Fibrates Affect Levels of Phosphorylated p38 in Intestinal Cells in a Differentiation-Dependent Manner

Abstract

Fibrates are widely used hypolipidaemic agents that act as ligands of the peroxisome proliferator-activated receptor α (PPARα). p38 is a protein kinase that is mainly activated by environmental and genotoxic stress. We investigated the effect of the PPARα activators fenofibrate and WY-14643 and the PPARα inhibitor GW6471 on the levels of activated p38 (p-p38) in the colorectal cancer cell lines HT-29 and Caco2 in relation to their differentiation status. Fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased. HT-29 cells showed p-p38 predominantly in the cytoplasm, whereas Caco2 cells showed higher nuclear positivity. The effect of fibrates may depend on the differentiation status of the cell, as differentiated HT-29 and undifferentiated Caco2 cells share similar characteristics in terms of villin, CYP2J2, and soluble epoxide hydrolase (sEH) expression. In human colorectal carcinoma, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium, p-p38 showed nuclear positivity. The decrease in p-p38 positivity was associated with a decrease in sEH, consistent with in vitro results. In conclusion, fibrates affect the level of p-p38, but its exact role in the process of carcinogenesis remains unclear and further research is needed in this area.

Keywords: colorectal carcinoma; epoxyeicosatrienoic acids; fibrates; p38 MAPK; peroxisome proliferator-activated receptor α.

Figure 1

Changes in expression levels of phosphorylated p38 (p-p38) after treatment with fenofibrate, WY-14643, and GW6471. Parts (A,C,E,G) Relative expression of p-p38 compared to control was measured by In-Cell ELISA. Results are expressed as mean ± SD (n = 8). The red dotted lines represent control cells (100%). Statistically significant results compared to control cells are marked with (*): * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Parts (B,D,F,H) Subcellular localisation of p-p38 after fenofibrate, WY-14643 and GW6471 treatment obtained by immunocytochemical staining. p-p38 was evaluated separately in the nucleus and cytoplasm of the cells. The results are presented as histoscores. Cells were counted in 5 different fields of vision (magnification ×200). Microphotographs show control cells and cells treated with 150 μM (HT-29) or 200 μM (Caco2) fenofibrate, 200 μM WY-14643 and 10 μM GW6471. All images are at the same magnification (200×).

Figure 2

Comparison of undifferentiated and differentiated HT-29 and Caco2 cells. The relative expression of villin, p-p38, CYP2J2 and sEH was measured by in-cell ELISA. Results are presented as mean ± SD (n = 8). The expression of the proteins of interest in HT-29 cells was used as a control (100%). Statistically significant results compared to control cells are indicated with (*): * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. Note comparable levels of p-p38 and sEH, increased levels of villin and decreased levels of CYP2J2 in undifferentiated Caco2 cells compared to differentiated HT-29, suggesting that undifferentiated Caco2 are more similar to differentiated HT-29 than undifferentiated HT-29 (red rectangles).

Figure 3

Immunohistochemical staining for p-p38, CYP2J2, and sEH in normal and colorectal carcinoma samples. Representative micrographs of grade 2 tumours and corresponding normal tissue samples were obtained from the same patient (male, 55 years old). Magnification ×100, white line represents 100 μm. Graphs show immunohistochemical staining intensities (n = 18): columns show medians, dots show individual samples. Immunostaining intensities were scored semiquantitatively as: 0 negative staining, 1 weak staining, 2 moderate staining, 3 strong staining. Immunostaining for p-p38 was scored separately for nucleus and cytoplasm, CYP2J2 and sEH were scored in cytoplasm. Comparison of staining intensity between normal and carcinoma tissues was performed by the Wilcoxon signed rank test at a significance level of p ≤ 0.05. Statistically significant results are marked with *, p-values are given directly in the graphs, ns—nonsignificant result.