Enzalutamide and abiraterone in the treatment of patients with metastatic castration-resistant prostate cancer treated previously with chemotherapy.

BACKGROUND: The evaluation of treatment outcomes and toxicity in patients with metastatic castration-resistant prostate cancer (mCRPC) treated by enzalutamide or abiraterone after previous docetaxel. PATIENTS AND METHODS: We analyzed 66 patients with mCRPC treated by enzalutamide (55 patients) or abiraterone (11 patients) after previous therapy with docetaxel. The median follow-up was 31.2 months. Enzalutamide and abiraterone were administered in daily doses of 160 mg and 1,000 mg per day, respectively. The progression free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier analysis. The prognostic influence of the factors on OS was evaluated by regression analysis. RESULTS: The progression was observed in 55 (83%) patients, and mPFS was 12.1 (95% CI 7.7-16.4) months. In total, 43 patients died, and he median OS was 21.9 (95% CI 12.2-31.7) months. In the regression analysis, we observed statistical favorable influence of the following factors on OS: PSA decrease ≥ 50%, in patients with early decrease of prostatic specific antigen (PSA) ≥ 50% in 3 months after initiation of enzalutamide or abiraterone treatment, in patients with visceral metastatic sites, in patients treated with only one regimen of previous chemotherapy and in those without anemia. We observed the toxicity grades 3-4 in 45.5% and 36.3% patients treated with enzalutamide and abiraterone, respectively. CONCLUSION: Our analysis demonstrated efficacy and good tolerance in patients with mCRPC treated with enzalutamide and abiraterone after previous docetaxel therapy.

New planar light source for the induction and monitoring of photodynamic processes in vitro.

We recently developed a new light source that allows for the continuous monitoring of light-induced changes using common spectrophotometric devices adapted for microplate analyses. This source was designed primarily to induce photodynamic processes in cell models. Modern light components, such as LED chips, were used to improve the irradiance homogeneity. In addition, this source forms a small hermetic chamber and thus allows for the regulation of the surrounding atmosphere, which plays a significant role in these light-dependent reactions. The efficacy of the new light source was proven via kinetic measurements of reactive oxygen species generated during the photodynamic reaction of chloroaluminium phthalocyanine disulfonate (ClAlPcS2) in three cell lines: human melanoma cells (G361), human breast adenocarcinoma cells (MCF7), and human fibroblasts (BJ).

Effect of ClAlPcS(2) photodynamic and sonodynamic therapy on HeLa cells.

Photodynamic therapy (PDT) uses photosensitive substance to provoke a cytotoxic reaction causing a cell damage or cell death. The substances, photosensitizers, are usually derivates of porphyrine or phtalocyanine. Photosensitizers must be activated by light in order to produce reactive oxygen species, mainly singlet oxygen. Sonodynamic therapy (SDT) utilizes ultrasound to enhance a cytotoxic effects of compounds called sonosensitizers. In this study we investigated photodynamic and sonodynamic effect of chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) on HeLa cells. DNA damage, cell viability and reactive oxygen species (ROS) production were assessed to find whether the combination of PDT and SDT inflicts HeLa cells more than PDT alone. We found that the combined therapy increases DNA fragmentation, enhances ROS production and decreases cell survival. Our results indicate that ClAlPcS(2) can act as a sonosentitiser and combined with PDT causes more irreversible changes to the cells resulting in cell death than PDT alone.

[Human alveolar echinococcosis and an overview of the occurrence of Echinococcus multilocularis in animals in the Czech Republic]. / Humánní alveolární echinokokóza a prehled výskytu tasemnic Echinococcus multilocularis u zvírat v Ceské republice.

Human alveolar echinococcosis (AE) is caused by larval stages of the tapeworm Echinococcus multilocularis. In the Czech Republic, screening tests to detect the specific infectious agent have been performed since 1998. The first AE cases were diagnosed in 2007, and until 2014, a total of 21 diseases were recorded. In accordance with radiological, histological, and/or PCR data, serological examinations of 699 individuals helped to reveal 15 additional AE cases in the period of 2015-2016. From the cumulative data for 1998-2016, it appears that of 2,695 patients examined, 36 (18 men and 18 women) were diagnosed with AE. Their age at diagnosis ranged from 20 to 82 years and was lower for women (mean 43.7, median 39.5) than for men (50.9 and 57.5, respectively), but the difference was not statistically significant. In the period of 2007-2016, the mean annual incidence rate was 0.034 cases/100 000 population. Our study indicates an ongoing increase in AE cases. The disease can be autochthonous in nature, as evidenced not only by some case history data but also by the detection of the larval stages in wild boar (Sus scrofa). AE risk to humans in the Czech Republic is discussed in the context of the known data on the presence of various parasite developmental stages in animals.

The in vitro cytotoxicity of metal-complexes of porphyrin sensitizer intended for photodynamic therapy.

The sulphonated derivatives of porphyrins (e.g. TPPS4) are hydrophilic photosensitizers and have certain advantages like fully known structures and the possibility of synthetic production. The aim of this work was to study in vitro cytotoxicity and to compare the new photosensitizer MgTPPS4 with TPPS4 and its other metal-complexes (ZnTPPS4, PdTPPS4) on human skin melanom and mouse fibroblast cell lines. A photodynamic treatment was induced by light emitting diodes with three different total doses (1, 5 and 10J/cm(2)). For proper analysis and understanding of cell behavior after the administration of sensitizers, a complex battery of in vitro tests including the production of reactive oxygen species, the MTT viability test, a comet assay, a cell cycle and a type of cell death determination were used. We discovered that the most suitable photosensitizer is ZnTPPS4 because it had the biggest lethal influence on melanoma cells and the lowest lethal influence on fibroblast cells. The second most effective photosensitizer seemed to be MgTPPS4. On the basis of our results we can also assume that there is a higher accumulation of photosensitizer in a tumorous cell line. The higher concentration of photosensitizer and light dose resulted in more reactive oxygen species production and found more cells undergoing necrosis.

Lipoprotein lipase deficiency: clinical, biochemical and molecular characteristics in three patients with novel mutations in the LPL gene.

Lipoprotein lipase (LPL) deficiency, caused by mutations in the LPL gene, is a rare autosomal recessive disorder manifesting in early childhood with recurrent abdominal pain, hepatosplenomegaly, acute pancreatitis, lipaemia retinalis and eruptive xanthomas. Typical laboratory findings are lactescent serum, extreme hypertriglyceridaemia and hypercholesterolaemia. The diagnostics is based on postheparin serum LPL assay and DNA analyses of the LPL gene. We report clinical, biochemical and molecular data of three children with LPL deficiency. One child manifested since the first week of life with recurrent abdominal pain (Patient 1), the second with abdominal distension and hepatosplenomegaly since the second month of life (Patient 3) and patient 2, asymptomatic younger brother of patient 1, was diagnosed in the first week of life. Lipaemia retinalis and splenomegaly were present in two symptomatic children, hepatomegaly in patient 3 and acute pancreatitis in patient 1. All children had lactescent serum, profound hypertriglyceridaemia (124 ± 25 mmol/l; controls < 2.2), hypercholesterolaemia (22.8 ± 7.3 mmol/l, controls < 4.2) and their LPL immunoreactive mass in serum did not increase after heparin injection. Molecular analyses revealed that both siblings are homozygous for novel mutation c.476C > G in the LPL gene changing the conserved amino acid of the catalytic centre. The third patient is a compound heterozygote for mutations c.604G>A and c.698A>G in the LPL gene, both affecting highly conserved amino acids. We conclude that LPL deficiency must be considered in neonates and young infants with abdominal pain and hypertriglyceridaemia because early treatment might prevent development of life-threatening acute pancreatitis.

Predicting axillary sentinel node status in patients with primary breast cancer.

The aim of this study is to determine the combination of characteristics in early breast cancer that could estimate the risk of occurrence of metastatic cells in axillary sentinel lymph node(s). If we were able to reliably predict the presence or absence of axillary sentinel involvement, we could spare a considerable proportion of patients from axillary surgery without compromising therapeutic outcomes of their disease. The study is based on retrospective analysis of medical records of 170 patients diagnosed with primary breast cancer. These women underwent primary surgery of the breast and axilla in which at least one sentinel lymph node was obtained. Logistic regression has been employed to construct a model predicting axillary sentinel lymph node involvement using preoperative and postoperative tumor characteristics. Postoperative model uses tumor features obtained from definitive histology samples. Its predictive capability expressed by receiver operating characteristic curve is good, area under curve (AUC) equals to 0.78. The comparison between preoperative and postoperative results showed the only significant differences in values of histopathological grading; we have considered grading not reliably stated before surgery. In preoperative model only the characteristics available and reliably stated at the time of diagnoses were used. The predictive capability of this model is only fair when using the data available at the time of diagnosis (AUC = 0.66). We conclude, that predictive models based on postoperative values enable to reliably estimate the likelihood of occurrence of axillary sentinel node(s) metastases. This can be used in clinical practice in case surgical procedure is divided into two steps, breast surgery first and axillary surgery thereafter. Even if preoperative values were not significantly different from postoperative ones (except for grading), the preoperative model predictive capability is lower compared to postoperative values. The reason for this worse prediction was identified in imperfect preoperative diagnostic.

[Characteristics and prognosis of malignant disease of the breast in women of very low age]. / Vlastnosti a prognóza zhoubného onemocnení prsu u zen velmi nízkého veku.

OBJECTIVE: This study aims to describe characteristics of malignant diseases of the breast in women of very low age (thus with breast cancer diagnosed before 35th year of age) and compare those characteristics with the phenotype of "average" breast cancer in Czech female population. METHODS: 98 women diagnosed with breast cancer before the age of 35 and treated between 2001 and 2010 in private medical center Medicon Praha s.r.o, were enrolled to this retrospective study. The control group of 100 women was constituted by random choice from patients older than 35 years at the time of diagnosis treated in the same time period. RESULTS: Size of the tumors at presentation were similar in both study groups. Tumors in younger group exhibited higher proliferative activity, higher grade and lower count of estrogen receptors. On the contrary, in the group of older women was significantly higher percentage of lobular type of cancer and also the proportion of in situ carcinomas. The number of multifocal tumors, positivity of HER-2/neu and progesterone receptors were all without statistically significant difference. In younger women neoadjuvant chemotherapy has been used more frequently. Prognosis of the disease did not differ in both groups. CONCLUSION: Tumors diagnosed in women younger than 35 years can be considered more aggressive. However, using adequate treatment makes the prognosis comparable in both age groups.

Phototoxic effect of TPPS4 and MgTPPS4 on DNA fragmentation of HeLa cells.

Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O(2) which converts to cytotoxic (1)O(2) and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS(4) and MgTPPS(4) at the concentrations higher than 5 µM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm(-2) is sufficient enough to provoke the DNA fragmentation.

Effects of tandem shock waves combined with photosan and cytostatics on the growth of tumours.

Shock waves, pressure waves manifested as a sharp increase in positive pressure followed by a decrease and the negative part of the wave, are not only used to treat concrements in medicine. Recently, research has been focused on the possibility of their use for damaging the tumour tissue. In contrast to concrements, which are different from the surrounding tissue by their acoustic impedance, the tumour tissue has the same acoustic impedance as the surrounding soft tissue. Therefore, we have developed a new source of shock waves, which is based on the principle of multichannel discharge. This new source generates two successive shock waves (tandem shock waves). The first shock creates acoustic non-homogeneity and cavitations in the tissue, and the second shock is damped in it. In this work we demonstrated the effect of tandem shock waves on the muscle tissue in depth. The damage is shown on the images from the magnetic resonance imaging and histological sections. In the further part of the experiment, we investigated the in vivo effects of tandem shock waves in combination with Photosan and cisplatin on the tumour tissue. The application of tandem shock waves resulted in the inhibition of tumour growth, compared with controls, in both parts of the experiment. The largest inhibition effect was observed in the groups of tandem shock waves combined with Photosan and in the second part with cisplatin.

The expression of NADPH oxidases and production of reactive oxygen species by human lung adenocarcinoma epithelial cell line A549.

Controlled production of reactive oxygen species (ROS) by NADPH oxidases in non-phagocytic cells has recently been suggested to participate in the regulation of cellular functions. Due to the role of ROS in control of cellular functions, precise and accurate detection of ROS is of essential importance. However, various methodological approaches currently used for ROS determination vary in sensitivity, specificity, as well as in requirements for specialized equipment. In this study, human lung epithelial cell line A549 was screened for expression of NADPH oxidases NOX1, NOX2, NOX4, NOX5, DUOX1 and DUOX2 by quantitative RT-PCR. Fluorometric, colorimetric, and chemiluminometric methods were applied to determine ROS production. A549 cells were found to significantly express NOX1, NOX2, DUOX1 and DUOX2. ROS production by A549 cells was detected with fluorometric probes 2',7'-dichlorofluorescein- diacetate, dihydroethidium, and amplex red or colorimetric probe nitrobluetetrazolium. The production of ROS detected by these probes was partially reduced by NADPH oxidase inhibitor diphenyleneiodonium. The inhibitory effect of diphenyleneiodonium was the most significant regarding amplex red detection of phorbol myristate acetateactivated ROS production. In contrast to other probes, neither cytochrome c colorimetric determination nor luminol- and L-012-amplified chemiluminescence, regardless of the addition of horseradish peroxidase, exerted sufficient sensitivity to detect ROS production by A549. The results revealed differences among methods used for ROS formation measurement by human lung epithelial cell line A549 and highlighted the sensitivity of fluorometric determination for this purpose.

Photodynamic properties of ZnTPPS(4), ClAlPcS(2) and ALA in human melanoma G361 cells.

Photodynamic therapy (PDT) has been approved as proper and effective kind of treatment for certain types of cancer and non-malignant diseases. We tested photodynamic effects on G361 human melanoma cells sensitized by zinc-5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrine (ZnTPPS(4)), chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) and 5-aminolevulinic acid (ALA). In particular, we examined the PDT efficiency depending on applied light dose (0.8; 1.7; 3.3; 6.6; 13.2; 26.4Jcm(-2)). The DNA gel electrophoresis, methylthiazol tetrazolium bromide (MTT) viability test, fluorescent microscopy using calcein AM and propidium iodide (PI) staining, and rhodamine 123 mitochondrial membrane potential assay were performed to detect and evaluate the cell death process. We also measured the time course of reactive oxygen species (ROS) production and its dependence on sensitizer concentration within continuously irradiated sensitized cells. In conclusion, these results demonstrate most significant phototoxic effect of ClAlPcS(2)-PDT in spite of significantly higher ROS production induced by ZnTPPS(4)-PDT on G361 cells. On the other hand, ALA-PDT has a minimal photoeffect and induces negligible ROS formation in G361 cells at the conditions described below.

Study of cytotoxic effect of photodynamically and sonodynamically activated sensitizers in vitro.

High resolution imaging of biological structures and their changes induced by different agents such as drugs are commonly performed by confocal and electron microscopy. The past decade has witnessed an emersion of the atomic force microscopy (AFM) from solid-state physics into cell biology and even medical applications. For these reasons, we used this relatively new microscopic technique to study the morphology of cell lines. We imaged the cells by atomic force microscopy before and after the photodynamic therapy (PDT) using the photosensitizer ClAlPcS(2). We also compared the impact of the photosensitizer in combination with silymarin antioxidant on cancer and non-cancer cell lines by measuring the kinetic production of reactive oxygen species (ROS). PDT was induced by LED source with total irradiation dose of 15 J cm(-2) and SDT was induced by therapeutic ultrasound with frequency of 1 MHz, intensity 2 W cm(-2) and time of exposition 10 min. The results show ROS kinetic production within the cells during PDT, sonodynamic therapy (SDT) and modification of morphological features investigated by AFM. The combination of a sensitizer and the specific light source can lead to the loss of surface rigidity and eventually to dramatic changes of the cell shape, which we can study by AFM.

Production of reactive oxygen species after photodynamic therapy by porphyrin sensitizers.

The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.

Photodynamic effect on melanoma cells investigated by atomic force microscopy.

Atomic force microscopy (AFM) is a modern experimental method for imaging of conducting or non-conducting samples. New trends in the application of scanning probe microscopy (SPM) give us the ability to scan live cells directly in their ingenuous surroundings or in air. Our apparatus was replenished with an inverse optical microscope, so we could observe the position of the scanning tip in every individual cell. The aim of the presented study is to picture the cell surface in air. A dry scanner in non-contact or tapping mode was used in the biological application of AFM. In our work the cell line G361 was used as a biological sample. We imaged the cell line before and after induction of a photodynamic effect (PDE) by irradiation of ZnTPPS4-loaded cells with a light dose of 15 J/cm(2). Individual cells before PDE induction had a smooth surface without protrusion on the entire surface. Cells after PDE induction did not have a smooth surface but their surface was rough with protrusion and in some places cleaved.

In vitro approaches to evaluation of Sun Protection Factor.

The efficacy of sunscreen products has been recognized as an important public health issue. Adequate methods for assessment of the level of protection should be developed and standardised. While the SPF COLIPA testing method in vivo has been used for years, preference should be given to in vitro testing methods as in vivo methods raise ethical concern. The present study aims to assess possible in vitro approaches based on diffuse transmission spectroscopy, published previously by Diffey, and two methods based on measurements of UVB transmission through a defined layer of a sunscreen product applied on various UV-transparent substrates. The attenuated UVB intensity, using different UV light sources, is detected radiometrically and transformed to real SPF value by means of a calibration curve, which is based on an extensive number of measurements performed using both in vivo and in vitro method The outcome of the three in vitro methods employed in the study showed great differences in the obtained SPF values in comparison with reference SPF determined by means of the COLIPA method in vivo. The high variability of in vitro results suggests that main attention should be focused on substrate selection simulating the human skin surface and homogenous product application. The in vitro screening methods may represent a fast and reasonable tool reducing the number of in vivo experiments and risks related to UV exposure of human subjects, when the technical test parameters are adjusted and optimized.

Sensitivity of different cell lines to phototoxic effect of disulfonated chloroaluminium phthalocyanine.

Photodynamic therapy (PDT) is a treatment for cancer involving three key components: sensitizer, light and tissue oxygen. A sensitizer is a chemical compound that can be excited by light of a specific wavelength. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on NIH3T3 (mouse fibroblasts), B16 (mouse melanoma), MCF7 (human breast adenocarcinoma) and G361 (human melanoma) cell lines. A semiconductor laser was used as a source for evocation of the photodynamic effect. We report the influence of various concentrations of the sensitizer in combination with laser irradiation on the photodamage of cells. Viability of cells was determined by means of molecular probes (Calcein AM and ethidium homodimer) for fluorescence microscopy. The quantitative changes of cell viability in relation to sensitizer concentrations and laser irradiation were proved by fluorometric measurement. We detected phototoxicity evoked by laser irradiated sensitizer in all studied cell lines. In addition, the viability studies showed that G361 melanoma cells and MCF7 breast adenocarcinoma cells were more sensitive than NIH3T3 mouse fibroblasts and B16 mouse melanoma to photodynamic damage induced by ClAlPcS(2).

Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests.

The aim of this study was to clarify the differences in the phototoxicity of bergamot oil obtained from four different suppliers. Spectral and chemical analyses were performed to identify presence of photoactive compounds in the test samples. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D PT). Confirmatory photopatch tests in a group of volunteers were performed using the first non-phototoxic concentration determined in the H3D PT. The spectral and chemical analyses revealed, that two samples of bergamot oil exhibited a potential for photoactivation. These oils were subsequently classified as phototoxic in the 3T3 NRU PT, however, only on the basis of borderline results and depending on the solvent used. H3D PT revealed clear classifications, correlating well with the findings of spectral and chemical analysis. The test was, however, not yet capable of precise prediction of safe, non-phototoxic concentrations. Additional endpoints, e.g. interleukin determination might be employed to increase the sensitivity of the test. Although the study showed the usefulness of the tiered testing strategy, currently, the extrapolation of in vitro results to human situation may be performed only to a limited extent.

Comparison of sensitizers by detecting reactive oxygen species after photodynamic reaction in vitro.

The production of reactive oxygen species (ROS) has a crucial effect on the result of photodynamic therapy (PDT). Because of this fact, we examined the ROS formation by means of three porphyrin sensitizers (TPPS(4), ZnTPPS(4) and PdTPPS(4)) and compared their effectivity for induction of cell death in the G361 (human melanoma) cell line. The porphyrins used are very efficient water-soluble aromatic dyes with a potential application in photomedicine and have a high tendency to accumulate in the membranes of intracellular organelles such as lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to the production singlet oxygen and other reactive oxygen species to induce cell death. Production of ROS was investigated by molecular probe CM-H(2)DCFDA. Our results demonstrated that ZnTPPS(4) induces the highest ROS production in the cell line compared to TPPS(4) and PdTPPS(4) at concentrations of 1, 10, and 100 microM and light dose of 1 J cm(-2). We also observed a consequence between ROS production and cell survival. In conclusion, these results demonstrate that photodynamic effect depends on sensitizer type, its concentration and light dose.

In vitro study of reactive oxygen species production during photodynamic therapy in ultrasound-pretreated cancer cells.

Several recent studies bring evidence of cell death enhancement in photodynamic compound loaded cells by ultrasonic treatment. There are a number of hypotheses suggesting the mechanism of the harmful ultrasonic effect. One of them considers a process in the activation of photosensitizers by ultrasonic energy. Because the basis of the photodynamic damaging effect on cells consists in the production of reactive oxygen species (ROS), we focused our study on whether the ultrasound can increase ROS production within cancer cells. Particularly, we studied ROS formation in ultrasound pretreated breast adenocarcinoma cells during photodynamic therapy in the presence of chloroaluminum phthalocyanine disulfonate (ClAlPcS2). Production of ROS was investigated by the molecular probe CM-H2DCFDA. Our results show that ClAlPcS2 induces higher ROS production in the ultrasound pretreated cell lines at a concentration of 100 microM and light intensity of 2 mW/cm2. We also observed a dependence of ROS production on photosensitizer concentration and light dose. These results demonstrate that the photodynamic effect on breast cancer cells can be enhanced by ultrasound pretreatment.