一.過去研究利用MTT assay以非小細胞肺癌細胞 (non-small cell lung cancer, NSCLC) 為平台篩選了逾五百種小分子化合物，發現含有indole 及quinoline這兩種官能基的小分子化合物compound A，於低劑量下，會抑制A549及H460這兩株NSCLC細胞生長速率及抑制A549腫瘤生長，但對抑癌基因p53缺失的H1299細胞株則無影響，接著使用western blot, MTT及flow cytometry，證實此藥物會誘導表現野生型p53的細胞產生凋亡，但是p53缺失的H1299細胞對此藥物的敏感度較低，而且不會出現凋亡。本論文內容是繼續探討此藥物對NSCLC細胞的調控生長機制與p53基因型的相關性。實驗主要是將p53缺失的H1299，分別轉殖入p53 DNA-binding domain 位點突變的p53R267P以及野生型p53的細胞株，其次再使用MTT 實驗，發現以此藥物10 µM處理H1299轉殖野生型p53細胞48小時後的生長速率較轉殖p53R267P敏感度高35％，繼續使用propidium iodide及Annexin V進行實驗，證實轉殖野生型p53細胞生長率的降低，是細胞凋亡造成，但以此藥物10 µM處理H1299/p53R267P細胞48小時後，細胞凋亡數目比例較H1299/p53細胞降低39.2％，最後由western blot確認細胞凋亡與p53及其下游poly (ADP-ribose) polymerase (PARP) 裂解及caspase-3活化的蛋白質相關。這些實驗結果證明此藥物對表現野生型p53的H1299/p53細胞有最佳的治療效果，且野生型p53功能在此藥物誘導的凋亡機制中扮演了重要角色。未來實驗中將持續探討compound A如何降低腫瘤細胞生長，實驗會以癌症類幹細胞為模式，持續研究抑制腫瘤生長及轉移的機制。

二.中草藥療法在近幾年被重視，因為以中草藥搭配放射療法能減低癌症病患副作用，提升體質。本實驗以可能具有較高轉移能力及抗藥性的肺癌類幹細胞為模式，研究中藥苦參 (Sophora flavescens) 、鴨膽子 (Brucea javanica)、夏枯草 (Prunella vulgaris) 及西藥teroxirone 如何抑制肺癌類幹細胞生長。本實驗觀察幹細胞表面標誌Nanog，確認肺癌類幹細胞平臺的建立，接續使用螢光顯微鏡觀察幹細胞在這些藥物處理下，H460, H1299/p53及A549肺癌類幹細胞會隨藥物濃度及時間增加，其數目及形狀明顯的變少及變小，表示具有抑制肺癌類幹細胞生長的潛力，最後再以western blot實驗確認teroxirone會誘導H460肺癌類幹細胞走向p53依賴的細胞凋亡路徑。於先前研究中已知夏枯草可降低肝癌細胞的轉移力，苦參及鴨膽子可抑制A549腫瘤生長，此外，低劑量的teroxirone可抑制肺癌細胞生長及誘導細胞凋亡，所以日後將持續以此類模式探討這些藥物是否能抑制肺癌類幹細胞生長以及降低轉移能力。

一.Previously, we have screened more than five hundred compounds with small molecular weight through MTT assay. The findings have identified that lower concentrations of a small molecular compound A containing functional groups indole and quinoline was effective to inhibit the growth rate of wild-type p53 NSCLC cells and suppress tumor growth, but the drug is less sensitive to p53-null H1299 cells. All western blot, MTT, and flow cytometry experiments showed that the induced apoptosis in NSCLC cells involved wild-type tumor suppressor genes p53. The cells without p53 were less sensitive to compound A.The experiments keep on exploring the mechanism on how compound A regulates the growth and association with p53 status in cells. We have further tested the effect of the compound in p53-null H1299 cells with stable expression of mutated (H1299/p53R267P) and wild-type p53 (H1299/p53), respectively. Through MTT assay, after treated 10 µM compound A 48 hours, the data was shown more drug effective in H1299/p53 cells than H1299/p53R267P cells. The results proved that the efficacy is related to p53 genotype. Using flow cytometry and western blot experiments, we found that compound A can reduce proliferation rate through apoptotic cell death in the H1299/p53 cells, and the apoptosis percent of H1299/p53 cells is 39.2％ higher than H1299/p53R267P cells. We have also shown that the p53-dependent apoptotic cell death pathway began with activating p53, attenuating Bcl-2, activating caspase family proteins, and finally cleaving poly (ADP-ribose) polymerase. These results demonstrate that the cells with wild-type p53 were more sensitive to compound A. In the future, we will continue to explore the mechanisms on how compound A inhibits the growth and metastasis in lung cancer stem-like models.

二.In recent years, the Chinese herb medicine therapy is taken seriously because this therapy combine with Radiotherapy can reduce side effect and improve constitution in cancer patients. The experiment based on the lung cancer stem-like cells model, which may be higher metastasis and resistance. To evaluate Chinese herb medicine Sophora flavescens , Brucea javanica , Prunella vulgaris and triep teroxirone can potentially eradicate lung cancer stem-like cells, we used human lung cancer stem-like cells as established from the parental cells. First, we used fluorescence microscopy to confirm that H460, A549 and H12PP/p53 stem-like cells changed in size and in numbers after the drugs treated. Finally, we used western blot experiment to confirm that teroxirone reduced the H460 stem-like cells numbers resulted from apoptotic cell death, and the apoptosis pathway is associated with p53and apoptosis proteins. Previously, we showed that the Chinese herb medicine Prunella vulgaris inhibited the metastasis capacity in liver cancer cells. Besides, Sophora flavescens and Brucea javanica inhibited A549 tumors growth.We also showed that teroxirone inhibited NSCLC cell growth that was caused by apoptotic cell death. In this work, we continued to use stem-like models to evaluate whether these drugs inhibit cells growth and metastatic property of the stem-like cells.

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