Carcinogenesis

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Carcinogenesis, Vol. 24, No. 3, 443-451, March 2003
© 2003 Oxford University Press

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION

Mesalazine causes a mitotic arrest and induces caspase-dependent apoptosis in colon carcinoma cells

A. Reinacher-Schick¹, A. Schoeneck¹, U. Graeven¹, I. Schwarte-Waldhoff¹ and W. Schmiegel^1,2,1

¹ Department of Medicine, Ruhr-University Bochum, Knappschaftskrankenhaus,
² Department of Gastroenterology and Hepatology, Bergmannsheil, Germany

	Abstract

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Non-steroidal anti-inflammatory drugs (NSAID) may inhibit colon cancer development through affecting proliferation and apoptosis. However, their use in cancer chemoprevention is still limited due to toxicities. There is longstanding clinical experience with the aminosalicylate mesalazine in the treatment of patients with inflammatory bowel disease with very few side effects. So far, most studies on the cellular effects of mesalazine were focused on its anti-inflammatory properties. Recent data, however, indicate that mesalazine may also reduce cell growth in vivo. We therefore investigated the growth inhibitory effect of mesalazine on human colon cancer cells in vitro compared with established chemopreventive agents. We also wished to determine the underlying cellular mechanisms of the effect. Here we show that mesalazine dose- and time-dependently inhibited the proliferation of colon cancer cells. Mesalazine was less potent in reducing cell growth than sulindac sulfide or indomethacin but growth effective mesalazine concentrations were comparable with concentrations achievable in vivo under standard mesalazine treatment. While other NSAID induced a robust G₁ arrest, mesalazine specifically blocked cells in mitosis although microtubule polymerization or spindle orientation was not affected. In addition, mesalazine induced apoptosis in colon cancer cells possibly through activation of caspase-3 whereas the levels of bcl-2 family proteins were not altered. We conclude that mesalazine inhibits growth of colon cancer cells largely through a mitotic arrest, which has not been reported for NSAID so far. Mesalazine also induces apoptosis through partial activation of caspases similar to, although weaker than, established chemopreventive agents. These findings may suggest a potential of mesalazine as a chemopreventive agent for colorectal cancer.

Abbreviations: CRC, colorectal cancer; COX, cyclooxygenase enzymes; IBD, inflammatory bowel disease; MI, mitotic index; MoAb, monoclonal antibody; NSAID, non-steroidal anti-inflammatory drugs; PARP, poly-ADP-ribosepolymerase; PBS, phosphate-buffered saline; TUNEL, terminal transferase dUTP nick end labeling assay.

	Introduction

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Colorectal cancer (CRC) is the second leading cause of cancer death in the western world. Although much has been learned about the molecular events leading to CRC there is still no cure for advanced and metastatic stages. Therefore, improved strategies for prevention of the disease are warranted. Findings from epidemiological and clinical studies as well as animal models of colon carcinogenesis implicate that non-steroidal anti-inflammatory drugs (NSAID) may be effective against the development of colorectal cancer (for review see ref. 1). Among the best-studied agents of the NSAID group of compounds are aspirin and sulindac. Pharmacologically, these drugs inhibit cyclooxygenase enzymes (COX) in various cell types. Whereas the COX-1 isoform is constitutively expressed, it is the inducible COX-2 isoform, which is thought to play a prominent role in colon carcinogenesis (2). Thus, COX-2 inhibition is believed to underlie the chemopreventive effect of NSAID possibly through reducing cell proliferation, inducing apoptosis or modulating angiogenesis (3). Although the chemopreventive efficacy of NSAID has been clearly established, their wide spread use is limited due to their frequent and often severe side effects including gastrointestinal bleeding and renal damage (4,5). In order to reduce these side effects, compounds have been developed which specifically inhibit the inducible COX-2 isoform. However, selective COX-2 inhibitors still retain the renal toxicities of their parent compounds (6). Furthermore, there is growing evidence that the chemopreventive effect of NSAID may also work through COX-independent mechanisms (7). Thus, efforts to identify additional agents with chemopreventive efficacy seem sensible and should not be confined to potent COX-2 inhibitors only.

Epidemiological data suggest that aminosalicylates may reduce the risk of colorectal cancer in patients with ulcerative colitis (8,9). The aminosalicylate mesalazine (5-aminosalicylate, 5-ASA), a weak COX and lipoxygenase inhibitor, has long been used in the treatment and prophylaxis of patients with inflammatory bowel disease (IBD). It is characterized by low systemic resorption and very few side effects (10,11). The molecular mechanisms underlying mesalazine’s potential chemopreventive effect are not fully understood. Most studies on mesalazine in colon epithelial cells were focused on its anti-inflammatory properties and the cellular effects of the compound were mainly evaluated in response to pro-inflammtory stimuli in order to mimick the conditions in the inflamed mucosa of patients with IBD. There, mesalazine was found to specifically inhibit the NFkB pathway or scavenge free radicals both mechanisms sustaining the chronic inflammatory processes in the gut (12–14). There are limited data on the effect of aminosalicylates in the prevention of CRC unassociated with IBD with differing results (15,16). Some of these reports suggest that aminosalicylates may act by inducing apoptosis in vivo in colonic epithelial cells similar to the effect of other NSAID (17,18). We therefore wished to investigate whether mesalazine possesses growth inhibitory activity in colon epithelial cells independent of its anti-inflammatory effects. Mesalazine was tested in direct comparison to well established chemopreventive agents like sulindac, indomethacin and a selective COX-2 inhibitor. We were particularly interested to determine whether mesalazine affected proliferation and apoptosis of colon epithelial cells in the absence of pro-inflammatory cytokines. We show here that mesalazine affects proliferation of colon cancer cells. It was much less potent than established chemopreventive compounds but its growth effective concentrations in vitro were comparable with concentrations achievable in vivo under standard mesalazine treatment. It does so through the specific induction of a mitotic arrest, a novel mechanism not described for NSAID so far. In addition, mesalazine moderately induces apoptosis in a caspase-dependent manner similar to other well established chemopreventive agents.

	Materials and methods

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Cell lines
HT29 colorectal cancer cells were obtained from ATCC. Culture medium consisted of DMEM (Dulbeco’s Modified Eagle Medium) supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM sodium pyruvate and sodium glutamate (all Gibco BRL, Gaithersburg, MD). Cells were grown in monolayers, incubated at 37°C in 10% CO₂ and 90% relative humidity and routinely passaged using trypsin–EDTA 0.025%.

Treatment with NSAID
For treatment with NSAID exponentially growing cells were trypsinized and seeded in culture medium (see above) supplemented with 2% FCS at the following densities: 10 000 cells/well in 96-well plates; 50 000 cells/well in 24-well plates, 200 000 cells/well in 6-well plates and 1x10⁶ cells in 10 cm dishes (all cell culture plates were obtained from Becton Dickinson, NJ). Cell monolayers remained subconfluent at the end of the treatment interval in all experiments. Mesalazine (5-aminosalicylate; 5-ASA, Sigma, Deisenhofen, Germany) was dissolved as a 40 mM stock solution in culture medium supplemented with 2% FCS with continuous stirring at 37°C. The pH of the drug solution was adjusted to 7.4 with NaOH. All assays testing mesalazine were protected from light. Sulindac sulfide and NS-398 (Biomol Res. Labs, PA) were made up in DMSO as 100 mM stock solutions, indomethacin (Biomol Res Labs) as a 500 mM stock solution in DMSO. The required final concentrations were obtained by diluting aliquots of the stock solution in cell culture medium supplemented with 2% FCS. To control for an effect of the solvent DMSO or a potential effect of the osmolarity of the mesalazine solution (up to 40 mM) control samples were treated with equivalent concentrations of DMSO or equivalent osmolar concentrations of mannitol in parallel.

The assessment of cell proliferation
The effect of NSAID on cell proliferation was determined colorimetrically using the WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. This assay assesses cell proliferation colorimetrically by measuring the metabolic activity of cultured cells. We first validated the assay by using standard cell counting assays in parallel in pilot experiments. Thereby, the range where the extinction in the WST-1 assay and actual cell numbers measured by cell counting correlated linearly was determined. The assay was found linear between absorbance values of 1 and 2.5. Then, cells were seeded onto 96-well plates and allowed to adhere for 24 h. The respective compound was added at increasing concentrations and cells were incubated for an additional 24, 48 or 72 h. All treatment and control conditions were tested at least in triplicates (between three and five wells per assay condition). Cells were then washed twice with phosphate-buffered saline (PBS), the tetrazolium salt WST-1 (dissolved 1:10 in cell culture media) was added and the plates were incubated at 37°C. Cell proliferation was assessed by measuring the amount of the formazan dye formed through cleavage of the WST-1 substrate using an ELISA reader. The respective WST-1 substrate incubation times varied depending upon the absolute extinction values measured (target range between 1 and 2.5, see above) and ranged between 45 min and 4 h depending for example on treatment intervals. The ID₅₀ value was determined as the inhibitory concentration of the respective compound which reduced the absorbance in the WST-1 assay by 50% compared with controls. For reversibility assays, cells were incubated with the respective compound at increasing concentrations for 48 h and then washed twice with PBS to remove the test agent. Cells were trypsinized and equal cell counts from each condition were replated onto 96-well plates in triplicates and incubated in standard culture medium supplemented with 10% FCS for 24 and 48 h. Cell proliferation was then determined using the WST-1 assay as described above.

Analysis of cell-cycle distribution after NSAID treatment
The cell-cycle distribution for control and drug-treated cells was determined after 48 h of treatment. Cells were seeded onto 6-well plates and allowed to adhere for 24 h. Cells were then incubated with the respective compound for 48 h and harvested by trypsinization. Nocodazole (Sigma Aldrich Chemie GmbH) was used to block cells in mitosis (400 nM concentration, 24 h). At the end of the treatment period cells were fixed in 100% methanol for 30 min at –20°C, centrifuged for 10 min at 2000 r.p.m., resuspended in 0.1% Triton-X 100 in PBS containing propidium iodide (40 µg/µl; Sigma Chemicals, Deisenhofen, Germany) and RNase (60 µg/µl; Sigma Chemicals) and incubated at 4°C for a minimum of 1 h. Subsequently, DNA content was measured using a flow cytometer (Beckman Coulter, Krefeld, Germany) and the cell-cycle distribution was calculated using the Phoenix Multicycle for Windows cell-cycle analysis software. A minimum of 10 000 events were measured for each sample.

Statistical analysis
For statistical analysis the two-tailed unpaired t-test was used to compare the effect of different mesalazine concentrations on cell proliferation and cell-cycle distribution. Statistical significance was assumed if the P value was <0.05.

Quantification and characterization of mitosis
Assessment of the mitotic index (MI).
To quantify the fraction of cells undergoing mitosis after NSAID treatment fluorescence microscopy was performed after staining DNA with bis-benzimide (Hoechst 33342, Sigma Aldrich Chemicals). Cells were treated with the respective compound for 48 h and the adherent and floating cells were harvested and pooled. Untreated control cells (culture media containing DMSO at equivalent concentrations to treatment conditions or mannitol at equivalent osmolar concentrations) were run in parallel. Cells were centrifuged onto glass slides in a cytospin centrifuge at 300 g for 10 min. Samples were fixed in 4% paraformaldehyde (PFA; Sigma) in PBS, pH 7.4, washed twice in PBS for 5 min each and then stained with Hoechst 33342 (100 µg/ml) for 15 min at room temperature. After washing once in PBS slides were mounted and cells visualized using a fluorescence microscope. At least 300 cells per experimental condition were counted by two independent observers. Cells with the typical morphological features of mitosis (condensed chromosomes, segregating chromosomes) were counted and the MI was calculated (number of mitotic cells per 100 cells counted, expressed in percent).

Characterization of mitosis.
To further characterize cells undergoing mitosis cells were double labeled with an anti-ß-tubulin antibody to visualize mitotic spindles combined with Hoechst 33342 to stain DNA. In brief, cytospins prepared as described above were fixed in 3% PFA in PBS, pH 7.4 for 10 min at room temperature, blocked in 1% bovine serum albumine (BSA) in PBS for 30 min at room temperature and incubated with the primary antibody for 1 h (MoAb anti-ß-tubulin, Sigma) at a dilution of 1:200 in 1% BSA–PBS. After three washes in PBS for 5 min each, the samples were incubated with secondary antibody (Donkey-anti-mouse-FITC conjugated, Jackson Immuno Res., West Grove, PA; diluted 1:200) for 30 min at room temperature. After three additional washes, cells were counterstained with Hoechst 33342 (100 µg/ml in PBS) for 15 min at room temperature, washed again and mounted with Vectashield mounting medium (Vector, Burlingame, CA). Cells were visualized using a fluorescence microscope (Leica, Germany).

Quantification and characterization of apoptosis
Assessment of the apoptotic index (AI).
Cells undergoing apoptosis were identified by typical morphology (nuclear and cytoplasmic shrinkage, condensation of chromatin, cytoplasmic blebbing) after staining DNA with Hoechst 33342 (100 µg/ml) and labeling DNA strand breaks using a terminal transferase dUTP nick end labeling assay (TUNEL assay; Roche Diagnostics GmbH). Cytospins were prepared as described above after incubation with the respective compound for 72 h and fixed for 1 h at room temperature in 4% PFA, pH 7.4, washed twice in PBS and permeabilized in 0.1% Triton-X 100 and 0.1% sodium citrate buffer for 2 min on ice. After two more washes in PBS, samples were incubated in 50 µl of TUNEL reaction mix (5 µl enzyme solution containing terminal deoxynucleotidyl transferase (TdT) from calf thymus in storage buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate) and 45 µl label solution containing FITC labeled dUTP nucleotides in reaction buffer (3 nM FITC–dUTP, 2 µl CoCl₂) for 1 h at 37°C in the dark. Omission of TdT provided the negative control for the assay, pre-incubation of cells with 10 µg/ml DNase I in 50 mM Tris–HCl, pH 7.4, 1 mM MgCl₂ and 1 mg/ml BSA for 10 min at room temperature to induce DNA strand breaks artificially served as a positive control. Cells were then washed twice in PBS and stained with Hoechst 33342 (Sigma) at 100 µg/ml for 15 min at room temperature in the dark. After two additional washes slides were mounted with mounting medium (Vectashield, Vector) and sealed with nail polish. Cells were then visualized under a fluorescence microscope. At least 500 cells/sample were evaluated by two independent observers. Apoptosis was assessed as the fraction of cells displaying morphological changes characteristic for apoptosis (nuclear and cytoplasmic shrinkage, condensation of chromatin, cytoplasmic blebbing) in combination with positive TUNEL labeling. Only cells displaying both features of apoptosis were counted as apoptotic. TUNEL positive cells without morphological characteristics of apoptosis were considered necrotic and omitted.

Detection of protein expression by western blotting
Cells were plated onto 10 cm dishes and incubated with the respective compound after 24 h. Adherent cells were lysed in buffer containing 25 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA and 1% NP-40 after an additional 48 h [note that after treatment with the respective compound for 48 h only a few cells (<5%) detached from the culture plate]. Protease inhibitor cocktail Type II (Roche Diagnostics GmbH) was added to lysates according to the manufacturer’s instructions and PMSF was added at a final concentration of 0.017 mg/ml. Lysates were resolved by SDS–PAGE and blotted onto polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were probed with primary antibodies at the following concentrations: monoclonal antibody (MoAb) anti-PARP, 1:500 (BD PharMingen, Heidelberg, Germany); MoAb anti-bak, 1:500 (BD PharMingen); MoAb anti-bax, 1:250 (BD Transduction Laboratories, Heidelberg, Germany); MoAb anti-p21 1:500 (BD PharMingen) and MoAb anti-ß-tubulin 1:1000 (Sigma). Blots were washed and probed with secondary antibody (anti-mouse-IgG-horseradish peroxidase conjugated, Santa Cruz, Heidelberg, Germany) and developed using the ECL chemoluminescence kit (Amersham, Piscataway, NJ).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Mesalazine inhibits proliferation of colon cancer cells
First, we evaluated the effect of mesalazine on the proliferation of colon cancer cells in vitro in direct comparison with other known chemopreventive NSAID. We chose the colon carcinoma cell line HT29 because it is among the most widely used cell lines for in vitro analyses of potential chemopreventive agents. HT29 cells were treated with increasing concentrations of mesalazine, sulindac sulfide, indomethacin (all non-selective COX inhibitors) or NS-398 (a selective COX-2 inhibitor) for 72 h and cell proliferation was measured with a colorimetric assay. In pilot experiments, this assay was validated by standard cell counting experiments (see Materials and methods). Mesalazine doses of 20 mM significantly reduced proliferation of HT29 cells compared with controls (P = 0.0081) whereas mesalazine concentrations of 30 mM reduced OD values of HT29 cells by 50% (ID₅₀) compared with untreated controls (Figure 1A). The ID₅₀ for indomethacin was 400 µM (Figure 1B), the ID₅₀ for sulindac sulfide was 80 µM (Figure 1C) and the ID₅₀ of NS-398 125 µM (Figure 1D). Similar molar concentrations of mannitol (control for osmolarity of solutions) and up to 0.4% of DMSO (solvent for sulindac sulfide, indomethacin and NS398) had no effect on the proliferation of HT29 cells (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1. (A–D) Mesalazine decreases proliferation of colon carcinoma cells in vitro. HT29 cells were treated with increasing concentrations of mesalazine (A), indomethacin (B), sulindac sulfide (C) or NS-398 (D) and cell proliferation was determined colorimetrically by WST-1 assay after 72 h. Cell proliferation was inhibited significantly at concentrations of 20 mM mesalazine (P = 0.0081) and most significantly at 40 mM (P < 0.0001). (E and F) The mesalazine effect is time-dependent and reversible. (E) HT29 cells were treated with increasing concentrations of mesalazine and cell proliferation was measured by WST-1 assay after 24, 48 and 72 h. Cell proliferation at 24 h was inhibited significantly by concentrations of 20 mM mesalazine (P = 0.0008) and was inhibited most significantly at 35 mM (P < 0.0001). (F) HT29 cells were treated with mesalazine at the indicated drug concentrations for 48 h. The drug was then removed and the cells replated at equal cell counts, re-fed with standard medium and allowed to grow for an additional 48 h. Proliferation was assessed by WST-1 assay. (Stars above bars indicate level of significance of the inhibition of cell proliferation at the respective mesalazine concentrations compared with controls. *P = 0.01–0.05, significant; **P = 0.001–0.01, very significant; ***P < 0.001, extremely significant.)

 
Next we characterized the time course of the mesalazine effect on cell proliferation. HT29 colon cancer cells were treated with increasing concentrations of mesalazine and proliferation was measured colorimetrically after 24, 48 and 72 h. As shown in Figure 1E, mesalazine inhibited cell proliferation of colon cancer cells in a time-dependent manner with a reduction in proliferation compared with controls detectable as early as 24 h after initiation of treatment. Inhibition of cell proliferation at 24 h was significant at a mesalazine concentration of 20 mM (P = 0.0008). In addition, mesalazine concentrations of 35 mM for 72 h lead to a reduction in OD values below starting OD values of untreated cells suggesting that an induction of cell death may contribute to the observed effect.

The effect of mesalazine on colon cancer cells is reversible
We then asked whether the effect of mesalazine on cell proliferation was reversible or whether cells were irreversibly damaged by the compound. Therefore, cells were treated with increasing concentrations of mesalazine for 48 h, then washed to remove the test compound and counted. Identical cell numbers from each sample condition were reseeded in 96-well plates, re-fed with standard cell culture medium and allowed to grow for an additional 48 h. As shown in Figure 1F, mesalazine-treated cells readily recovered upon removal of the drug, including those pre-treated with the highest concentrations of mesalazine (i.e. 40 mM), and subsequently proliferated almost as rapidly as untreated control cells.

Mesalazine treatment causes an accumulation of cells in G₂/M
The NSAID sulindac sulfide and indomethacin are known to accumulate cells in the G₁ phase of the cell cycle and to reduce the proportion of cells in S phase. To further explore the mechanism responsible for the antiproliferative effect of mesalazine its effect on the cell division cycle of HT29 cells was studied by flow cytometry. For comparison, the effect of indomethacin was assessed in parallel. The cell-cycle distribution of HT29 cells incubated with increasing concentrations of indomethacin and mesalazine is shown in Table I and illustrated in Figure 2. Indomethacin induced a non-linear progressive accumulation of cells in the G₀/G₁ phase and decreased the proportion of cells in the S and G₂/M phases of the cell cycle (Figure 2A and B and Table I), as has been reported previously (19). The effect of mesalazine differed markedly (Figure 2C). Mesalazine increased the fraction of cells in G₂/M compared with controls in a moderately dose-dependent manner and reduced cells in the G₁ phase of the cell cycle. The increase of cells in the G₂/M cell-cycle phase was significant compared with controls at a mesalazine concentration of 30 mM (P = 0.0329) and was most significant at 40 mM (P = 0.0004). Also, the increase in the fraction of G₂/M cells under 40 mM measlazine was significantly larger compared with 30 mM mesalazine (P = 0.0087) suggesting moderate dose-dependency.

View this table: [in this window] [in a new window]   Table I. Cell-cycle distribution of HT29 cells after treatment with mesalazine or indomethacin

 

View larger version (20K): [in this window] [in a new window]   Fig. 2. The effect of mesalazine on cell-cycle distribution differs from the effect of indomethacin. HT29 cells were treated with increasing concentrations of mesalazine or indomethacin and cell-cycle distribution was determined by flow cytometry after 48 h. The graphs show representative images of the cell-cycle distribution of controls (A), cells treated with 600 µM indomethacin (B) or 40 mM mesalazine (C). Quantification of cell-cycle distribution and statistical analysis is presented in Table I. Numbers represent means ± standard deviations of three independent assay samples (of a triplicate experiment). (D) The difference in cell-cycle distribution is reflected on a molecular level. HT29 cells were treated with increasing concentrations of mesalazine (0–40 mM) or indomethacin (0–600 µM) and protein levels of the cell-cycle inhibitor p21 were determined after 48 h by western blotting. Untreated control cell lysates were loaded in parallel (lanes 1 and 5). Probing for ß-tubulin served as loading control.

 
We were further interested whether the different effects of mesalazine versus indomethacin on cell-cycle distribution were reflected on a molecular level. To that aim, we investigated proteins implicated in cell-cycle regulation by western blotting. As shown in Figure 2D, indomethacin robustly induced expression of the cell-cycle inhibitor p21, a protein which is known to affect G₁ to S phase progression (20). In contrast, mesalazine had no effect on p21 expression.

Mesalazine specifically arrests cells in mitosis but does not influence spindle formation
The G₂/M cell-cycle fraction represents cells in G₂ as well as cells undergoing mitosis. Thus, mesalazine may block cells in G₂ or alternatively arrest cells during mitosis. To differentiate between the two, we used immunofluorescence microscopy of Hoechst-stained cells to study nuclear chromatin patterns and identify mitotic figures. The MI was calculated as the fraction of mitotic cells per 100 cells counted. The incubation of HT29 cells with 40 mM mesalazine for 48 h markedly increased the fraction of cells undergoing mitosis from 2% (range 0–3%) in controls to 25% (range 18–33%) in mesalazine-treated samples. Representative images of control and mesalazine-treated HT29 cells are shown in Figure 3A and B.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Mesalazine arrests cells in mitosis. Representative microscopic images of control and mesalazine-treated cells. Cells displaying the typical morphological features of mitosis are indicated by arrows. HT29 cells were treated with 40 mM mesalazine or treatment medium only for 48 h. Cells undergoing mitosis were visualized by fluorescence microscopy after chromatin stain with Hoechst 33342.

 
To test the specificity of the mitotic arrest induced by mesalazine we blocked HT29 cells in mitosis by the spindle toxin nocodazole. Cells blocked in mitosis by nocodazole and released into standard culture media rapidly entered G₁ and displayed a cell-cycle distribution similar to untreated cells as early as 24 h after nocodazole treatment (Figure 4A–D). In contrast, nocodazole-treated cells released into mesalazine-containing media remained largely arrested in mitosis throughout the observation period (Figure 4A,E,F,G). Thus, the strong shift in cell-cycle distribution of mesalazine is probably due to a specific block in mitosis.

View larger version (21K): [in this window] [in a new window]   Fig. 4. The mitotic arrest through mesalazine is specific. HT29 cells were arrested in mitosis with nocodazole for 24 h and subsequently released into control media (B–D) or 40 mM mesalazine containing media (E–G). Cell-cycle distribution was determined before (A) and after 2 (B and E), 6 (C and F) and 24 h (D and G). Images are representative of three independent experiments.

 
Nocodazole is known to block cells in the prophase of mitosis by strongly interfering with microtubule assembly. To further investigate whether mesalazine similarly affected the mitotic spindle apparatus or potentially the segregation of chromosomes we studied mesalazine treated cells by indirect immunostaining for ß-tubulin (mitotic spindle) in combination with chromatin staining to identify mitotic cells. Nocodazole treatment strongly blocked HT29 cells in the prophase of mitosis compared with untreated cells as visualized by Hoechst stain (Figure 5A and C). Nocodazole arrested cells did not stain for ß-tubulin indicating that microtubule polymerization was completely inhibited through nocodazole treatment as reported previously (Figure 5D). Mesalazine, in contrast, did not interfere with microtubule organization or spindle polarization in cells blocked in mitosis as visualized by immunofluorescence microscopy (Figure 5E and F). Mesalazine also did not arrest cells in a distinct phase of mitosis as cells in all stages of mitosis were detected after mesalazine treatment.

View larger version (93K): [in this window] [in a new window]   Fig. 5. Mesalazine does not affect spindle assembly or polarization. HT29 cells were treated with mesalazine (E and F) for 48 h and cells arrested in mitosis were evaluated by fluorescence microscopy. Untreated cells (A and B) and nocodazole-treated cells (C and D) served as negative and positive controls, respectively. Cells were simultaneously stained with Hoechst 33342 to visualize mitotic figures and by antibodies for ß-tubulin to visualize microtubules. In cells blocked in mitosis by nocodazole no intact microtubules were detected (C and D). In contrast, mesalazine-treated cells displayed undisturbed assembly of microtubules as well as normal polarization of the spindle apparatus in virtually all mitotic cells analyzed (E and F).

 
Mesalazine induces apoptosis in colon cancer cells
One of the key mechanisms of the chemopreventive effect of sulindac sulfide or indomethacin is thought to be a robust induction of apoptosis in colon epithelial cells in vitro and in vivo (19,21,22). Mesalazine has also been observed to increase cell death in preliminary studies in vivo (17,18). We therefore wished to determine the proapoptotic capacity of mesalazine in colon epithelial cells in direct comparison with other NSAID. We were also interested in the pathways leading to this effect. Cells undergoing apoptosis were identified morphologically and biochemically through simultaneous staining of DNA with Hoechst 33342 and detecting DNA strand breaks by TUNEL. Only cells displaying the typical morphological criteria of apoptosis and stained positive by TUNEL assay were counted as apoptotic. At least 500 cells/sample were evaluated by two independent investigators to determine the AI. As shown in Figure 6G, sulindac sulfide (at concentrations from 0 to 120 µM) or indomethacin (at concentrations from 0 to 600 µM) dose-dependently increased the fraction of cells undergoing apoptosis. Similarly, increasing concentrations of mesalazine (from 0 to 40 mM) also increased apoptosis, although the fraction of apoptotic cells never exceeded 10% of cells analyzed. Representative images for control cells and indomethacin-treated cells are shown in Figure 6A–D. The images of mesalazine-treated cells are shown in Figure 6E and F. Thus, mesalazine was less potent in inducing apoptosis compared with indomethacin or sulindac sulfide at respective growth inhibitory concentrations.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Mesalazine induces apoptosis in HT29 cells. HT29 cells were treated with indomethacin (C and D) or mesalazine (E and F) for 48 h and apoptosis was measured by fluorescence microscopy after double labeling chromatin with Hoechst 33342 (A,C,E) and DNA strand breaks by TUNEL assay (B,D,F). (A and B) Images of cells incubated with treatment medium only. Apoptotic cells are indicated by arrows, mitotic cells are indicated by arrow heads. (G) Graph showing quantification of apoptosis after treatment with the respective compounds at increasing concentrations. After treatment with mesalazine a smaller increase in apoptosis was observed compared with sulindac sulfide and indomethacin (numbers indicate mesalazine at millimolar concentrations and sulindac sulfide and indomethacin at micromolar concentrations).

 
Progammed cell death may be mediated through activation of death receptors (death receptor pathway) or through the release of cytochrome c from mitochondria (mitochondrial pathway) involving members of the bcl-2 family of proteins. Activation of effector caspases like caspase-3 is thought to be a highly specific key event during apoptosis responsible for the subsequent cleavage of the vast majority of polypeptides that undergo proteolysis in apoptotic cells. Also, activation of caspase-3 has been described previously for sulindac in colon cancer cells (23). We therefore evaluated a potential activation of caspases after treatment with mesalazine and other NSAID through western blotting for poly-ADP-ribosepolymerase (PARP) cleavage products. Cleavage products of PARP are commonly used as a marker for an activation of the effector caspases caspase-3 and 7. Treatment with indomethacin or sulindac sulfide caused strong cleavage of PARP suggesting a robust activation of caspases by these compounds. Mesalazine, similarly, also resulted in caspase activation although the effect was much less pronounced in comparison with treatment with indomethacin or sulindac sulfide (Figure 7A).

View larger version (38K): [in this window] [in a new window]   Fig. 7. (A) Mesalazine treatment is associated with the activation of caspases whereas levels of bcl-2 family members are unchanged. HT29 cells were incubated with increasing concentrations of mesalazine (0–40 mM), indomethacin (0–800 µM) or sulindac sulfide (0–160 µM) and protein levels were evaluated by western blotting for PARP after 48 h. Cleavage of PARP indicates activation of caspases. Mesalazine induced weak cleavage of PARP whereas the cleavage of PARP after indomethacin or sulindac sulfide was more robust. (B) Mesalazine treatment has no effect on expression of bcl-2 family proteins. HT29 cells were treated with increasing concentrations of mesalazine (0–40 mM), indomethacin (0–600 µM) or sulindac sulfide (0–120 µM) and levels of members of the bcl-2 protein family were determined by western blotting. ß-Tubulin served as loading control.

 
Apoptosis may in part be regulated by influencing the expression levels of various pro- and anti-apoptotic members of the bcl-2 family of proteins (mitochondrial pathway). We therefore determined the levels of proapoptotic bcl-2 family proteins after treatment with mesalazine, indomethacin or sulindac sulfide. As shown in Figure 7B, neither mesalazine nor indomethacin or sulindac markedly changed the levels of the apoptosis-associated proteins bak and bax. As reported before, we were unable to detect the anti-apoptotic protein bcl-2 in HT29 cells.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Here we show that the aminosalicylate mesalazine, a weak COX and lipoxygenase inhibitor well known for its low systemic toxicity, reduces cell growth of colon carcinoma cells in vitro. The anti-proliferative effect is presumably mediated through a specific accumulation of cells in mitosis. This represents a novel mechanism which has not been reported for other NSAID so far and which differs significantly from the G₁ arrest induced by well-established chemopreventive agents like sulindac sulfide or indomethacin. Mesalazine also increases apoptosis in HT29 cells—presumably through activation of caspases—although to a lesser extent than sulindac sulfide or indomethacin.

Mesalazine reversibly decreases cell proliferation in HT29 cells
Mesalazine significantly reduced cellular proliferation of HT29 cells in a dose- and time-dependent manner with an ID₅₀ of 30 mM at 72 h. Mesalazine is thus much less potent on a molar basis in reducing cell growth of HT29 cells than other known chemopreventive agents which we demonstrated directly by testing other compounds in parallel. The growth inhibitory concentrations (ID₅₀) we determined for HT29 cells using the known chemopreventive agents sulindac sulfide, indomethacin and NS-398 were in close range of those reported by others before [100 µM after 72 h for sulindac sulfide (24); 200 µM for indomethacin at 72 h (25) and 82 µM for NS-398 (26)]. Importantly, however, mesalazine concentrations which were effective in vitro closely correspond to concentrations of mesalazine achievable in the bowel in vivo under standard oral mesalazine treatment (27) and may even be increased after rectal administration of the compound as has been suggested by recent studies (28). Furthermore, the drug apparently did not irreversibly damage colon epithelial cells as the effect of mesalazine—even at highest concentrations used—was almost fully reversible. This may implicate that mesalazine acts through specific cellular mechanisms.

Mesalazine causes a mitotic block, which differs from the effect of other NSAID
Surprisingly, we found HT29 cells to be accumulated in the G₂/M phase after treatment with mesalazine. This differs markedly from the observations by us and others for indomethacin and sulindac sulfide both of which induce a robust G₁ arrest in various colon carcinoma cell lines (19,24,25,29) and Figure 2 presumably through the induction of the cell-cycle inhibitor p21 (29,30). The different effect of mesalazine on the cell cycle compared with established compounds seemed reflected on a molecular level since we observed robust p21 induction after indomethacin treatment whereas mesalazine had no influence on p21 levels.

We propose that the effect of mesalazine on growth of colon cancer cells is mainly due to a block in mitosis and may represent a specific anti-mitotic activity of mesalazine since cells released from a nocodazole block into mesalazine remained largely arrested in mitosis whereas control cells rapidly resumed progression throughout the cell cycle.

We presently do not know the underlying mechanisms responsible for the anti-mitotic activity of mesalazine. The precise regulation of mitosis is only beginning to be elucidated (31). Apparently, mitosis can only be completed with the mitotic spindle apparatus being intact. Some established anti-mitotic agents like nocodazole or members of the taxane or vincaalcaloid family act through targeting microtubule polymerization or depolymerization and thus interfere with normal spindle assembly (32). Mesalazine, in contrast, does not target the spindle apparatus in any obvious way. Cells in all phases of mitosis were detected by fluorescence microscopy and mitotic spindle formation was seemingly undisturbed. Certainly, more subtle effects on microtubule assembly and spindle orientation not assessable by immunofluorescence microscope cannot be excluded at present and need further investigation.

Mesalazine induces apoptosis in HT29 cells
The classic chemopreventive NSAID sulindac sulfide or indomethacin and the new selective COX-2 inhibitors have been found to affect net cell growth in part through inducing apoptosis in colon cancer cells in vitro (19,25,26), and possibly in vivo (21). Mesalazine also increased apoptosis in colon cancer cells albeit to a lesser extent than other compounds tested. It is notable that the extent of apoptosis we measured in HT29 cells for other compounds is very similar to results obtained by others (e.g. ref. 33), further indicating that our system is suitable to directly compare mesalazine effects with effects of established chemopreventive agents. Mesalazine seemed to act through mechanisms specific for apoptosis. Mesalazine like indomethacin or sulindac sulfide appeared to activate caspase-3, an enzyme that is known to be turned on early and specifically during apoptosis. We can presently not determine whether mesalazine acts through the death receptor pathway of apoptosis similar to various chemotherapeutic agents or whether it affects the mitochondrial pathway of apoptosis. Our finding, that expression of members of the bcl-2 family of proteins (as a means to regulate the mitochondrial pathway) remains unchanged after mesalazine treatment may suggest that additional pathways may be involved.

Mesalazine did not induce a subdiploid (sub-G₁) peak upon cell-cycle analysis—a method frequently used as an alternative marker for apoptosis induction—whereas sulindac sulfide and indomethacin did (data not shown). The occurrence of a subdiploid cell fraction correlates with the induction of the so-called ‘typical’ apoptosis pattern characteristic for drugs like sulindac sulfide. A so-called ‘atypical’ apoptosis pattern has been described to occur in cells treated with acetylsalicylate or aspirin a compound structurally related to mesalazine (24,25). Cells undergoing ‘atypical’ apoptosis lack the classical features of apoptosis such as an increase in the subdiploid fraction upon cell-cycle analysis or the occurrence of DNA laddering detected by gel electrophoresis. In contrast, ‘atypical’ apoptotic cells show specific DNA strand breaks detected by TUNEL in combination with classic morphological criteria of apoptosis (34), which we consistently saw after treatment with mesalazine.

Mesalazine as a candidate chemopreventive agent
Mesalazine may be an attractive candidate for chemoprevention of colorectal cancer particularly because there are strong limitations to the use of aspirin or sulindac as chemopreventive agents due to their frequent and often severe gastrointestinal and renal side effects (4,5). Although the introduction of selective COX-2 inhibitors for chemoprevention seems promising recent data suggest that these compounds retain the renal toxicity of classic NSAID (6). Furthermore, data on the long-term safety of this new class of drugs is still missing. Also, it is still unclear whether selectively targeting COX-2 isoforms is the best strategy for chemoprevention of colorectal cancer since not all colorectal tumors overexpress COX-2 (36). Furthermore, classic NSAID may act through COX-independent pathways (7). Through long standing experience with mesalazine in the treatment of patients with IBD we have learned that the drug is well tolerated with limited adverse effects and no gastrointestinal toxicity (11). Mesalazine acts predominantly in the intestine with little systemic availability (10). Mesalazine thus lacks the well-known toxicity of long-term NSAID use.

Most studies investigating the effect of mesalazine on colon epithelial cells have focused on its anti-inflammatory properties mainly because the drug is most recognized for its effect in the treatment of inflammatory bowel disease. Thereby, mesalazine has been shown to inhibit NFkB activation, a pathway, which is highly implicated in the responses of various cell types to inflammatory stimuli (13,14,37). Most of these studies investigated the effect of mesalazine in response to pro-inflammatory cytokines like TNF-alpha or interleukin-1 (13,14,38) or the cytotoxic agent peroxynitrite (39). The later study found a reduction in apoptosis of colon cancer cells after mesalazine treatment, which seemingly differs from our results. However, that study tested mesalazine after exogenous administration of peroxynitride, after a much shorter time interval and at lower concentrations than what we used in our study.

Much less is known about a potential growth inhibitory effect of mesalazine on colon epithelial cells. Data from epidemiological studies suggest that aminosalicylates like mesalazine may have chemopreventive properties in vivo (8,9) in patients with inflammatory bowel disease, findings, which may not be readily transferrable to all patients at risk for CRC. There is recent evidence, however, that aminosalicylates may also inhibit growth in non-inflamed colon mucosa in vivo. In two rodent models for colon carcinogenesis the aminosalycilate balsalazide effectively reduced tumor formation (15). Preliminary data suggest, that a decrease in proliferation and the induction of apoptosis may be detected in colon epithelial cells after administration of aminosalicylates although the underlying mechanism remains unclear (15,17,18). Here, we present experimental evidence that the aminosalicylate mesalazine decreases net cell growth of colon carcinoma cells in vitro predominantly through a specific arrest of colon cancer cells in mitosis, an effect that differs significantly from the effect of other NSAID in vitro. In addition, mesalazine mediates a moderate induction of apoptosis in a caspase-dependent manner with no apparent effect on mitochondria associated proteins of the bcl-2 family. An induction of apoptosis and inhibition of proliferation is widely recognized as a potential mechanism for chemoprevention of colorectal cancer. The finding that mesalazine inhibits growth in vitro may suggest a potential of the drug for the prevention of colorectal cancer in vivo.

We are presently conducting a multicenter placebo-controlled trial in patients with sporadic adenoma of the large bowel investigating whether long-term mesalazine treatment reduces the risk of adenoma recurrence after polypectomy as a means of primary prevention of CRC (German 5-ASA Polyp Prevention Study). This study should help to answer more conclusively whether mesalazine is effective in chemoprevention of CRC.

	Notes

 
¹ To whom correspondence should be addressed Email: wolff.schmiegel{at}ruhr-uni-bochum.de

	Acknowledgments

 
This study was supported by the Forschungsfoerderung der Ruhr-Universität Bochum (FoRUM), Germany. The authors would also like to thank Marc Zapatka for help with statistical analysis and members of the IMBL for lively discussion and support.

	References

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Received June 6, 2002; revised October 1, 2002; accepted November 11, 2002.

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