| BackgroundOral Submucous Fibrosis(OSF) is a kind of chronic insidious disease and it predisposes to cancer. The rate of OSF carcinogenesis reach up to 7.6%.Arecanut chewing is one of the primer atiological factors for OSF and carcinogenesis. Arecanut is indentified as first class carcinogen by IARC. Histological, the majority of OSF is characterized by epithelial atrophy and progressive accumulation of collagen fibers in the lamina propria while minority is characterized by epithelial atypical hyperplasia. In OSF carcinogenesis tissue epithelial atrophy and atypical could exist or coexist. There are abnormal protein expression which relate to cell cycle arrest and apotosis in OSF and carcinogenesis tissue. The role of arecoline in epithelial atrophy and epithelial hyperplasia and the mechanisms needs further investigation.ObjectFirst of all, human keratinocyte cells of the Hacat cell line and human embryo lung fibroblasts of the Hel cell line were treated with high doses of arecoline in a short time to observe survival rate, cycle and apotosis of the two cells.The molecular mechanism of epithelium atrophy induced by arecoline was then discussed. Secondly, the Hacat cells were treated with low doses of arecoline in a long time to observe the change of cell cycle and cell migration which relate to malignant transformation,which provided a theoretical basis for arecoline-induced OSF and its carcinogenesis.Methods1.Firstly, the cytotoxic effects stimulated by short-term high-dose arecoline on Hacat cells and Hel cells were studied. Then cell growth and proliferation were detected by MTT, cell cycle arrest and apotosis were examined by flow cytometry. Secondly, Western blot analysis and the reverse transcription-polymerase chain reaction technique were performed to study protein expression and gene transcription which relate to cell cycle. Apotosis protein and DNA fragmentation were detected by Western blot analysis and agarose gel electrophoresis, respectively. After that CyclinD1 was chosen as the delegate to report the effects of arecoline on CyclinD1 promoter activity through gene assay as well as its effects on CyclinD1 ubiquitination regulation through dual luciferase reporter assay and CO-IP assay2.Finally, the changes in the cycle and migration of epithelial cells after long-term and low doses of arecoline exposure were examined by means of molecular biological methods, which was related to malignant transformation and carcinogenesis.Cell growth and proliferation were analyzed by MTT assays. Dual luciferase reporter assay and Western blot dectected promoter activity and protein expression of CyclinD1, respectively.MMP9 protein expression was detected by Western blot. Transwell migration experiment were performed to test whether the low concentrations of arecoline promoted the migration of Hacat cells. Transcription factors of increased MMP-9 expression induced by arecoline were detected by RNA interference test.Results1.The results of MTT showed that short-term(24h) high-dose (over 50μg/ml)arecoline inhibited Hacat cells growth obviously (P<0.05) but exerted little effect on Hel(over 100μg/ml), and long-term(2m,3m,4m, 5m) low-dose(20μg/ml) arecoline promoted Hacat cell growth (P<0.05).2. Flow cytometry revealed that short-term (12h)high-dose arecoline(75, 100,125μg/ml)arrested Hacat cells at G1/S phase, but had no effects on Hel. Hacat cells apoptosis induced by arecoline was dose-and time-dependent.3.Western blot analysis and RT-PCR revealed that short-term(24h) high-does(0,25,50,75,100,125μg/ml) arecoline declined protein expression and gene transcripition of CyclinD1,CDK4 which related to early-G1 and CDK2,E2F1 which related to late-G1,but induced CyclinA,CyclinE which related to late-Gl,while had no obvious effects on CyclinB/CDC2 related to G2/M.4.The results of dual luciferase reporter assay showed that the promoter activity of CyclinDl was inhibited remarkably after short-term(24h) and high doses(50,75,100,125μg/ml) of arecoline exposure (P<0.05), but it was enhanced gradually after long-term(4m, 5m)and low doses(20μg/ml) of arecoline exposure (P<0.05)5.CO—immunoprecipitation assay confirmed that the CyclinD1 protein ubiquitination was induced in Hacat cells after short-term(24h)and high doses(0,25,50,75,100,125μg/ml) of arecoline exposure, and arecoline could promote CyclinD1 degradation via ubiquitination.6.Western blot analysis indicated that arecoline(75,100,125μg/ml) could promote proapoptotic-Bax and inhibit the expression of Bcl-2 at 24h. Arecoline(25,50,75,100,125/ml)could activate caspase-3,8,9 and induce apoptosis at 24h7.Western blot analysis revealed that arecoline(50,75,100,125μg/ml) could raise the expression of NF-κB,AP-1,Ets-1 and MMP-9 in Hacat cells at 24h. As detected by RNAi test,arecoline(50μg/ml)affected MMP-9 by transcription factors at 24h, such as NF-κB,AP-land Ets-1,and especially NF-κB.8.Transwell migration experiment indicated that arecoline could promote the migration of Hacat cells after long-term(2m,3m,4m,5m)and low doses(20μg/ml) of exposure.Conclusion1.Short-term high-dose arecoline exerted little effect on Hel, but inhibited Hacat cells growth obviously and arrested Hacat cells at G1/S phase, which was mainly through declined protein expression and gene transcripition of CyclinD1,CDK4,CDK2,E2Fl,but have no obvious effects on CyclinB/CDC2 which related to G2/M. On the other hand, short-term high doses of arecoline inhibited the CyclinD1 promoter activity and promoted its protein ubiquitination. Short-term and high doses arecoline also promoted the protein expression and activation of caspase-3,8,9 and induced Hacat cells apoptosis. So, epithelial atrophy in OSF is mainly attibuted to cell cycle arrest and imbalance of proliferation and apoptosis induced by arecoline.2. Arecoline can speed up Hacat cell cycle progress through up-regulate CyclinD1 promoter activity gradually after long-term and low doses of exposure. Arecoline up-regulate MMP-9 mainly by NF-κB transcription in a dose- and time-dependent manner, and enhance the migration of Hacat cell. The maliqnancy proficiency of Hacat such as cell proliferation, invasion and migration induced by long-term and low doses of arecoline exposure may cause the epithelial atypical hyperplasia and carcinogenesis in OSF...
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