Oral submucous fibrosis (OSF) affects an estimated 2.5 million people, mostly in the Indian subcontinent. Limitation of oral opening resulting in difficulty in eating is the main presenting feature. Although nutritional deficiencies and immunological processes may play a part in the pathogenesis, the available epidemiological evidence indicates that chewing betel quid (containing areca nut, tobacco, slaked lime or other species) is an important risk factor for OSF. Genetically determined susceptibility could explain why only a small fraction of those using betel quid develop the disease. In OSF there is an incidence of oral cancer of 7.6 per cent for a median 10-year follow-up period. Risk markers for malignant transformation in OSF include epithelial dysplasia, silver binding nucleolar organizer region counts, and sister-chromatid exchange frequencies; p53 tumour suppressor gene mutations may be involved in these potentially malignant changes.

Oral submucous fibrosis―a review

Background IARC has classified the betel nut as a human environmental carcinogen. Previous studies have found that arecoline (AR) is the major alkaloid present in the saliva of betel quid chewers. Saliva contains a large content of AR which has been further shown to cause mutation of oral mucosa cells, resulting in oral cancer. Whereas, to date, there are only few studies reported the hepatotoxicity associated with arecoline and betel nut chewing. Therefore, the main purpose of this study was to determine the toxic effects of AR and its oxidative metabolite, arecoline N-oxide (ARNO), in normal liver cell lines. Methods The cytotoxic, genotoxic, and mutagenic effects were detected by crystal violet staining, alkaline comet assay, and Salmonella mutagenicity test, respectively. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2-DCFDA assay. Results Our results demonstrated that ARNO exerted higher cytotoxicity, DNA damage, and mutagenicity than its parent compound arecoline in liver cells. Antioxidants, such as N-acetylcysteine, Trolox, and penicillamine, strongly protected liver cells from ARNO-induced DNA damage and ROS production. Furthermore, co-treatment with Mito-TEMPO also effectively blocked ARNO-induced ROS production in liver cells. Besides antioxidants, co-treatment with 1-aminobenzotriazole and methimazole nearly completely suppressed ARNO-induced ROS production in liver cells. Conclusions Our data suggest that arecoline ingested from the habit of chewing betel quid can be primarily oxidized to ARNO, thereby enhancing its toxicity through increased ROS production. Considering the excellent protective effects of both mitochondria-targeted antioxidant and CYP450 inhibitor on ARNO-induced ROS production in liver cells, mitochondria CYP450-mediated metabolism of ARNO may be a key mechanism. Collectively, our results provide novel cellular evidence for the positive connection between habitual betel quid chewing and the risk for liver damage.

CYP450-mediated mitochondrial ROS production involved in arecoline N -oxide-induced oxidative damage in liver cell lines

Total glucosides of paeony (TGP) inhibits the production of inflammatory cytokines in oral lichen planus by suppressing the NF-κB signaling pathway.

Protective effect of Shaoyao Decoction against colorectal cancer via the Keap1-Nrf2-ARE signaling pathway.

Oral submucous fibrosis (OSF) induced by chewing of the areca nut has been considered to be a precancerous lesion with a high probability of developing oral squamous cell carcinoma. Tanshinone (TSN) is the main component extracted from Salvia miltiorrhiza, a traditional Chinese medicine, which was found to have diverse pharmacological effects, such as anti-inflammatory and antitumor. In the current study, we aimed to identify the inhibitory effects and the underlying mechanism of TSN on OSF progress. We found that treatment with TSN inhibited the arecoline-mediated proliferation of primary human oral mucosal fibroblasts and reversed the promotive effects of arecoline on the EMT process. By RNA deep sequencing, we screened two possible targets for TSN: LSD1 and p53. We confirmed that p53 is much lower in OSF than in normal mucous tissues. In addition, p53 and its downstream molecules were decreased by arecoline treatment in oral mucosal fibroblasts, which was reversed by treatment with TSN in a dose-dependent manner. Our results also revealed that arecoline stimulation resulted in hypermethylation of the promoter of TP53 and subsequent downregulation of p53 levels, which was reversed by TSN. Furthermore, we identified that LSD1 could epigenetically activate TP53 by recruiting H3K27me1 and H3K4m2 to its promoter. Our findings provide new insights into the mechanism by which TSN influences arecoline-induced OSF and rationale for the development of clinical intervention strategies for OSF and even oral squamous cell carcinoma.

Tanshinone Suppresses Arecoline-Induced Epithelial-Mesenchymal Transition in Oral Submucous Fibrosis by Epigenetically Reactivating the p53 Pathway.

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as a significant etiological factor for disease manifestation. However, the underlying molecular mechanisms regarding areca nut chewing-induced OSF are only partially understood. Herein, we reported that arecoline markedly induced morphologic change in HaCaT epithelial cells, but had no obvious effect on Hel fibroblast cells. MTS assay revealed that arecoline significantly suppressed HaCaT cell viability. Moreover, flow cytometric analysis indicated that arecoline substantially promoted HaCaT cell, but not Hel cell apoptosis in a dose-dependent manner. Furthermore, arecoline-induced HaCaT cell apoptosis was found to be associated with increased expression and activation of cleaved-Bid, cleaved-PARA and cleaved-caspase-3. Collectively, our results suggest that HaCaT epithelial cells are more sensitive than Hel fibroblast cells to arecoline-induced cytotoxicity, which may be involved in the pathogenesis of OSF.

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Ji-Jia Li

Papers

Arecoline inhibits epithelial cell viability by upregulating the apoptosis pathway: implication for oral submucous fibrosis.