The effectiveness of chemotherapy and electrochemotherapy on ovarian cell lines in vitro.

The presented study aimed to evaluate in vitro the effectiveness of improvement standard chemotherapy with bleomycin by electroporation in two various ovarian cancer cell lines. Two human ovarian cell lines OvBH-1 and SKOV-3 were used. The lines were selected because of their resistance to several therapeutic methods. As anticancer drug we use range of concentrations of bleomycin. In EP and ECT experiments different voltage values: from 0 to 1200 V/cm, 8 pulses with duration of 100µs and intervals between pulses 1s long were used. The cells viability after applied treatments was evaluated by MTT assay. The expression of heat shock proteins - HSP27 was examined by immunocytochemical ABC method.The cytotoxicity with different concentrations of bleomycin alone was not significantly decrease in both cell lines. It confirms resistance of these cells to conventional chemotherapy. The highest decrease of cell proliferation was observed after EP with bleomycin after 48h of incubation for 1000 V/cm. The intensity of expression of small heat shock proteins HSP27 slightly increased after ECT in both treated cell lines, in particular in OvBH-1. The presented study indicated that application of electroporation may effectively enhance chemotherapy with bleomycin, particularly in the case of treating ovarian cancer resistant to standard therapy.

Cellular stress induced by photodynamic reaction with CoTPPS and MnTMPyPCl5 in combination with electroporation in human colon adenocarcinoma cell lines (LoVo and LoVoDX).

Two porphyrins, CoTPPS and MnTMPyPCl5, were tested for their photodynamic activity and potential novel use in a therapy of human cancers. We investigated an effect of photodynamic reaction (PDR), electroporation (EP) and their combination (electro-photodynamic reaction [EP-PDR]) on human colon adenocarcinoma cell lines (LoVo and resistant to doxorubicin LoVoDX), human breast adenocarcinoma (wild type MCF-7/WT and resistant to doxorubicin MCF-7/DOX), and human melanoma (Me45). The efficiency of macromolecules transport was examined with cytofluorymetry by assessing the degree of propidium iodide (PI) penetration. Additionally, cellular ultrastructure after EP was evaluated. We determined cyto- and photo-cytotoxic effect on the cells viability (MTT assay) after standard PDR and PDR combined with EP. Intracellular distribution and mitochondrial colocalization of both porphyrins was also performed. The experiments proved that both complexes exhibit desirable photodynamic properties on LoVo LoVoDX cells, and EP effectively supports photodynamic method in this type of cancer. The application of EP provided shorter time of incubation (only 10 min) and enhanced effect of applied therapy. The porphyrins did not affect the MCF-7 and Me45 cell lines.

Cytotoxic potential of vasoconstrictor experimental gingival retraction agents: in vitro study on primary human gingival fibroblasts.

The aim of this in vitro study was to evaluate the cytotoxic effects of the vasoconstrictor experimental gingival retraction agents (VEGRAs) in a dynamic setting. The strong cytotoxic effects of the astringent-based conventional gingival retraction agents (ACGRAs) on human gingival fibroblasts (HGFs) in vitro was our motivation to evaluate the biocompatibility of the vasoconstrictor-based experimental gingival retraction agents (VEGRAs) for the selected minimally invasive chemical agent. These agents were used to create three self-made retraction gels. Human gingival fibroblasts (HGFs) were treated with two groups of retraction agents: 1) three α- and ß-adrenergic agents (VEGRA-αß-s) based on 0.1%, 0.01% and 0.05% HCl-epinephrine, and 2) seven α-adrenergic agents (VEGRA-α-s), including two commercially available 0.05% HCl-tetrahydrozoline solutions, one 0.05% HCl-oxymetazoline solution, 10% HCl-phenylephrine solution, and three new self-made experimental 0.05% HCl-tetrahydro zoline-based gels. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was performed to determine the oxidoreductive mitochondrial function after 3, 5, 10 min and 24 h of incubation. The cytotoxic effect, measured by cell viability lower than the 50% threshold, was not observed at any time period, even 24 h after application of 0.05% HCl-tetrahydrozolinebased self-manufactured retraction gels. High cell viability values of human gingival fibroblasts after the treatment with the three self-made 0.05% HCl-tetrahydrozoline- based gels may serve as a basis for further studies aimed at selecting the best retraction agents biocompatible with gingival margin tissues.

The influence of electroporation on in vitro photodynamic therapy of human breast carcinoma cells.

Phototoxicity of drugs used in cancer photodynamic therapy could be augmented by increased accumulation of a photosensitizer in target cells. The intracellular delivery mode that enhances drug transportation could facilitate therapy by reducing the exposure time. Doses of the administered drug and related side effects could be lowered, whilst maintaining the same therapeutic efficiency. Electroporation supports transport of many drugs by creating electric field-induced transient nanopores in the plasma membrane. In this study, the electroporation- assisted transport of a photosensitizer was tested in vitro in human breast carcinoma cell lines: wild-type (MCF-7/WT) and doxorubicin-resistant (MCF-7/DOX). The efficacy of photodynamic therapy alone and in combination with electroporation was evaluated by cell viability with MTT test, using a haematoporphyrin derivative as a model. The data presented show up to 10-fold greater efficacy of the combined method, with very significantly reduced drug exposure times.

Cyanines as efficient photosensitizers in photodynamic reaction: photophysical properties and in vitro photodynamic activity.

The purpose of the present study was to explore the potential application of cyanines in photodynamic treatment. The photophysical features of four cyanines (KF570, HM118, FBF-749, and ER-139) were investigated by elemental and spectral analyses. Two malignant cell lines (MCF-7/WT and MCF-7/DOX) were used to test the potential for use in the photodynamic therapy. The cytotoxic effects of these dyes were determined by the MTT assay after 4 and 24 h of incubation with the cyanine. KF570 and HM118 were irradiated with red light (630-nm filter) and FBF-749 and ER-139 with green light (435-nm filter). The results showed that the cyanine HM118 demonstrated a major phototoxic effect. It was also noted that the efficiency of photodynamic therapy was higher in the doxorubicin-resistant cell line (MCF-7/DOX).

The influence of phenothiazine derivatives on doxorubicin treatment in sensitive and resistant human breast adenocarcinoma cells.

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.

Dynamic oxidoreductive potential of astringent retraction agents.

The aim of this study was to evaluate the dynamics of the cytotoxicity of gingival margin retraction astringents based on aluminium chloride, aluminium sulphate, and ferric sulphate (solutions and gels) in human fibroblasts isolated from gingiva. The cytocompatibility of ten astringent-based chemical retraction agents: Gingiva Liquid, Alustin, Racestypine, Orbat sensitive, Astringedent®, Alustat, Hemostat, Racécord, Gel cord and ViscoStat®, in dilutions of 1 : 10 and 1 : 20, with human gingival fibroblasts was investigated. The MTT assay was performed to determine oxidoreductive mitochondrial function after 3, 5, 10 min and 24 h of incubation. Cell viability was determined according to the chemical group, concentration, exposure time, and the clinical form of the gingival retraction agents. Ferric sulphate- based agents were the most cytotoxic, followed by aluminium chloride and aluminium sulphate. The form of the astrigents influenced cell viability. The evaluated astringents may have cytotoxic potential for gingival margin tissues under clinical conditions.

Photo-oxidative action in cervix carcinoma cells induced by HPD - mediated photodynamic therapy.

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.

Photooxidative action in cancer and normal cells induced by the use of photofrin in photodynamic therapy.

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.

Intracellular distribution of Photofrin in malignant and normal endothelial cell lines.

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.

The influence of photodynamic therapy on apoptosis in human melanoma cell line.

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.

Levels of lipid peroxidation in A549 cells after PDT in vitro.

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.

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis.

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.

Immunocytochemical evaluation of reorganisation of keratinocyte cytoskeleton induced by change in Ca2+ concentration in culture medium.

Ca2+ level-induced changes in the arrangement of cytoskeleton of cultured keratinocytes were estimated immunocytochemically, by evaluating expression of specific cytokeratins, desmoplakin and tubulin. Keratinocytes were isolated from fragments of skin of dead human foetuses. Culture of epidermal cells was performed in two phases: phase I yielded cells of high proliferation abilities in serum-free Keratinocyte SFM of low Ca2+ level (0.03 mM); in phase II differentiated cells were obtained in Dulbecco medium of a high Ca2+ concentration (1.2 mM). Immunocytochemical evaluation of phase I and II cells revealed an array of differences which involved mainly expression and distribution of specific cytokeratins, distribution of tubulin, testifying to a different microtubule arrangement and distribution of desmoplakin and indicating a tendency to form desmosomes. The changes were induced by the changes in Ca2+ level in the culture medium.

Variability of the reproductive system of Piscicola fasciata (Kollar, 1842 (Hirudinea, Piscicolidae).

The studies on the reproductive system of P. fasciata were based on 43 specimens. Two main types of the reproductive system can be distinguished in the studied population. Two types of reproductive system were the most frequent (20.93% each): with symmetrical ejaculatory ducts without a loop and symmetrical seminal vesicles reaching the end of the first pair of testes, situated next to the testes (adult individuals) (fig. 23) and with symmetrical ejaculatory ducts without a loop (fig. 21) and symmetrical poorly developed seminal vesicles situated asymmetrically anterior to the first pair of testes (juvenile individuals). The structure of the reproductive system corresponding with the description of the type specimen (BRUMPT, 1900) was not observed in the studied population.

Variability of the reproductive system of Piscicola respirans (Troschel, 1850) (Hirudinea, Piscicolidae).

The studies on the reproductive system of P. respirans, based on 115 specimens, have revealed its considerable variability. Two main types of the system can be distinguished: 1) ovaries reaching the first pair of testes, ejaculatory ducts symmetrical, no seminal vesicles (juvenile individuals), 2) ovaries reaching the first pair of testes, ejaculatory ducts symmetrical, seminal vesicles reaching the ends of ovaries (adults individuals). The structure of the reproductive system is correlated with the individual size and age. Because the reproductive system of P. respirans as described by BRUMPT (1900) and SKET (1968) based on type specimens designated by them, is found only in a small fraction (c. 4-8%) of specimens, a redescription of the species is necessary, best based on a neotype series.