| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 97 === In Taiwan, there are approximately 2 million people having betel chewing habit. The stadarized mortality of oral caner is increasing from 1998 to 2007 while the standardized incidence of oral cancer of Taiwanese male shows a highly climbing rate from 1979 to 2004, and the oral cancer of male is 4th or 5th in the rank of top ten malignant tumors. Numerous epidemiological studies have demostrated that betel chewing is the major risk of oral precancerous lesions, oral precancerous conditions, and oral cacner, especially subject is in lon-term exposure or high-dosage consumption. By the year 2004, the IARC has declared that chewing of betel quid, by itself, to be a Group 1 carcinogen and the areca nut to be, correspondingly, a Group 1 carcinogen.
However，there is no sufficient evidence in the constituents of areca nut for tumorgenesis or carcinogenesis in human. The major alkoid, arecoline, not only show cytotoxicity in oral cell, but also has cell transformation and genotoxicity in experimental studies. This may imply that arecoline might be a carcinogen, but so far no evidence of arecoline-like DNA adducts is found. In this study, therefore, we use the technologies of transciptome which is based on toxicology as a strategy to screen the susceptibility genes of arecoline-induced cytotoxicity or genotoxicity on normal oral cells, including xenobiotic biotransformation (metabolism and detoxification), oncogene and turmor suppressor gene, stress response and inflammation, and TGF-β signaling related genes. The cytotoxicity of cell morphology and gene expression level was observed on human gingival fibroblasts-1 (HGF-1) treated with 100 μg/mL of arecoline in 24 hour. Subsequently, the dye-swap cDNA microarray and real-time polymerase chain reaction were performed to screen 13 differentially expressed genes, including 5 upregulated genes (PTGS2, HSPA1, DNAJA1, DDIT4, and GDF15) and 7 downregulated genes (CHAF1A, CHAF1B, FANCG, BRCA1, S100A12, CYP26B1, and GSS). We also observed the gene expression changes of these candidate gens were increased in a dose-dependent manner in a dose range of 50-150μg/mL of arecoline on a primary cultured HGF, and this may prove that these were actually susceptibility to cytotoxic effects of arecoline. Based on toxicology, we construct a putative pathway of above findings to explore the potential roles of these candidate genes on the development of betel-related oral lesions.
In epidemiological surveys, we note that some subjects with substance use of alcohol drinking, betel chewing and cigarette smoking (ABC) did not develop toward oral precancerous lesions, oral precancerous conditions, or oral cacner. We therefore speculate that individual genome may affect the result due to diverse genetic variations on the special susceptibility genes. In this part, we estimated the risk of cyclooxygenase-2 (COX-2/PTGS2) promoter polymorphisms and substance use of ABC by a gene-enviroment interaction model in a case-control study. The findings showed that the COX-2 1195A allele was positively associated with the risk of oral cancer, and subject with COX-2 -1195A/A has a minor risk (OR=1.55) to be an oral cancer. The 1195 A/A homozygote presents a stronger risk of oral cacner than A/G and G/G genotypes, especially when joined with the habit of betel chewing (aOR=79). Furthermore, in betel nonchewers, the strongest joint effect is also seen in A/A homozygote carriers who have a combined use of alcohol drinking and cigarette smoking (aOR=32). Intriguingly, the 1195A allele may create a c-myb binding site on COX-2 promoter region and the 1195A allele reveals a higher promoter activity and COX-2 mRNA expression, compared with the 1195G allele. Nevertheless, whether the c-myb will also be induced by areca nut, or higher expression level of c-myb will be observed in tumor site of oral caner, it need further studies to ascertain this puzzle.
Due to the previous finding on the arecoline-induced mRNA expression of cyclooxygenase-2 (COX-2) in human gingival fibroblasts, we further investigated the mRNA expression level of COX-2 and its upstream effectors in three oral epithelial carcinoma cell lines (KB, SAS, and Ca9-22) by areca nut extrat (ANE) and saliva-reacted ANE. This is not only to explore the difference of cytotoxicity on inflammation between ANE and sANE, but also to ascertain the possible upstream signaling pathway to COX-2 induction. The finding showed a higher expression level of inflammation-related genes on sANE treatement than ANE. Also, it is suggested that COX-2 induction might be mostly elicited from alternative NF-κB activation than classical. In addition, both extracts can induce higher overall inflammation on KB cells (COX-2 -1195 A/A) than Ca9-22 cells (G/G) and SAS cells (A/G). Unexpectedly, we can not conclude the A/A effects on mRNA expression level of clinical oral tumor tissue. Although it indicated that COX-2 is up-regulated in malignant tissues, the accumulative dosage and combination of substance use and tumor histologic grade lead to much more complexity on elucidation of COX-2 -1195 variants into the role of inflammatory mechanisms.
Based on both in vitro experiments and population association study, we hope to find candidate pathogenic genes associated with betel-related oral lesions and/or oral cancer. Also, in clinical practice, these candidate makers need to be validated outright, and they may be useful in diagnosing the ouccurence of oral cancer in the furthure.
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