3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium and Breast-Neoplasms

Concern with increasing levels of emerging contaminants exists on a global scale. Three commonly observed emerging environmental contaminants: triclosan (2,4,4-trichloro-2'-hydroxydiphenyl ether), a synthetic, broad-spectrum antibacterial agent, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), used in stain- and water-resistant treatments, have become distributed ubiquitously across ecosystems and have been detected in wildlife and humans. MCF-7 BOS human breast cancer cells were used to investigate the potential for cytotoxicity, estrogenicity and anti-estrogenicity of these three compounds at environmentally relevant concentrations using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay (MTS) and the E-SCREEN bioassay. The doses used were 0.002-200 µg ml(-1) for triclosan and 0.03-30 µg ml(-1) for PFOS and PFOA. Quantitative results from the MTS assay revealed no significant cytotoxicity at lower concentrations for any of the test compounds; however, both triclosan and PFOA were cytotoxic at the highest concentrations examined (100-200 and 30 µg ml(-1), respectively), while PFOS showed no significant cytotoxicity at any of the concentrations tested. Positive estrogenic responses (P < 0.05) were elicited from the E-SCREEN at all concentrations examined for triclosan and PFOA and at 30 µg ml(-1) for PFOS. Further, significant anti-estrogenic activity (P < 0.05) was detected for all compounds tested at all concentrations when cells were co-exposed with 10(-9) m 17-β estradiol (E(2)). The overall results demonstrated that triclosan, PFOS and PFOA have estrogenic activities and that co-exposure to contaminants and E(2) produced anti-estrogenic effects. Each of these compounds could provide a source of xenoestrogens to humans and wildlife in the environment. Published 2011. This article is a US Government work and is in the public domain in the USA.

Topics: Alkanesulfonic Acids; Breast Neoplasms; Caprylates; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Environmental Pollutants; Estrogen Antagonists; Estrogens, Non-Steroidal; Female; Fluorocarbons; Humans; Tetrazolium Salts; Thiazoles; Triclosan

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Count; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorimetry; Culture Media, Serum-Free; Deoxyuridine; DNA; Drug Design; Estradiol; Estrogens; Fingolimod Hydrochloride; Fluorescent Dyes; Fulvestrant; Humans; Isopropyl Thiogalactoside; Membrane Potential, Mitochondrial; Mitochondria; Propylene Glycols; Reproducibility of Results; Sphingosine; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p14ARF

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0-15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1-10 μM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose-apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.

Topics: Annexin A5; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cytological Techniques; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Microscopy, Confocal; Phosphatidylserines; Staurosporine; Surface Plasmon Resonance; Tetrazolium Salts; Thiazoles

Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study.. The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways.. Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay.. Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC(50) values 620+/-12.7 and 515+/-10.1 microg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells.. This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Humans; Lung Neoplasms; Phytotherapy; Rheum; Tetrazolium Salts; Thiazoles

There are several scientific approaches for the determination of cellular growth influences of known or novel substances under in vitro conditions, among which colourimetric absorption measurement is considered to be one of the convenient methods. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay is one of the commonly used colourimetric absorption assays based on the ability of dehydrogenase from viable cells to produce the brown soluble formazan detectable at 490 nm. Here we have tested the possible growth influence of iron (II) sulphate on two human cancer cell lines, the K562 chronic myelogenous leukaemia and T47D breast carcinoma cells, based on the MTS assay. We found that iron (II) sulphate possessed an inhibitory effect when added at 16- to 125-microM concentrations, but iron (II) sulphate became growth stimulatory when its concentration was further increased to 1000 microM. In addition, a dose-dependent increase in absorbance at the same wavelength was observed when we repeated the experiments without the addition of MTS and phenazine methosulfate. When we further repeated the cell growth determinations using adenosine triphosphate content assay for K562 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for T47D, iron (II) sulphate showed a consistent dose-dependent growth inhibitory effect. Morphological investigation after methylene blue staining clearly demonstrated that iron (II) sulphate, at a concentration of 1000 microM, is cytotoxic to T47D cells. Interestingly, a consistent increment for the absorbance at 490 nm was further observed with increased iron (II) sulphate concentration either in the presence or absence of MTS even in a cell-free environment. Thus we conclude that iron (II) sulphate is actually growth inhibitory and even cytotoxic at high concentrations towards the K562 and T47D cancer cells and the paradoxical proliferative activity of iron (II) sulphate on these two cancer cell lines using the MTS assay was solely due to the oxidation of initial pale green iron (II) to brownish iron (III) during incubation in the aqueous condition.

Topics: Breast Neoplasms; Carcinoma; Cell Proliferation; Colony-Forming Units Assay; Humans; Iron Compounds; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplasms; Sulfates; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured