Tetrazolium Bromide Test Research Articles

Inhibitory effects of bark extracts from Ulmus laevis on endometrial carcinoma: an in-vitro study.

Anti-inflammatory effects of elm tree have been shown in several studies. Besides this, protective effects of components of elm bark on damaged tissue have also been described. This study was carried out to investigate the antitumour potential of an ethanolic extract isolated from Ulmus laevis in the hormone-dependent endometrial carcinoma cell line RL95-2. A range of 2.5-500 microg/ml of elm bark extract was used as standard concentrations. The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionization mass spectrometry. The influence of the bark extracts was determined on cell vitality [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test], cell proliferation (5-bromo-2-deoxyuridine test) and cytotoxicity (lactate dehydrogenase test) in the human endometrial carcinoma cell line RL 95-2. By pyrolysis-field ionization mass spectrometry, the main substance classes of the extract as a composition of sterols/triterpenes, free fatty acids and a group of phenols, lignin monomers and flavonoids was identified. Our study showed a significant inhibition of cell vitality and proliferation measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test up to 5 microg/ml extract and up to 100 microg/ml according to the 5-bromo-2-deoxyuridine test. Concentrations of 500 microg/ml induced a significant inhibition of cell vitality up to 80% and cell proliferation up to 81.5%. A significant cytotoxity was not observed. The results lead to the assumption that the bark extract from Ulmus laevis has antiproliferation and anticancer potential in hormone-dependent endometrial carcinoma cells.

Effect of Dy3+on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement, mineralized function, Oil Red O stain and measurement were employed to assess the effect of Dy3+ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts (OBs). The results showed that Dy3+ had no effect on BMSC proliferation at concentrations of 1×10−8 and 1×10−5 mol/L, but inhibited BMSC proliferation at other concentrations. Dy3+ had no effect on OB proliferation at concentrations of 1×10−10 and 1×10−9 mol/L, but inhibited OB proliferation at other concentrations. Dy3+ had no effect on the osteogenic differentiation of BMSCs at concentrations of 1×10−9 and 1×10−7 mol/L, and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day. The osteogenic differentiation of BMSCs was inhibited by Dy3+ at concentration of 1×10−5 mol/L at the 14th day, but promoted osteogenic differentiation of BMSCs at concentrations of 1×10−9, 1×10−8, 1×10−7 and 1×10−6 mol/L with the maximal effect at concentration of 10−6 mol/L. Dy3+ promoted mineralized function of BMSCs at any concentration. Dy3+ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10−7 mol/L, but inhibited adipogenic differentiation of BMSCs at other concentrations. Dy3+ inhibited adipocytic trans-differentiation of OBs at any concentration, suggesting that Dy3+ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of OBs which may promote differentiation and mineralization of OBs. These results may be valuable for better understanding the mechanism of the effect of Dy3+ on pathogenesis of osteoporosis.

AC Magnetic-Field-Induced Heating and Physical Properties of Ferrite Nanoparticles for a Hyperthermia Agent in Medicine

AC magnetic-field-induced heating, cytotoxicity, and bio-related physical properties of two kinds of spinel ferrite nanoparticles, soft (NiFe2O4) and hard (CoFe2O4), with different mean particle sizes were investigated in this paper to confirm the effectiveness for an in vivo magnetic nanoparticle hyperthermia agent in biomedicine. AC magnetically induced heating temperature of the nanoparticles measured both in a solid and an agar state at different applied magnetic fields and frequencies clarified that the maximum heating temperature of NiFe2O4 nanoparticles is much higher than that of CoFe2O4 nanoparticles. In addition, it was demonstrated that solid-state NiFe2O4 nanoparticles with 24.8 and 35 nm mean particle size exhibited a promisingly high heating temperature (21.5degC-45degC) for a hyperthermia agent in the physiologically tolerable range of the ac magnetic field with less than 50 kHz of applied frequency. According to the magnetic and physical analysis results, the superior ac magnetically induced heating performance of NiFe2O4 nanoparticles was primarily due to their higher magnetic susceptibility (permeability) that directly induces a larger magnetic minor hysteresis loop area at the low magnetic field. Cytotoxicity test results, quantitatively estimated by methylthiazol tetrazolium bromide test method, verified that uncoated NiFe2O4, chitosan-coated NiFe2O4, and CoFe2O4 showed a noncytotoxicity, which is clinically suitable for a hyperthermia agent application.

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10 −9 mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10 −11 and 10 −10 mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.

Effects of small interfering RNA magnetic nanoparticles combination with external magnetic fields on survivin gene expression of bladder cancer cells and apoptosis

To investigate the effects of the small interfering RNA plasmid-dextran magnetic nanoparticles (siRNA-DMN) combination with external magnetic fields on silencing survivin gene expression of bladder cancer cells and apoptosis when DMN used as gene carrier to transfer siRNA-survivin recombinant plasmid in vivo. The siRNA-survivin recombinant plasmid specific targeted survivin was synthesized in previous experiment. DMN were prepared by chemical coprecipitation method and used as gene carrier. The siRNA-DMN were constructed by static electricity of polylysine and transferred into human bladder cancer BIU-87 cells with the help of external magnetic fields. The growth inhibiting rate (IR) of BIU-87 cells was observed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide's test and the apoptosis index (AI) was detected by transferase-mediated dUTP nick end labeling method. The relatively transcription levels of survivin mRNA and protein expression were respectively detected by semi-quantitive reverse transcription polymerase chain reaction and Western Blotting techniques. The diameter, effective diameter and saturation magnetization of DMN-siRNA were about 10 - 12 nm, 94.8 nm and 0.19 emu/g, respectively. The IR (39.60%) and AI (28.72%), the relative expression of survivin mRNA and protein of siRNA-DMN combination with external magnetic fields on BIU-87 cells were significantly higher and lower than those in the control group and single siRNA-DMN group, respectively (P < 0.05). The siRNA-survivin plasmid-DMN combination with external magnetic fields could effectively inhibit survivin expression and induce BIU-87 cells apoptosis which provided experimental basis for the magnetic targeting gene therapy of bladder tumor.

Continuous cytokine exposure of colonic epithelial cells induces DNA damage

Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducible nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells. A colonic cell line (HT29) was stimulated for 1-10 weeks with interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) or both and compared with unstimulated cells or cells stimulated for 48 h. Cells were co-incubated with a selective iNOS inhibitor (N-monomethyl-L-arginine (L-NMMA)) in some experiments. Viability was assessed by the dimethylthiazol diphenyl tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis. Continuously stimulated colonic cells had increased ROS production, especially those stimulated with TNF-alpha or IFN-gamma/TNF-alpha (P<0.001). The ROS production could be inhibited by L-NMMA co-incubation, indicating that iNOS is responsible for the up-regulation (P<0.05). Continuously stimulated cells had increased DNA instability (P<0.002), whereas short-term stimulated cells did not. The DNA instability was inhibited by L-NMMA co-incubation (P<0.05). Continuous cytokine exposure induces an iNOS dependent up-regulation of ROS production and DNA instability. This mechanism could be involved in carcinogenesis in chronic inflammatory diseases of the intestinal tract.

Photobiological and Ultrastructural Studies of Nanoparticles of Poly(lactic-co-glycolic acid)-Containing Bacteriochlorophyll-a as a Photosensitizer Useful for PDT Treatment

The interaction of polymeric nanoparticles formulated from the biodegradable polymer poly(DL-lactide-co-glycolide) loaded with bacteriochlorophyll-a was studied in homogeneous solution and in vitro in the presence of a macrophage cell line (P388-D1-ATCC). Photodynamic therapy (PDT) activity after different laser doses also was investigated. Scanning electron microscopy analysis of cell phagocyte nanoparticles showed that after 30 min of incubation most of the nanoparticles are in a clear adhesion process to the cell surface. The majority of nanoparticles became phagocytic after 2 hr of incubation time. After laser irradiation of the dye-containing system a total photodamage by nanoparticle phagocyte cells was observed and the cell survival was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Our results indicate that polymeric nanoparticles work as an efficient drug delivery system for PDT drugs. This approach can be widely used for many other hydrophobic photosensitizers with higher aggregation tendency in neoplastic cell treatment.

Presence of CCK-A, B receptors and effect of gastrin and cholecystokinin on growth of pancreatobiliary cancer cell lines

To investigate the effects of gastrin and cholecystokinin (CCK) and their specific antagonists on the growth of pancreatic and biliary tract cancer cell lines. Five pancreatic and 6 biliary cancer cell lines with 2 conrtol cells were used in this study. Cell proliferation study was done using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test and direct cell count method. Reverse transcription-polymerase chain reaction (RT-PCR) and slot blot hybridization were performed to examine and quantify the expression of hormonal receptors in these cell lines. SNU-308 showed a growth stimulating effect by gastrin-17, as did SNU-478 by both gastrin-17 and CCK-8. The trophic effect of these two hormones was completely blocked by specific antagonists (L-365, 260 for gastrin and L-364, 718 for CCK). Other cell lines did not respond to gastrin or CCK. In RT-PCR, the presence of CCK-A receptor and CCK-B/gastrin receptor mRNA was detected in all biliary and pancreatic cancer cell lines. In slot blot hybridization, compared to the cell lines which did not respond to hormones, those that responded to hormones showed high expression of receptor mRNA. Gastrin and CCK exert a trophic action on some of the biliary tract cancers.

CYTOTOXICITY INDUCED BY NIVALENOL, DEOXYNIVALENOL, AND FUMONISIN B1 IN THE SF-9 INSECT CELL LINE

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.

Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines.

Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration-dependent manner that was already evident 24 h after CBD exposure, with an apparent IC(50) of 25 microM. The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; SR2) and alpha-tocopherol. By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation, or pertussis toxin did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists. Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion, the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent.

Characterization of heme oxygenase 1 (heat shock protein 32) induction by atrial natriuretic peptide in human endothelial cells.

Atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory and cytoprotective potential. The aim of this study was to characterize induction of heme oxygenase (HO)-1 by ANP in human umbilical vein endothelial cells (HUVEC). HUVEC were treated with ANP, 8-bromo-cyclic GMP (cGMP), or cANF in the presence or absence of various inhibitors. HO-1 was determined by Western blot and RT-PCR, c-jun N-terminal kinase (JNK) and ERK by the use of phospho-specific antibodies. Activator protein (AP)-1 activation was assessed by gelshift assay. Reporter gene assays were performed using native or mutated AP-1 binding sites of the HO-1 promoter. TNF-alpha-induced cell death was investigated by Hoechst staining, fluorescence-activated cell sorting analysis, caspase-3-measurement, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. ANP (10(-9)-10(-6) mol/liter) induced the expression of HO-1 protein and mRNA. Induction was mediated via the guanylate-cyclase-coupled receptor because 8-Br-cGMP mimicked the effect of ANP, whereas the clearance receptor agonist cANF did not induce HO-1. Endogenously produced cGMP also induced HO-1 because phosphodiesterase inhibition markedly elevated HO-1. The lack of effect of the cGMP-dependent protein kinase inhibitor 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-pCT-cGMPS) suggested no involvement for this cGMP effector pathway in the signal transduction. ANP lead to activation of the transcription factor AP-1, and subsequently of JNK, as well as of ERK. Cotreatment of the cells with U0126 or SP600125, as well as reporter gene assays revealed the involvement of AP-1/JNK activation in HO-1 induction. Abrogation of HO-1 induction by PD-98059 showed also a role for ERK. Treatment of HUVEC with ANP did not protect from TNF-alpha-induced apoptosis. This work characterizes the induction of HO-1 by ANP in HUVEC, which is shown to be mediated via JNK/AP-1 and ERK pathways. ANP-induced HO-1 does not confer protection against TNF-alpha-induced apoptosis.

Edaravone, a radical scavenger, may enhance or produce antiproliferative effects of fluvastatin, amlodipine, ozagrel, GF109203X and Y27632 on cultured basilar artery smooth muscle cells.

Proliferation of vascular smooth muscle cells stimulated by reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. To clarify mechanisms by which ROS promote vascular atherogenesis, effects of fluvastatin, amlodipine, ozagrel (thromboxane synthetase inhibitor), GF109203X (a protein kinase C inhibitor) and Y27632 (a ROCK inhibitor) on the proliferation of guinea-pig basilar artery smooth muscle cells (GBa-SM3) in a 5% FBS culture medium were studied over 3 d in the presence or absence of a free radical scavenger, edaravone. Viability of cells at the end of incubation was measured by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. Results demonstrated that fluvastatin and amlodipine by themselves possess antiproliferative effects on the GBa-SM3 cells at 10-100 microM and 0.1-1 microM, respectively. While edaravone possessed no antiproliferative effect by itself at 100 microM, it significantly (p<0.05) augmented the antiproliferative effects of fluvastatin and amlodipine. In addition, ozagrel, GF109203X and Y27632 possessed no appreciable effects on the cell growth by themselves. However, coincubation of edaravone at 100 microM with these agents elicited significant antiproliferative effects for ozagrel, GF109203X and Y27632 at 10-100 microM, 1-10 microM and 0.1-1 microM, respectively. In conclusion, edaravone may have clinically beneficial interactions with fluvastatin, amlodipine and ozagrel regarding the prevention of vascular atherosclerosis. The interactions between edaravone and the inhibitors of protein kinase C and ROCK were suggestive of possible contributions of ROS-triggered intracellular signals associated with these enzymes to vascular atherogenesis, but further studies are required for confirmation.

Inhibition of LPS-induced nitric oxide and TNF-alpha production by alpha-lipoic acid in rat Kupffer cells and in RAW 264.7 murine macrophages.

The activation of Kupffer cells represents a central mechanism of inflammatory liver injury involving the production of two important inflammatory mediators, nitric oxide and TNF-alpha. The aim of this study was to investigate the effect of the hepatoprotective compound alpha-lipoic acid (thioctic acid) on the production of nitric oxide and TNF-alpha in isolated rat Kupffer cells and RAW 264.7 macrophages. Isolated rat Kupffer cells or RAW 264.7 were either untreated, treated with alpha-lipoic acid (500 micro g/mL), or activated with 1 micro g/mL of lipopolysaccharide in the presence or absence of alpha-lipoic acid (0.2-500 micro g/mL). After 20 h the accumulation of nitrite was measured by the Griess assay. Tumour necrosis factor-alpha secretion was quantified after 4 h by L929 bioassay. Cell viability was determined by mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) DNA binding activity by gelshift assays. Treatment of Kupffer cells and RAW 264.7 with alpha-lipoic acid alone had no effect on basal nitric oxide production. However, alpha-lipoic acid significantly inhibited lipopolysaccharide-induced nitrite accumulation. alpha-Lipoic acid did not alter basal TNF-alpha secretion in Kupffer cells, whereas it significantly inhibited lipopolysaccharide-induced TNF-alpha production. alpha-Lipoic acid attenuated the activation of nuclear factor-kappaB and AP-1, two transcription factors pivotal in induction of inducible nitric oxide synthase and TNF-alpha. alpha-Lipoic acid significantly inhibits lipopolysaccharide-induced macrophage production of nitric oxide and TNF-alpha via an attenuated activation of NF-kappaB and activator protein-1. The reduced production of nitric oxide and TNF-alpha in Kupffer cells may be involved in the hepatoprotective action conveyed by alpha-lipoic acid.

Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 ("Iressa").

ZD1839 (‘Iressa’) is an orally active, selective epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR–TKI), which blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. Permanent downstream activation of the mitogen-activated protein kinase pathway can theoretically bypass the upstream block of epidermal growth factor receptor-dependent mitogen-activated protein kinase activation at the epidermal growth factor receptor level. We investigated the impact of epidermal growth factor receptor content, p53 status and mitogen-activated protein kinase signalling status on ZD1839 sensitivity in a panel of human tumour cell lines: seven head and neck cancer cell lines and two colon cancer cell lines (LoVo, HT29) with derivatives differing only by a specific modification in p53 status (LoVo p53 wt + p53 mut cells, HT29 p53 mut + p53 wt rescued cells). The antiproliferative activity of ZD1839 was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. ZD1839 concentrations ranged from 0.2–200 μM (48 h exposure). Epidermal growth factor receptor expression, p53 status and p42/p44 (for testing a constitutively active mitogen-activated protein kinase pathway status) were determined by competition analysis (Scatchard plots), denaturing gradient cell electrophoresis and Western blot, respectively. Epidermal growth factor receptor levels ranged from 388 to 33794 fmol mg−1 protein, a range that is similar to that found in head and neck tumours. The IC50 values for cell sensitivity to ZD1839 ranged from 6 to 31 μM and a significant inverse correlation (P=0.022, r=0.82) between IC50 values and epidermal growth factor receptor levels was observed. There was no influence of p53 status on the sensitivity to ZD1839. In two head and neck cancer cell lines with comparably elevated epidermal growth factor receptor expression, a two-fold higher ZD1839 IC50 value was found for the one with a constitutively active mitogen-activated protein kinase. In conclusion, ZD1839 was active against cells with a range of epidermal growth factor receptor levels, although more so in cells with higher epidermal growth factor receptor expression. Activity was unaffected by p53 status, but was reduced in cells strongly dependent on epidermal growth factor receptor signalling in the presence of an intrinsically activated mitogen-activated protein kinase pathway.British Journal of Cancer (2002) 86, 1518–1523. DOI: 10.1038/sj/bjc/6600299 www.bjcancer.com© 2002 Cancer Research UK

Chemical and biological characteristics of low-temperature plasma treated ultra-high molecular weight polyethylene for biomedical applications.

Several low-temperature radio-frequency (RF) plasma surface treatments were performed on ultra-high molecular weight polyethylene (UHMWPE) used in biomedical applications. Process gases included Ar, C3F6, CH4, hexamethyldisiloxane (HMDSO), and NH4. These treatments were carried out at pressures in the range of 64-400 mTorr, RF powers of 240-1200 W, and temperatures well below the melting point of UHMWPE. X-ray photoelectron spectroscopy (XPS) was used to obtain information about the surface characteristics of UHMWPE treated with the HMDSO, C3F6, and CH4 gases as a function of treatment conditions. XPS spectra of UHMWPE treated with C3F6 and CH4 and exposed to a laboratory environment for different time periods were examined in order to assess the stability of these treatments. It was found that for the C3F6 process gas the amount of fluorine at the surface decreased over time, whereas the oxygen content of the CH4 treated samples increased as a function of time. In vitro cytotoxicity of Ar, C3F6, CH4, and NH4 plasma treated samples was studied in light of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test results. The hemolytic nature of the various plasma treatments was evaluated using standard hemolysis tests. All of the samples tested in this study exhibited no cytotoxic and negligible hemolytic effects. The process parameters for several low-temperature plasma treatments demonstrating chemical and structural stability and good biocompatibility are discussed in conjunction with the broad applicability to other biomedical polymers.

Functional assessment in vitro of human-complement-dependent antibody-induced cytotoxicity of neoplastic cells.

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.

Effects of cerivastatin on human arterial smooth muscle cell proliferation and migration in transfilter cocultures.

Statins competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity reducing mevalonate synthesis. In this study, antiproliferative and antimigratory effects of the new compound cerivastatin were analyzed and compared with classic statins of the first and second generation using mono- and cocultures of human arterial smooth muscle (haSMC) and endothelial (haEC) cells. Effects on the mitotic index and mitochondrial activity of haEC and haSMC monocultures were tested using BrdU enzyme-linked immunosorbent assay (ELISA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) tests, respectively. In lactate dehydrogenase (LDH) assays, cytotoxicity of statins was studied. Transfilter cocultures were performed for 14 days to evaluate haSMC growth under the stimulatory effect of proliferating haEC, which release growth factors [e.g., platelet-derived growth factor (PDGF)]. The hydrophobic statins simvastatin, lovastatin, and atorvastatin significantly inhibited haSMC and haEC growth in monocultures at 0.5-50 microM. However, most potent effects were exerted by cerivastatin in 10- to 30-fold lower doses without any significant cytotoxicity. More important, cerivastatin showed also significant effects on haSMC proliferation and migration in transfilter cocultures at extremely low doses (IC50, 0.04-0.06 microM), even when applied exclusively to the endothelial side and in the presence of low-density lipoprotein (LDL). Addition of mevalonate abolished the effects of cerivastatin completely. Even in the presence of growth-stimulating haEC and LDL, cerivastatin was found to be the most potent inhibitor of haSMC proliferation and migration in doses that also can be reached in human serum after oral drug administration. The results support the concept that statins seems to influence additional cellular mechanisms beyond cholesterol reduction, which might also have a relevance for the prevention of restenosis.

In Vitro Cytotoxicity of Various Forms of Cobalt for Rat Alveolar Macrophages and Type II Pneumocytes

It has been shown that cobalt (Co) and the mixture cobalt–tungsten carbide (CoWC) are cytotoxic for alveolar macrophages (AM) and alveolar type II cells (AT-II), but the exact mechanisms of toxicity are not entirely elucidated. In this study, we exposed primary cultures of AT-II and AM, in vitro, to different forms of Co (Co particles, CoWC particles, CoCl2) and assessed cell damage using the dimethylthiazol diphenyl tetrazolium bromide test. In some experiments, inserts were used to separate the particles from the cells. The results show that AT-II are twice as sensitive to the effects of 100 μg Co particles/well (1.88 cm2) than AM. For this latter cell type, the presence of WC almost doubled (at 25 μg Co/well) the toxic effects compared to pure Co, but this synergy between Co and WC only occurred if the particles were in close contact with the cells. Lactalbumin and, to a lesser degree, EDTA were able to reduce the toxicity of Co, CoWC, and CoCl2 for AT-II and AM. CoCl2 showed a similar toxicity for AT-II and AM. The use of Co-conditioned medium revealed that Co particles are “aged” after having been incubated for 24 h in an aqueous medium and are then no longer able to cause the same degree of cell damage as fresh Co particles (71 versus 15% viability for 100 μg Co/well). The time course of the toxicity of the different forms of Co for AT-II and AM showed different patterns in causing cell damage, suggesting different action mechanisms. Evaluation of cell damage by electron microscopy was consistent with biochemical indices. Overall, our results indicate that the Co ion does play a role in the toxicity of both Co particles and CoWC particles.

Viability of oospores of Phytophthora infestans under field conditions in northeastern North America

Studies were conducted in potato fields on Prince Edward Island (PE) and New Brunswick (NB) to determine the overwintering viability, germination, and infectivity of oospores of Phytophthora infestans. Oospore viability was determined using plasmolysis (4 M NaCl) and tetrazolium bromide (MTT) tests. After seven months exposure to winter conditions in potato fields, the viability of oospores buried on PE and NB ranged from 2.5 to 13% and from 1 to 28%, respectively, with the plasmolysis test and from 13 to 44% and from 2.5 to 40%, respectively, with the MTT test. In other studies on PE, oospore viability ranged from 5 to 15% 12 months after burial. In laboratory studies, oospores exposed to temperatures of -20 and -50°C for up to 90 days had viabilities greater than 50%, whereas exposure to 0, 4, or 15°C resulted in average viabilities from 23 to 36%. The lowest viability, 22%, was recorded at the highest temperature tested, 36°C. Oospore germination was low, ranging from 6 to 19%. However, this level was sufficient to demonstrate that oospores could be a source of inoculum. A potato soil leachate medium was superior to others for stimulating oospore germination. Oospores of P. infestans present in potato stems and infested farm soil, maintained underground for seven months, were able to infect potato leaf discs in the laboratory. Although all studies were conducted in essentially gnotobiotic conditions, they demonstrate that oospores cannot only survive the winter conditions in northeastern North America but can also germinate and infect new potato tissue in the following growing season. While it would be useful to confirm these findings with new studies under native soil conditions, this is not practical at this time. The presence of oospores also poses a new disease risk that needs to be addressed in future late blight management strategies.

Embryotoxicity Screening Using Embryonic Stem Cells in vitro: Correlation to in vivo Teratogenicity

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analyzed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 cells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibition concentrations for differentiation (ID₅₀) and cytotoxicity (IC₅₀D3 and IC₅₀3T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg(IC₅₀D3), the lg(IC₅₀3T3) and the relative distance between IC₅₀3T3 and ID₅₀, that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development. Unlike the orginal PM, the improved PM incorporates as one variable the relative distance between IC₅₀3T3 and ID₅₀, instead of the ratio ID₅₀/IC₅₀D3 that was used previously.