Elżbieta Anuszewska

Assessment of the cytotoxicity of aluminium oxide nanoparticles on selected mammalian cells

The rapid development of nanotechnology raises both enthusiasm and anxiety among researchers, which is related to the safety use of the manufactured materials. Thus, the aim of this study was to investigate the effect of aluminium oxide nanoparticles on the viability of selected mammalian cells in vitro. The aluminium oxide nanoparticles were characterised using SEM and BET analyses. Based on Zeta (ζ) potential measurements and particle size distribution, the tested suspensions of aluminium oxide nanoparticles in water and nutrient solutions with or without FBS were classified as unstable. Cell viability, the degree of apoptosis induction and nanoparticles internalization into the cells were assessed after 24 h of cell exposure to Al2O3 nanoparticles. Our results confirm the ability of aluminium oxide nanoparticles to penetrate through the membranes of L929 and BJ cells. Despite this, there was no significant increase in apoptosis or decrease in cell viability observed, suggesting that aluminium oxide nanoparticles in the tested range of concentrations has no cytotoxic effects on the selected mammalian cells.

New approach for amino acid profiling in human plasma by selective fluorescence derivatization

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.

Modification of a micellar system for amino acid separation by MEKC—Application for amino acid profiling in formulations for parenteral use

The paper proposes a new method for amino acid determination which can be applied for amino acid profiling in solutions for parenteral nutrition. The MEKC method based on a mixed micellar system was developed for the separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. Background electrolyte was based on tris-borate buffer with high alkaline pH. Sodium dodecyl sulfate micelles were modified using 1,2-hexanediol as a co-surfactant. The effect of the modifier on amino acid migration was studied with respect to hydrophobicity of the analytes. The modifier appeared to be suitable to improve the separation of AQC-tagged amino acids without an adverse effect on buffer ionic strength or EOF velocity. The method was successfully validated and applied for amino acid profiling in medicinal preparations for parenteral nutrition. The results obtained were compared with a reference chromatographic method (amino acid analyser).

Effect of structural modification at the 4, 3', and 2' positions of doxorubicin on topoisomerase II poisoning, apoptosis, and cytotoxicity in human melanoma cells.

Abstract.Introduction:The mechanism of the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth inhibition seems to be highly specific and dependent on cell type and anthracycline drug. Resistance and the high cardiotoxicity of anthracyclines have stimulated many studies aimed at identifying critical substituents required for optimal activity. Many authors point to the fact that the double-strand breaks, the consequence of the activity of topoisomerase II poisons, and the inability of cells to repair the DNA lesions are the signal for apoptosis. The aim of this study was to define the influence of 4-demetoxy 2′-halogenated analogs with altered basicity at the 3′-position on topoisomerase II and the relationship of that interaction with apoptosis and the cytotoxicity of these novel anthracyclines. Parental human ME18 melanoma cells and the ME18/R subline, obtained experimentally, resistant to doxorubicin (DOX), exposed to 1.7 and 8.6 µM DOX or its analogs, annamycin and WP903 (both 0.3 and 3.0 µM) were studied.Materials and Methods:The MTT test was used to assay cytotoxicity. Interaction of the drugs with topoisomerase II and apoptosis were done by Western blot and fluorescence microscopy using Hoechst 33342.Results:The structural changes at positions 4, 2′, and 3′ can influence topoisomerase II interaction and apoptotic activity, although correlation between these events and cytotoxic consequences has not been proved.Conclusions:The biological response of the cells to the structurally similar anthracyclines may be variable and probably depends on the cell type which seems to be an additional problem in the multifactorial resistance of tumor cells to anthracyclines.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Selenitetriglycerides—Redox-active agents

BACKGROUND Human prostate cancer (hPCa) is the most commonly diagnosed cancer in elderly men and is the second leading cause of male cancer death. Data from epidemiological, eco-environmental, nutritional prevention and clinical trials suggest that selenium Se(IV) can prevent prostate cancer. Selol, a new organic semisynthetic derivative of Se(IV), is a mixture of selenitetriglycerides. This mixture is non-toxic and non-mutagenic, and after po treatment - 56-times less toxic (in mice) than sodium selenite. It exhibits strong anti-cancer activity in vitro in many cancer cell lines and can overcome the cell resistance to doxorubicin. Selol seems a promising compound for prostate cancer therapy. MATERIALS AND METHODS The aim of the present study is the evaluation of Selols influence on intracellular redox state (Eh) of prostatic tumors and the liver in androgen-dependent hPCa-bearing mice, and extracellular redox state in serum of these mice. RESULTS AND CONCLUSIONS The anticancer activity of Selol involves perturbation of the redox regulation in the androgen dependent hPCa (LNCaP) cells, but not in healthy cells. After Selol treatment, intracellular Eh has increased in tumors from -223 mV to -175 mV, while in serum it has decreased (-82 mV vs -113 mV). It shows significant changes Eh in the extra- and intracellular environment. The difference decreases from 141 mV to 62 mV. The changes suggest that a tumor cell was probably directed toward apoptosis. This is exemplified in a significant decrease in cancer tumor mass by approx. 17% after the three weeks of Selol administration.

Isothiocyanate–drug interactions in the human adenocarcinoma cell line Caco-2

Isothiocyanates, among which alyssin is counted, are the compounds that have proved chemopreventive properties and the ability to induce the 2 and the 3 detoxification phase by affecting the transcription factor nuclear erythroid 2-related factor (Nrf2). Having a positive effect on the human body, these compounds are used as dietary supplements. Because of the observed increase in the consumption of dietary supplements taken along with the drugs routinely used in medical practice, this study examined the possibility of interactions between alyssin and drugs, which could have an impact on cell metabolism. We have determined the effects of the tested substances and their interactions on the expression and activity of the phase 2 genes, as well as on the drug transport, which could be influenced by affecting the expression of transport proteins that belong to the 3 phase of metabolism. It was also studied whether the transcription factor Nrf2 is responsible for the interactions that occurred. The results showed that the interactions between alyssin and the tested drugs strengthen or weaken the effect of the drugs given separately depending on the concentration of alyssin and the type of drug. Even though Nrf2 is involved in the interaction, it seems that it is not the only factor regulating the interactions between the tested medications.

Intracellular glutathione level and efflux in human melanoma and cervical cancer cells differing in doxorubicin resistance.

INTRODUCTION Drug resistance continues to be a major problem in cancer treatment. Occurrence of this phenomenon is often associated with altered levels of glutathione (GSH) and GSH-related enzymes. The aim of the study was to evaluate the possible involvement of GSH and GSH-related enzymes in doxorubicin (DOX) resistance in two types of cancer cells of different etiology, from both parental and DOX-resistant sublines. MATERIALS AND METHODS The human melanoma (ME18 and ME18/R) and cervical cancer cells (HeLa and KB-V1) were tested in terms of their DOX sensitivity (EZ4U test), GSH level (HPLC) and its efflux (spectrofluorometrically). The effects of inhibition of the GSH-related enzymes γ-glutamylcysteine synthetase (γ-GCS) and glutathione S-transferase (GST) were also evaluated. RESULTS Exposure to DOX caused an increase of GSH levels in all tested cells except for HeLa cells. However, depletion of GSH did not have a significant influence on the sensitivity of the cells to DOX. Inhibition of the activity of GST also did not have a major effect on DOX sensitivity, although it caused changes of the GSH content. Our attempts to use the spectrofluorometric method for measurements of GSH efflux were not successful. It could be suggested that in ME18 and HeLa cells treated with DOX, GSH efflux does occur. DISCUSSION The obtained results seem to refute the hypothesis of a central role of GSH in DOX resistance of the tested cells. Despite observations of different effects related to GSH, they do not seem to be essential in terms of DOX resistance. The mechanisms underlying DOX resistance are highly cell-specific.

Role of thiamine in Huntington’s disease pathogenesis: In vitro studies

BACKGROUND Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

Comparison of selected gene expression profiles in sensitive and resistant cancer cells treated with doxorubicin and Selol

Aim of the study Cellular resistance is strongly correlated with the risk of failure in doxorubicin (DOX) treatment, and the knowledge of the mechanisms of resistance and its possible modulation is still very limited. Material and methods In this study, we assessed the effect of 5% Selol and DOX on the expression of genes that affect cell proliferation in the resistant KB-V1 and sensitive HeLa cell lines, using RT2 ProfilerTM PCR Array matrix “Human Cancer Drug Resistance and Metabolism” (SABiosciences). Results We showed that HeLa and KB-V1 cell lines, characterised by varying susceptibility to DOX, have different genetic profiles as regards the studied genes. KB-V1 cells show overexpression of MYC and BCL2 genes, which encode proteins with anti apoptotic properties. Selol, when used in KB-V1 cells, reduced the expression of MYC and BCL2 genes, suggested as a new therapeutic target in the treatment of cancers resistant to cytostatic drugs. Conclusions The results suggest that Selol could be used as a modulator that enhances the cytotoxic effects of doxorubicin, particularly in cells resistant to this drug.