of 8-IC on HDF-1 cells showed cytotoxic effects, while treating these cells with 8-GC and 8- GCC did not show any significant cytotoxicity on these normal human fibroblasts. Assessing the ability of 4-MMPB (as a specific inhibitor of 15-LOX-1), 8-GC, and 8-GCC compounds to inhibit SLO revealed that these compounds exerted strong 15-LOX-1 inhibitory activity, while 8-IC and 8-FCC had a weak inhibitory effect and also 8-ICC showed no inhibitory effect on SLO enzyme. In addition, flow cytometric analysis by FITC (fluorescein isothiocyanate)- annexin V and propidium iodide showed that treatment with IC50 values of 8-GC and 8-GCC induced apoptosis in 35.2% and 30.8% of PC-3 cells, respectively. CONCLUSION: Thus, 8-GC and 8-GCC can be introduced as effective anticancer agents with apoptosis-inducing properties. Furthermore, our results suggest that the cytotoxic effects of these compounds might be related to the inhibition of 15-LOX-1 enzyme in PC-3 cells. On the other hand, the cytotoxic effects of 8-IC might be due to the inhibition of other signaling pathways in PC-3 cells. However, further in vivo experiments are required to determine the exact mechanisms involved in the anticancer effects of these coumarin compounds.

Identification of Zirconia Particle Uptake in Human Osteoblasts by ToF-SIMS Analysis and Particle-Size Effects on Cell Metabolism.

As the use of zirconia-based nano-ceramics is rising in dentistry, the examination of possible biological effects caused by released nanoparticles on oral target tissues, such as bone, is gaining importance. The aim of this investigation was to identify a possible internalization of differently sized zirconia nanoparticles (ZrNP) into human osteoblasts applying Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), and to examine whether ZrNP exposure affected the metabolic activity of the cells. Since ToF-SIMS has a low probing depth (about 5 nm), visualizing the ZrNP required the controlled erosion of the sample by oxygen bombardment. This procedure removed organic matter, uncovering the internalized ZrNP and leaving the hard particles practically unaffected. It was demonstrated that osteoblasts internalized ZrNP within 24 h in a size-dependent manner. Regarding the cellular metabolic activity, metabolization of alamarBlue by osteoblasts revealed a size- and time-dependent unfavorable effect of ZrNP, with the smallest ZrNP exerting the most pronounced effect. These findings point to different uptake efficiencies of the differently sized ZrNP by human osteoblasts. Furthermore, it was proven that ToF-SIMS is a powerful technique for the detection of zirconia-based nano/microparticles that can be applied for the cell-based validation of clinically relevant materials at the nano/micro scale.

Synthesis and in vitro studies for structure-based design of novel chalcones as antitubercular agents targeting InhA.

Background: The authors aimed to estimate the therapeutic potential of novel chalcones against tuberculosis. Methods: 11 synthesized compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis (H37RV; American Type Culture Collection number: 27294) using the microplate alamarBlue assay. Molecular docking and pharmacokinetic parameter analyses were then performed. Results: The most potent compounds, (2E)-1-(4-bromophenyl) (2E)-1-(2-nitrophenyl) prop-2-en-1-one, -3-(2-nitrophenyl) prop-2-en-1-one (4-bromophenyl) (2E)-1-(3-phenoxyphenyl)prop-2-en-1-one, 3-(phenoxyphenyl)prop-2-en-1-one (4-bromophenyl) prop-2-en-1-one and (2E)-1-(4-bromophenyl)-3-(5-chloro-2-hydroxyphenyl)-prop-2-en-1-one, showed in vitro activity, with a minimum inhibitory concentration (MIC) of 6.25 µg/ml. Conclusion: Compounds LSD2, LSD12, LSD13 and LSD15 showed strong in vitro antimycobacterial activity at a concentration of 6.25 µg/ml.

Detecting Ethambutol Resistance in Mycobacterium tuberculosis Isolates in China: A Comparison Between Phenotypic Drug Susceptibility Testing Methods and DNA Sequencing of embAB.

With the increasing incidence of drug-resistant tuberculosis (DR-TB), determining a rapid and accurate drug susceptibility testing (DST) method to identify ethambutol (EMB) resistance in Mycobacterium tuberculosis has become essential for patient management in China. Herein, we evaluated the correlation between three phenotypic DST methods, namely, proportion method (PM), MGIT 960 system, and microplate alamar Blue assay (MABA), and DNA sequencing of embAB in 118 M. tuberculosis isolates from China. When the results of the phenotypic DST methods were compared with those of DNA sequencing, the overall agreement and kappa values of the PM, MGIT 960 system, and MABA were 81.4% and 0.61, 77.1% and 0.55, and 84.7% and 0.67, respectively. The agreement for EMB resistance between MABA and PM was significantly higher than that between the MGIT 960 system and PM (P = 0.02). Moreover, among the isolates with detectable embAB mutations, 97.2% (70/72 isolates) harbored mutations in embB. The analysis of embB mutations predicted EMB resistance with 81.3% sensitivity, 86.8% specificity, and 83.1% accuracy. Thus, MABA may be a better phenotypic DST method for detecting EMB resistance. DNA sequencing of embB may be useful for the early identification of EMB resistance and the consequent optimization of the treatment regimen.

Cell proliferation assessment of human gingival fibroblasts treated with Clinacanthus nutans using alamarBlue assay

@#Clinacanthus nutans (C. nutans), a well-known ethnopharmacological plant consumed for its medicinal purposes by Southeast Asian communities. C. nutans is said to possess antipyretic, inflammatory, antiedemic as well as analgesic properties and used traditionally in treating various skin ailments, Herpes infection, cancer and diabetes. The young leaves of this C. nutans are consumed in Malaysia for maintaining health. In this study, the proliferative activity of human gingival fibroblast cells (HGF-1, ATCC®CRL-2014™, USA) treated with the ethanol extract obtained from C. nutans leaves at three different concentrations (250, 125 and 62.5 µg/ml) was compared with the untreated cells using alamarBlue assay. The proliferative activity of HGF-1 using alamarBlue assay showed that the cells treated with 62.5 μg/ml of ethanolic extract of C. nutans leaves exhibited increased proliferation compared to the other groups and hence does not exhibit any cytotoxicity on HGF-1.

The Dispersion State of Tangled Multi-Walled Carbon Nanotubes Affects Their Cytotoxicity.

The medical applications of carbon nanotubes (CNTs) have garnered much attention. However, evaluating the safety of CNTs remains difficult, and no consensus has been reached. Moreover, assessing the biosafety of multi-walled CNTs (MWCNTs), which can become tangled during manufacturing, is challenging because they do not readily disperse. We studied how the dispersion state of tangled MWCNTs affects their cytotoxicity, using three sonicators. Flotube 9110 (FT9110), tangled MWCNTs, were dispersed in two dispersants (fetal bovine serum and polysorbate 80) using a new type of sonicator (PR-1) and two conventional sonicators. The size and cytotoxicity of the dispersed FT9110 were measured using the BEAS-2B human bronchial epithelial cell line. The PR-1 dispersed the FT9110 to agglomerates <200 nm in diameter; FT9110 dispersed with the PR-1 did not show cytotoxicity regardless of dispersant. The other sonicators dispersed the FT9110 to particles >1000 nm in diameter, and cytotoxicity depended on the dispersant. We found that excluding cells adhered to agglomerated FT9110 before evaluating cytotoxicity can lead to false-positive results. The PR-1 sonicator dispersed tangled FT9110 to many single fibers, which showed lower cytotoxicity than conventionally-sonicated MWCNTs. We suggest that dispersion state should be accounted for when evaluating the cytotoxicity of MWCNTs.

HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF-7 Breast Cancer Cells in Vitro.

Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, ß-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normal-phase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γ-tocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0-100 µM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 µM) than that of nonoxidized γ-tocotrienol (134 µM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro.

In vitro antimycobacterial activity of six Cameroonian medicinal plants using microplate alamarBlue assay.

OBJECTIVE/BACKGROUND: The latest incidence of tuberculosis (TB) (per 100,000 people) in Cameroon was 243.00 as of 2011. Over the past 21 years, the value for this indicator has fluctuated between 112.00 in 1990 and 320.00 in 2003. Worldwide, this incidence has also increased, bringing back TB as a reemerging disease. On the same note, resistance to anti-TB drugs has increased, urging the search for new molecules. METHODS: This study was carried out to evaluate the antimycobacterial activity of six medicinal plants on the virulent strain, H37Rv, using the microplate alamarBlue assay. Mycobacterium tuberculosis (H37Rv strain) was incubated with decreased concentrations of six plant extracts, ranging from 250 µg/mL to 31.25 µg/mL. After 7 days of incubation at 37 °C, the effects of these plant extracts on the viability of the mycobacteria were evaluated. For each plant extract, the minimal inhibitory concentration was determined. RESULTS: The results showed that the compounds MBC1, MBC24, MBC68, MBC81, MBC117, and MBC118 were the best candidates with minimal inhibitory concentrations of 31.25, 62.5, 125, 62.5, and 125 µg/mL, respectively. CONCLUSION: These results confirm and validate the traditional use of these plants to treat respiratory diseases, which could be good sources and alternatives of plant metabolites for anti-TB-drug development.

A high-throughput screening assay for fungicidal compounds against Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)-infected individuals. Although amphotericin B is the "gold standard" treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.

Human epithelial tissue culture study on restorative materials.

OBJECTIVES: Health condition of the gingival tissues contacting the surfaces of fixed prostheses is a result of multiple etiologic factors. The aim of the investigation discussed here was to evaluate the attachment and proliferation rate of cultured human epithelial cells on three commonly used restorative materials under in vitro conditions. METHODS: Morphological and chemical structure of polished lithium-disilicate (IPS e.max Press, Ivoclar Vivadent AG, Germany), yttrium modified zirconium dioxide (5-TEC ICE Zirkon Translucent, Zirkonzahn GmbH Srl, Germany) and cobalt chromium alloy (Remanium star, Dentaurum GmbH & Co. KG, Germany) discs were examined by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and atomic force microscopy (AFM). Human epithelial cells harvested and cultured from one donor, were applied to investigate cell attachment (24h observation) and proliferation (72h observation) via dimethylthiazol-diphenyl tetrazolium bromide (MTT) and AlamarBlue(®) (AB) assays on control surface (cell-culture plate) and on the restorative materials (n=3×20 specimens/material). RESULTS: SEM and AFM revealed typical morphology and roughness features for the materials. Zirconia presented significantly higher Ra value. EDS confirmed typical elements on the investigated restorative materials: lithium-disilicate (Si, O); Zirconia (Zi, Y, O); CoCr (Co, Cr, W). All surfaces except CoCr exhibited significant cell proliferation according to MTT and AB assays after 72h compared to 24h. Among the restorative materials, CoCr samples showed the highest cell attachment as indicated by MTT assay. AB results showed that attachment and proliferation of human epithelial cells is supported more on lithium-disilicate. Both assays indicated the lowest value for zirconia. CONCLUSIONS: The results indicate that the restorative materials examined are equally suitable for subgingival restorations. Lithium-disilicate exhibited the best biocompatibility. CLINICAL SIGNIFICANCE: The examined materials are indicated for use in restorative procedures, directly contacting the sulcular epithelial tissues. Thus it is essential to monitor the biological acceptibility of these materials in order to better understand their clinical properties. The results indicate that Lithium-disilicate is a suitable material for such purposes.