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Carcinogenesis Advance Access originally published online on May 20, 2008
Carcinogenesis 2008 29(6):1258-1266; doi:10.1093/carcin/bgn122
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mesalazine negatively regulates CDC25A protein expression and promotes accumulation of colon cancer cells in S phase

Carmine Stolfi, Daniele Fina, Roberta Caruso, Flavio Caprioli, Massimo Claudio Fantini, Angelamaria Rizzo, Massimiliano Sarra, Francesco Pallone and Giovanni Monteleone*

Dipartimento di Medicina Interna, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy

* To whom correspondence should be addressed. Tel: +39 06 72596158; Fax: +39 06 72596391; Email: gi.monteleone{at}med.uniroma2.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Regular consumption of mesalazine has been associated with a reduced risk of colorectal cancer (CRC) in patients with inflammatory bowel disease. The molecular mechanisms underlying the antineoplastic effect of 5-aminosalicylic acid remain, however, poorly characterized. In this study, we examined whether mesalazine affects cell cycle progression and analyzed specific checkpoint pathways in experimental models of CRC. Mesalazine inhibited the growth of HCT-116 and HT-29 cells, two CRC cell lines that express either a wild-type or mutated p53. Cell cycle analysis revealed that mesalazine induced cells to accumulate in S phase. This effect was associated with a sustained phosphorylation of the cyclin-dependent kinase (CDK)2 at threonine 14 and tyrosine 15 residues, an event that inactivates the CDK2–cyclin complex and blocks S–G2 phase cell cycle transition. Consistently, mesalazine reduced the protein content of CDC25A, a phosphatase that regulates CDK2 phosphorylation status. Analysis of upstream kinases that negatively control CDC25A expression showed that mesalazine enhanced the activation of CHK1 and CHK2. However, silencing of CHK1 and CHK2 did not prevent the mesalazine-induced CDC25A protein downregulation. In contrast, CDC25A protein ubiquitination and degradation and accumulation of cells in S phase following mesalazine exposure were reverted by proteasome inhibitors. Notably, mesalazine also inhibited CDC25A in human CRC explants. Finally, we showed that mesalazine downregulated CDC25A in CT26, a murine CRC cell line, and prevented the formation of CT26-derived tumors in mice. Data show that mesalazine negatively regulates CDC25A protein expression, thus delaying CRC cell progression.

Abbreviations: ATR, ATM- and Rad3-related; CDK, cyclin-dependent kinase; CRC, colorectal cancer; IBD, inflammatory bowel disease; PBS, phosphate-buffered saline; PI, propidium iodide; siRNA, small interfering RNA; Thr-14, threonine 14; Tyr-15, tyrosine 15


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Patients with Crohn’s disease and patients with ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs) in man, are at an increased risk of developing colorectal cancer (CRC) (1,2). Present CRC prevention in IBD patients depends on the detection of precancerous dysplasia or CRC during scheduled screening and surveillance colonoscopy. When dysplasia or CRC is identified, proctocolectomy is performed to remove the at-risk organ. Because of the risk, expense and sampling error of endoscopic surveillance, this prevention strategy does not seem to be an ideal approach to control IBD-related CRC (1,2). The development of safe and effective measures for reducing the risk of CRC would thus be of substantial benefit to IBD patients.

Mesalazine or 5-aminosalicylic acid is the drug of choice in the maintenance of remission and treatment of mildly flare-ups of IBD. Mesalazine is characterized by low systemic resorption after oral and rectal administration and very few adverse effects. More recently, epidemiological studies have suggested that mesalazine therapy is chemopreventive for CRC development in patients with ulcerative colitis, even though some publications documented no benefit (3–6). Additionally, in vitro studies have documented important regulatory effects of mesalazine on human CRC cell lines. In particular, it has been shown that mesalazine blocks CRC cell growth and promotes apoptosis, as well as it reduces frameshift mutations at a (CA)13 microsatellite (7,8). Consistently, we have shown previously that mesalazine inhibits epidermal growth factor receptor activation, a transmembrane tyrosine kinase that triggers mitogenic signaling in CRC cells (9). Mesalazine interferes also with other signaling pathways that sustain CRC cell activity. Indeed, Bos et al. (10) showed that mesalazine affects the Wnt/β-catenin pathway in CRC cells via the inhibition of the phosphatase 2A and degradation of β-catenin. Moreover, Rousseaux et al. (11) demonstrated that mesalazine interacts with and enhances the expression and activation of peroxisome proliferator activated receptor-{gamma}, a negative regulator of colonic inflammation and cancer. Despite these advances, the basic mechanism of the antimitogenic effect of mesalazine on CRC cells remains unclear.

In eukaryotic cells, the initiation of DNA replication and entry into mitosis are orchestrated by cyclin/cyclin-dependent kinase (CDK) complexes, which are formed at specific stages of the cell cycle, and whose activities are necessary for progression through S phase and mitosis. CDK1 is necessary for mitotic onset and shares an overlapping role with CDK2 in controlling S phase initiation. Activation of both CDK1 and CDK2 is strictly dependent on the activity of a specialized phosphatase, denominated CDC25 (12). There are three members of the human CDC25 family. CDC25A controls progression through S phase, whereas CDC25B and CDC25C are involved in the control of the transition from G2 to mitosis. CDC25A dephosphorylates and activates cyclin E–CDK2, cyclin A–CDK2 and cyclin B–CDK1, whereas CDC25B and CDC25C target cyclin B–CDK1 (13–15).

In the present study, we have examined whether mesalazine interferes with CRC cell cycle and analyzed specific checkpoint pathways using experimental models of colon carcinogenesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell cultures
Mesalazine (Giuliani S.p.A., Milan, Italy) was dissolved as a 100 mM stock solution in culture medium. The pH of the drug solution was adjusted to 7.4 with NaOH, and experiments carried out were protected from light. All reagents were from Sigma–Aldrich (Milan, Italy), unless specified. The human CRC cell lines, HCT-116 and HT-29, were maintained in McCoy’s 5A medium and the murine CRC cell line, CT26, in RPMI 1640 medium, both supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

To examine whether mesalazine affects the phosphorylation of CDK2 at threonine 14 (Thr-14) and tyrosine 15 (Tyr-15) residues, the expression of CDC25 family members and the activation status of CHK1 and CHK2 serum-starved cells were cultured in appropriate medium containing 0.05% bovine serum albumin, in the presence or absence of mesalazine (5–30 mM) or mannitol (30 mM) for 1–44 h. Additionally, cells were treated with campothecin (40 nM) as a positive control for CHK1/CHK2 activation. In parallel, to examine the role of CHK1 and CHK2 on cell cycle distribution and CDC25A protein, cells were cultured with or without human CHK1, CHK2 or control small interfering RNA (siRNA) according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA). After 32 h, cells were treated with or without mesalazine for further 32 h, and cell cycle distribution was then analyzed by flow cytometry. Moreover, to evaluate the role of CDC25A on cell cycle progression, cells were transfected with human CDC25A or control siRNA (Santa Cruz Biotechnology) and then either left untreated or treated with 15 mM mesalazine for the indicated time points.

To determine whether the effect of mesalazine on CDC25A was reversible, cells were cultured in the presence or absence of mesalazine (5 and 15 mM) for 16 h (first culture). Afterward, cells were either left untreated or extensively washed and cultured with medium in the absence of mesalazine for additional 16 h (second culture). Additionally, cells were preincubated with proteasome inhibitors, such as N-p-tosyl-L-phenylalanine chloromethyl ketone (5 µM), lactacystin (5 mM), MG115 (25 µM) + MG132 (25 µM) (Inalco, Milan, Italy) or dimethyl sulfoxide (vehicle) for 30 min prior to adding mesalazine (15 mM) for further 1–32 h.

Cell proliferation, death and cycle
Cell proliferation was assessed by using carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR), according to the manufacturer’s instruction. Briefly, cells were incubated with carboxyfluorescein diacetate succinimidyl ester for 30 min, the medium was then removed and fresh media containing 0.05% bovine serum albumin and the desiderated concentrations of the test compounds were added and incubated for further 24–32 h. Cells were then collected, washed twice with phosphate-buffered saline (PBS) and incubated with 5 µg/ml of propidium iodide (PI) for 15 min. Carboxyfluorescein diacetate succinimidyl ester and PI-positive cells were determined by flow cytometry (FACSCalibur; Becton Dickinson, Milan, Italy).

To score cell death, cells were either left untreated or treated with increasing doses of mesalazine (5–30 mM) for 32–96 h. Cells were then collected, washed twice in PBS 1X, stained with fluorescein isothiocyanate–annexin V (1:100 final dilution) according to the manufacturer’s instructions (Becton Dickinson) and incubated with 5 µg/ml PI for 30 min at 4°C, and their fluorescence was measured using FL-1 and FL-2 channels of FACSCalibur using CellQuest Pro software. Annexin V/PI-negative cells were considered as viable cells.

For analysis of cell cycle distribution, serum-starved cells were cultured in medium containing 0.05% bovine serum albumin and the desiderated concentrations of the test compounds for 32 h. At the end, cells were collected, washed twice with PBS, fixed in 70% cold ethanol and stored at –20°C for at least 3 h. Samples were washed twice with PBS, treated with 20 µg/ml RNase A and 100 µg/ml PI and analyzed by flow cytometry.

Organ culture
CRC mucosal explants were taken from five patients undergoing colon resection for primary adenocarcinoma. Patients received neither radiotherapy nor chemotherapy prior to undergoing surgery. Explants were cut in small pieces (2–3 mm) and placed on steel grids in an organ culture chamber at 37°C in a 5% CO2/95% O2 atmosphere in serum-free media with or without 25 mM mesalazine for 18 h.

Immunoprecipitation and western blotting
Protein extracts were prepared and run as described previously (9). Blots were incubated with CDC25A (sc-7389), CDC25B (sc-5619), CDC25C (sc-13138), p-CDK2 (Thr-14/Tyr-15) (sc-28435-R) and CDK2 (sc-6248) antibodies (1:500 final dilution, all from Santa Cruz Biotechnology) followed by a secondary antibody conjugated to horseradish peroxidase. After analysis, each blot was stripped and incubated with a mouse antihuman monoclonal β-actin antibody (1:5000) to ascertain equivalent loading of the lanes.

To analyze p-CHK1 and p-CHK2, blots were incubated with a rabbit antihuman p-CHK1 antibody (1:1000 final dilution) or a rabbit antihuman p-CHK2 antibody (1:1000) (both from Cell Signaling, DBA Italia, Milan, Italy), which specifically recognize phosphorylation of CHK1 and CHK2 on serine 317 and threonine 68 residues, respectively. Phosphorylation of such residues reflects activation of these two kinases (16,17). Afterward, blots were stripped and incubated with antibodies recognizing total CHK1 and CHK2 (Cell Signaling).

For immunoprecipitation, the protein lysates were prepared from cells either left untreated or treated with proteasome inhibitor mix or vehicle for 1 h followed by 15 mM mesalazine treatment for further 1–3 h. Proteins were immunoprecipitated with 2 µg of antiubiquitin (Santa Cruz Biotechnology) or control isotype antiserum for 2 h followed by incubation with protein A/G agarose beads overnight. The resulting immunoprecipitates were washed thoroughly four times with cold lysis buffer, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with antibodies against CDC25A. At the end, blots were stripped and incubated with anti-Smad3 antibody (Santa Cruz Biotechnology) to ascertain equivalent loading of the lanes.

Computer-assisted scanning densitometry (Total Laboratory, AB.EL Science-Ware Srl, Rome, Italy) was used to analyze the intensity of the immunoreactive bands.

RNA extraction, complementary DNA preparation and real-time polymerase chain reaction
Total RNA was extracted from cells by using TRIzol reagent, according to the manufacturer’s instructions (Invitrogen, Milan, Italy). A constant amount of RNA (1 µg per sample) was retro-transcribed into complementary DNA, and 1 µl of complementary DNA/sample was then amplified using the following conditions: denaturation 1 min at 95°C, annealing 30 s at 58°C for CDC25A and/or 30 s at 62°C for β-actin, followed by 30 s of extension at 72°C. Primer sequence of CDC25A was forward: 5'-GTACAAAGAGGAGGAAGAGC-3'; reverse: 5'-GATGCCAGGGATAAAGACTG-3'. Real-time polymerase chain reaction was performed using the IQ SYBR Green Supermix (Bio-Rad Laboratories, Milan, Italy). β-Actin (forward: 5'-AAGATGACCCAGATCATGTTTGAGACC-3' and reverse: 5'-AGCCAGTCCAGACGCAGGAT-3') was used as an internal control.

Effects of mesalazine on the in vivo formation of CT26-derived tumors
CT26 (105 cells/300 µl PBS) were injected subcutaneously into the flank of Balb/c mice whose fur was shaved and depilated. Three groups of 10 mice each were implanted with CT26. The first group received daily subcutaneously 30 mM mesalazine in 300 µl PBS, starting the same day (day 0) of CT26 injection. In this case, mesalazine administration was started 10 h after CT26 injection. The second group received daily subcutaneously 30 mM mesalazine in 300 µl PBS starting 3 days after the CT26 injection. The last group received daily 300 µl PBS (control). Both mesalazine and PBS were injected into a site adjacent to that of CT26 inoculation. After 2 weeks, mice were killed, tumors were photographed, then excised, and their mass was calculated as described elsewhere (18). Studies were approved by the Local Ethical Committee.

Statistical analysis
Values are expressed as means ± SD or SEM. To evaluate the difference in means between groups, the Student’s t-test was used and significance was defined as P values <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Mesalazine inhibits CRC cell growth
As a starting point for in vitro studies, we evaluated the effect of mesalazine on the proliferation of two CRC cell lines, which express either a wild-type (HCT-116) or mutated (HT-29) p53. As shown in Figure 1A, mesalazine significantly and dose dependently inhibited the growth of both cell lines after 32 h treatment. Importantly, the percentage of viable cells was not changed by 32 h treatment with mesalazine independently of the cell line used (Figure 1B). Analysis at later time points revealed no change in HT-29 cell viability after 48 h treatment with mesalazine. A significant increase in death was seen in HT-29 cells treated with 30 mM mesalazine for 72 h and with 15 and 30 mM mesalazine for 96 h (Figure 1B, lower panel). In contrast, mesalazine significantly reduced the viability of HCT-116 at 48 h when used at 30 mM but not 5 or 15 mM. After 72 h culture, the percentage of viable HCT-116 cells was significantly decreased by 15 and 30 mM mesalazine while the three doses of drug were effective in enhancing cell death after 96 h culture (Figure 1B, upper panel). These results collectively suggest that the mesalazine-mediated block in cell growth is not secondary to the induction of death. Studies were next done to examine the mechanism by which mesalazine affects cell proliferation.


Figure 1
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  		Fig. 1. (A) Mesalazine dose dependently inhibits the growth of HCT-116 and HT-29 cells. Carboxyfluorescein diacetate succinimidyl ester-labeled cells were either left untreated (Untr) or treated with mesalazine. After 32 h, the percentage of proliferating cells was evaluated by flow cytometry. Data indicate mean ± SD of three different experiments (untreated versus mesalazine-treated cells; *P < 0.005, **P < 0.001). Right insets show the representative flow cytometry profiles of carboxyfluorescein diacetate succinimidyl ester-labeled HCT-116 cells either left untreated or treated with 15 mM mesalazine. Numbers above lines indicate the percentages of proliferating cells. (B) Mesalazine induces CRC cell death in a time- and dose-dependent fashion. HCT-116 and HT-29 cells were either left untreated or treated with mesalazine for the indicated time points. Data indicate the percentage of viable cells as assessed by flow cytometry analysis of annexin V and PI-negative cells and are expressed as mean ± SD of three experiments. (HCT-116: 48 h, untreated versus 30 mM mesalazine-treated cells, *P = 0.01; 72 h, untreated versus 15 and 30 mM mesalazine-treated cells, **P < 0.001; 96 h, untreated versus mesalazine-treated cells, **P < 0.001 and HT-29: 72 h, untreated versus 30 mM mesalazine-treated cells, *P < 0.01; 96 h, untreated versus 15 and 30 mM mesalazine-treated cells, *P < 0.001).

 
Mesalazine induces CRC cells to accumulate in S phase
We next assessed whether mesalazine induced any change on cell cycle progression. Increasing concentrations of the drug caused a progressive accumulation in the numbers of cells in S phase and a decrease of cells with G2/M and G1 phase DNA content. This effect was seen in both HCT-116 and HT-29 (Figure 2A and B), thus suggesting that DNA replication is lowed down in these cell types upon treatment with mesalazine.


Figure 2
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  		Fig. 2. Mesalazine promotes CRC cells accumulation in S phase. HCT-116 (A) and HT-29 (B) cells were either left untreated (Untr) or treated with mesalazine for 32 h, and the cell cycle distribution was analyzed as indicated in Materials and Methods. Values are the percentages of cells in the different phases of cell cycle and indicate mean ± SD of four experiments. A significant increase in the number of cells that accumulate in S phase was seen after mesalazine treatment (untreated versus mesalazine-treated cells; *P < 0.04, **P < 0.006). Right insets show a representative flow cytometry profile of the cell cycle in HCT-116 either left untreated or treated with 15 mM mesalazine.

 
Mesalazine enhances the phosphorylation of CDK2 at Thr-14 and Tyr-15 residues and downregulates CDC25A protein expression
Progression through the cell cycle is regulated by CDKs, which associate with activating partners, named cyclins, to phosphorylate substrates efficiently. The enzymatic activity of CDKs is also modulated by both inhibitory and activating phosphorylations. In particular, it is known that the CDK–cyclin complex can be inhibited by phosphorylation of Thr-14 and Tyr-15 residues within the adenosine triphosphate-binding pocket of the CDK. Among CDK molecules, CDK2 seems to play a central role in the control of S phase. Indeed, CDK2 can bind to either cyclin E or cyclin A and regulate the G1–S transition and S phase progression, respectively (19). Therefore, we then evaluated whether the mesalazine-induced accumulation of cells in S phase was associated with enhanced phosphorylation/inactivation of CDK2. Phosphorylation of CDK2 is regulated by CDC25 phosphatases (20). So, we next evaluated whether mesalazine interfered with the protein expression of CDC25 family members. Time course studies revealed a decrease in CDC25A protein expression, which occurred as early as 4 h after mesalazine exposure (Figure 3A and B). The effect of mesalazine on CDC25A was dose dependent (Figure 3C) and reversible (Figure 3D). No change in CDC25A protein expression was seen in mannitol-treated cells (Figure 3C). In contrast, mesalazine did not alter the protein levels of CDC25B and C, and this was evident at early (i.e. 1–12 h, Figure 3A) and later time points (i.e. 20–28 h, data not shown). To ascertain whether the downregulation of CDC25A by mesalazine was stable over the time, we evaluated CDC25A protein expression in cells cultured with or without mesalazine for 20 and 28 h. Data in Figure 3E show that treatment of CRC cells with mesalazine caused a persistent reduction in the level of CDC25A. As expected, downregulation of CDC25A by mesalazine was followed by enhanced phosphorylation of CDK2 at Thr-14 and Tyr-15 residues (Figure 3E, lower blots). To confirm the functional relevance of CDC25A protein downregulation in the accumulation of CRC cells in S phase, we specifically knocked down the expression of CDC25A by siRNA and then cultured these cells with or without mesalazine. As shown in Figure 3F, transfection of CRC cells with CDC25A but not control siRNA reduced the protein level of CDC25A, without altering the rate of cell death (data not shown). Either CDC25A siRNA- or mesalazine-treated cells accumulated in S phase. Notably, exposure of CDC25A-deficient cells to mesalazine did not increase further the fraction of cells accumulated in S phase (Figure 3G).


Figure 3
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  		Fig. 3. (A) Mesalazine downregulates CDC25A in a time-dependent fashion without affecting CDC25B and CDC25C expression. HCT-116 cells were either left untreated (Untr) or treated with 15 mM mesalazine for the indicated time points. CDC25A, CDC25B, CDC25C and β-actin were analyzed by western blotting. One of four representative western blots is shown. (B) Quantitative analysis of CDC25A to β-actin protein ratio in total extracts of HCT-116 cells either left untreated or treated with 15 mM mesalazine for the indicated time points, as measured by densitometry scanning of western blots. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of four experiments (untreated versus mesalazine-treated cells; *P = 0.04, **P = 0.02, ***P < 0.001). (C) Representative western blots for CDC25A and β-actin in extracts of HCT-116 cells either left untreated or treated with graded doses of mesalazine or mannitol (30 mM) for 8 h. One of three separate experiments is shown. (D) The mesalazine effect on CDC25A is reversible. Representative western blots for CDC25A and β-actin in extracts of HCT-116 cells cultured in the presence or absence of mesalazine (5–15 mM) for 16 h (first culture). Afterward, cells were either left untreated or extensively washed and then cultured with fresh medium in the absence of mesalazine for additional 16 h (second culture). One of three separate experiments is shown. (E) The mesalazine-induced downregulation of CDC25A protein expression is stable over the time and is followed by enhanced phosphorylation of CDK2 at Thr-14 and Tyr-15 residues. HCT-116 cells were either left untreated or treated with 15 mM mesalazine for the indicated time points. CDC25A, β-actin, p-CDK2 (Thr-14/Tyr-15) and total CDK2 were analyzed by western blotting. One of four representative western blots is shown. (F) Representative western blots for CDC25A, β-actin, p-CDK2 and total CDK2 in extracts of HCT-116 cells either left unstimulated or transfected with CDC25A or control siRNA for 2 days and then either left untreated or treated with 15 mM mesalazine for 12 h. One of three separate experiments is shown. (G) Representative histograms showing the cell cycle distribution of HCT-116 cells transfected as indicated in (F) and either left untreated or treated with 15 mM mesalazine for 32 h. Data indicate mean ± SD of three different experiments.

 
CHK1/CHK2 are not involved in the mesalazine-mediated CDC25A protein downregulation
CDC25A protein expression can be regulated by two upstream kinases, CHK1 and CHK2, which phosphorylate CDC25A, thus promoting its degradation (21). To assess whether these two kinases were involved in the mesalazine-induced CDC25A protein downregulation, we initially analyzed the activation status of CHK1/CHK2 by western blotting. Mesalazine enhanced CHK1 phopshorylation in a time-dependent fashion (Figure 4A). However, densitometric analysis of p-CHK1 and total CHK1 blots revealed that CHK1 phosphorylation was significantly increased as early as 24 h after mesalazine treatment, and such an increase persisted over the time (Figure 4A, right inset). In contrast, a pronounced phosphorylation of CHK2 was seen within 30 min of treatment with mesalazine (data not shown) and persisted up to 24 h (Figure 4B). Therefore, the onset of decline in CDC25A protein expression (Figure 3A) was preceded by activation of CHK2 but not of CHK1.


Figure 4
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  		Fig. 4. CHK1 and CHK2 are not involved in the mesalazine-mediated CDC25A protein downregulation. (A) Representative western blots showing CHK1 phosphorylation at serine 317 residue (pS317) and total CHK1 in total extracts of HCT-116 cells either left untreated (Untr) or treated with 15 mM mesalazine for the indicated time points. Camptothecin (Cpt) was used as a positive control for CHK1 activation. Right panel shows quantitative analysis of CHK1 pS317 to total CHK1 protein ratio in extracts of HCT-116 cells either left untreated or treated with 15 mM mesalazine, as measured by densitometry scanning of western blots. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of three experiments (untreated versus mesalazine-treated cells, *P < 0.04). (B) Representative western blots showing CHK2 phosphorylation at threonine 68 residue (pT68) and total CHK2 in extracts of HCT-116 cells either left untreated or treated with 15 mM mesalazine. Camptothecin (Cpt) was used as a positive control for CHK2 activation. Right panel shows quantitative analysis of CHK2 pT68 to total CHK2 protein ratio in extracts of HCT-116 cells either left untreated or treated with 15 mM mesalazine, as measured by densitometry scanning of western blots. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of three experiments (untreated versus mesalazine-treated cells, *P < 0.01). (C) Representative western blots for CHK1, CHK2, CDC25A and β-actin in extracts of HCT-116 cells either left unstimulated or transfected with CHK1, CHK2 or control siRNA for 2 days and then either left untreated or treated with mesalazine for 12 h. One of three separate experiments is shown. (D) Representative histograms showing the cell cycle distribution of HCT-116 cells transfected as described above and then either left untreated or treated with 15 mM mesalazine for 32 h. Data indicate mean ± SD of three different experiments.

 
Subsequently, we evaluated whether targeted silencing of CHK1 or CHK2 blocked the mesalazine-mediated CDC25A protein reduction. Data in Figure 4C show the marked suppression of either CHK1 or CHK2 protein expression in cells treated with specific siRNA. Notably, neither CHK1 nor CHK2 siRNA prevented the mesalazine-mediated downregulation of CDC25A (Figure 4C). Consistently, mesalazine induced cells to accumulate in S phase independently of the presence of CHK1 or CHK2 (Figure 4D). Overall, these data indicate that CHK1/CHK2 do not have a causative role in CDC25A protein downregulation in our system, even though this pathway is activated following exposure to mesalazine.

Inhibitors of proteasome pathway prevent the mesalazine-mediated CDC25A protein downregulation
Studies carried out in other cell systems have shown previously that intracellular levels of CDC25A can be regulated by stimuli, which either enhance or inhibit gene transcription (22). Therefore, to examine if mesalazine regulated CDC25A at the transcriptional level, total RNA was extracted from cells cultured with or without mesalazine for different time points and analyzed for CDC25A by real-time polymerase chain reaction. Treatment of cells with mesalazine for 1–4 h did not modify the content of CDC25A RNA transcripts. In contrast, starting from 6 h, the treatment with mesalazine resulted in an increased and persistent CDC25A RNA expression (Figure 5A). Taken together, these data indicate that the mesalazine-mediated downregulation of CDC25A protein expression is not secondary to a block in cdc25A gene transcription.


Figure 5
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  		Fig. 5. Mesalazine regulates CDC25A protein expression by ubiquitin-dependent proteasome pathway. (A) Mesalazine does not affect CDC25A RNA expression in HCT-116. Cells were treated with or without 15 mM mesalazine for the indicated time points, and CDC25A transcripts were then evaluated by real-time polymerase chain reaction. Levels are normalized to β-actin. Values are mean ± SD of three experiments. (B) Representative expression of ubiquitinated CDC25A protein in HCT-116 extracts. HCT-116 cells were cultured in the presence or absence of MG115 and MG132 for 30 min and then stimulated with 15 mM mesalazine for the indicated time points. Proteins were immunoprecipitated by an antihuman ubiquitin or control isotype (Ve–) antibody and then subjected to immunoblot analysis using an anti-CDC25A antibody. After analysis of ubiquitinated CDC25A, the blot was stripped and incubated with an anti-Smad3 antibody to ascertain the equivalent loading of the lanes. (C) The mesalazine-mediated CDC25A protein downregulation is reverted by inhibitors of the proteasome pathway. Representative western blots for CDC25A and β-actin in extracts of HCT-116 cells either left untreated or treated with 15 mM mesalazine in the presence or absence of lactacystin, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) or dimethyl sulfoxide (DMSO) (vehicle) for 12 h. One of three experiments in which similar results were obtained is shown. (D) Both lactacystin and N-p-tosyl-L-phenylalanine chloromethyl ketone revert the mesalazine-mediated accumulation of HCT-116 cells in S phase. Cells were either left untreated or treated with 15 mM mesalazine in the presence or absence of lactacystin, N-p-tosyl-L-phenylalanine chloromethyl ketone or dimethyl sulfoxide for 32 h, and the cell cycle distribution was analyzed as indicated in Materials and Methods. Values are the percentages of cells in S phase and indicate mean ± SD of four experiments (mesalazine versus mesalazine + lactacystin, *P = 0.002, mesalazine versus mesalazine + N-p-tosyl-L-phenylalanine chloromethyl ketone, **P < 0.001).

 
CDC25A protein levels are also controlled by ubiquitin-dependent proteasome pathway (13,14). Therefore, we first looked at the ubiquitination status of CDC25A in immunoprecipitates from cells either left untreated or treated with mesalazine. As shown in Figure 5B, two bands corresponding to the ubiquitinated CDC25A were clearly seen in mesalazine-treated but not untreated cells. Importantly, the mesalazine-induced ubiquitination of CDC25A was largely prevented by preincubation of cells with proteasome inhibitors. No band was detected when an irrelevant antibody was used in the immunoprecipitation. Second, we determined whether proteasomal degradation is involved in mesalazine regulation of CDC25A. Treatment of cells with proteasome inhibitors, namely N-p-tosyl-L-phenylalanine chloromethyl ketone or lactacystin, prevented the mesalazine-mediated CDC25A inhibition (Figure 5C). Consistently, N-p-tosyl-L-phenylalanine chloromethyl ketone and lactacystin reverted the mesalazine-induced accumulation of CRC cells in S phase (Figure 5D).

Mesalazine reduces CDC25A in human CRC tissue and CT26 cell line and inhibits the in vivo formation of CT26-derived tumors in mice
To extend our observations to primary CRC cells, mesalazine was added to organ cultures of CRC explants. After 18 h, CDC25A was evaluated by western blotting. Data in Figure 6A show that mesalazine significantly reduced CDC25A expression.


Figure 6
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  		Fig. 6. Mesalazine reduces CDC25A in human CRC tissue and CT26 cell line and inhibits the in vivo formation of CT26-derived tumors in mice. (A) Mesalazine negatively regulates CDC25A protein expression in primary CRC cells. Freshly obtained CRC explants were cultured in the absence (Untr, untreated) or presence of 25 mM mesalazine for 18 h. One of three representative western blots is shown. Right inset shows the quantitative analysis of CDC25A to β-actin protein ratio in extracts of CRC explants cultured as indicated above and measured by densitometry scanning of western blots. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of two separate experiments analyzing in total five CRC samples (untreated versus mesalazine-treated samples, *P < 0.01). (B) Mesalazine dose dependently inhibits the growth of CT26 cells. Carboxyfluorescein diacetate succinimidyl ester-labeled cells were either left untreated or treated with mesalazine. After 24 h, the percentage of proliferating cells was evaluated by flow cytometry. Data indicate mean ± SD of three experiments (untreated versus mesalazine-treated cells; *P = 0.04, **P < 0.005, ***P < 0.001). (C) Mesalazine promotes CT26 cells accumulation in S phase. Cells were either left untreated or treated with mesalazine for 24 h, and the cell cycle distribution was analyzed as indicated in Materials and Methods. Values are the percentages of cells in the different phases of cell cycle and indicate mean ± SD of four experiments. A significant increase in the number of cells that accumulate in S phase was seen after mesalazine treatment (untreated versus cells treated with 15 and 30 mM mesalazine, P < 0.02). (D) Mesalazine dose dependently reduces CDC25A protein expression in CT26 cells. Cells were either left untreated or treated with mesalazine for 12 h. One of four representative western blots is shown. (E) Administration of mesalazine reduces the in vivo formation of CT26-derived tumors. CT26 were inoculated into the flank of Balb/c mice, and animals were then treated daily with subcutaneous injection of mesalazine (30 mM) starting the same day (day 0) of or 3 days (day 3) after the CT26 injection. Control mice received daily PBS. Representative photographic images of CT26 xenografts (arrows) developed in control (Ctr) and mesalazine-treated mice are shown. Right inset shows the tumor mass of CT26-derived xenografts in control and mesalazine-treated mice. Data indicate mean ± SD of all experiments (control versus mesalazine, **P < 0.01).

 
To assess further the effect of mesalazine on CRC cell growth, we used an in vivo syngeneic CRC model in mice. Tumors were generated by injecting the murine CRC cell line, CT26, into Balb/c mice. Initially, we showed that mesalazine significantly reduced the proliferation of cultured CT26 cells, caused an accumulation of such cells in S phase and downregulated CDC25A protein expression (Figure 6B–D). Moreover, mesalazine reduced the viability of CT26 cells, but this effect was seen after 32 and 48 h treatment (data not shown), thus confirming the data obtained with human CRC cells. Subsequently, we tested the capability of mesalazine in inhibiting CT26 tumorigenicity in vivo. To this end, mice were injected daily subcutaneously with mesalazine starting from the same day of or 3 days after the CT26 inoculation. This later time was selected on the basis of preliminary experiments showing that a microscopically evident CT26-derived tumor was already evident at this time point (data not shown). No body weight loss was observed during the study in both control and drug-treated groups, and all animals survived until the end of study. Fourteen days after injection, CT26-derived tumors were evident in 10 of 10 control mice, 8 of 10 mice treated with mesalazine from day 0 and 8 of 10 mice treated with mesalazine from day 3. Moreover, mice treated with mesalazine exhibited a significant decrease in the tumor mass in comparison with controls, regardless of whether the drug administration was started at day 0 or day 3 (Figure 6E, P < 0.01).


   Discussion
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 Materials and methods
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We here show that mesalazine inhibits proliferation of CRC cells and reduces the in vivo formation of murine CRC cell (CT26)-derived tumors. The antineoplastic effects of mesalazine were seen with a concentration of drug which is in a range similar to that reached within the gut tissue under standard oral treatment (23). The mesalazine doses we selected for our experiments are similar to those used by other workers to test the biological effects of this drug in vitro (7,8,10). We demonstrate also that mesalazine affects cell cycle progression by increasing the proportion of CRC cells in S phase. Analysis of regulatory components of the S phase pathway revealed that mesalazine dose dependently reduced the expression of the phosphatase CDC25A. This inhibitory effect was reversible, as substantiated by the demonstration that cells fully recovered the CDC25A protein content upon removal of the drug from the culture. Altogether, these findings indicate that block of cell cycle progression and downregulation of CDC25A expression by mesalazine are not secondary to a toxic effect of the drug and that CDC25A is rapidly synthesized in untreated CRC cells. The effect of mesalazine on CDC25A would seem to be specific, as the abundance of other members of CDC25 family, namely CDC25B and CDC25C, was not affected by this drug. CDC25A has traditionally been assigned the role of promoting entry into S phase by dephosphorylating CDK2 on residues Thr-14 and Tyr-15, thereby promoting the activation of the CDK2–cyclin E and CDK2–cyclin A complexes (13,14). In line with this, we showed that mesalazine enhanced the phosphorylation of CDK2 at Thr-14 and Tyr-15 residues. The involvement of CDC25A in the mesalazine-induced block of progression through S phase is also supported by the demonstration that silencing of CDC25A induced CRC cells to accumulate in S phase, and that mesalazine did not alter the cell cycle progression in CDC25A-deficient CRC cell.

In response to DNA damage and/or replication block, cell cycle arrest or delay is imposed by ATM- and rad3-related (ATR) and ataxia-telangiectasia mutated (ATM) protein kinases that activate CHK1 and/or CHK2 (24). Activated CHK1 and CHK2 phosphorylate CDC25A, thus promoting its degradation. Although, CHK2 and CHK1 were activated by mesalazine, silencing of either CHK1 or CHK2 in CRC cells did not overcome the mesalazine-mediated CDC25A protein downregulation. Notably, knock down of either CHK1 or CHK2 did not alter the level of CDC25A in untreated CRC cells. This would seem to suggest that both CHK1 and CHK2 play no role in the control of CDC25A in our cell system. Another possibility is that the contribution of CHK1 to the control of CDC25A in untreated CRC cells is partially or totally redundant with CHK2, and therefore, the activity of either CHK1 or CHK2 is sufficient to maintain a fixed level of CDC25A. While our study was ongoing, Luciani et al. (25) reported that mesalazine affects cell cycle progression by inducing CRC cells to accumulate in S phase. These authors elegantly showed that mesalazine facilitates the recruitment of the sensor kinase ATR and of the adapter molecule claspin onto chromatin, enhances phosphorylation of proteins involved in the ATR-dependent S phase checkpoint response, such as CHK1 and Rad17, and delays the completion of DNA synthesis. However, these effects were mostly seen in cells exposed to 40 mM mesalazine, a concentration that is higher than that used in our studies. In this context, it is also noteworthy that no functional study was conducted to mechanistically link the activation of ATR–CHK1 pathway with the accumulation of cells in S phase. It is thus plausible that, depending on the dose, mesalazine can activate different pathways that ultimately induce accumulation of CRC cells in S phase and reduce the rate of DNA synthesis.

Downregulation of CDC25A and impairment of cell cycle progression have been recently linked to p53 activity (26). However, the fact that the mesalazine-mediated CDC25A inhibition occurs in HT-29, a CRC cell line that does not express a functional p53, would argue against the involvement of p53 in the control of CDC25A in our system.

cdc25A is a direct transcriptional gene target of the proto-oncogene c-myc (27). Although it has been recently shown that mesalazine can inhibit c-myc in CRC cell lines (28), it is unlikely that this is the mechanism responsible for the CDC25A protein downregulation. In fact, exposure of CRC cells to mesalazine for a time shorter (i.e. 1–3 h) than that required to induce CDC25A protein decline (i.e. 4 h) did not alter the content of CDC25A RNA transcripts. Moreover, analysis at later time points showed that mesalazine enhanced CDC25 RNA expression. This later finding could reflect the attempt of CRC cell to restore the CDC25A protein content.

Our results are compatible with the possibility that mesalazine induces an acceleration of CDC25A degradation via the proteasome pathway. Indeed, mesalazine-treated cells exhibited enhanced ubiquitination of CDC25A, and the addition of proteasome inhibitors to mesalazine-treated CRC cell cultures largely prevented the ubiquitination and degradation of CDC25A protein. This confirms and expands on previous data showing that CDC25A levels may be regulated by the proteasome pathway (14,29). Ubiquitination and proteasome-mediated CDC25A degradation usually occur in a phosphorylation-dependent manner. Although, serine 123 was the first site reported to regulate CDC25A stability, recent studies have shown that serines 75, 178, 278 and 292 may also contribute to control CDC25A protein levels depending on the cell context analyzed (14,30). However, commercially available antibodies that specifically recognize these residues are not yet available. Therefore, we do not know whether mesalazine facilitates the proteasome-mediated CDC25A protein degradation by promoting CDC25A phosphorylation at specific serine sites. However, a recent study has identified a non-phosphorylated motif in the CDC25A protein sequence that could be targeted for proteasomal degradation (31). Further studies will be necessary to determine the molecular mechanisms by which CDC25A is labeled for degradation through the proteasome pathway after mesalazine treatment.

Our results would seem also to suggest that mesalazine may directly interfere with CDC25A expression rather than acting on primary S phase components that in turn control CDC25A stability. This hypothesis is supported by the demonstration that prevention of CDC25A protein degradation by proteasome pathway inhibitors markedly reduced the mesalazine-induced accumulation of cells in S phase. In this contest, it is, however, noteworthy that inhibitors of proteasome pathway could also interfere with other proteins that are responsible for the progression through the cell cycle.

We here confirm and expand on previously published studies showing that mesalazine enhances CRC cell apoptosis (7). In response to mesalazine treatment, cell death occurred at later time points (e.g. 48 h) and was preceded by cell cycle arrest, thus suggesting that induction of cell death is driven by cell cycle blockage via inhibition of CDC25A. However, it is likely that mesalazine can also trigger directly apoptotic signals in CRC cells. It is also conceivable that CDC25A depletion by itself may promote cell death because CDC25A is known to interact with and negatively regulate the activity of kinases (e.g. extracellular signal-regulated kinase 1/2) that stimulate proapoptotic programs (32–34).

The functional relevance of our data relates to the demonstration that a subset of cancers have been found to display increased levels of CDC25A, and that deregulation of CDC25A activity seems to contribute to the unrestrained proliferation of tumor cells (13,35,36). Indeed, a number of recent publications witness the collective drug discovery effort being directed toward the identification of novel compounds that not only inhibit CDC25A in cultured cells but also exert antineoplastic effects in vivo. Results of our study would seem to indicate that mesalazine fulfills these objectives, because we show that mesalazine inhibits CDC25A both in CRC mucosal explants and cell lines, and reduces the development of CRC in vivo. In this context, we would like, however, to emphasize that the in vivo antineoplastic effects of mesalazine could be also due to its ability to interfere with other biological pathways that sustain CRC growth (9–11).

An important consideration in the design of our in vivo study was the selection of the route of mesalazine administration. Our initial attempts to inhibit CT26-derived tumors by intraperitoneal administration of mesalazine were, however, unsuccessful (C.Stolfi, G.Monteleone, personal observation), probably due to the rapid inactivation and/or elimination of the drug from the circulation (37,38). Therefore, we selected the subcutaneous route of administration in order to achieve optimal concentration of the drug in the site of tumor growth. This was also suggested by the fact that, after oral or rectal administration, mesalazine acts locally and that its effectiveness depends on the luminal rather than the systemic concentration of the drug.

In conclusion, this study indicates that mesalazine negatively regulates CDC25A protein expression and induces accumulation of CRC cells in S phase, substantiating further the antineoplastic properties of this drug.


   Funding
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Fondazione ‘Umberto Di Mario’, Rome; Associazione Italiana per la Ricerca sul Cancro; Giuliani S.p.A., Milan, Italy.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Obrador A, et al. Review article: colorectal cancer surveillance in ulcerative colitis—what should we be doing? Aliment. Pharmacol. Ther. (2006) 24:56–63.[CrossRef][Web of Science][Medline]
  2. Jess T, et al. Risk of intestinal cancer in inflammatory bowel disease: a population-based study from olmsted county, Minnesota. Gastroenterology (2006) 130:1039–1046.[CrossRef]
  3. Eaden J, et al. Colorectal cancer prevention in ulcerative colitis: a case control study. Aliment. Pharmacol. Ther. (2000) 14:145–153.[Web of Science][Medline]
  4. Van Staa TP, et al. 5-Aminosalicylate use and colorectal cancer risk in inflammatory bowel disease: a large epidemiological study. Gut (2005) 54:1573–1578.[Abstract/Free Full Text]
  5. Rutter M, et al. Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis. Gastroenterology (2004) 126:451–459.[CrossRef][Web of Science][Medline]
  6. Rubin DT, et al. Aminosalicylate therapy in the prevention of dysplasia and colorectal cancer in ulcerative colitis. Clin. Gastroenterol. Hepatol. (2006) 11:1346–1350.
  7. Reinacher-Schick A, et al. Mesalazine causes a mitotic arrest and induces caspase-dependent apoptosis in colon carcinoma cells. Carcinogenesis (2003) 24:443–451.[Abstract/Free Full Text]
  8. Gasche C, et al. Mesalazine improves replication fidelity in cultured colorectal cells. Cancer Res. (2005) 65:3993–3997.[Abstract/Free Full Text]
  9. Monteleone G, et al. Silencing of SH-PTP2 defines a crucial role in the inactivation of epidermal growth factor receptor by 5-aminosalicylic acid in colon cancer cells. Cell Death Differ. (2006) 13:202–211.[CrossRef][Web of Science][Medline]
  10. Bos CL, et al. Protein phosphatase 2A is required for mesalazine-dependent inhibition of Wnt/beta-catenin pathway activity. Carcinogenesis (2006) 27:2371–2382.[Abstract/Free Full Text]
  11. Rousseaux C, et al. Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-gamma. J. Exp. Med. (2005) 201:1205–1215.[Abstract/Free Full Text]
  12. Karlsson-Rosenthal C, et al. Cdc25: mechanisms of checkpoint inhibition and recovery. Trends Cell Biol. (2006) 16:285–292.[CrossRef][Web of Science][Medline]
  13. Busino L, et al. Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis. Oncogene (2004) 23:2050–2056.[CrossRef][Web of Science][Medline]
  14. Kristjansdottir K, et al. Cdc25 phosphatases and cancer. Chem. Biol. (2004) 11:1043–1051.[CrossRef][Web of Science][Medline]
  15. Singh SV, et al. Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C. J. Biol. Chem. (2004) 279:25813–25822.[Abstract/Free Full Text]
  16. Sancar A, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. (2004) 73:39–85.[CrossRef][Web of Science][Medline]
  17. Buscami G, et al. DNA damage-induced cell cycle regulation and function of novel chk2 phosphoresidues. Mol. Cell. Biol. (2006) 26:7832–7845.[Abstract/Free Full Text]
  18. Tomayko MM, et al. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol. (1989) 24:148–154.[CrossRef][Web of Science][Medline]
  19. Shapiro GI. Cyclin-dependent kinase pathways as targets for cancer treatment. J. Clin. Oncol. (2006) 24:1770–1783.[Abstract/Free Full Text]
  20. Sampath D, et al. Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: dysregulation by 7-hydroxystaurosporine. Mol. Pharmacol. (2002) 62:680–688.[Abstract/Free Full Text]
  21. Rudolph J. Cdc25 phosphatases: structure, specificity, and mechanism. Biochemistry (2007) 46:3595–3604.[CrossRef][Web of Science][Medline]
  22. Rodrigues S, et al. Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells. Oncogene (2006) 25:6628–6636.[CrossRef][Web of Science][Medline]
  23. Frieri G, et al. Rectal and colonic mesalazine concentration in ulcerative colitis: oral vs oral plus topical treatment. Aliment. Pharmacol. Ther. (1999) 13:1413–1417.[CrossRef][Web of Science][Medline]
  24. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer (2003) 3:155–168.[CrossRef][Web of Science][Medline]
  25. Luciani MG, et al. Mesalazine affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint. Gastroenterology (2007) 132:221–235.[CrossRef][Web of Science][Medline]
  26. Rother K, et al. p53 downregulates expression of the G(1)/S cell cycle phosphatase Cdc25A. Oncogene (2007) 26:1949–1953.[CrossRef][Web of Science][Medline]
  27. Galaktionov K, et al. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature (1996) 382:511–517.[CrossRef][Medline]
  28. Chu EC, et al. Mesalazine downregulates c-Myc in human colon cancer cells. A key to its chemopreventive action? Aliment. Pharmacol. Ther. (2007) 25:1443–1449.[Web of Science][Medline]
  29. Busino L, et al. Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature (2003) 426:87–91.[CrossRef][Medline]
  30. Neely KE, et al. Cdc25A regulation: to destroy or not to destroy-is that the only question? Cell Cycle (2003) 2:455–457.[Medline]
  31. Kanemori Y, et al. Beta-TrCP recognizes a previously undescribed nonphosphorylated destruction motif in Cdc25A and Cdc25B phosphatases. Proc. Natl Acad. Sci. USA (2005) 102:6279–6284.[Abstract/Free Full Text]
  32. Wang Z, et al. Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5. J. Cell. Physiol. (2005) 204:437–444.[CrossRef][Web of Science][Medline]
  33. Ollinger R, et al. Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle (2007) 6:3078–3085.[Web of Science][Medline]
  34. Lee SH, et al. Induction of apoptotic cell death by 2'-hydroxycinnamaldehyde is involved with ERK-dependent inactivation of NF-kappaB in TNF-alpha-treated SW620 colon cancer cells. Biochem. Pharmacol. (2005) 70:1147–1157.[CrossRef][Web of Science][Medline]
  35. Brezak MC, et al. Inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of CDC25 phosphatases. Mol. Cancer Ther. (2005) 4:1378–1387.[Abstract/Free Full Text]
  36. Kar S, et al. PM-20, a novel inhibitor of Cdc25A, induces extracellular signal-regulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo. Mol. Cancer Ther. (2006) 5:1511–1519.[Abstract/Free Full Text]
  37. Myers B, et al. Metabolism and urinary excretion of 5-amino salicylic acid in healthy volunteers when given intravenously or released for absorption at different sites in the gastrointestinal tract. Gut (1987) 28:196–200.[Abstract/Free Full Text]
  38. Bondesen S, et al. Pharmacokinetics of 5-aminosalicylic acid in man following administration of intravenous bolus and per os slow-release formulation. Dig. Dis. Sci. (1991) 36:1735–1740.[CrossRef][Web of Science][Medline]
Received January 4, 2008; revised April 15, 2008; accepted May 15, 2008.


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