咔唑衍生物促使人類神經膠質瘤細胞自噬及增強temozolomide作用機轉之研究

第一部份咔唑衍生物促使人類神經膠質瘤細胞自噬及增強temozolomide作用機轉之研究 Glioblastoma multiforme (GBM, 多型性神經膠母細胞癌)是一個最常見也是最惡性的腦部癌症，其預後相當的差且對治療上有極大的抗藥性。從天然咖哩葉中萃取出來的咔唑化合物廣泛應用於抗癌上，在之前的研究中，我們利用咔唑的骨架加上一系列的官能基修飾發展出一系列的咔唑衍生物，用以評估在其對於GBM的影響。結果發現咔唑衍生物能有效的抑制GBM增生，其中以BC3EE2,9B效果最佳。我們進一步發現BC3EE2,9B也能有效的抑制primary human GBM，但對正常細胞株則無顯著的影響。BC3EE2,9B能使GBM細胞株的細胞週期停滯於G1期，且能抑制細胞遷移及細胞侵襲的能力。而在BC3EE2,9B合併GBM臨床用藥temozolomide處理上有協同性加成，並能誘發自噬性細胞死亡而不是細胞凋亡。我們進一步的發現其自噬性細胞死亡的訊息傳遞路徑可能透過抑制AKT蛋白的活性、增加AMPK的活性，而使其下游蛋白mTOR的活性減少，進而增加LC3-II蛋白的表現量增加。總括來說，透過BC3EE2,9B合併temozolomide而誘導自噬性細胞死亡，可能是一個有效對抗GBM的方式。第二部份五葉松萃取物白楊素抑制人類神經膠質瘤細胞增生機轉之初步研究 Glioblastoma multiforme (GBM, 多型性神經膠母細胞癌)是一個最常見也是最惡性的腦部癌症，其預後相當的差且對治療上有極大的抗藥性。松樹萃取物廣泛應用於抗癌上，五葉松為台灣特有的松樹種類，在台灣廣泛的當作健康食品使用，在之前的研究中，我們發現五葉松的粗萃取物能有效的抑制HL-60細胞增生，並發現水相中含有大量的生物類黃酮活性成分，目前我們使用這幾種生物類黃酮在抗癌上做進一步評估。初步結果發現Chrysin能有效的抑制GBM8901細胞，並在高劑量時可能會造成細胞凋亡，而在較低劑量的時候會誘發細胞自噬。我們進一步的發現其細胞自噬的訊息傳遞路徑可能透過誘導AMPK的活性，進而增加LC3-II蛋白的表現量增加，並抑制Caspase 3的活性。第三部份 Mevastatin藉由細胞自噬減少β-Amyloid造成細胞毒性之初步研究阿茲海默症(AD)被認為是一種神經退化性的疾病，主要起始分子為Aβ(β-Amyloid, β型類澱粉蛋白)，臨床上的統計發現一個有趣的現象，服用降血脂藥物的病人能顯著改善阿茲海默症的病況。因此本研究藉由臨床使用的statin類降血脂藥物Mevastatin，來探討其與阿茲海默症的關連性。初步結果發現Mevastatin能有效保護SK-N-MC細胞受到Aβ的損害。我們進一步的發現加入Aβ後細胞自噬受到抑制，而加入Mevastatin則能活化細胞自噬並可能將Aβ清除。其細胞自噬的訊息傳遞路徑可能透過抑制AKT進而增加LC3-II蛋白的表現量而誘發細胞自噬產生。

關鍵字

多型性神經膠母細胞癌； carbazole ； temozolomide ；自噬性細胞死亡； AKT ； AMPK ； mTOR ； LC3-II ；五葉松；生物類黃酮； Chrysin ；細胞凋亡；細胞自噬； Casepase 3 ；阿茲海默症； Aβ ； statin ； Mevastatin

並列摘要

Part 1 Mechanisms of Autophagy-inducing Effects and Sensitizing Temozolomide of Carbazole Derivatives in Human Glioblastoma Cells　 Glioblastoma multiforme (GBM) is the most common malignant brain tumor, notoriously poor prognosis and resistant to therapy. Recently, the naturally occurring carbazole alkaloids from curry leaf have attracted great attention due to their wide range of anticancer properties. In the present study, a series of carazole derivatives, obtained by introducing substituted different moieties on a carbazole backbone, was evaluated for the anti-GBM profile in vitro. Our results demonstrated that a number of derivatives showed a good antiproliferative activity. The most promising derivative in this series was BC3EE2,9B, which showed excellent antiproliferative and on both GBM8401 and GBM8901 Taiwan GBM cell lines. The BC3EE2,9B derivative significantly arrested GBM cell cycle at G1 stage and inhibited cancer cell invasion/migration abilities. Furthermore, we showed that via lysosomal signaling, BC3EE2,9B derivative combined with temozolomide induced autophagic cell death instead of apoptosis. BC3EE2,9B derivative combined with temozolomide significantly increased LC3-II level by immunoblotting. The possible mechanisms of autophagy may through suppress AKT activity which stimulating AMPK and attenuating mTOR downstream signaling. Taken together, our results suggest that the BC3EE2,9B derivative combined with the temozolomide, are likely to obtain excellent anti-GBM compounds endowed with a autophagy-inducing capability. Part 2 Preiminary data：Studies on the Anti-cancer Effects of Pinus Morrisonicola Hay Extract, Chrysin, in Human Glioblastoma Cells Glioblastoma multiforme (GBM) is the most common malignant brain tumor, notoriously poor prognosis and resistant to therapy. Pine extracts have wide range of anticancer properties. Pinus Morrisonicola Hay as a characterized pine in Taiwan and use extensively as a healthy food. In the present study, we found that the Pinus Morrisonicola Hay crude extract has an antiproliferative activity on HL-60 cells, and the water phase had a large amount of bioflavonoids. Our preliminary results demonstrated that bioflavonoids showed an antiproliferative activity on GBM8901. On of the most promising bioflavonoids was Chrysin. Chrysin showed apoptotic effects in high dose but induced autophagy in low dose. Further, we suggested that chrysin-induced autophagy signaling may result from enhancement of AMPK activity stimulation of LC3-II level and attenuation of caspase 3 activity. Part 3 Preliminary data：Studies on the Mevastatin Attenuates β-Amyloid Induced Neurotoxicity by Enhancing Cell Autophagy Alzheimer's disease (AD) is considered to be a neurodegenerative disease and Aβ(beta-amyloid) is the potential initiation molecule. Clinical statistics find an interesting phenomenon: pateient taking hyperlipidemia can apparently to improve AD’s condition. Therefore, we study the connection of AD and hyperlipidemia by mevastatin, one kind of statins for hyperlipidemia. Our preliminary results showed that mevastatin can attenuate the Aβ-induced cell toxicity by enhancing autophagy-degradation pathway. Forthermore, we showed that the autophagy-degradation pathway may be through attenuating AKT activity and stimulating downstream LC3-II level.

並列關鍵字

Glioblastoma multiforme ； carbazole ； temozolomide ； autophagic cell death ； AKT ； AMPK ； mTOR ； LC3-II ； Pinus Morrisonicola Hay ； bioflavonoids ； Chrysin ； apoptosis ； autophagy ； Casepase 3 ； Aβ ； statin ； Mevastatin

參考文獻

14. Math, M. and P. Balasubramaniam, Curry leaves (Murraya Koenigii spreng) and halitosis. BMJ South Asia edition, 2003. 19(3): p. 211.

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咔唑衍生物促使人類神經膠質瘤細胞自噬及增強temozolomide作用機轉之研究

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