CANCER GENOMICS & PROTEOMICS 10: 225-232 (2013) The Influence of Selol on the Expression of Oxidative Stress Genes in Normal and Malignant Prostate Cells IZA KSIAZEK1, KAROLINA SITARZ1, MAGDALENA ROSLON1, ELZBIETA ANUSZEWSKA1, PIOTR SUCHOCKI2,3 and JADWIGA DUDKIEWICZ WILCZYNSKA1 Departments of 1Biochemistry and Biofarmaceuticals and 3Pharmaceutical Chemistr, National Medicines Institue, Warsaw, Poland; 2Department of Bioanalysis and Drug Analysis, Medical University of Warsaw, Warsaw, Poland Abstract. Selol is a mixture of selenitriglycerides, obtained Using the Ames test, Rahden-Staroń et al. demonstrated, that by the chemical modification of sunflower oil, which contain Selol has no mutagenic effect on strains of Salmonella selenium at the +4 oxidation state. The aim of the present typhimurium (2). Our own investigations into the cytotoxicity study was to describe the changes in the expression of genes of 5% Selol showed that it is toxic to malignant HeLa cells related to oxidative stress caused by Selol in prostate cells: (3) as well as HL-60 human promyelocytic leukaemia cells, both normal (PNT1A) and malignant (LNCaP). The changes both susceptible and multidrug resistant (4), dependent on in gene expression in PNT1A and LNCaP cell lines under the dosage and incubation time. Work carried out with BJ normal influence of Selol were measured using a 96-well RT2 human fibroblasts and PNT1A normal human prostate Profiler ™PCR Array: Human Oxidative Stress and epithelial cells (data not published) revealed the significantly Antioxidant Defense, which arrayed 84 genes functionally lower toxic effect of Selol on these cell lines when compared involved in the cellular oxidative stress response. Based on to malignant HeLa cells. It was also shown that due to being the obtained data, LNCaP cells exhibited a significantly a mixture of triglycerides, Selol exhibits much lower levels lower potential for antioxidant defence when compared to of in vitro cytotoxicity when compared to sodium selenite, an PNT1A cells. The response of the malignant LNCaP cells to inorganic selenium compound (3). This effect has also been exposure to Selol was significantly different from that of the confirmed through in vivo experiments (5). normal PNT1A cells, especially after 48 h of incubation. In Due to its antioxidant properties, as well as its presence the case of LNCaP cells, Selol causes down-regulation of the as part of certain enzymes, selenium takes part in metabolic expression of many vital genes. Under in vitro conditions, the processes at the cellular level, protecting cellular membranes efficacy of Selol slightly changes with increasing from free radicals, preventing lipid peroxidation, protein concentration, but significantly increases when the oxidation, and DNA and RNA damage, all of which may incubation time is lengthened. lead to a lowered risk of malignant changes taking place (6). On the other hand, a high intracellular selenium The ability of malignant cells to acquire multidrug resistance, concentration leads to the formation of reactive oxygen along with the powerful side-effects caused by chemotherapy species (ROS), which in turn leads to apoptosis due to the are the driving forces behind the constant search for new, imbalance between ROS generation and the antioxidant more efficient and selective therapeutic compounds. potential of the cell (7). One of these potential anticancer agents is Selol [Patent, As part of ongoing research into the mechanism of action Pol. PL 176530 (Cl.A61K31/095)], an organic selenium (IV) of Selol, this article aims to describe the changes in the compound consisting of a mixture of selenotriglycerides expression of genes related to oxidative stress caused by obtained by the chemical modification of sunflower oil (1). Selol in prostate cells, both normal (PNT1A) and malignant (LNCaP). Prior to undertaking molecular analysis, the cytotoxic influence of 5% Selol on selected prostate cells was Correspondence to: Iza Ksiazek, Department of Biochemistry and measured using the salt 3-(4,5-dimethylthiazol-2-yl)-2,5- Biofarmaceuticals, National Medicines Institue, Chelmska 30/34, diphenyltetrazolium bromide (MTT assay). 00-725 Warsaw, Poland. E-mail: [email protected] The levels of gene expression were determined using real- Key Words: LNCaP, PNT1A, Selol, gene expression, oxidative time polymerase chain reaction (PCR) using a commercially stress. available array containing 84 genes related to cellular 1109-6535/2013 225 CANCER GENOMICS & PROTEOMICS 10: 225-232 (2013) responses to oxidative stress. The array was selected based to exposure to the compounds of interest using the conditions on the pro-oxidative properties of selenium described in the described above, in order to reach a similar level of confluence literature. It was assumed that the toxic properties of Selol, (approx. 70%). observed in cytotoxic studies, are due in part to its ability to Incubation with Selol. For the purposes of investigating gene disrupt the cellular oxidoreductive balance, leading to the expression in PNT1A and LNCaP cells, the concentration of Selol induction of apoptosis and subsequent cell death (7, 8). was chosen based on a previously performed cytotoxicity assay A complex statistical analysis was carried out on genes (MTT). The concentration chosen was one that caused an which displayed changes in expression level, which then led approximately 30% reduction in the number of live LNCaP cells in to conclusions concerning the cellular processes taking place culture following a 24-h incubation, i.e. 150 μM with respect to under the influence of Selol. selenium. The same concentration was used for PNT1A cells. The impact of Selol on gene expression was observed in both cell lines Materials and Methods following 24 and 48 h of incubation. A culture of appropriate cells in medium was used as a control. Compounds and reagents. Selol was synthesized at the Department of Analysis of Medicines at the Medical University of Warsaw, as RNA isolation and quantification. Total RNA was isolated from cells described in the patent (1). A micellar solution of Selol was used using the GeneMatrix Universal RNA/miRNA Purification Kit. The (based on lecithin, water and Selol), with a declared selenium isolation was performed in three independent replicates for each concentration of 5% (w/v). experimental system. The quantity and quality of isolated RNA was The following reagents were used: phosphate buffered saline determined using a NanoDrop spectrophotometer (Thermo (PBS) without Mg2+ and Ca2+ ions (Institute of Immunology and Scientific, Wilmington, USA). The isolated RNA was stored at Experimental Therapy, Wroclaw, Poland), sterile water (Polfa, −70˚C with added RiboLock™ RNase Inhibitor (Fermentas). Lublin, Poland), RPMI and fetal bovine serum (FBS) (Lonza, Verviers, Belgium), antibiotics: penicillin, streptomycin, PCR array. The investigation of gene expression in the chosen amphotericin B (Lonza, Walkersville MD, USA), trypsin-EDTA and experimental systems was carried out using RT2 Profiler ™ PCR Array: glucose (Sigma, St. Louis MD, USA), HEPES, sodium pyruvate and Human Oxidative Stress and Antioxidant Defense (Qiagen). The (Sigma, Irvine, USA), GeneMatrix Universal RNA/miRNA efficiency of cDNA synthesis along with the quality of the resulting Purification Kit (EURx, Gdansk, Poland), RT2 First-Strand Kit material were examined using RT2 RNA QC PCR Array (Qiagen). (Qiagen, Maryland, USA), RT2 SYBR Green/ROX qPCR Master Mix (Qiagen) and RiboLock™ RNase Inhibitor (Fermentas, ABO, Measuring gene expression using real-time PCR. The experiment Gdansk, Poland). consisted of the following steps: a) Clean-up of the isolated RNA in order to remove contaminating DNA followed by cDNA synthesis Cell culture. The model system described in literature for using RT2 First Strand Kit. b) Confirmation of the presence of cDNA investigating the properties of selenium is a pair of prostate cell in the samples using Human RT2 RNA QC PCR Array. c) Quantitative lines, one normal (PNT1A) and one malignant (LNCaP) (9, 10). The analysis of gene expression in the samples using RT2 Profiler™ PCR LNCaP cell line was first derived from prostate metastases in the Array: Human Oxidative Stress and Antioxidant Defense, using an superclavicular lymph nodes (11). In vitro testing showed that this MX3005p cycler (Stratagene) and RT2 SYBR Green/ROX qPCR line has a doubling time longer than most malignant cell lines, of Master Mix. d) Data analysis using the ΔΔCt method, using RT2 approximately 60 h. LNCaP cells present with morphology Profiler PCR Array Data Analysis software version 3.5 characteristic of malignant epithelial cells, including the expression (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). of specific cytokeratins 8 and 18, as well as a lack of desmin and Quantitative assessment of gene expression is usually carried out factor VIII (12). Due to high sensitivity and low adherence using real-time PCR, where gene expression is measured through following splitting, they require a 48-hour incubation period to quantitative and qualitative changes in mRNA caused by an external properly adhere to the culture flask. The PNT1A normal cell line factor (14). The changes in gene expression in PNT1A and LNCaP was derived from the prostate of a 25-year-old man and cell lines under the influence of Selol were measured using a 96- immortalised by transfection with a plasmid containing the SV40 well RT2 Profiler ™PCR Array: Human Oxidative Stress and viral