Mechanism of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction

Abstract: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.     

Cytotoxic Amyloid Peptides Inhibit Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction by Enhancing MTT Formazan Exocytosis

Abstract: Amyloid β peptide (Aβ) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of Aβ toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that Aβ and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.     

Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.     

Steroid Hormones Block Amyloid Fibril-Induced 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Formazan Exocytosis: Relationship to Neurotoxicity

Abstract: Perhaps the most reproducible early event induced by the interaction of amyloid β peptide (Aβ) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as Aβ and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with β-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by Aβ. NAC35, an amyloidogenic fragment of α-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the β-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with β-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute Aβ neurotoxicity.     

The Intracellular Component of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Is Specifically Inhibited by β-Amyloid Peptides

Abstract: In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of β-amyloid peptide (β-AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the β-AP fragments β1–40, β1–39, and β25–35, but not β1–28, were found to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In both cell lines, the β-AP-sensitive component represented ∼70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3α-naphthyl-2-phenyl-5-(4-nitrophenyl)-2 H -tetrazolium chloride (NTV) was also found to be sensitive to β25–35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT-formazan product formation in PC12 and HeLa cells following β25–35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of β-AP toxicity.     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

β-Amyloid-Induced Cell Toxicity: Enhancement of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Dependent Cell Death

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of β-amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

The formation of 2-hydroxy-4-hydroxymethyl-3-(indol-3-yl)-cyclopent-2-enone derivatives from ascorbigens

A facile preparation is described of 3-(indol-3-yl)-2-hydroxy-4-hydroxymethylcyclopent-2-enone and its N-derivatives in 15-40% yields by the degradation of ascorbigen or its N-derivatives in a warm solution of L-ascorbic acid through a sequential domino reaction. The same cyclopentenone derivatives were obtained in 30-40% yields by the condensation of (N-alkylindol-3-yl)glycolic acids with ascorbic acid. 2,6-Dihydroxy-1-(indol-3-yl)hexa-1,4-diene-3-one and 2-hydroxy-4-hydroxymethyl-5-(indol-3-yl)cyclopent-2-enone were identified as intermediates in this reaction.     

Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G₁ phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21^Waf1/Cip1 and p27^Kip1; and knockdown of p27^kip1 with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.     

Synthesis of Base Substituted 2-Hydroxy-3-(purin-9-yl)-propanoic Acids and 4-(Purin-9-yl)-3-butenoic Acids

Abstract

Alkylation of 6-chloropurine and 2-amino-6-chloropurine with bromoacetaldehyde diethyl acetal afforded 6-chloro-9-(2,2-diethoxyethyl)purine (3a) and its 2-amino congener (3b). Treatment of compounds 3 with primary and secondary amines gave the N⁶-substituted adenines (5a–5c) and 2,6-diaminopurines (5d–5f). Hydrolysis of 3 resulted in hypoxanthine (6a) and guanine (6b) derivatives, while their reaction with thiourea led to 6-sulfanylpurine (7a) and 2-amino-6-sulfanylpurine (7b) compounds. Treatment with diluted acid followed by potassium cyanide treatment and acid hydrolysis afforded 6-substituted 3-(purin-9-yl)- and 3-(2-aminopurin-9-yl)-2-hydroxypropanoic acids (8–10). Reaction of compounds 3 with malonic acid in aqueous solution gave exclusively the product of isomerisation, 6-substituted 4-(purin-9-yl)-3-butenoic acids (15).     

Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway

In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple and efficient structure-based design. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent activity against human chronic myeloid leukemia (CML) cell line K562, but very weak or no cellular toxicity through monitoring the growth kinetics of K562 cell during a period of 72 h using the real-time live-cell imaging. Among these compounds, 1-(5-((6-((3-morpholinopropyl) amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (7) exhibited the least cellular toxicity and better biological activity in cellular assays (K562, IC₅₀: 0.038 μM). Compound 7 also displayed very good induced-apoptosis effect for human CML cell line K562 and exerted its effect via a significantly reduced protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea derivatives are lead molecules for further development as treatment of chronic myeloid leukemia and cancer.     

A new 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay for testing macrophage cytotoxicity to L1210 and its drug-resistant cell lines in vitro

Summary Activated by interferon (IFN)(50 U/ml) and lipopolysaccharide (50 ng/ml), mouse peritoneal macrophages were cocultured with the L1210 parental cell line (L1210/PRT) and its Adriamycin-, cisplatin-resistant cell lines (L1210/ADM, L1210/CDDP) for 24 h at effector: target (E:T) ratios of 10:1, 5:1 and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT)-cleavage assay, a new improved indirect MTT assay, and the colony-formation assay were used to quantify macrophage-mediated suppression of these non-adherent tumour targets. The results showed that the macrophages can produce formazan at a high level, which can interfere with the final results of a direct MTT assay. The new indirect MTT assay can avoid such interference because the effectors are separated from the targets before the assay is performed, so the real viability of the targets is reflected. An indirect MTT assay, as developed in this study, could be better than the direct assay for examining the suppressive effect of activated macrophages on non-adherent tumour cells in vitro. This study also revealed that all the L1210 cell lines can be suppressed significantly by the macrophages at E:T ratios of 10:1 and 5:1 while the two drug-resistant cell lines have lower survival rates at an E:T ratio of 10:1, indicating that they are more susceptible than their parental cell line.Supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture, Japan     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.     

Isolation and identification of urinary metabolites of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) in rabbits

After oral administration of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) to rabbits, the two unique metabolites, M-I and M-II, were isolated from the urine. M-I, yellow needles of mp 117°, was identified as a new type metabolite of nitrofuran derivative, 2-(β-carboxypropionyl)-3-(5-methylthio-2-furyl) acrylamide by its mass, ir and nmr spectrometries. M-II, yellow solid, appears to be cis-trans isomer of M-I considering from its uv and mass spectral data, and the behavior on tlc.     

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.     

Biosynthesis and biological activity of β-(5-methylisoxazolin-3-on-2-yl)alanine in higher plants

Enzyme preparations from Leucaena seedlings catalysed the formation of β-(5-methylisoxazolin-3-on-2-yl)alanine (MIA) by using 3-hydroxy-5-methylisoxazole (HMI) and O-acetyl-L-serine. Some properties of this enzyme are described. The β-substituted alanine synthases from Pisum and Citrullus seedlings could not catalyse the synthesis of MIA. The phytotoxic effect of HMI on rice seedlings is reduced by alanylation.     

Synthesis of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazines that have antibacterial activity and also inhibit inorganic pyrophosphatase

Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.     

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.