- 學位論文
異甘草素藉由抑制ATM訊息途徑誘導口腔鱗狀細胞癌細胞週期停滯與細胞凋亡
Isoliquiritigenin induced cell cycle arrest and apoptosis by ATM signaling pathway suppression in oral squamous cell carcinoma
摘要
異甘草素(Isoliquiritigenin，ISL)。萃取自甘草，屬黃酮類化合物，具有抗病毒、抗氧化、抗發炎、抗潰瘍和抗腫瘤的活性。異甘草素已被應用在前列腺癌和乳腺癌等研究上，但在口腔癌的研究上仍不廣泛。使用三株口腔鱗癌狀細胞oral squamous cell carcinoma (OSCC) ：HSC-3、SAS、OECM-1，兩株正常人類口腔細胞口腔纖維母細胞(oral fibroblast，OF)、口腔牙齦上皮細胞，(SG)，以MTT [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide]測試細胞存活率，細胞週期和細胞凋亡比例由流式細胞儀(flow cytometry)分析，以RT-PCR和西方墨點法偵測Ataxia-telangiectasia mutated (ATM)之mRNA和蛋白質表現量，藉由Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay)與phospho-Histone H2AX (p-H2AX)證實異甘草素造成DNA損傷，抑制惡性腫瘤特性由群落形成能力(colony formation)、細胞移行(migration)、侵襲(invasion)、非貼附性生長能力(anchorage independent growth)測試，以動物模型進行異甘草素對口腔鱗狀細胞癌之體內試驗。研究結果顯示異甘草素對OSCC毒性較OF、SG強，使細胞週期停滯在G2期且誘導癌細胞凋亡，並確認G2期相關蛋白phospho-Checkpoint kinase 1 (p-Chk1)、p-cdc25c、cdc2、cyclin A表現量減少，細胞凋亡相關蛋白Cysteine aspirate protease (Caspase)-9/-3、Poly (ADP-ribose) polymerase (PARP)表現量增加，DNA fragmentation與p-H2AX蛋白質表現量增加確認DNA damage，ATM蛋白質表現減少經由RT-PCR及Dual-GloR luciferase assay證明與mRNA、 promoter無關，而是藉由Caspase-3的活化造成ATM蛋白質表現減少影響DNA修復機制無法啟動，異甘草素在不造成OSCC毒殺的劑量也可抑制其惡性腫瘤特性；從動物模型顯示異甘草素可抑制腫瘤的生長，減小腫瘤體積。因此認為異甘草素是藉由抑制DNA修復ATM訊息途徑誘導口腔鱗狀癌細胞細胞週期停滯與細胞凋亡。
並列摘要
Objectives: Isoliquiritigenin (ISL) was extracted from licorice. It has anti-virus, anti-oxidation, anti-inflammation, anti-ulcer and anti-cancer activity. It is also utilized in cancer research, such as prostate and breast cancer. However there were a few literature about oral cancer. Methods: The oral squamous cell carcinoma (OSCC) cell lines: HSC3, SAS, and OECM-1 were used for anti-oral cancer drugs screening, and oral fibroblast (OF), human gingival epithelial S-G cells (SG) were used for control. Cell viability was detected by MTT assay. Cell cycle and apoptosis were analyzed by flow cytometry. The mRNA and protein expression were detected by RT-PCR and western blotting, respectively. The DNA damage was analyzed by TUNEL assay and phospho-Histone H2AX (p-H2AX) protein expression. The cell colony formation, migration, invasion and anchorage independent growth ability were used for malignant phenotype inhibition tests. In vivo, we used xenograft model. Results: the result showed ISL inhibited OSCC cells proliferation, but in OF and SG were not. ISL induced OSCC cell cycle arrest at G2 phase and apoptosis in dose dependent manner. DNA damage was confirmed by DNA fragmentation and p-H2AX expression increased. Ataxia-telangiectasia mutated (ATM) protein expression decreased that were not related with mRNA or promoter activity. ISL induced Cysteine aspirate protease-3 (Caspase-3) activated that inhibited ATM DNA repair pathway. OSCC cell line colony formation, migration, invasion, anchorage independent growth were inhibited after low dosage ISL treated. In vivo, the result showed ISL can inhibit tumor growth and decreased tumor volume. Conclusion: ISL induced cell cycle arrest and apoptosis by ATM pathway suppression in oral squamous cell carcinoma.