Publications by authors named "Anna Choromańska"

33 Publications

Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer.

Pharmaceutics 2020 Dec 24;13(1). Epub 2020 Dec 24.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process's effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E is not related to the change of cell membrane permeability.
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http://dx.doi.org/10.3390/pharmaceutics13010019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823502PMC
December 2020

Effect of diallyl disulfide and garlic oil on different human astrocytoma cell lines.

Biomed Rep 2020 Oct 31;13(4):32. Epub 2020 Jul 31.

Department of Histology and Embryology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, Poland.

Gliomas are a group of malignant brain tumors. Despite significant efforts to optimize treatment options for patients with high-grade glioma, the prognosis of the overwhelming majority of patients remain poor. This bleak prognosis despite treatment of the glioma, is partly due to the tendency of therapeutics to diffusely penetrate into the neighboring brain tissues, but also due to the innate resistance of these tumors to chemotherapy and radiation. Garlic contains water-soluble and oil-soluble sulfur compounds. The oil-soluble compounds, including diallyl sulfide, diallyl disulfide (DADS), diallyl trisulfide and ajoene, are more effective potential anti-cancer treatments than the water-soluble compounds. There are several studies examining the effects of oil-soluble compounds on various types of cancer cells, although, to the best of our knowledge, there are no studies examining the effects of these compounds on glioma cells. The aim of the present study was to investigate the potential anti-glioma properties of DAD and garlic oil on proliferation and induction of apoptosis in four different types of glioma cell lines representative of different grades of the disease. The results showed that garlic oil exhibits favorable anti-cancer potential towards gliomas of various degrees of differentiation.
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http://dx.doi.org/10.3892/br.2020.1339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412714PMC
October 2020

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis Against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1.

Int J Mol Sci 2020 Jun 29;21(13). Epub 2020 Jun 29.

Department of Dental Surgery, Wroclaw Medical University, 50425 Wroclaw, Poland.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.
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http://dx.doi.org/10.3390/ijms21134611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370030PMC
June 2020

Caffeic Acid Phenethyl Ester Assisted by Reversible Electroporation-In Vitro Study on Human Melanoma Cells.

Pharmaceutics 2020 May 24;12(5). Epub 2020 May 24.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Melanoma is one of the most serious skin cancers. The incidence of this malignant skin lesion is continuing to increase worldwide. Melanoma is resistant to chemotherapeutic drugs and highly metastatic. Surgical resection can only be used to treat melanoma in the early stages, while chemotherapy is limited due to melanoma multi-drug resistance. The overexpression of glutathione -transferase (GST) may have a critical role in this resistance. Caffeic acid phenethyl ester (CAPE) is a natural phenolic compound, which occurs in many plants. Previous studies demonstrated that CAPE suppresses the growth of melanoma cells and induces reactive oxygen species generation. It is also known that bioactivation of CAPE to its corresponding quinone metabolite by tyrosinase would lead to GST inhibition and selective melanoma cell death. We investigated the biochemical toxicity of CAPE in combination with microsecond electropermeabilization in two human melanoma cell lines. Our results indicate that electroporation of melanoma cells in the presence of CAPE induced high oxidative stress, which correlates with high cytotoxicity. Moreover, it can disrupt the metabolism of cancer cells by inducing apoptotic cell death. Electroporation of melanoma cells may be an efficient CAPE delivery system, enabling the application of this compound, while reducing its dose and exposure time.
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http://dx.doi.org/10.3390/pharmaceutics12050478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284363PMC
May 2020

Proapoptotic activity induced by photodynamic reaction with novel cyanine dyes in caspase-3-deficient human breast adenocarcinoma cell lines (MCF/WT and MCF/DX).

Photodiagnosis Photodyn Ther 2020 Jun 21;30:101775. Epub 2020 Apr 21.

Wroclaw Medical University, Department of Molecular and Cellular Biology, Wroclaw, Poland.

Photodynamic therapy (PDT) is currently one of the cancer treatment options. PDT requires the application of a photosensitizer (such as: porphyrins, chlorines, and phthalocyanines) that selectively targets malignant cells. It is a dilemma to find a proper photosensitizer. In our study, we have tested a new in-vitro group of cyanine dyes. These dyes are widely applied in biotechnology as fluorescent markers. Two malignant adenocarcinoma cell lines (MCF-7/WT and MCF-7/DOX) were investigated using photodynamic reaction (PDR) with four cyanine dyes (KF-570, HM-118, FBF-749, and ER-139). KF-570 and HM-118 were irradiated with red light (630 nm), whereas FBF-749 and ER-139 with green light (435 nm). To evaluate PDR efficiency, a clonogenic test was conducted. Apoptosis was investigated by TUNEL and NCA (neutral comet) assays. Proteins selected as indicators of the apoptotic pathway (AIF, sPLA2, Smac/Diablo) and intracellular response markers (SOD-1 and GST-pi) were detected using western blot. The highest number of apoptotic cells (ca. 100%) was observed after PDR with HM-118 and KF-570 in both conducted tests, in both cell lines. The results showed that HM-118 and KF-570 cyanine dyes demonstrated a major phototoxic effect causing apoptosis in doxorubicin-resistant and sensitive cell lines.
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http://dx.doi.org/10.1016/j.pdpdt.2020.101775DOI Listing
June 2020

Cisplatin and Vinorelbine -Mediated Electrochemotherapeutic Approach Against Multidrug Resistant Small Cell Lung Cancer (H69AR) .

Anticancer Res 2019 Jul;39(7):3711-3718

Department of Thoracic Surgery, Wroclaw Medical University, Wroclaw, Poland.

Background/aim: Small cell lung cancer (SCLC) originates from neuroendocrine branchial cells (15-20%). It is regarded as distinct from other lung cancers due to its biological and clinical features. In most cases of SCLC, surgery or radiotherapy alone is not an effective cure. The aim of our study was to examine the cytotoxic effects of chemotherapy supported by electroporation (EP) on a resistant SCLC model, in vitro.

Material And Methods: The multidrug resistant small lung cell line H69AR was used to evaluate the cytotoxic effects of cisplatin (CPPD) and vinorelbine (Navirel®; NAV) at lower doses when used with EP. Cells were treated with different concentrations of CPPD and NAV, alone or in combination with the following EP parameters: 400-1200 V/cm, 8 pulses of 100 μs duration, at 1Hz. The cell viability was estimated by MTT assay after 24 and 48 h. Apoptotic cells were detected by neutral comet assay and immunofluorescence assay with PARP-6.

Results: CPPD and NAV alone showed a dose-dependent effect on cell viability. Cytostatic drugs combined with EP revealed increased anticancer activity. Lower doses of CPPD or NAV delivered by EP were as effective as higher doses of these drugs without EP. The electrochemotherapeutic protocols increased the number of apoptotic cells and increased immunoreactivity of PARP-6. Our results indicated higher sensitivity of H69AR cells to NAV supported by EP.

Conclusion: In SCLC cells, an increased anticancer activity was potentiated by exposure of cells to high intensity electric pulses and low drug doses. It is suggested that this method could be effectively applied in the treatment of lung cancer.
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http://dx.doi.org/10.21873/anticanres.13519DOI Listing
July 2019

Chemical Composition of East Asian Invasive Knotweeds, their Cytotoxicity and Antimicrobial Efficacy Against Cariogenic Pathogens: An In-Vitro Study.

Med Sci Monit 2019 May 4;25:3279-3287. Epub 2019 May 4.

Department of Pharmaceutical Biology and Botany, Wrocław Medical University, Wrocław, Poland.

BACKGROUND Giant knotweeds originating from East Asia, such as Reynoutria japonica, and Reynoutria sachalinensis, and their hybrid such as Reynoutria x bohemica, are invasive plants in Europe and North America. However, R. japonica is also a traditional East Asian drug (Polygoni cuspidati rhizoma) used in Korean folk medicine to improve oral hygiene. The aim of this study was to evaluate the antibacterial activity of acetone extracts of Reynoutria species against dominant caries pathogen such as Streptococcus mutans and alternative pathogens, as well as characterize the phytochemical composition of extracts and examine their cytotoxicity. MATERIAL AND METHODS Ultrasonic extraction was used to obtain polyphenol-rich extracts. The extracts were characterized by HPLC-DAD-ESI-MS. To test bacterial viability, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans, S. salivarius, S. sanguinis, and S. pyogenes were determined. The cytotoxicity of the extracts to human fibroblasts derived from gingiva was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS The R. japonica extract had the highest bacteriostatic and bactericidal activity against pathogens causing caries, mainly dominant caries pathogen S. mutans (mean MIC 1000 μg/mL and MBC 2000 μg/mL), which was most likely associated with a higher content of stilbene aglycons and anthraquinone aglycons in the extract. Moreover, the R. japonica extract demonstrated the lowest cytotoxic effect on human fibroblasts and exhibited cytotoxic activity only at the concentration causing the death of all S. mutans. CONCLUSIONS The results indicate that the R. japonica acetone extract can be considered as a natural, antimicrobial agent for caries control.
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http://dx.doi.org/10.12659/MSM.913855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512351PMC
May 2019

Effect of electrochemotherapy with betulinic acid or cisplatin on regulation of heat shock proteins in metastatic human carcinoma cells in vitro.

Oncol Rep 2019 Jun 8;41(6):3444-3454. Epub 2019 Apr 8.

Department of Molecular and Cellular Biology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, 50‑556 Wroclaw, Poland.

Betulinic acid (BTA) is naturally occurring triterpene that has received interest as a novel therapeutic substance with cytotoxicity towards a number of cancer cell lines. Despite the wide spectrum of biological and pharmacological effects, its effect may be limited its lipophobic properties. Therefore, strategies to improve the access of BTA to the cells are required to enhance the anticancer effects. Electroporation (EP) enables increased inflow of drugs into cancer cells, even at low doses, which may reduce the side effects caused by high doses of chemotherapy. The potential application of BTA in electrochemotherapy (ECT) in metastatic type of cancers was investigated in the present study. The efficacy of BTA with EP was estimated using a cell survival assay (MTT assay), microscopical morphology analysis and the immunocytochemical expression of heat shock proteins (HSPs). HSPs are molecules that protect the cell from harmful environmental, chemical and physical stresses, and ensure cell survival, recovery and proper functioning. HSP expression is induced various stress factors. Therefore, the expression of HSP27 and HSP70 was evaluated after cells were exposed to an external pulsed electric field and anticancer drugs. Facilitated drug delivery and the anticancer effect on metastatic tumor cells were evaluated in vitro. The effect of BTA was compared with cisplatin (CP), a standard cytostatic agent. Two different metastatic cancer cell lines were used, an ovary adenocarcinoma cell line (SW626) and melanoma cell line (Me45). BTA combined with EP exhibited similar efficacy to CP with EP after 24 and 48 h in SW626 and Me45 cancer cells. Me45 cells also had high HSP27 and low HSP70 immunosignals post‑ECT treatment. ECT caused increased expression of HSP27 and HSP70 proteins in SW626 cells, which were less sensitive to ECT than the Me45 melanoma cell line. The results indicate that BTA may be efficiently applied instead of CP in ECT approaches, but its activity differs between tumor cell lines.
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http://dx.doi.org/10.3892/or.2019.7103DOI Listing
June 2019

Valspodar-modulated chemotherapy in human ovarian cancer cells SK-OV-3 and MDAH-2774.

Bosn J Basic Med Sci 2019 Aug 20;19(3):234-241. Epub 2019 Aug 20.

Department of Gynecology and Obstetrics, Wroclaw Medical University, Wroclaw, Poland.

Overcoming drug resistance in ovarian cancer is the overarching goal in gynecologic oncology. One way to increase drug cytotoxicity without increasing the drug dose is to simultaneously apply multidrug resistance modulator. Valspodar is the second generation P-glycoprotein 1 modulator capable of reversing multidrug resistance in different cancers. In this study, we evaluated the effect of valspodar and cisplatin co-treatment on cell viability, cell death and oxidative status in ovarian cancer cells. Two human ovarian cancer cell lines SK-OV-3 and MDAH-2774 were treated with cisplatin, valspodar, or cisplatin + valspodar for 24 or 48 hours. Untreated cells were used as control group. Cell viability was evaluated by MTT assay. Cell death was assessed by TUNEL and comet assay. Lipid peroxidation (malondialdehyde) and protein thiol groups were analyzed as oxidative stress markers. The expression of mitochondrial superoxide dismutase (MnSOD) was assessed by immunocytochemistry. Valspodar effectively reduced the resistance of SK-OV-3 cells to cisplatin, as demonstrated by increased oxidative stress, decreased cell viability and increased apoptosis in SK-OV-3 cells co-treated with valspodar and cisplatin compared to other groups. However, valspodar did not significantly affect the resistance of MDAH-2774 cells to cisplatin. Stronger staining for MnSOD in MDAH-2774 vs. SK-OV-3 cells after co-treatment with cisplatin and valspodar may determine the resistance of MDAH-2774 cell line to cisplatin.
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http://dx.doi.org/10.17305/bjbms.2019.4073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716097PMC
August 2019

Calophyllum inophyllum in vaginitis treatment: Stimulated by electroporation with an in vitro approach.

Adv Clin Exp Med 2019 Feb;28(2):223-228

Department of Molecular and Cellular Biology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland.

Background: Vaginitis is one of the most common problems in clinical medicine and is cited most often during visits to obstetricians and gynecologists. Most of the inflammation cases are caused by candidiasis trichomoniasis and bacterial vaginosis. Therefore, treatment of vaginal infections must use antibiotic or antifungal drugs, which often provide quick relief to the patient. The real cause of the problem - disrupting the ecosystem of the vagina - remains unchanged. Thus, new therapeutic compounds are being explored.

Objectives: The aim of our study was to evaluate the effect of a natural substance: tamanu oil, an extract from the plant Calophyllum inophyllum, applied to the human fibroblast cell line (normal human dermal fibroblasts - NHDFs) and to the isolated human fibroblasts from the vagina (human vaginal fibroblasts - HVFs) in vitro.

Material And Methods: We evaluated the viability of cells by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay after incubation only with tamanu oil and with electroporation (EP). We also examined the immunocytochemical reaction of collagen type III and mitochondrial superoxide dismutase (MnSOD) under established conditions.

Results: Tamanu oil increased the proliferation of cells and the amount of collagen III. It has been shown that the C. inophyllum extract stimulates the proliferation of commercial fibroblasts. For direct application in patients, one should use C. inophyllum extract in the range of 1:10-1:100 (saline dilution).

Conclusions: The use of this extract (at concentrations indicated by the studies presented here) stimulates the healing processes (increased expression of collagen type III), and has anti-inflammatory, analgesic and antiseptic qualities.
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http://dx.doi.org/10.17219/acem/87045DOI Listing
February 2019

A Deep Unsupervised Learning Approach Toward MTBI Identification Using Diffusion MRI.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:1267-1270

Mild traumatic brain injury is a growing public health problem with an estimated incidence of over 1.7 million people annually in US. Diagnosis is based on clinical history and symptoms, and accurate, concrete measures of injury are lacking. This work aims to directly use diffusion MR images obtained within one month of trauma to detect injury, by incorporating deep learning techniques. To overcome the challenge due to limited training data, we describe each brain region using the bag of word representation, which specifies the distribution of representative patch patterns. We apply a convolutional auto-encoder to learn the patch-level features, from overlapping image patches extracted from the MR images, to learn features from diffusion MR images of brain using an unsupervised approach. Our experimental results show that the bag of word representation using patch level features learnt by the auto encoder provides similar performance as that using the raw patch patterns, both significantly outperform earlier work relying on the mean values of MR metrics in selected brain regions.
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http://dx.doi.org/10.1109/EMBC.2018.8512556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686862PMC
July 2018

Mechanisms of antimelanoma effect of oat β-glucan supported by electroporation.

Bioelectrochemistry 2018 Oct 6;123:255-259. Epub 2018 Jun 6.

Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368 Wroclaw, Poland; Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland. Electronic address:

There are still not specified mechanisms how beta-glucan molecules are transported into cells. Supposing, beta-glucan toxicity against tumor cells may be related to the overexpression of the transporter responsible for the transport of glucose molecules in the cells. In this case, glucans - polymers composed of glucose units are much more up-taken by tumor than normal cells. Increased GLUT1 (Glucose Transporter Type 1) expression has been demonstrated earlier in malignant melanomas. GLUT1 expression promotes glucose uptake and cell growth in that cells. Also, in human melanoma tissues a significant correlation between GLUT1 expression and mitotic activity was found. The aim of the study was to verify if oat β-glucan (OβG) is delivered into cells by GLUT-1 membrane protein. To check it out we blocked GLUT1 transporters by an inhibitor WZB117 and then we investigated cells viability with and without reversible electroporation (EP). The obtained results bring us to elucidate the mechanism of transport of the OβG into the cells is GLUT-1 dependent and moreover can be supported by EP method.
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http://dx.doi.org/10.1016/j.bioelechem.2018.06.005DOI Listing
October 2018

ANTICANCER ACTIVITY OF OAT β-GLUCAN IN COMBINATION WITH ELECTROPORATION ON HUMAN CANCER CELLS.

Acta Pol Pharm 2017 Mar;74(2):616-623

The currently available data suggest that natural products may exert significant cytotoxic and immunomodulatory effects. Plant-derived chemotherapeutic agents such as taxol, etoposide or vincristine, currently used in cancer therapy, are prominent examples in this regard. However, there is a need for new and nat- ural anticancer compounds with low or without toxicity to normal cells. One of the active compounds responsible for the immune effects is β-glucan derived from cereals, fungi, seaweeds, yeasts and bacteria. The recent data suggest that β-glucans are potent immunomodulators with anticancer properties. Antitumor properties of fungi and yeast derived β-glucans have been widely recognized, but those polysaccharides are mostly insoluble, creating several problems especially in topical formulation. To overcome the issue of low water solubility, in the current study a more soluble β-glucan type from oats was chosen for the investigation of its antitumor activities. Cytotoxic effects were studied using a human melanoma cell line (Me45). The effect of electroporation on the antitumor activity of oat β-glucan was investigated as well. Cellular viability assessment, immuno-cytochemistry and immunofluochemistry were employed to evaluate biologic effects. Our results indicate strong anticancer properties of oat β-glucan, enhanced by electroporation.
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March 2017

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

Adv Anat Embryol Cell Biol 2017 ;227:39-58

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, 205 Route de Narbonne, BP 64182, F-31077, Toulouse, France.

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.
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http://dx.doi.org/10.1007/978-3-319-56895-9_3DOI Listing
May 2018

High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung Cancer.

Pathol Oncol Res 2018 Jul 29;24(3):583-592. Epub 2017 Jul 29.

Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368, Wroclaw, Poland.

Beta-glucans are widely used in treatment, cosmetics, and the food industry. Glucans play a significant role in activation of the immune and antioxidant system and inhibiting tumor proliferation. In the current study the antitumor activities of new high and low molecular weight beta-glucan derived from oats were investigated in two human lung cancer cell line (A549, H69AR) and normal keratinocytes (HaCaT). The effect of high and low molecular weight beta-glucan from oat was evaluated by cellular viability assessment, lipid peroxidation and manganese superoxide dismutase evaluation and cytoskeleton visualisation. Additionally the level of red blood cells hemolysis was performed. Our results indicate strong anti-tumor properties of new beta-glucan from oat and at the same time no toxicity for normal cells.
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http://dx.doi.org/10.1007/s12253-017-0278-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972159PMC
July 2018

[Mechanisms of signaling associated with reactive nitrogen and oxygen in apoptosis].

Pol Merkur Lekarski 2016 May;40(239):277-82

Department of Medical Biochemistry, Medical University of Wrocław, Poland.

The knowledge of apoptotic mechanisms is essential in many biologic aspects related to both normal and neoplastic cells. Cell death by apoptosis is a very desirable way to eliminate unwanted cells: prevents release of the cellular content, which, in contrast to necrosis, provides no activation of inflammatory reactions. Apoptosis is a multistep process in where an extremely important role is played by caspases. Functions of caspases and their modifications are fundamental to understanding the signaling pathways responsible for regulation of apoptosis. These enzymes belong to a family of cysteine proteases that have the potential to destroy the enzymatic and structural proteins, and in the final stages of apoptosis, to lead to the disintegration of the cell. Apoptosis can be modulated by certain signaling pathway.
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May 2016

Preparation and characterization of new zinc(II) phthalocyanine - Containing poly(l-lactide)-b-poly(ethylene glycol) copolymer micelles for photodynamic therapy.

J Photochem Photobiol B 2016 Jul 16;160:185-97. Epub 2016 Apr 16.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland. Electronic address:

Poly(l-lactide)-b-poly(ethylene oxide) block copolymer (mPEG-b-PLLA) micelles were fabricated and applied as a new biodegradable and biocompatible nanocarrier for solubilization of hydrophobic zinc (II) phthalocyanine (ZnPc). The nanocarrier demonstrated a good colloidal stability and its in vitro sustained cargo release profile was assessed. Photobleaching of ZnPc, both in its native form and encapsulated in the obtained polymeric micelles, was studied by means of spectroscopic measurements. The photodynamic reaction (PDR) protocol for cyto- and photocytotoxicity was performed on metastatic melanoma cells (Me45), normal human keratinocytes (HaCaT) being used for comparison. The intracellular accumulation of free and encapsulated ZnPc was visualized at various time periods (1, 3 and 24h). The proapoptotic potential of the encapsulated phthalocyanine was evaluated by monitoring DNA double strand break damage fragmentation (TUNEL assay) and caspase 3/7 activity. In addition, in vitro biocompatibility studies were conducted by determining hemolytic activity of Zn-Pc-loaded mPEG-b-PLLA micelles and their lack of cytotoxicity against macrophages (P388/D1) and endothelial cells (HUV-EC-C). Our results suggest that the PDR using Zn-Pc-loaded mPEG-b-PLLA micelles can be effective in inhibiting tumor cell growth and apoptosis induction with higher responses, observed for Me45 cells. Additionally, the ZnPc-loaded micelles appear to be hemato-biocompatible and safe for normal keratinocytes, macrophages and endothelial cells.
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http://dx.doi.org/10.1016/j.jphotobiol.2016.04.018DOI Listing
July 2016

Anticancer properties of low molecular weight oat beta-glucan – An in vitro study.

Int J Biol Macromol 2015 Sep 16;80:23-8. Epub 2015 Jun 16.

Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368 Wroclaw, Poland.

Anticancer properties of 1-3, 1-4 oat beta glucan are under intensive investigation now. Antitumor characteristic of fungi and yeast beta-glucans have been widely recognized, but those polysaccharides are mostly insoluble which creates several problems especially in topical formulation. Also high molecular weight oat beta-glucans reveal high viscosity which restricts its application. According to those problems in the current study the antitumor activities of low molecular weight beta-glucan derived from oats were investigated in cancer cells: Me45, A431 and normal HaCaT and murine macrophages P388/D1. The low molecular weight beta-glucan from oat significantly deceased cancer cells viability, while for the normal cells it was non-toxic. It was observed that with the increasing incubation time and the beta-glucan concentration the cancer cells viability significantly deceased. Furthermore for the normal cells the low molecular weight beta-glucan from oat was non-toxic. Immunocytochemical ABC analysis showed that beta-glucan induced strong expression of caspase-12 in both cancer cell lines, while in HaCaT cells ABC reaction was significantly lower and in P388/D1 cell line ABC reaction was negative. Our preliminary studies show strong anti-tumor properties of new low molecular weight beta-glucan from oat and at the same time no toxicity for normal cells.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.05.035DOI Listing
September 2015

The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.

Biomed Pharmacother 2015 May 13;72:91-7. Epub 2015 Apr 13.

Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wroclaw, Poland.

Background: Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications.

Objective: To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells.

Methods: In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100μM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically.

Results: The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7μM) and ornithine (IC50=10μM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain.

Conclusions: The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.
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http://dx.doi.org/10.1016/j.biopha.2015.04.003DOI Listing
May 2015

Oxidative modification induced by photodynamic therapy with Photofrin®II and 2-methoxyestradiol in human ovarian clear carcinoma (OvBH-1) and human breast adenocarcinoma (MCF-7) cells.

Biomed Pharmacother 2015 Apr 19;71:30-6. Epub 2015 Feb 19.

Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wrocław, Poland. Electronic address:

Ovarian cancer is among the most lethal cancers in women. The successful anticancer treatment depends on the effectiveness of cytotoxic effect of applied therapeutic procedures either alone or in combination with other treatments. Photodynamic therapy (PDT) is a relatively new method of anticancer therapy. Its dominant mechanism of action is the over-production of reactive oxygen species induced by oxidative stress in malignant cells, which attack lipid membranes, proteins and nucleic acids. One of the important mechanisms is induction of unfolded protein response, ubiquitin-proteasome pathway of protein degradation. The aim of this study was to evaluate the cytotoxic effect of various protective enzymes in ovarian carcinoma clear cell line in comparison to the model breast cell line after photodynamic reaction and photodynamic reaction with 2-methoxyestradiol (2-Me). Human malignant ovarian cell line (OvBH-1) was used and human breast adenocarcinoma cells (MCF-7) were used as a control. Photodynamic reaction (PDR) with Photofrin(®)II and Ph(®)II with 2-Me was performed. The expression of protective proteins by immunocytochemistry (HSP70 and iNOS) and western blot (Hsp27 and Hsp70) methods was evaluated directly, 3 and 6 h after PDR. The changes in cells' cytoskeleton were evaluated using immunofluorescence by confocal microscopy. The expression of iNOS was observed for both experiments with differential intensity and quantity. A higher expression of Hsp70 in MCF-7 cells was observed than in OvBh-1 cells. The reorganization of cytoskeleton and nucleus was observed after 3 and 6 h after exposition to light.
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http://dx.doi.org/10.1016/j.biopha.2015.02.008DOI Listing
April 2015

Effects of electrophotodynamic therapy in vitro on human melanoma cells--melanotic (MeWo) and amelanotic (C32).

Melanoma Res 2015 Jun;25(3):210-24

aDepartment of Medical Biochemistry bDepartment of Histology and Embryology, Wroclaw Medical University cDepartment of Animal Developmental Biology, University of Wroclaw dFaculty of Fundamental Problems of Technology, Institute of Biomedical Engineering and Instrumentation, Wroclaw University of Technology, Wroclaw, Poland.

Photodynamic therapy has been considered ineffective for melanomas because of the competition between the absorbance of melanin from the melanoma and the absorbance of photosensitizers at the photosensitizer excitation light wavelength. Melanomas show considerable heterogeneity and resistance to phototherapy. The effectiveness of photodynamic therapy could be intensified by electroporation for enhanced transport of a photosensitizer by transient pores in the membrane. In this study, photodynamic therapy combined with electroporation was tested in vitro on the human melanoma cell lines melanotic melanoma (MeWo) and amelanotic melanoma (C32). Control experiments were conducted on human keratinocytes (HaCaT). Photofrin was used as a photosensitizer. Photosensitizer distribution, cloning efficacy test, comet assay, and assessment of apoptotic proteins were performed. Melanin levels were determined before and after photodynamic therapy. The experiments indicated that electroporation effectively supports the photodynamic method. It was found that photodynamic therapy with electroporation efficiently induces apoptosis in melanotic and amelanotic melanoma cells.
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http://dx.doi.org/10.1097/CMR.0000000000000153DOI Listing
June 2015

The photodynamic effect of far-red range phthalocyanines (AlPc and Pc green) supported by electropermeabilization in human gastric adenocarcinoma cells of sensitive and resistant type.

Biomed Pharmacother 2015 Feb 24;69:145-52. Epub 2014 Nov 24.

Institute of Pathology, Charité Universitätsmedizin, Berlin, Germany.

Introduction: Electroporation (EP) is commonly applied for effective drug transport thorough cell membranes based on the application of electromagnetic field. When applied with cytostatics, it is called electrochemotherapy (ECT) - a quite new method of cancer treatment. A high-voltage pulse causes the formation of temporary pores in the cell membrane which create an additional way for the intracellular drug transport. In the current work, EP was effectively merged with the already known photodynamic therapy (PDT) to selective photosensitizers' delivery to diseased tissue. The application of electroporation can reduce the dose of applied drug.

Research Objective: The aim of research was to evaluate the effectiveness of photodynamic reaction using two near infrared cyanines (AlPc and Pc green) combined with electroporation in two human gastric adenocarcinoma cell lines.

Materials And Methods: Two human cell lines - EPG85-257P (parental) and EPG85-257RDB (resistant to daunorubicin) - of gastric cancer were used. The effect of two photosensitizers (aluminum 1,8,15,22-tetrakis(-phenylthio)-29H,31H-phthalocyanine chloride and Phthalocyanine green) was investigated. The efficiency of EP parameters was assessed by propidium iodide uptake. The viability assay was applied to analyse EP, PDT and EP-PDT effect. Cyanine localization was determined by confocal microscopy. Immunocytochemical evaluation of manganese superoxide dismutase and glutathione S-transferase-pi was determined after applied therapies.

Results: PDT in combination with EP affected the viability of EPG85-257P and EPG85-257RDB cells negatively while both cyanine were used. The most evident changes were observed in the following concentrations: 15, 10 and 5μM. The optimal field strength for enhanced EP-PDT was 800 and 1200V/cm. AlPc distributed selectively in the lysosomes of parental cell line.

Conclusions: PDT, enhanced by EP, caused decreased viability when compared to the application of PDT alone. Both phthalocyanines found to be more effective after electroporation. Due to the low concentration of light-sensitive compounds and safety of electroporation itself, a treatment plan can be an alternative therapeutic modality against gastric adenocarcinomas.
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http://dx.doi.org/10.1016/j.biopha.2014.11.017DOI Listing
February 2015

Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro.

Biomed Pharmacother 2014 Jun 12;68(5):573-80. Epub 2014 Jun 12.

Department of Medical Biochemistry, Wroclaw Medical University, 10, street Chalubinskiego, 50-368 Wroclaw, Poland. Electronic address:

High electric field, applied to plasma membrane, affects organization of the lipid molecules, generating transient hydrophilic electropores. The application of the cell membrane electroporation in combination with cytotoxic drugs could increase the drug transport into cells. This approach is known as electrochemotherapy (ECT). Our work shows new data concerning the influence of electrochemical reaction with cisplatin or with 5-fluorouracil (5-FU) on cancer ovarian cells resistant to standard therapy with cisplatin, in comparison to ECT effect on human primary fibroblasts. We investigated the effect of electroporation and electrochemotherapy with 5-FU and cisplatin on human ovarian clear-cell carcinoma cell line (OvBH-1) and epithelial ovarian carcinoma cell line (SKOV-3) - both resistant to cisplatin typically used in ovarian cancers. As control cells, human gingival fibroblasts (HGF's) from primary culture were used. Electropermeabilization efficiency was determined by FACS analysis with iodide propidium. Efficiency of electrochemotherapy was evaluated with viability assay. The cytotoxic effect was dependent on the electroporation parameters and on drug concentration. Electroporation alone only insignificantly decreased cells proliferation in OvBH-1 line; SKOV-3 line was more sensitive to the electrical field. Electrochemotherapy with cisplatin and 5-FU showed promising effects on both ovarian cell lines with recovery of normal cells revealed after 72 hours.
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http://dx.doi.org/10.1016/j.biopha.2014.05.005DOI Listing
June 2014

Doxorubicin delivery enhanced by electroporation to gastrointestinal adenocarcinoma cells with P-gp overexpression.

Bioelectrochemistry 2014 Dec 4;100:96-104. Epub 2014 Apr 4.

Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-367 Wroclaw, Poland.

Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 μs each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 μM). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects.
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http://dx.doi.org/10.1016/j.bioelechem.2014.03.013DOI Listing
December 2014

The influence of retraction agents on cytoskeleton reorganization and oxidative stress in primary human gingival fibroblasts (HGFs).

Arch Oral Biol 2014 Mar 8;59(3):341-8. Epub 2014 Jan 8.

Department of Medical Biochemistry, Wroclaw Medical University, Poland. Electronic address:

Objective: Contemporary gingival retraction chemicals are not without disagreeable side-effects; there appears to be no best gingival retraction agent. The aim of this research was to select the most biocompatible retraction agents based on examination of the parameters of oxidative stress in fibroblasts derived from human primary cell culture.

Design: In this in vitro study we evaluated parameters of oxidative stress after treatment with retraction agents. Visine, Afrin, Neosynephrin, Strazolin and Adrenaline were the commercial products studied as gingival retraction agents. Additionally we examined three experimental agents. We determined lipid peroxidation and protein damage and monitored changes in cellular cytoskeleton proteins. Proliferative and survival efficiency were also evaluated.

Results: Oxidative changes included by evaluated retraction agents were at the lowest level in the case of the experimental gels. Also cytoskeleton observations suggest that the experimental agents did not degrade the cellular structure of human gingival fibroblasts (HGFs).

Conclusions: The current study was performed because of a need to project new nontoxic and save retraction agents for peridontological therapeutic usage. We suggest that the new investigational gels are most biocompatible with periodontal tissues and can be applied as new vasoconstrictor chemical retraction agents.
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http://dx.doi.org/10.1016/j.archoralbio.2013.12.011DOI Listing
March 2014

The potential role of photodynamic therapy in the treatment of malignant melanoma--an in vitro study.

Adv Clin Exp Med 2012 Mar-Apr;21(2):179-85

Department of Medical Biochemistry, Wroclaw Medical University, Poland.

Background: Melanoma is the most severe of skin neoplasms as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens up new prospects in the treatment of this tumor. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cellular and molecular mechanisms which mediate oxidative stress in cells.

Objectives: The aim of this study was to evaluate in vitro the influence of photodynamic therapy on the human melanoma Me45 cell line.

Material And Methods: Photofrin (Ph) was used as a photosensitizer.

Results: Viability studies have shown that there are significant differences between cells after PDT and cells without irradiation. After 24 hours of incubation with a 20 microg/ml concentration of Ph and with irradiation, less than 20% of the cells survived. In the control (without PDT), 65% of the cells survived.

Conclusions: The mitochondrial localization of Ph is significant, as it may lead to disturbances of mitochondrial transmembrane potential and finally to apoptotic cell death. The expressions of manganese superoxide dismutase and heme oxygenase and the level of carbonyl and thiol groups are indicating factors for oxidative stress in Me45 cells.
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January 2013

Automatic reconstruction of neural morphologies with multi-scale tracking.

Front Neural Circuits 2012 25;6:25. Epub 2012 Jun 25.

Department of Electrical Engineering, Columbia University New York, NY, USA.

Neurons have complex axonal and dendritic morphologies that are the structural building blocks of neural circuits. The traditional method to capture these morphological structures using manual reconstructions is time-consuming and partly subjective, so it appears important to develop automatic or semi-automatic methods to reconstruct neurons. Here we introduce a fast algorithm for tracking neural morphologies in 3D with simultaneous detection of branching processes. The method is based on existing tracking procedures, adding the machine vision technique of multi-scaling. Starting from a seed point, our algorithm tracks axonal or dendritic arbors within a sphere of a variable radius, then moves the sphere center to the point on its surface with the shortest Dijkstra path, detects branching points on the surface of the sphere, scales it until branches are well separated and then continues tracking each branch. We evaluate the performance of our algorithm on preprocessed data stacks obtained by manual reconstructions of neural cells, corrupted with different levels of artificial noise, and unprocessed data sets, achieving 90% precision and 81% recall in branch detection. We also discuss limitations of our method, such as reconstructing highly overlapping neural processes, and suggest possible improvements. Multi-scaling techniques, well suited to detect branching structures, appear a promising strategy for automatic neuronal reconstructions.
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http://dx.doi.org/10.3389/fncir.2012.00025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3385559PMC
October 2012

Photo-oxidative action in MCF-7 cancer cells induced by hydrophobic cyanines loaded in biodegradable microemulsion-templated nanocapsules.

Int J Oncol 2012 Jul 2;41(1):105-16. Epub 2012 May 2.

Department of Chemistry, Wroclaw University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wroclaw, Poland.

Searching for photodynamic therapy-effective nanocarriers which enable a photosensitizer to be selectively delivered to tumor cells with enhanced bioavailability and diminished dark cytotoxicity is of current interest. We have employed a polymer-based nanoparticle approach to encapsulate the cyanine-type photosensitizer IR-780 in poly(n-butyl cyanoacrylate) (PBCA) nanocapsules. The latter were fabricated by interfacial polymerization in oil-in-water (o/w) microemulsions formed by dicephalic and gemini saccharide-derived surfactants. Nanocarriers were characterized by SEM, AFM and DLS. The efficiency of PBCA nanocapsules as a potential system of photosensitizer delivery to human breast cancer cells was established by dark and photocytotoxicity as the function of the cellular mitochondria. The photodynamic effect of cyanine IR-780 was determined by investigation of oxidative stress markers. The nanocapsules were the main focus of our studies to examine their cellular uptake and dark and photocytotoxicity as the function of the cellular mitochondria as well as oxidative stress markers (i.e., lipid peroxidation and protein damage) in MCF-7/WT cancer cells. The effects of encapsulated IR-780 were compared with those of native photosensitizer. The penetration of the nanocapsules into cancer cells was visualized by CLSM and their uptake was estimated by FACS analysis. Cyanine IR-780 delivered in PBCA nanocapsules to MCF-7/WT cells retains its sensitivity upon photoirradiation and it is regularly distributed in the cell cytoplasm. The intensity of the photosensitizer-generated oxidative stress depends on IR-780 release from the effective uptake of polymeric nanocapsules and seems to remain dependent upon the surfactant structure in o/w microemulsion-based templates applied to nanocapsule fabrication.
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http://dx.doi.org/10.3892/ijo.2012.1458DOI Listing
July 2012

Electroporation-induced changes in normal immature rat myoblasts (H9C2).

Gen Physiol Biophys 2012 Mar;31(1):19-25

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2). The electric field was applied in short pulses of 25-3250 V/cm, 50 µs each. The viability of cells was tested by MTT assay after 24 hours. For detection of DNA fragmentation, associated with apoptosis, alkaline and neutral comet assays were performed after EP. Additionally phase contrast images of cells obtained directly after EP were analyzed. Although cell images indicated disruption of cell membranes after EP with high intensities, only a few percent of apoptotic cells and no necrotic effects in the cell nucleus could be observed in comet assay tests performed 2 hours post EP. MTT viability test showed that pulse intensities above 375 V/cm are destructive for myocytes viability.
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http://dx.doi.org/10.4149/gpb_2012_003DOI Listing
March 2012

Comparison of the influence of photodynamic reaction on the Me45 and MEWO cell lines in vitro.

Contemp Oncol (Pozn) 2012 6;16(3):240-3. Epub 2012 Jul 6.

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

Aim Of The Study: Photodynamic therapy (PDT) is an approved, minimally invasive and highly selective therapeutic approach to a variety of tumors. It is based on specific photosensitizer accumulation in the tumor tissue, followed by irradiation with visible light. The photochemical interactions of the photosensitizer, light and molecular oxygen produce singlet oxygen and other reactive oxygen forms. The imbalance between ROS generation and antioxidant capacity of the body gives rise to oxidative stress in the cell, which initiates cell death in PDT. The aim of this study was to investigate the effect of photodynamic reactions in human melanoma cell lines.

Material And Methods: Photofrin(®) (Ph) was used for the photodynamic reaction in vitro as a photosensitizer. The primary cell line was MEWO cell line (granular fibroblasts), derived from a human melanoma. As a recurrent cell line we used Me45 cell line, derived from a lymph node metastasis of skin melanoma. We compared cell viability (MTT assay) to determine the effectiveness of applied therapy. The intracellular distribution of photosensitizer (Photofrin) and localization of mitochondria (Mito-Tracker Green) were detected by confocal microscopy.

Results: We observed that Me45 and MEWO cell viability was dependent on the time of incubation after irradiation. In the recurrent cell line Ph accumulated mainly in the mitochondrial membranes and in MEWO cells also in the cytoplasm. The primary melanoma cell line exhibited significantly reduced cellular proliferation (below 50%) after photodynamic reaction with Ph.

Conclusions: The applied photodynamic reaction was more effective in primary melanoma cells. Additionally, mitochondrial localization of Ph can lead to disturbances of mitochondrial transmembrane potential and finally to release of apoptotic proteins.
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http://dx.doi.org/10.5114/wo.2012.29292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687420PMC
June 2013