| Abstract: | 口腔黏膜下纖維化症(oral submucous fibrosis; OSF)為一種口腔癌前期病變，主要 與檳榔的嚼食習慣有關，而口腔黏膜下纖維化症大約在3到16年間就有可能會發展成口 腔癌。而造成口腔黏膜下纖維化症及口腔癌的主要原因之ㄧ是細胞外基質中膠原蛋白異 常的堆積及分解所導致的。基質金屬蛋白水解酶 (matrix metalloproteinases; MMPs)及纖 維蛋白溶解系統(plasminogen activator (PA)/plasmin system)在細胞外基質的組成及分解 過程中扮演很重要的角色。而本實驗室一直致力於結締組織內膠原蛋白相關的分解基因 如MMPs 及其抑制劑 TIMPs 及胞漿素原活化劑的抑制劑(PAIs)與口腔相關疾病的研 究，尤其是在口腔黏膜下纖維化症與口腔癌的致病機轉。 碳酸酐異構酶 (carbonic anhydrases; CAs;)是一種 zinc metalloenzyme，主要與pH值 的調節作用有關，可是近年來有越來越多文獻發現CA isoform尤其是CA IX 及CA XII 與癌症的發展有密切的相關性。而本實驗室在之前的國科會計畫中，也發現位於細胞膜 上的碳酸酐異構酶 IX及XII (CA IX, CA XII)，與口腔黏膜下纖維化症及口腔癌的致病機 轉扮演著很重要的角色，目前已有兩篇相關論文發表在PLoS ONE 及Oral Oncology 期 刊中。但是除了位於細胞膜上的CA IX及CA XII之外，本實驗室之前也發現位於細胞質 的CA I、CA II及CA III與非小細胞肺癌、肝癌都有相關性。但是，細胞質的CA I、CA II 及CA III與口腔癌的相關性，除了本實驗室最近利用tissue array探討CA I及C AII與口腔 癌組織的相關性之外(已發表在Journal of Oral Pathology Medicine)，細胞質的CA I、CA II 及CA III在細胞層次或CAIII與口腔癌檢體或口腔黏膜下纖維化的相關性卻完全無相關 文獻探討。因此，本計劃擬探討細胞質中碳酸酐異構酶 CA I, II及III在口腔黏膜下纖維 化及口腔癌的表現。目前本實驗室積極建立CA III大量表現的穩定口腔癌細胞株及CA III 的siRNA系統，而我們在CA III大量表現的口腔癌細胞株(SCC-9)中，發現其轉移能力有 明顯的促進效果。而我們更進一步利用real-time PCR方式篩選與轉移相關的MMPs家 族，發現在CA III大量表現的口腔癌細胞株中，其MMP-2基因則有明顯的促進效果。綜 合以上結果，我們初步發現CA III在口腔癌細胞株的轉移過程扮演著重要的角色。因此， 這促使我們想要利用口腔癌檢體組織及口腔癌細胞實驗來進一步釐清CA III所扮演的角 色。因此，本計劃擬於第一年利用已建立好的CA III stable clones 口腔癌細胞株，除了 觀察其促進口腔癌細胞轉移的詳細機制之外，我們擬進一步觀察其細胞移動、侵襲、貼 附和轉移能力及基質分解相關蛋白酶活性的影響之分析，並以動物模式，分析其對於接 種不同的人類口腔癌細胞株，如SCC-9、SAS、HSC-3、OECM-1的裸鼠其腫瘤大小、重 量及轉移至肺部的能力，以評估應用的可行性。而第二年我們擬應用到檢體方面，先觀 察細胞質的CA I、CA II及CA III在人類正常頰黏膜及口腔黏膜下纖維化症的差異性表 現。我們擬進一步利用arecoline及檳榔萃取物探討其對人類正常頰黏膜纖維母細胞其CA I、CA II及CA III的影響機制及其相關的訊息傳遞路徑。而我們也擬於第三年收集較多的 人類口腔癌檢體及口腔黏膜下纖維化的檢體，分析其細胞質的CA I、CA II及CA III的表 現量，更進一步分析其表現量與口腔癌及口腔黏膜下纖維化的發生原因，並與口腔癌的 轉移與否及一些臨床數據做進一步的分析。
Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). Thus, matrix metalloproteinases (MMPs) and plasminogen activator (PA)/plasmin system play an important role in the pathogenesis of OSF. In our present studying, we emphasize the proteinase and inhibitor expression in OSF and oral cancer. Carbonic anhydrases (CAs), zinc metalloenzymes, mainly involve in the regulation of pH level. However, a lot of recently published studies have revealed that certain CA isoforms, especially CA IX and CA XII, closely implicate in the cancer development. Results from our prior NSC projects also revealed that CA IX and CA XII, located on cellular membrane, played a vital role in the pathogenesis of OSF and oral carcinogenesis. Two papers written based on these results have been published in PLoS ONE and Oral Oncology. In addition, we also discovered that cytosolic CA I, CA II and CA III are related to non-small cell lung cancer and hepatocellular carcinoma. With tissue array analysis, the relationship between CAI and CAII was studied and published in Journal of Oral Pathology Medicine. However, the involvement, cellular or tissue levels, of cytosolic CA I, CA II, and CA III in oral carcinogenesis or OSF have not been studied by us or others. Therefore, the aim of this proposal is to investigate the expression levels of cytosolic CA I, CAII, and CA III. Based on the high metastasis potential of SCC-9 with a high expression level of CA III, oral cancer cell lines with a stable CA III expression or siRNA for CA III will be established to study the effect of CA III on metastasis. Furthermore, real time-PCR has been conducted to screen metastasis-related MMPs to reveal that MMP-2 may enhance the metastasis potential of oral cancer cells with high level of CA III. Thus, in this study, we propose to study the expression of cytosolic CA I, II and III complex in human OSF and oral cancer. Therefore, in the first year of this study, we propose to analyze the effect of CA III RNAi system and CA III overexpression system and on viability, invasion and ECM degraded proteinase activities of oral cancer cell. Furthermore, to investigate the signal pathways and MMP-2 involved in induced invasiveness by CA III overexpression system. We also propose to determine the effect of CA III RNAi and overexpression system on metastasis of oral cancer cell inoculated mice via an animal model. In the second year of this study, we propose to analyze the expressions of cytosolic CA I, II and III from cell extracts from OSF and normal buccal mucosa fibroblast. Furthermore, to verify whether arecoline or areca nut extracts could affect cytosolic CA I, II and III production by human buccal mucosa fibroblasts and further investigate the signal pathways for regulated arecoline and areca nut extracts affect cytosolic CA I, II and III production. In the third year of this study, we propose to examine mRNA and protein expression of cytosolic CA I, II and III in 100 OSF patients and 100 paired human surgical tumor/adjacent normal specimens of oral cancer. To further study the relationship between the tumor progression or tumor stages and cytosolic CA I, II and III expression. |
