探討三環藥物Teroxirone及吲哚基喹嚀藥物抑制人類非小細胞肺癌類幹細胞增生的機制

Mechanisms of Teroxirone and Indolylquinoline Compound That Impair Growth in Cancer Stem-Like Cells of Human Non-Small-Cell-Lung-Cancer

倪玉玲(Ni Yu-Ling)

指導教授 ： 方剛

國立臺灣師範大學/理學院/生命科學系/碩士(2016年)

https://doi.org/10.6345/NTNU202204046

摘要

先前研究結果顯示三環衍生物teroxirone (1, 3, 5-triazine-2, 4, 6 (1H, 3H, 5H)-tri-one-1, 3, 5-tri-(oxiranylmethyl))及吲哚基喹嚀藥物EMMQ (3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline)具有抑制人類非小細胞肺癌生長的效果，雖然它的反應機制是透過細胞凋亡，然而卻因為殘存的癌幹細胞阻礙了藥物的完整活性。所以本論文的主要目的是想透過肺癌類幹細胞的培養模式探討teroxirone及EMMQ是否能夠根除潛在的肺癌幹細胞。首先須建立無血清以及低貼附性的特殊環境培養出肺癌類幹細胞球再進行後續的實驗。利用BrdU螢光染色和soft agar assay發現teroxirone與EMMQ可以抑制球狀細胞的生長、數量以及形成群落的能力。而以內差法計算soft agar assay的結果顯示，肺癌類幹細胞球所需的IC50遠高於貼附性細胞先前於MTT assay結果中所需的IC50，證明肺癌類幹細胞球確實具有較高的抗藥性。次以反轉錄聚合酶連鎖反應、西方墨點法及免疫螢光染色法證實teroxirone和EMMQ抑制肺癌類幹細胞的自我更新能力相關的幹細胞標誌基因，例如Nanog和ALDH1A1等。此外，TUNEL assay和西方墨點法的結果也顯示，teroxirone及EMMQ會誘發球狀細胞產生凋亡，最後以裸鼠皮下注射之腫瘤異種移植模式探討EMMQ之治療效果。根據論文的結論，teroxirone與EMMQ除了可以抑制貼附性肺癌細胞生長，若能適當地提高濃度也可以藉由細胞凋亡清除肺癌幹細胞。

關鍵字

肺癌 ； 癌類幹細胞 ； teroxirone ； EMMQ ； 細胞凋亡

並列摘要

We showed before that teroxirone and EMMQ inhibited the growth of human non-small-cell-lung-cancer cells (NSCLC) and the effects dose- and time-dependent. The apoptotic cell death accounted for cell death. However, the residual drug resistance hampers complete drug activity because of residual stem cells. Cancer stem cells (CSCs) are resistant to chemotherapy treatments, and accentuate disease progression despite therapeutic intervention. The work is aimed to override the drug resistance of teroxirone and EMMQ by targeting their corresponding CSC cells as enriched from the parental cells. To know if the chemical drugs can potentially eradicate lung CSC cells, we have established the spheroids of human NSCLC cells under serum-free condition. Experiments with fluorescence microscopy and soft-agar assay showed that the colony sizes and numbers of spheroids were suppressed after treatment with higher teroxirone and EMMQ concentrations. The formation of spheres, the expression of self-renewal related genes, such as, Nanog and ALDH1A1 stemness markers, were repressed as confirmed by RT-PCR, western blot analysis and immunofluorescence staining. The results proved that the chemical drugs affected the self-renewal ability of the spheroid cells. Furthermore, we demonstrated that higher concentrations of teroxirone and EMMQ caused apoptotic cell death of spheres as confirmed by western blot and TUNEL assay. At last, EMMQ suppressed the growth of H460 spheroid cells in xenograft tumors by exhibiting apoptosis characteristics. As a potential therapeutic agent by restraining cell growth through apoptotic death in spheroids, teroxirone and EMMQ promised an addition to the current list in complete eradication of human lung cancer.

並列關鍵字

lung cancer ； cancer stem-like cells ； teroxirone ； EMMQ ； apoptosis

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