低葉酸微環境藉由幹性訊息路徑促進非小細胞肺癌非貼附依賴性腫瘤球生長

Low folate Microenvironment Promotes Anchorage-independent Oncosphere Formation of Human NSCLC Through Stemness-signaling Pathway

陳婉菁(Wan-Jing Chen) ； 許瑞芬(Rwei-Fen S. Huang)

《台灣營養學會雜誌》 43卷2期 (2019/06) Pp. 32-46

https://doi.org/10.6691/NSJ.201906_43(2).0001

摘要

近年研究指出營養微細環境會影響非小細胞肺癌（non-small cell lung cancer, NSCLC）幹性特徵表現，但詳細機制仍不清楚。本研究模擬臨床病人短期和長期葉酸營養不良的情況，探討時間在低葉酸微環境中對NSCLC癌幹細胞特性的影響。4、8、12天的10 nM低葉酸培養基培養肺腺癌細胞株（A549），分析細胞存活率、上皮細胞間質轉化（epithelial-mesenchymal transition, EMT）、sonic hedgehog（Shh）、癌幹細胞標誌物、腫瘤球形成和添加抑制劑探討肺癌幹性與自我更新能力以及相關機制。結果顯示，相較於正常葉酸和短期葉酸營養不良，長期低葉酸促進A549細胞株死亡和凋亡率，且中長期葉酸營養不良的細胞展現EMT特性，包含細胞型態拉伸延長、下調E-cadherin，增上調Vimentin, Hif1α蛋白表達。幹性標誌物：ALDH1A1, Sox2, Oct4和胚胎幹性路徑標誌物：Shh, Gli1於短期和中長期低葉酸都被上調。觀察腫瘤球形成發現，低葉酸比控制組形成更多和大的腫瘤球。最後拮抗葉酸、阻斷mTOR和Shh訊號傳遞均有效抑制腫瘤球生長，但與SIRT1無關。綜合上述，葉酸營養不良刺激NSCLC表現癌幹細胞特徵且與Shh和mTOR路徑相關。

關鍵字

低葉酸微環境 ； sonic hedgehog ； 幹細胞特性 ； 腫瘤球 ； mTOR signaling

並列摘要

Recent studies have confirmed that the nutritional microenvironment affects the stem characteristics of non-small cell lung cancer (NSCLC), but the mechanisms remain unclear. This study simulates short- and medium to long-term folate dystrophy in clinical patients and explores the effect of time on the stemness of NSCLC cells in a low-folate microenvironment. The A549 cell line was cultured with low folate medium (10 nM) for 4, 8 and 12 days. Analysis of cell viability, epithelial-mesenchymal transition (EMT), Sonic hedgehog (Shh), cancer stem cell markers, anchorage-independent oncosphere formation, and the addition of inhibitors to examine self-renewal ability, stemness and mechanisms in NSCLC. The results showed that short- and medium to long-term low folic acid all had no significant cytotoxicity against A549 cell line. The results showed that compared with normal folate and short-term folate dystrophy, long-term low folate promoted the death and apoptosis rate of A549 cell line, and the medium and long-term folate dystrophic cells exhibited EMT characteristics, including cell type stretch extension and down-regulation of E- cadherin, up-regulated Vimentin, Hif1α protein expression. Stemness markers: ALDH1A1, Sox2, Oct4 and embryonic signaling pathway markers: Shh, Gli1 is up-regulated in both short- and medium to long-term. Moreover, low folate formed more and larger oncospheres than the control group. Finally, antagonizing folic acid, blocking mTOR and Shh signaling were effective in inhibiting tumor sphere growth, but not SIRT1. The folate malnutrition trigger NSCLC to exhibit cancer stem cell characteristics and is associated with Shh and mTOR pathways.

並列關鍵字

Low folate microenvironment ； sonic hedgehog signaling ； stemness ； oncosphere ； mTOR signaling