- 學位論文
吳茱萸鹼對於具有喜樹鹼抗藥性卵巢癌細胞之毒殺作用機制探討
Cytotoxic Effects of Evodiamine on Camptothecin-Resistant Ovarian Carcinoma Cells and the Mechanism Investigation
摘要
拓撲異構酶(topoisomerase)參與DNA拓撲結構的改變，許多抗腫瘤化療藥物皆以拓撲異構酶為作用標靶，而喜樹鹼(camptothecin, CPT)是已用於臨床治療的拓撲異構酶I (TopoI)抑制劑，但其抗藥性及嚴重副作用阻礙其應用性。AMP-activating protein kinase (AMPK)是細胞內主要的能量狀態偵測器，當細胞內ATP含量降低，AMP含量相對升高時，能透過AMPK的活化，產生ATP以維持能量平衡。近年來已有研究發現AMPK與癌細胞的增生與凋亡有關，當AMPK被活化時，會促使癌細胞進行細胞自噬，對細胞產生保護作用而不走向細胞凋亡。本研究室先前發表一從吳茱萸植物所萃取出的吳茱萸鹼(EVOdiamine, EVO)，結構類似於Topo I抑制劑CPT，可藉由將DNA與TopoI停留在TopoI-DNA cleavage complex而造成TopoI poison。本研究中，我們針對吳茱萸之有效成分EVO對CPT抗藥株A2780R2000人類卵巢癌細胞毒殺作用的機制進行探討，同時比較CPT抗藥株在分別處理CPT以及EVO後，各項分子調控機制的差異。並且進一步釐清AMPK的活化以及細胞自噬(autophagy) 對於CPT抗藥株細胞A2780R2000抗藥性的影響。我們發現EVO會造成細胞DNA damage，同時透過caspase-3的活化，以及MAPK（ERK、JNK、p38）蛋白的抑制而促使細胞走向細胞凋亡(apoptosis)。以CPT處理細胞則發現MAPK蛋白質被活化以維持細胞的存活而不走向細胞凋亡。此外我們也觀察到細胞處理CPT後，可能會藉由AMPK的持續活化來促使細胞自噬的發生，進而保護細胞免於凋亡的命運；以EVO處理細胞時AMPK有短暫的活化保護，但隨著加藥時間增加，仍會促使細胞走向細胞凋亡。因此由以上實驗推論，AMPK和細胞自噬與細胞的抗藥性有關，而EVO能夠有效毒殺對CPT產生抗藥性的卵巢癌細胞。
並列摘要
The regulation of DNA topology by topoisomerase I (TopoI) is essential for DNA replication, transcription, and recombination events. Camptothecins (CPTs) are family of compounds that specifically target TopoI. These compounds ‘poison’ TopoI by stabilize reversible TopoI–DNA cleavage complex and prevent the religation of DNA. TopoI activity is elevated in various cancer cells, thus TopoI has been identified as an important therapeutic target in cancer chemotherapy. However drug resistance is the main hurdle in the successful treatment of cancer. AMP-activated protein kinase（AMPK）is a key regulator of energy homeostasis, and regulates a variety of cell functions including proliferation and apoptosis. Our previous studies have shown that evodiamine (EVO), a major constituent of Chinese herb Evodiae fructus, exhibited anti-tumor activities by inhibit TopoI activity. In our present study, we determined the cytotoxic effects of evodiamine on camptothecin-resistant ovarian carcinoma cells (A2780R2000). EVO exhibited dose-dependent cytotoxicity for A2780R2000 cells whereas CPT showed much less cytotoxicity. In addition, EVO effectively induced cell apoptosis and DNA tailing in the Comet assay. It downregulated MAPKs protein expression, and caused an increase in the ratio of pro-apoptotic/anti-apoptotic protein, which was associated with the increase in caspase activity. In contrast to EVO, MAPKs was activated in CPT treated cells and caused an decrease in the ratio of pro-apoptotic/anti-apoptotic protein. Furthermore, we found that phosphorylated AMPK level was increased transiently upon EVO treatment, while phosphorylated AMPK expression was endured upon CPT treatment. In conclusion, these data suggest that activation of AMPK and autophagy has protective effect on camptothecin-resistant ovarian carcinoma cells, and EVO is able to sustain the cytotoxic effect against the CPT-resistant cancer cells.
參考文獻
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