formazans and Lung-Neoplasms

The use of well-characterized human lung cancer cell lines has allowed for new opportunities in preclinical and clinical drug evaluation. Development of semiautomated tests of in vitro cytotoxicity such as the MTT assay, which utilizes the formazan salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), has allowed for preclinical evaluation of novel chemotherapeutic agents and drug combinations. In addition, techniques such as this make possible the testing of sufficient data sets to allow determination of true biochemical drug synergy. Assessment of drug combinations which possess in vitro synergy or supraadditive effects can suggest chemotherapeutic regimens for further clinical testing. Using the MTT assay in conjunction with isobolographic analysis, it is possible to test commonly used regimens which are based on presumed or apparent in vivo drug synergy, such as the combination of etoposide and cis-platinum. This frequently prescribed combination was found to lack in vitro biochemical synergy when tested with human lung cancer cell lines, indicating that the observed clinical benefits of this drug combination may be due to factors in the tumor microenvironment, drug metabolism, or non-overlapping toxicities. Finally, although it remains to be determined if a significant role for in vitro drug testing will be found in direct clinical applications, preclinical drug evaluation during the drug development process using cultured tumor cell lines may ultimately allow for disease or patient specific therapies for testing.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Coloring Agents; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Formazans; Humans; Lung Neoplasms; Mice; Tetrazolium Salts; Tumor Cells, Cultured; Tumor Stem Cell Assay

We hypothesized that solid tumors rarely occur in patients with hydatid disease. We obtained the serum of 14 patients diagnosed with hydatid disease, the serum of 10 patients who did not have a history of hydatid disease, and the hydatid cyst fluid from six patients. These sera and fluid samples were added at different concentrations to NCI-H209/An1 human lung small cell carcinoma cells and L929 mouse fibroblasts as a control group. Sera of patients with hydatid diseases had cytotoxic effects on NCI-H209/An1 cells, but they did not have cytotoxic effects on fibroblast cells. Sera from healthy subjects did not have a cytotoxic effect on the tumor cell line or control fibroblasts. Cyst fluid, also, did not have toxic effects on the NCI-H209/An1 cell line, but was toxic to fibroblasts up to a 1:32 dilution. Sera from patients with hydatid disease had cytotoxic effects on human small cell lung cancer cells in vitro.

Topics: Animals; Cell Line, Tumor; Cell Survival; Echinococcosis, Pulmonary; Fibroblasts; Formazans; Humans; Lung Neoplasms; Mice; Microscopy; Serum; Statistics, Nonparametric; Tetrazolium Salts

Recently, miR-451 as a tumor suppressor has been reported in other studies. However, whether miR-451 can affect the sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin (DDP) remains unclear. The aim of this study is to evaluate the roles of miR-451 in the sensitivity of NSCLC cells to DDP.. Quantitative RT-PCR assay was performed to detect the expression of miR-451 in 10 pairs of NSCLC and noncancerous tissue samples. pcDNA-GW/EmGFP-miR-451 was stably transfected into NSCLC cell line (A549). Then, the effects of miR-451 upregulation on growth, colony formation and apoptosis of A549 cells were investigated. Finally, the effects of miR-451 upregulation on in vitro and in vivo sensitivity of A549 cells of DDP were also determined.. The level of miR-451 expression in NSCLC tissues was significantly higher than that in corresponding noncancerous tissues. Ectopic overexpression of miR-451 could significantly inhibit growth and induce apoptosis of A549 cells. Moreover, ectopic overexpression of miR-451 could sensitize A549 cells to DDP possibly by increasing DDP-induced apoptosis which might be associated with the inactivation of Akt signaling pathway.. This study demonstrated for the first time that combination of DDP application with miR-451 upregulation might be a potential strategy for the treatment of human NSCLC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Female; Formazans; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; MicroRNAs; Signal Transduction; Tetrazolium Salts; Transfection; Up-Regulation

Cisplatin-induced nephrotoxicity has been accepted as an important obstacle for efficient cisplatin-based chemotherapy. Silymarin from seeds of milk thistle [Silybum marianum L. (Asteraceae)] has been shown to possess various potential pharmacological properties; however, whether or not this agent selectively protects renal cells from cisplatin-induced cell death with no interfering effect on cancer cells is not clear.. Potential of silymarin in protection of cisplatin-induced renal cell death without compromising effect on anticancer activity of cisplatin was demonstrated in this study.. Cisplatin-induced cell death was evaluated in human proximal tubular HK-2, lung carcinoma H460, and melanoma G361 cells using MTT, Hoechst 33342, and propidium iodide assays.. Cisplatin induced both apoptosis and necrosis in HK-2 cells and caused a decrease in cell viability by ~40% and 60% at the doses of 25 and 100 µM, respectively. Pretreatment with 25-200 µM of silymarin significantly protected against cisplatin-induced cell death in a dose-dependent manner. In contrast, pretreatment of silymarin (25-100 µM) caused no significant change on cisplatin-induced cell death in H460 cells but significantly potentiated cisplatin-induced apoptosis in G361 cells.. These findings reveal the selectivity of silymarin in protection of renal cells from cisplatin-induced cell death and could be beneficial for the development of this considerately safe compound as a renoprotective agent.

Topics: Acute Kidney Injury; Antineoplastic Agents; Antioxidants; Apoptosis; Benzimidazoles; Cell Death; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Formazans; Humans; Kidney; Lung Neoplasms; Melanoma; Propidium; Protective Agents; Silybum marianum; Silymarin; Tetrazolium Salts; Tumor Cells, Cultured

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC(50) of approximately 6.64+/-1.84 microM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 microM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.

Topics: Adenocarcinoma; Annexin A5; Antineoplastic Agents; Apoptosis; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinases; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Formazans; G1 Phase; Humans; Inhibitory Concentration 50; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Tetrazolium Salts; Time Factors; Uncoupling Agents

Recently, we have introduced [tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772, FFC24) as a new lanthanum compound which has promising anticancer properties in vivo and in vitro. Aim of this study was to investigate the impact of ABC transporter-mediated multidrug resistance (MDR) on the anticancer activity of KP772. Here, we demonstrate that all MDR cell models investigated, overexpressing ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance protein 1), or ABCG2 (breast cancer resistance protein) either due to drug selection or gene transfection, were significantly hypersensitive against KP772. Using ABCB1-overexpressing KBC-1 cells as MDR model, KP772 hypersensitivity was demonstrated to be based on stronger apoptosis induction and/or cell cycle arrest at unaltered cellular drug accumulation. KP772 did neither stimulate ABCB1 ATPase activity nor alter rhodamine 123 accumulation arguing against a direct interaction with ABCB1. Accordingly, several drug resistance modulators did not sensitize but rather protect MDR cells against KP772-induced cytotoxicity. Moreover, long-term KP772 treatment of KBC-1 cells at subtoxic concentrations led within 20 passages to a complete loss of drug resistance based on blocked MDR1 gene expression. When exposing parental KB-3-1 cells to subtoxic, stepwise increasing KP772 concentrations, we observed, in contrast to several other metallo-drugs, no acquisition of KP772 resistance. Summarizing, our data demonstrate that KP772 is hyperactive in MDR cells and might have chemosensitizing properties by blocking ABCB1 expression. Together with the disability of tumor cells to acquire KP772 resistance, our data suggest that KP772 should be especially active against notoriously drug-resistant tumor types and as second line treatment after standard chemotherapy failure.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Carcinoma, Small Cell; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Formazans; HL-60 Cells; Humans; Lanthanum; Lung Neoplasms; Molecular Structure; Neoplasm Proteins; Organic Anion Transporters; Organometallic Compounds; Phenanthrolines; Sensitivity and Specificity; Tetrazolium Salts

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands have been shown to inhibit human lung cancers by inducing apoptosis and differentiation. In the present study, we elucidated the apoptotic mechanism of PPARgamma activation in human lung cancers by using a novel PPARgamma agonist, 1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1H-indene-2-carboxylic acid ethyl ester (KR-62980), and rosiglitazone. PPARgamma activation selectively inhibited cell viability of non-small-cell lung cancer with little effect on small-cell lung cancer and normal lung cells. The cell death induced by PPARgamma activation presented apoptotic features of oligonucleosomal DNA fragmentation in A549 human non-small-cell lung cancer cell line. Reactive oxygen species (ROS) production was accompanied by increased expression of proline oxidase (POX), a redox enzyme expressed in mitochondria, upon incubation with the agonists. POX RNA interference treatment blocked PPARgamma-induced ROS formation and cytotoxicity, suggesting that POX plays a functional role in apoptosis through ROS formation. The apoptotic effects by the agonists were antagonized by bisphenol A diglycidyl ether, a PPARgamma antagonist, and by knockdown of PPARgamma expression, indicating the involvement of PPARgamma in these actions. The results of the present study suggest that PPARgamma activation induces apoptotic cell death in non-small-cell lung carcinoma mainly through ROS formation via POX induction.

Topics: Apoptosis; Benzhydryl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Epoxy Compounds; Formazans; Humans; Indenes; Lung Neoplasms; Morpholines; PPAR gamma; Proline Oxidase; Reactive Oxygen Species; RNA Interference; Rosiglitazone; Tetrazolium Salts; Thiazolidinediones

The effects of a bombesin/gastrin releasing peptide (BB/GRP) receptor antagonist, PD176252, and histone deacetylase (HDAC) inhibitor, MS-275, were investigated on human lung cancer cell lines. Using the MTT assay, PD176252 and MS-275 inhibited the proliferation of NCI-H1299 cells with IC50 values of 7 and 5 microg/mL, respectively. Using MS-275 and PD176252 together, the ability to inhibit lung cancer cellular growth increased significantly. The combination index for MS-275 and PD176252 was <0.2, indicating that the compounds are highly synergistic in inhibiting lung cancer cellular growth. Also, MS-275 and PD176252 together strongly inhibited the clonal growth of NCI-H345 or NCI-H1299 cells. MS-275 had little effect on the expression of lung cancer cellular GRP or GRP receptors, but increased expression of transforming growth factor-beta receptor II (TGF-beta RII). These results indicate that GRP receptor antagonists may potentiate the action of histone deacetylase inhibitors on lung cancer cellular proliferation by increasing expression of tumor suppressor genes.

Topics: Animals; Benzamides; Cell Line, Tumor; Cell Proliferation; Formazans; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Indoles; Lung Neoplasms; Pyridines; Receptors, Bombesin; Tetrazolium Salts

Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.

Topics: Adenocarcinoma; Antineoplastic Agents; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Coumarins; Formazans; Humans; In Vitro Techniques; Lung Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Tumor Cells, Cultured; Umbelliferones

Using a model with external ligation of the thigh, the effect of ischemia-reperfusion injury on tumor growth and the activity of lung metastasis was investigated in mice inoculated a spontaneous murine osteosarcoma cell line (POS-1) in vivo. POS-1 cell suspension was inoculated into the right hind footpad of 70 mice. Four weeks after inoculation, the ipsilateral thigh was ligated for 3 h in 15 mice and the contralateral thigh in 15 mice. Another ten mice were inoculated with POS-1 without ligating the thigh. The number of metastatic foci on the lung surface 6 weeks after inoculation was 2.29+/-0.98 (mean+/-SE) foci/lungs in mice with ipsilateral ligation and 6.25+/-2.41 in mice with contralateral ligation, which were significantly lower than control (13.40+/-1.42 in mice no ligation) (P<0.01). The number of metastatic foci on the lung surface in mice with intraperitoneal injection of superoxide dismutase (SOD) and catalase was 3.25+/-0.65 (mean+/-SE) foci/lungs in mice with ligation which was significantly greater than that in mice without SOD and catalase injection 1.29+/-0.97 (P=0.04). Cell viability was 9.12+/-4.07% with 100 microM H(2)O(2) in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. It revealed that at concentrations of 100 microM H(2)O(2) or higher was cytotoxic to POS-1. In cell invasion assay, the number of invading cells with 10 microM H(2)O(2) was 2.80+/-0.53 cells/field, which was significantly lower than control (5.93+/-0.18) (mean+/-SE), indicating that low-dose H(2)O(2) suppressed invasion of POS-1. These results suggested that reperfusion injury had selective cytotoxicity to POS-1 through producing reactive oxygen species. Activated oxygen was considered to inhibit the regional growth and the ability of lung metastasis of POS-1 cells.

Topics: Animals; Bone Neoplasms; Catalase; Cell Division; Cell Survival; Disease Models, Animal; Formazans; Hydrogen Peroxide; Ligation; Lung Neoplasms; Male; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Osteosarcoma; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tetrazolium Salts; Thigh; Tumor Cells, Cultured

7-ethyl-10-[4-(1-piperidyl)-1-piperidyl] carbonyloxy-camptothecin, a topoisomerase I (topo I) inhibitor, is one of the most active agent against lung cancer, and its radiosensitizing effect has been reported recently. We evaluated a combination in vitro effect of irradiation and 7-ethyl-10-hydroxy-CPT (SN-38), an active metabolite of 7-ethyl-10-[4- (1-piperidyl)-1-piperidyl] carbonyloxy-camptothecin, on a human small cell lung cancer cell line (SBC-3) and its cisplatin-resistant subline (SBC-3/CDDP). Growth-inhibitory effects of irradiation with or without SN-38 were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A modified isobologram method was used to evaluate the treatment interaction. The combination of irradiation and SN-38 showed a synergistic inhibitory effect on the growth of SBC-3/CDDP despite its cross-resistance to irradiation and SN-38. In contrast, the same combination showed only an additive effect on the growth of parental SBC-3 cells. There was no significant difference in topo I protein expression between these two cell lines. In SBC-3 cells, topo I catalytic activity was suppressed by 4 Gy of irradiation, without a decrease of nuclear topo I protein, whereas the exposure of SBC-3 cells to 1 microM SN-38 subsequent to irradiation showed no remarkable additional effects on both topo I activity and protein content. On the other hand, in SBC-3/CDDP cells, topo I activity was unchanged by irradiation, but the subsequent exposure to SN-38 gave rise to a decrease in topo I activity, which was accompanied by a significant decrease in the topo I protein content (P = 0.02). These observations may indicate that SN-38 induces sequestration of topo I onto DNA in radiation-treated SBC-3/CDDP cells and suggest that the synergistic effect of irradiation and SN-38 in SBC-3/CDDP cells was considered attributable to DNA repair-related enhanced recruitment of topo I onto the damaged DNA.

Topics: Antineoplastic Agents; Camptothecin; Carcinoma, Small Cell; Cell Survival; Cisplatin; Combined Modality Therapy; DNA Topoisomerases, Type I; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Formazans; Humans; Immunoblotting; Irinotecan; Lung Neoplasms; Tetrazolium Salts; Topoisomerase I Inhibitors; Tumor Cells, Cultured

Lovastatin, the drug used for the treatment of hypercholesterolemia, has previously been reported to exert antitumor activity in experimental murine models. Butyrate and butyric acid derivatives are well known to induce differentiation and apoptosis of tumour cells and also have recently gained acceptance as potential anticancer agents. In this study, we examined the antitumor effects of the combination of lovastatin and butyrate or its prodrug tributyrin in vitro and in vivo against a murine Lewis lung carcinoma (3LL). This combination therapy showed synergistic antitumor activity against 3LL cells in vitro. These effects were at least in part due to apoptosis induction that occurred after 12 hr of incubation with lovastatin and butyrate and was preceded by changes in cell cycle distribution of treated cells and expression of p21, p53 and cyclin D1. Remarkably, a systemic treatment of syngeneic mice inoculated with 3LL cells with both drugs resulted in significant tumour growth retardation.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Lewis Lung; Cell Cycle; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Drug Therapy, Combination; Female; Flow Cytometry; Formazans; Lovastatin; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Tetrazolium Salts; Triglycerides; Tumor Suppressor Protein p53

The absence or the presence of low levels of the Coxsackievirus and adenovirus receptor (CAR) on several tumor types might limit the efficacy of recently proposed tumor-specific or conditionally replicative adenoviruses (CRAds). To address this issue, we used a genetic modification of the fiber knob in the context of an E1A-defective CRAd to allow CAR-independent target cell infection as a means to enhance oncolytic potency. Such infectivity-enhanced CRAd showed higher replication, more efficient infection, and lysis of tumor cells in vitro. Of note, the improved antitumor effect of the fiber-modified CRAd could be demonstrated in vivo. We conclude that the combination of genomic modification to achieve tumor-selective replication and capsid modification to enhance infectivity yields more potent oncolytic adenoviruses for use in cancer treatment.

Topics: Adenoviridae; Adenovirus E1A Proteins; Animals; Cell Division; Female; Formazans; Genetic Vectors; Humans; Immunologic Tests; In Vitro Techniques; Luciferases; Lung Neoplasms; Male; Mice; Mice, Nude; Oligopeptides; Prostatic Neoplasms; Retinoblastoma Protein; Time Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Virus Replication; Viruses

Human non-small cell lung cancer (N-SCLC), a common malignancy generally unmanageable by conventional cytotoxic chemotherapy, represents a major world health burden. Suramin, a polyanionic drug which appears to interfere with growth-factor/receptor interaction, has recently been shown to be cytostatic for small cell lung cancer cells; it may also be effective for N-SCLC. As insulin-like growth factor I (IGF-I) is a known progression agent for N-SCLC, we have examined the effects of suramin on the 'IGF-I system' in a panel of human N-SCLC cell lines. Colorimetric and thymidine incorporation assays were used to assess cell chemosensitivity whereas a radio-receptor assay was employed to evaluate IGF-I/receptor binding. Suramin reversibly reduced, in a concentration- and time-dependent manner, the growth of each N-SCLC cell line examined either cultured in serum-containing or serum-free medium. Furthermore, suramin caused a concentration-related inhibition of labeled IGF-I peptide specific binding on all cell lines studied. Suramin caused a significant reduction in the Bmax values with only weak variations in the affinity constants (Kd). We hypothesize that suramin interference with IGF-I mitogenic activity is a pathway by which this drug produces its effect in vitro. These data indicate further studies on the mechanism of action and pharmacology of suramin in vivo are warranted.

Topics: Adenocarcinoma; Binding, Competitive; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Division; DNA; Dose-Response Relationship, Drug; Formazans; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Mitogens; Radioligand Assay; Suramin; Tetrazolium Salts; Thymidine; Tumor Cells, Cultured

The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.

Topics: Cell Count; Cell Line; Colorimetry; Coloring Agents; Culture Media; Formazans; Humans; Hydrogen-Ion Concentration; Interferon Type I; Lung Neoplasms; Receptors, Immunologic; Receptors, Interferon; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.

Topics: Azo Compounds; Buffers; Cell Count; Coloring Agents; Culture Media; Doxorubicin; Drug Screening Assays, Antitumor; Formazans; Humans; Hydrogen-Ion Concentration; Lung Neoplasms; Oxidation-Reduction; Spectrum Analysis; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vincristine

Formazans can be lost from tissue sections when the incubation medium is removed at the end of a dehydrogenase reaction. The loss is not uniform and will clearly affect the interpretation of results. This suggests that both the qualitative evaluation of the reaction and the measurement of enzyme activity should be made at the time of incubation.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Animals; Azo Compounds; Formazans; Histocytochemistry; Humans; Liver; Lung Neoplasms; Male; Oxidoreductases; Plants; Rats