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PubMed2005

[Identification of antiviral activity of Toddalia asiatica against influenza type A virus].

OBJECTIVE: To identify antiviral activity of Toddalia asiatica against influenza virus type A in vitro. METHOD: More than two hundred Chinese medicinal herb extracts were screened for antiviral activities against influenza A/PR/8/34 (H1N1) virus using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for virus induced cytopathic effect (CPE) in a primary screening. Positive samples were picked up and were subjected to quantitative real-time polymerase chain reaction (PCR) to quantify reduction of H1N1 virus genomic RNA. RESULT: Toddalia asiatica showed potent antiviral activities against H1N1 virus, with 50% effective concentration (EC50) value of 4.7 mg x L(-1) in MTS assay and 0.9 mg x L(-1) in quantitative PCR assay respectively. The cytotoxicity test of Toddalia asiatica generated a CC50 value of 187.2 mg x L(-1) and a selective index (SI) larger than 206 in quantitative PCR. Although the best antiviral activity of Toddalia asiatica was observed with co-treatment of influenza virus infection, it remained effective even when administrated 24 h before and after the initiation of infection. CONCLUSION: The results suggested that Toddalia asiatica compound extract could be a candidate for anti-H1N1 virus agent in the treatment of influenza.

Animals↗

PubMed1999

[In vitro evaluation of potentially effective gemcitabine combination therapy for exocrine pancreatic carcinoma].

The aim of the present study was to define potentially synergistic gemcitabine drug combinations for the treatment of pancreatic adenocarcinoma by using a tumour-specific in vitro screening system. The anticancer drug screening system used for these experiments consisted of four different established human pancreatic adenocarcinoma cell lines (BxPc-3, Panc-1, ASPC-1, Ca-pan-1) and the Microculture Tetrazolium (MTT) Assay for quantification of cytotoxic drug effects. To define single agent activities, dose-response curves, IC 50 values, and in order to validate the test system, in a first step gemcitabine and several conventional anticancer drugs including 5-FU, cisplatin, epirubicin, and mitomycin C were tested at 10 different concentrations ranging from 0.001 to 100 micrograms/ml. The effectiveness of various gemcitabine combinations was subsequently determined by using clinically relevant in vitro drug concentrations, and was rated as synergistic, additive or subadditive according to the criteria of Aapro. Overall, a heterogeneous chemosensitivity pattern was noted within the four tested cell lines. In agreement with the known chemotherapeutic refractoriness of pancreatic cancer, major cytotoxic effects were only seen with use of rather high drug concentrations. Investigation of various drug exposure times revealed a superior antiproliferative activity of gemcitabine and the other compounds in case of prolonged incubation. During subsequent drug combination experiments, gemcitabine + cisplatin and gemcitabine + epirubicin resulted in synergistic activity in 2/4 cell lines each. As opposed to the poor activity of single agents, a > 50% growth inhibition (in vitro response) was noted in 3 and 2 cell lines, respectively. Experimental data obtained with this pancreatic cancer specific in vitro screening system suggest that dose escalation or prolonged administration of gemcitabine, as well as the combination of this drug with cisplatin or epirubicin might result in improved therapeutic results. Encouraging preliminary results obtained in phase II studies seem to support the potential clinical relevance of the described disease-oriented screening system.

Adenocarcinoma↗

PubMed1986

Stimulation of astrocytes affects cytotoxic brain edema.

Cytotoxic brain edema has been produced in rats by subacute intoxication with triethyltin (TET). Some animals were allowed to recover spontaneously, others were post-treated with an extract of Ginkgo biloba (EGB) for 1 to 4 weeks, beginning 3 days after intoxication was stopped. The time course of the resolution of the edema was studied biochemically and morphologically by light microscopy, histochemistry and electron microscopy (EM). Morphometric evaluation showed that the spontaneous reabsorption of TET-induced edema was very slow: it was evident only 2 weeks after ending TET administration and it required more than 4 weeks to be completed. EGB therapy markedly decreased the vacuolation, as well as the abnormal levels of water and sodium contents, 1 week after beginning the treatment. Less influence of EGB was observed at the later stages. During spontaneous recovery, astroglial cells in the edematous white matter of TET-intoxicated animals showed short and swollen processes containing few organelles, low levels of NADH- and NADPH-tetrazolium reductase activities and glial fibrillary acidic protein (GFAP)-immunofluorescence for about 2 weeks. During EGB therapy the astrocytes regained their cellular processes, containing intense oxidative enzyme activities and GFAP-immunofluorescence as early as after 1 week of treatment. In the EM, astrocytes often appeared hypertrophic, surrounding myelin vacuoles and displaying phagocytosis of myelin debris. We conclude that EGB can accelerate the reabsorption of TET-induced cerebral edema and improve the astroglial reaction.

Animals↗

PubMed2001

Psychosine is as potent an inducer of cell death as C6-ceramide in cultured fibroblasts and in MOCH-1 cells.

Cytotoxic capacity of psychosine (galactosylsphingosine) was evaluated in comparison with C6-ceramide in cultured fibroblasts and the glia-derived MOCH-1 cells that have characteristics of myelinating cells (1). Psychosine caused cytotoxic cell death and DNA fragmentation at concentrations similar to C6-ceramide and MOCH-1 cells were substantially more sensitive to their cytotoxic effects than fibroblasts. In this system, pretreatment with GM1-ganglioside failed to protect the cells from the deleterious effects of these compounds. These findings are consistent with the hypothesis that psychosine is the cytotoxic metabolite that causes apoptotic death of the oligodendrocyte in globoid cell leukodystrophy (Krabbe disease). They further suggest that the protective capacity of GM1-ganglioside is unlikely to be the explanation for the paradoxical improvement of the phenotype of globoid cell leukodystrophy in the mouse simultaneously deficient in two lysosomal beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.

Animals↗

PubMed1997

Synergistic effects of curcumin on all-trans retinoic acid- and 1 alpha,25-dihydroxyvitamin D3-induced differentiation in human promyelocytic leukemia HL-60 cells.

Treatment of human promyelocytic leukemia HL-60 cells with 10 muM curcumin for 48 h inhibited cellular proliferation and induced small increases in differentiation (100-200%) as measured by the proportion of cells that reduced nitroblue tetrazolium (NBT) and expressed Mac-1. Synergistic induction of differentiation as measured by the above markers was observed when 1-10 muM curcumin was combined with 10-100 nM all-trans retinoic acid (RA) or with 100 nM 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3). Cell morphology and flow cytometric studies (with the monocytic surface antigen CD14) indicated that combinations of RA and curcumin stimulated differentiation predominantly to granulocytes whereas combinations of vitamin D3 and curcumin stimulated differentiation predominantly to monocytes. Studies on cell cycle kinetics indicated that treatment of HL-60 cells with a combination of RA and curcumin for 48 or 96 h reduced the proportion of cells in the S phase of the cell cycle and increased the proportion of cells in the G0/G1 phase of the cell cycle to a greater extent than occurred for cells treated with either compound alone. Combinations of vitamin D3 and curcumin did not alter cell cycle kinetics to a greater extent than was observed for either compound alone. Combinations of RA and curcumin or vitamin D3 and curcumin inhibited the proliferation of HL-60 cells to a greater extent than was observed for either compound alone. The results indicate that curcumin is a weak stimulator of differentiation in HL-60 cells and that is has synergistic effects when combined with RA or vitamin D3. Combinations of curcumin and RA have a particularly potent inhibitory effect on the proliferation of HL-60 cells.

Antigens, Differentiation, Myelomonocytic↗

PubMed1999

Inhibition of activated human mesangial cell proliferation by the natural product of Cordyceps sinensis (H1-A): an implication for treatment of IgA mesangial nephropathy.

Cordyceps sinensis (CS) is a parasitic fungus that has been used as a Chinese medicine for a long time in the treatment of nephritis. Today, the hypothesis about the pathogenesis of immunoglobulin A nephropathy (IgAN) is that nephritogenic IgA immune complexes (IgAIC) go to the kidney to stimulate resting mesangial cells to release cytokines and growth factors. These cytokines and growth factors cause mesangial cell proliferation and release matrix, chemical mediators that lead to the glomerular injury. However, nephritogenic IgAIC in humans is still unknown. To solve this problem previously, we established an in vitro model that showed that cultured human mesangial cells (HMC) stimulated with interleukin-1 (IL-1) plus IL-6 can cause mesangial cell proliferation, increasing production of chemical mediators and superoxide anion. An in vivo model also proved that this culture medium may lead to renal injury with hematuria and proteinuria. Therefore, to fractionate the crude components that can be used in the treatment of patients with IgAN, we cultured HMC, and then an HMC activating model with HMC incubated with IL-1 and IL-6 was established. We fractionated the crude methanolic extracts from fruiting bodies of CS with the use of this in vitro inhibition of HMC activation model as our assay method. In brief, the fruiting bodies were extracted by silica gel column chromatography. One out of 6 column fractions, F-2, significantly inhibited the HMC activation by IL-1 plus IL-6. The acute toxicity test with male Institute of Cancer Research mice showed no liver toxicity or mutagenicity. Then we established an IgAN animal model with R36A (Pneumococcal C-polysaccharide purified from Streptococcus pneumoniae) as antigen and anti-R36A IgA monoclonal antibody to form nephritogenic IgA-IC, which can induce hematuria and proteinuria in mice with IgA deposition in the mesangial area. The mice in the IgAN model fed with 1% F-2 in diet had significant reduction of hematuria and proteinuria together with histopathologic improvement. Therefore this fraction was then purified by silica gel column chromatography and high-performance liquid chromatography, which got a purified compound H1-A, which can suppress the activated HMC and alleviate IgAN (Berger's disease) with clinical and histologic improvement. These results give us a new regimen for the treatment of patients with IgAN in the future.

Adolescent↗

PubMed2006

DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl2.

Mercuric chloride (HgCl(2)) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl(2) on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 microM HgCl(2), either in the absence or in the presence of 60 microM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl(2) exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl(2) and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl(2).

Adenosine Triphosphate↗

PubMed1993

Reduction of myocardial infarct size by ramiprilat is independent of angiotensin II synthesis inhibition.

The angiotensin-converting enzyme inhibitor ramiprilat, the angiotensin II receptor antagonist losartan, angiotensin II, ramiprilat plus angiotensin II, or saline (N = 6 each group), were administered i.v. in anesthetized, open-chest rabbit preparations of acute myocardial ischemia. Animals were instrumented for measurement of systemic hemodynamics and left ventricular +dP/dtmax, then subjected to 30 min of left anterior descending coronary artery occlusion (marginal branch) followed by 2 h of reperfusion. Ramiprilat (50 micrograms/kg), losartan (10 mg/kg), or saline were administered prior to reperfusion, and angiotensin II (2.5 ng/kg per min) was infused 15 min prior to occlusion and throughout the remainder of the experiment. Losartan was supplemented (10 mg/kg) after 1 h of reperfusion. These non-hypotensive doses of ramiprilat and losartan were demonstrated to significantly antagonize the systemic pressor effects of i.v. challenge with angiotensin I (15% of control, maximum) and II (5% of control, maximum), respectively, for the duration of the experiment. Systemic hemodynamic and +dP/dtmax changes due to occlusion/reperfusion or drug administration were similar between treatment groups. Infarct size was measured post-experimentally using tetrazolium staining and is reported as a percent of area at risk. Infarct size/area at risk (%) was significantly lower in rabbits administered ramiprilat only (20 +/- 6%*) or ramiprilat plus angiotensin II (26 +/- 5%*), compared to those receiving saline (41 +/- 6%), angiotensin II (51 +/- 4%), or losartan (52 +/- 4%, mean +/- S.E.M., * P < 0.05). These data indicate that direct angiotensin II receptor stimulation or receptor antagonism does not alter the degree of myocardial necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1995

Down-regulation of NADPH-diaphorase (nitric oxide synthase) may account for the pharmacological activities of Cu(II)2 (3,5-diisopropylsalicylate)4.

Purposes of this work were to develop an enzyme system as an in vitro model of the NADPH-dependent component of nitric oxide synthase (NOS) and examine the plausible down-regulation of this system and brain NOS by copper (II)2(3,5-diisopropylsalicylate)4[Cu(II)2(3,5-DIPS)4] as a mechanism accounting for its analgesic, anticonvulsant, and other pharmacological activities. Porcine heart diaphorase (PHD) was found to oxidize 114 microM NADPH with the corresponding reduction of an equivalent amount of 2,6-dichlorophenolindophenol (DCPIP). Addition of Cu(II)2(3,5-DIPS)4 to the reaction mixture decreased the reduction of DCPIP without substantially affecting the oxidation of NADPH. The IC50 for Cu(II)2(3,5-DIPS)4 in inhibiting the reduction of DCPIP was 1.5 microM. Mechanistically, this inhibition of DCPIP reduction was found to be due to the ability of Cu(II)2(3,5-DIPS)4 to serve as a catalytic electron acceptor for reduced PHD, which was enhanced by the presence of a large concentration of DCPIP and inhibited by a large concentration of NADPH. Oxidation of NADPH by PHD in the absence of DCPIP was linearly related to the concentration of Cu(II)2(3,5-DIPS)4 through the concentration range of 5-25 microM Cu(II)2(3,5-DIPS)4 with 50% recovery of NADPH oxidation by PHD at a concentration of 16 microM Cu(II)2(3,5-DIPS)4. Whole rat brain tissue sections incubated in medium containing an NADPH-generating system and nitroblue tetrazolium chloride (NBT) were less intensely stained when Cu(II)2(3,5-DIPS)4 was added to the medium. It is concluded that Cu(II)2(3,5-DIPS)4 serves as an electron acceptor in down-regulating PHD reduction of DCPIP and in down-regulating NOS in brain tissue sections. A decrease in NO synthesis in animal models of seizure, pain, and other disease states with Cu(II)2(3,5-DIPS)4 may account for the anticonvulsant, analgesic, and other pharmacological activities of this complex.

Animals↗

PubMed2003

Immunomodulatory effects of docetaxel on human lymphocytes.

Docetaxel is an antineoplastic taxoid that interferes with microtubule polymerization dynamics and is used clinically to treat advanced cancers. Because microtubules play significant roles in T lymphocyte activation and function we characterized the in vitro immunomodulatory properties of docetaxel. Effects of docetaxel on lectin-induced peripheral blood mononuclear cell (PBMC) proliferation were measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and proliferating cell nuclear antigen (PCNA) staining. In addition, apoptosis was measured by annexin V staining and cell activation by determination of CD25 and CD71 cell surface expression. Intracellular calcium kinetics in lectin-activated Jurkat T lymphocytes exposed to docetaxel were investigated. Th1 cytokine production was assessed in T lymphocytes by intracellular cytokine staining. Docetaxel significantly inhibited PBMC proliferation and promoted apoptosis of lectin-activated PBMCs. Docetaxel significantly decreased expression of CD71 but not that of CD25. Docetaxel altered intracellular calcium homeostasis but did not affect Th1 cytokine production in T lymphocytes. In conclusion we demonstrate that docetaxel, although exerting significant antiproliferative effects on lymphocytes and promoting activation-induced apoptosis does affect only partially lymphocyte activation and function and does not affect Th1 cytokine production. These results suggest maintenance of lymphocyte functions important for host tumor surveillance and suggest that this compound may have a role in the treatment of cancer arising organ transplant recipients.

Adjuvants, Immunologic↗

PubMed2004

Nitric oxide donating nonsteroidal anti-inflammatory drugs induce apoptosis in human prostate cancer cell systems and human prostatic stroma via caspase-3.

PURPOSE: New nitric oxide (NO) donating nonsteroidal anti-inflammatory drugs (NSAIDs) have been synthesized to counteract the side effects of conventional NSAIDs. Mounting evidence suggests that NSAIDs may have a possible chemopreventative/therapeutic role in prostate cancer. NO is a powerful biological messenger with multiple cellular effects. We established the effects of 2 of these new drugs in prostate cell systems. MATERIALS AND METHODS: We studied the effects of NO-ibuprofen (NCX 2111) and NO-aspirin (NCX 4060) on hormone sensitive (LNCap) and insensitive (PC3) prostate cancer epithelial cell lines as well as primary cultures of prostatic stroma. Proliferation was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazalium bromide) assay to examine proliferation. Subsequently flow cytometry, cell cycle analysis and TUNEL staining were used to look for apoptosis. Caspase-3 expression was also examined in treated cell types. RESULTS: NCX 2111 and NCX 4060 were found to be potent inhibitors of proliferation in a dose dependent fashion. The 2 drugs induced apoptosis, as seen by flow cytometry, cell cycle analysis and TUNEL staining, at doses between 10 and 100 microM. These NO-NSAIDs increased caspase-3 expression. NCX 4060 was more effective at lower concentrations (10 microM) but each compound was much more potent than conventional ibuprofen and aspirin at inducing apoptosis and inhibiting proliferation. CONCLUSIONS: NO-NSAIDs are potent antiproliferative pro-apoptotic compounds in prostate cell systems. This pro-apoptotic effect is mediated via caspase-3 and it is independent of the type of prostate cell used. These findings have ramifications for the use of these new drugs in prostate cancer chemoprevention or treatment.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1990

Isobestic wavelength determination for succinic dehydrogenase-nitroblue tetrazolium-derived formazans in autonomic neurons.

Two methods have been used to determine the isobestic (equiconcentration) wavelength of formazans derived from nitroblue tetrazolium-succinate dehydrogenase (EC 1.3.99.1) activity in cryostat sections of the rat superior cervical ganglion prior to microdensitometric measurements. Both methods indicate that maximal absorbance of the final reaction product is at a wavelength of 540-550 nm. This wavelength differs by 35-45 nm from that used to measure the same reaction product in sections of other rat tissues such as liver and implies that it may be unwise to adopt a "standard" wavelength for a particular reaction product when making microdensitometric measurements in relation to quantitative enzyme histochemistry.

Animals↗

PubMed2001

Expression of peroxisome proliferator-activated receptor gamma in renal cell carcinoma and growth inhibition by its agonists.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. It plays a role in both adipocyte differentiation and tumorgenesis. Up-date, the up-regulation of PPAR-gamma expression is a frequent occurrence in a variety of different malignant tumors. In this study, we investigated the expression of PPAR-gamma in human renal cell carcinoma (RCC) tissues, and the role of PPAR-gamma in cell growth in human RCC-derived cell lines. Immunohistochemistry showed a strong immunoreactive expression of PPAR-gamma in all slides from cancer specimens. RT-PCR and Western blot analysis showed 3 RCC cell lines expressed PPAR-gamma mRNA and its protein. MTT assay in 3 RCC cells showed that the synthetic PPAR-gamma agonists thiazolidinedione compounds (pioglitazone and troglitazone) and the endogeneous PPAR-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) inhibited the growth of the RCC cells. These results suggest that PPAR-gamma may become a new target in the treatment of RCC.

Antineoplastic Agents↗

PubMed1985

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.

5'-Nucleotidase↗

PubMed2001

Hypercapnic hypoxia compromises bactericidal activity of fish anterior kidney cells against opportunistic environmental pathogens.

Acute hypoxia can cause massive fish and shellfish mortality. Less clear is the role that chronic sublethal hypoxia might play in aquatic animal health. This study tested whether production of reactive oxygen species (ROS) and bactericidal activity of fish phagocytic cells are suppressed under the conditions of decreased oxygen and pH and increased carbon dioxide which occur in the blood and tissue of animals exposed to sublethal hypoxia. Anterior head kidney (AHK) cells of the mummichog, Fundulus heteroclitus, were exposed in parallel to normoxic (pO2=45 torr, pCO2=3.8 torr, pH=7.6) or hypoxic (pO2=15 torr, pCO2=8.0 torr, pH=7.0) conditions and stimulated with a yeast cell wall extract, zymosan. or live Vibrio parahaemolyticus. Hypercapnic hypoxia suppressed zymosan-stimulated ROS production by 76.0% as measured in the chemiluminescence assay and by 58.5% in the nitroblue tetrazolium (NBT) assay. The low O2, high CO2 and low pH conditions also suppressed superoxide production by 75.0 and 47.3% as measured by the NBT assay at two different challenge ratios of cells:bacteria (1:1 and 1:10, respectively). In addition to its effects on ROS production, hypercapnic hypoxia also reduced bactericidal activity by 23.6 and 72.5% at the 1:1 and 1:10 challenge ratios, respectively. Low oxygen levels alone (pO2=15 torr, pCO2=0.76 torr, pH=7.6) did not significantly compromise the killing activity of cells challenged with equal numbers of V. parahaemolyticus. At the higher 1:10 AHK:bacteria challenge ratio, low oxygen caused a small (26.3%) but significant suppression of bactericidal activity as compared to aerial conditions (pO2=155 torr, pCO2=0.76 torr, pH=7.6). This study demonstrates that while hypoxia alone has detrimental effects on immune function, suppression of phagocytic cell activity is compounded by naturally occurring conditions of hypercapnia and low pH, creating conditions that might be exploited by opportunistic pathogens. These results indicate that the adverse health effects of chronic hypercapnic hypoxia might greatly exceed the effects of low oxygen alone.

Animals↗

PubMedDate not supplied

Copper and zinc uptake and hsp70 expression in HepG2 cells.

The aim of this work is to study the accumulation in HepG2 cells of two essential metals with toxic potency and to analyse the induction of the heat shock protein 70 kDa (hsp70) consequent to metal exposure. Cu and Zn were the metals considered and were analysed both as single compounds and in combination in order to evidence synergic effects of the mixture. The use of HepG2 cells provided an in vitro system that retains morphological and metabolic properties and the expression of specific genes typical of liver parenchymal cells. Moreover, the hepatic cells represent a suitable model for their susceptibility to metal toxicity since liver, gastrointestinal tract and renal tubular cells are involved in the uptake, transport, detoxification and secretion of these compounds. The uptake of Cu and Zn followed a time-dependent accumulation when they were used separate. The combination of the two metals produced a higher accumulation of Zn. The stress protein hsp70 was expressed before the metals accumulated within the cells, as shown by the measures obtained with the ICP-AES technique. Moreover, the accumulation of hsp70 by a sublethal shock provided a protective mechanism against metal cytotoxicity.

Cell Survival↗

PubMed1996

Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

3T3 Cells↗

PubMed2005

Amantadine and memantine induce the expression of the glial cell line-derived neurotrophic factor in C6 glioma cells.

Aminoadamantanes are commonly used in the treatment of Parkinson's and Alzheimer's diseases. While these drugs are shown to antagonise ionotropic glutamate receptors on neuronal cells, additional mechanisms could contribute to their neuroprotective properties. The aim of the present study was to investigate the effect of aminoadamantanes on the production of the glial cell line-derived neurotrophic factor (GDNF) in glial cells. For this purpose, we measured the modulation of GDNF release in C6 glioma cell cultures treated for 24 h with amantadine and memantine. Both drugs dose-dependently increased GDNF level in the culture medium with similar potency (submicromolar range) and efficacy (three to four-fold induction). RT-PCR studies revealed that both compounds also increased GDNF mRNA levels and their influence on the GDNF gene transcription was further evidenced using a rat GDNF promoter luciferase reporter assay. Together, these results demonstrate that the neuroprotective effect of amantadine and memantine could involve the regulation of GDNF production by glial cells.

5' Untranslated Regions↗