p-Hydroxylcinnamaldehyde induces the differentiation of oesophageal carcinoma cells via the cAMP-RhoA-MAPK signalling pathway

Abstract

p-Hydroxylcinnamaldehyde (CMSP) has been identified as an inhibitor of the growth of various cancer cells. However, its function in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. The aim of the present study was to characterize the differentiation effects of CMSP, as well as its mechanism in the differentiation of ESCC Kyse30 and TE-13 cells. The function of CMSP in the viability, colony formation, migration and invasion of Kyse30 and TE-13 cells was determined by MTS, colony-formation, wound healing and transwell assays. Western blotting and pull-down assays were used to investigate the effect of CMSP on the expression level of malignant markers of ESCC, as well as the activity of MAPKs, RhoA and GTP-RhoA in Kyse30 and TE-13 cells. We found that CMSP could inhibit proliferation and migration and induce Kyse30 and TE-13 cell differentiation, characterized by dendrite-like outgrowth, decreased expression of tumour-associated antigens, as well as the decreased expression of malignant markers. Furthermore, increased cAMP, p-P38 and decreased activities of ERK, JNK and GTP-RhoA, were detected after treatment with CMSP. These results indicated that CMSP induced the differentiation of Kyse30 and TE-13 cells through mediating the cAMP-RhoA-MAPK axis, which might provide new potential strategies for ESCC treatment.

Figure 1. Chemical structure of CMSP and its inhibitory effect on ESCC cells.

(A) Structure of CMSP. (B,C) The inhibition effects of CMSP isolated from CMS on the number and morphology of ESCC and normal oesophageal cells were determined. The data presented are means ± SD from at least three independent experiments (×100). **P < 0.01, compared with the control group.

Figure 2. Effects of CMSP on the viability and morphology change in Kyse30 and TE-13 cells.

(A) The effect of 10–40 μg/ml CMSP on the viability of Kyse30 and TE-13 cells was detected by the MTS assay. The data presented are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group. (B) Morphology change and differentiated cell rate caused by CMSP or ATRA, as determined by Giemsa staining. Representative histograms as the percentage of dendrite-like cells in CMSP-treated Kyse30 and TE-13 cells (×100). The data presented are means ± SD from at least three independent experiments. **P < 0.01, compared with the control group. (C) The morphology change in Kyse30 and TE-13 cells was investigated by scanning electron microscopy.

Figure 3. Effects of CMSP on tumour-related antigen secretion and malignant marker expression of Kyse30 and TE-13 cells caused by CMSP.

The content of CEA, SCC, IL-6 and MIC-1 in the supernatants of Kyse30 and TE-13 cells was determined using ELISA. The data presented are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group.

Figure 4. Effects of CMSP on the colony formation, migration and invasion of Kyse30 and TE-13 cells.

(A) Significant inhibition of cell colony formation was observed upon treatment with CMSP (20 μg/ml) for 10 days. The number of colonies was calculated and plotted on the histogram (n = 3). **P < 0.01, compared with the control group. (B) Effect of CMSP on the migration and invasion ability following exposure to different concentrations (10, 20 or 40 μg/ml) for 24 h was investigated by the transwell assay. The number of migrated and invaded cells was calculated (n = 3). **P < 0.01, compared with the control group. (C) Effect of CMSP on the mobility ability of Kyse30 and TE-13 cells after exposure for 48 h was investigated by the wound healing assay. The wounds were photographed, and the wound closure percentage from a representative experiment (n = 3) was temporally measured using Axio Vison software. **P < 0.01, compared with the control group. (D) The protein levels of the EMT-related proteins MMP2, E-cadherin N-cadherin, and vimentin, as measured by western blotting. β-Actin served as a loading control. The data presented are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group.

Figure 5. Effects of CMSP on cAMP production, RhoA and GTP-RhoA expression and the phosphorylation of MAPKs in Kyse30 and TE-13 cells.

(A) The content of cAMP in the supernatants of Kyse30 and TE-13 cells treated with CMSP was assayed by ELISA. The data presented are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group. (A) Kyse30 and TE-13 cells were treated with 20 μg/ml CMSP for 30, 60 and 120 min. The GTP-RhoA, phosphorylated ERK, p38, and JNK kinase proteins in the cell lysate were assayed by western blotting. β-Actin was used as an internal control. The data presented are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, compared with the control group.

Figure 6. Inhibition of Kyse30 cell growth in vivo by treatment with CMSP.

(A) CMSP treatment inhibited tumour growth of Kyse30 cells in vivo. Left: Representative images of tumours formed in nude mice. Right: Tumour growth curves of xenograft tumours and measurement of the tumour weight (n = 6 for each group). (B) Histological analysis of the tumour tissues of mice in all of the groups. Representative H&E images are shown (×400). (C) Representative immunohistochemical images showing the expression levels of C-myc and N-myc in the tumour tissues of both groups (×400). (D) The content of CEA, SCC, IL-6 and MIC-1 in the serum of nude mice treated with CMSP was assayed by ELISA. The data presented are means ± SD from at least three independent experiments. **P < 0.01, compared with the control group. (E) Histological analysis of sections from the spleen and livers of mice treated with CMSP or untreated mice. Representative H&E images are shown (×400).