Selenomethionine induces sustained ERK phosphorylation leading to cell-cycle arrest in human colon cancer cells
Anne-Christine Goulet,
Marianne Chigbrow,
Peter Frisk and
Mark A. Nelson1
Department of Pathology, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
1 To whom correspondence should be addressed Email: mnelson{at}azcc.arizona.edu
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Abstract
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Selenomethionine (SeMet) is being tested alone and in combination with other agents in cancer chemoprevention trials. However, the molecular targets and the signaling mechanism underlying the anticancer effect of this compound are not completely clear. Here, we provide evidence that SeMet can induce cell-growth arrest and that the growth inhibition is associated with SG2/M cell-cycle arrest. Coincidentally with the cell-cycle arrest, we observed a striking increase in cyclin B as well as phosphorylation of the cyclin-dependent kinase Cdc2. Since activation of the mitogen-activated protein kinase (MAPK) cascade has been associated with cell-cycle arrest and growth inhibition, we evaluated the activation of extracellular signal-regulated kinase (ERK). We found that SeMet induced phosphorylation of the MAPK ERK in a dose-dependent manner. We also demonstrate phosphorylation of ribosomal S6 kinase (p90RSK) by SeMet. Additionally, we show phosphorylation of histone H3 in a concentration-dependent manner. Furthermore, the phosphorylation of p90RSK and histone H3 were both antagonized by the MEK inhibitor U0126, implying that SeMet-induced phosphorylation of p90RSK and histone H3 are at least in part ERK pathway dependent. Based on these results, we propose that SeMet induced growth arrest and phosphorylation of histone H3 are mediated by persistent ERK and p90RSK activation. These new data provide valuable insights into the biological effects of SeMet at clinically relevant concentrations.
Abbreviations: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; RSK, ribosomal S6 kinase; SeMet, L-selenomethionine; SRB, sulforhodamine B
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Introduction
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Currently, high selenium containing yeast is being investigated in a phase II double-blind, placebo-controlled, trial designed to assess the effect of selenium and celecoxib, either individually or in combination, on colon polyp recurrence (1). The launching of this trial is largely driven by the seminal findings of Clark et al. (2) that high selenium containing yeast supplementation was capable of significantly reducing the incidence of colon (RR = 0.42), lung (RR = 0.54) and prostate cancers (RR = 0.37). The colon prevention trials with selenium provide the establishment of a repository of colon biopsy tissue and hematopoeitic cell types. These materials are archived for research discoveries in the future. One of the secondary objectives of this trial is to study the cellular and molecular biomarkers using the banked samples and to elucidate their relevance with respect to colon carcinogenesis and drug effects. Despite the considerable public interest in the potential benefit of selenium chemoprevention of colon cancer, little information is currently available on the molecular targets or the signaling mechanisms underlying the anticancer effects of high selenium containing yeast. Our present study was aimed at addressing this gap of knowledge with the use of human colon cancer cell lines.
Analytical speciation studies showed that the bulk of the selenium in selenized-yeast is in the form of selenomethionine (SeMet) (85%) (3). Thus, the anticancer effects of high selenium containing yeast may be due in large part to SeMet. SeMet represents an organic form of selenium and is more bioavailable than an inorganic form such as Na2SeO3 (4). Substitution of methionine by SeMet represents a mechanism for non-specific incorporation of selenium into proteins in vivo. SeMet is metabolized primarily in the liver to a monomethylated intermediate for the expression of its anticancer activity and epithelial tissues typically retain a low capacity to generate a mono-methylated Se-metabolite form SeMet (59). Consequently concentrations of SeMet that are 20100 times above the physiological levels have been used in cultured cells not relevant to physiological concentrations of selenium. In the present study, we developed a low-dose multi-treatment protocol using SeMet within the physiological range of selenium in the circulation and tissues.
Although extracellular signal-regulated kinase (ERK) is thought to play a key role in the proliferative process, recent studies suggest that persistent activation of ERK might mediate cell-cycle arrest and differentiation (10,11). After activation, phospho-ERK is translocated from the cytoplasm to the nucleus, where it can phosphorylate and activate multiple nuclear substrates such as transcription factors and other kinases leading to altered gene expression. More recently, it has become clear that chromatin structure plays an important role in eukaryotic gene regulation. An increasing body of evidence indicates that the MAP kinase cascades direct the phosphorylation of upstream transcription factors and co-activators controlling intermediate early genes. They also act directly on chromatin proteins such as histone H3 and the high-mobility group protein HMG-14 to modify chromatin concomitant with gene induction (1217).
In this study, we first examine the dose-dependent effect of SeMet on the growth of HCT116 and SW48 human colon cancer cells. We then show that growth inhibition by SeMet is probably attributable to an effect on the cell cycle. We also demonstrate alterations in the expression of the SG2/M cyclins concomitant with SeMet induced cell-cycle effects. Next, we examine the activation of ERK pathway to gain further insight into the signaling pathways that may play a role in the regulation of these cellular events. Finally, we report that phosphorylation of histone H3 is associated with increased treatment with SeMet, which might further lead to nucleosomal modifications. From these findings, we are able to develop a model of the signal events that might explain the action of SeMet in blocking cell-cycle progression in colon cancer cells.
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Materials and methods
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Cell culture
The DNA mismatch repair deficient human colorectal carcinoma cell lines, HCT116 and SW48 were both obtained from the American Type Culture Collection (Rockville, MD). HCT116 has been reported to have a transversion in the mismatch repair gene hMLH1, producing a premature stop codon (1820). SW48 lacks MLH1 expression because of hypermethylation of the hMLH1gene promoter (21). HCT116 cells were maintained in monolayer culture in McCoy's 5A media and SW48 in RPMI 1640 (Cellgro Mediatech, Herndon, VA), supplemented with 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA), 2 mM L-glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin (Gibco/Invitrogen, Carlsbad, CA) in a humidified incubator with 95% air and 5% CO2 at 37°C. Subculturing was done at subconfluent densities with a solution of 0.25% trypsin (Gibco/Invitrogen).
Antibodies and chemicals
The mouse monoclonal cyclin A (BF683), mouse monoclonal cyclin B1 (GNS1), mouse monoclonal Cdc2 p34, mouse monoclonal p-ERK (E-4) Tyr-204, mouse monoclonal p-JNK (G-7) Thr-183 and Tyr-185, rabbit polyclonal p-p38 Tyr-182, mouse monoclonal p-Elk-1 (B-4) Ser-383, were all bought from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit phospho-cdc2 (Tyr-15) and p44/42 MAP kinase rabbit polyclonal antibody were obtained from Cell Signaling Technology (Beverly, MA). The rabbit anti-phospho-MSK1 (S-376)/MSK2 (S-360) antibody and rabbit anti-phospho-ribosomal S6 kinase (RSK) (S-380) antibody were bought from R&D Systems (Minneapolis, MN). The mouse monoclonal anti-phospho-Histone H3 (Ser-10) antibody and the MEK1/2 inhibitor U0126 were purchased from Upstate Biotechnology (Lake Placid, NY). Alpha-Tubulin (Ab-1) monoclonal antibody was obtained from Oncogene Research Products/EMD Biosciences (San Diego, CA). The secondary horseradish peroxidase-linked antibodies (anti-mouse and anti-rabbit) were purchased from Sigma Chemical (St Louis, MO).
Multi-treatment protocol
Cells in the log phase were harvested by trypsinization and plated at a density of 2 x 104 cells/plate (HCT116) or 4 x 104 cells/plate (SW48) in 100 mm tissue culture plates. Cells were allowed to adhere to the plates for 24 h before treatment. Thereafter cells were treated with 522 µM of SeMet (Sigma). Cells were treated for up to 8 or 12 days. Once treated, cells were either collected (removed by trypsinization) or re-treated by adding fresh media and fresh SeMet every 2 days out to day 8 or to day 12. The rationale for using a multi-treatment protocol was to investigate a low concentration of SeMet representing a supplementation. The U0126 drug treatment (10 µM) was done at day 8 for 1 h at 37°C with or without FBS, with or without SeMet replenishment. Stock solutions for the pharmacological inhibitor were prepared in DMSO at a concentration of
1000-fold. The final concentration of DMSO in the culture medium was always 0.1%.
Sulforhodamine B (SRB) assay
Details on cell growth determination utilizing the sulforhodamine B (SRB, Sigma) colorimetric assay have been described previously by our group (22). Briefly, the optimum cell number per well is determined for each cell line over a 7-day period. Ninety-six-well microtiter plates are used for plating the cell lines and the cells are subsequently incubated for 24 h prior to the addition of drugs to be tested. SeMet was solubilized in media. In some experiments SeMet was tested over a concentration of 8-fold dilutions to find the IC50. In other studies the concentration range was 522 µM. After an additional 6 days of culture, viable cells were fixed to the bottom of each well with cold 50% trichloroacetic acid at a final concentration of 10%. The plates were kept at 4°C for 1 h, the supernatant was then aspirated and the plates were washed with deionized water. SRB solution was prepared to 0.4% (w/v) in 1% acetic acid. SRB (50 µl) was added to each well and the cells stained for 10 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid followed by air-drying. Bound stain was solubilized with 1 M unbuffered Tris and optical density was measured with an EL311 microplate autoreader (Biotek) at a single wavelength of 540 nm.
Cell number determination
In assessing total cell number, HCT116 and SW48 cells following treatment with SeMet were collected and counted every 2 days. Counts were determined by trypan blue (0.4%) dye exclusion (Sigma). The effect of the treatment was studied in at least three independent experiments and each experiment was evaluated in quadruplicate. The results of all experiments were combined for the purpose of statistical analysis.
Cell-cycle distribution
Following SeMet treatment, cells were harvested and stained with propidium iodide using a protocol described (23,24). Cells were counted using trypan blue and 1.0 x 106 cells were pelleted. Pellets were then washed with a phosphate-buffered saline solution (PBS), re-suspended in 1 ml of 1x PBS and fixed by addition of 3 ml of ice-cold 100% ethanol. Samples were stored at 20°C until the day of the analysis. Fixed cells were pelleted, washed with 1x PBS and stained with 1 ml of Krishan's buffer (0.1% sodium citrate, 0.02 mg/ml RNase A, 0.3% v/v NP-40 and 50 µg/ml propidium iodide) at a pH of 7.4. Samples were vortexed and stored at 4°C, wrapped in foil until analysis. Cell samples were sent to the Flow Cytometry Service of the Arizona Cancer Center for analysis of cell-cycle phases. Results are expressed as percentages.
RNase protection assay
Cells were homogenized using Trizol reagent (Gibco/Invitrogen) as described by the manufacturer and as carried out previously by our group (25). RNA was separated using Trizol and chloroform. Precipitation was achieved using isopropyl alcohol followed by an ethanol wash. The resulting RNA product was re-dissolved in RNase free water. Analysis of cyclin expression was performed using a commercially available RNase protection assay. An hcyc-1 probe (BD PharMingen, San Diego, CA) was synthesized using the protocol described by the manufacturer. Probe labeled with [
-32P]UTP (ICN, Irvine, CA) was allowed to hybridize 510 µg RNA overnight. The hybridized RNA was subjected to RNase treatment, purified and protected RNA fragments were resolved on a 6% acrylamide gel for 10 min at 1200 V, dried for 1 h at 80°C without vacuum. Cyclin RNA levels were analyzed by autoradiography and compared with internal standard L32 expression.
Real-time PCR
SeMet (22 µM) treated and non-treated HCT116 cells were collected at day 8 and total RNA was isolated. To limit the possibility of detection of genomic DNA, total RNA was subjected to DNase treatment. The cDNA was then produced by omniscript reverse transcriptase (Qiagen, Valencia, CA) and quantified using Taqman Universal Master Mix (Applied Biosystems, Foster City, CA) on a Gene Amp 5700 sequence detector (Applied Biosystem). Primers and probes for Wee1 kinase and GAPDH were design by Applied Biosystems. Each sample was run in triplicate on two sets of RNA on two independent real-time PCR reactions. Differences among the treated groups were determined using the threshold cycle (CT) method outlined by Miura et al. (26). Means ± SD values were determined and these values were subsequently used in two-tailed t-test to determine statistical significance.
Protein extraction and western blotting
Treated and control cells were washed with cold 1x PBS, lysed and sonicated in cold RIPA buffer [10 mM TrisHCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% v/v Nonidet P-40, 1% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma)] on ice for 30 min. Lysates were cleared by centrifugation at 13 000 g for 30 min at 4°C. Equal amounts of cell extracts (50 µg) (bicinchoninic acid assay, BCA, Pierce, Rockford, IL) were resolved on SDSpolyacrylamide gels and then transferred onto a polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). After blocking membranes in 5% non-fat dry milk, they were probed overnight with the primary antibody. Membranes were then washed three times in 1x PBS containing 0.5% Tween 20 (PBS-T) for 20 min before being incubated with the secondary horseradish peroxidase-linked antibody diluted in 1% bovine serum albumin PBS-T for 1 h. Detection was performed using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). Relative protein blot intensities were determined using one-dimensional Image Analysis Software (Kodak). Graphs were constructed using the sum intensity representing the sum of all the pixel intensities in the band rectangle.
Statistical analyses
All experiments were repeated on at least three separate occasions. Values are reported as means ± SD. Differences between means were compared using the Student's t-test and P values of <0.05 were considered significant.
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Results
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SeMet effects on cell crowth and apoptosis
To understand how SeMet and, therefore, high selenized yeast may be acting as an anti-proliferative agent, a repeated low-dose model was established to mimic more closely the dosing of patients (Figure 1). Cell counting was done to quantify any growth effect caused by SeMet. We observed a time-dependent growth inhibition in both HCT116 and SW48 cells treated with SeMet at 22 µM (Figure 2). The effects of SeMet on the growth of these tumor cells were obvious at days 6 and 8. HCT116 cells (Figure 2A) exhibit an inhibition of cell growth at day 6 with a 43% decrease (P = 1.12, E-05) and at day 8 with a 38% decrease (P = 6.35, E-04) in the number of treated cells compared with control cells. The effect of SeMet on SW48 (Figure 2B) was also significant with a decrease in growth inhibition of 51% at day 6 (P = 1.47, E-03) and of 77% at day 8 (P = 2.55, E-06). To further investigate the role of SeMet in cell growth inhibition, cell death was evaluated by 7 amino-actinomycin D (7-AAD) staining. There was no difference in the number of cells undergoing apoptosis between control and SeMet-treated HCT116 and SW48 cells (data not shown). These findings were confirmed by morphological analysis (data not shown). Thus, increased apoptosis cannot account for the growth inhibitory effects of SeMet in HCT116 and SW48 colon cancer cells at 22 µM.

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Fig. 1. Design of the 12-day in vitro repeated dosing experiments for administering SeMet to colon cancer cells.
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Fig. 2. The effects of SeMet on colon cancer cell growth. Growth curve for HCT116 cells (A) and for SW48 cells (B) treated with 22 µM SeMet. Colon cancer cell lines were treated with SeMet (square) or non-treated, control (triangle) for up to 8 days as described under multi-treatment protocol in the Materials and methods. Cell number was enumerated using trypan blue exclusion. Mean ± SD (n = 3).
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Cell-cycle arrest induced by SeMet
To explore the role of SeMet in the cell cycle, HCT116 cells were treated with 22 µM SeMet for up to 8 days and then analyzed for cell-cycle alterations by flow cytometry (Figure 3). We observed a decrease in the percentage of cells in G0/G1 treated with SeMet at days 6 and 8. Whereas HCT116 cells demonstrated an increase in the number of cells in the SG2/M phase of the cell cycle when treated with SeMet, with respect to S-phase, HCT116 controls at days 6 and 8 had 16 and 10% of cells in S phase. In comparison, SeMet-treated cells had almost double with 34 and 17%, respectively. With regard to mitosis, the vehicle-control cells had 8 and 5% at days 6 and 8, respectively, whereas SeMet treatment had 12 and 10% in the G2/M phase of the cell cycle. We observed a similar effect on the cell cycle in SW48 cells. However, this effect was not as obvious compared with HCT116 cells (data not shown).

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Fig. 3. The cell-cycle distribution of colon cancer cells treated with SeMet. HCT116 cells were treated with SeMet for the indicated time points and stained with propidium iodide followed by flow cytometry analysis. Results are expressed as percentage of cells in G0/G1, S and G2/M phases of the cell cycle. The data are representative of three independent experiments.
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SeMet affects cyclin RNA and protein expression
RNase protection assays were conducted to analyze cells for alterations in cyclin RNA expression that may explain the observed growth inhibition and SG2/M arrest. RNA levels for both cyclin A and cyclin B were elevated in SeMet-treated cells compared with control cells (Figure 4A). Elevated cyclin RNA levels were seen in SeMet-treated HCT116 cells both at days 6 and 8.
To evaluate cyclins at the protein level, we performed western blot analysis on SeMet-treated cells. We observed only a slight increase in cyclin A protein levels at the time points tested, whereas there was an obvious increase in cyclin B protein levels in SeMet treated cells at day 8 (Figure 4B).
Phosphorylation of Cdc2 on Tyr-15 coincides with SG2/M cell-cycle arrest
The phosphorylation of Cdc2 on Tyr-15 causes cells to arrest in the G2/M transition phase of the cell cycle (27,28). To determine if the accumulation of cells in the SG2/M phase observed by flow cytometry was associated with phosphorylation of Cdc2, we examined the phosphorylation status of Cdc2 at Tyr-15 by western analysis. After treatment with 22 µM SeMet we observed an increase in Cdc2 phosphorylation (taken at days 6 and 8) compared with control cells (Figure 4C, upper panel). There were no obvious changes in non-phosphorylated Cdc2 levels (Figure 4C, third panel).
Wee1 kinase level is increased by SeMet
Cdc2 is heavily regulated through phosphorylation. Its activity is suppressed through phosphorylation on Tyr-15 by Wee1 kinase. We investigated the role of Wee1 in SeMet-treated HCT116 cells and found that the RNA level of Wee1 measured by real-time PCR was significantly increased (P < 0.05) at day 8 (Figure 4D). This finding suggests a potential role of Wee1 kinase in SeMet treatment.
Effects of SeMet on the mitogen-activated protein kinase (MAPK) pathway
To evaluate the effect of SeMet in HCT116 in greater detail, we assessed the impact on the MAPK signaling pathways. Activation of both p38 and ERK has been associated with G2/M arrest (10,29). We evaluated the phosphorylation status of JNK, p38 and ERK. We found no differences between control and SeMet-treated cells with respect to JNK and p38 phosphorylation status (data not shown). However, there were differences in ERK activation. Treatment with SeMet increased ERK phosphorylation on Tyr-204 in HCT116 at days 6 and 8 (Figure 5A, upper panel). Densitometric scan of p-ERK normalized with
-tubulin showed an increase of p-ERK1 p44 (a) and p-ERK2 p42 (b) with SeMet at both days (Figure 5C). In contrast, no change in total ERK (p42/p44) was observed during SeMet stimulation at days 6 and 8 in HCT116 cells (Figure 5B). Pharmacological treatment of HCT116 with the MEK1/2 inhibitor U0126 (10 µM) for 1 h at day 8 decreased the phosphorylation of ERK induced by SeMet in HCT116 (Figure 5D). The decrease in phosphorylation was obtained in the presence and in the absence of FBS. Moreover, U0126 had no effect on the expression of ERK1 and ERK2 demonstrating the specificity of the inhibitor on phosphorylation (Figure 5E). Taken together, the data support the notion that G2/M arrest of tumor cells after SeMet treatment involves activation of the MAP kinase ERK.
SeMet-induced ERK activation is related to p90RSK phosphorylation
The p90RSK family of serine/threonine kinases are known to be activated by ERK in response to stimuli (11). Thus, the fact that SeMet induced ERK activation prompted us to examine whether p90RSK was involved in SeMet actions. We compared the effects of SeMet on RSK1/2 phosphorylation at serine residue 380 because the phosphorylation at Ser-380 residue is thought to be related to ERK kinase activity (30). We found that SeMet stimulated RSK phosphorylation at Ser-380 at days 6 and 8 (Figure 6A). To examine the relationship between RSK and ERK phosphorylation, we treated cells with U0126 to determine whether this MEK inhibitor could reverse the effect of SeMet on RSK phosphorylation. Figure 6B shows that U0126 effectively antagonized SeMet-mediated RSK phosphorylation, suggesting that persistent ERK activation by SeMet leads to RSK phosphorylation and activation. Although MSK1/2 are also downstream of ERK (31), we did not observe phosphorylation of MSK by SeMet in these experiments (data not shown).

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Fig. 6. SeMet induced phosphorylation of RSK is ERK dependent. (A) HCT116 cells were treated with 22 µM SeMet for up to 6 and 8 days. At the indicated time point, cell extracts were prepared and immunoblotted with anti-phospho-RSK antibody. (B) HCT116 cells were subjected to SeMet for up to 8 days. The cells were then treated with the MEK inhibitor U0126 (in serum-free conditions) for 1 h. Cell lysates were immunoblotted with anti-phospho-RSK antibody. Consistency in loading was assessed by immunoblotting for -tubulin.
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Histone H3 is phosphorylated in SeMet-treated cells
Previous reports have suggested roles for both MSK and RSK in histone phosphorylation downstream of MAP kinases (14,15,32). Histone H3 is highly phosphorylated on condensed chromosomes during mitosis (14,16,17). The presence of Ser-10 histone H3 phosphorylation is considered an excellent mitosis-specific marker and is also indicative of cell-cycle arrest. Our experiments showed that SeMet induces an increase in the phosphorylation state of histone H3 on Ser-10 after 6 and 8 days of consecutive treatment in both HCT116 cells and SW48 cells (Figure 7A). The MEK inhibitor U0126 reduces the phosphorylation state of histone H3 in SeMet-treated HCT116 cells (Figure 7B). This suggests that SeMet induced ERK activation can lead to histone H3 phosphorylation. No phosphorylation of Elk-1 on Ser-383, which was another possible effector of ERK, was observed (data not shown).

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Fig. 7. The effects of SeMet on histone H3 phosphorylation are antagonized by the MEK inhibitor U0126. (A) HCT116 and SW48 cells were treated with 22 µM SeMet for up to 8 days. Cell lysates were prepared at the indicated time points and immunoblotted for histone H3 phosphorylation status. (B) HCT116 cells were treated with SeMet 22 µM for up to 8 days. Then, the cells were treated with the MEK inhibitor U0126 (in serum-free conditions) for 1 h. Cell lysates were immunoblotted with anti-phospho-histone H3 Ser-10 antibody. Uniform loading of lysates was confirmed by immunoblotting for -tubulin.
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SeMet induces ERK, RSK and histone H3 phosphorylation at physiological concentrations
We first investigated the ability of SeMet to inhibit HCT116 cell growth by the SRB assay (7 days) at a concentration range between 5 and 22 µM. We found that a single dose of SeMet inhibited tumor cell growth in a dose-dependent manner, being significant at 10 µM (P = 0.03) and 22 µM (P = 0.02) (Figure 8A). Next, we examined the extent of ERK phosphorylation in cells treated up to 12 days with 5, 10 or 22 µM SeMet. We found that SeMet caused a concentration-dependent activation of ERK (Figure 8B, upper panel). We also observed phosphorylation of RSK and histone H3 in low-dose SeMet-treated cells (Figure 8B).

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Fig. 8. Growth inhibition by SeMet at physiological concentrations. (A) HCT116 cells were treated only once with SeMet 522 µM up to 7 days. Cell viability was measured using the SRB assay as described in the Materials and methods. (B) SeMet induced phosphorylation of ERK, RSK and histone H3 is concentration dependent. HCT116 cells were treated with SeMet (522 µM) for up to 12 days. Cell extracts were prepared and immunoblotted with phospho-ERK, phospho-RSK and phospho-histone H3 antibodies. Uniform loading of lysates was confirmed by immunoblotting for -tubulin.
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Discussion
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Colorectal cancer remains the second leading cause of cancer mortality in the USA (33). Risk factors include a combination of genetic and environmental (nutrition and physical activity) variables. Surveillance, early detection and prevention are central to the reduction of incidence and mortality from this disease (34). Although current colorectal screening methods such as fecal occult blood testing and screening colonoscopy are known to reduce mortality from this disease, patient compliance with screening recommendations remains low (3538). In patients that comply, the removal of precursor lesions (adenomas) can reduce the incidence of subsequent colorectal cancer (39,40). However, the colonoscopic polypectomy has an associated miss-rate (41), and the polyp recurrence rate is often high (41). Thus, there is considerable need to develop dietary and chemopreventive intervention strategies that can be applied to large populations to reduce adenoma recurrence and colorectal cancer risk.
In human clinical trials, selenium, in the form of high selenium containing yeast has been shown in secondary analysis of a phase II study to reduce the incidence and mortality from colon cancer, and experiments in laboratory animals have demonstrated that various selenium compounds inhibit colorectal carcinogenesis (2). Recently a multi-center phase II double-blind, randomized, factorial trial to determine the efficacy of celecoxib and selenium supplementation in the form of high selenium containing baker's yeast separately and combined in reducing colorectal adenoma recurrence in patients with sporadic has been undertaken (1). SeMet is the major selenium compound in the selenized yeast (3). However, the mechanism of action of SeMet remains obscure. Furthermore, the optimal concentration of selenium supplementation and exactly what groups will benefit from selenium supplementation remain to be determined.
Previous studies by our group showed that SeMet is able to block cell-cycle progression at specific checkpoints, which might be explained by a decrease in cyclin B and Cdc2 kinase activity (25). In studies by other groups, SeMet at a concentration of 500 µM induced cell-cycle arrest and phosphorylation of Cdc2 at Tyr-15 in prostate tumor cells (42). At 150 µM SeMet was also shown to be able to inhibit the growth of LNCaP and decrease cyclin D1 and cyclin D3, which were associated with a G1 phase arrest (43). All of these experiments were conducted with very high dose of SeMet, sometimes being 10 times higher than what is found in the circulation under normal physiological conditions. For this reason, we developed a low-dose multi-treatment protocol for in vitro studies. In the present study, we demonstrate that SeMet inhibits the growth of colon cancer cells by cell-cycle blockade (SG2/M phase) and not by apoptosis. We recognize that a concentration of 22 µM is slightly higher than physiologic concentration of selenium (i.e. 10 µM) seen in clinical trials; however, this concentration represents the inhibitory concentration for 50% cell growth for the HCT116 cells. Also, this concentration may not be out of the physiological range in clinical studies where 400 µg and up to 800 µg of selenized yeast are being evaluated (44). Thirdly, the concentration of 10 µM represents a physiologically achievable plasma concentration of selenium (45). It is well accepted that drug concentrations can be higher in the target organ or tissue than the plasma concentration. To our knowledge no studies have definitively determined the physiologic concentration of selenium in a target tissue (i.e. the colon or prostate) in clinical trials. Finally, in the present study, we repeated our experiments at lower concentration (i.e. 5 and 10 µM) and observed dose-dependent effects.
Coincidentally, with the observed growth arrest and cell-cycle alterations, we observed a slight increase in cyclin A and marked increase in cyclin B in SeMet-treated cells. Conversely, the levels of both cyclin A and B decreased in control cells. Our data in the control cells are consistent with previous studies. It is well known that as cells continue to proliferate and progress through the cell cycle, the levels of the cyclins decrease at specific phases of the cell cycle. For example, the levels of cyclin B decrease when cells exit mitosis (46,47). Our results have similarities to the mitotic responses in other organism upon stress. In cells of the Drosophila gastrula, cyclin A is stabilized during metaphaseanaphase delay (48). In budding yeast, destruction of the anaphase inhibitor Pds1 is normally achieved by the anaphase-promoting complex/cyclosome in conjunction with Cdc20 and is required for anaphase progression. The destruction of mitotic cyclins allows mitotic exit. The presence of DNA damage induces mitotic cell-cycle arrest and inhibition of cyclin destruction by stabilization of Pds1 (49).
In the present study we also saw increased phosphorylation of Cdc-2 on Tyr-15 by SeMet. Activation of the p34cdc2 kinase (CDK1) is the universal event controlling the onset of mitosis. By being phosphorylated, Cdc2 is maintained in an inactive state. Complex formations predominantly with cyclin B/Cdc2 and to a lesser extent with cyclin A/Cdc2 have been described as essential to process the cell cycle. The inactive cyclin B/Cdc2 complex accumulates during G2 until it is activated by dephosphorylation of Thr-14 and Tyr-15 by CDC25 phosphatase, which promotes entry into mitosis. Our result is consistent with previous studies using high concentrations of SeMet (500 µM) in prostate cancer cells (42). The phosphorylation of Cdc2 on Tyr-15 has been implicated in the G2/M checkpoint (27,28). Wee1 kinase is known to phosphorylate Cdc-2 at Tyr-15. We also observed an increase in Wee 1 RNA levels in SeMet-treated cells during growth arrest. In Xenopus egg extracts, G2 arrest by Wee 1 is associated with p42 MAPK (50). We speculate that if ERK activates Wee1 it is through an indirect mechanism involving induction of Wee1 at the transcriptional level. Although Wee1 kinase RNA levels were found to be increased in our studies, whether it is ERK-related and leads to subsequent phosphorylation of Cdc2 requires further study. The possible role of the endoplasmic reticulum- and Golgi complex-bound protein kinase MYT1 as another candidate responsible for phosphorylation of Cdc2 (51,52) and the destruction of CDC25 phosphatases, which have been studied previously in response to mitotic DNA damage and H3 phosphorylation (53), are also under current investigation in relation to SeMet.
Although ERK is thought to mediate mitogenic responses, there are several reports indicating that persistent ERK activation can lead to cell-cycle arrest (54,55). In the present study we provide evidence that ERK activation is associated with SeMet induced cell growth inhibition. A role for MAPK pathway signaling in growth arrest or cellular differentiation is not unprecedented. For example, a synthetic vitamin K analog compound 5 (Cpd 5) or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone is an inhibitor of protein phosphatase Cdc25A and causes persistent activation of ERK and cell growth inhibition in Hep3B hepatoma cells (11). ERK activation also appears to mediate cell-cycle arrest and apoptosis after DNA damage independent of p53 (10).
We observed phosphorylation of histone H3 at Ser-10 upon SeMet treatment and U0126 caused a partial reduction of histone H3 phosphorylation. Stimulation of the ERK pathway results in an increase in phosphorylation of histone H3 during the activation of immediate early genes expression. These genes are activated directly and require no new transcription or translation for their induction. Phosphorylation at Ser-10 on the N-terminal tail of histone H3 has a role in stabilizing the three-dimensional fiber structure. It is though to destabilize higher-order compaction of the chromatin fiber. Morever, the phosphorylation of histone H3 at serine 10 is associated with cell-cycle arrest at mitosis (16,17). Although phosphorylation of histone H3 is now proven, the precise identity of the kinase that phosphorylates it has been contentious (14). RSK, MSK, IKK-
and aurora kinase have been identified as responsible kinases for histone H3 phosphorylation (31,56,57). In our studies we observed RSK phosphorylation upon SeMet treatment. Furthermore, U0126 suppressed phosphorylation of RSK. We did not see phosphorylation of MSK. The data suggest that SeMet induced ERK activation leads to RSK phosphorylation and activation. However we cannot completely exclude aurora kinase or IKK-alpha activation at this time.
In summary, we demonstrate that SeMet activation of ERK leads to histone H3 phosphorylation via p90RSK in HCT116 cells. We hypothesize that histone H3 phosphorylation leads to chromatin remodeling, which leads to changes in gene expression, and in turn ultimately results in growth inhibition. We also show for the first time a dose-dependent activation of this pathway with physiologic concentrations of SeMet seen in human clinical trials. However, the molecular mechanism leading to ERK activation by SeMet (at physiologic concentrations) will require further study.
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Acknowledgments
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Work supported in part by the American Institute for Cancer Research and CA 72008.
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References
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Received May 28, 2004;
revised September 16, 2004;
accepted October 1, 2004.