Microculture Tetrazolium Research Articles

Halogenated 2-amino-4H-pyrano[3,2-h]quinoline-3-carbonitriles as antitumor agents and structure–activity relationships of the 4-, 6-, and 9-positions

A series of halogenated 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives were prepared via interaction of 8-hydroxyquinoline, 5-chloro-8-hydroxyquinoline, and 8-hydroxy-2-methylquinoline with various α-cyanocinnamonitriles. The assignments of the structure of all synthesized compounds were based on spectral data. The cytotoxic activities of the synthesized compounds against four human tumor cell lines MCF-7, HCT-116, HepG-2, and A549 in comparison with the reference drugs Vinblastine and Colchicine were determined by microculture tetrazolium assay. Several compounds showed significant cytotoxic activity. The structure–activity relationship studies reported that the substitution at 4-, 6-, and 9-positions in several 2-amino-4H-pyrano[3,2-h]quinoline nucleus with the specific halogen atom and lipophilicity increases the ability of the molecule against the different cell lines.

Evaluation of the Cytotoxicity Effect of Chaerophyllum Extract on Leishmania major and J774 Cell Line in Vitro

Background: Cutaneous Leishmaniasis (CL) is a serious threat to many endemic regions of the world, which may lead to some complications such as secondary infections, permanent scars, etc. Although there are available drugs, i.e. pentavalent antimony compounds for the treatment of CL, they are expensive, have side effects and/or there may be some drug-resistant species. Therefore, there is a tantalizing field of study to discover novel compounds against CL. In this study, we evaluated the leishmanicidal effects of Chaerophyllum plant extract on Leishmania major promastigotes in vitro. Methods: In each sterile 96-well plate, a volume of 100 λ of promastigotes was added. The three first wells lacked drug but contained drug solvents. Then, various concentrations of Chaerophyllum extract (6.25, 12.5, 25, 50, 100 and 200 µg/mL) were added to the parasite culture. Subsequently, the percentage of cytotoxicity of promastigotes and J774 macrophages were evaluated by the Microculture Tetrazolium Test (MTT) method. Also, the percentage of J774 macrophage apoptosis was assessed by flow cytometry. Results: The IC50 of Chaerophyllum extract was 80.62 µg/mL after 24 hours. The percentage of cytotoxicity of promastigotes and J774 macrophages after adding Chaerophyllum extract was 91% and 45%, respectively, by MTT. The percentage of apoptosis (early and late apoptosis) in macrophages induced by 50 and 100 μg/mL of Chaerophyllum extract after 24 hours was 0.01%, 0.13%, 0.01% and 0.1%, respectively. Conclusions: According to the MTT test, the concentration of 200 μg/mL of Chaerophyllum extract with 90% cytotoxicity was promising against L. major promastigotes and further studies are recommended to evaluate the effects of this plant.

Development of bioceramic bone scaffolds by introducing triple liquid phases

Abstract

Iridoid glycosides from Callicarpa nudiflora Hook

Four new iridoid glycoside derivatives (1–4), along with ten known iridoid glycosides (5–14), were isolated from Callicarpa nudiflora Hook et Arn. The structure of the new iridoid glycosides was elucidated as 5″-methoxy-ampicoside (1), 6″-O-trans-caffeoylcatalpol (2), 6″-O-trans-feruloylcatalpol (3) and 3″-methoxy-agnucastoside C (4) on the basis of spectroscopic analysis. Compounds 1–11 were reported from this plant for the first time. The cytotoxic activity of the isolated compounds against human cervical carcinoma Hela cells and ovarian carcinoma HeyA8 cells was evaluated using the microculture tetrazolium assay. Compounds 4, 5, 8, 12 and 13 showed cytotoxic activity against the Hela cell line with IC50 values of 25.3, 48.1, 17.3, 38.3 and 28.2 μM, respectively. While only compound 8 showed cytotoxicity against the HeyA8 cell line, with an IC50 of 35.5 μM

Synthesis, Characterization, and Cytotoxicity of a Novel Gold(III) Complex with O,O′-Diethyl Ester of Ethylenediamine-N,N′-Di-2-(4-Methyl)Pentanoic Acid

A novel gold(III) complex, [AuCl2{(S,S)-Et2eddl}]PF6, ((S,S)-Et2eddl = O,O′-diethyl ester of ethylenediamine-N,N′-di-2-(4-methyl)pentanoic acid) was synthesized and characterized by IR, 1D (1H and 13C), and 2D (H,H-COSY and H,H-NOESY) NMR spectroscopy, mass spectrometry, and elemental analysis. Density functional theory calculations confirmed that (R,R)-N,N′ diastereoisomer was energetically the most stable isomer. In vitro antitumor action of ligand precursor [(S,S)-H2Et2eddl]Cl2 and corresponding gold(III) complex was determined against tumor cell lines: human adenocarcinoma (HeLa), human colon carcinoma (LS174), human breast cancer (MCF7), non-small cell lung carcinoma cell line (A549), and non-cancerous cell line human embryonic lung fibroblast (MRC-5) using microculture tetrazolium test (MTT) assay. The results indicate that both ligand precursor and gold(III) complex have showed very good to moderate cytotoxic activity against all tested malignant cell lines. The highest activity was expressed by [AuCl2{(S,S)-Et2eddl}]PF6 against the LS174 cells, with IC50 value of 7.4 ± 1.2 µM.

Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage.

Manganese (Mn) complexes are widely studied because of their important catalytic properties in synthetic and biochemical reactions. A Mn (III) complex of an amidoamine ligand was synthesized using a tetradentate amidoamine ligand. In this study, the Mn (III) complex was evaluated for its biological activity by measuring its cytotoxicity in human breast adenocarcinoma cell line (MCF-7). Cytotoxic effects of the Mn (III) complex were determined using established biomarkers in an attempt to delineate the mechanism of action and the utility of the complex as a potential anticancer drug. The Mn (III) complex induces cell death in a dose- and time-dependent manner as shown by microculture tetrazolium assay, a measure of cytotoxic cell death. Our results demonstrated that cytotoxic effects were significantly increased at higher concentrations of Mn (III) complex and with longer time of treatment. The IC50 (Inhibitor concentration that results in 50% cell death) value of Mn (III) complex in MCF-7 cells was determined to be 2.5 mmol/L for 24 hours of treatment. In additional experiments, we determined the Mn (III) complex-mediated cell death was due to both apoptotic and nonspecific necrotic cell death mechanisms. This was assessed by ethidium bromide/acridine orange staining and flow cytometry techniques. The Mn (III) complex produced reactive oxygen species (ROS) triggering the expression of manganese superoxide dismutase 1 and ultimately damaging the mitochondrial function as is evident by a decline in mitochondrial membrane potential. Treatment of the cells with free radical scavenger, N, N-dimethylthiourea decreased Mn (III) complex-mediated generation of ROS and attenuated apoptosis. Together, these results suggest that the Mn (III) complex-mediated MCF-7 cell death utilizes combined mechanism involving apoptosis and necrosis perhaps due to the generation of ROS.

Synthesis of novel 17-(4′-formyl)pyrazolylandrosta-5,16-dienes and their derivatives as potent 17α-hydroxylase/C17,20-lyase inhibitors or antiproliferative agents depending on the substitution pattern of the heteroring

A series of novel 17-(4′-formyl)pyrazolylandrosta-5,16-dienes were efficiently synthesized in two steps from pregnadienolone acetate with monosubstituted hydrazines via the cyclization/formylation sequence of the primarily formed hydrazones on treatment with the Vilsmeier-Haack reagent. The products were further transformed by deacetylation and subsequent reduction in order to enlarge the compound library available for pharmacological studies. Moreover, 4′-formylpyrazoles containing H or Me on the heteroring-N were subjected to oxime formation and Ac2O-induced dehydration to furnish the corresponding 4′-cyano derivatives in good yields. The antiproliferative activities of the structurally related steroidal 17-exo-pyrazole derivatives were tested in vitro on four human adherent breast cancer cell lines (MCF7, T47D, MDA-MB-231 and MDA-MB-361): the microculture tetrazolium assay revealed that seven compounds exerted better cell growth-inhibitory effects on some or all these cell lines than those of the reference cisplatin. With regard to the well-known structural features that a potent C17,20-lyase inhibitor should possess, some relevant derivatives were tested in vitro from the aspects of their inhibitory effects on rat testicular enzyme, and one of them proved to exert noteworthy enzyme-inhibitory action, with an IC50 (26 nM) of the same order of magnitude as that of abiraterone.

Thin Degradable Coatings for Optimization of Osteointegration Associated with Simultaneous Infection Prophylaxis

The colonization of biomaterials with bacteria represents the main cause of implant-associated infections. Both an antibiotic prophylaxis and a faster osteointegration can be obtained by incorporation of bactericidal active metals in degradable CaP coatings. At present there is no reliable method on the basis of thermal spraying to get thin homogeneous layers containing silver, copper and bismuth in bacteriostatic / bactericidal concentrations. The aim of the study was the development and optimization of high-velocity suspension flame spraying (HVSFS) process for producing thin resorbable bioactive ceramics coatings on the basis of degradable calcium phosphates. In these layers the bacteriostatic / bactericidal effective metal copper should be integrated. Cells were grown on the materials for 3, 7, 14, and 21 days. Live/dead assay was used to measure cell viability. The in vitro cytotoxicity was determined by the microculture tetrazolium (WST) assay. Cell morphology, cell attachment, and cell spreading were investigated using laser scanning microscopy and raster scanning electron microscopy. All substrates supported sufficient cellular growth for 21 days and showed no cytotoxicity. On each material an identical cell colonisation of well communicating, polygonal, vital cells was verified

Chemical composition and anti-proliferative effect of Oman's Ganoderma applanatum on breast cancer and cervical cancer cells

Abstract Objectives To evaluate the anti-proliferative potential of Oman's Ganoderma applanatum on triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HEp-2) and Vero cells (normal) and its chemical profiling of G. applanatum. Methods The cytotoxicity of the methanolic extract of G. applanatum was tested on MDA-MB-231 cells, HEp-2 cells and Vero cells by microculture tetrazolium (MTT) assay. The anti-cancer properties of G. applanatum were characterized using gas chromatography coupled with gas chromatography mass spectrometry (GCMS) analysis. Results The methanolic extract of G. applanatum elicited dose-dependent cell death on both MDA-MB-231 and HEp-2 cells after 24 h of treatment, with an IC 50 value of 84.6 μg/ml and 43.2 μg/ml, respectively. Normal vero cells were able to significantly withstand the treatment. Furthermore, seven molecules were identified from G. applanatum by GCMS analysis, and five of them were present in considerable amounts, namely, γ-terpinene (30.3%), d -limonene (23.6%), cis-2-methyl-4-pentylthiane-s,s-dioxide (15.3%), β-cymene (12.7%) and α-terpinolene (8.1%). Conclusion In this study, surprisingly, G. applanatum exhibited toxicity particularly towards tumour cells (MDA-MB-231 and HEp-2) compared to normal vero cells. This tumour-specific chemo-sensitivity of G. applanatum is unclear, and the mechanism of cancer cell death induced by G. applanatum should be studied in detail. In addition, the observed anti-cancer activity of G. applanatum might be due to the synergistic effects of the identified properties. This would pave the way for isolation and characterization of the potential anti-cancer molecule from Oman's G. applanatum .

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo.

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16β-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone.The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65μM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4μM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Konsentrasi Aman Kurkumin dan PGV-0 terhadap Sel Vero Berdasarkan Hasil Uji Sitotoksik

Curcumin (1,7-bis(3-methoxyphenyl 4'hidroksi) -1.6 heptadien, 3,5-dione) is the yellow pigment of Curcuma longa. Curcumin has various biological activities such as antioxidant, antibacterial, antifungal, antiprotozoa l and antivirus. Various benefits of curcumin can not be separated from the weakness which is not stable to pH and light. Pentagamavunon-0 (PGV-0) were made by changing the β diketone group on cluster analog of curcumin into monoketon while eliminating active methylene group so it is more stable to pH and light. PGV-0 also has potential as a stronger antioxidant and antiinflammatory agent than other curcumin analogues. The objective of this research was to determine the safe concentrations of curcumin and PGV-0 on vero cells due to the increased use of the two compounds through the cytotoxic test. This research includes experimental research. Cytotoxic test performed with microculture tetrazolium technique ( MTT ) method. Use of MTT to evaluate the cytotoxic is based on changes of tetrazolium salt into formazan crystals by mitochondrial enzyme succinate dehydrogenase with the help of cellular NADH. The results showed that the safe concentrations of curcumin and PGV-0 on vero cells respectively are 6.25 and 1.5625 ppm. Based on the cytotoxic test the secure concentration of curcumin was higher than PGV-0.

A biocompatibility study of injectable poly(caprolactone-trifumarate) for use as a bone substitute material

The need for bone graft alternatives has led to the development of numerous bone graft substitutes. Here, the authors have synthesized a biodegradable poly(caprolactone-trifumarate) (PCLTF) polymer solution that could be injected into any bony defect. This polymer solution was synthesized using polycaprolactone-triol and fumaryl chloride (FCl). PCLTF is a multiple-branching, unsaturated and cross-linkable in situ material. The surface microstructure of PCLTF was investigated using a field emission scanning electron microscope. The incorporation of double bonds originating from FCl into the poly(caprolactone) backbone was confirmed in the Fourier transform infrared spectra. The in vitro cytotoxic effects of PCLTF, its leachable extracts and degradation products were evaluated in direct and indirect contact tests against human oral fibroblasts. Cell viability was evaluated using the microculture tetrazolium assay and cytotoxicity evaluations of PCLTF were tested in accordance with ISO 10993-5 standards. The results showed that there was evidence of reasonable cell growth, good cell viability and intact cell morphology after exposure to PCLTF, its extracts and degradation products. There was no evidence of critical cytotoxic effects.

Calcium sulfate bone scaffolds with controllable porous structure by selective laser sintering

The calcium sulfate (CaSO4) bone scaffolds with high porosity and interconnectivity and controllable pore size were prepared by using selective laser sintering. The phase composition, micro morphology and biocompatibility were investigated by using X-ray diffraction, scanning electron microscopy and microculture tetrazolium test. The results showed that the CaSO4 powders fused better and a more compact structure was built due to the decrease of holes in the scaffold at laser power of 7 W compared with 6 W or lower. At this time, both compressive strength and fracture toughness were optimal. While CaSO4 decomposed and resulted in the mechanical properties decreasing when laser power further increased. Consequently, the mechanical properties of the scaffolds decreased. Moreover, the osteoblast-like cells attached on the scaffolds were obtained by cell culture in vitro. The results revealed that the cells could adhere and grow well on the scaffolds.

MiR-183 promotes growth of non-small cell lung cancer cells through FoxO1 inhibition.

Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLCs often appear to be fast growing, which renders comprehension of the mechanisms underlying the growth of NSCLC extremely critical. Previous study has addressed a role of microRNA (miR) family member, miR-183, in the regulation of the invasiveness of NSCLC, whereas the role of miR-183 in the growth control of NSCLC is not clear. Here, we analyzed the regulation of FoxO1 by miR-183 in vitro using luciferase-reporter assay. We also analyzed the effects of miR-183 on NSCLC cell growth in vitro using a microculture tetrazolium (MTT) assay and in vivo by visualizing tumor growth using bioluminescence assay. We found that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3'untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC resulting in a significant decrease in NSCLC growth. Taken together, our data demonstrate that miR-183/FoxO1 axis may be a novel therapeutic target for regulating the growth of NSCLC.

The Anticancer Activity of Marine Sponge Cinachyrella sp. (Family Tetillidae)

Cinachyrella sp. marine sponge produce many kinds of secondary metabolites. The purpose of this study was to examine the cytotoxic and anti-proliferative activity of marine sponge Cinachyrella sp. The sponge was extracted with 96 % ethanol. Ethanol extract cytotoxicity assay were performed with MTT method (Microculture Tetrazolium) against to cell lines of HeLa, T47D, WiDr and Vero. The results show that of the ethanol extract were only toxic to cell lines of HeLa IC50 897.809 μg/mL but did not toxic to cell lines of T47D, WiDr and normal cell lines of Vero. Fractionation of the ethanol extract was conducted by a Vacuum Column Chromatography (VCC) and have 4 fraction that were F1, F2, F3 and F4. Cytotoxicity and cell proliferation inhibitory were tested of fraction F1, F2, F3 and F4 against cell lines of T47D. The values IC50 of F1; F2; F3 and F4 against cell lines of T47D were 82.744; 163.679; 66.522 and 333.026 μg/mL and fraction F3 concentration 31.5 µg/mL inhibits cell proliferation cell lines of T47D at 48 hours of incubation

Silibinin, up-regulates chemokine receptor expression in MDA-MB-231 breast cancer cell line

Introduction: Silibinin, a polyphenolic flavonoid isolated from the milk thistle plant (Silybummarianum), has various applications in cancer therapy. This investigation aimed to examine the effects of silibinin on proliferation and chemokine receptor expression on MDA-MB-231 cells, a highly metastatic human breast cancer cell line.Methods: The cytotoxic effect of silibinin on MDA-MB-231 cells was determined by micro-culture tetrazolium test (MTT) assay. In addition, the expression of chemokine receptors CXCR3, CCR5 and CCR7 genes in response to silibinin was evaluated by Real-Time PCR.Results: Data analysis from MTT assay showed that silibinin had dose-dependent and time-dependent inhibitory effects on MDA-MB-231 cell line. Moreover, Real-Time PCR analysis showed that silibinin not only had no inhibitory effects on CXCR3, CCR5 and CCR7 gene expressions, but also could increase significantly the expression of these genes in a dose and time-dependent manner.Conclusion: These results revealed for the first time the increased possibility of CXCR3, CCR5 and CCR7 genes expression in response to silibinin in human breast cancer cells.Bangladesh Journal of Medical Science Vol.14(2) 2015 p.190-195

Synthesis of novel 17-(5′-iodo)triazolyl-3-methoxyestrane epimers via Cu(I)-catalyzed azide–alkyne cycloadditon, and an evaluation of their cytotoxic activity in vitro

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 3-methoxyestrane 17α- and 17β-azide epimers (3 and 5) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a–f and 11a–f). If the Ph3P in the classical CuAAC process was replaced by Et3N, the formation of small quantities of 5-iodotriazoles (9a–f and 11a–f) was observed. For the preparation of 5-iodo-1,2,3-triazoles (9a–f and 11a–f), an improved method was developed, directly from steroidal azides and terminal alkynes, in reactions mediated by CuI and ICl as iodinating agents. The antiproliferative activities of the structurally related triazoles were determined in vitro with the microculture tetrazolium assay on six malignant human cell lines of gynecological origin (HeLa, A2780, MCF7, MDA-MB-231, MDA-MB-361 and T47D). X-ray analysis revealed the presence of the iodo substituent on the 1,2,3-triazole ring.

Lasallia pustulata lichen as possible natural antigenotoxic, antioxidant, antimicrobial and anticancer agent.

The methanol extract of the lichen Lasallia pustulata was tested for genotoxic, antioxidant, antimicrobial and anticancer activities. We did this using a cytokinesis block micronucleus (MN) assay on peripheral blood lymphocytes, by measuring free radical and superoxide anion scavenging activity, reducing power, determining of total phenolic compounds and determining the total flavonoid content, measuring the minimal inhibitory concentration by the broth microdilution method against five species of bacteria and five species of fungi and by using the microculture tetrazolium test on FemX (human melanoma) and LS174 (human colon carcinoma) cell lines. As a result of this study, we found that the methanol extract of L. pustulata did not modify the frequency of the MN and nuclear division index in comparison to untreated cells (p>0.05). These results revealed that the methanol extract had moderate free radical scavenging activity with IC50 values of 395.56μg/mL. Moreover, the extract tested had effective reducing power and superoxide anion radical scavenging. The values of the minimum inhibitory concentration against the tested microorganisms ranged from 0.625 to 20mg/mL. In addition, the extract tested had strong anticancer activity against both cell lines with IC50 values of 46.67 and 71.71μg/mL.