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1.
EJNMMI Res ; 9(1): 55, 2019 Jun 21.
Article in English | MEDLINE | ID: mdl-31227938

ABSTRACT

BACKGROUND: This study investigated the noninvasive assessment of tumor vascularization with clinical F-18-fluorodeoxyglucose positron emission tomography/computed tomography and contrast-enhanced computed tomography ([18F]FDG-PET/CT and CE-CT) in experimental human xenograft tumors with modifiable vascularization and compared results to histology. Tumor xenografts with modifiable vascularization were established in 71 athymic nude rats by subcutaneous transplantation of human non-small-cell lung cancer (NSCLC) cells. Four different groups were transplanted with two different tumor cell lines (either A549 or H1299) alone or tumors co-transplanted with rat glomerular endothelial (RGE) cells, the latter to increase vascularization. Tumors were assessed noninvasively by [18F]FDG PET/CT and contrast-enhanced CT (CE-CT) using clinical scanners. This was followed by histological examinations evaluating tumor vasculature (CD-31 and intravascular fluorescent beads). RESULTS: In both tumor lines (A549 and H1299), co-transplantation of RGE cells resulted in faster growth rates [maximal tumor diameter of 20 mm after 22 (± 1.2) as compared to 45 (± 1.8) days, p < 0.001], higher microvessel density (MVD) determined histologically after CD-31 staining [171.4 (± 18.9) as compared to 110.8 (± 11) vessels per mm2, p = 0.002], and higher perfusion as indicated by the number of beads [1.3 (± 0.1) as compared to 1.1 (± 0.04) beads per field of view, p = 0.001]. In [18F]FDG-PET/CT, co-transplanted tumors revealed significantly higher standardized uptake values [SUVmax, 2.8 (± 0.2) as compared to 1.1 (± 0.1), p < 0.001] and larger metabolic active volumes [2.4 (± 0.2) as compared to 0.4 (± 0.2) cm3, p < 0.001] than non-co-transplanted tumors. There were significant correlations for vascularization parameters derived from histology and [18F]FDG PET/CT [beads and SUVmax, r = 0.353, p = 0.005; CD-31 and SUVmax, r = 0.294, p = 0.036] as well as between CE-CT and [18F]FDG PET/CT [contrast enhancement and SUVmax, r = 0.63, p < 0.001; vital CT tumor volume and metabolic PET tumor volume, r = 0.919, p < 0.001]. CONCLUSIONS: In this study, a human xenograft tumor model with modifiable vascularization implementable for imaging, pharmacological, and radiation therapy studies was successfully established. Both [18F]FDG-PET/CT and CE-CT are capable to detect parameters closely connected to the degree of tumor vascularization, thus they can help to evaluate vascularization in tumors noninvasively. [18F]FDG-PET may be considered for characterization of tumors beyond pure glucose metabolism and have much greater contribution to diagnostics in oncology.

2.
Anticancer Res ; 32(4): 1239-43, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22493354

ABSTRACT

BACKGROUND/AIM: This study specifies a strategy to improve radiotherapy by partial synchronization of p53-deficient cancer cells (FaDu and H1299) in mitosis using taxol, with protecting p53 wild-type cells (A549) by the prior administration of cytostatic compounds. Cytotoxic and cytostatic effects of ionizing radiation, cisplatin, doxorubicin and taxol, administrated alone or in combination were investigated in vitro by flow cytometry. RESULTS: A protective effect of doxorubicin but not cisplatin was found after administration of triple sequence with ionizing radiation and taxol. It was found that preliminary administration of doxorubicin induced growth arrest and protected A549 cells from the taxol/radiation treatment, while simultaneously killing FaDu and H1299 cells. CONCLUSION: The proposed therapeutic strategy allows protection of p53 wild-type cells and selectively increases radiosensitivity of p53-deficient cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Lung Neoplasms/metabolism , Radiation Tolerance/drug effects , Tumor Suppressor Protein p53/metabolism , Cell Line, Tumor , Cisplatin/pharmacology , Doxorubicin/pharmacology , Flow Cytometry , Humans , Lung Neoplasms/pathology
4.
Nutr Cancer ; 62(6): 795-801, 2010.
Article in English | MEDLINE | ID: mdl-20661829

ABSTRACT

This study specifies the basic principles to selectively kill p53-deficient cells (H1299, FaDu) by taxol and to protect p53 wild type cells (A549) by the prior administration of structurally related flavonoids (apigenin, genistein, and quercetin). Cytotoxic and cytostatic properties of flavonoids were investigated in vitro by flow cytometry and were compared to known anticancer drugs (cisplatin, doxorubicin, etoposide). It was confirmed that doxorubicin induced growth arrest and protected A549 cells from taxol while simultaneously killing or blocking H1299 and FaDu cancer cells. It was found that doxorubicin could be successfully substituted in this way by the isoflavone genistein used at physiologically relevant concentrations. The other compounds analyzed revealed less selectivity (apigenin, cisplatin) or demonstrated higher toxicity (cisplatin, etoposide, and quercetin). We concluded that genistein-based therapy may have antagonistic effects when combined with mitotic poisons. The proposed therapeutic strategy allows protection of p53 wild type cells from taxol and selectively increases apoptosis in p53-deficient cells. This strategy exploits the naturally occurring compound that can be used without significant toxicity in rather high concentrations as present in common diets.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Genistein/pharmacology , Lung Neoplasms/drug therapy , Paclitaxel/pharmacology , Tumor Suppressor Protein p53/physiology , Cell Cycle/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Etoposide/pharmacology , Humans , Lung Neoplasms/pathology
5.
J Biomed Biotechnol ; 2010: 580531, 2010.
Article in English | MEDLINE | ID: mdl-20368799

ABSTRACT

Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC) xenografts in a nude rat model irradiation (2 + 2 Gy) from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours.


Subject(s)
Carcinoma, Non-Small-Cell Lung/immunology , Disease Models, Animal , Immunosuppression Therapy/methods , Lung Neoplasms/immunology , Whole-Body Irradiation/methods , Xenograft Model Antitumor Assays/methods , Analysis of Variance , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Disease Progression , Humans , Lung Neoplasms/pathology , Male , Monte Carlo Method , Phantoms, Imaging , Rats , Rats, Nude , X-Rays
6.
BMC Cancer ; 10: 57, 2010 Feb 23.
Article in English | MEDLINE | ID: mdl-20178585

ABSTRACT

BACKGROUND: Mutations within the tumor suppressor TP53 gene are one of the most common genetic alterations present at high frequency in human tumors and have been shown to be associated with resistance to radio-chemotherapy. The lack of the wild type TP53 gene in cancer cells could be exploited for therapeutic advantage using a sequence of two antagonistic drugs. The aim of this study was to selectively kill p53 deficient cells (FaDu and H1299) by taxol and to protect p53 wild type cells (A549) by the prior administration of nutlin-3 in comparison to certain known anticancer drugs (5-fluorouracil, camptothecin, roscovitine). METHODS: Cytotoxic and cytostatic properties of 5-fluorouracil, camptothecin, roscovitine and nutlin-3 administrating alone or in combination with taxol were investigated in vitro by flow cytometry. RESULTS: It was found that nutlin-3 induced growth arrest and protected A549 cells from taxol. FaDu and H1299 cells responded to the same treatments with mitotic arrest and massive apoptosis. Other compounds (5-fluorouracil, camptothecin and roscovitine) revealed weaker selectivity and elevated toxicity in comparison to nutlin-3. CONCLUSIONS: We propose a therapeutic strategy protecting normal cells from taxol while increasing apoptosis selectively in p53-deficient cells using nutlin-3.


Subject(s)
Gene Expression Regulation, Neoplastic , Imidazoles/administration & dosage , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Piperazines/administration & dosage , Tumor Suppressor Protein p53/physiology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis , Camptothecin/administration & dosage , Cell Line, Tumor , Drug Screening Assays, Antitumor , Flow Cytometry/methods , Fluorouracil/administration & dosage , Humans , Models, Genetic , Purines/administration & dosage , Roscovitine , Tumor Suppressor Protein p53/genetics
7.
Int J Radiat Biol ; 85(9): 747-51, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19444764

ABSTRACT

PURPOSE: The aim of our study was to develop a method for the fusion of images received after repeated staining of the same sample taking into account spatial differences between the images. MATERIAL AND METHODS: A method of objective fusion performance was investigated on the images receiving during multistep staining of the xenograft tumour cross-sections. RESULTS: It was shown that several images receiving from different steps of staining procedures may be successfully fused by fluorescent marking of slide position with Trout red blood cells before analysis. CONCLUSIONS: Proposed technique provides an accurate rigid fusion of light and fluorescent images receiving during multistep image analysis under microscope and may be applied for study of neovascularisation.


Subject(s)
Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/pathology , Staining and Labeling/methods , Animals , Cell Line, Tumor , Humans , Male , Microscopy, Fluorescence , Neoplasm Transplantation , Rats , Rats, Nude
8.
Mol Cells ; 24(2): 255-60, 2007 Oct 31.
Article in English | MEDLINE | ID: mdl-17978579

ABSTRACT

Bone marrow (BM) derived mesenchymal stem cells (MSC) are pluripotent cells which can differentiate into osteogenic, adipogenic and other lineages. In spite of the broad interest, the information about the changes in BM cell composition, in particularly about the variation of MSC number and their properties in relation to the age of the donor is still controversial. The aim of this study was to investigate the age associated changes in variations of BM cell composition, phenotype and differentiation capacities of MSC using a rat model. Cell populations were characterized by flow cytometry using light scattering parameters, DNA content and a set of monoclonal antibodies. Single cell analysis was performed by conventional fluorescent microscopy. In vitro culture of MSC was established and their phenotype and capability for in vitro differentiation into osteogenic and adipogenic cells was shown. Age related changes in tibiae and femurs, amount of BM tissue, BM cell composition, proportions of separated MSC and yield of MSC in 2 weeks of in vitro culture were found. At the same time, neither change in phenotype no in differentiation capacities of MSC was registered. Age-related changes of the number of MSC should be taken into account whenever MSC are intended to be used for investigations.


Subject(s)
Aging/physiology , Bone Marrow Cells/cytology , Cell Differentiation , Mesenchymal Stem Cells/cytology , Adipogenesis , Animals , Cell Count , Cell Proliferation , Cell Separation , Femur/cytology , Male , Organ Size , Osteogenesis , Phenotype , Rats , Rats, Wistar , Tibia/cytology
9.
Stem Cells Dev ; 16(3): 439-46, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17610374

ABSTRACT

Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into endothelial, osteogenic, adipogenic, and other lineages. In spite of the broad interest, little is known about the variation of MSC number in relation to the age of the donor. The aim of this study was to investigate the age-associated variations of bone marrow (BM) MSCs using a rat model. Cell populations were characterized by flow cytometry using light-scattering parameters, DNA content and a set of monoclonal antibodies and detected by magnetic resonance imaging (MRI). Single-cell analysis was performed by conventional fluorescent microscopy. BM mononucleated cells (MNCs) were isolated, in vitro culture of MSCs was established, and endothelial cells differentiation and intracellular magnetic labeling was shown. The amount of BM tissue obtainable from femurs and tibiae increased with age and reached a maximum in 8- to 12-week-old rats. At the same time, the proportional number of MNCs containing MSCs decreased. As a result, after 2 weeks of culture, the maximum yield of MSC number was registered from the youngest age group (4 weeks). MSCs were differentiated into endothelial cells by administration of vascular endothelial growth factor (VEGF) and subsequently revealed immunocytochemical and morphological characteristics of endothelial cells. The results of our study are the basis for further experiments with MSCs and their endothelial descendants, which may be labeled with different agents for cell tracking and detection experiments, but age-related changes in MSCs number should be taken into account whenever these cells are considered for practical applications.


Subject(s)
Aging/physiology , Bone Marrow Cells/physiology , Mesoderm/cytology , Pluripotent Stem Cells/physiology , Animals , Antigens, CD/metabolism , Bone Marrow Cells/cytology , Cell Separation , Cells, Cultured , Flow Cytometry , Humans , Male , Pluripotent Stem Cells/cytology , Rats , Rats, Wistar
10.
J Exp Zool B Mol Dev Evol ; 308(2): 127-38, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-16788897

ABSTRACT

Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.


Subject(s)
Cichlids/metabolism , Lectins/metabolism , Oryzias/metabolism , Spermatogenesis/physiology , Testis/cytology , Animals , Boron Compounds , Cells, Cultured , Extracellular Fluid/metabolism , Flow Cytometry , Fluorescein-5-isothiocyanate , Histocytochemistry , Male , Microscopy, Confocal , Protein Binding , Testis/metabolism
11.
Biol Chem ; 386(3): 279-83, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15843173

ABSTRACT

The metabolism of the flavonol quercetin in human leukaemia (HL-60) cells was investigated. The fluorescence that is elicited by quercetin upon binding to a target protein was quickly attenuated in vital cells, while apoptotic cells showed persistent fluorescence. The dynamics of induction and loss of fluorescence in the cells were quantified by flow cytometry. Several potential metabolites of quercetin, apart from isorhamnetin, had weak or no fluorogenic properties with test proteins. HPLC analysis showed that quercetin was metabolised to several substances, among them glycosylated metabolites. The loss of fluorescence in vital cells offers the unique opportunity to directly observe the metabolic conversion of quercetin in human cells.


Subject(s)
Apoptosis , Quercetin/metabolism , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology
12.
J Agric Food Chem ; 52(10): 2915-23, 2004 May 19.
Article in English | MEDLINE | ID: mdl-15137835

ABSTRACT

Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.


Subject(s)
Cinnamates/metabolism , Cyclopentanes/pharmacology , Lavandula/metabolism , Antioxidants/analysis , Caffeic Acids/analysis , Caffeic Acids/metabolism , Cells, Cultured , Cinnamates/analysis , Cinnamates/chemistry , Culture Media, Conditioned , Depsides , Gas Chromatography-Mass Spectrometry , Hydrogen-Ion Concentration , Oxygen/administration & dosage , Oxylipins , Rosmarinic Acid
13.
Pharmacology ; 71(1): 46-56, 2004 May.
Article in English | MEDLINE | ID: mdl-15051922

ABSTRACT

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4'-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4',7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 micromol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (DeltaPsim) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules.


Subject(s)
Cell Division/drug effects , Flavanones/chemical synthesis , Flavanones/toxicity , Benzimidazoles/pharmacology , Breast Neoplasms/pathology , Carbocyanines/pharmacology , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/physiology , Dose-Response Relationship, Drug , Flavanones/pharmacology , Fluorescent Dyes/pharmacology , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Germany, East , HL-60 Cells , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mitochondria/drug effects , Mitochondria/physiology , Quercetin/pharmacology , Reactive Oxygen Species/antagonists & inhibitors , Time Factors , Vitamin K 3/pharmacology
14.
Environ Res ; 94(2): 145-51, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14757377

ABSTRACT

The aim of this study was to demonstrate the expression of heat shock (HS) genes in human cells in response to extremely low-frequency electromagnetic fields (ELF-EMF) alone and in combination with thermal stress. After exposing human myeloid leukemia (HL-60) cells to the stressor(s) for 30 min we quantified the expression of the HS genes HSP27, HSP60, HSP70 (A, B, and C), HSC70, HSP75, HSP78, and HSP90 (alpha and beta) by RT-PCR. The results clearly show that HS genes, in particular the three HSP70 genes (A, B, and C), are induced by ELF-EMF, a reaction that is enhanced by simultaneous HS (43 degrees C for 30 min). The results show similarities and some significant differences to previous experiments in which transgenic nematodes were used to monitor the induction of the HSP70 gene under similar stress conditions. We also studied the effect of different flux densities on gene expression in the range of 10-140 microT. Even the lowest dose tested (10 microT) resulted in a significant induction of the genes HSP70A, HSP70B, and HSP70C. The reaction to ELF-EMF shows a maximum at a flux density of 60-80 microT. The unusual dose-response relation reveals an interesting difference to other stressors that elicit the HS response.


Subject(s)
Electromagnetic Fields/adverse effects , Gene Expression Regulation , Heat-Shock Proteins/biosynthesis , Leukemia, Myeloid/pathology , Acute Disease , Blotting, Western , Flow Cytometry , Heat-Shock Proteins/analysis , Humans , Reverse Transcriptase Polymerase Chain Reaction , Temperature , Tumor Cells, Cultured
15.
J Agric Food Chem ; 52(2): 239-45, 2004 Jan 28.
Article in English | MEDLINE | ID: mdl-14733502

ABSTRACT

The biological effects of epicuticular substances in farinose exudates accumulated on inflorescence shafts and calyces of Primula denticulata on human acute myeloid leukemia cells (HL-60) were analyzed. The crude material possessed little antioxidative capacity but strong cytostatic properties. Some of its known components (5-hydroxyflavone, 2'-hydroxyflavone, 5,2'-dihydroxyflavone, and 5,8-dihydroxyflavone) were further tested to identify the biologically active compounds. The effects of these flavones on cell cycle progression, mitochondrial membrane potential, and reactive oxygen species have been investigated by flow cytometry. The flavonol quercetin was included in the study as reference compound because of its known cytostatic properties and its activity as radical scavenger. Compared to quercetin the flavones induced little apoptosis (up to 40 microM), but despite their low toxicity, the Primula flavonoids possessed strong cytostatic properties even at low concentrations. The cell cycle distribution showed a characteristic time-dependent shift, giving evidence of a generally short-lived effect of the test compounds in the exposed cells. The antioxidative properties quantified according to two different methods correlated with the number of hydroxyl groups. Whereas quercetin strongly affected the mitochondrial membrane potential, none of the Primula flavones showed a comparable effect.


Subject(s)
Cell Division/drug effects , Flavonoids/pharmacology , Leukemia/pathology , Primula/chemistry , Antioxidants/pharmacology , Apoptosis/drug effects , Flavonoids/isolation & purification , Free Radical Scavengers/pharmacology , HL-60 Cells , Humans , Membrane Potentials/drug effects , Mitochondria/ultrastructure , Quercetin/pharmacology
16.
Cell Prolif ; 36(2): 101-11, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12680877

ABSTRACT

Stress-induced effects in human acute leukaemia cells (HL-60) were studied by flow cytometry using the fluorescent dye carboxyfluorescein succinimidyl ester which allows the analysis of several successive cell generations for up to 10 days. Asynchronously cycling cells subjected to heat shock (30 min at 41 degrees C) responded in two distinct ways: while one fraction of the cell population (about 15%) re-entered the cell cycle after a short delay, other cells became arrested at different phases of the cell cycle and remained arrested for up to several days and finally underwent apoptosis. Weak electromagnetic fields (60 micro T, 50 Hz) alleviated the heat-induced block and the fraction of arrested cells was significantly smaller.


Subject(s)
Cell Cycle , Electromagnetic Fields , Heat-Shock Response , Cell Division , Flow Cytometry , Fluoresceins , Fluorescent Dyes , HL-60 Cells , Humans , Kinetics , Succinimides
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