NFD誘導肺癌細胞凋亡及抑制轉移作用機制探討

The Mechanism of NFD Induced Apoptosis and Migration Inhibition in Human Non-small Cell Lung Cancer

吳品萱(Wu, Pin-Hsuan)

指導教授 ： 邱建智

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2012年)

https://doi.org/10.6832/KMU.2012.00120

摘要

Naphtho [ 1,2-b ]furan-4,5-dione (NFD) 在1985年從植物海茄冬 ( Avicennia marina )中被分離出來，隨著化學合成技術的精進，現今NFD已可以由全合成的方式生成。NFD 已被報導能誘導多種癌細胞產生細胞凋亡現象。在我們實驗室先前的研究發現NFD抑制人類非小細胞肺癌: H1299、H1437以及A549的生長，但是對於正常的肺纖維母細胞MRC-5沒有明顯抑制，顯示NFD對於人類非小細胞肺癌具有選擇性地胞殺能力。此外在Annexin V染色標定細胞內膜外翻實驗中證實超過細胞毒殺性劑量的NFD會誘導癌細胞凋亡。在免疫螢光及西方點墨法實驗觀察到高劑量NFD 處理後偵測到顯著的H2AX磷酸化情形，並且在即時定量PCR中觀察到DNA損傷修復蛋白ATM及ATR的mRNA表現量，顯示NFD可能造成DNA的損傷。在Dihydroethidium染色實驗中發現高劑量NFD處理會提高細胞內生性ROS含量，並且抑制第二型DNA拓樸異構酶 (Topoisomerase II) 的活性。在以ELISA為基礎的轉錄因子活性測試實驗中也發現到負責調控與生長、免疫高度相關的轉錄因子NF-κB在NFD處理過後活性明顯受到抑制。這些結果顯示NFD所導致的生長抑制及細胞凋亡現象可能是透過ROS的產生與抑制第二型拓樸異構酶的活性造成DNA雙股螺旋斷裂，以及調控NF-κB訊息傳遞路徑共同導致的結果。此外，我們也觀察以對細胞不具毒殺劑量的NFD能明顯地抑制細胞移行及穿孔能力。即時定量PCR實驗中顯示NFD降低細胞中細胞移行相關蛋白質MMP-2、MMP-9以及β-catenin的mRNA表現量。在西方點墨法實驗中觀察到NFD降低MMP-2、MMP-9以及其上游調控轉錄因子SP-1的蛋白質表現量，更進一步在Gelatin zymography實驗中證實NFD減弱MMP-2及MMP-9酵素活性。另外，我們使用ERK、JNK及p38 MAPK訊息傳遞路徑的抑制劑證明了NFD能藉由調控JNK/p38 MAPK 訊息傳遞路徑抑制細胞的轉移。顯示對細胞不具細胞毒性劑量的NFD抑制細胞轉移可以透過降低MMP-2及MMP-9的活性與調控β-catenin、JNK/p38訊息傳遞路徑共同導致的結果。綜合以上實驗，高劑量的NFD能夠造成DNA雙股螺旋斷裂誘發細胞凋亡，而低劑量NFD調控MMP-2及MMP-9抑制細胞轉移能力，顯示NFD能藉由兩種不同劑量抑制肺癌細胞生長及轉移。同時，因其結構的簡單具有可塑性，未來能以NFD為骨架做延伸發展出更多功能且具專一性的抗肺癌藥物。

關鍵字

肺癌細胞

並列摘要

Naphtho[1,2-b]furan-4,5-dione (NFD), which has been proven to exerts the cytotoxicity to a variety of tumor cells. Previous studies indicated that NFD inhibits cell proliferation and induces cell apoptosis in different types of tumors. In our preliminary study, we observed that NFD exerts the anti-proliferation and apoptosis-inducing potential against human non-small cell lung cancer (NSCLC), but not normal lung fibroblast MRC-5, indicating NFD is potent and specific against NSCLC tumor cells. The immunofluorescence and western blot further showed that the marker of DNA damage, γ-H2AX was activated following NFD administration. Additionally, the increased transcription levels of DNA damage-sensors ATM and ATR were observed following NFD treatment. Furthermore, the intracellular ROS were increased under cytotoxic dose of NFD administration. The analysis of molecular docking and topoisomerase II (topo II) assay suggested that NFD could be a novel topo II inhibitor. The enzyme-linked immunosorbent assay (ELISA) - based binding assay showed the activity of transcription factor NF-κB was dramatically reduced by NFD. Taken together, our present work demonstrated that NFD induces DNA damage and apoptosis of NSCLC cells may through inhibition of topo II activity and NF-κB signaling pathway in NSCLC tumor cells. Besides, in wound healing and Boyden’s chamber assay, we observed that non-cytotoxic dose of NFD efficiently inhibits the migration and invasion of H1299 cells. Moreover, qRT-PCR result demonstrated that the mRNA level of MMP-2, MMP-9 and β-catenin were dramatically decreased. The western blot and gelatin zymography analysis data further showed that NFD down-regulates the protein levels and enzyme activities of MMP-2 and MMP-9. Moreover, three MAPK signaling pathway inhibitors were used to confirm that NFD inhibits cell metastasis via regulating the JNK/p38 MAPK signaling pathway. These results indicated that NFD inhibits cells migration may through inhibiting MMP-2 and MMP-9 activity and modulating the JNK/p38 MAPK and β-catenin signaling pathway in NSCLC. In conclusion, this study we evaluated NFD have dual potent function in NSCLC: cytotoxic dose of NFD performs anti-proliferation and apoptosis-inducing capacity. On the other hand, non-cytotoxic dose of NFD exerts effective anti-migration ability. With the dual function on NSCLC, NFD might provide a new version on naphthoquinone-based anti-cancer drug development.

並列關鍵字

NFD ； Human Non-small Cell Lung Cancer

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