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Different effect of sodium butyrate on cancer and normal prostate cells
Toxicology in Vitro 27 (2013) 1489–1495
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Different effect of sodium butyrate on cancer and normal prostate cells Lenka Paskova a,⇑, Katerina Smesny Trtkova a,b, Barbora Fialova a, Andrea Benedikova c, Katerina Langova d, Zdenek Kolar a,b a Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic b Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Hnevotinska 5, 775 20 Olomouc, Czech Republic c Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Hnevotinska 5, 775 20 Olomouc, Czech Republic d Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic
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Article history: Received 12 November 2012 Accepted 10 March 2013 Available online 20 March 2013 Keywords: Androgen receptor Coregulators HDAC inhibitor Sodium butyrate Acetylation Prostate cells
a b s t r a c t Sodium butyrate, as a naturally occurring inhibitor of histone deacetylases (HDACI), is a non-toxic agent, with an ability to change histone acetylation and expression of large number genes. This study shows different effects of sodium butyrate on expression and transcription activity of the androgen receptor in cancer (LNCaP, C4-2) and normal (RWPE-1) prostate cells. Moreover, we studied the coregulator expressions and histone acetylation alteration in cancer and normal cells. Coregulators, coactivators as well as corepressors, play an important role in AR-mediated growth and progression of prostate cancer. There is a competition between coactivators and corepressors for binding on the AR and therefore the changes in coregulators expression and ratio could be important for prostate cancer survival. Our study was focused on two coregulators, SMRT and p300, which interact with AR in multiprotein complex and affect the AR transcription activity. Our data indicate that sodium butyrate has an effect on AR coregulators expression, transcription activity and histone acetylation in cancer cells, but there is only minimal effect in normal cells. In addition, the results of changes in acetylation level on lysine residues of histone H4 after sodium butyrate treatment confirm its epigenetic effect on prostate cancer cells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Prostate cancer (CaP) is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males (Jemal et al., 2011). In development and progression of CaP are involved many molecular mechanisms such as genetic alterations and epigenetic changes. The essential role in development and growth of normal prostate as well as prostate cancer progression plays androgen receptor (AR) together with androgens. AR is a ligand-inducible transcription factor which belongs to the superfamily of steroid hormonal nuclear receptors. It is expressed in almost all mammalian organs except spleen and bone marrow (Rahman et al., 2004). However, AR can be activated in non-androgen fashion by other alternative mechanisms (Culig et al., 2002). The nuclear receptors (NR) can both activate as well as inhibit expression of appropriate target genes (Glass and Rosenfeld, 2000). Due to the fact that transcription activity of AR is very important for growth and progression
⇑ Corresponding author. Tel.: +420 585 632 263; fax: +420 585 632 063. E-mail address: [email protected] (L. Paskova). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.03.002
of prostate cancer, many studies are focused on the regulation of AR transcription activity in different ways. One of the possibilities of regulation the transcription activity of NRs is the recruitment of different coregulators (Fu et al., 2003). The coregulators (transcription cofactors) are biological macromolecules which interact with NRs and can increase (coactivators) or decrease (corepressors) transcription activity of NR (Rahman et al., 2004). Both coregulator types are important for effective modulation of target genes transcription which is mediated through the NRs (Gao et al., 2002). The expression of selective coregulators may provide growth advantages for tumor cells during androgen ablation or antiandrogen therapy (Rahman et al., 2004). Coregulators are organized in multiprotein complexes which affect the access of NRs and RNA polymerase II into the target DNA sequence via chromatin remodelation and histone modifications (Ishizuka et al., 2005). Coactivators are molecules with histone acetyltransferase (HAT) activity which interact with NRs in ligand-dependent manner and increased its transcription activity (Gao et al., 2002). The overexpression of coactivators may lead to increase sensitivity of AR onto low level of androgens or to increase the ligand specifity of AR (Taplin and Balk, 2004). In the activation and to induce transcription
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activity of androgen receptor plays an important role coactivator p300. The p300 mediated acetylation of AR increases potential of this receptor to induce proliferation of prostate cells (Fu et al., 2003; Debes et al., 2005). Genetic, biochemical and functional data suggest that some of coregulators may be important regulators for some groups of NR-regulated genes (Glass and Rosenfeld, 2000), e.g. prostate specific antigen (PSA) gene whose expression is regulated by AR. Compared to coactivators, corepressors are molecules which interact with NRs in hormone-absence or in antiandrogens presence and they can block transcription activity of NRs (Rahman et al., 2004). One of the best described corepressors is nuclear protein SMRT (silencing mediator for retinoid and thyroid hormone receptors). SMRT play an important role in development, differentiation and tumorigenesis of prostate cancer (Yoon and Wong, 2006). This corepressor can interact by means of specific motifs not only with non-liganded AR, but it may interact as well with ligand-binding AR (Liao et al., 2003). Because that in ligand-dependent manner could be binding both coactivators and corepressors to the AR, it is the competition between coactivators and corepressors for binding to the AR (Yoon and Wong, 2006; Liao et al., 2003; Dotzlaw et al., 2002). Within this competition the corepressor SMRT is binding to AR only when it is overexpressed (Liao et al., 2003; Dotzlaw et al., 2002). This AR-SMRT interaction may subsequently prevent binding of coactivators to AR (Liao et al., 2003) thus leading to inhibition of transcription activity of the androgen receptor (Yoon and Wong, 2006). The other mechanism for regulation of gene transcription is epigenetic modification of histones, concretely posttranslation acetylation and deacetylation of histones which affect condensation of chromatin. Histone acetylation is associated with release chromatin structure, creation of specific binding sites for transcription coregulators, transcription activation, DNA damage repair etc. On the other site, histone deacetylation leads to chromatin condensation and transcription repression (Ducasse and Brown, 2006; Popov et al., 2007). Histone acetylation/deacetylation is mediated by group of enzymes called histone acetyltransferases (HAT) and histone deacetylases (HDAC). Both HAT and HDAC are involved to mechanism of activation as well as repression and operate in complexes formed from many subunits. These enzymes secure dynamical equilibrium between acetylation and deacetylation of lysines in vivo (Minucci and Pelicci, 2006). For decrease the level of histone acetylation is possible to use chemotherapeutic agents belong to the inhibitors of histone deacetylases (HDACIs). These agents can modify expression of different genes, induce cell cycle arrest, stimulate differentiation and/ or induce apoptosis of transformed cells both in vitro and in vivo (Johnstone, 2002; Gu et al., 2006). Sodium butyrate (NaB) is a non-toxic short-chain fatty acid which is a member of group HDACIs, potential anticancer therapeutics for prostate cancer (Gu et al., 2006). NaB may modify histone acetylation and thereby affect the expression large number of genes and hence also coregulators (Yuan et al., 2004). 2. Materials and methods 2.1. Cell lines The two human prostate cancer cell lines (LNCaP, C4-2) and one normal prostate cell line (RWPE-1) were used in this study. LNCaP cell line was derived from a needle aspiration biopsy of lymph node metastatic lesion of a 50-year-old white man. LNCaP cells express AR which contains a T877A mutation. Cell line C4-2 was derived from chimeric LNCaP/MS (human osteosarcoma cell line) tumors. C4-2 cell line expresses PSA and AR mRNA, but the level of AR is lower than in LNCaP cell line (Sobel and Sadar, 2005a).
The third cell line RWPE-1 was derived from epithelial cells taken from the peripheral zone of a non-neoplastic adult human prostate and these cells were immortalized with HPV-18 (Sobel and Sadar, 2005b). Cell line LNCaP was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and C4-2 cell line was obtained from UroCor Labs (Oklahoma City, OK). RWPE-1 cell line was provided by Department of Experimental Biology Masaryk University (Brno, CZ). Cell lines LNCaP and C4-2 were cultivated in RPMI 1640 medium (Gibco) supplemented with final 10% concentration of fetal bovine serum (FBS), 0.01% antibiotics (penicillin, streptomycin), 2 mM L-glutamine and 1 mM sodium pyruvate. The normal cell line RWPE-1 was cultivated in Keratinocyte-SFM Medium (Kit) with L-glutamine, human recombinant epidermal growth factor (EGF) and bovine pituitary extract (BPE) (Gibco) supplemented with final 0.01% concentration combination of two antibiotics (penicillin, streptomycin), 0.01% concentration of amfomycin and 0.005% concentration of gentamycin. All cell lines were cultivated at 37 °C in atmosphere consisting of 5% CO2 and 95% air. 2.2. Assessment of cell viability MTT assay was conducted to determine the cell viability after treatment with sodium butyrate (NaB). The cells were seeded in a 96-well plates at a density 5000 cells per well for C4-2 and LNCaP and 6500 cells per well for cell line RWPE-1. After a 2-day (LNCaP and C4-2) or 3-day (RWPE-1) cultivation, cells with confluency about 60% were treated with 0.5 mM, 1 mM, 2.5 mM and 5 mM NaB. Control cells were treated only with 0.1% DMSO. Cells were than incubated for 24 or 48 h. After incubation 10 ll of 0.5% MTT (3-[4,5-dimethylthiaolyl]-2,5-diphenyltetrazolium bromide) was added to each well of the 96-well plates and incubation was prolonged for another 4 h at the same conditions. Then 100 ll of 10% SDS was added to each well to dissolve the formazan. The absorbance (A) was measured at 570 nm using Microplate spektrofotometr PowerWave XS (BioTek, Winooski, VT, USA). The percentage of viable cells was calculated as follows: (the average of absorbance of experimental cells/the average absorbance of control cells) 100%. All experiments were performed in triplicate and they were repeated three times. 2.3. Treatment with HDAC inhibitor for total RNA and total protein isolation Cells were seeded at a density 1 106 (C4-2), 1.1 106 (LNCaP) or 1.4 106 (RWPE-1) per 100-mm plate. Cells with confluency of about 70% and were treated with sodium butyrate (NaB) (Sigma, St. Louis, MO) for 24 or 48 h. NaB was dissolved in 10% DMSO and added to the media at final concentration of 1 mM or 5 mM of NaB. Control cells received the same amount of DMSO (0.1% final concentration) as treated cells. 2.4. Total RNA isolation and RT-PCR Total RNA from LNCaP, C4-2 and RWPE-1 cells after 24 or 48 h treatment with NaB or 0.1% DMSO (control cells) was isolated using High Pure RNA Isolation Kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer´s specifications. Two micrograms of total RNA from each sample were used for reverse transcription by Transcriptor First Strand cDNA Synthesis Kit Roche Diagnostics, Basel, Switzerland). 2.5. Real-time PCR 1 ll of cDNA from each sample was used for quantitative realtime PCR analysis using Taq-Man probes labeled with hexafluores-
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cein. Real-time PCR analyzes were performed with Thermo-Start DNA Polymerase (AB gene) using the real-time PCR analyzer Rotor-Gene RG-3000 (Corbett Research, San Francisco, CA). The primers and probes were: AR forward 50 -ATCCCAGTCCCACTTGTGTC-30 ; AR reverse 50 -GGTCTTCTGGGGTGGAAAGT-30 ; AR probe 50 -AAGCGAAATGGGCCCTGGA-30 ; PSA forward 50 -CGGAGAGCTGTGTCACCAT-30 ; PSA reverse 50 -CACAATCCGAGACAGGATGA-30 ; PSA probe 50 -CGTGGATTGGTGCTGCACCC-30 ; SMRT forward 5´-GTGTACAAAGACCGCCAGGT-30 ; SMRT reverse 50 -CAGCCACTGTCTTCCTCTCC-30 ; SMRT probe 50 -TCCGGGAGAAGTTCATGCAGCA-30 ; p300 forward 50 -CAAACGCCGAGTCTTCTTTC-30 ; p300 reverse 50 -GTTGAGCTGCTGTTGGCATA-30 ; p300 probe 50 -CCAGTGTGCCCTCCCTGGGT-30 ; GAPDH forward 50 -GAAGATGGTGGGGATTTC-30 ; GAPDH reverse 50 -GAAGGTGAAGGTCGGAGT-30 ; GAPDH probe 50 -CAAGCTTCCCGTTCTCAGCC-30 . All experiments were prepared in triplicates and all measures were performed at least three times independently. Values were normalized for GAPDH content. Relative expression levels and SD values were calculated using the comparative method. 2.6. Western blot analysis Total proteins were prepared from cell lines after 24 or 48 h cultivation with 0.1% DMSO, 1 mM NaB and 5 mM NaB. The cells were washed once with cold PBS on plates, harvested from plates and lysed in cell lysis buffer (2.5% solution of protease inhibitor cocktail (Complete, Mini, Roche Applied Science), 1% Nonidet P-40). Total cell lysates were incubated 1 h on ice and centrifuged at 14,000g at 4 °C for 30 min. The protein concentration of each sample was determined using the Bradford assay. Thirty micrograms of the total proteins from each sample was separated by 10% SDS–PAGE and transferred onto nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). After 2 h blocking with the 5% non-fat dried milk at room temperature, the membranes were incubated at 4 °C overnight with the specific primary antibodies including anti-AR (clone 441, Santa Cruz), anti-AMACR (P504S, Chemicon) to determine the PSA expression, anti-acetyl-histone H4 (Lys12) (#2591, cell signaling), anti-acetyl-histone H4 (Lys8) (#2594, cell signaling). Anti-a-tubulin (clone AA12, Santa Cruz) was used as a loading control. Following this the membranes were washed for 1 h and incubated for 30 min. at room temperature with secondary antibodies including appropriate peroxidase-conjugated goat antimouse or goat anti-rabbit antibody (both Santa Cruz). Then the membranes were washed again for 1 h. Antibody labeling was detected using with Supersignal West Duro Chemiluminescent Substrate (Pierce, Rockford, IL). Western blot analysis for all proteins after NaB treatment in all three cell lines was repeated at least two times.
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LNCaP and C4-2 and normal prostate cell line RWPE-1. Cells were exposed to these NaB concentrations for 24 or 48 h and the absorbance was measured using MTT assay. As shown in Fig. 1, there was significantly visible decrease in cell viability, especially in LNCaP and C4-2 cell lines after 48 h treatment in comparison with normal cell line RWPE-1. 3.2. Morphology changes in human prostate cell lines in vitro The photos of prostate cells (Fig. 2) demonstrate dose-dependent changes in cell morphology and cell viability (cell number) after 48 h cultivation with different concentrations of sodium butyrate. Morfological (phenotypic) changes in LNCaP and C4-2 cells were following: extension of pseudopodia, round off the cells and lower adhesion of cells to the plate surface. These changes were not observed in normal cell line RWPE-1. Changes of cell number in prostate cancer cell lines visible on the microphotographs are comparable with the results of MTT assay of these cell lines. 3.3. Expression of androgen receptor coregulators in vitro To investigate the effect of different concentrations NaB on expression of two selected coregulators – SMRT and p300, we used real-time RT-PCR with specific primers and Taq-Man probes. Compared with RWPE-1 cell line, in LNCaP and C4-2 prostate cancer cell lines treated with NaB for 24 h, we found the same level of increased expression of both coregulators SMRT and p300 (Fig. 3a). In contrast, 48 h treatment with NaB was followed again by increased expression of SMRT and p300 (Fig. 3b) in both prostate
2.7. Statistical analysis The data were represented as means ± SD of determinations from at least 3 independent experiments. Statistical differences between the untreated control (0.1% DMSO) and treated cells were determined using Kruskal–Wallis tests followed by multiple comparison Mann–Whitney U-tests with Bonferroni correction. Analyzes were performed with the SPSS software version 15 (SSPS, Inc., Chicago, IL, USA) and statistical significance of differences was set as p < 0.05. 3. Results 3.1. Butyrate-induced decrease of cell viability especially in prostate cancer cell lines in vitro Increasing concentration of sodium butyrate: 0.5 mM, 1 mM, 2.5 mM and 5 mM NaB were applied onto prostate cancer cell lines
Fig. 1. Effect of sodium butyrate (NaB) on the cell viability of LNCaP, C4-2 and RWPE-1 cell lines. The cells were treated with various concentrations of NaB (0.5 mM, 1 mM, 2.5 mM and 5 mM) for 24 (a) and 48 h (b). The cell viability was measured by MTT assay. Percentage of viable cells was calculated as compared with DMSO-treated cells (100% survival) and it is the means ± SD from three triplicate experiments. Statistical significance: p < 0.01; p < 0.001.
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Fig. 2. Changes in cell morphology and number after sodium butyrate treatment in vitro. After 48 h incubation of cells with 0.1% DMSO (a), 1 mM NaB (b) and 5 mM NaB (c) on plates were made a photos. Morfological (phenotypic) changes in LNCaP and C4-2 cells were following: extension of pseudopodia (marked by arrows) and/or round off the cells (marked by circles). The total magnification of the microscope (Nikon Eclipse TS 1000) was 100 and the total magnification of the camera was 4,9. All photos were done at the same magnification of both microscope and camera. Scale bar = 20 lm.
cancer cell lines – but the level of SMRT expression was almost double that of p300. Considerable increasing of gene expression of both coregulators (SMRT and p300) was in C4-2 cells in comparison with both other cell lines – prostate cancer cell line LNCaP and normal prostate cell line RWPE-1.
of NaB. Decrease of AR expression was particularly evident in both prostate cancer cell lines after treatment with 5 mM NaB, both after 24 h and 48 h (Fig. 5). In normal cell line RWPE-1 there was no observed changes in AR expression both after 24 h and 48 h (Fig. 5).
3.4. Differences in androgen receptor (AR) and prostate specific antigen (PSA) gene expression
3.6. Sodium butyrate has a very weak effect on the expression level of protein PSA
Real-time RT-PCR analysis showed various level of decrease in AR and PSA gene expression. Dose-dependently treatment with sodium butyrate for 24 h (Fig. 4a) as well as for 48 h (Fig. 4b) led to decreasing of AR and PSA gene expression (compared with control cells treated by 0.1% DMSO) in both LNCaP and C4-2 prostate cancer cell lines. In most cases only higher 5 mM concentration of NaB induced significant decreasing of AR and PSA genes expression compared to the lower 1 mM concentration of NaB. In C4-2 prostate cancer cell line led treatment with 1 mM concentration of NaB for 48 h to increasing of PSA expression, but treatment with 5 mM concentration of NaB for 48 h led to very substantial decrease of PSA expression. There were certain differences between decrease of AR and PSA gene expression under the same cell line and the same treatment. The results of AR and PSA expression are not shown, because their expression level detected by realtime PCR was in all cases too low and therefore non-evaluated.
The expression of protein PSA was detected in the same way as the expression of AR. In prostate cancer cell line LNCaP was shown no effect of NaB on the PSA expression level in all cases (Fig. 5). In normal cell line RWPE-1 the PSA expression was very weak (Fig. 5). In C4-2 cell line was detected no effect of different concentrations of NaB on PSA expression after 24 h treatment (Fig. 5). However, in this cell line there was a moderate increase of PSA expression after 48 h from treatment with 5 mM NaB (Fig. 5).
3.5. Sodium butyrate significantly suppress AR expression on protein level in prostate cancer cell lines but not in normal cell line The results of Western blot analysis showed that sodium butyrate (NaB) has an effect only on prostate cancer cell lines LNCaP and C4-2 in a dose-dependent manner. As shown in Fig. 5, the level of AR expression decreased according to increasing concentration
3.7. Increase of H4 acetylation after sodium butyrate treatment According to Western blot analysis of total cell lysates of LNCaP, C4-2 and RWPE-1 cell lines, sodium butyrate treatment increase the acetylation on two chosen lysines (Lys 8 (K8) and Lys 12 (K12)) of histone H4. In both prostate cancer cell lines LNCaP and C4-2, the acetylation on Lys 8 as well as on Lys 12 were markedly increased after 24 h and 48 h treatments with both different NaB concentrations (Fig. 5). However, the acetylation level of Lys 12 increased gradually with rising concentration of NaB whereas Lys 8 acetylation level increased without noticeable dependence on NaB concentration. As shown Fig. 5, in normal cell line RWPE-1 was shown the same character of the Lys 8 and Lys 12 increase acetylation, but extent of increase was not as high as in prostate cancer cell lines.
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Fig. 3. Variation in expressions of SMRT and p300 coregulators after 24 (a) and 48 h (b) NaB treatment. Two prostate cancer cell lines (LNCaP and C4-2) and one normal prostate cell line (RWPE-1) were exposed to different concentrations of NaB for 24 and 48 h prior to isolation of total RNA. The SMRT and p300 gene expressions were analyzed by real-time RT-PCR. Relative changes in gene expressions compare to the control group (DMSO treated cells) were calculated using comparative DDCt method. The GAPDH gene was used for normalization. The columns represent means ± SD from at least three measures. Statistical significance: p < 0.05.
4. Discussion The HDAC inhibitor sodium butyrate as a naturally occurring potential anticancer agent from the group of HDACIs became a subject of interest in various researches (Gu et al., 2006). Many reports concern an impact of different concentrations of the sodium butyrate on prostate cancer cells, but its effect on normal prostate cells was not sufficiently evaluated. Therefore, the goal of our study was to evaluate the effect of sodium butyrate simultaneously on normal and prostate cancer cells. We determined the AR and PSA gene expressions and changes in acetylation of lysine residues of histone H4 after NaB treatment and its effect on p300 coactivator and SMRT corepressor expressions. The cell survival of LNCaP and C4-2 cell lines after treatment with 5 mM concentrations of NaB was lower in comparison with RWPE-1 cells, particularly after 48 h (Fig. 1b). These data are consistent with previous findings (Kuefer et al., 2004), now we present novel data demonstrating NaB effect on prostate cells derived from normal non-tumor prostate. This finding could bring a new perspective in assessing of sodium butyrate toxicity. Zhuang et al. (2008) described similar effect of NaB on cells derived from gastric tumor. The changes in cell morphology and growth patterns are dose-dependent. Sodium butyrate as a typical inhibitor of HDACs (Gozzini et al., 2003) shows the significant effect on cancer cells, with minimal toxicity on normal cells. However, in comparison to other inhibitors of HDACs with a broad spectrum of epigenetic activities, such as Vorinostat or SAHA (suberoylanilide hydroxamic acid), which have an effect in micromolar concentrations, sodium butyrate was only effective at millimolar concentrations (Minucci and Pelicci, 2006). In connection with this fact, our study presents findings that the effective concentration of NaB (up to 5 mM) does not show so strong toxicity on normal prostate cells as on prostate cancer cells.
Further, the NaB treatment affects expressions of corepressor SMRT and coactivator p300. Real-time RT-PCR analysis showed that especially the 48 h treatment with 5 mM concentration of NaB increases expressions of both above mentioned coregulators in cancer cells. It is known that the coregulators affect the transcriptional activity of AR (Glass and Rosenfeld, 2000) due to the competition between coactivators and corepressors for binding to the AR (Liao et al., 2003). Individual activities of the coregulators or a ratio of their occurrence could play an important role in regulation of the AR (Rahman et al., 2004). We found that expression of SMRT is almost twice as high as expression level of p300 (Fig. 3b). These results suggest that NaB may via increase of corepressor expression lead to decrease transcription activity of AR. Expression analysis focused on AR and PSA genes showed considerable differences in expression level of both these genes. In prostate cancer cells cultivated with 5 mM concentration of NaB we found decreases of AR and PSA gene expression compared with control DMSO-treated cells (Fig. 4). The reduction of the AR expression was confirmed also on protein level (Fig. 5). Compared to that, in the normal cell line RWPE-1 no significant changes of PSA or AR on protein level were detected. However, some controversial results confirming NaB effect on the androgen receptor expression in different prostate cancer cells have been published. Frigo and McDonnell (2008) showed that both 2 mM and 5 mM NaB treatment for 24 or 72 h decrease AR expression in LNCaP and LAPC4 prostate cancer cells. As well Trtkova et al. (2010a) confirmed that NaB reduces AR expression in LNCaP and C4-2 after treatment with 1 mM and 5 mM NaB for 24 or 48 h. In contrast to this, Kim et al. (2007) reported that treatment LNCaP cells with 5 mM or 10 mM NaB for 24 or 48 h led to reduce of AR expression. In the other cell lines, concretely DU-145 and PC-3, treatment with 2 mM NaB for 24 h increased AR expression (Huang et al., 2010).
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sodium butyrate could have the same effect on histone acetylation on both cell types. We found, increase of H4(Lys8) and H4(Lys12) acetylation after NaB treatment which was intensive in both prostate cancer cell lines compared to normal cells (Fig. 5). In conclusion, our results show, that the sodium butyrate, a naturally occurring inhibitor of histone deacetylases, has an effect on prostate cancer cells but it has not considerable effect on normal RWPE-1 cells. This finding may be important for potential introduction of NaB into prostate cancer therapy.
Fig. 4. Changes in expression of androgen receptor (AR) gene and prostate specific antigen (PSA) gene after 24 h (a) and 48 h (b) NaB treatments. Two prostate cancer cell lines (LNCaP and C4-2) exposed to different concentrations of NaB for 24 h and 48 h prior to isolation of total RNA. The AR and PSA gene expression was analyzed by real-time RT-PCR. Relative increase of these genes expression in compare to the control group was calculated using comparative DDCt method. The GAPDH gene was using for normalization. The columns represent means ± SD from at least three measures. Statistical significance: p < 0.05.
We suppose that determined decrease of PSA gene expression (Fig. 4) may be associated with higher expression of corepressor SMRT compared with level of coactivator p300. Androgen receptor, as a transcription factor, forms a multiprotein transcription complex including coregulatory proteins and this complex binds to the specific promoter region (ARE) of AR target genes (Taplin and Balk, 2004). The transcription activity of AR is affected by coregulators and depending on coregulator type – coactivator or corepressor, this transcription complex represses or activates expression of target genes (Glass and Rosenfeld, 2000; Kinoshita et al., 2005; Trtkova et al., 2010b). High doses of NaB promotes expression of corepressor SMRT (Trtkova et al., 2010b), which is a part of the transcription complex with AR, then the transcriptional activity of AR as well as subsequent expression of PSA gene, as a target gene of the AR can be suppressed. Finally, the acetylation of lysine residues at histone tails is one of the most studied post-translation histone modification. Increased H4 acetylation is associated with transcription activation of many genes involved in the suppression of tumor growth (Advani et al., 2010). Chen et al. (2011) showed that valproic acid, belongs to HDACI as well as NaB, stimulates the acetylation level of H4 in prostatic cancer cells. Therefore we examined whether
Fig. 5. Protein expression analysis using antibodies against AR – 110 kDa, PSA – 47 kDa, H4 (Lys 8) – 11 kDa and H4 (Lys 12) – 11 kDa after treatment of LNCaP, C4-2 and RWPE-1 prostate cell lines with sodium butyrate. a-Tubulin – 55 k Da was served as a loading control. Total cell protein lysates were harvested after 24 h and 48 h after treatment with 0.1% DMSO (D), 1 mM NaB (1) and 5 mM NaB (5). Proteins were separated by electrophoresis, transferred to a membrane, hybridized with primary and appropriate secondary antibody. Detection was performed using Supersignal West Duro Chemiluminescent Substrate.
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Conflict of interest None. Acknowledgements We thank Jana Holínková, Eva Pimrová, Ondrˇej Bláha for technical assistance and Karel Soucˇek for cell line RWPE-1. This study was supported by Grants NS10262-3/2009 from the Czech Ministry of Health, Grant Agency of the Czech Republic 303/09/H048, LF_2012_019 and CZ.1.05/2.1.00/01.0030. References Advani, A.S., Gibson, S.E., Douglas, E., Jin, T., Zhao, X., Kalaycio, M., Copelan, E., Sobecks, R., Sekeres, M., Sungren, S., Hsi, E.D., 2010. Histone H4 acetylation by immunohistochemistry and prognosis in newly diagnosed adult acute lymphoblastic leukemia (ALL) patients. BMC Cancer 10, 387. Chen, X., Wong, J.Y.C., Wong, P., Radany, E.H., 2011. Low-dose valproic acid enhances radiosensitivity of prostate cancer through acetylated p53-dependent modulation of mitochondrial membrane potential and apoptosis. Mol. Cancer Res. 9, 448–461. Culig, Z., Klocker, H., Bartsch, G., Hobisch, A., 2002. Androgen receptor in prostate cancer. Endocrinol.-Relat. Cancer 9, 155–170. Debes, J.D., Comuzzi, B., Schmidt, L.J., Dehm, S.M., Culig, Z., Tindall, D.J., 2005. p300 regulates androgen receptor-independent expression of prostate-specific antigen in prostate cancer cells treated chronically with interleukin-6. Cancer Res. 65, 5965–5973. Dotzlaw, H., Moehren, U., Mink, S., Cato, A.C.B., Iñiguez Lluhí, J.A., Baniahmad, A., 2002. The amino terminus of the human AR is target for corepressor action and antihormone agonism. Mol. Endocrinol. 16, 661–673. Ducasse, M., Brown, M.A., 2006. Epigenetic aberrations and cancer. Mol. Cancer 5, 1–10. Frigo, D.E., McDonnell, D.P., 2008. Differential effects of prostate cancer therapeutics on neuroendocrine transdifferentiation. Mol. Cancer Ther. 7, 659–669. Fu, M., Rao, M., Wang, Ch., Sakamaki, T., Wang, J., Di Vizio, D., Zhang, X., Albanese, Ch., Balk, S., Chang, Ch., Fan, S., Rosen, E., Palvimo, J.J., Jänne, O.A., Muratoglu, S., Avantaggiati, M.L., Pestell, R.G., 2003. Acetylation of the androgen receptor enhances coactivator binding and promotes prostate cancer cell growth. Mol. Cell. Biol. 23, 8563–8575. Gao, X., Loggie, B.W., Nawaz, Z., 2002. The roles of sex steroid receptor coregulators in cancer. Mol. Cancer 1, 1–7. Glass, Ch.K., Rosenfeld, M.G., 2000. The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121–141. Gozzini, A., Rovida, E., Sbarba, P.D., Galimbert, S., Santini, V., 2003. Butyrates, as a single drug, induce histone acetylation and granulocytic maturation: possible selectivity on core binding factor-acute myeloid leukemia blasts. Cancer Res. 63, 8955–8961. Gu, J., Zhao, X., Spanjaard, R.A., Chen, T.C., Flanagan, J.N., Boosalis, M., Perrine, S.P., Faller, D.V., 2006. Histone deacetylase-inhibitors sensitize human prostate cancer cell lines to growth suppression and apoptosis by retinoids. J. Cancer Mol. 2, 25–36.
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Huang, K.-H., Chiou, S.-H., Chow, K.-C.h., Lin, T.-Y., Chang, H.-W., Chiang, I.-P., Lee, M.-C.h., 2010. Overexpression of aldo-keto reductase 1C2 is associated with disease progression in patients with prostatic cancer. Histopathology 57, 384– 394. Ishizuka, M., Kawate, H., Takayanagi, R., Ohshima, H., Tao, R.-H., Hagiwara, H., 2005. A zinc finger protein TZF is a novel corepressor of androgen receptor. Biochem. Biophys. Res. Commun. 331, 1025–1031. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global cancer statistics. CA-Cancer J. Clin. 61, 69–90. Johnstone, R.W., 2002. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat. Rev. Drug Discov. 1, 287–299. Kim, J., Park, H., Im, J.Y., Choi, W.S., Kim, H.S., 2007. Sodium butyrate regulates androgen receptor expression and cell cycle arrest in human prostate cancer cells. Anticancer Res. 27, 3285–3292. Kinoshita, M., Nakagawa, T., Shimizu, A., Katsuoka, Y., 2005. Differently regulated androgen receptor transcriptional complex in prostate cancer compared with normal prostate. Int. J. Urol. 12, 390–397. Kuefer, R., Hofer, M.D., Altug, V., Zorn, C., Genze, F., Kunzi-Rapp, K., Hautmann, R.E., Gschwend, J.E., 2004. Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer. Brit. J. Cancer. 90, 535–541. Liao, G., Chen, L.-Y., Zhang, A., Godavarthy, A., Xia, F., Ghosh, J.-C.h., Li, H., Chen, J.D., 2003. Regulation of androgen receptor activity by the nuclear receptor corepressor SMRT. J. Biol. Chem. 278, 5052–5061. Minucci, S., Pelicci, P.G., 2006. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 6, 38–51. Popov, V.M., Wang, Ch., Shirley, L.A., Rosenberg, A., Li, S., Nevalainen, M., Fu, M., Pestell, R.G., 2007. The functional significance of nuclear receptor acetylation. Steroids 72, 221–230. Rahman, M., Miyamoto, H., Chang, Ch., 2004. Androgen receptor coregulators in prostate cancer: mechanisms and clinical implications. Clin. Cancer Res. 10, 2208–2219. Sobel, R.E., Sadar, M.D., 2005a. Cell lines used in prostate cancer research: a compendium of old and new lines – Part 1. J. Urol. 173, 342–359. Sobel, R.E., Sadar, M.D., 2005b. Cell lines used in prostate cancer research: a compendium of old and new lines – Part 2. J. Urol. 173, 360–372. Taplin, M.-E., Balk, S.P., 2004. Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J. Cell. Biochem. 91, 483–490. Trtkova, K., Paskova, L., Matijescukova, N., Strnad, M., Kolar, Z., 2010a. Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB. Neoplasma 57, 406–414. Trtkova, K., Paskova, L., Matijescukova, N., Kolar, Z., 2010b. Formation of AR-SMRT binding in prostate cancer cells treated with natural histone deacetylase inhibitor. Cancer Biomark. 7, 79–90. Yoon, H.-G., Wong, J., 2006. The corepressors silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor are involved in agonist- and antagonist-regulated transcription by androgen receptor. Mol. Endocrinol. 20, 1048–1060. Yuan, H., Liddle, F.J., Mahajan, S., Frank, D.A., 2004. IL-6-induced survival of colorectal carcinoma cells is inhibited by butyrate through down-regulation of the IL-6 receptor. Carcinogenesis 25, 2247–2255. Zhuang, Y.-Q., Li, J.-Y., Chen, Z.-X., Wang, X.-Z., 2008. Effects of sodium butyrate on proliferation and differentiation of human gastric carcinoma cell line AGS. Chin. J. Cancer Res. 27, 135–139.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Different effect of sodium butyrate on cancer and normal prostate cells Lenka Paskova a,⇑, Katerina Smesny Trtkova a,b, Barbora Fialova a, Andrea Benedikova c, Katerina Langova d, Zdenek Kolar a,b a Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic b Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Hnevotinska 5, 775 20 Olomouc, Czech Republic c Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Hnevotinska 5, 775 20 Olomouc, Czech Republic d Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic
a r t i c l e
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Article history: Received 12 November 2012 Accepted 10 March 2013 Available online 20 March 2013 Keywords: Androgen receptor Coregulators HDAC inhibitor Sodium butyrate Acetylation Prostate cells
a b s t r a c t Sodium butyrate, as a naturally occurring inhibitor of histone deacetylases (HDACI), is a non-toxic agent, with an ability to change histone acetylation and expression of large number genes. This study shows different effects of sodium butyrate on expression and transcription activity of the androgen receptor in cancer (LNCaP, C4-2) and normal (RWPE-1) prostate cells. Moreover, we studied the coregulator expressions and histone acetylation alteration in cancer and normal cells. Coregulators, coactivators as well as corepressors, play an important role in AR-mediated growth and progression of prostate cancer. There is a competition between coactivators and corepressors for binding on the AR and therefore the changes in coregulators expression and ratio could be important for prostate cancer survival. Our study was focused on two coregulators, SMRT and p300, which interact with AR in multiprotein complex and affect the AR transcription activity. Our data indicate that sodium butyrate has an effect on AR coregulators expression, transcription activity and histone acetylation in cancer cells, but there is only minimal effect in normal cells. In addition, the results of changes in acetylation level on lysine residues of histone H4 after sodium butyrate treatment confirm its epigenetic effect on prostate cancer cells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Prostate cancer (CaP) is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males (Jemal et al., 2011). In development and progression of CaP are involved many molecular mechanisms such as genetic alterations and epigenetic changes. The essential role in development and growth of normal prostate as well as prostate cancer progression plays androgen receptor (AR) together with androgens. AR is a ligand-inducible transcription factor which belongs to the superfamily of steroid hormonal nuclear receptors. It is expressed in almost all mammalian organs except spleen and bone marrow (Rahman et al., 2004). However, AR can be activated in non-androgen fashion by other alternative mechanisms (Culig et al., 2002). The nuclear receptors (NR) can both activate as well as inhibit expression of appropriate target genes (Glass and Rosenfeld, 2000). Due to the fact that transcription activity of AR is very important for growth and progression
⇑ Corresponding author. Tel.: +420 585 632 263; fax: +420 585 632 063. E-mail address: [email protected] (L. Paskova). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.03.002
of prostate cancer, many studies are focused on the regulation of AR transcription activity in different ways. One of the possibilities of regulation the transcription activity of NRs is the recruitment of different coregulators (Fu et al., 2003). The coregulators (transcription cofactors) are biological macromolecules which interact with NRs and can increase (coactivators) or decrease (corepressors) transcription activity of NR (Rahman et al., 2004). Both coregulator types are important for effective modulation of target genes transcription which is mediated through the NRs (Gao et al., 2002). The expression of selective coregulators may provide growth advantages for tumor cells during androgen ablation or antiandrogen therapy (Rahman et al., 2004). Coregulators are organized in multiprotein complexes which affect the access of NRs and RNA polymerase II into the target DNA sequence via chromatin remodelation and histone modifications (Ishizuka et al., 2005). Coactivators are molecules with histone acetyltransferase (HAT) activity which interact with NRs in ligand-dependent manner and increased its transcription activity (Gao et al., 2002). The overexpression of coactivators may lead to increase sensitivity of AR onto low level of androgens or to increase the ligand specifity of AR (Taplin and Balk, 2004). In the activation and to induce transcription
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activity of androgen receptor plays an important role coactivator p300. The p300 mediated acetylation of AR increases potential of this receptor to induce proliferation of prostate cells (Fu et al., 2003; Debes et al., 2005). Genetic, biochemical and functional data suggest that some of coregulators may be important regulators for some groups of NR-regulated genes (Glass and Rosenfeld, 2000), e.g. prostate specific antigen (PSA) gene whose expression is regulated by AR. Compared to coactivators, corepressors are molecules which interact with NRs in hormone-absence or in antiandrogens presence and they can block transcription activity of NRs (Rahman et al., 2004). One of the best described corepressors is nuclear protein SMRT (silencing mediator for retinoid and thyroid hormone receptors). SMRT play an important role in development, differentiation and tumorigenesis of prostate cancer (Yoon and Wong, 2006). This corepressor can interact by means of specific motifs not only with non-liganded AR, but it may interact as well with ligand-binding AR (Liao et al., 2003). Because that in ligand-dependent manner could be binding both coactivators and corepressors to the AR, it is the competition between coactivators and corepressors for binding to the AR (Yoon and Wong, 2006; Liao et al., 2003; Dotzlaw et al., 2002). Within this competition the corepressor SMRT is binding to AR only when it is overexpressed (Liao et al., 2003; Dotzlaw et al., 2002). This AR-SMRT interaction may subsequently prevent binding of coactivators to AR (Liao et al., 2003) thus leading to inhibition of transcription activity of the androgen receptor (Yoon and Wong, 2006). The other mechanism for regulation of gene transcription is epigenetic modification of histones, concretely posttranslation acetylation and deacetylation of histones which affect condensation of chromatin. Histone acetylation is associated with release chromatin structure, creation of specific binding sites for transcription coregulators, transcription activation, DNA damage repair etc. On the other site, histone deacetylation leads to chromatin condensation and transcription repression (Ducasse and Brown, 2006; Popov et al., 2007). Histone acetylation/deacetylation is mediated by group of enzymes called histone acetyltransferases (HAT) and histone deacetylases (HDAC). Both HAT and HDAC are involved to mechanism of activation as well as repression and operate in complexes formed from many subunits. These enzymes secure dynamical equilibrium between acetylation and deacetylation of lysines in vivo (Minucci and Pelicci, 2006). For decrease the level of histone acetylation is possible to use chemotherapeutic agents belong to the inhibitors of histone deacetylases (HDACIs). These agents can modify expression of different genes, induce cell cycle arrest, stimulate differentiation and/ or induce apoptosis of transformed cells both in vitro and in vivo (Johnstone, 2002; Gu et al., 2006). Sodium butyrate (NaB) is a non-toxic short-chain fatty acid which is a member of group HDACIs, potential anticancer therapeutics for prostate cancer (Gu et al., 2006). NaB may modify histone acetylation and thereby affect the expression large number of genes and hence also coregulators (Yuan et al., 2004). 2. Materials and methods 2.1. Cell lines The two human prostate cancer cell lines (LNCaP, C4-2) and one normal prostate cell line (RWPE-1) were used in this study. LNCaP cell line was derived from a needle aspiration biopsy of lymph node metastatic lesion of a 50-year-old white man. LNCaP cells express AR which contains a T877A mutation. Cell line C4-2 was derived from chimeric LNCaP/MS (human osteosarcoma cell line) tumors. C4-2 cell line expresses PSA and AR mRNA, but the level of AR is lower than in LNCaP cell line (Sobel and Sadar, 2005a).
The third cell line RWPE-1 was derived from epithelial cells taken from the peripheral zone of a non-neoplastic adult human prostate and these cells were immortalized with HPV-18 (Sobel and Sadar, 2005b). Cell line LNCaP was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and C4-2 cell line was obtained from UroCor Labs (Oklahoma City, OK). RWPE-1 cell line was provided by Department of Experimental Biology Masaryk University (Brno, CZ). Cell lines LNCaP and C4-2 were cultivated in RPMI 1640 medium (Gibco) supplemented with final 10% concentration of fetal bovine serum (FBS), 0.01% antibiotics (penicillin, streptomycin), 2 mM L-glutamine and 1 mM sodium pyruvate. The normal cell line RWPE-1 was cultivated in Keratinocyte-SFM Medium (Kit) with L-glutamine, human recombinant epidermal growth factor (EGF) and bovine pituitary extract (BPE) (Gibco) supplemented with final 0.01% concentration combination of two antibiotics (penicillin, streptomycin), 0.01% concentration of amfomycin and 0.005% concentration of gentamycin. All cell lines were cultivated at 37 °C in atmosphere consisting of 5% CO2 and 95% air. 2.2. Assessment of cell viability MTT assay was conducted to determine the cell viability after treatment with sodium butyrate (NaB). The cells were seeded in a 96-well plates at a density 5000 cells per well for C4-2 and LNCaP and 6500 cells per well for cell line RWPE-1. After a 2-day (LNCaP and C4-2) or 3-day (RWPE-1) cultivation, cells with confluency about 60% were treated with 0.5 mM, 1 mM, 2.5 mM and 5 mM NaB. Control cells were treated only with 0.1% DMSO. Cells were than incubated for 24 or 48 h. After incubation 10 ll of 0.5% MTT (3-[4,5-dimethylthiaolyl]-2,5-diphenyltetrazolium bromide) was added to each well of the 96-well plates and incubation was prolonged for another 4 h at the same conditions. Then 100 ll of 10% SDS was added to each well to dissolve the formazan. The absorbance (A) was measured at 570 nm using Microplate spektrofotometr PowerWave XS (BioTek, Winooski, VT, USA). The percentage of viable cells was calculated as follows: (the average of absorbance of experimental cells/the average absorbance of control cells) 100%. All experiments were performed in triplicate and they were repeated three times. 2.3. Treatment with HDAC inhibitor for total RNA and total protein isolation Cells were seeded at a density 1 106 (C4-2), 1.1 106 (LNCaP) or 1.4 106 (RWPE-1) per 100-mm plate. Cells with confluency of about 70% and were treated with sodium butyrate (NaB) (Sigma, St. Louis, MO) for 24 or 48 h. NaB was dissolved in 10% DMSO and added to the media at final concentration of 1 mM or 5 mM of NaB. Control cells received the same amount of DMSO (0.1% final concentration) as treated cells. 2.4. Total RNA isolation and RT-PCR Total RNA from LNCaP, C4-2 and RWPE-1 cells after 24 or 48 h treatment with NaB or 0.1% DMSO (control cells) was isolated using High Pure RNA Isolation Kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer´s specifications. Two micrograms of total RNA from each sample were used for reverse transcription by Transcriptor First Strand cDNA Synthesis Kit Roche Diagnostics, Basel, Switzerland). 2.5. Real-time PCR 1 ll of cDNA from each sample was used for quantitative realtime PCR analysis using Taq-Man probes labeled with hexafluores-
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cein. Real-time PCR analyzes were performed with Thermo-Start DNA Polymerase (AB gene) using the real-time PCR analyzer Rotor-Gene RG-3000 (Corbett Research, San Francisco, CA). The primers and probes were: AR forward 50 -ATCCCAGTCCCACTTGTGTC-30 ; AR reverse 50 -GGTCTTCTGGGGTGGAAAGT-30 ; AR probe 50 -AAGCGAAATGGGCCCTGGA-30 ; PSA forward 50 -CGGAGAGCTGTGTCACCAT-30 ; PSA reverse 50 -CACAATCCGAGACAGGATGA-30 ; PSA probe 50 -CGTGGATTGGTGCTGCACCC-30 ; SMRT forward 5´-GTGTACAAAGACCGCCAGGT-30 ; SMRT reverse 50 -CAGCCACTGTCTTCCTCTCC-30 ; SMRT probe 50 -TCCGGGAGAAGTTCATGCAGCA-30 ; p300 forward 50 -CAAACGCCGAGTCTTCTTTC-30 ; p300 reverse 50 -GTTGAGCTGCTGTTGGCATA-30 ; p300 probe 50 -CCAGTGTGCCCTCCCTGGGT-30 ; GAPDH forward 50 -GAAGATGGTGGGGATTTC-30 ; GAPDH reverse 50 -GAAGGTGAAGGTCGGAGT-30 ; GAPDH probe 50 -CAAGCTTCCCGTTCTCAGCC-30 . All experiments were prepared in triplicates and all measures were performed at least three times independently. Values were normalized for GAPDH content. Relative expression levels and SD values were calculated using the comparative method. 2.6. Western blot analysis Total proteins were prepared from cell lines after 24 or 48 h cultivation with 0.1% DMSO, 1 mM NaB and 5 mM NaB. The cells were washed once with cold PBS on plates, harvested from plates and lysed in cell lysis buffer (2.5% solution of protease inhibitor cocktail (Complete, Mini, Roche Applied Science), 1% Nonidet P-40). Total cell lysates were incubated 1 h on ice and centrifuged at 14,000g at 4 °C for 30 min. The protein concentration of each sample was determined using the Bradford assay. Thirty micrograms of the total proteins from each sample was separated by 10% SDS–PAGE and transferred onto nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). After 2 h blocking with the 5% non-fat dried milk at room temperature, the membranes were incubated at 4 °C overnight with the specific primary antibodies including anti-AR (clone 441, Santa Cruz), anti-AMACR (P504S, Chemicon) to determine the PSA expression, anti-acetyl-histone H4 (Lys12) (#2591, cell signaling), anti-acetyl-histone H4 (Lys8) (#2594, cell signaling). Anti-a-tubulin (clone AA12, Santa Cruz) was used as a loading control. Following this the membranes were washed for 1 h and incubated for 30 min. at room temperature with secondary antibodies including appropriate peroxidase-conjugated goat antimouse or goat anti-rabbit antibody (both Santa Cruz). Then the membranes were washed again for 1 h. Antibody labeling was detected using with Supersignal West Duro Chemiluminescent Substrate (Pierce, Rockford, IL). Western blot analysis for all proteins after NaB treatment in all three cell lines was repeated at least two times.
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LNCaP and C4-2 and normal prostate cell line RWPE-1. Cells were exposed to these NaB concentrations for 24 or 48 h and the absorbance was measured using MTT assay. As shown in Fig. 1, there was significantly visible decrease in cell viability, especially in LNCaP and C4-2 cell lines after 48 h treatment in comparison with normal cell line RWPE-1. 3.2. Morphology changes in human prostate cell lines in vitro The photos of prostate cells (Fig. 2) demonstrate dose-dependent changes in cell morphology and cell viability (cell number) after 48 h cultivation with different concentrations of sodium butyrate. Morfological (phenotypic) changes in LNCaP and C4-2 cells were following: extension of pseudopodia, round off the cells and lower adhesion of cells to the plate surface. These changes were not observed in normal cell line RWPE-1. Changes of cell number in prostate cancer cell lines visible on the microphotographs are comparable with the results of MTT assay of these cell lines. 3.3. Expression of androgen receptor coregulators in vitro To investigate the effect of different concentrations NaB on expression of two selected coregulators – SMRT and p300, we used real-time RT-PCR with specific primers and Taq-Man probes. Compared with RWPE-1 cell line, in LNCaP and C4-2 prostate cancer cell lines treated with NaB for 24 h, we found the same level of increased expression of both coregulators SMRT and p300 (Fig. 3a). In contrast, 48 h treatment with NaB was followed again by increased expression of SMRT and p300 (Fig. 3b) in both prostate
2.7. Statistical analysis The data were represented as means ± SD of determinations from at least 3 independent experiments. Statistical differences between the untreated control (0.1% DMSO) and treated cells were determined using Kruskal–Wallis tests followed by multiple comparison Mann–Whitney U-tests with Bonferroni correction. Analyzes were performed with the SPSS software version 15 (SSPS, Inc., Chicago, IL, USA) and statistical significance of differences was set as p < 0.05. 3. Results 3.1. Butyrate-induced decrease of cell viability especially in prostate cancer cell lines in vitro Increasing concentration of sodium butyrate: 0.5 mM, 1 mM, 2.5 mM and 5 mM NaB were applied onto prostate cancer cell lines
Fig. 1. Effect of sodium butyrate (NaB) on the cell viability of LNCaP, C4-2 and RWPE-1 cell lines. The cells were treated with various concentrations of NaB (0.5 mM, 1 mM, 2.5 mM and 5 mM) for 24 (a) and 48 h (b). The cell viability was measured by MTT assay. Percentage of viable cells was calculated as compared with DMSO-treated cells (100% survival) and it is the means ± SD from three triplicate experiments. Statistical significance: p < 0.01; p < 0.001.
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Fig. 2. Changes in cell morphology and number after sodium butyrate treatment in vitro. After 48 h incubation of cells with 0.1% DMSO (a), 1 mM NaB (b) and 5 mM NaB (c) on plates were made a photos. Morfological (phenotypic) changes in LNCaP and C4-2 cells were following: extension of pseudopodia (marked by arrows) and/or round off the cells (marked by circles). The total magnification of the microscope (Nikon Eclipse TS 1000) was 100 and the total magnification of the camera was 4,9. All photos were done at the same magnification of both microscope and camera. Scale bar = 20 lm.
cancer cell lines – but the level of SMRT expression was almost double that of p300. Considerable increasing of gene expression of both coregulators (SMRT and p300) was in C4-2 cells in comparison with both other cell lines – prostate cancer cell line LNCaP and normal prostate cell line RWPE-1.
of NaB. Decrease of AR expression was particularly evident in both prostate cancer cell lines after treatment with 5 mM NaB, both after 24 h and 48 h (Fig. 5). In normal cell line RWPE-1 there was no observed changes in AR expression both after 24 h and 48 h (Fig. 5).
3.4. Differences in androgen receptor (AR) and prostate specific antigen (PSA) gene expression
3.6. Sodium butyrate has a very weak effect on the expression level of protein PSA
Real-time RT-PCR analysis showed various level of decrease in AR and PSA gene expression. Dose-dependently treatment with sodium butyrate for 24 h (Fig. 4a) as well as for 48 h (Fig. 4b) led to decreasing of AR and PSA gene expression (compared with control cells treated by 0.1% DMSO) in both LNCaP and C4-2 prostate cancer cell lines. In most cases only higher 5 mM concentration of NaB induced significant decreasing of AR and PSA genes expression compared to the lower 1 mM concentration of NaB. In C4-2 prostate cancer cell line led treatment with 1 mM concentration of NaB for 48 h to increasing of PSA expression, but treatment with 5 mM concentration of NaB for 48 h led to very substantial decrease of PSA expression. There were certain differences between decrease of AR and PSA gene expression under the same cell line and the same treatment. The results of AR and PSA expression are not shown, because their expression level detected by realtime PCR was in all cases too low and therefore non-evaluated.
The expression of protein PSA was detected in the same way as the expression of AR. In prostate cancer cell line LNCaP was shown no effect of NaB on the PSA expression level in all cases (Fig. 5). In normal cell line RWPE-1 the PSA expression was very weak (Fig. 5). In C4-2 cell line was detected no effect of different concentrations of NaB on PSA expression after 24 h treatment (Fig. 5). However, in this cell line there was a moderate increase of PSA expression after 48 h from treatment with 5 mM NaB (Fig. 5).
3.5. Sodium butyrate significantly suppress AR expression on protein level in prostate cancer cell lines but not in normal cell line The results of Western blot analysis showed that sodium butyrate (NaB) has an effect only on prostate cancer cell lines LNCaP and C4-2 in a dose-dependent manner. As shown in Fig. 5, the level of AR expression decreased according to increasing concentration
3.7. Increase of H4 acetylation after sodium butyrate treatment According to Western blot analysis of total cell lysates of LNCaP, C4-2 and RWPE-1 cell lines, sodium butyrate treatment increase the acetylation on two chosen lysines (Lys 8 (K8) and Lys 12 (K12)) of histone H4. In both prostate cancer cell lines LNCaP and C4-2, the acetylation on Lys 8 as well as on Lys 12 were markedly increased after 24 h and 48 h treatments with both different NaB concentrations (Fig. 5). However, the acetylation level of Lys 12 increased gradually with rising concentration of NaB whereas Lys 8 acetylation level increased without noticeable dependence on NaB concentration. As shown Fig. 5, in normal cell line RWPE-1 was shown the same character of the Lys 8 and Lys 12 increase acetylation, but extent of increase was not as high as in prostate cancer cell lines.
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Fig. 3. Variation in expressions of SMRT and p300 coregulators after 24 (a) and 48 h (b) NaB treatment. Two prostate cancer cell lines (LNCaP and C4-2) and one normal prostate cell line (RWPE-1) were exposed to different concentrations of NaB for 24 and 48 h prior to isolation of total RNA. The SMRT and p300 gene expressions were analyzed by real-time RT-PCR. Relative changes in gene expressions compare to the control group (DMSO treated cells) were calculated using comparative DDCt method. The GAPDH gene was used for normalization. The columns represent means ± SD from at least three measures. Statistical significance: p < 0.05.
4. Discussion The HDAC inhibitor sodium butyrate as a naturally occurring potential anticancer agent from the group of HDACIs became a subject of interest in various researches (Gu et al., 2006). Many reports concern an impact of different concentrations of the sodium butyrate on prostate cancer cells, but its effect on normal prostate cells was not sufficiently evaluated. Therefore, the goal of our study was to evaluate the effect of sodium butyrate simultaneously on normal and prostate cancer cells. We determined the AR and PSA gene expressions and changes in acetylation of lysine residues of histone H4 after NaB treatment and its effect on p300 coactivator and SMRT corepressor expressions. The cell survival of LNCaP and C4-2 cell lines after treatment with 5 mM concentrations of NaB was lower in comparison with RWPE-1 cells, particularly after 48 h (Fig. 1b). These data are consistent with previous findings (Kuefer et al., 2004), now we present novel data demonstrating NaB effect on prostate cells derived from normal non-tumor prostate. This finding could bring a new perspective in assessing of sodium butyrate toxicity. Zhuang et al. (2008) described similar effect of NaB on cells derived from gastric tumor. The changes in cell morphology and growth patterns are dose-dependent. Sodium butyrate as a typical inhibitor of HDACs (Gozzini et al., 2003) shows the significant effect on cancer cells, with minimal toxicity on normal cells. However, in comparison to other inhibitors of HDACs with a broad spectrum of epigenetic activities, such as Vorinostat or SAHA (suberoylanilide hydroxamic acid), which have an effect in micromolar concentrations, sodium butyrate was only effective at millimolar concentrations (Minucci and Pelicci, 2006). In connection with this fact, our study presents findings that the effective concentration of NaB (up to 5 mM) does not show so strong toxicity on normal prostate cells as on prostate cancer cells.
Further, the NaB treatment affects expressions of corepressor SMRT and coactivator p300. Real-time RT-PCR analysis showed that especially the 48 h treatment with 5 mM concentration of NaB increases expressions of both above mentioned coregulators in cancer cells. It is known that the coregulators affect the transcriptional activity of AR (Glass and Rosenfeld, 2000) due to the competition between coactivators and corepressors for binding to the AR (Liao et al., 2003). Individual activities of the coregulators or a ratio of their occurrence could play an important role in regulation of the AR (Rahman et al., 2004). We found that expression of SMRT is almost twice as high as expression level of p300 (Fig. 3b). These results suggest that NaB may via increase of corepressor expression lead to decrease transcription activity of AR. Expression analysis focused on AR and PSA genes showed considerable differences in expression level of both these genes. In prostate cancer cells cultivated with 5 mM concentration of NaB we found decreases of AR and PSA gene expression compared with control DMSO-treated cells (Fig. 4). The reduction of the AR expression was confirmed also on protein level (Fig. 5). Compared to that, in the normal cell line RWPE-1 no significant changes of PSA or AR on protein level were detected. However, some controversial results confirming NaB effect on the androgen receptor expression in different prostate cancer cells have been published. Frigo and McDonnell (2008) showed that both 2 mM and 5 mM NaB treatment for 24 or 72 h decrease AR expression in LNCaP and LAPC4 prostate cancer cells. As well Trtkova et al. (2010a) confirmed that NaB reduces AR expression in LNCaP and C4-2 after treatment with 1 mM and 5 mM NaB for 24 or 48 h. In contrast to this, Kim et al. (2007) reported that treatment LNCaP cells with 5 mM or 10 mM NaB for 24 or 48 h led to reduce of AR expression. In the other cell lines, concretely DU-145 and PC-3, treatment with 2 mM NaB for 24 h increased AR expression (Huang et al., 2010).
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sodium butyrate could have the same effect on histone acetylation on both cell types. We found, increase of H4(Lys8) and H4(Lys12) acetylation after NaB treatment which was intensive in both prostate cancer cell lines compared to normal cells (Fig. 5). In conclusion, our results show, that the sodium butyrate, a naturally occurring inhibitor of histone deacetylases, has an effect on prostate cancer cells but it has not considerable effect on normal RWPE-1 cells. This finding may be important for potential introduction of NaB into prostate cancer therapy.
Fig. 4. Changes in expression of androgen receptor (AR) gene and prostate specific antigen (PSA) gene after 24 h (a) and 48 h (b) NaB treatments. Two prostate cancer cell lines (LNCaP and C4-2) exposed to different concentrations of NaB for 24 h and 48 h prior to isolation of total RNA. The AR and PSA gene expression was analyzed by real-time RT-PCR. Relative increase of these genes expression in compare to the control group was calculated using comparative DDCt method. The GAPDH gene was using for normalization. The columns represent means ± SD from at least three measures. Statistical significance: p < 0.05.
We suppose that determined decrease of PSA gene expression (Fig. 4) may be associated with higher expression of corepressor SMRT compared with level of coactivator p300. Androgen receptor, as a transcription factor, forms a multiprotein transcription complex including coregulatory proteins and this complex binds to the specific promoter region (ARE) of AR target genes (Taplin and Balk, 2004). The transcription activity of AR is affected by coregulators and depending on coregulator type – coactivator or corepressor, this transcription complex represses or activates expression of target genes (Glass and Rosenfeld, 2000; Kinoshita et al., 2005; Trtkova et al., 2010b). High doses of NaB promotes expression of corepressor SMRT (Trtkova et al., 2010b), which is a part of the transcription complex with AR, then the transcriptional activity of AR as well as subsequent expression of PSA gene, as a target gene of the AR can be suppressed. Finally, the acetylation of lysine residues at histone tails is one of the most studied post-translation histone modification. Increased H4 acetylation is associated with transcription activation of many genes involved in the suppression of tumor growth (Advani et al., 2010). Chen et al. (2011) showed that valproic acid, belongs to HDACI as well as NaB, stimulates the acetylation level of H4 in prostatic cancer cells. Therefore we examined whether
Fig. 5. Protein expression analysis using antibodies against AR – 110 kDa, PSA – 47 kDa, H4 (Lys 8) – 11 kDa and H4 (Lys 12) – 11 kDa after treatment of LNCaP, C4-2 and RWPE-1 prostate cell lines with sodium butyrate. a-Tubulin – 55 k Da was served as a loading control. Total cell protein lysates were harvested after 24 h and 48 h after treatment with 0.1% DMSO (D), 1 mM NaB (1) and 5 mM NaB (5). Proteins were separated by electrophoresis, transferred to a membrane, hybridized with primary and appropriate secondary antibody. Detection was performed using Supersignal West Duro Chemiluminescent Substrate.
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