Anna Choromańska

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 (HGFs) 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.

Can photodynamic therapy be an alternative method in melanoma treatment

Melanoma is the most severe of all skin neoplasms as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this tumour. Photodynamic therapy is an effective local cancer treatment that induces cytotoxicity through intercellular generation of reactive oxygen species. The disintegration of cellular structures and modulation of genetic information induced by PDT direct cancer cells to a death pathway. Numerous studies suggest that the exposure of tumour cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. In contrast to necrosis, apoptosis is an energy dependent, distinct form of cell death that follows a sequence of genetically programmed events and proceeds without inflammation. This study presents the mechanism of photodynamic therapy and its potential in melanoma treatment. SŁOWA KLUCZOWE: terapia przeciwnowotworowa, apoptoza, reaktywne formy tlenu, stres oksydacyjny, rak skory.

Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy

Tumor markers are measurable biochemicals that are associated with a malignancy. They are either produced by tumor cells (tumor-derived) or by the body in response to tumor cells (tumor-associated). They are usually substances that are released into the circulation and then measured in the blood sample. There are a few exceptions to this, such as tissuebound receptors that must be measured in a biopsy from the solid tumor or proteins that are secreted into the urine. Despite the fact that tumor markers are hardly ever specific enough to be used alone to diagnose cancer, they do have a number of clinical applications. They can be used to stage cancer, to indicate a prognosis, to monitor treatment, or in follow-up to watch for cancer recurrence. Changes in some tumor markers have been sensitive enough to be used as targets in clinical trials. Tumor markers for diagnosis are used in combination with other clinical parameters such as biopsy and radiological findings. Although there are a multitude of tumor markers, very few of them have found their way into clinical practice because of their lack of specificity. However, some of these non-specific markers have found a place in monitoring cancer treatment rather than in diagnosis.

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

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.

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

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.