摘要:流行病學研究指出,臺灣地區口腔癌發生與嚼檳榔有密切關係。根據衛生署癌症登記報告指出,口腔癌在臺灣男性十大癌症中死亡率與發生率皆位居第五位,口腔癌年增率也是臺灣地區年增率最高之癌症。可見問題之嚴重。雖然檳榔致癌的細胞學及分子生物學證據日漸充足,但檳榔致癌機制仍未詳細闡明。我們最近以Microarray 分析的方法,比較人類正常牙齦上皮細胞株SG細胞,口腔癌TW2.6細胞株,經檳榔鹼arecoline 處理以及未處理之間基因表現的差異。 再經西方墨點法分析,確認 arecoline 處理可使SOX9的量增加。 SOX-9屬於SOX(SRY-related high mobility group box)基因superfamily的一員。參與諸如軟骨生成、胚胎性別分化、呼吸上皮發育、黑素細胞分化等多種胚胎發育過程。由於SOX9參與許多發育過程,近年有研究發現SOX9與惡性腫瘤發生有關。又由於arecoline可誘導 SOX-9的產生, 因此我們先利用免疫組織染色方法,探討SOX-9於口腔癌、口腔上皮變異(OED)及正常口腔黏膜(NOM)中的表現。結果發現SOX9在NOM的平均labeling index(LI) 為4%,輕度、中度、重度OED平均LI分別為9%、18%、29%,口腔癌組平均LI為 48%,癌化過程中,SOX-9的表現有增加的情形,差異並達統計意義(P < 0.001) 。SOX-9的表現且和腫瘤大小、淋巴結轉移與否、癌症臨床分期有統計相關。經過 Cox regression model 進行多變數分析以後, SOX-9 LI為影響存活時間的獨立因子(p <0.05 )。已知嚼檳榔與口腔癌發生有密切關係, 但其致癌機轉並未完全明暸。我們初步研究成果發現檳榔鹼arecoline可以誘導口腔上皮細胞 SOX9 的產生,口腔癌檢體中有SOX9 的過度表現,且為影響存活時間的獨立因子。SOX9 在人類癌症致癌機轉僅有三篇零星報告,並未完全明暸。且它在不同的組織中,它調控的下游標的(target) 基因並不相同,都還有待進一步研究。 SOX9在口腔癌扮演的角色,還有其致癌機轉,至今仍未見任何文獻報告。檳榔鹼arecoline透過何種機轉誘導口腔上皮細胞產生SOX9目前也並不清楚。所以,我們提出本三年計劃擬於三年內逐步探討SOX-9在口腔癌的致癌機轉及arecoline誘導口腔上皮細胞產生SOX-9的 基因調控。 SOX-9 在口腔癌扮演致癌的角色。 為達到此目標,本計劃包含以下數個研究主題:我們第一年擬 1. 選殖人類SOX9基因 2. 將 SOX9 轉殖到 SOX9 表現量較低的口腔癌細胞株TW2.6中,選取兩株 SOX9 蛋白表現量高於控制組的細胞株將其命名 TW2.6/ SOX9#1,SOX9#2。3. 由於先導實驗發現SOX-9的表現和腫瘤大小、淋巴結轉移與否、癌症臨床分期有統計相關。 因此我們擬先於 In Vitro 比較TW2.6/ SOX9#1,SOX9#2 和控制組細胞株其生長速率、移動能力、侵襲能力的改變, 然後以動物實驗觀察比較其生長速率、促血管新生能力及存活時間的改變。 4.先前肺腺癌研究發現SOX-9表現細胞株的生長速率比對照組快,但其作用機轉僅知增加CDK4 的表現及減少CDK4抑制者P21cip1。 我們擬進一步比較觀察TW2.6/ SOX9#1 ,SOX9#2 和控制組細胞株細胞形態, 細胞週期, 細胞週期蛋白, 細胞週期相關蛋白(Aurora-B, INCENP, DNA-PK, pericentrin, Eg5, NuPARP) 的不同。 5. 測試SOX9的大量表現是否可減輕藥物(如cisplatin, 5-FU, taxol)所引起的細胞凋亡。以確定SOX-9的表現是否會改變口腔癌細胞抗細胞凋亡的能力,而與其致癌力,抗藥性的獲得及惡性度的改變有關??。6. 以Microarray Analysis的方法,比較TW2.6/Neo和TW2.6/ SOX9#1 、TW2.6/ SOX9#2基因表現的差異。本計劃第二年擬以 shRNA 技術刪除SOX9表現量較高的SAS口腔癌細胞株SOX9表現,先於 In Vitro比較SAS-AS-SOX9#1,SAS-AS-SOX9#2和控制組SAS/Puro 之間生長速率、移動能力、侵襲能力的改變, 然後以動物實驗觀察比較接種刪除SOX9的SAS xenograft 和控制組在SCID 小鼠體內腫瘤的體積大小、促血管新生能力及存活時間的改變。2. 擬比較觀察SAS-AS-SOX9#1, SAS-AS-SOX9#2和控制組細胞株細胞形態, 細胞週期, 細胞週期蛋白, 細胞週期相關蛋白(Aurora-B, INCENP, DNA-PK, pericentrin, Eg5, NuPARP) 的不同。3. 擬以微陣列分析的方法,比較控制組和刪除SOX9的SAS細胞株基因表現的差異。將這個結果和第一年過度表現微陣列分析結果比較。挑選具有相同差異未被報告的基因幾個,做進一步測試。 4. 觀察刪除SOX9是否可增加藥物所引起的細胞凋亡,以tunnel assay分析SAS-AS- SOX-9#1, SAS-AS-SOX-9#2組的腫瘤是否凋亡細胞較多? 未來或許可提供口腔癌病人新的治療方向,提高口腔癌病人對抗癌藥的敏感度。由於我們初步發現SOX9 表現類似於癌基因,本計劃第三年延續第二年計劃擬繼續探討 SOX9在口腔癌進展過程所扮演的角色。 擬繼續進一步研究第二年以微陣列分析的方法挑選出來SOX9調控的基因,並探究其可能調控機轉。並擬探討檳榔鹼arecoline誘導口腔上皮細胞產生SOX9的基因調控機轉。 1.我們擬以人類正常牙齦上皮細胞株SG細胞及口腔癌TW2.6探討arecoline 對 SOX9 基因調控的機轉。首先由於 arecoline是muscarinic cholinergic receptor之agonist,因此我們擬加入Carbachol (muscarinic receptors agonist) ,Atropine (muscarinic receptor antagonist) ,4-DAMP (M3 antagonist) ,Gallamine (M1 antagonist) ,Tropicamide (M4 antagonist)。 探討 arecoline 是否經由muscarinic receptor 誘導口腔上皮細胞產生SOX9。 2. arecoline誘導許多反應都和活性氧有關。 因此我們擬加入細胞內各種產生活性氧路徑的抑制劑,例如: ketoconazole, ETYA, DPI, L-NAME, baicalein, MK886, PD146176, ibuprofen 探討arecoline 是否經由產生活性氧誘導細胞產生SOX9及產生活性氧的路徑。 3. 擬加入細胞內p38 MAP kinase, JNK, ERK, NF-κB,PI3K等等的抑制劑,探討arecoline 是否經由上述路徑誘導細胞產生SOX9。 4. 擬加入細胞內Rac1,Rho-associated protein kinases,geranylgeranyltransferase,farnesyl transferase 抑制劑,探討arecoline 是否經由Rho GTPase路徑誘導細胞產生SOX9。5. 以得知的誘導路徑尋找可抑制arecoline誘導細胞產生SOX-9的化學預防藥物。
Abstract: In Taiwan, oral cancer ranks as the seventh most prevalent cancer in both sexes and account for the fourth most common cancers in men in 2008. The regular use of areca nut (AN, Areca catechu) preparations has long been associated with the high incidence of oral submucous fibrosis and oral cancer in south-central Asia (including Taiwan). Although advanced surgical techniques and new anticancer drugs are developed constantly, the overall postoperative survival of oral cancer patients did not improve much. Arecoline, a major areca nut alkaloid, was found to have genotoxic and cytotoxic effects in various human cells. However, the molecular mechanisms involved in the AN-induced oral cancer remain largely unknown. A full understanding of the mechanism underlying AN-induced oral cancer may help to design a more effective strategy to treat oral cancer and prolong life.SOX9 is an important transcription factor required for development and has been implicated in several types of cancer. However, SOX9 has never been linked to oral cancer to date. We previously used immunohistochemical staining to examine the SOX9 expression in oral squamous cell carcinoma (OSCC), specimens of oral epithelial dysplasia (OED), and normal oral mucosa (NOM). The labeling indices of SOX-9 significantly increased from NOM (4%), through mild dysplasia (9%), moderate dysplasia (18%), and severe dysplasia (29%) to OSCC (48%) (p<0.001). The expression of SOX-9 was correlated with tumor size, lymph node status and clinical stage (p<0.05). Univariate analysis showed that tumor size, lymph node status, clinical stage and SOX-9 LI are related to the survival time (p<0.05). Multivariate analysis demonstrated that lymph node metastasis, clinical stage and SOX-9 LI were independent predictors for the patients’ survival (p<0.05). We also found that treatment of normal gingival keratinocyte SG cells with arecoline induced increase in SOX9 expression.Here, we propose a novel mechanism for the development of areca nut-induced oral cancer in Taiwan.The specific aims for the proposed three-year study are:(1) To examine whether SOX9 exhibits the hallmarks of an oncogene in oral cancer .(2) To explore whether overexpressed SOX9 cell has the ability to promote cancer metastasis both in vitro and in vivo.(3) To explore whether overexpressed SOX9 cell has the ability to resist anti-cancer drug induced apoptosis both in vitro and in vivo.(4) To explore the genes involves in cell cycle, apoptosis, angiogenesis and metastasis that are regulated by SOX9.(5) To explore the signaling pathways of arecoline-induced SOX9 expression in oral keratinocytes.(6) To explore the chemotherapeutic or chemopreventive agents that can inhibit the SOX9 overexpression in oral keratinocytes and oral cancer cells.Significance:Finding the SOX9 and its down-stream proteins that participate in the proliferation, apoptosis, angiogenesis and metastasis of oral cancer will help us to design new drugs for molecular target therapy and may apply to the clinical treatment in oral cancer in the future.