2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38687Oral 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 CA IX and CA XII expression in OSF and oral cancer. Carbonic anhydrase (CA, 4.2.1.1), a family of zinc metalloenzymes, can efficiently catalyze the reversible processes of hydration-dehydration of CO2 and HCO3-. Over the past decades, extensive efforts has been made to find the role of CAs in tumor progression, either as frequently investigated in variety of tumor cells and cell lines, but it has been difficult to find a clear-cut relationship between the expression of CA isozymes in normal and malignant cell. However, little evidence of a direct relationship between OSF, oral cancer and CAs expression. It appears that only the recently characterized isoform CAIX (MN protein) and CAXII are exceptional, as it expression can be associated with tumorgenesis. Thus, in this study, we propose to study the expression of CA IX and CA XII in human normal buccal mucosal and oral submucous fibrosis and oral cancer. With a few established techniques, including zymography, western blotting, RT-PCR and EMSA for MMPs, PAI-1 and Ca IX and CA XII, our Lab is very capable of performing studies on genes relevant to ECM-degradation, such as MMPs, TIMPs, and PAI-1 to clarify their involvements in the pathogenesis of OSF and oral cancer. Therefore, in the first year of this study, we propose to (1) analyze the activity of CA IX and CA XII from cell extracts from OSF and normal buccal mucosa fibroblast via western blotting assay. (2) analyze the mRNA levels of Ca IX and CA XII from OSF and normal buccal mucosa fibroblast via RT-PCR assay. (3) Using immunohistochemistry to investigate the expression of CA IX and CA XII from OSF and normal buccal mucosa. (4) To verify whether arecoline or areca nut extracts could affect CA IX and CA XII production by human buccal mucosa fibroblasts used western blots and RT-PCR. (5)further investigate the signal pathways for regulated arecoline and areca nut extracts affect CA IX and CA XII production by human buccal mucosa fibroblasts, including MAPK family, PI3K/Akt pathway, and finially (6) further investigate the regulation of transcription factors via western blotting and EMSA assay to determine the transactivity of these transcription factors. In the second year of this study, we propose to (1) examine the total CA activity in 100 paired human surgical tumor/adjacent normal specimens of oral cancer; (2) examine each 100 paired oral cancer specimens with the tumor progression or tumor stages related to CA IX and CA XII expression. (3) analyze the mRNA levels of CA IX and CA XII from 100 paired human surgical tumor/adjacent normal specimens of oral cancer via RT-PCR assay. (4) Using immunohistochemistry to investigate the expression of CA IX and CA XII of 100 paired human surgical tumor/adjacent normal specimens of oral cancer. In the third year of this study, we propose to (1) analyze the effect of CA inhibitors (acetazolamide 及sulfonamides) on viability and invasion of oral cancer cell SCC-4. (2) investigate the mechanism involved in reduced invasiveness by CA inhibitors, including changed adhesion capability and invasion ability and ECM degraded proteinase (MMPs, u-PA) activities. (3) analyze the effect of CA IX and CA XII RNAi on viability, invasion and ECM degraded proteinase (MMPs, u-PA) activities of oral cancer cell SCC-4. (4) further investigate the signal pathways (MAPK family ERK 1/2、p38 and JNK 1/2、phosphatidylinositol 3-kinase （PI3K）/Akt pathway) involved in reduced invasiveness by CA inhibitors. (5) investigate the regulation of transcription factors(CREB, AP-2, SP-1, NF-κB, c-Jun, c-Fos, SP-1 and Ets-1) via western blotting and EMSA assay to determine the transactivity of these transcription factors. (6) via an animal model, we propose to determine the effect of CA inhibitors (acetazolamide and sulfonamides) on metastasis of oral cancer cell inoculated mice.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後大約在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 anhydrase; CA; EC4.2.1.1.) 是一種 zinc metalloenzyme，其 分子量約為30 KDa 左右，主要是催化CO2+ H2O 􀀧 HCO3 -+ H+這一個可逆性的水解反 應。而CAs 雖然與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。而本實驗室之前發現口腔黏膜 下纖維化症病人的纖維母細胞較人類正常頰黏膜組織有著大量PAI-1 及HIF-1α 的表 現。而又有許多的文獻發現 HIF-1α會調控著CA IX 與CA XII 的表現。因此本計畫擬 探討碳酸酐異構酶 IX, XII 在口腔黏膜下纖維化及口腔癌的表現及其相關訊息傳遞。 直至目前，本實驗室已經建立了MMPs 之 zymography 技術，TIMPs、PAI-1 及CA IX, CA XII 之 Western blotting 技術，CA 活性的測定方式以及 RT-PCR、EMSA 等技 術，以從事相關口腔黏膜下纖維化及口腔癌相關基因的探討。因此，本計畫擬於第一 年進行【1】利用Western blotting 分析人類正常頰黏膜及口腔黏膜下纖維化所培養的纖 維母細胞其CA IX 及 CA XII 的蛋白表現。並利用酵素呈色方式分析其total CA 的活 性表現。【2】利用RT-PCR 分析人類正常頰黏膜及口腔黏膜下纖維化纖維母細胞其CA IX 及CA XII 的mRNA 基因層次表現。【3】利用immunohistochemistry 分析人類正常 頰黏膜及口腔黏膜下纖維化組織切片上CA IX 及CA XII 的差異性變化。【4】在人類 正常頰黏膜纖維母細胞中，分別加入arecoline 及 areca nut extracts，利用Western blotting 及RT-PCR 的技術分析 arecoline 及areca nut extracts 對人類正常頰黏膜纖維母 細胞中CA IX 及CA XII 的表現為何? 【5】進一步利用訊息傳遞路徑的抑制劑探討 arecoline 及areca nut extracts 對誘導人類正常頰黏膜纖維母細胞中CA IX 及CA XII 的 相關訊息傳遞路徑及其蛋白變化，如MAPK family、PI3K/Akt pathway。【6】以EMSA 的分析方式探討arecoline 及areca nut extracts 是否影響CA IX 及CA XII 的相關轉錄因 子表現或與其target binding sequence 結合能力。本計畫擬於第二年進行【1】擬收集100 位口腔癌病人的手術切除組織。以酵素呈色方式分析口腔癌病人其手術切除癌組織及 其週遭較正常組織中total CA 的活性表現。並進一步以病患同意授權之資料，對口腔 癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【2】擬收集100 位口 腔癌病人的手術切除組織。以Western blotting 方式分析口腔癌病人其手術切除癌組織 及其週遭較正常組織中CA IX 及 CA XII 的蛋白表現。並對口腔癌病人的發生原因、 轉移與否及一些臨床症狀做進一步的分析。【3】擬收集100 位口腔癌病人的手術切除 組織。以其手術切除癌組織及其週遭較正常組織中，取出組織並以RT-PCR 及即時 RT-PCR (Real time-RT PCR)分析其組織中CA IX 及CA XII 的mRNA level 的表現。並 對口腔癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【4】擬收集100 位口腔癌病人的手術切除組織檢體，製作成病理切片。利用以immunohistochemistry (免 疫組織染色方式)分析口腔癌病人其切片組織中CA IX 及 CA XII 的蛋白表現。並對口 腔癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。本計畫擬於第三年 進行【1】以CAs inhibitors (acetazolamide 及sulfonamides)處理人類口腔癌細胞株SCC-4 以改變細胞株CAs 活性，並觀察CA IX 及CA XII 的表現情形，更進一步利用migration/ invasion assay、adhesion assay 探討口腔癌細胞株在處理CA inhibitors 的細胞其移動、 侵襲和轉移能力之變化情形。【2】以CAs inhibitors (acetazolamide 及sulfonamides)處理 人類口腔癌細胞株SCC-4 以改變細胞株CAs 活性，進一步利用zymography、Western blotting 及 RT-PCR 等技術，探討口腔癌細胞株在處理CA inhibitors 的情況下，其細胞 分泌基質分解相關蛋白酶，如 MMP-2、MMP-9、u-PA、t-PA 和基質分解相關蛋白酶 抑制劑，如TIMP-1、TIMP-2、PAI-1 及PAI-2 之變化情形。【3】構築CA IX 及CA XII 的RNAi vectors 以liposome 或adenovirus 將RNAi 殖入口腔癌細胞中以改變細胞株中 CAs 活性，進一步觀察處理CA IX 及CA XII 的RNAi vectors 的情況下， 其口腔癌細 胞移動、侵襲和轉移能力之變化情形，及細胞分泌基質分解相關蛋白酶，如 MMP-2、 MMP-9、u-PA、t-PA 和基質分解相關蛋白酶抑制劑，如TIMP-1、TIMP-2、PAI-1 及PAI-2 之變化情形。【4】進一步觀察CA inhibitors 及RNAi 系統改變口腔癌細胞分泌ECM 分 解酵素和ECM 分解酵素抑制劑的機制分析，如影響侵襲能力的基質金屬蛋白水解酶活 性有關的訊息傳遞MAPK family ERK 1/2、p38 及JNK 1/2、phosphatidylinositol 3-kinase （PI3K）/Akt pathway。【5】以electrophoretic mobility shift assay（EMSA）、Western blotting 探討CA inhibitors 及RNAi 系統是否抑制上述基因的相關轉錄因子表現，或與其target binding sequence 的結合能力，如：MMP-2 的轉錄因子CREB、AP-2、SP-1，MMP-9 的轉錄因子AP-1、NF-κB、c-Jun、c-Fos、SP-1 及u-PA 的轉錄因子Ets-1、NF-κB 等。 【6】選用CAs inhibitors (acetazolamide 及sulfonamides)，以動物模式，分析其對於接 種口腔癌細胞株（SCC-4）的老鼠其腫瘤大小、重量及轉移至肺部的能力，以評估應用 的可行性。Project