No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Abstract
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.
... As a result, a total of 10 chromanone derivatives (1-10), including two new compounds (1 and 2), were isolated from the fruits of the EtOAc-soluble fraction of C. thesiodes fruits, and their structures were identified based on UV, IR, MS and NMR spectroscopic data, and comparison with those of reference compounds ( Figure 1). Moreover, compounds 1-10 were evaluated for their antiangiogenic activity by an MTT assay in vitro using axitinib as a positive control [8]. It was found that 1, 2, 3 and 9 exhibited important antiangiogenic activity. ...
... In the same way, the sub-fraction C-3 was applied to a silica gel CC (200-300 mesh) eluting with ether/EtOAc (8:1→6:1→4:1) to afford three fractions: C-3a (5.65 g), C-3b (7.55 g), and C-3c (5.02 g). Antiangiogenic assay: Compounds 1-10 were evaluated for their antiangiogenic activity by an MTT assay to determine whether they decreased VEGF-mediated cell proliferation in human umbilical vascular endothelial cells (HUVECs) using axitinib as a positive control [8]. The HUVECs were fostered in 10% fetal bovine serum and Dulbecco modified Eagle medium with 100 μg/mL penicillin/streptomycin in a humidified atmosphere in 5% CO 2 at 37°C, and they were seeded in 96-well plates containing 10 ng/mL vascular endothelial growth factor [14]. ...
... In this work, two new chromanone derivatives (1 and 2) and eight known ones (3)(4)(5)(6)(7)(8)(9)(10) were isolated from C. thesiodes fruits. Among them, 1, 2, 3 and 9 exhibited important antiangiogenic activity with IC 50 values of 13.8, 11.9, 43.6 and 74.8 μM, respectively. ...
A total of 10 chromanone derivatives (1-10), including two new compounds (1 and 2), were isolated from the fruits of Cynanchum thesiodes, and their structures were identified based on UV, IR, MS and NMR spectroscopic data, and comparison with those of reference compounds. The isolated compounds (1-10) were evaluated for their antiangiogenic activity by MTT assay using axitinib as a positive control. As a result, 1, 2, 3 and 9 exhibited important antiangiogenic activity with IC50 values of 13.8, 11.9, 43.6, and 74.8 μM, respectively.
... Sample supernatant (100 µL) obtained as described above, or serum (150 µL) were mixed with 500 µL PBS pH 7.4 plus 0.05% Tween-20 and centrifuged for 30 min at 12,000× g, 4 °C and the supernatant recovered for cytokine assay. Cytokines (VEGF, TNF-α, TGF-β) were determined by immunoassay using anti-mice DuoSet ® , R&D Immunoassay Kit (Minneapolis, MN, USA), as described by Teixeira and coworkers [59]. ...
... The cytotoxic effect of P1G10 cells was evaluated by the MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide) assay [59]. Briefly, exponentially growing cells, seeded in 96-well plates (10 3 cells/well), were incubated with different concentrations (0.01-100 µg/mL) of sterile P1G10 for 72 h. ...
The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases OPEN ACCESS Int. J. Mol. Sci. 2015, 16 7028 act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.
... Following the removal of the supernatant, thiazole blue tetrazolium bromide was added to each of the incubations (MTTassay). 11 After 4 hours of exposure to MTT, the optical density of the cell lines was measured to determine cell survival. 11 Cell-free supernatants from the medium alone and the 1.544-mM concentration of all 3 Mn oxidation states were removed at 24 hours. ...
... 11 After 4 hours of exposure to MTT, the optical density of the cell lines was measured to determine cell survival. 11 Cell-free supernatants from the medium alone and the 1.544-mM concentration of all 3 Mn oxidation states were removed at 24 hours. The amount of tumor necrosis factorα (TNFα),interleukin1(IL-1),andIL-6 in each supernatant was measured using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). ...
Manganese chloride (MnCl2) 2.5% is included in the extended metals patch test series to evaluate patients for contact hypersensitivity to this metal salt.
The objective of this study was to prospectively determine the rate of allergic and irritant patch test reactions to MnCl2 (Mn(II)), Mn2O3 (Mn(III)), and KMnO4 (Mn(VII)) in a cohort of patients undergoing patch testing.
Fifty-eight patients were patch tested with MnCl2, Mn2O3, and KMnO4, each at 2.5% in petrolatum. Patch readings were taken at 48, and 72 or 96 hours, and scored using standard methods. Cultured monolayers of keratinocytes (KCs) were exposed to MnCl2, Mn2O3, and KMnO4 in aqueous culture medium, and cell survival and cytokine release were studied.
MnCl2 caused irritant patch test reactions in 41% of the cohort, whereas Mn2O3 and KMnO4 caused a significantly lower rate of irritant reactions (both 3%). No allergic morphologies were observed. Similarly, in cultured KC monolayers, only MnCl2 was cytotoxic to KC and induced tumor necrosis factor α release.The oxidation state of manganese used for patch testing affects the irritancy of this metal salt, as Mn(II) caused an unacceptably high rate of irritant reactions in a cohort of patients. In vitro studies confirmed these clinical data, as only Mn(II) was cytotoxic to cultured monolayers of KC.
... Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) with slight modifications [56]. A cell proliferation kit (MTT, # 11465007001) was purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). ...
MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.
... Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay (30). Cells were seeded into 96-well plates (4x10 3 cells/100µl) and incubated with CITs (0.01-3 µM) or DMSO in quintuplicates for 72 h. ...
Introduction:
Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death.
Methods:
The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death.
Results:
CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs.
Discussion:
CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.
... In situ hybridization for genes in NK(0) or A-NK (0) cells rorul-expression of cytokine. IL-2R or perforin tured with X i -I tumor rells. ...
Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.
... Cell viability assay. An MTT (2-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay was used to determine the cytotoxicity of lapcin towards diverse cancer cell lines 45 . Lapcin was dissolved in DMSO to make a 3.2 mg/mL working solution. ...
In natural product discovery programs, the power of synthetic chemistry is often leveraged for the total synthesis and diversification of characterized metabolites. The synthesis of structures that are bioinformatically predicted to arise from uncharacterized biosynthetic gene clusters (BGCs) provides a means for synthetic chemistry to enter this process at an early stage. The recent identification of non-ribosomal peptides (NRPs) containing multiple ρ-aminobenzoic acids (PABAs) led us to search soil metagenomes for BGCs that polymerize PABA. Here, we use PABA-specific adenylation-domain sequences to guide the cloning of the lap BGC directly from soil. This BGC was predicted to encode a unique N-acylated PABA and thiazole containing structure. Chemical synthesis of this structure gave lapcin, a dual topoisomerase I/II inhibitor with nM to pM IC50s against diverse cancer cell lines. The discovery of lapcin highlights the power of coupling metagenomics, bioinformatics and total chemical synthesis to unlock the biosynthetic potential contained in even complex uncharacterized BGCs. Chemical synthesis of secondary metabolites isolated from nature, and derivatives thereof, is still a paradigm of significance to drug development. Here the authors instead use bioinformatics to analyze a biosynthetic gene cluster found in the soil metagenome, and chemical synthesis of its predict product to produce lapcin, a dual topoisomerase I/II inhibitor with promising activity against cancer cell lines.
... The final concentration of DMSO in the medium did not exceed 0.1%. Following 24 h or 48 h incubation, 50 µL of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added into each well to achieve a final concentration of 0.5 mg/mL and then incubated for an additional 4 h [25]. MTT was procured from Molecular Probes Inc. (Eugene, OR, USA). ...
The rhizomes of Alpinia galanga (Thai ginger) have been used extensively as a spice in Southeast Asian and Arabian cuisines and reported to possess a wide range of biological properties, such as antioxidant, antimicrobial, and antibacterial. However, the specific molecular and cellular mechanisms underlying the anti-tumor effects induced by Thai ginger and its corresponding active compounds have been poorly characterized. We found that upon EtOH extraction, Thai ginger extract exhibits cytotoxic activity (IC50 < 10 μg/mL) and triggers cell death via caspase-dependent apoptosis in human ovarian cancer cells. Among the three major compounds isolated from the extract, 1′-acetoxyeugenol acetate (AEA) exhibited potent cytotoxic activity in human ovarian cancer cells, SKOV3 and A2780. AEA induced apoptotic cell death through the activation of caspases-3 and -9. Notably, AEA enhanced the intracellular levels of reactive oxygen species (ROS), and the application of an antioxidant markedly reversed AEA-induced apoptosis of ovarian cancer cells. The knockdown of p47phox, a subunit of NADPH oxidase, suppressed both the pro-apoptotic and ROS-inducing effects of AEA. Additionally, the activation of the mitogen-activated protein kinase (MAPK) pathway by AEA through ROS regulation was found to be involved in AEA-induced apoptosis. Altogether, these results suggest that AEA exhibits potent apoptosis-inducing activity through the activation of the intrinsic pathway via ROS-mediated MAPK signaling in human ovarian cancer cells.
... The acute cytotoxicity effect of MPs and SPs was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assay (Heo et al., 1990). Cells were seeded at a density of 5 × 10 4 cells/mL in a 96-well plate and treated with 38.5 μg/well of 5-FU and 1 mg/well of MPs and SPs for 24, 48 and 74 h at 37 • C. Cell medium was changed to serum free medium (SFM) and 100 μL of MTT solution (Sigma Aldrich Corporation) was added. ...
Head and neck squamous cell carcinoma (HNSCC), a prevalent cancer worldwide, has a high incidence of loco-regional dissemination, frequent recurrence, and lower 5-year survival rates. Current gold standard treatments for advanced HNSCC rely primarily on radiotherapy and chemotherapy but with limited efficacy and significant side effects. In this study, we characterized a novel 5-fluorouracil (5-FU) carrier composed of chitosan solution (CS) and polycaprolactone (PCL) microparticles (MPs) in HNSCC preclinical models. The designed MPs were evaluated for their size, morphology, drug entrapment efficiency (EE%) and in vitro drug release profile. The anti-cancer activity of 5-FU-loaded particles was assessed in HNSCC human cell lines (CAL27 and HSC3) and in a preclinical mouse model (AT84) utilizing cell proliferation and survival, cell motility, and autophagy endpoints. The results demonstrated a 38.57% in 5-FU entrapment efficiency associated with reduced 5-FU in vitro release up to 96 h post-exposure. Furthermore, CS-decorated PCL MPs were able to promote a significant inhibition of cancer cell proliferation based on the metabolic and colony formation assays, in comparison to controls. In contrast, CS-decorated PCL MPs did not influence the pharmacological efficacy of 5-FU to inhibit in vitro cancer cell migration. Last, cell protein analysis revealed a significant increase of autophagy and cell death evaluated by LC3-II expression and PARP1 cleavage, respectively. In summary, these results support the potential utility of CS-decorated PCL MPs as an effective 5-FU-delivery carrier to improve HNSCC therapeutic management.
... The cytotoxic effect of PET and PEP in 4T1 (murine breast carcinoma), CT26.WT (murine colon carcinoma) and L929 (murine fibroblast) cells was evaluated by the MTT [3-(4,5)-dimethylthiazol(-z-yl)-3,5-diphenyltetrazolium bromide] assay (Heo et al., 1990). Briefly, exponentially growing cells, seeded in 96-well plates (2 × 10 3 cells/well), were incubated with different concentrations (0.01-500 μg mL − 1 ) of sterile PET and PEP. ...
Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L − 1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g − 1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.
... The cytotoxic effect of compounds 3 and 5 in 4T-1 (murine breast carcinoma), CT26.WT (murine colon carcinoma), and L-929 (murine fibroblasts) cells was evaluated by the MTT assay (Heo et al. 1990). Exponentially growing cells seeded in 96-well plates (2 × 10 3 cells/well) were incubated with different concentrations (0.01-500 μg/ml) of 3 and 5. Doxorubicin (0.01-50 μg/ml) was used as positive control. ...
Dipteryx lacunifera Ducke, Fabaceae, popularly known as “fava-de-morcego” and “garampara,” comprises pleasant tasting kernels and is commonly consumed by inhabitants northeast of Brazil. In the present study, among six flavonoids, two are reported for the first time as natural products, 6,3′,4′-trihydroxyflavone (2) and 4-O-metilsulfuretin (4); two reported for the first time in the species isoliquiritigenin (1) and homobutein (6); and two already known flavonoids, 7,3′,4′-trihydroxyflavone (3) and (−)-butin (5), were isolated from fruits and kernels of D. lacunifera. The compounds were isolated by chromatographic techniques and elucidated by spectrometric data (1D and 2D NMR and mass). Compounds 1–3 and 5 showed interesting cytotoxicity against tumor cell lines with inhibitory concentration (IC50) values ranging from 21.13 to 2.47 μg/ml. In addition, compounds 1 and 2 showed antimalarial activity against P. falciparum with 81.25 and 88.98% of parasitemia reduction, respectively.Graphical Abstract
.
... Cytotoxicity (MTT) assay is among the first in vitro bioassay methods used to predict toxicity of substances to various tissues [32]. In vitro cytotoxicity testing provides a crucial means for safety assessment and screening, and also for ranking compounds. ...
... Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the influence of microencapsulation on the toxicity of POH. 42 For this, nontumorigenic HEK293 were maintained in high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL of streptomycin. HEK293 cells were seeded at a density of 1.5 Â 10 4 cells/well in 96-well culture plates and were allowed to attach for 24 h at 37 C in a humidified atmosphere with 5% CO 2 . ...
Perillyl alcohol (POH) is a natural compound that has attracted a significant interest due to its potent antitumor activity. However, clinical trials have exhibited poor tolerance by oral administration, mainly due to gastrointestinal side effects. We propose the entrapment of POH into poly(D,L-lactic acid)-block-poly(ethylene glycol) (PLA-b-PEG) as delivery platform (entrapment efficiency of 63 - 68%). The influence of different concentrations of the tensoactives poly(vinyl alcohol) (PVAl) and sodium cholate (SC) on shear strength and morphology was evaluated by confocal laser scanning microscopy and interfacial tension studies. Only the microcapsules formulated with SC maintained their sphericity when submitted to shear stress. These results indicate that the interface is better organized with SC, conferring mutual stacked packing that is able to better stabilize the organic drop. The in vitro release profile of the drug from the microcapsules was correlated with pore formation and polymer degradation, best fitted to the Baker-Lonsdale model. The loaded microcapsules showed an IC50 equivalent to that of the free drug (80 μg/mL) after 72 h of exposure. However, after 24 h of exposure, loaded microcapsules showed an IC50 almost two-fold higher (220 μg/mL) suggesting gradual release.
... The cell proliferation efficiency was assessed using the MTT test. This test is based on the ability of mitochondrial dehydrogenases of live cells to convert the yellow MTT (3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide) (PanEco) into blue formazan that is insoluble in aqueous solutions [41]. Cells were plated in 96-well plates (5000 per well) in 90 μL of standard culture medium. ...
Adult male mice C57BL/6 (n = 105) were divided into five groups. The first group served as a control. In the 2nd–5th groups, the animals were treated subcutaneously with 40 mg/kg of proneurotoxin MPTP (methylphenyltetrahydropyridine), which forms a state similar to the initial stage of Parkinson’s disease over a 2-week period. Mice of groups 3–5 daily received an additive along with their food: one of three extracts of the biomass of the litter beetle Alphitobius diaperinus. In 2 weeks, all animals were tested for motor disorders in the vertical bar test; they were then euthanized and histochemical analysis of the dopamine-containing brain regions was performed. In addition, the same extracts were tested for counteraction to MPP⁺ toxin in cultured neuroblastoma cells. It was found that the primary aqueous and, especially, secondary water–methanol extracts had a powerful protective effect against the neurotoxic effect judging by the results of both the behavioral test and morphological control. Arginine was found at substantial concentrations in both effective extracts. An in vitro study confirmed the protective effect of the primary aqueous extract.
... 289][290].Les extraits sont d'abord dilués dans du DMSO pour avoir des concentrations de 10 mg/ml.Les cellules sont ensemencées dans des plaques à 96 puits (5000 cellules par puit) avec 100 µl de milieu (DMEM ou RPMI1640) et 10% de FBS pendant 24 heures. Ensuite, 200 µl de milieu fraichement préparé contenant 50 µg/ml d'extrait sont ajoutés à chaque puits. ...
Le genre Eryngium L. (Apiaceae, Saniculoideae) comprend plus de 250 espèces utilisées en médecine traditionnelle à travers le monde. En Tunisie il existe seulement huit espèces : E. barrelieri Boiss., E. campestre L., E. dichotomum Desf., E. glomeratum Lamk., E. ilicifolium Lamk., E. maritimum L., E. tricuspidatum L. et E. triquetrum Vahl. ; ces espèces sont en majorité peu étudiées du point de vue phytochimique. La présente étude a été effectuée sur la totalité des Eryngium qui poussent en Tunisie dans le but d’évaluer leurs activités biologiques, essentiellement antimicrobienne contre des microorganismes multirésistants et producteurs de béta-lactamases à spectres étendus (BLSE), mais aussi phototoxique et cytotoxique ainsi que la variabilité chimique par analyse par GC-FID et GC-MS des extraits les plus actifs.Toutes les espèces étudiées étaient dotées d’un pouvoir antimicrobien (1,25 à 0,07 mg/mL) et cytotoxique (24,4 à 0,32 µg/mL) considérable. Le criblage de l’activité phototoxique a permis de mettre en évidence la richesse de ces plantes en composés photoréactifs antimicrobiens potentiellement intéressants pour leur efficacité d’action et l’élargissement du spectre d’activité antimicrobienne.L’analyse des extraits actifs apolaires a permis d’étudier la variabilité chimique entre les différentes espèces et la mise en évidence de la présence majoritaire de composés antimicrobiens notamment des sesquiterpènes oxygénés tels que le spathulénol, le lédol, l’α-bisabolol et le cubénol, et des sesquiterpènes hydrocarbonés comme le β-bisabolène et le copaène ; et cytotoxiques tel que le falcarinol.Une étude phytochimique approfondie a été réalisée sur les racines d’E. triquetrum afin d’extraire, isoler par des essais bio-guidés et identifier les composés actifs. Le fractionnement a été optimisé par des chromatographies sur colonnes, CPC et CLHP. Parmi les composés identifiés deux polyacétylènes, le panaxydiol et le falcarinol, ont montré un fort pouvoir antimicrobien et une spécificité d’action notamment contre les souches de Pseudomonas aeruginosa BLSE et multirésistantes, avec des CMI allant jusqu’à 0,25 ng/mL et une activité en majorité bactéricide.
... The conversion of blue tetrazolium into purple formazan by metabolically active cells indicates the extent of cell viability. Measure cells viability was done by quantification of formazan dye using a colorimetric method (Heo et al. 1990). MTT assay and colorimetric method were used to evaluate the effects of P. viridis extracts on cell proliferation. ...
The phytochemical study of hexane, chloroform and methanol extracts from leaves of Psychotria viridis resulted in the identification of: the pentacyclic triterpenes, ursolic and oleanolic acid; the steroids, 24-methylene-cycloartanol, stigmasterol and β-sitosterol; the glycosylated steroids 3-O-β-D-glucosyl-β-sitosterol and 3-O-β-D-glucosyl-stigmasterol; a polyunsaturated triterpene, squalene; the esters of glycerol, 1-palmitoylglycerol and triacylglycerol; a mixture of long chain hydrocarbons; the aldehyde nonacosanal; the long chain fat acids hentriacontanoic, hexadecanoic and heptadenoic acid; the ester methyl heptadecanoate; the 4-methyl-epi-quinate and two indole alkaloids, N,N-dimethyltryptamine (DMT) and N-methyltryptamine. The chemical structures were determined by means of spectroscopic (IR, 1H and 13C NMR, HSQC, HMBC and NOESY) and spectrometric (CG-MS and LCMS-ESI-ITTOF) methods. The study of biologic properties of P. viridis consisted in the evaluation of the acetylcholinesterase inhibition and cytotoxic activities. The hexane, chloroform, ethyl acetate and methanol extracts, the substances 24-methylene-cycloartanol, DMT and a mixture of 3-O-β-D-glucosyl-β-sitosterol and 3-O-β-D-glucosyl-stigmasterol showed cholinesterase inhibiting activity. This activity induced by chloroform and ethyl acetate extracts was higher than 90%. The methanol and ethyl acetate extracts inhibit the growth and/or induce the death of the tumor cells strains B16F10 and 4T1, without damaging the integrity of the normal cells BHK and CHO. DMT also demonstrated a marked activity against tumor cell strains B16F10 and 4T1.
... The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, it is one of the most convenient and popular methods for the detection of cell proliferation inhibitory activity (Heo et al., 1990). The purity of all biologically evaluated compounds was determined to be >95% by HNMR analysis. ...
A series of dithiocarbamate-3-epi-jaspine B hybrids have been designed and synthesized from D-xylose based on the introduction of long lipophilic alkyl dithiocarbamate moieties at the C4 position of 3-epi-jaspine B. The anticancer activities of these compounds have been evaluated against two selected tumor cell lines (MGC-803 and B16-F10 cells), with most of the synthesized compounds exhibiting moderate activity. Among them, compounds 20d and 20f showed the highest levels of inhibitory activity of all the compounds tested against MGC-803 and B16-F10 cells with IC50 values of 16.39 and 14.83 µM, respectively. This work therefore represents the first reported account of the synthesis and in vitro cytotoxic evaluation of dithiocarbamate-3-epi-jaspine B hybrids.
... Cell proliferation assay. Cell lines were cultured in the presence of various concentrations of rhIL-2 and their proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described elsewhere [18]. Briefly, cells (2 Â 10 3 /well) were cultured in 96-well tissue culture dishes. ...
Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.
... DMSO 能溶解甲瓒, 用酶联免疫 检测仪测定 570 nm 处的吸光度(OD 值), 在一定细胞数 范围内, 吸光度与活细胞数量成正比, 由此能间接反映 活细胞的数量. MTT 法是操作简便、使用频率高的用于 检测细胞增值抑制活性的方法之一 [15] . ...
29 novel 3-substituted-benzoyl-4-substituted-thienyl-pyrrole compounds 2a~5c were synthesized via aldol condensation and Van Leusen pyrrole reaction using substituted acetophenone and substituted 2-thenaldehyde as raw materials, and the cell proliferation inhibition efficacy of 2a~5c against human colon cancer (HCT-116), human gastric adenocarcinoma (SGC-7901), human cervical carcinoma (Hela) and human umbilical vein endothelial (HUVEC) cell lines were estimated. Then MTT assay was used to evaluate the anti-proliferative activity. The result indicated that some target compounds exhibited strong (IC50≤20 μmol·L-1) or moderate (20 μmol·L-150≤50 μmol·L-1) proliferation inhibition efficacy against tumor cells, meanwhile didn't have significate inhibition effect on HUVEC. Compounds 2c, 2i, 3i, 3j, 4a~4f and 5c showed strong or moderate inhibition efficacy against HCT-116. The IC50 value of 2j was 4.3 μmol·L-1 against Hela, and compounds 2i and 3j exhibited moderate inhibition efficacy against Hela. The IC50 value of 3j was 10.5 μmol·L-1 against SGC-7901, and compounds 2i indicated moderate inhibition efficacy against SGC-7901. Compounds 2i and 3j showed broad anti-proliferative activity against all selected tumor cells.
... Cell proliferation assay. Cell lines were cultured in the presence of various concentrations of rhIL-2 and their proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described elsewhere [18]. Briefly, cells (2 Â 10 3 /well) were cultured in 96-well tissue culture dishes. ...
Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for b and c subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P < 0:01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.
... The Chromium-51-( 51 Cr-) release assay, first described in 1968 [10], is still the gold-standard but has the drawback of being radioactive and consequently hazardous. Newer nonradioactive assays using vital dyes [11], fluorescent dyes [12,13], and combinations thereof [14] as well as bioluminescencebased assays [15,16] have various disadvantages ranging from suboptimal labelling of targets to spontaneous release by leaky cells and inacceptable labor intensiveness [14,17,18]. ...
Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput
in vitro
cytotoxicity assays that efficiently analyze immune effector functions. The gold standard
51
Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase
in vitro
transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the
51
Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.
... The other group was kept at 37 ºC. The two groups were incubated at 37 ºC in the incubator for three days, and the MTT assay was performed as before [6]. The optical density was then measured at 590 nm wavelength, and the percentage of cell viability was calculated and plotted against drug concentration. ...
The exploitation of folate receptor as Trojan horse to deliver folate-targeted liposomes bearing diverse cargo represents a novel therapeutic strategy to target folate receptor-expressing cells. In this paper, thermosensitive liposomes made of synthetic lipids (distearolphosphatidylcholine, DSPC and dipalmitoylphosphatidylcholine, DPPC) showing gel to liquid phase transition at 41º C, were used for encapsulation of methotrexate. The liposomes were prepared by thin film hydration method, the liposome binding constant K b for the drug was measured using a spectroscopic assay and was found to be 57.18 (mg/ml) –1. We have studied the liposome-mediated delivery of methotrexate to breast tumor cells in vitro, the cell sensitivity study was performed at normal physiological temperature (37º C) as well as hyperthermia increased temperature of 42º C. Moreover, a targeting moiety was used by modifying the liposome surface with folate using polyethyleneglycol (PEG) as a spacer. The ability of methotrexate to inhibit tumor cell growth is increased dramatically when encapsulated in targeted (folated) thermosensitive liposomes, but decreased when encapsulated in liposomes deficient folate. The index of cell Killing expressed as IC 50 was reduced dramatically from 7 µg/ml to 0.864 µg/ml upon using folate as a targeting moiety. Hyperthermia was not effective when used with non-specific targeted liposomes. However, the cytotoxicity of the drug increased dramatically upon heating folate targeted thermosensitive liposomes (the IC 50 was reduced to 0.34 µg /ml).
... Mitochondrial dehydrogenases at the sites of cytochromes b and c in viable cells convert the yellow form of the salt to an insoluble, intracellular purple formazan. Solubilized formazan can then be quantitated spectrophotometrically and the results related to the proportion of viable tumor cells in a population incubated with antitumor effectors (Heo et al., 1990). ...
Gmelina arborea roxb commonly known as ‘Gambhari’ tree, the various parts of the plants are widely used in diarrhoea, anti-pyretic, thirst, anemia, leprosy, ulcers, consumption, strangury and vaginal discharges. We tested the cytotoxic potential of Gmelina arborea roxb in HL-60 cells. Aqueous Extract of Gmelina arborea roxb (AEGA) was tested at the various concentrations 5, 10, 15 and 20 mg in MTT assay, Cell Viability assays and clonogenic assay. Our study shows that AEGA inhibits cell growth and decrease the cell viability. The AEGA inhibits cell proliferation at a dose and time dependent manners measured by MTT assay. The AEGA very significantly decreased the cell viability of HL-60 Cells after 24 and 48 h compared to the control cells. In the semisolid culture, the number of colonies decreased significantly (p<0.01) in a dose-dependent manner. Overall, AEGA has shown a substantial and significant anti cancer activity in all the models. This protective effect might have been mediated by apoptosis mechanisms.
... For this purpose, cytotoxic and cellar response tests were carried out in vitro using the Methyl thiazolyl tetrazoliun (MTT) method [24,25]. A number of rectangular samples were used in this preliminary biocompatibility testing. ...
... The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity of the CTLs in each group. This is a standard colorimetric assay for determining in vitro cytotoxicity of CTLs against tumor cells in experiments [16,17] . As negative control groups, an equal number of target cells were cultured alone and an equal number of effector cells were cultured without target cells in a total of 200 μL. ...
AIM: To investigate whether tumor debris created by high-intensity focused ultrasound (HIFU) could trigger antitumor immunity in a mouse hepatocellular carcinoma model.
METHODS: Twenty C57BL/6J mice bearing H22 hepatocellular carcinoma were used to generate antitumor vaccines. Ten mice underwent HIFU ablation, and the remaining 10 mice received a sham-HIFU procedure with no ultrasound irradiation. Sixty normal mice were randomly divided into HIFU vaccine, tumor vaccine and control groups. These mice were immunized with HIFU-generated vaccine, tumor-generated vaccine, and saline, respectively. In addition, 20 mice bearing H22 tumors were successfully treated with HIFU ablation. The protective immunity of the vaccinated mice was investigated before and after a subsequent H22 tumor challenge. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the cytotoxicity of splenic lymphocytes co-cultured with H22 cells was determined in vitro before the tumor challenge, and tumor volume and survival were measured in vivo after the challenge in each group. The mechanism was also explored by loading the vaccines with bone marrow-derived dendritic cells (DCs).
RESULTS: Compared to the control, HIFU therapy, tumor-generated and HIFU-generated vaccines significantly increased cytolytic activity against H22 cells in the splenocytes of the vaccinated mice (P 0.05).
CONCLUSION: Tumor debris remaining after HIFU can improve tumor immunogenicity. This debris releases tumor antigens as an effective vaccine to develop host antitumor immune response after HIFU ablation.
... The FLK-1 promoter EGFP transgenic zebrafish (FLK-1: EGFP) was used to investigate the in vivo antiangiogenic activity of SKLB-287 as previously described [30]. 10 embryos per experimental group were used in our study, and each experiment was performed in 3 replicates. ...
Colorectal cancer (CRC) is the third common cancer and most of the chemotherapies of CRC currently used often suffer limited efficacy and large side effects. Targeted small-molecule by anti-tumor drugs are thought a promising strategy for improving the efficacy and reducing the side effects. In this investigation, we report a novel multikinase inhibitor, termed SKLB-287, which was discovered by us recently. SKLB-287 could efficiently inhibit the activation of endothelial growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2). It displayed very good anti-proliferative activity against LoVo CRC cells and considerable antiangiogenic potency in transgenic zebrafish embryos. Oral administration of SKLB-287 resulted in dose-dependent suppression of tumor growth in LoVo xenograft mouse model. Immunohistochemistry was adopted to examine the in vivo anti-tumor mechanism of action of SKLB-287. Keywords: SKLB-287, EGFR, antiangiogenesis, colorectal cancer.
... Finally, the absorbance at 570 nm (test wavelength) and with a reference filter of 630 nm was measured. All experiments were performed three times and the viability was calculated and showed in Figure 6(14–15). ...
In this study, we was tried to prepare a nano compound with a new way in functionalization as anti gastric cancer candidate.
Functionalization of nanotubes is a useful route for modification of their biologic properties. (3-oxoindolin-2-ylidene) urea is a chemical compound that made of isatin and urea that can be useful in cancer study.
MWNT-COOH was functionalized by this compound with one-step reaction that is a new class in modification. Product has been investigated by FT-IR, Raman and SEM. Anti cancer investigation with human gastric cells and MTT assay test for measurement of viable cell numbers were also performed.
The two bands at around 2800-2900 cm(-1) which are seen in functionalized product are attributed to the CH stretching of MWNT-COOH defects.
Cellular results demonstrated that the functionalized nano-tube is a more toxic agent compared to other samples for cancer cells and can be used as a candidate material for chemotherapy.
... Cell proliferation assays. Cell proliferation was measured using MTT assay (Sigma, USA) as previously described 23 . SKOV3, A2780, A549, and L-02 cells were treated with indicated concentrations of poly Ab (0.08-2.56 mg/ml) for 48 h. ...
Most women with ovarian cancer are diagnosed at an advanced stage and there are few therapeutic options. Recently, monoclonal antibody therapies have had limited success, thus more effective antibodies are needed to improve long-term survival. In this report, we prepared polyclonal rabbit anti-ovarian cancer antibody (Poly Ab) by immunizing rabbits with the human ovarian cancer cell line SKOV3. The Poly Ab bound to SKOV3 and inhibited the cancer cells proliferation. Western blot analysis was conducted, which indicated that Poly Ab inhibited cancer cells through apoptosis involving the caspase signaling pathway including caspase-3 and caspase-9. Finally, compared with the control antibody, administration of Poly Ab reached 64% and 72% tumor inhibition in the subcutaneous and intraperitoneal xenograft mouse model, respectively. Our findings suggest that Poly Ab is an effective agent for apoptosis induction and may be useful as a safe anticancer agent for ovarian cancer therapy.
... The cell proliferation evaluation of the HUVECs was measured using MTT as previously described. 35 The HUVECs were exposed to a series of free PL and PL micelles for 48 h, respectively. Each assay was replicated 3 times. ...
Piperlongumine (PL) shows an inhibitory effect on tumor growth; however, lipophilicity has restricted its further applications. Nanotechnology provides an effective method to overcome the poor water solubility of lipophilic drugs. Polymeric micelles with small particle size can passively target tumors by the enhanced permeability and retention (EPR) effect, thus improving their anti-tumor effects. In this study, to improve the water solubility and anti-tumor activity of PL, PL encapsulated polymeric micelles (PL micelles) were prepared by a solid dispersion method. The prepared PL micelles showed a small particle size and high encapsulation efficiency, which could be lyophilized into powder, and the re-dissolved PL micelles are homogenous and stable in water. In addition, a sustained release behavior of PL micelles was observed in vitro. Encapsulation of PL into polymeric micelles could increase the cytotoxicity, cellular uptake, reactive oxygen species (ROS) and oxidized glutathione (GSSG), and reduce glutathione (GSH) levels in vitro. Encapsulation of PL into polymeric micelles enhanced its inhibitory effect on neovascularization both in vitro and in vivo. Compared with free PL, PL micelles showed a stronger inhibitory effect on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). Additionally, in a transgenic zebrafish model, embryonic angiogenesis was inhibited by PL micelles. Furthermore, PL micelles were more effective in inhibiting tumor growth and prolonging survival in a subcutaneous CT-26 murine tumor model in vivo. Therefore, our data revealed that the encapsulation of PL into biodegradable polymeric micelles enhanced its anti-angiogenesis and anti-tumor activities both in vitro and in vivo.
... The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the proliferation rate of the cells as described previously [28]. After irradiation, cells were immediately plated in 96-well plated. ...
Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. Tumor cells genetically modified to secrete immune activating cytokines have been proved to be more immunogenic. IL-18 could augment proliferation of T cells and cytotoxicity of NK cells. GM-CSF could stimulate dendritic cells, macrophages and enhance presentation of tumor antigens. In our study, we used mouse GM-CSF combined with IL-18 to modify Lewis lung cancer LL/2, then investigated whether vaccination could suppress tumor growth and promote survival.
The Lewis lung cancer LL/2 was transfected with co-expressing mouse GM-CSF and IL-18 plasmid by cationic liposome, then irradiated with a sublethal dose X ray (100Gy) to prepare vaccines. Mice were subcutaneously immunized with this inactivated vaccine and then inoculated with autologous LL/2 to estimate the antitumor efficacy.
The studies reported here showed that LL/2 tumor cell vaccine modified by a co-expressing mouse GM-CSF and IL-18 plasmid could significantly inhibit tumor growth and increased survival of the mice bearing LL/2 tumor whether prophylactic or adoptive immunotherapy in vivo. A significant reduction of proliferation and increase of apoptosis were also observed in the tumor treated with vaccine of co-expressing GM-CSF and IL-18. The potent antitumor effect correlated with higher secretion levels of pro-inflammatory cytokines such as IL-18, GM-CSF, interferon-gamma in serum, the proliferation of CD4+IFN-gamma+, CD8+ IFN-gamma+ T lymphocytes in spleen and the infiltration of CD4+, CD8+ T in tumor. Furthermore, the mechanism of tumor-specific immune response was further proved by 51Cr cytotoxicity assay in vitro and depletion of CD4, CD8, NK immune cell subsets in vivo. The results suggested that the antitumor mechanism was mainly depended on CD4+, CD8+T lymphocytes.
These results provide a new insight into therapeutic mechanisms of IL-18 plus GM-CSF modified tumor cell vaccine and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.
... Dye was released from intact, adherent cells by the addition of 0.8 ml of 50% ethanol and optical density (OD) measured at 570 nm. Percentage cytotoxicity was calculated using the formula 100 Â (A À [B À C])/D where A is OD of MCF7 cells after mixing to remove non-adherent cells, B is OD of experimental wells, C is OD of adherent YT2C2-PR cells and D is OD of MCF7 cells without mixing to remove those that are non-adherent (Chong and Parish, 1985;Heo et al, 1990;Gondolf et al, 1996;Adrián-Cabestré et al, 1999). ...
Background:
Sialophorin is a transmembrane sialoglycoprotein. Normally, the molecule is only produced by white blood cells where it regulates functions such as intercellular adhesion, intracellular signalling, apoptosis, migration and proliferation.
Methods:
Normal breast tissue and primary breast tumours were analysed by immunohistochemistry for sialophorin expression. The sialophorin-positive breast cancer cell line MCF7 was engineered to stably express either non-targeted or sialophorin-targeted small interfering RNA (siRNA). Assays were then performed in vitro to assess apoptosis, intracellular adhesion, transendothelial migration and cytotoxicity. An orthotopic mouse model assayed ability to produce tumours in vivo.
Results:
Normal breast epithelial cells exhibit expression of the N-terminal domain of sialophorin in the cytoplasm but not the nucleus. The majority of these normal cells are also negative for expression of the C-terminal domain. In contrast, malignant breast epithelial cells exhibit N-terminal expression both in the cytoplasm and nucleus and the majority express the C-terminus in the nucleus. Using differential patterns of intracellular expression of the N and C termini of sialophorin, we define six subtypes of breast cancer that are independent of histological and receptor status classification. Targeting sialophorin with siRNA resulted in the MCF7 breast cancer cell line exhibiting increased homotypic adhesion, decreased transendothelial migration, increased susceptibility to apoptosis, increased vulnerability to lysis by natural killer cells and decreased ability to produce tumours in mice.
Conclusion:
Our results indicate that intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and that sialophorin represents a novel therapeutic target.
... For this purpose, cytotoxic and cellar response tests were carried out in vitro using the Methyl thiazolyl tetrazoliun (MTT) method [24,25]. A number of rectangular samples were used in this preliminary biocompatibility testing. ...
This study presents the design, processing, properties and potential applications of a novel layered bio-ceramic composites consisting of three different micro-porous calcium phosphate coatings on strong zirconia cores manufactured using a recently developed slip coating-deposition and coating-substrate co-sintering technique. Detailed microstructures of the three graded micro-porous calcium phosphate coatings, and the coating/substrate interface have been investigated. Also, the flexural strength of the bio-ceramic composite and the bonding state between the coatings and zirconia substrate have been characterized. A preliminary and limited in vitro cell test indicates that the new scaffold composite has no cytotoxicity to the fibroblasts which can attach, proliferate and grow on the coating surfaces. Because of the combination of bio-function and strength, such layered load-bearing bio-ceramic composites are a potential candidate for large-scale head bone repairs.
The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.
Two new flavones, namely 5-hydroxy-2-(4′-hydroxyphenyl)-6,12,13-trimethoxypyrano-xanthene-4,9-dione (1) and 5-hydroxy-2-(3′-methoxy-4′-hydroxyphenyl)-6-methoxy-11-isopropyl-pyranochromene-4,9-dione (2), were isolated from Salvia plebeia. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR spectral data. Compounds 1 and 2 were evaluated for their anti-angiogenic activities by the MTT assay with axitinib as positive control, and they displayed moderate anti-angiogenic activities with IC50 values of 14.07 and 11.25 μM, respectively.
A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2–dependent NK-92 and IL-2–independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 andneor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/106 cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.
Objectives Cardamonin (CD), an active chalconoid, has been extensively studied in a wide variety of human tumors. However, the effects and underlying mechanism of cardamonin on 5-fluorouracil (5-FU)-resistant gastric cancer (GC) remain largely unclear. This study aimed to investigate the antitumor effects of cardamonin on 5-FU-resistant GC cells and explore the molecular mechanisms underlying its therapeutic potential. Methods The antitumor activities of cardamonin, 5-FU and their combination against BGC-823 and BGC-823/5-FU cells were determined using cytotoxicity assay, flow cytometry-based cell cycle analysis and Annexin V apoptosis assay. The effect of cardamonin on P-glycoprotein activity was assessed by Rh123 uptake assay. Real-time PCR, Western blotting and Co-immunoprecipitation analysis were carried out to assess the inhibition of Wnt/β-catenin signaling pathway. A xenograft mouse model was established using BALB/c nude mice to examine the combinatorial effects of cardamonin and 5-FU on tumor growth. Results Our data provided the first demonstration that cardamonin significantly enhanced the chemosensitivity of 5-FU in GC cells via suppression of Wnt/β-catenin signaling pathway. Additionally, the combination of cardamonin and 5-FU might result in the apoptosis and cell cycle arrest of BGC-823/5-FU cells, accompanied by the downregulated expression levels of P-glycoprotein, β-catenin and TCF4. More importantly, our results demonstrated that cardamonin specifically disrupted the formation of β-catenin/TCF4 complex, leading to TCF4-mediated transcriptional activation in 5-FU-resistant GC cells. Besides, through a xenograft mouse model, co-administration of cardamonin and 5-FU significantly retarded tumor growth in vivo, thus, confirming our in vitro findings. Conclusions Overall, this study revealed that cotreatment of cardamonin and 5-FU could strongly potentiate the antitumor activity of 5-FU, and put forth cardamonin as a rational therapeutic strategy for drug-resistant GC treatment.
A new phenylpropanoid glycoside, named α-L-rhamnopyranosyl-(1↔2)-β-D-[4″-(8E)-7-(3,4-dihydroxyphenyl)-8-propenoate, 1″-O-(7S)-7-(3,4-dihydroxyphenyl)-7-methoxy-ethyl]-glucopyranoside (1), together with nine known compounds (2–10) were isolated from the active fraction (n-Butanol fraction) of Gynura cusimbua for the first time. The known compounds (2–10) were identified as phenylpropanoid glycosides on the basis of extensive spectral data and references. The antiangiogenic activities of compounds (1–10) were evaluated by MTT assay on HUVECs and wild-type zebrafish in vivo model assay. As a result, compounds 1, 6, 7, 8 and 10 exhibited certain antiangiogenic activities.
The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.
Medicago sativa L. is the most important cultivated herbage known as "the king of forage" and "feed queen" in the world. Eight new chalcones (1-8), and twelve known chalcones (9-20) were isolated from the aerial parts of M. sativa for the first time. Their structures were identified by extensive spectral data and references. The hypolipidemic and anti-angiogenic activities of compounds (1-20) were evaluated for the first time. Compounds 3, 4, 11, 12, and 20 (10 μM) exhibited significant hypolipidemic activities by measuring the triglyceride content in HepG2 cells with Simvastatin as positive control. Moreover, compounds 6, 8, 18, and 19 (μM) exhibited moderate anti-angiogenic activities, which inhibited VEGF-induced HUVEC proliferation in vitro with IC50 values of 13.86 ± 0.43, 15.53 ± 0.19, 39.52 ± 0.24, and 45.04 ± 0.51 μM, respectively. These research results may guide the search for new natural products with hypolipidemic and anti-angiogenic attributes.
AIM: For the need of cell model in drugs research that may have effect on amyloid β-protein in Alzheimer's disease processing, the cells coexpressed with human amyloid protein precursor (APP) 695 gene and human nicotinic acetylcholine receptor (nAChR) α4β2 subunit genes were constructed. METHODS: Liposome was used to transfect human APP 695 gene into SH-EP1 cells which stably expressed nAChR α4β2 genes. Neomycin (500 mg·L-1) was used to ensure stable expression of APP 695, and limiting dilution assay was used to obtain single transfected cell clones. RT-PCR and Western blot were used to verify the clones. The highly expressed cell clones were selected, and the activity of nAChR α4β2 was justified by patch clamp. RESULTS: Cell clone with stable coexpression of APP 695 gene and nAChR α4β2 genes was constructed successfully, and the activity of α4β2 receptors in the cell clone was confirmed by patch clamp. CONCLUSION: Coexpression cell model of human APP 695 and nAChR α4β2 genes was constructed successfully, which may provide good method for pharmaceutical experiments on effects of nAChR α4β2 on amyloid processing.
Nowadays, cancer is one of the main reasons of death between nations caused by different elements such as presence of mutagenic and cancer causing materials in the environment. Isatin and Carbon nanotubes can be used to deliver their cargoes to cells and organs. The potential use of them to treat several types of cancer, with minimal or no toxic effects to normal cells. In the present study, 1,4-diazabicyclo [2.2.2] octane (DABCO) was used as a catalyst for one-pot reaction. Three-component condensation reactions consisting of isatin or bromo-isatin, 4-amino-3-nitrophenol and MWNTCOOH in hot water under mild conditions at 85°C, to afford the corresponding MWNTCO-4-hydroxy-2-nitrophenylimino-indolin-2-one derivatives. This method has the advantages of a simple operation and mild reaction conditions, by using low cost chemical as a catalyst. Products have been investigated by FT-IR, Raman, SEM, TEM and Ultra Violent spectroscopy. Biological activity with human gastric cells and MTT assay test for measurement of viable cell numbers were also performed.
Melanoma is a type of cancer arising from the melanocytes, which are the cells that make up the pigment melanin and are derived from the neural crest. There is no particularly effective therapy once the disease is metastatic, highlighting the need for discovery of novel potent agents. In this investigation, we adopted a zebrafish embryonic pigmentation model to identify antimelanoma agents by screening an in-house small molecule library. With this assay, we found that a small molecule compound, SKLB226, blocked zebrafish pigmentation and pigment cell migration. Mechanism of action studies showed that SKLB226 downregulated MITF mRNA level in both zebrafish embryos and mammalian melanoma cells. Further studies showed that it could efficiently suppress the viability and migration of mammalian melanoma cells. In summary, SKLB226 can be used as a chemical tool to study melanocyte development as well as an antimelanoma lead compound that should be subjected to further structural optimization.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) are completely different viruses with similar natural histories of infection. Both target the liver and cause chronic, probably immunopathological diseases. The morphogenesis of HBV and bovine viral diarrhea virus (BVDV) is more dependent on the glucosidase step of glycoprocessing than most host functions. Because BVDV is similar with respect to its morphogenesis to HCV, it is speculated that inhibitors of glucosidases will have broad therapeutic value. Glucosidases mediate the first step of glycoprocessing that is necessary for the proper folding and trafficking of some glycoproteins. Viruses that acquire their lipid envelopes and glycoproteins from intracellular membranes-such as the ER and Golgi-are extremely dependent on the ER glucosidase. It is less conventional to propose targeting a host enzyme for the treatment of a viral infection. The ultimate usefulness of these compounds as therapeutics depends on their safety profiles.
Chemical investigation of the whole plants of Salvia substolonifera E.Peter yielded seven germacrane sesquiterpenoids, substolides A-G (1-7), an ethoxylated artifact (8), and two known analogues, 6β-tigloyloxyglechomafuran (9) and castanin F (10). Four germacrane 8-acetylation derivatives (1a-4a) were obtained by chemical transformation. Their structures and relative or absolute configurations were elucidated by intensive spectroscopic methods, and single-crystal X-ray diffraction analysis. Compounds 1a-4a, and 5-10 were evaluated for their in vitro anti-angiogenic effects. Compounds 7 and 9 significantly inhibited VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation in vitro, with IC50 values of 16.15±0.19, and 4.03±0.26μM, respectively. The structure activity relationship of these compounds is discussed.
AIM: To investigate the effects of 5-Fluorouracil (5-FU) on modulation of pro-inflammatory and anti-inflammatory cytokines in acute pancreatitis and the mechanism of it in the treatment of acute pancreatitis.
METHODS: Male Sprague Dawley rats were assigned to 3 Groups: Group A, sham operated rats as controls (n = 7); Group B, acute pancreatitis induced by ductal injection with 5% sodium cholate at a volume of 1.0 mL/kg without any other treatment; Group C, after the pancreatitis was induced as in Group B, the rats were injected intravenously with 5-FU 40 mg/kg. The animals in Groups B and C were killed at 2, 6 and 24 h after operation (n = 7), and blood samples were taken for measurement of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6) (by bioassay), and interleukin-10 (IL-10), transforming growth factor-β (TGF-β) (by ELISA). The wet weight of pancreatic tissue, serum amylase levels and white blood cells were also measured.
RESULTS: Four rats in Group B and one in Group C died after pancreatitis was induced. Both pro-inflammatory cytokines (TNF-α, IL-1, IL-6) at the 2 and 6 h period and the anti-inflammatory cytokines (IL-10, TGF-β) at 24 h increased significantly (P < 0.05) in rats of Group B. After treatment with 5-FU, TNF-α, IL-1, and IL-6 in serum of rats of Group C were inhibited at 2 and 6 h after operation (P < 0.05), and IL-10, TGF-β were inhibited at 24 h compared to Group B (P < 0.05). Obvious improvements in the severity of the acute pancreatitis, including the amylase levels, wet weight of pancreatic tissue and neutrophil counts, were also observed after treatment with 5-FU.
CONCLUSION: 5-FU is an anti-metabolic and immunosuppressive agent which can minimize the abnormal immune cytokine response and relieve the pathophysiological disorders associated with experimental acute pancreatitis.
JK184 could specially inhibit Gli in Hedgehog (Hh) pathway, which showed great promise in cancer therapeutics. For developing aqueous formulation and improving anti-tumor activity of JK184, we prepared JK184 encapsulated MPEG-PCL micelles by solid dispersion method without using any surfactants or toxic organic solvent. The cytotoxicity and cellular uptake of JK184 micelles were both increased compared with free drug. JK184 micelles induced more apoptosis and blocked proliferation of Panc-1 and BxPC-3 tumor cells. Besides, JK184 micelles exerted a sustained in vitro release behavior and had a stronger inhibitory effect on proliferation, migration and invasion of HUVECs than free JK184. Furthermore, JK184 micelles had stronger tumor growth inhibiting effects in subcutaneous Panc-1 and BxPC-3 tumor models. Histological analysis showed that JK184 micelles improved anti-tumor activity by inducing more apoptosis, decreasing microvessel density and reducing expression of CD31, Ki67, and VEGF in tumor tissues. JK184 micelles showed a stronger inhibition of Gli expression in Hh signaling which played an important role in pancreatic carcinoma. Furthermore, circulation time of JK184 in blood was prolonged after entrapped into polymeric micelles. Our results suggested that JK184 micelles were a promising drug candidate for treating pancreatic tumors with high inhibitory effect on Hh activity.
Novel antidepressants or treatment strategies that may offer a more rapid onset of action, improved efficacy, and greater tolerability are in desperate need. Because current clinically utilized antidepressants, which target high-affinity transporters for serotonin and norepinephrine, fail to provide satisfactory treatment outcomes for quite a portion of patients. In recent investigations, a low-affinity but high-capacity transporter organic cation transporter 2 (OCT2, SLC22A2) has been proposed as an important postsynaptic determinant of aminergic tonus and mood-related behaviors, a complementary system to the high-affinity transporters. In order to evaluate whether OCT2 inhibition may at least in part contributes to the pharmacological effects of antidepressants, several typical antidepressant compounds of various mechanism categories were employed to inhibit OCT2 activity in cells stably overexpressing OCT2. The tested antidepressant agents included selective serotonin reuptake inhibitors (SSRIs, fluoxetine, sertraline and paroxetine), tricyclic antidepressants (TCAs, amitriptyline, imipramine, desipramine), monoamine oxidase inhibitor (MAOI, moclobemide), serotonin-norepinephrine reuptake inhibitor (SNRI, venlafaxine) and reported antidepressant alkaloid piperine. Piperine was screened through synaptosomes before cell experiments, without the interference of monoamine oxidase. All of the nine antidepressant compounds showed moderate inhibitory effects on OCT2-mediated metformin, serotonin and/or norepinephrine uptake. Sertraline and desipramine tended to inhibit OCT2 activity via a competitive mechanism. The fact could be easily belied, since passive diffusion dominated the influx process. It remains to be seen whether OCT2 inhibition plays a role to the overall therapeutic effects in clinical practice.
The uncontrolled progression of the inflammatory cascade is the main cause underlying the development of multiple organ dysfunction syndrome (MODS) in acute pancreatitis. In this study, we investigated the effects of several immunosuppressants on mitigating the systemic inflammatory reaction syndrome (SIRS) and the compensatory anti-inflammatory response syndrome (CARS) associated with acute pancreatitis. A total of 93 male Sprague Dawley rats were divided into 5 groups: group 1 was the sham group and group 2 underwent laparoscopic intrapancreatic duct injection of sodium taurocholate to induce pancreatitis. The remaining 3 groups were the same as group 2, with the addition of methylprednisolone, cyclophosphamide or methotrexate treatment (metastab, CTX or MTX groups, respectively). Following establishment of the acute pancreatitis model, the serum levels of inflammatory and anti-inflammatory cytokines in groups 2, 3, 4 and 5 were found to be significantly elevated. Following immunosuppressant administration, the levels of all inflammatory and anti-inflammatory cytokines investigated in groups 3, 4 and 5 were decreased compared to those in group 2. The pancreatic amylase levels and pancreatic wet weight (PWW) were also decreased in groups 3, 4 and 5 compared to those in group 2. Therefore, immunosuppressants may reduce inflammation-related cytokine levels in acute pancreatitis and relieve disease progression.
Silibinin (silybin A and silybin B), the primary active component of Silybum marianum has antiviral activity against herpes simplex virus, type 2 (HSV-2). The fifty percent cytotoxic dose (CD50) of silibinin was 378 μg/ml. By plaque reduction assay silibinin had a weak antiviral effect against HSV-2 with a fifty percent inhibitory concentration (IC50) of 100 μg/ml and therapeutic index of 3.8. As a virucide, silibinin was more potent with an IC50 of 5 μg/ml and therapeutic index of 76. Further investigations of the mechanisms of action of silibinin against herpes simplex viruses and subsequent silibinin monotherapy or combination therapy with other flavonoids as a topical treatment for genital herpes are warranted.
Human carcinomas were found to express IL-2 R and to produce, but not to secrete, IL-2. Intermediate affinity IL-2Rβγ detected on the surface and in the cytoplasm of carcinoma cells binds exogenous IL-2 at the nanomolar or micromolar concentrations and mediates cell cycle arrest (CCA) possibly through the upregulation of the CDK inhibitor p27kipl expression. In contrast, IL-2Rα is modestly expressed on the cell surface, and it may be involved in the intracrine pathway of delivering endogenous IL-2 to the cell surface. IL-2 is a growth factor for human carcinomas, and as it binds to the high-affinity IL-2R, it promotes cellular proliferation by suppressing expression of p27kipl. It also protects tumour cells from apoptosis. The presence in carcinomas and in normal tissue cells of two IL-2/IL-2R pathways regulating cellular growth and survival is a novel finding. Both the endogenous and exogenous IL2/IL2R pathways could become therapeutic targets in the future and could be explored to obtain insights into the mechanisms of tumour growth control as well as to modulate its sensitivity to other therapies.
Twenty-one head and neck squamous cell carcinoma (HNSCC) cell lines were established from 89 fresh tumor specimens in order to study the biology of HNSCC lines, establish tumors in nude mice, and evaluate the sensitivity to immunological effector cells of these tumors in vitro and in vivo in nude mice. The lines were established from explants using differential trypsinization and culture for 2 to 20 mo. The explants were derived from 11 different sites. Three pairs of lines were derived from both the primary tumor and metastatic lymph nodes in the same patients. All cultures grew as either compact or diffuse adherent monolayers, and they had a median doubling time of 86 h (range, 33 to 531 h). DNA fingerprinting confirmed that the HNSCC lines were individual isolates. Thirteen of 14 lines tested induced tumors in athymic mice. The histology of each line growing in nude mice was similar to that of the original tumor tissue. Immunocytochemistry showed keratin production in all lines tested. Aneuploidy (36 to 87 chromosomes) was present in all 16 lines studied; the median chromosome number for lines derived from primary tumors was 70, whereas for lines originating from metastatic or recurrent tumors, it was 54. Karyotypic analysis showed deletion of the short arm of chromosome 3 (3p-) in 12 of 16 cell lines and trisomy 6 in 12 of 16 lines. In addition, translocations between chromosomes 9 and 11 or 9 and 12 were each present in five of 16 lines tested. The HNSCC lines were resistant to lysis by natural killer cells, but were efficiently lysed by lymphokine-activated killer cells in 4-h 51Cr release assays. These new lines have allowed us to establish a model of local adoptive immunotherapy of HNSCC in tumor-bearing nude mice, and they provide a resource for future studies of the biology of HNSCC.
For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.
The in vitro chemosensitivity of 11 human colorectal cell lines to seven chemotherapeutic agents was determined using a semiautomated tetrazolium-based colorimetric assay (MTT assay). Four of the cell lines were from primary tumors and seven from metastases. Eight lines were from patients with no prior chemotherapy. From assay results, we predict 5-fluorouracil (5-FU) to be the sole active agent of the seven tested. This is based on two observations: the range of drug concentrations which produced 50% inhibition of cell growth was greatest with 5-FU (388-fold versus 5- to 30-fold with the other six agents); and the area under the curve (AUC) which produced 50% growth inhibition was within a clinically achievable range only for 5-FU. Since the assay AUC of 5-FU at 50% inhibition was in a clinically achievable range for only two of the 11 cell lines, we performed a multivariate analysis to explore parameters which predict 5-FU sensitivity. In the best fitting model, sensitivity was positively correlated with cloning efficiency in media and with cell surface TAG-72 (a tumor-associated glycoprotein found on epithelial tumors of ovary, lung, colon, and breast origin) expression. If validated with an in vivo test such as the nude mouse model, the MTT assay could be very useful in new drug screening for colorectal carcinoma, for examining combination chemotherapy for synergy, for exploring strategies for biochemical modulation, and perhaps in individualizing therapy when cell lines can be established from a patient.
Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.
A sensitive enzyme-release assay for natural cytotoxicity is described. The kinetic determination of the amount of the enzyme lactate dehydrogenase (LDH) released from lysed target cells was determined to provide a sensitive and precise measure of natural cytotoxicity when used in conjunction with appropriate controls and calculational methods. Values for the percentage of cytotoxicity or kinetic parameters determined by this method were identical, within experimental error, to values determined in parallel 51Cr release assays. Moreover, it was found that the spontaneous release of LDH from the target cells tested was considerably lower than the spontaneous release of 51Cr. This enzyme-release cytotoxicity assay is convenient, inexpensive, and precise, and should be applicable to the study of other cytotoxicity reactions, including antibody-dependent and T-cell mediated reactions.
We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.
The significance, specificity and high sensitivity of a new method to determine the natural killer cell cytolysis of europium diethylenetriaminepentaacetate (EuDTPA)-labelled target cells has been confirmed. The targets used in this release assay were the NK sensitive cell line K-562 and the resistant cell line Raji. The released EuDTPA was detected by a method based on time-resolved fluorometry. The specific EuDTPA release was higher than specific 51chromium (51Cr) release. Competitive assays, where half of the target cells were labelled with EuDTPA and the other half with 51Cr and the use of double labelled target cells showed that the results were identical with those of single labelled cells. The reliability of the EuDTPA release assay was further confirmed by performing experiments using NK cells from a patient whose complete lack of NK activity had earlier been demonstrated with the 51Cr release assay. Furthermore, our studies show that the amount of incorporated EuDTPA was directly proportional to the concentration of marker used. Due to the proportional incorporation of EuDTPA the labelling conditions can be chosen to obtain a sensitivity which allows even single cells to be detected.
Several different techniques are used to assess the expression of cellular immunity (reviewed in Ref. 1). In this article Benjamin Bonavida and Thomas Bradley discuss ways of measuring one aspect of cellular immunity-the activity of cytotoxic cells on target cells.
Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.
A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.
Recent evidence indicates that both natural killer and killer cells active in antibody-dependent cell-mediated cytotoxicity are cell types that bear Fc receptors. Natural killer and antibody-dependent cell-mediated cytotoxicity have also been shown to be in a SRBC-rosetting subpopulation. Human mononuclear peripheral blood leukocytes were cultured under various conditions to examine natural killing and antibody-dependent cell-mediated cytotoxicity. It was of interest to determine if these activities would be maintained or augmented under various culture conditions. Before culture, normal mononuclear peripheral blood leukocytes were tested in a 4-hr 51Cr release assay against K-562 to measure natural killer activity, and against antibody-coated Chang liver cells to measure antibody-dependent cell-mediated cytotoxicity. Fc receptor-bearing cells were removed by adsorption to immobilized antigen-antibody complexes. When lymphocytes were cultured alone, we often observed what we call cytotoxicity from cultured cells (CCC). The amount of cytotoxicity against K-562 and in antibody-dependent cell-mediated cytotoxicity was dependent on the presence of FBS and proportional to its concentration in the cultures. The presence of FCS resulted in high levels of CCC whereas human serum demonstrated low or negligible activities. This CCC was not directed against FBS-associated antigens. These CCC cannot be called NK since their extensive characteristics are not known. As was characteristic on day 0 for natural killing and antibody-dependent cell-mediated cytotoxicity, the cultured effector cells also contained an Fc receptor.
Therefore, the presence of CCC with features of natural killer and killer cell activities and its regeneration in culture indicate that it must be distinguished from specific tumor reactivity or alloreactivity in mixed lymphocyte and mixed lymphocyte-tumor reactions, particularly when tumor cell lines are used as targets.
Recent advances in understanding of cytokine function have indicated the need for more stringent cytokine assays, and have at the same time provided the information and tools necessary for establishing monospecific assays for a number of T cell-derived cytokines. Monoclonal antibodies are invaluable for improving the specificity of bioassays, both by removing unwanted activities, and in the most stringent assays, for confirming the identity of the cytokine detected. Several cytokines can also be directly measured by ELISA techniques using monoclonal antibodies.
We have developed an in vitro assay for Natural Killer (NK) cell cytolysis of and binding to substrate attached human glioma and fetal brain cells. The monolayer cells were labeled with [51Cr] and the effectors were directly sedimented onto these substrate attached target cells. Using this method we screened several glioma and fetal brain cell lines. The results indicate that the majority of gliomas are NK resistant, however two of the tested lines (U251MG and BN3) were relatively sensitive as were the fetal brain cell lines (CHI and CHII). We conclude that this monolayer assay for NK cytotoxicity and binding of glioma targets is a reproducible and valid method for assessing NK sensitivity, and should have applications in the study of other cultured solid tumors and substrate attached cells.
Early events of cellular infiltration during allograft rejection involve interactions between alloreactive lymphocytes and vascular endothelium. These interactions have been studied in an in vitro model of lymphocyte adherence to human arterial endothelial cell (HAEC) monolayers. Alloreactive lymphocytes primed against donor HLA antigens (DPL) or unrelated HLA antigens from a third party (TPL) were studied for their adherence to HAEC derived from organ transplant donors. DPL exhibited a degree of adherence to donor HAEC which was nearly twice that of the TPL population and developed a morphologically blastoid appearance. DPL adherence was dependent on the recognition of donor HLA antigens expressed on the HAEC surface and investigation of DPL and TPL adherence at various lymphocyte concentrations showed that the binding of DPL and TPL to HAEC was saturable. Monoclonal antibody (MoAb) directed against HLA class I antigens showed inhibition of only DPL adherence to HAEC monolayers expressing donor class I HLA antigens, whereas MoAb directed against other HAEC surface antigens failed to inhibit either the DPL or TPL populations. The results of this study suggest that in a transplant situation, lymphocyte activation and adherence to the graft endothelium is increased in the context of allorecognition.
IFN-gamma has been shown to reduce the sensitivity of tumor cells to lysis by NK cells. The close relationship between NK cells and lymphokine-activated killer (LAK) cells has prompted us to investigate whether IFN-gamma pre-treatment also affects the sensitivity of tumor cells to lysis by LAK. We have shown previously that IFN-gamma can induce a significant reduction in the sensitivity of both cultured and fresh (surgically obtained) human tumor cells to lysis by LAK. Herein we show that changes in the sensitivity to LAK lysis of cultured human tumor cells can be induced by as little as 1 to 10 U/ml of IFN-gamma; a dose well within the range that can be achieved in vivo. Protection is induced within hours after treatment with IFN-gamma and is dependent on the continued presence of IFN-gamma. Tumor cells cultured in IFN-gamma for several days remain less sensitive to lysis and do not become refractory to IFN-gamma-mediated protection. In the absence of IFN-gamma, treated tumor cells regain "normal" sensitivity to lysis within 48 to 72 h. We have also investigated the mechanisms by which IFN-gamma reduces tumor cell sensitivity to LAK lysis using cold target competition, monolayer depletion, direct binding, and kinetic assays. IFN-gamma pre-treatment does not alter the kinetics of tumor cell lysis by LAK. Our data are most compatible with a model in which IFN-gamma reduces the ability of a subpopulation of tumor cells to induce the LAK effector cell to initiate lysis. These results are closely parallel to observations made on the IFN-mediated protection of targets from NK lysis and support the notion that NK- and LAK-mediated lysis are closely related. These results may have significance in vivo because high levels of IFN-gamma may be present at the tumor site or may be induced after therapeutic immunomodulation.
We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.
An in vitro tetrazolium dye (MTT) reduction technique was modified and evaluated for use in the large-scale screening of anticancer compounds by examining the activity of ten clinically used drugs against 16 different human and murine cell populations. Cell populations included colon and mammary adenocarcinomas, melanomas, leukemias, and freshly isolated normal cells. Cell lines were grown in microtiter plates for 18-20 hours prior to a 72-hour continuous exposure to the drugs. Cultures were initiated at cell densities which maximized both the difference in dye reduction and the number of cell doublings between the beginning and end of the drug exposure period. Drug potency, expressed as the 50% inhibitory concentration (IC50), was comparable whether the effect on cell doublings or dye reduction was determined. There was good agreement between this method and the more labor-intensive, conventional method of counting trypan blue dye-excluding cells in a hemacytometer. Implemented as a large-scale, high-capacity system, our adaptation of the MTT technique is a rapid, sensitive, reproducible first-line screening device for detecting anticancer compounds with cytostatic or cytocidal activity.
After cellular immunoassays are compared with classical bioassays, conventional methods and consequent problems of data analysis for cytolysis assays are reviewed and a new solution is proposed. This solution incorporates new methods, called dose-response surface assays and analysis (DRSA), which estimate cytolytic activity coefficients on a surface in a three-dimensional space with two dose variables (killers and targets) and one response variable (counts). These new methods based on dose-response surfaces are demonstrated to be more informative and reliable than classical methods based on dose-response curves. In a test of the methods' robustness (sensitivity of parameter estimates to changes in the dose levels of the assay design), cytolytic activity coefficients estimated by DRSA varied by less than or equal to 30% over a reduction of three to four orders of magnitude in the dose levels. This remarkable robustness should be compared with the corresponding figures of as much as 500% over less than 1 order of magnitude for previously published results of coefficients estimated by conventional methods. DRSA is distinguished from replot-of-plots methods such as those used for enzyme inhibition assays in biochemistry, and is recommended as a more efficient method that should replace replot-of-plot methods now antiquated by the advent of microcomputers. DRSA can be applied to any experimental system that requires an activity coefficient to be estimated on a dose-response surface in a space of greater than or equal to 3 dimensions (greater than or equal to 2 dose variables and one response variable), regardless of the mathematical model and statistical estimators used to analyze the dose-response interaction. Finally, DRSA is compared with the methods known as response surface methodology (RSM), and is described as a new class of methods to be added to those that constitute RSM.
51Cr release assay showed that peripheral blood lymphocytes from all of 21 normal individuals had cell mediated cytotoxic reactivity against several human lymphoid tissue culture cell lines. Lysis by the normal donor lymphocytes of the various cell lines varied markedly among lines; lysis of a given cell line by an individual's lymphocytes also varied at different times. For example, human lymphoid cell lines, including F-230, F-265, and Raji, were destroyed by lymphocytes of all individuals tested. Other lines including SS-5 and NC-37 were lysed by lymphocytes from most, but not all, normal donors. Finally, cell lines SS-4 and Stuck were not destroyed by lymphocytes from any normal donors tested, or their antigenicity fluctuated from positive to negative at different times. Lymphocytes of 9 of 13 individuals had significant cytotoxic reactivity against established autologous lymphoid cell lines. Lysis by lymphocytes from immunodeficient patients and patients receiving immunosuppressive drugs was less than that by lymphocytes from normal individuals. Agents such as X irradiation, actinomycin D, 3',5' cyclic AMP, and concanavalin A completely or partially blocked the cytolytic activity of lymphocytes against F-265 cells. Target cell inhibition tests revealed that addition of graded doses of unlabeled F-265 cells always inhibited the release of 51Cr from labeled F-265 cells. Similarly, cytotoxicity of F-265 was inhibited by some other cultured lymphoid cells, including NC-37, but was not inhibited by at least one other cultured lymphoid cell line (Stuck). Normal human lymphocytes stimulated in vitro by phytohemagglutinin (PHA) did not inhibit lysis of F-265, nor were they directly lysed by lymphocytes of normal individuals. F-265 cells grown in media containing human serum instead of fetal bovine serum (FBS) were effective targets in the assay. The results suggested that true cell mediated immunologic reactions caused the lysis of human tissue cultured lymphoid cells by normal donor lymphocytes. The relevant antigen(s) appeared to be neither associated with or possessed by PHA stimulated lymphocytes nor related to antigens of FBS, but may be antigen(s) expressed by lymphocytes in prolonged tissue culture.
The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of 51Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.
An improved colorimetric assay for estimation of cytotoxic T (Tc) cells is described. The method involves staining thioglycollate-induced macrophage targets with the dye neutral red prior to addition of cytotoxic T cells and estimating macrophage survival at the end of the assay by measuring dye remaining in viable targets. The method using macrophage targets is more sensitive than the 51Cr release assay employing macrophages or a variety of other targets. It may be used to detect alloreactive and H-2 restricted Tc cells in both short-term (4 h) and long-term (24 h) assays and overcomes some variability encountered with a previously described colorimetric procedure. Furthermore, the method is cheap, fast, reliable and avoids the use of radioactivity.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
A rapid and simple technique for the isolation of viable tumor cells from human and mouse solid neoplasms is described. It consists of a 5 to 10-min treatment with trypsin-collagenase-DNase mixture, followed by mechanical disaggregation of the tumor tissue and subsequently by a brief centrifugation on a discontinuous Percoll gradient. With the tumors employed, this procedure usually requires less than 1 hr and results in preparations comprising greater than 80% tumor cells with viability of 80-90%. Cell-mediated cytotoxic response was measured with: (a) unsensitized lymphocytes freshly obtained from tumor-bearing hosts; (b) lymphocytes propagated in culture with T cell growth factor; and (c) lymphocytes stimulated in cocultures with autologous or syngeneic tumor cells. The cytotoxic activity was assessed in a modified [51Cr]-release assay adapted for solid tumor cells, allowing a long incubation period (24 hr) and the use of a low number (200-1000) of highly labeled target cells (2-10 counts/min/cell).
On analysis of in vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations-the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant "dose-response" curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.
Testing of LAK cell activity using a tetrazolium-based colorimetrie (MTT) assay
- J G Park
- O J Kwon
- S W Kim
- D S Choi
- J P Kim
Park, J. G., Kwon, O. J., Kim. S. W.. Choi, D. S., and Kim, J. P. Testing of LAK cell activity using a tetrazolium-based colorimetrie (MTT) assay. J. Korean Cancer Assoc., 19: 68-78, 1987.
cancerres.aacrjournals.org Downloaded from MIT COLORIMETRV COMPARED WITH "CR RELEASE target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence
- American Association For Cancer Research
- Copyright
American Association for Cancer Research
Copyright © 1990
on July 10, 2011
cancerres.aacrjournals.org
Downloaded from
MIT COLORIMETRV COMPARED WITH "CR RELEASE
target cells in an assay of natural killer cell activity. I. A novel non-radioactive
method based on time-resolved fluorescence. J. Immunol. Methods. 86: 225-
229. 1986.
An enzyme-release assay for T cell cytotoxicity
- Callewaert C Korzeniewski
Korzeniewski. C., and Callewaert, D. M. An enzyme-release assay for T cell
cytotoxicity. J. Immunol. Methods, 64: 313-320. 1983.