TW-01在人類非小型肺癌細胞 H460 之凋亡作用機制探討

Studies on TW-01-Induced Apoptotic Mechanism in Human Non-Small Cell Lung Cancer Cells (H460)

張珍瑜(Chen-Yu Chang)

指導教授 ： 徐鳳麟 ； 林建煌

臺北醫學大學/藥學院/生藥學研究所/碩士(2004年)

摘要

根據衛生署統計顯示，癌症依然是台灣地區的主要死因，且肺癌為造成癌症死亡的重要原因之一。而針對肺癌的化學治療仍未能達到預期的治癒率，因此，發展新一代的化學治療製劑並應用於臨床治療肺癌將是一重要課題。本論文將探討TW-01(新合成的化合物)在人類非小型肺癌細胞H460中的細胞凋亡機制。根據MTT與流式細胞儀分析結果顯示，TW-01能夠時間與劑量依循性地減少H460細胞存活率及增加凋亡細胞百分比。利用DNA斷片和TUNEL染色分析細胞核濃染，更進一步地證實TW-01引起H460細胞的死亡是經由細胞凋亡而來。我們也發現TW-01引起H460細胞凋亡過程中，伴隨著粒線體膜電位喪失，cytochrome c和Smac的釋放以及 caspase 3的活化。同時，z-VAD-fmk (非選擇性caspase 抑制劑)也可以減輕TW-01的細胞凋亡現象。我們的實驗結果也指出，TW-01會引起Bax的表現量增加；然而，Bcl-2與Bcl-XL的含量卻呈現減少的現象。西方墨點法分析的結果顯示，SB 203580 (選擇性p38 MAPK抑制劑)可有效抑制TW-01在細胞凋亡過程中所誘導的p53磷酸化和Bax表現增加的作用。此外，在TW-01刺激下，p38 MAPK的磷酸化亦即活化也會增加。綜合以上實驗結果，我們推測TW-01在H460的細胞凋亡作用主要是透過活化p38 MAPK/p53/Bax/caspase訊息傳遞路徑。

關鍵字

細胞凋亡 ； 粒線體

並列摘要

Results from department of heath have shown that cancer is main cause of death, and lung cancer is the leading cause of cancer death in Taiwan area. Since the treatment of lung cancer using chemotherapy is limited success, novel chemotherapeutic agents are needed to be defined for clinical applications. The apoptotic mechanisms of TW-01, a newly synthetic compound, were studied in H460 human non-small lung cancer cells. MTT and flowmetric analysis showed the ability of TW-01 to decrease cell viability via apoptosis in time- and concentration-dependent manners. TW-01-mediated cell apoptosis was also demonstrated by DNA laddering and nuclear condensation using TUNEL analysis. Apoptosis in H460 cells elicited by TW-01 was accompanied by the collapse of mitochondrial transmembrane potential, the release of cytochrome c, Smac, and the activation of caspase 3. The apoptotic effect of TW-01 was diminished by the presence of z-VAD-fmk (non-selective caspase inhibitor). Our results also revealed that the expression of Bax protein was elevated, however, the content of Bcl-2 and Bcl-XL proteins were decreased by TW-01. Western blotting indicates the inhibitory effect of SB203580 (selective p38 MAPK inhibitor) on p53 phosphorylation and Bax protein expression in H460 cells undergoing apoptosis triggered by TW-01. Moreover, the phosphorylation of p38 MAPK was also increased upon TW-01 treatment. Taken together, we demonstrated the apoptotic effect of TW-01 in H460 cells primarily relied on activation of p38 MAPK/p53/Bax/caspase signaling cascade.

並列關鍵字

apoptosis ； mitochondria

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