ABSTRACT
Malignant melanoma is a highly metastatic tumour, resistant to treatment. Serotonin type-3 (5-HT3) receptor antagonists, such as tropisetron and ondansetron, are well-tolerated antiemetic drugs commonly used to prevent nausea caused by chemotherapy or radiotherapy. We investigated the anticancer effects of these drugs on melanoma cancer cell lines WM-266-4 and B16F10 with or without paclitaxel. We constructed IC50 curves and performed Chou-Talalay analysis, using data obtained with the MTT assay. Flow cytometry and fluorescent microscopy were used to examine characteristics of the cell cycle, cell death and cytoskeleton changes. Protein levels and activation were analysed by western blotting and molecular docking studies carried out. Data were analysed by one way ANOVA and post hoc testing. Ondansetron and tropisetron showed selective concentration-dependent cytotoxicity in melanoma cell lines WM-266-4 and B16F10. The effect in combination with paclitaxel was synergistic. The drugs did not cause cell cycle arrest but did promote characteristics of classical apoptosis, including accumulation of subG1 DNA, cleaved caspase-3, mitochondrial membrane permeability and phosphatidylserine exposure. As well, the cytosolic calcium level in the melanoma cells was enhanced, phosphorylated ERK1/2 induced and NF-κB inhibited. Finally, the formation of microtubules was shown to be impaired in melanoma cells treated with ondansetron or tropisetron. Docking studies were used to predict that these drugs could bind to the colchicine binding site on the tubulin molecule. Antiemetic drugs, already given in combination with chemotherapy, may enhance the cytotoxic effect of chemotherapy, following successful delivery to the tumour site.
Subject(s)
Antiemetics , Melanoma , Humans , Antiemetics/adverse effects , Ondansetron/adverse effects , Tropisetron/adverse effects , Serotonin/adverse effects , NF-kappa B , Vomiting/chemically induced , Vomiting/drug therapy , Vomiting/prevention & control , Down-Regulation , Molecular Docking Simulation , Melanoma/drug therapy , Apoptosis , Paclitaxel/pharmacologyABSTRACT
Pinnatoxin G is a cyclic imine neurotoxin produced by dinoflagellates that has been reported in shellfish. Like other members of the pinnatoxin family, it has been shown to have its effects via antagonism of the nicotinic acetylcholine receptors, with preferential binding to the α7 subunit often upregulated in cancer. Because increased activity of α7 nicotinic acetylcholine receptors contributes to increased growth and resistance to apoptosis, the effect of pinnatoxin G on cancer cell viability was tested. In a panel of six cancer cell lines, all cell types lost viability, but HT29 colon cancer and LN18 and U373 glioma cell lines were more sensitive than MDA-MB-231 breast cancer cells, PC3 prostate cancer cells, and U87 glioma cells, correlating with expression levels of α7, α4, and α9 nicotinic acetylcholine receptors. Some loss of cell viability could be attributed to cell cycle arrest, but significant levels of classical apoptosis were found, characterized by caspase activity, phosphatidylserine exposure, mitochondrial membrane permeability, and fragmented DNA. Intracellular Ca2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.
Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Imines/pharmacology , Nicotinic Antagonists/pharmacology , Spiro Compounds/pharmacology , Calcium , Cell Line, Tumor , Humans , Marine Toxins/pharmacology , Molecular Structure , Receptors, NicotinicABSTRACT
PURPOSE: Anti-helmintic drugs mebendazole and albendazole are commonly used to treat a variety of parasitic infections. They have recently shown some promising results in pre-clinical in vitro and in vivo anti-cancer studies. METHODS: We compare their efficacy in breast and colon cancer cell lines as well as in non-cancerous cells and elucidate their mechanism of action. The drugs were screened for cytotoxicity in MDA-MB-231, MCF-7 (breast cancer), HT-29 (colorectal cancer), and mesenchymal stromal cells, using the MTT assay. Their effects on the cell cycle, tubulin levels, and cell death mechanisms were analysed using flow cytometry and fluorescent microscopy. RESULTS: Mebendazole and albendazole were found to selectively kill cancer cells, being most potent in the colorectal cancer cell line HT-29, with both drugs having IC50 values of less than 1 µM at 48 h. Both mebendazole and albendazole induced classical apoptosis characterised by caspase-3 activation, phosphatidylserine exposure, DNA fragmentation, mitochondrial membrane permeability, and reactive oxygen species production. Cell cycle arrest in the G2/M phase was found, and tubulin polymerisation was disrupted. CONCLUSION: Mebendazole and albendazole were shown to cause selective cancer cell death via a mechanism of classical apoptosis and cell cycle arrest, involving the destabilisation of microtubules.
Subject(s)
Albendazole/pharmacology , Anthelmintics/pharmacology , Apoptosis , Breast Neoplasms/drug therapy , Colonic Neoplasms/drug therapy , Mebendazole/pharmacology , Breast Neoplasms/pathology , Cell Cycle , Cell Proliferation , Colonic Neoplasms/pathology , Female , Humans , Tumor Cells, CulturedABSTRACT
TIMP-3 is one of four tissue inhibitors of matrix metalloproteinases, the endogenous inhibitors of the matrix metalloproteinase enzymes. These enzymes have an important role in metastasis, in the invasion of cancer cells through the basement membrane and extracellular matrix. TIMP-1, -2 and -4 both promote and inhibit tumour development, in a context-dependent manner, however TIMP-3 is consistently anti-tumourigenic. TIMP-3 is also the only insoluble member of the family, being either bound to the extracellular matrix or the low density lipoprotein-related protein-1, through which it can be endocytosed. Levels of TIMP-3 have also been shown to be regulated by micro RNAs and promoter hypermethylation, resulting in frequent silencing in many tumour types, to the extent that its expression has been suggested as a prognostic marker in some tumours, being associated with lower levels of metastasis, or better response to treatment. TIMP-3 has been shown to have anti-metastatic effects, both through inhibition of matrix metalloproteinases and ADAM family members and downregulation of angiogenesis. This occurs via interactions with receptors including VEGF, via modulation of signaling pathways and due to protease inhibition. TIMP-3 has also been shown to reduce tumour growth rate, most often by inducing apoptosis by stabilisation of death receptors. A number of successful mechanisms of delivery of TIMP-3 to tumour or inflammatory sites have been investigated in vitro or in animal studies. It may therefore be worthwhile further exploring the use of TIMP-3 as a potential anti-metastatic or anti-tumorigenic therapy for many tumour types.
Subject(s)
Carcinogenesis/pathology , Neoplasm Metastasis/pathology , Tissue Inhibitor of Metalloproteinase-3/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis/genetics , Basement Membrane/metabolism , Carcinogenesis/genetics , DNA Methylation , Disease Models, Animal , Down-Regulation , Epigenesis, Genetic , Extracellular Matrix/metabolism , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/metabolism , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Invasiveness/prevention & control , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/genetics , Prognosis , Promoter Regions, Genetic , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/therapeutic use , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/therapeutic useABSTRACT
Mesenchymal stem cells home towards inflammatory microenvironments, such as the tumour stroma, where they have been shown to have both pro- and anti-tumorigenic effects. Here, we demonstrate that the aspartic acid protease cathepsin D is part of the chemoattraction process. Using a Boyden chamber co-culture system, the migration of the mesenchymal stem cells and their invasion through Matrigel increased in the presence of breast cancer MDA-MB-231â¯cells, colon cancer HT29â¯cells or their conditioned media. Mesenchymal stem cell movement was reduced by protease inhibitors of matrix metalloproteinases and by pepstatin A, an inhibitor of cathepsin D. We confirmed a role for cathepsin D through addition of recombinant protein, upregulation of cathepsin D release using chloroquine and knockdown of cathepsin D expression. While all cell types expressed active cathepsin D, enzymatically inactive precursor procathepsin D was expressed only at low levels by mesenchymal stem cells. Expression in mesenchymal stem cells was increased following co-culture with cancer cells. The chemoattractive effect of cathepsin D required its enzymatic activity, but not changes in mesenchymal stem cell proliferation or adhesion rates. In conclusion, cathepsin D and its precursors enhance mesenchymal stem cell homing towards tumour sites, most likely by enzymatic mechanisms.
Subject(s)
Breast Neoplasms/pathology , Cathepsin D/metabolism , Cell Movement , Colonic Neoplasms/pathology , Enzyme Precursors/metabolism , Mesenchymal Stem Cells/pathology , Tumor Microenvironment , Breast Neoplasms/metabolism , Cell Adhesion , Cell Proliferation , Cells, Cultured , Coculture Techniques , Collagen , Colonic Neoplasms/metabolism , Culture Media, Conditioned , Drug Combinations , Female , Humans , Laminin , Mesenchymal Stem Cells/metabolism , ProteoglycansABSTRACT
Honey has been shown to have a range of therapeutic effects in humans, with anti-inflammatory and anti-bacterial effects among those previously characterised. Here, we examine the possibility of New Zealand thyme, manuka and honeydew honeys, and their major sugar and phenolic components, reducing the development of metastatic cancer. Their activity was examined in vitro, in PC3 and DU145 prostate cancer cell lines, through measuring the compounds' effects on the metastatic characteristics of migration, invasion and adhesion. First, the phenolic compounds gallic acid, caffeic acid, quercetin, kaempferol and chrysin were quantified in the honeys using high performance liquid chromatography, and found in nanomolar concentrations. In a Boyden chamber-based migration assay, non-toxic concentrations of thyme and honeydew honeys reduced cell migration by 20%, and all phenolic compounds except caffeic acid also lowered migration, although a mixture of only the sugars found in honey had no effect. All of the honeys, phenolics and the sugar-only mixture reduced invasive movement of cells through extracellular matrix by up to 75%. Most notably, each of the three honeys and the sugar-only mixture reduced cell adhesion to collagen I by 90%. With the exception of quercetin, phenolic compounds did not reduce adhesion. Therefore, honey and its sugar and phenolic components can lower the metastatic properties of cancer cells, and may do this by preventing effective cell adhesion to the extracellular matrix. The sugars and phenol compounds of honey are much more effective in combination than individually.
ABSTRACT
Honey is a complex biological substance, consisting mainly of sugars, phenolic compounds and enzymes. Using five quick and accessible assays for measuring honey's cytotoxicity in vitro, we found honey is cytotoxic towards prostate cancer cells PC3 and DU145. However, the level of cell death varied with assay. The MTT assay was confounded by the reduction of the MTT reagent by honey's reducing sugars and phenolic compounds, and the lactate dehydrogenase assay was invalidated by honey oxidising the enzyme cofactor NADH. The sulforhodamine B assay gave valid results, but measures only protein content, providing no information about cell death in the remaining cells. The trypan blue assay and a microscope-based propidium iodide/Hoechst staining assay assess only late stage membrane permeability. However, the propidium iodide/Hoechst assay gives morphological information about cell death mechanism. A combination of the sulforhodamine B and propidium iodide/Hoechst assays would provide the most accurate quantification of honey cytotoxicity.
Subject(s)
Honey , Prostatic Neoplasms , Cell Line, Tumor , Cell Survival , Humans , Indicators and Reagents , Male , Trypan BlueABSTRACT
Fibroblast activation protein (FAP), an integral membrane serine protease, is found on fibro- and osteo-sarcoma and on myofibroblasts in epithelial carcinoma, but rarely on other adult tissue. FAP has been demonstrated to be an excellent target for tumor imaging in clinical trials, and antibodies and other FAP-targeting drugs are in development. Here we have shown that FAP overexpression increased the growth of HT1080 fibrosarcoma cells in vitro and in vivo, and found that the expression of FAP affects response to chemotherapy. When treated with doxorubicin, expression of FAP increased susceptibility to the drug. In spite of this, FAP-HT1080 cells had fewer markers of classical apoptosis than HT1080 cells and neither necrosis nor necroptosis were enhanced. However, levels of early mitochondrial and lysosomal membrane permeability markers were increased, and autophagy switched from a protective function in HT1080 cells to part of the cell death mechanism with FAP expression. Therefore, FAP may affect how the tumor responds to chemotherapeutic drugs overall, which should be considered in targeted drug development. The overexpression of FAP also alters cell signaling and responses to the environment in this cell line. This includes cell death mechanisms, changing the response of HT1080 cells to doxorubicin from classical apoptosis to an organelle membrane permeability-dependent form of cell death.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Fibrosarcoma/enzymology , Gelatinases/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Autophagy/drug effects , Cell Line, Tumor , Doxorubicin/pharmacology , Endopeptidases , Fibrosarcoma/drug therapy , Fibrosarcoma/genetics , Fibrosarcoma/physiopathology , Gelatinases/genetics , Humans , Membrane Proteins/genetics , Serine Endopeptidases/geneticsABSTRACT
Cytotoxic amides have been isolated from the fruits of the endemic New Zealand medicinal plant kawakawa, Macropiper excelsum (Piperaceae). The main amide was piperchabamide A and this is the first report of this rare compound outside the genus Piper. Eleven other amides were purified including two new compounds with the unusual 3,4-dihydro-1(2H)-pyridinyl group. The new compounds were fully characterized by 2D NMR spectroscopy, which showed a slow exchange between two rotamers about the amide bond, and they were chemically synthesized. In view of the antitumor activity of the related piperlongumine, all of these amides plus four synthetic analogs were tested for cytotoxicity. The most active was the piperine homolog piperdardine, with an IC50 of 14 µM against HT 29 colon cancer cells.
Subject(s)
Alkaloids/chemistry , Amides/chemistry , Benzodioxoles/chemistry , Piperaceae/chemistry , Piperidines/chemistry , Plant Extracts/chemistry , Polyunsaturated Alkamides/chemistry , Alkaloids/isolation & purification , Alkaloids/pharmacology , Amides/isolation & purification , Amides/pharmacology , Benzodioxoles/isolation & purification , Benzodioxoles/pharmacology , Cell Survival/drug effects , Fruit/chemistry , HT29 Cells , Humans , Inhibitory Concentration 50 , Molecular Structure , Piperidines/isolation & purification , Piperidines/pharmacology , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plants, Medicinal , Polyunsaturated Alkamides/isolation & purification , Polyunsaturated Alkamides/pharmacologyABSTRACT
The serine protease fibroblast activation protein (FAP) is selectively expressed on tumour-associated fibroblasts in most human epithelial tumours, as well as on some mesenchymal tumours such as sarcoma. High FAP expression is most often associated with poor outcome and increased metastasis. Here, we compare the in vitro metastatic potential of HT1080 fibrosarcoma cells with and without FAP expression in order to elucidate the mechanism by which FAP may influence metastasis. In the presence of FAP, cells were more adhesive to extracellular matrix proteins and migrated and invaded through Matrigel to a greater degree. The anti-FAP antibody ESC11, which caused internalization of FAP, decreased adhesion and migration, but only when cells expressed FAP. It was also found that blocking activity of integrins ß1 and αvß3 reduced both cell adhesion and migration and this effect was much more marked in FAP-expressing HT1080 cells than mock-transfected HT1080 cells. The expression or activation of intracellular proteins that form part of the downstream signaling of integrins, including integrin-linked kinase, Rac1 and focal adhesion kinase, was also upregulated when FAP was expressed, suggesting that FAP not only upregulates metastatic-like cell behaviours through interaction with integrins, but also influences the intracellular signaling of integrins. This was confirmed using both PI3 kinase and Src kinase inhibitors, which decreased adhesion and migration in FAP-expressing cells, but did not affect mock-transfected HT1080 cells. FAP is therefore a useful target for anti-cancer therapy, as not only is its expression tumour-selective, but its downregulation has the potential to reduce incidence of metastasis.
Subject(s)
Cell Movement , Cell Proliferation , Fibrosarcoma/metabolism , Fibrosarcoma/secondary , Gelatinases/metabolism , Integrin alphaVbeta3/metabolism , Integrin beta1/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Antibodies, Monoclonal/pharmacology , Blotting, Western , Cell Adhesion , Endopeptidases , Flow Cytometry , Gelatinases/antagonists & inhibitors , Gelatinases/immunology , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/immunology , Microscopy, Fluorescence , Neoplasm Invasiveness , Serine Endopeptidases/immunology , Tumor Cells, CulturedABSTRACT
Mesenchymal stem cells (MSCs) form part of tumor stroma, and are typically considered to be pro-tumorigenic, promoting continuing tumor growth and metastasis. Here, we describe a mechanism by which MSCs may be anti-tumorigenic, through inhibition of breast cancer cell migration and invasion, an important part of metastasis. MDA-MB-231 and T47D cells were co-cultured in a Transwell insert above MSCs or MSC conditioned media and their migration or invasion through Matrigel measured. The conditioned media was found to inhibit breast cancer cell movement. TIMP-1 and TIMP-2, inhibitors of matrix metalloproteinases (MMPs), were identified as candidates for this inhibition and enhanced secretion of MMPs was not sufficient to counter the anti-migratory effects of TIMP expression. Inhibition of TIMP activity showed that TIMP-1 and TIMP-2 together were largely responsible for the reduction of migration and invasion by MSCs. Therefore, MSCs may play anti-tumorigenic, anti-metastatic roles in tumor development, with the overall outcome dependent upon the balance of pro-and anti-tumorigenic molecules secreted.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Movement/physiology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Cell Line, Tumor , Coculture Techniques/methods , Collagen/metabolism , Culture Media, Conditioned/metabolism , Drug Combinations , Female , Humans , Laminin/metabolism , Proteoglycans/metabolismABSTRACT
The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Pancreatic Neoplasms/therapy , Radiopharmaceuticals , Xenograft Model Antitumor Assays , Animals , Antibodies, Monoclonal/pharmacokinetics , Drug Evaluation, Preclinical , Female , Humans , Iodine Radioisotopes , Mice , Mice, Nude , Pancreas/immunology , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Pentetic Acid/analogs & derivatives , Pentetic Acid/pharmacokinetics , Radioimmunoassay , Receptors, Vitronectin/immunology , Receptors, Vitronectin/metabolism , Tissue Distribution , Tumor Cells, CulturedABSTRACT
OBJECTIVES: Plasma neopterin is a clinical marker of inflammation. Interferon-gamma triggers 7,8-dihydroneopterin and its oxidation product, neopterin, to be released from macrophages. 7,8-dihydroneopterin is a potent antioxidant which can protect macrophages from oxidative damage in vitro. This study examined whether 7,8-dihydroneopterin/neopterin levels reach sufficient concentrations in human pus to provide antioxidant activity and be the source of plasma neopterin. DESIGN AND METHODS: Pus was removed by needle aspiration from 19 patients and examined for total neopterin, protein-bound DOPA, dityrosine, alpha-tocopherol, lipid oxidation and protein carbonyls. RESULTS: Total neopterin was detected between 50 nM and 1.2 microM, with an average concentration of 0.51 microM. Significant quantities of oxidized proteins and lipids were detected. alpha-Tocopherol concentrations positively correlate with total neopterin levels. CONCLUSIONS: Total neopterin levels found in the pus was up to 100 times higher than that reported in plasma, suggesting plasma neopterin originates from inflammatory sites. The concentration of total neopterin suggests that 7,8-dihydroneopterin could act as an antioxidant during inflammation.
Subject(s)
Abscess/metabolism , Inflammation/physiopathology , Neopterin/blood , Oxidative Stress , Suppuration/metabolism , Adult , Aged , Aged, 80 and over , Dihydroxyphenylalanine/analysis , Female , Humans , Male , Middle Aged , Neopterin/analogs & derivatives , Neopterin/analysis , Tyrosine/analogs & derivatives , Tyrosine/analysis , alpha-Tocopherol/analysisABSTRACT
The adenoviral E1A CR2 mutant dl922-947 has potent activity in ovarian cancer. We have used Renilla luciferase bioluminescence imaging to monitor viral E1A expression and replication and [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to quantify the activity of dl922-947 in vivo. We created dlCR2 Ren, with the same E1A CR2 deletion as dl922-947 and the luciferase gene from Renilla reniformis downstream of E1. Light emitted from s.c. and i.p. IGROV1 ovarian carcinoma xenografts was measured following treatment with dlCR2 Ren. Mice bearing s.c. IGROV1 xenografts were injected with 2.96 to 3.7 MBq of [18F]FLT 48 and 168 hours following i.t. injection of dl922-947 or control virus Ad LM-X. The presence of Renilla luciferase in dlCR2 Ren did not reduce in vitro nor in vivo potency compared with dl922-947. Light emission correlated closely with E1A expression in vitro and peaked 48 hours after dlCR2 Ren injection in both s.c. and i.p. IGROV1 xenografts. It diminished by 168 hours in s.c. tumors but persisted for at least 2 weeks in i.p. models. Normalized tumor [18F]FLT uptake at 60 minutes (NUV60), fractional retention, and area under radioactivity curve all decreased marginally 48 hours after dl922-947 treatment and significantly at 168 hours compared with controls. There was a close linear correlation between NUV60 and both tumor proliferation (Ki67 labeling index) and thymidine kinase 1 expression. Renilla luciferase bioluminescence and [18F]FLT-PET imaging are capable of quantifying the activity and effectiveness of E1A CR2-deleted adenoviral mutants in ovarian cancer.
Subject(s)
Adenoviridae/physiology , Adenovirus E1A Proteins/biosynthesis , Adenovirus E1A Proteins/genetics , Dideoxynucleosides , Luciferases, Renilla/metabolism , Ovarian Neoplasms/virology , Adenoviridae/genetics , Adenoviridae/metabolism , Adenovirus E1A Proteins/metabolism , Animals , Cell Line, Tumor , Conserved Sequence , Female , Fluorine Radioisotopes , Gene Deletion , Humans , Luciferases, Renilla/biosynthesis , Luciferases, Renilla/genetics , Luminescent Measurements , Mice , Mice, Inbred BALB C , Oncolytic Virotherapy/methods , Ovarian Neoplasms/diagnostic imaging , Ovarian Neoplasms/therapy , Positron-Emission Tomography/methods , Radiopharmaceuticals , Virus Replication/physiology , Xenograft Model Antitumor AssaysABSTRACT
The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions.
Subject(s)
Lysosomes/metabolism , Metallothionein/metabolism , Oxidative Stress , Animals , Apoptosis , Autophagy , Cell Line , Iron/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Necrosis , Oxidation-Reduction , Oxidative Stress/drug effects , Protein Binding , Up-Regulation/drug effects , Zinc/pharmacologyABSTRACT
The atherosclerotic plaque is an inflammatory site where macrophage cells are exposed to cytotoxic oxidised low density lipoprotein (oxLDL). Interferon-gamma released from T-cells results in macrophage synthesis of 7,8-dihydroneopterin which has antioxidant and cytoprotective activity. Using the human derived monocyte-like U937 and THP-1 cell lines, we examined whether 7,8-dihydroneopterin could inhibit the cytotoxic effect of oxLDL. In U937 cells, oxLDL caused a dramatic loss of cellular glutathione and caspase independent cell death associated with phosphatidylserine exposure on the plasma membrane. 7,8-Dihydroneopterin completely blocked the cytotoxic effect of oxLDL. In contrast, oxLDL initiated THP-1 cell apoptosis with reduction in cellular thiols, caspase-3 activation and plasma membrane phosphatidylserine exposure. 7,8-Dihydroneopterin was unable to alter these processes or restore the THP-1 cellular thiol content. 7,8-Dihydroneopterin did provide some protection to both THP-1 cells and U937 cells from AAPH derived peroxyl radicals. The preincubation of oxLDL with 7,8-dihydroneopterin did not reduce cytotoxicity, suggesting that 7,8-dihydroneopterin may be acting in U937 cells by scavenging intracellular oxidants generated by the oxLDL. The data show that muM levels of 7,8-dihydroneopterin may prevent oxLDL mediated cellular death within atherosclerotic plaques.
Subject(s)
Apoptosis/drug effects , Atherosclerosis/metabolism , Lipoproteins, LDL/antagonists & inhibitors , Macrophages/metabolism , Neopterin/analogs & derivatives , Analysis of Variance , Caspase 3 , Caspases/metabolism , Cell Membrane/metabolism , Chromatography, High Pressure Liquid , Fluorescence , Glutathione/metabolism , Humans , Interferon-gamma/metabolism , Neopterin/pharmacology , Phosphatidylserines/metabolism , Sulfhydryl Compounds/metabolism , U937 CellsABSTRACT
Monocytic cell lines have been extensively used to characterize and model various features of the atherogenic process. We found striking differences in the apoptotic pathways of U937 cells and THP-1 cells exposed to copper-oxidized LDL. While phosphatidylserine exposure occurred in both lines, caspase activation was only apparent in the THP-1 cells. OxLDL caused caspase activity to decrease below that seen in untreated U937 cells, and this corresponded with a loss in intracellular thiols. In conclusion, exposure of U937 cells to oxLDL did not trigger a conventional apoptosis response, but still resulted in phosphatidylserine externalization.
Subject(s)
Caspases/metabolism , Lipoproteins, LDL/pharmacology , Monocytes/drug effects , Phosphatidylserines/metabolism , Apoptosis/physiology , Cell Line , Cell Shape , Cell Survival , Enzyme Activation , Humans , Monocytes/cytology , Monocytes/metabolism , Sulfhydryl Compounds/metabolismABSTRACT
Increasing evidence supports a role of cellular iron in the initiation and development of atherosclerosis. We and others reported earlier that iron-laden macrophages are associated with LDL oxidation, angiogenesis, nitric oxide production and apoptosis in atherosclerotic processes. Here we have further studied perturbed iron metabolism in macrophages, their interaction with lipoproteins and the origin of iron accumulation in human atheroma. In both early and advanced human atheroma lesions, hemoglobin and ferritin accumulation correlated with the macrophage-rich areas. Iron uptake into macrophages, via transferrin receptors or scavenger receptor-mediated erythrophagocytosis, increased cellular iron and accelerated ferritin synthesis at both mRNA and protein levels. The binding activity of iron regulatory proteins was enhanced by desferrioxamine (DFO) and decreased by hemin and iron compounds. Iron-laden macrophages exocytosed both iron and ferritin into the culture medium. Exposure to oxidized low-density lipoprotein (oxLDL, >or=50 microg/mL) resulted in <20% apoptosis of iron-laden human macrophages, but cells remained impermeable after a 24 h period and an increased excretion of ferritin could be observed by immunostaining techniques. Exposure to high-density lipoprotein (HDL) significantly decreased ferritin excretion from these cells. We conclude: (i) erythrophagocytosis and hemoglobin catabolism by macrophages contribute to ferritin accumulation in human atherosclerotic lesions and; (ii) iron uptake into macrophages leads to increased synthesis and secretion of ferritin; (iii) oxidized LDL and HDL have different effects on these processes.