1 Modulatory effect of selenium on cell-cycle regulatory genes in the 2 prostate adenocarcinoma cell line 3 4 Agnieszka Wanda Piastowska-Ciesielska1*, Małgorzata Gajewska2, Waldemar 5 Wagner3, Kamila Domińska1, Tomasz Ochędalski1 6 7 1Department of Comparative Endocrinology, Faculty of Biomedical Sciences and 8 Postgraduate Training, Medical University of Lodz, Poland 9 2Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of 10 Life Sciences - SGGW, Poland. 11 3Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of 12 Sciences, Lodz, Poland. 13 14 * Corresponding author: Dr Agnieszka Wanda Piastowska-Ciesielska 15 Department of Comparative Endocrinology, Faculty of Biomedical Sciences and 16 Postgraduate Training, Medical University of Lodz 17 Zeligowskiego 7/9, Lodz 90-752, POLAND 18 [email protected] 19 Tel/fax +48 42 677 93 18 20 1 1 Summary 2 Epidemiological data indicate that selenium status is inversely connected with cancer 3 risk. Animal and human studies have demonstrated that most inorganic and organic 4 forms of selenium compounds have anticancer action. This work investigated the 5 impact of organic selenium on multiple signaling pathways involved in the inhibition of 6 prostate cancer cells viability. Prostate adenocarcinoma cells (PC-3) were incubated 7 with seleno-L-methionine (SeMet) at four concentrations, cell viability and apoptosis 8 was determined by the WST-1, BrdU assays and Tali image based cytometer. The 9 expression of chosen cell-cycle regulatory genes was determined by Real-time RT– 10 PCR analysis and confirmed at the protein level. SeMet treatment of PC-3 cells 11 resulted in an inhibition of cell proliferation in a dose- and time-dependent manner. 12 The inhibition of proliferation correlated with the up-regulation of gene expression 13 and the protein levels of CCNG1, CHEK1, CDKN1C and GADD45A, whereas SeMet 14 down-regulated the expression of CCNA1 and CDK6 genes. Therefore SeMet 15 inhibits the proliferative activity of prostate cancer cells by a direct influence on the 16 expression of genes involved in the regulation of cell cycle progression. 17 18 Key words: selenium; prostate; neoplasm; gene expression; cells viability 19 2 1 INTRODUCTION 2 Prostate cancer (PCa) is the second most commonly diagnosed cancer in the 3 Western countries and the second leading cause of cancer deaths in men in Poland 4 (Malvezzi et al. 2011). PCa expected mortality rates for 2011 vary between 12.6 and 5 8.1/100,000 men in Europe, reaching the highest values in Poland and the UK, and 6 the lowest in Italy (Malvezzi et al. 2011). Surgical treatment and radiation therapy are 7 successful for a local disease, but there is no effective treatment approach for 8 metastatic or refractory PCa (Wang et al. 2011). Chemotherapy can lengthen the 9 lives of men with highly advanced PCa, but it is also associated with dose restrictive 10 toxicity (Mahal et al. 2004). With advances in the understanding of molecular 11 pathways involved in prostate cancer progression, targeted therapies intended to 12 interfere with the way cancer cells grow and survive create new expectations in 13 prostate cancer therapeutics (Liu et al. 2010b). Trace mineral selenium is an 14 essential nutrient of fundamental importance to human biology (Liu et al. 2010a). 15 Some selenium compounds, when administered in supranutritional doses, produce 16 significant health benefits, such as improvement in the immune system and male 17 fertility (Mahn et al. 2009). Epidemiological data indicate that selenium status is 18 inversely connected with cancer risk, and the results of some, but not all, nutritional 19 studies show that high selenium intakes greatly reduce the risk of mammary, 20 prostate, lung, colon, and liver cancers (Zeng et al. 2009). Numerous case control 21 studies have verified a negative correlation between a low serum selenium 22 concentration and the risk of developing PCa (Nomura et al. 2000, Zhang et al. 2009, 23 Zeng et al. 2009, Brooks et al. 2001, Nomura et al. 1987). 3 1 Various in vitro studies have suggested that the possible mechanisms of the 2 antiproliferative effect of selenium formulations in PCa cells are caused by cell cycle 3 arrest and the induction of apoptosis. This work investigated the impact of organic 4 selenium on genes involved in the regulation of tumour cell proliferation. 5 6 7 MATERIALS AND METHODS 8 Cell cultures 9 The study was conducted on the PC-3 metastatic prostate adenocarcinoma cell line 10 obtained from the American Type Culture Collection (ATCC, LGC Standards, 11 Poland). The PC-3 cells are androgen-insensitive cells derived from a grade 4 human 12 prostate adenocarcinoma (Montejo et al. 2010), and are shown to posses high 13 metastatic potential (Simon et al. 2009). The cell cultures were incubated at 37°C 14 and 5% CO2 in a humidified incubator. They were maintained in an RPMI-1640 (Life 15 Technologies Corporation) with 10% fetal bovine serum (FBS) (Life Technologies 16 Corporation). The growth medium was changed three times a week and when the 17 cells reached 70% to 80% confluence, the cultures were split using 0.25% Trypsin 18 (Life Technologies Corporation). 19 20 Cell viability and cell proliferation assays 21 Cell viability was evaluated using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)- 22 2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche Applied Science, Poland), 23 according to the manufactures’ instruction. WST-1 is a tetrazolium salt that is cleaved 24 to formazan by mitochondrial dehydrogenases in contact with metabolically active 4 1 cells. To test the effect of seleno-L-methionine (SeMet) (Sigma-Aldrich, Poland) on 2 cell viability, the PC-3 cells were plated in 96 well plates at a density of 1000 3 cells/well and allowed to adhere to the bottom of the wells overnight before the 4 beginning of treatment. In an initial phase of the study (data not shown), the cells 5 were exposed to increasing concentrations of SeMet for 12 to 72 h (1–200 µmol). 6 The chosen dosages of SetMet represent the levels according to the 7 recommendation of Peternac at al. (Peternac et al. 2008). SeMet was dissolved in 8 culture medium. The results of the preliminary experiment enabled us to choose four 9 concentrations of SeMet (10, 30, 50 and 100 µmol), which were used in the 10 presented study. The PC-3 cells were exposed to the increasing concentrations of 11 SeMet for 12, 24 and 36 hours. Simultaneously the viability of non-treated control 12 cells was assessed. At the end of the exposure period, the medium was replaced 13 with 100 μl of the (1:10 dilution) WST-1 in fresh medium in each well and incubated 14 for two hours. Absorbance was measured on an ELISA plate reader (BioTeck, 15 Germany) at 450 nm with reference at 655 nm. The effect of SeMet was expressed 16 as: (OD of treated cells/OD of non-treated cells)×100. IC50 (inhibition concentration 17 50%) was also calculated by plotting the log of percentage of inhibition values versus 18 the SeMet concentrations assayed (Montejo et al. 2010). The analysis was 19 performed in three independent experiments. 20 Cellular proliferation was measured using a colorimetric immunoassay based on 21 bromodeoxyuridine (BrdU) incorporation into the cellular DNA, following the 22 instructions recommended by the manufacturer (Roche Applied Science, Poland). 23 The experimental design was parallel to the experiments set for the WST-1 assay. 5 1 The cells were incubated with a BrdU labeling reagent for 4 h, followed by fixation in 2 a FixDenat solution for 30 min at room temperature. Then, the cells were incubated 3 with a 1:100 dilution of anti- BrdU-POD for 2h at room temperature. Finally, their 4 immune reaction was examined by adding the substrate solution, and the developed 5 color was measured at 450 and 650 nm in a microplate reader (BioTeck, Germany). 6 The effect of SeMet on cell proliferation was calculated as described previously (in 7 the WST-1 assay), and expressed as a percentage of cell proliferation measured in 8 the non-treated cells. The analysis was performed in three independent experiments. 9 10 Image based cytometer analysis for apoptosis determination 11 Annexin-V/propidium iodide double assay was performed using the Tali™ Apoptosis 12 kit (Life Technologies Corporation). Following treatment (as described previously), 13 cells were released from the 6 well plates with trypsin and centrifuged and the 14 supernatant was discarded. 1 × 106 cells were resuspended in 100 μL Annexin 15 binding buffer and stained with 5 μL of Annexin V Alexa Fluor® 488 according to the 16 manufacturer's instructions. 1 μL of Tali™ Propidium Iodide was added to each 100 17 μL sample and allowed to incubate with cells for 5 min at room temperature in the 18 dark. After this time 25 μL of the stained cells were loaded into a Tali™ Cellular 19 Analysis Slide and analysis using Tali™ Image Based Cytometer. The data were 20 analyzed using Tali™ data acquisition and analysis software (Life Technologies 21 Corporation). 22 23 Real-Time Reverse Transcription PCR 6 1 Total RNA was extracted from the control and SeMet-treated PC-3 cells using using 2 the Trizol® (Life Technologies Corporation). The isolated RNA samples were 3 dissolved in RNase-free water, and the RNA quantity was measured with the use of 4 NanoDrop (Thermo Fisher Scientific, USA). cDNA synthesis for Real-Time Reverse 5 Transcription PCR. Samples with an adequate amount of RNA were treated with 6 DNase I to eliminate DNA contamination, and then purified using an RNeasy 7 MiniElute Cleanup Kit (Qiagen, Poland). cDNA was synthesized from 10 µg of total 8 RNA at a volume of 100 µl using ImProm RT-IITM (Promega, Poland).