ABSTRACT
The original version of this article unfortunately contained a mistake in the author group section. Author A. Bronowicka-Szydelko's surname was inadvertently interchanged to "Szydelko-Bronowicka".
ABSTRACT
The most abundant proteins in the arteries are those of extracellular matrix, ie. collagen and elastin. Due to their long half-lifes these proteins have an increased chance to undergo glycation. The aim of this study was to determine relationship between the content of the main extracellular matrix proteins and the advanced glycation end products (AGEs) in arteries. In this study 103 fragments of aorta were analyzed by ELISA and immunobloting for the content of collagens type I, III and IV and elastin and the content of advanced glycation end-products (AGE). A negative correlation between the content of collagens type III and IV and AGE (r = -0,258, p = 0,0122, and a weak negative correlation between collagen type III and age of the sample donor (r = 0,218, p = 0,0262) were demonstrated. This result comes as a surprise and it contradicts an intuitive assumption that with more glycation substrate, i.e. matrix proteins, more AGE products are expected. We have concluded that the results of the ELISA tests must have been influenced by the glycation. As a consequence, either modified protein molecules were not being recognized by the antibodies, or the glycation, and formation of crosslinks have blocked access of the antibodies to the antigen. It will conceal the effect of the linear dependence between the result (absorbance/densitometry) from the quantity of protein to which the antibody is directed.
Subject(s)
Artifacts , Glycation End Products, Advanced/immunology , Immunoenzyme Techniques/standards , Adult , Aged , Aorta/chemistry , Collagen/analysis , Collagen/immunology , Elastin/analysis , Elastin/immunology , Female , Humans , Male , Middle AgedABSTRACT
Breast cancer is commonly treated by various combinations of surgery, radiation therapy, chemotherapy and hormone therapy. Most cancers either are increasingly resistant to any initial treatment or acquire resistance to a broad spectrum of anticancer drugs over time. Combination of more than one drug or combination with multidrug resistance (MDR) modifiers will possibly support the efficiency of the applied therapy. Understanding the MDR mechanisms in malignancies is crucial for developing novel strategies for treatment. The main goal of our study was to determine the cytostatic effect of doxorubicin in combination with phenothiazine derivatives (PD; promazine and triflupromazine) in doxorubicin-sensitive (MCF-7/WT) and -resistant (MCF-7/DOX) human breast adenocarcinoma cell lines. We determined cytotoxicity of the investigated compounds (MTT assay) after 24 and 48 h. The effect of phenothiazine derivatives was evaluated and doxorubicin localization was performed using confocal microscopy. The mode of the cell death was examined by the comet assay. We also determined the expression of P-glycoprotein (P-gp), which is a membrane-associated protein responsible for the multidrug resistance.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm , Phenothiazines/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cell Line, Tumor , Comet Assay , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Female , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Phenothiazines/pharmacologyABSTRACT
UNLABELLED: Photodynamic therapy leads to oxidative stress through the generation of free radicals. Oxidative stress causes damage to cellular macromolecules such as nucleic acids, proteins and lipids. AIM: To examine the hematoporphyrin derivative (HpD) - mediated photodynamic effect on cervical adenocarcinoma cell line HeLa. METHODS: The HpD localization in HeLa cells was analyzed by confocal microscopy with epi-fluorescence system. Lipid peroxidation (LPO) was estimated by measurement of the concentration of malondialdehyde, protein degradation - by modified Ellman's method, superoxide dysmutase (SOD) - using Ransod Kit. The expression of inducible nitric oxide synthase (iNOS) was detected by immunocytochemical staining. RESULTS: The HpD was distributed all over the cytoplasm with preferential localization in the inner side of the plasma membrane and around the nuclear envelope. The process of photosensitizer distribution was time dependent. PDT-HpD increased the level of malonodialdehyde (MDA), SOD activity and the expression of iNOS in HeLa cells. However, PDT induced the decrease in the level of protein-associated thiol groups. CONCLUSIONS: Our study showed the important role of PDT-mediated oxidative stress in HeLa cells. HpD-PDT might be alternative and less invasive approach for treatment of patients with cervical cancer resistant for standard chemotherapy and radiotherapy.
Subject(s)
Adenocarcinoma/therapy , Hematoporphyrin Derivative/pharmacology , Photochemotherapy/methods , Uterine Cervical Neoplasms/therapy , Adenocarcinoma/metabolism , Female , HeLa Cells , Humans , Immunohistochemistry , Lipid Peroxidation/drug effects , Microscopy, Confocal , Nitric Oxide Synthase Type II/drug effects , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/drug effects , Superoxide Dismutase/drug effects , Superoxide Dismutase/metabolism , Uterine Cervical Neoplasms/metabolismABSTRACT
Photofrin-mediated PDT was applied to malignant (A549 and MCF-7) and normal (HUV-EC-C) cells. The cells were incubated for different lengths of time after PDT. The cell responses to the therapy were examined by changes in SOD activity, phototoxicity, and mode of the cell death. PDT induced dynamic changes in SOD activity. Initially, an increase in SOD activity was observed, and after 6 hours of culture it decreased to the control level. Results obtained from MTT and the comet assay indicate that PDT caused immediate cell death via apoptosis in the A549, MCF-7, and HUV-EC-C cell lines. Our studies confirm that SOD is involved in the response of both cancer and normal cells to PDT.
Subject(s)
Dihematoporphyrin Ether/pharmacology , Light , Neoplasms/drug therapy , Neoplasms/pathology , Photochemotherapy , Aged , Cell Line, Tumor , Comet Assay , Female , Formazans , Humans , Male , Middle Aged , Oxidation-Reduction/radiation effects , Photosensitizing Agents/pharmacology , Superoxide Dismutase/metabolism , Tetrazolium SaltsABSTRACT
Compared to current treatments including surgery, radiation therapy, and chemotherapy, PDT offers the advantage of an effective and selective method of destroying diseased tissues without damaging surrounding healthy tissues. One of the aspects of antitumour effectiveness of PDT is related to the distribution of photosensitizing drugs. The localization of photosensitizers in cytoplasmic organelles during PDT plays a major role in the cell destruction; therefore, intracellular localization of Ph in malignant and normal cells was investigated. The cell lines used throughout the study were: human malignant A549, MCF-7, Me45 and normal endothelial cell line HUV-EC-C. After incubation with Ph cells were examined using fluorescence and confocal microscopy to visualize the photosensitizer accumulation. For cytoplasm and mitochondria identification, cells were stained with CellTracker Green and MitoTracker Green, respectively. Distribution of Ph was different in malignant and normal cells and dependent on the incubation time. The maximal concentration of Ph in two malignant cell lines (A549 and MCF-7) was observed after 4 hours of incubation, and the most intensive signal was observed around the nuclear envelope. Intracellular distribution of Ph in the Me45 cell line showed that the fluorescence emitted by Ph overlaid that from MitoTracker. This indicates preferential accumulation of the sensitizer in mitochondria. Our results based on the mitochondrial localization support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.
Subject(s)
Dihematoporphyrin Ether/metabolism , Endothelial Cells/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Adult , Aged , Biological Transport , Endothelial Cells/cytology , Female , Humans , Male , Microscopy, Confocal , Microscopy, Fluorescence , Middle Aged , Tumor Cells, CulturedABSTRACT
Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegröm Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.
Subject(s)
Apoptosis/drug effects , Melanoma/drug therapy , Melanoma/pathology , Photochemotherapy , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Nucleus/pathology , Comet Assay , DNA Damage , Dihematoporphyrin Ether/pharmacology , Dihematoporphyrin Ether/therapeutic use , HumansABSTRACT
Photodynamic therapy (PDT) is an increasingly used treatment for various types of cancer. The principle of PDT involves the administration of a photosensitizer, followed by a distribution interval, and subsequent illumination of tumour area with light of an appropriate wavelength to excite the sensitizer to its triplet state. The aim of the study was to determine the level of lipid peroxidation and the level of thiol groups (-SH) in A549 cells after PDT. The final product of fatty acid peroxidation--malondialdehyde--was quantified Spectrophotometrically, based on a set of MDA standards of known concentration. Protein damage was based on Ellman's method. The level of lipid peroxidation was significantly higher for cells after PDT, comparing to control cells. We observed much lower concentrations of -SH groups for cells after PDT treatment, in comparison with respective values in control cells. In conclusion, PDT with Ph II induces lipid peroxidation with accompanying protein damage in A549 cells, what can lead to distinct epidemiological, pathological and clinical features.
Subject(s)
Dihematoporphyrin Ether/pharmacology , Lipid Peroxidation/drug effects , Photochemotherapy , Cell Line, Tumor , Humans , Lung Neoplasms , Malondialdehyde/metabolismABSTRACT
Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.