Comparison Between 5-Aminosalicylic Acid (5-ASA) and Para-Aminosalicylic Acid (4-PAS) as Potential Protectors Against Mn-Induced Neurotoxicity

Manganese (Mn) is an essential metal for biological systems; however, occupational or clinical exposure to high levels of Mn can produce a neurological disorder called manganism. Oxidative stress and neuroinflammation play major roles in the Mn-induced neurodegeneration leading to dysfunction of the basal ganglia. We investigated the toxic effects of MnCl₂ in an immortalized rat brain endothelial cell line (RBE4) and the protective effects of the radical scavenging aminosalicylic acids, 5-aminosalicylic acid (5-ASA) and 4-aminosalicylic acid (4-PAS). Mn cytotoxicity was determined with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) activity. A significant decrease in MTT reduction concomitant with increased LDH release was noted in RBE4 cells exposed for 24 h to MnCl₂ (600 and 800 μM; p?<?0.0001). Our results establish that compared to 4-PAS, 5-ASA has greater efficacy in protecting RBE4 cells from Mn-induced neurotoxicity after preexposure to MnCl₂ 800 μM (p?<?0.0001).     

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto-phosphatase activity.

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane-bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco-2 cells. ALP activity expressed on the apical surface of blood-brain barrier cells, and its relationship with (125)I-insulin internalization were investigated under physiological conditions using p-nitrophenylphosphate (p-NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and (125)I-insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto-oriented active site which was functional at physiological pH. Orthovanadate (100 microM), an inhibitor of phosphatase activities, decreased both RBE4-ALP activity and (125)I-insulin internalization. In the presence of L-arginine (1 mM) or adenosine (100 microM) RBE4-ALP activity and (125)I-insulin, internalization were significantly reduced. However, D-arginine (1 mM) had no significant effect. Additionally, RBE4-ALP activity and (125)I-insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 microM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 microM and 100 microM), beta-estradiol (100 microM), iron (100 microM) or in the presence of all-trans retinoic acid (RA) (10 microM). The ALP inhibitor levamisole (500 microM) was able to reduce (125)I-insulin internalization to 69.1 +/- 7.1% of control. Our data showed a positive correlation between ecto-ALP activity and (125)I-insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood-brain barrier may be modulated through ALP.     

Adropin reduces paracellular permeability of rat brain endothelial cells exposed to ischemia-like conditions

Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16 h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40 kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100 ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.     

Leptin-derived peptide, a targeting ligand for mouse brain-derived endothelial cells via macropinocytosis

Leptin is an appetite regulatory hormone that is secreted into the blood circulation by adipose tissue, and functions in the central nerve system (i.e. hypothalamus) by crossing the blood brain barrier (BBB). In the present study, we investigated the function of a leptin-derived peptide (Lep_70-89) as a ligand for mouse brain-derived endothelial cells (MBEC4). Lep_70-89-modified liposomes, prepared with a polyethyleneglycol (PEG) spacer (Lep_70-89-PEG-LPs) exhibited a significantly higher cellular uptake than peptide-unmodified liposomes (PEG-LPs). Furthermore, cellular uptake was inhibited by amiloride, while no significant inhibitory effect was observed by the presence of chlorpromazine and filipin III, suggesting that macropinocytosis largely contributed to the cellular uptake of Lep_70-89-PEG-LPs. Imaging studies revealed that Lep_70-89-PEG-LPs were not colocalized with endosome/lysosomes, whereas neutral dextran (70 kDa) was predominantly colocalized with these compartments. This indicates that Lep_70-89-PEG-LPs are taken up via macropinocytosis and are subject to non-classical intracellular trafficking, resulting in the circumvention of lysosomal degradation in endothelial cells.     

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na(+)-independent and shows a Michaelis-Menten constant of approx. 20 microM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 microM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na(+)-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na(+)-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Human lymphocyte subpopulations identified by bacterial adherence are functionally different.

Previously, two B (B₁ and B₂)- and four T (T₁, T₂, T₃, T₄)-lymphocyte subpopulations have been identified in human blood smears by bacterial adherence. Here, to study the functional differences between these subpopulations the T₁T₂ cells were separated from T₃T₄ cells by selective adherence to Escherichia coli-2⁴ monolayers. The adherent cells (T₁T₂ cells) responded well to concanavalin A in 3-day cultures and in mixed lymphocyte culture (MLC) in 6-day cultures and developed into cells specifically cytotoxic for allogeneic lymphocytes. The nonadherent cells (T₃T₄ cells) cultured for the same length of time were poorly responsive to concanavalin A, variably responsive in MLC, and poorly active in specific cytotoxicity. The T₃T₄ cells were naturally cytotoxic for allogeneic lymphocytes and for a normal lymphoblastoid cell line. We concluded that the T cells that bind E. coli-2 (T₁T₂ cells) are functionally different from those that do not bind (T₃T₄ cells).     

Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (γGTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed γCTP and ALP activities. Such structures, associated with high levels of γCTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related γCTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier. © 1994 wiley-Liss, Inc.     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

Samarium doped titanium dioxide nanoparticles as theranostic agents in radiation therapy

PurposeTitanium dioxide nanoparticles (TiO₂ NPs) have been investigated for their role as radiosensitisers for radiation therapy. The study aims to increase the efficiency of these NPs by synthesising them with samarium.MethodsSamarium-doped TiO₂ NPs (Ti(Sm)O₂ NPs) were synthesised using a solvothermal method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDS) were performed for characterising of the Ti(Sm)O₂ NPs. The intracellular uptake and cytotoxicity were assessed in vitro using A549 and DU145 cancer cell lines. Furthermore, the effect of dose enhancement and generation of reactive oxygen species (ROS) in response to 6 MV X-rays was evaluated. Additionally, the image contrast properties were investigated using computed tomography (CT) images.ResultsThe synthesised Ti(Sm)O₂ NPs were about 13 nm in diameter as determined by TEM. The XRD pattern of Ti(Sm)O₂ NPs was consistent with that of anatase-type TiO₂. EDS confirmed the presence of samarium in the nanoparticles. At 200 μg/ml concentration, no differences in cellular uptake and cytotoxicity were observed between TiO₂ NPs and Ti(Sm)O₂ NPs in both A549 and DU145 cells. However, the combination of Ti(Sm)O₂ NPs and X-rays elicited higher cytotoxic effect and ROS generation in the cells than that with TiO₂ NPs and X-rays. The CT numbers of Ti(Sm)O₂ NPs were systematically higher than that of TiO₂ NPs.ConclusionsThe Ti(Sm)O₂ NPs increased the dose enhancement of MV X-ray beams than that elicited by TiO₂ NPs. Samarium improved the efficiency of TiO₂ NPs as potential radiosensitising agent.     

Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Cellular uptake and cytotoxicity of octahedral rhenium cluster complexes

Cellular uptake behavior of a novel class of octahedral rhenium cluster compounds, hexahydroxo complexes K₄[{Re₆S₈}(OH)₆] · 8H₂O (1) and K₄[{Re₆Se₈}(OH)₆] · 8H₂O (2), was evaluated in human cervical adenocarcinoma HeLa cells. Confocal microscopy and flow cytometry studies demonstrated that rhenium cluster 1 was not internalized into cell, while rhenium cluster 2 was. Conjugation of a polymer to rhenium cluster 1, namely the derivative K₄[{Re₆S₈}(OH)₅L] (3) (L is amphiphilic diblock copolymer MPEG550-CH₂CONH-GlyPheLeuGlyPheLeu-COO⁻), considerably enhanced cellular uptake in a concentration-dependent manner and was predominantly localized in the cytoplasm and nucleus upon incubation time. The uptake of rhenium cluster 2 was mediated by energy-dependent endocytosis, whereas rhenium cluster 3 was directly ingested into cells by cell-fusion-like mechanism. According to the cytotoxicity evaluation test, both rhenium clusters 2 and 3 did not exhibit acute cytotoxic effects up to 50 μM, at the practical concentration level of biological applications. It is, therefore, expected that the rhenium cluster complexes can be promising potential candidates as diagnostic agents for medical treatment.     

Differential Effect of Nimodipine in Attenuating Iron-Induced Toxicity in Brain- and Blood–Brain Barrier-Associated Cell Types

Metal homeostasis is increasingly being evaluated as a therapeutic target in stroke and neurodegenerative diseases. Metal dysregulation has been shown to lead to protein aggregation, plaque formation and neuronal death. In 2007, we first reported that voltage-gated calcium channels act as a facile conduit for the entry of free ferrous (Fe²⁺) ions into neurons. Herein, we evaluate differential iron toxicity to central nervous system cells and assess the ability of the typical L-type voltage-gated calcium channel blocker nimodipine to attenuate iron-induced toxicity. The data demonstrate that iron sulfate induces a dose-dependent decrease in cell viability in rat brain endothelial cells (RBE4; LC₅₀ = 150 μM), neuronal cells (Neuro-2α neuroblastoma; LC₅₀ = 400 μM), and in astrocytes (DI TNC1; LC₅₀ = 1.1 mM). Pre-treatment with nimodipine prior to iron sulfate exposure provided a significant (P < 0.05) increase in viable cell numbers for RBE4 (2.5-fold), Neuro2-α (~2-fold), and nearly abolished toxicity in primary neurons. Astrocytes were highly resistant to iron toxicity compared to the other cell types tested and nimodipine had no (P > 0.05) protective effect in these cells. The data demonstrate variable susceptibility to iron overload conditions in different cell types of the brain and suggest that typical L-type voltage-gated calcium channel blockers (here represented by nimodipine), may serve as protective agents in conditions involving iron overload, particularly in cell types highly susceptible to iron toxicity.     

Selective Induction of DNA Damage and Cell Cycle Arrest Mediated by Chromone-Triazole Dyads Derivatives: Effects on Breast and Prostate Cancer Cells

Chromones and triazoles are groups of heterocyclic compounds widely known to exhibit a broad spectrum of biological activities. The combination of these two pharmacophores could result in multiple mechanisms of action to increase the potency of anticancer drugs and reduce their side effects. The in vitro antitumor effect of eight chromone-based compounds was evaluated in breast (T-47D and MDA-MB-231) and prostate (PC3) cancer cell lines, and in non-cancerous human mammary epithelial cells (HuMEC) using a resazurin-based method. Flow cytometry was used to evaluate the cell cycle and cell death, and ɣ-H2AX detection to identify DNA damage. The compounds showed selective cytotoxicity against cancer cell lines, with (E)-2-(2-(5-(4-methoxyphenyl)-2H-1,2,3-triazol-4-yl)vinyl)-4H-chromen-4-one (compound 2 a ) being more potent in non-metastatic T-47D cells (IC₅₀ 0.65 μM). Replacing the hydrogen by a methyl group on the triazole ring in compound 2 b enhanced the cytotoxic activity up to IC₅₀ 0.24 μM in PC3, 0.32 μM in MDA-MB-231 and 0.52 μM in T-47D. Compound 2 b was 3-fold more potent than doxorubicin in PC3 (IC₅₀ 0.73 μM) and 4-fold in MDA-MB-231 (IC₅₀ 1.51 μM). The addition of tetrahydroisoindole-1,3-dione moiety in compound 5 did not improve its effectiveness in any of the cell lines but it exerted the lowest cytotoxic effect in HuMEC (IC₅₀ 221.35 μM). The compounds revealed different cytotoxic mechanisms: 2 a and 2 b induced G2/M arrest, and compound 5 did not affect the cell cycle.     

Heterogeneity of murine adherent interleukin-2-activated killer cells differential effect of prostaglandin E2 and forskolin

We have studied the relationship between cytotoxic activity, size and granularity of murine interleukin-2-activated adherent killer cells issued from spleen cells cultured with high levels of IL-2. The effects of prostaglandin E₂ (PGE₂) and forskolin upon these cells were assessed. All adherent spleen cells obtained after 5 days of culture were large granular lymphocytes but presented a heterogeneity in size and granularity. After fractionation on a discontinuous-density Percoll gradient, four cellular subpopulations were isolated. Fluorescence-activated cell sorting analysis showed that cells of the lightest fraction (F1) were the largest, while the cells found in the heaviest fraction (F4) were much more granular than the cells collected in the two intermediate fractions (F2 and F3). The serine esterases level was higher in F4 than in unfractionated cells and diminished to about 40% in cells of fractions F2 and F3, which expressed a cytotoxic activity against YAC-1 cells higher than that in unfractionated cells or in F1 or F4, which presented the lowest cytotoxic activity. When AK cells were cultured for 48 h in the presence of either PGE₂ or forskolin, which induce an intracellular increase of cAMP, we observed that PGE₂ (1M) inhibited the cytotoxic activity, but surprisingly forskolin (2M) exerted a stimulating effect on the induction of cytotoxic activity. After fractionation on a discontinuous Percoll gradient we observed the same cellular distribution among PGE₂ or forskolin-treated or-untreated cells, but PGE₂ induced an increase of size and granularity. This effect of PGE₂ was more potent on the cells collected in F4. However this variation of granularity was not associated with any variation in the serine esterase level. The cytotoxic activity of PGE₂- or forskolin-treated cells did not present any significant variation relative to the control for cells collected in F2 and F3: on the other hand, forskolin-treated cells collected in F4 showed a significantly higher cytotoxicity than did the corresponding untreated or PGE₂-treated cells.     

Regulation of prostate cancer cell division by glucose

Previous studies have shown that rapid cell proliferation is associated with elevated glucose consumption. However, those studies did not establish whether glucose is required for prostate cancer cell proliferation or define the molecular mechanisms by which glucose regulates cell division. We addressed these issues by studying two metastatic human prostate cancer cell lines: DU145, which is androgen independent and highly proliferative; and LNCaP, which is androgen dependent and relatively slow growing. We found that proliferation of DU145 cells was significantly inhibited by reduction of glucose in the medium to 0.5 g/L, which is half the physiologic concentration, whereas LNCaP cells grew at control rates even in the presence of only 0.05 g/L glucose. Glucose deprivation of DU145 cells caused a 90% reduction in DNA synthesis; a 10–20-fold reduction in cyclins D and E and CDK4 levels; and cell cycle arrest in G₀-G₁. However, glucose deprivation did not cause global inhibition of protein synthesis, since mutant p53 levels increased in glucose-deprived DU145 cells. This observed increase in mutant p53 levels was not associated with a rise in p21 levels. Glucose deprivation of DU145 cells also led to apparent dephosphorylation of mutant retinoblastoma (RB) protein. We conclude that: 1) high levels of glucose consumption are required for rapid proliferation of androgen-independent prostate cancer cells, 2) glucose may not be required for slow growth of androgen-dependent prostate cancer cells, and 3) glucose promotes passage of cells through early G₁ by increasing the expression of several key cell cycle regulatory proteins that normally inhibit RB function. J. Cell. Physiol. 180:431–438, 1999. © 1999 Wiley-Liss, Inc.     

TNF activates P-glycoprotein in cerebral microvascular endothelial cells.

BACKGROUND/AIMS: Multidrug resistance proteins (MDRs, including P-glycoproteins) are efflux pumps that serve important biological functions but hinder successful drug delivery to the CNS. Many chemotherapeutic agents, anti-epileptics, anti-HIV drugs, and opiates are substrates for MDRs. Therefore, understanding the regulation of MDRs in the endothelial cells composing the blood-brain barrier has therapeutic implications. METHODS: We used microarray, real time RT-PCR, Western blotting, and uptake of vinblastine by RBE4 cerebral endothelial cells to test the effects of tumor necrosis factor alpha (TNF) on the expression and functions of P-glycoprotein (MDR1). RESULTS: The proinflammatory cytokine TNF specifically induced the expression and enhanced the function of MDR1 in RBE4 cells. The persistent upregulation of MDR1 mRNA was shown by cDNA microarray at 6, 12, and 24 h after TNF treatment. This was confirmed by real-time RT-PCR between 2 and 24 h. MDR1 protein expression was increased 6 to 24 h after TNF treatment and resulted in a significant reduction in the cellular uptake of (3)H-vinblastine. CONCLUSION: The drug efflux transporter in cerebral endothelial cells can be upregulated by TNF. This suggests that adjunctive anti-TNF treatment has novel therapeutic potential in conditions such as brain cancer, epilepsy, neuroAIDS, and chronic pain.     

Malignant H1299 tumour cells preferentially internalize iron-bound inositol hexakisphosphate

In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP₆ (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP₆ complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP₆ but, interestingly, found that the cells are able to internalize metal-free and Cr³⁺-bound InsP₆. Thus, the inability of CaCo-2 cells to take up iron/InsP₆ complexes seems to be due to the iron-bound state of InsP_6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP_6, we examined whether these cells may be able to take up iron/InsP₆ complexes. Indeed, we found that InsP₆ dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP₆ is more effectively internalized than in the metal-free or Cr³⁺-bound state, indicating that H1299 cells preferentially take up iron/InsP₆ complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP₆/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP₆-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP_6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.     

Antitumor and toxicologic properties of the organometallic anticancer agent vanadocene dichloride

We report here on the antineoplastic, toxicologic, and transmembrane transfer properties of vanadocene dichloride (VDC), a representative metallocene dihalide. VDC is cytotoxic to HEp-2 human epidermoid carcinoma cells, in vitro, in a dose dependent manner, with a D_o value (dose increment reducing the survival fraction by 1/e) of 0.530 ± 0.005 μg/ml. Under similar experimental conditions, the D_o for cisplatin (CDDP) against these cells is 0.46 ± 0.08 μg/ml. In a murine mammary adenocarcinoma (TA3Ha) system, 125 μg/ml VDC inhibits the tumor-forming ability of 10⁵ cells upon i.p. inoculation into syngeneic Strain A mice. The transmembrane transfer rate constants for the metal uptake of VDC and CDDP by TA3Ha cells in vitro were found to be 3.3 ± 0.8 × 10⁻⁴ min⁻¹ and 12 ± 2.0 × 10^t-4 min⁻¹, respectively. In vivo studies with TA3Ha cells show that two i.p. treatments of 20, 40, and 60 mg/kg VDC increase the host survival by 30, 50, and 90%, respectively. Under similar conditions, 2, 4, and 6 mg/kg CDDP (equitoxic dose levels) prolong the host survival 50, 75, and 83%, respectively. Morphological, blood urea nitrogen level, and serum creatinine level data for Strain A mice treated with 60 mg/kg VDC give no evidence of renal or small intestinal damage. However, changes in the liver consistent with fatty cell degeneration are observed in these mice.