Abstract
A previous study showed that a downexpression of protease-activated receptor 4 (PAR4) is associated with the development of esophageal squamous cell carcinoma (ESCC). In this study, we explored the relationship between PAR4 activation and the expression of p16, and elucidated the underlying mechanisms in PAR4 inducing the tumor suppressor role in ESCC. ESCC cell lines (EC109 and TE-1) were treated with PAR4-activating peptide (PAR4-AP). Immunohistochemistry for DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) was performed in 26 cases of ESCC tissues. We found that DNMT1 and HDAC2 immunoreactivities in ESCC were significantly higher than those in adjacent noncancerous tissues. PAR4 activation could suppress DNMT1 and HDAC2, as well as increase p16 expressions, whereas silencing PAR4 dramatically increased HDAC2 and DNMT1, as well as reduced p16 expressions. Importantly, the chromatin immunoprecipitation-PCR (ChIP-PCR) data indicated that treatment of ESCC cells with PAR4-AP remarkably suppressed DNMT1 and HDAC2 enrichments on the p16 promoter. Furthermore, we demonstrated that activation of PAR4 resulted in an increase of p38/ERK phosphorylation and activators for p38/ERK enhanced the effect of PAR4 activation on HDAC2, DNMT1, and p16 expressions, whereas p38/ERK inhibitors reversed these effects. Moreover, we found that activation of PAR4 in ESCC cells significantly inhibited cell proliferation and induced apoptosis. These findings suggest that PAR4 plays a potential tumor suppressor role in ESCC cells and represents a potential therapeutic target of this disease.
Highlights
Protease-activated receptors (PARs), a superfamily of Gprotein-coupled receptors that are activated by thrombin, have been perceived in multiple cells affiliated with inflammatory reactions, such as macrophages, neutrophils, and mast cells [1]
DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) were mainly in nuclear staining in esophageal squamous cell carcinoma (ESCC) tissues and adjacent nontumorous tissues. Levels of both protein immunoreactivities in ESCC tissues were significantly higher than those in adjacent nontumorous tissues (Figure 1(a)). These suggest that interaction between DNMT1 and HDAC2 might be involved in ESCC carcinogenesis [20]
PD98059 or SB203580, inhibitors for ERK1/2 and p38, partially or completely blocked the increase of p16 protein expression, which in turn markedly reversed the downexpression of DNMT1 and HDAC2 proteins, compared with protease-activated receptor 4 (PAR4)-APonly groups (Figure 2(d)). These results indicated that the effect of PAR4-activating peptide (PAR4-AP) on DNMT1, HDAC2, and p16 expression is associated with MAPK signal pathways [22]
Summary
Protease-activated receptors (PARs), a superfamily of Gprotein-coupled receptors that are activated by thrombin, have been perceived in multiple cells affiliated with inflammatory reactions, such as macrophages, neutrophils, and mast cells [1]. The recent detection of PARs on various cancer cells suggests that PARs might be involved in inflammation, and in the development of cancers [2]. Several studies suggest that PARs play roles in cancer progression including tumor growth, invasion, migration, survival, and metastasis [3, 4]. Studies investigating the role of PAR4 in cancer have had conflicting results, as they were found to be overexpressed in several malignant tumors and implicated in tumor growth and cancer metastasis [4,5,6]. Studies demonstrated that mice with knockdown PAR4 gene could accelerate tumor growth [10] and reduce cardiomyocyte apoptosis [11]. The role of PAR4 in the progress of ESCC has not been defined
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