Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1

Abstract

Mesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APC(min) polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APC(min) polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action.

Fig. 1

Differential regulation of genes by 5-ASA. Two cell lines (HCT116, HT29) treated with 20 mM 5-ASA (8, 24 h) were used in the analysis. The graphs represent expression of genes representative of the three major pathways obtained after microarray data analyses as described in methods. (a) Wnt/β-catenin (b) MAPK signaling (c) Cell adhesion. (d) Validation of some targets by Western blot. 5-ASA inhibited PAK1 that mediates all three pathways. Pan-actin was used as a loading control.

Fig. 2

5-ASA increases cell adhesion. (a) Adhesion of HCT116 and HT29 upon 5-ASA treatment (20 mM; 24 h). The graph is representative of one of the three independent experiments, ±SD of quadruplicate samples. Statistical analysis revealed that 5-ASA increases cell adhesion in a dose-dependent manner (p = 0.005 for the linear contrast). This effect was more pronounced in HCT116 cells. (b) Electric cell substrate impedance sensing (ECIS) assay to monitor cell adhesion upon 5-ASA treatment. The graph shows an increase in impedance upon 5-ASA treatment. The cell attachment data was collected using AC current at 4000 Hz.

Fig. 3

5-ASA recruits E-cadherin and β-catenin to the cell membrane. (a) Western blot analyses of E-cadherin and β-catenin in total (RIPA) or membrane fractions of 5-ASA treated cells. The data is representative of three independent experiments (b) Immunofluorescent detection of E-cadherin expression. Arrows indicate low levels of membranous E-cadherin in untreated cells. (c) Immunofluorescent detection of β-catenin. Arrows indicate AJs. Cells were grown on coverslips and treated with or without 5-ASA (20 mM) for 24 h. An increase in intercellular membranous E-cadherin and β-catenin was observed upon 5-ASA treatment. The experiment was performed three times. (d) Protein profiles of cytoplasmic and nuclear β-catenin and phospho-β-catenin in cells treated with 5-ASA for indicated time intervals. (e) 5-ASA inhibits β-catenin signaling in CRC cells. Luciferase reporter assay was performed in cells transfected with the TCF reporter pTOPFLASH or pFOPFLASH and co-transfected with pCMV-Renilla luciferase. Following treatment with 5-ASA for 8 h and 24 h, luciferase was measured using a luminometer. All samples were taken in quadruplicates and independently repeated 2 times (p < 0.05). (f) CHIP assay showing downregulation of transcriptional targets of β-catenin on 5-ASA treatment. Error bars represent SEM.

Fig. 4

PAK1 mediates 5-ASA effects. (a) Western blot showing inhibition of PAK1by 5-ASA in cytoplasmic and nuclear fractions. Fibrillarin and tubulin were used as control for purity of cellular fractions. The blot represents one of the three independent experiments (b) Western blot to analyze PAK1 knockdown using siRNA. PAK1 depletion shows decreased level of PAK1 protein upon increasing siRNA in CRC cells. (c) The effect of PAK1 depletion (by siRNA) on cell adhesion. Linear trend analysis by ANOVA revealed a significant dose-dependent effect on cell adhesion upon silencing PAK1 (p = 0.032) which was more pronounced in HCT116 cells. (d) The effect of IPA3 on cell adhesion. A dose dependent increase in cell adhesion was observed upon pharmacological inhibition of PAK1 kinase activity. Linear trend analysis by ANOVA showed statistically significant dose dependence (p < 0.001) (e) PAK1 depletion increases membranous localization of E-cadherin and β-catenin. HCT116 cells stained with anti-E-cadherin or β-catenin were analyzed by confocal microscopy. The experiment was repeated three times.

Fig. 5

Effect of 5-ASA in vivo. (a) Intestinal polyps per mouse in untreated and 5-ASA treated APC^min mice. The total number of polyps significantly decreased upon treatment with 5-ASA for 12 weeks. For statistical analysis the nonparametric Mann–Whitney and Wilcoxon test was performed to compare untreated vs 5-ASA treated group (b) PAK1 expression was elevated in APC^min polyps (left) and 5-ASA activity effectively reduced its expression (right). (c) Immunoreactivity score (IRS) of PAK1 expression in APC^min polyps. Linear contrast analysis by ANOVA confirmed that 5-ASA treatment significantly reduced PAK1 expression (p = 0.015). This trend was most pronounced in small polyps (p = 0.029) (d) Number of APC^min polyps separated for size. There was a significant reduction in large polyps upon 5-ASA treatment. (e) Nuclear c-myc expression in polyps from untreated and 5-ASA treated APC^min mice. Treatment with 5-ASA siginficantly decreased nuclear c-myc staining within the polyps (p < 0.01). (f)Single polyp, represented by a single dot in the plot (n = 16), were scored for nuclear c-myc expression within the polyp (0, no staining; 1, low staining intenstiy; 2, medium staining instensity). Representative pictures were imaged at 200× magnification. (g) In APC^min polyps, E-cadherin was heterogeneously expressed in the epithelium that was distorted in the architecture (left). 5-ASA treatment was effective in restoring homogeneity of E-cadherin expression as well as its membranous localization. This was also reflected in re-organization of epithelial architecture (right). Image magnifications 100×.