Upregulated expression of MMP-9 in gingival epithelial cells induced by prolonged stimulation with arecoline

Abstract

Abstract. Betel quid chewing is implicated in the high prevalence of oral cancer in Southeast Asian countries. One of the major components of betel quid is arecoline. In the present study, in order to characterize the association between chronic arecoline stimulation and carcinogenesis the expression level of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA in human gingival epithelial progenitor cells (HGEPs) stimulated with arecoline was assessed. The HGEPs were alternated between 3 days of incubation with arecoline (50 µg/ml), and 3 days without arecoline, for up to 30 days. The expression levels of the MMPs and TIMPs in the cells stimulated with arecoline were evaluated by reverse transcription-quantitative polymerase chain reaction at 18 and 30 days. The expression of MMP-9 mRNA in the experimental group was significantly increased compared with in the control group (P<0.01). No significant differences in the expression of MMP-2, TIMP-1 or TIMP-2 mRNA were observed between the experimental and control groups. Using an MMP-9 activity assay, the levels of MMP-9 activity in the experimental group were demonstrated to be significantly higher than in the control group (P<0.05). To investigate associated cellular signaling pathways, PDTC [a nuclear factor (NF)-κB/inhibitor of NF-κB (IκB) inhibitor], PD98059 [a mitogen-activated protein kinase kinase (MAPKK)1 and MAPKK2 inhibitor], SB203580 (a p38 MAPK inhibitor) and 5,15-DPP [a signal transduction and activator of transcription (STAT) 3 inhibitor] were used. All inhibitors decreased the extent of MMP-9 upregulation induced by stimulation with arecoline. Based on the data, it is hypothesized that MMP-9 activity may be involved in the pathological alterations of oral epithelium induced by betel quid chewing, and that the NF-κB/IκB, MAPK, p38 MAPK and STAT3 signaling pathways may be involved in the production of MMP-9 induced by betel quid chewing.

DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing—a possible predictive biomarker for malignant transformation

“…Therefore, we employed an in vitro model of a daily BQ chewing habit that we showed previously to contain hypermethylated genes [23]. The cells were stimulated with arecoline, a major component of BQ, for a prolonged period according to our previous protocol [24]. We confirmed that significantly high level of methylation of SIRT1 was observed followed by downregulated expression of SIRT1 transcription and protein expression.…”

“…Following overnight incubation, the HGEPs were treated with arecoline at a concentration of 50 μg/mL. The concentration of arecoline used in this study was as described in previous experiments [24]. Briefly, arecoline at the concentration of 50 μg/mL had no cytotoxic effect on the cells stimulated, even for a prolonged period, the method of alternating between 3 days with 50 μg/mL of arecoline and 3 days without arecoline for 1 month was selected.…”

Network‑based identification of signature genes KLF6 and SPOCK1 associated with oral submucous fibrosis

“…It binds to the promoter region of MMP-9 (matrix metalloproteinase-9) and inhibits its expression in endothelial cells (61). Expression and activity levels of MMP-9 was significantly upregulated in primary keratinocytes derived from healthy gingival epithelium treated with arecoline (62). MMP-9 also shows positive stromal expression in OSF cases (63).…”

Sirtuin 1 and oral cancer (Review)

“…Recently, our group evaluated a significant association between arecoline and the expression of SIRT1 in oral epithelial cells. Arecoline, the major alkaloid in betel quid, has been reported to be involved in the pathogenesis of oral cancer by facilitating the cellular transformation and transcriptional repression of tumour suppressor genes (TSGs) (10,45,46). We discovered that arecoline significantly induced DNA hypermethylation, followed by downregulation of SIRT1 expression in oral epithelial cells.…”

“…Reactive oxygen species (ROSs) are the strongest risk factor associated with the pathogenesis of oral cancer (10). Habits such as betel quid chewing have been reported to induce generation of ROSs in the oral epithelium and lead to genetic instability by damaging DNA and other macromolecules (10,45). SIRT1 has been shown to increase the synthesis of antioxidant enzymes such as glutathione and superoxide dismutase 2 and prevent ROS-mediated genomic alterations (73).…”