EFFECT OF THE ZINC PHTHALOCYANINE MEDIATED PHOTODYNAMIC THERAPY ON CYTOSKELETAL APPARATUS OF HELA CELLS

This study deals with the utilization of photosensitizer (λmax ~ 660 nm) from the group of the phthalocyanines, in photodynamic therapy. Effect of the zinc phthalocyanine photosensitizer mediated photodynamic therapy was evaluated in vitro on the tumor cell line – HeLa (cervical cancer cells) using mass spectrometry and atomic force and fluorescent microscopy techniques

IMUNOFLUORESCENČNÍ ANALÝZA PROAPOPTICKÝCH SIGNÁLNÍCH MOLEKUL V BUŇKÁCH LIDSKÉHO MELANOMU PO FOTODYNAMICKÉ TERAPII

IMMUNOFLUORESCENCE ANALYSIS OF PROAPOPTOTIC SIGNALING MOLECULES IN HUMAN MELANOMA CELLS AFTER PHOTODYNAMIC TREATMENT. Photodynamic therapy (PDT) is connected with oxidative damage of biomolecules causing significant impairment of essential cellular functions that lead to cell death. It is the reason, why photodynamic therapy has also found its application in treatment of different oncological, cardiovascular, skin and eye diseases. The cell death after PDT is mediated by an apoptotic and/or necrotic process including activation of various biomolecules. In the presented study we have used immunofluorescence method to detect caspase 3 and 9, poly ADP-ribose polymerase (PARP) in their active forms, and release of the cytochrome c as the proapoptotic protein after photodynamic treatment of human melanoma cells

FOTOTOXICKÝ VLIV PORFYRINOVÝCH SENSITIZERŮ A VIDITELNÉHO ZÁŘENÍ NA GRAM-POZITIVNÍ METHICILIN-REZISTENTNÍ KMEN S. AUREUS

The use of antimicrobial photodynamic therapy (aPDT) as a therapeutic modality for the treatment of localized microbial infections represents an developing new field. The emergence of strains resistant to antibiotics has provided the necessary impulse for new drug or technology discoveries to combat these resistant compounds. Although the aPDT is still in infancy, its need is still growing. Like PDT, main components of antimicrobial photodynamic therapy are appropriate light, dye called photosensitizer and created reactive oxygen species. In this article photosensitizers TMPyP and ZnTPPS4 are investigated for antimicrobial photodynamic therapy. We tested these porphyrins on bacterial methicilin – resistant strain MRSA alone and bound in complex created with hp-β-cyclodextrin. The light emitting diodes (414 nm) were used at the doses 0 and 150 J/cm2. Tested concentrations were from 0.78 to 100 μM. This experimental work predicated that TMPyP is very successful compound in aPDT. In contrary to ZnTPPS4 which was efficient for eradication of tested gram-positive bacteria only in higher concentrations

VLIV ULTRAZVUKU NA ÚČINNOST FOTODYNAMICKÉ TERAPIE – IN VITRO STUDIE

Photodynamic therapy (PDT) belongs in perspective modalities of cancer treatment. It is based on the tumour-selective accumulation of a photosensitizer followed by irradiation with light of a specific wavelength. PDT is widely developed nowadays due to its high specificity and selectivity along with absence of the unadvisable side-effects. Sonodynamic therapy (SDT) exploits ultrasound to induce cytotoxic effect of sensitizer. In our study we tested the possibility of combination of this therapies and icrease of efficiency. Our results suggest that irradiation in combination with application of therapeutic ultrasound increases production of reactive oxygen species and reduces viability of tumour MCF7 cells, compared to irradiation of ZnTPPS4 only, especially in the case of higher therapeutic doses. In the future, the combination of PDT and SDT can bring a new treatment modality for malignant and also nonmalignant diseases

Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules

Biological Evaluation of Photodynamic Effect Mediated by Nanoparticles with Embedded Porphyrin Photosensitizer

Clinically approved photodynamic therapy (PDT) is a minimally invasive treatment procedure that uses three key components: photosensitization, a light source, and tissue oxygen. However, the photodynamic effect is limited by both the photophysical properties of photosensitizers as well as their low selectivity, leading to damage to adjacent normal tissue and/or inadequate biodistribution. Nanoparticles (NPs) represent a new option for PDT that can overcome most of the limitations of conventional photosensitizers and can also promote photosensitizer accumulation in target cells through enhanced permeation and retention effects. In this in vitro study, the photodynamic effect of TPP photosensitizers embedded in polystyrene nanoparticles was observed on the non-tumor NIH3T3 cell line and HeLa and G361 tumor cell lines. The efficacy was evaluated by viability assay, while reactive oxygen species production, changes in membrane mitochondrial potential, and morphological changes before and after treatment were imaged by atomic force microscopy. The tested nanoparticles with embedded TPP were found to become cytotoxic only after activation by blue light (414 nm) due to the production of reactive oxygen species. The photodynamic effect observed in this evaluation was significantly higher in both tumor lines than the effect observed in the non-tumor line, and the resulting phototoxicity depended on the concentration of photosensitizer and irradiation time

Analysis of DNA strand breaks by Comet assay.

<p>Comet assay of the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (B) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (C) for 30 min. After the treatment, U937 cells were stained by SYBR Green.</p

Determination of the U937 cell viability.

<p>The cell viability was determined 30 min after the addition of 5 mM H₂O₂ or Fenton reagent to the U937 cells. The results are normalized to control U937 cells. The data are presented as the mean and standard deviation of at least 3 measurements.</p

Detection of singlet oxygen by EPR spin-trapping spectroscopy.

<p>TEMPONE EPR spectra were measured in the control (A and B), the H₂O₂-treated (C and D) and the Fenton reagent-treated (E and F) U937 cells in the presence of 100 mM TEMPD. U937 cells were treated with no addition (A), 5 mM H₂O₂ (C) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (E) for a period indicated in Fig. In C and E, top traces show the simulation of TEMPONE EPR signal using hyperfine coupling constants <i>a</i>^N = 16 G. Chemical source of ¹O₂ (10 mM molybdic acid + 10 mM H₂O₂ measured right after the preparation) was used as a positive control (C,E). Bar graphs represent the hight of the middle peak of TEMPONE EPR signal in the control (B), the H₂O₂-treated (D) and the Fenton reagent-treated (F) U937 cells. Experimental EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1, gain 500. Bars represent 4000 (A) and 8000 (C and E) relative units. Data are presented as mean values and standard deviations. The mean value represent the average value from at least three measurements.</p

Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.

<p>Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.</p><p>Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.</p