Comparison of MTT and ATP-based assays for the measurement of viable cell number

Cell viability assays are widely used to assess the effect of chemotherapeutic drugs and other agents on cell lines and have shown promise for the prediction of tumour chemosensitivity. In this study we have compared two viability assays using Daudi and CCRF-CEM cell lines over a range of 1500–100,000 cells/well of a microplate. The ATP assay was able to detect the lower limit of 1563 cells/well with luminescence values at least 100× background readings, while the MTT assay could not detect less than 25,000 cells/well above background readings. The ATP assay also showed better reproducibility and sensitivity when cells were grown in microtitre plates over several days, and is particularly useful for the measurement of viability with low cell numbers.     

Electrochemical monitoring of anticancer compounds on the human ovarian carcinoma cell line A2780 and its adriamycin- and Cisplatin-resistant variants.

A novel electrochemical technique which detects and monitors real-time changes in cell behavior in vitro has been used to examine the effects of recognized anticancer drugs on the human ovarian carcinoma cell line A2780 and its adriamycin (A2780adr)- and cisplatin (A2780cispt)-resistant variants. These cells, adherent to gold electrodes or sensors, modify the extracellular microenvironment at the cell:sensor interface, producing an electrochemical potential that is different from that of the bulk culture medium. Confluent, adherent A2780 cells produced an electrochemical signal, measured as an open circuit potential (OCP), of approximately -100 mV compared to a cell-free value of approximately -15 mV. Exposure of A2780 cells to cisplatin (range 10(-4) to 10(-6) M), adriamycin (range 10(-5) to 10(-7) M), and vinblastine (10(-6) M) all produced positive shifts in the OCP signal relative to untreated control cells during 24 h of culture, but Taxotere (range 10(-5) to 10(-7) M) had no effect. These positive shifts in OCP signal were evident well before observations of reduced cellular adhesion and viability after 24 h, as judged in parallel cultures with a plastic substratum and by scanning electron microscopy. By contrast, the same treatments applied to the A2780adr and A2780cispt variants showed that each demonstrated different sensitivities to the same drugs applied to the parental A2780 cells. The effects of the same four anticancer drugs on ovarian carcinoma (A2780) and breast carcinoma (8701-BC) cell lines showed that the former was far more responsive to adriamycin and cisplatin. Such differences in drug sensitivities between the two cell lines were subsequently confirmed using the conventional MTT assay over 5 days. Although this electrochemical technology readily detects changes in cell adhesion and viability, the modified OCP signals recorded within a few hours of anticancer drug treatments are evident well before microscopic morphological changes become apparent. It is proposed that these early changes in OCP signals, relative to control untreated cells, reflect modifications of physiological/behavioral processes manifested at the cell surface.     

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.     

Antioxidant,antimicrobial and cytotoxicity potential of n-hexane extract of Cayratia trifolia L

It is of interest to analyze the antioxidant, antimicrobial and cytotoxicity activity of n-hexane extract of Cayratia trifolia L. (C. trifolia). The antimicrobial activity of n-hexane extract of C. trifolia was determined using disc diffusion method against six selected pathogenic microorganisms. The cytotoxicity potential of n-hexane plant extract was also studied against A2780 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results, n-hexane extract of C. trifolia possess significant antioxidant activity with significant IC50 values in radical scavenging assays. In antimicrobial studies, the maximum zone of inhibition was found in the range of 19.0 ± 0.1 to 22.0 ± 0.1 mm. In MTT assay, inhibition of cell growth with minimal IC50 values of 46.25±0.42μg/mL against A2780 cell lines was observed. Thus, n-hexane extract of C. trifolia is a possible antioxidant, antimicrobial and cytotoxicity agent.     

Evaluation of viability assays for anthocyanins in cultured cells

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC(50) of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended.     

Application of Colorimetric Assays to Assess Viability, Growth and Metabolism of Hydrogel-Encapsulated Cells

The applicability of the colorimetric 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays to measure cell growth and viability in hydrogel encapsulation systems was investigated using HepG2 liver cells encapsulated in alginate matrices. The MTT assay was effective in measuring viable cell density in alginate-encapsulated cell systems, demonstrating less variance and higher throughput capability than hemocytometry. The LDH assay was effective in measuring dead cell density in monolayer cultures and in alginate-encapsulated cells simply by measuring the LDH concentration secreted into the medium. Further validation of these assays was shown in two additional cell lines (rat muscle and mouse embryonic fibroblasts). The MTT and LDH assays are particularly significant in the rapid evaluation of in vitro cell encapsulation device design.     

Effect of green microalgal extracts exhibiting antibacterial activity on viability of human malignant and non‐malignant cells

Microalgae have been investigated for their ability to produce metabolites that exhibit antibacterial activity. However, as research on antibacterial activity progresses, the effect of microalgal extracts on mammalian cells needs to be also assessed. The in vitro effect of microalgal extracts with demonstrated antibacterial activity against the human opportunistic pathogen Staphylococcus aureus was examined on the viability of non‐malignant (MCF10A and 184B5 cells) and malignant human cell lines (A2780 and MCF7). Direct cell counts indicated that the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) proliferation assay was found to under/overestimate cell viability when specific microalgal extracts and/or concentrations were tested. From direct cell counts, the viability of non‐malignant cells was not reduced after exposure to the extracts, whereas the viability of malignant cells was affected by specific microalgal extracts and concentrations. The results suggest that green microalgae are worthy of further investigation as potential sources of antibiotics, since they did not show a negative effect on the non‐malignant cell lines, MCF10A and 184B5. Additional studies should evaluate the compounds responsible for the anti‐proliferative activity of specific microalgal extracts observed on the malignant cells A2780 and MCF7.     

Synthesis,docking studies,in vitro cytotoxicity evaluation and DNA damage mechanism of new tyrosine-based tripeptides

Peptides are one of the leading groups of compounds that have been the subject of a great deal of biological research and still continue to attract researchers' attention. In this study, a series of tripeptides based on tyrosine amino acids were synthesized by the triazine method. The cytotoxicity properties of all compounds against human cancer cell lines (MCF-7), ovarian (A2780), prostate (PC-3), and colon cancer cell lines (Caco-2) were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay method, and % cell viability and logIC50 values of the compounds were calculated. Significant decreases in cell viability were observed in all cells (p < 0.05). The comet assay method was used to understand that the compounds that showed a significant decrease in cell viability had this effect through DNA damage. Most of the compounds exhibited cytotoxicity by DNA damage mechanism. Besides, their interactions between investigated molecule groups with PDB ID: 3VHE, 3C0R, 2ZCL, and 2HQ6 target proteins corresponding to cancer cell lines, respectively, were investigated by docking studies. Finally, molecules with high biological activity against biological receptors were determined by ADME analysis.     

Studies of interaction of trichloro{eta2-cis-N,N-dimethyl-1-[6-(N',N'-dimethyl-ammoniummethyl)-cyclohex-3-ene-1-yl]-methylammonium}platinum(II) chloride with DNA: Effects on secondary and tertiary structures of DNA. Cytotoxic assays on human ovarian cancer cell lines, resistant and non-resistant to cisplatin

The studies of interaction with DNA and the cytotoxic activity of a new organometallic platinum(II) compound are presented. The ability of this new platinum complex to modify secondary DNA structure was explored by circular dichroism (CD). Electrophoretic mobility showed changes in tertiary DNA structure, and atomic force microscopy (AFM) revealed morphological changes of plasmid DNA (pBR322). This compound breaks the traditional structure-activity rules for cis-platinum compounds, but it could be of interest because of its different kinetics. An organometallic bond normally shows a trans-effect higher than that of an amine ligand, and that fact, a priori, could contribute to a higher DNA binding rate. Several ovarian cancer cell lines, resistant and non-resistant to cisplatin, were exposed to increasing concentrations of cisplatin and complex 5 for 24 h, after which time the cell number/viability was determined by the colorimetric MTT assay. A lower cytotoxicity but also a lower resistant factor was observed for organometallic compound 5 than for cisplatin, against A2780 and A2780cisR cell lines. This result is consistent with the DNA interaction degree observed by the aforementioned techniques.     

Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols

Background

The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC₅₀ concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature.

Methodology/Principal Findings

We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC₅₀ concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays.

Conclusions/Significance

These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.     

An in vitro testing of chemoresistance in leukaemic patients

The fact that leukaemic cells are primarily or secondarily resistant to cytostatics is a serious phenomenon, which leads to the failure of chemotherapy of malignant diseases in clinical practise. Some detoxification and transporting systems are responsible for the generation of chemoresistance on the cellular level and the decrease of effectiveness in treatment. In vitro testing of chemoresistance of leukaemic cells is presently an inseparable component of “tailoring” therapy in the developing field of predictive oncology. The aim of this work was to estimate profiles of drug resistance, based on the predictive in vitro test, and to help in choosing the most effective cytostatic. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoline (MTT) assay was used, based on the direct effect of cytostatics on the viability of leukaemic cells in vitro. The number of living leukaemic cells was evaluated by a computer program, where LC₅₀ (concentration of cytostatics lethal to 50% of leukaemic cells) was established from the achieved dose-relation curves. Seventy-one samples of leukaemic cells isolated from the patients’ peripheral blood or bone marrow were examined. All samples were tested to 3 cytostatics minimally. It was found by the in vitro assay, that resistance to dexamethasone, prednisolone, etoposide and vincristine is increased in patients with acute myeloid leukaemia disease, compared to the acute lymphoblastic leukaemia patients. In patients with a relapsed disease population, leukaemic cells are highly heterogeneous in the MTT assay. It was concluded that the MTT assay can be used to study drug interactions in vitro in leukaemia samples. The type of interaction was highly different between patients, and depended on drug concentrations.     

Extracellular ATP activates the PLC/PKC/ERK signaling pathway through the P2Y2 purinergic receptor leading to the induction of early growth response 1 expression and the inhibition of viability in human endometrial stromal cells

ATP is an extracellular signaling molecule that activates specific G protein-coupled P2Y receptors in most cell types to mediate diverse biological effects. ATP has been shown to activate the phospholipase C (PLC)/diacylglycerol/protein kinase C (PKC) pathway in various systems. However, little is known about the signaling events in human endometrial stromal cells (hESCs). The objective of this study was to examine the presence of the P2Y2 receptor and the effects of exogenous ATP on the intracellular mitogen-activated protein kinases (MAPKs) signaling pathway, immediate early genes expression, and cell viability in hESCs. Western blot analysis, gene array analysis, and MTT assay for cell viability were performed. The current study demonstrated the existence of the P2Y2 purinergic receptor in hESCs. UTP and ATP activated MAPK in a dose- and time-dependent manner. Suramin (a P2-purinoceptor antagonist), neomycin (a PLC inhibitor), staurosporin (a PKC inhibitor), and PD98059 (a MEK inhibitor) significantly attenuated the ATP-induced activation of MAPK. ATP activated ERK1/2 and induced translocation of activated ERK1/2 to the nucleus. The gene array for 23 genes associated with members of the mitogenic pathway cascade and immediate early genes revealed that the expression of early growth response 1 was increased. In addition, MTT assay revealed an inhibition effect of ATP on cell viability. ATP activated MAPKs through the P2Y2 purinoceptor/PLC/PKC/ERK signaling pathway and induced translocation of ERK1/2 into the nucleus. Further, ATP induced the expression of early growth response 1 and inhibited cell viability in hESCs.     

Enhancing Anti-Tumor Efficacy of Doxorubicin by Non-Covalent Conjugation to Gold Nanoparticles – In Vitro Studies on Feline Fibrosarcoma Cell Lines

BackgroundFeline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity.MethodsStability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA).ResultsA novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance).ConclusionsThe results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.     

Cosmc基因在卵巢癌中生物学功能的研究

目的:明确Cosmc基因在卵巢癌细胞中的生物学功能。方法:本研究采用慢病毒转染技术,在卵巢癌细胞A2780和SKOV3中过表达Cosmc基因,MTT实验、细胞凋亡实验以及Transwell侵袭实验对过表达Cosmc后卵巢癌细胞在生长、凋亡、侵袭等方面的影响。结果:慢病毒转染卵巢癌细胞A2780和SKOV-3后,Cosmc的蛋白表达显著提高;MTT结果显示,与空载体对照组相比,过表达Cosmc的卵巢癌细胞生长能力显著减弱;流式细胞术结果表明Cosmc过表达的卵巢癌细胞凋亡数较空载体对照组细胞显著增加;Transwell实验显示Cosmc过表达的细胞侵袭和迁移能力较空载体对照组显著减少。结论:卵巢癌细胞系中过表达Cosmc基因能抑制卵巢癌细胞的生长和侵袭并促进细胞凋亡。     

Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth

Summary Adenosine triphosphate is the primary energy unit for cells, and levels of this compound offer a potential marker for cell viability and growth. The availability of a bioluminescence assay allows for a rapid, sensitive, and reproducible measurement of ATP. A method is described for the quantification of intracellular ATP levels in human cancer cells. ATP levels were linearly related to the number of viable cells and increased with time in human cancer cell line cultures correlating with growth kinetics. The effect of 5-fluorouracil, doxorubicin, methotrexate, cytosine arabinoside, nitrogen mustard, melphalan, vinblastine, and cisplatin on the growth of human cancer cell lines was studied utilizing ATP levels. ATP levels and colony formation in agar of drug-exposed cells were compared. Overall there was a significant correlation between drug effects on colony formation and ATP levels. The ATP assay is rapid, simple, reproducible, and a relatively inexpensive method of quantifying drug effects on malignant cells. This makes it a potentially useful method for screening new anticancer drugs in human cancer cell lines.     

Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.     

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.     

The toxic effect of cytostatics on primary cilia frequency and multiciliation

The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple‐negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple‐negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

TCF4 promotes colorectal cancer drug resistance and stemness via regulating ZEB1/ZEB2 expression

The present study aims to investigate the roles of TCF4 and its underlying mechanism in colorectal cancer (CRC). Doxorubicin-resistant DLD-1 (DLD1 DR), TCF4 overexpression, and TCF4 knockdown cell lines were constructed. A flow cytometer was used to analyze frequencies of CD133+ cell in the DLD1 and DLD1 DR cells. Quantitative real-time PCR (qPCR) was used to determine the expressions of cancer stem cell (CSC) makers. Stemness of CRC cells were determined using tumorsphere formation assay. The correlation between TCF4 and ZEB1/ZEB2 were determined using public data from The Cancer Genome Atlas (TCGA) datasets. ZEB1/ZEB2 overexpression cell lines were constructed and cell viabilities were then determined using MTT and colony formation assays. TCF4 overexpression promoted proliferation of CRC cell lines and relative expressions of TCF4 were significantly increased in the DLD1 DR cells. TCF4 overexpression promoted CRC cell doxorubicin resistance, whereas TCF4 knockdown significantly decreased doxorubicin resistance. Additionally, TCF4 overexpression also significantly increased frequencies of CSC cells, expressions of CSC markers, and CRC ability to form tumorsphere. Furthermore, TCF4 promoted ZEB1 and ZEB2 expression, leading to CRC proliferation and doxorubicin resistance. TCF4 promoted CRC doxorubicin resistance and stemness by regulating expressions of ZEB1 and ZEB2.