探討Thiostrepton在肝癌細胞的毒性作用和潛在機制

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學研究所 > 博碩士論文 > Item 310902500/12323

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/12323

Title:	探討Thiostrepton在肝癌細胞的毒性作用和潛在機制 Explore the toxic effects and underlying mechanisms of thiostrepton in Hepatocellular carcinoma cells
Authors:	劉珮君 Liou, Pei-Jyun
Contributors:	中山醫學大學:醫學研究所;吳俊錡
Keywords:	Thiostrepton;肝癌細胞;細胞凋亡;活性氧分子;創?;-2 Thiostrepton;Hepatocellular carcinoma (HCC);apoptosis;Reactive oxygen species (ROS);Caspase-2
Date:	2015
Issue Date:	2015-09-21T03:31:53Z (UTC)
Abstract:	在台灣癌症死亡中排名第二位的是肝癌，目前治療肝癌的藥物有嚴重不良的副作用，預後仍然不佳，所以尋找更有效的治療藥物是當務之需的。thiostrepton是一種天然環肽抗生素，先前的研究表明thiostrepton誘導各種腫瘤細胞死亡。在本論文研究中，我們探討thiostrepton在肝癌細胞中的毒性作用及分子機轉。結果顯示，thiostrepton可以有效促進HepG2和Hep3B肝癌細胞株的死亡。進一步分析創酶活性，顯示了 thiostrepton 可以誘發caspase-2、caspase-3、caspase-8和caspase-9的活性。若加入caspase-2 、caspase-8和caspase-9的抑制劑可減少thiostrepton誘導的肝癌細胞死亡，表示caspase-2、caspase-8和caspase-9參與thiostrepton誘導肝癌細胞的凋亡。從Western blot分析中發現thiostrepton會改變tBid和Bax的表現量，以及DR4路徑的活化，這表明thiostrepton誘導的細胞凋亡可能是通過內源性和外源性的途徑。從流式細胞儀分析經JC-1處理後之細胞，其結果顯示thiostrepton會干擾肝癌細胞粒線體膜電位。處理caspase-2 、caspase-8和caspase-9的抑制劑結果發現這些抑制劑會減少thiostrepton對粒線體膜電位之破壞，表示caspase的活化是會影響thiostrepton對肝癌細胞之粒線體膜電位之變化。進一步分析細胞中ROS之變化，結果顯示thiostrepton會造成細胞內H2O2、OH含量的增加。處理抗氧化劑NAC後會減少thiostrepton誘發肝癌細胞之死亡，同時加了NAC會減少thiostrepton對肝癌細胞之粒線體破壞，表示ROS參與thiostrepton誘導肝癌細胞之死亡。實驗結果也發現thiostrepton會造成細胞DNA損傷，加了NAC後會降低thiostrepton對肝癌細胞DNA之損傷，本論文之研究結果顯示，在肝癌細胞中，thiostrepton可誘發ROS的產生，造成DNA與粒線體受損而引發一系列caspase活化，進而導致細胞凋亡。這些結果顯示誘導細胞凋亡可能是thiostrepton的抗癌特性之一。 Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in Taiwan. The current therapies for HCC patients exhibit serious adverse effects, and the prognosis is still poor, so searching more effectively therapeutic agents is urgently needed. Thiostrepton is a natural peptide antibiotic, previous study shows that thiostrepton induces various tumor cell death: however, the exact mechanisms remain largely unclear. In this study, the anti-proliferative effects of thiostrepton in HCC cells were examined. Our results showed that thiostrepton effectively inhibited the prolifreration of human HCC HepG2 and Hep3B cell lines in both dose- and time-dependent manners. Data from flow cytometry and JC-1 assay indicated that thiostrpton induced an apoptotic cell death, disrupted the mitochondrial membrane potential, and increased the activities of caspase-2, caspase-3, caspase-8, and caspase-9, suggesting that thiostrepton-induced apoptosis might be through both intrinsic and extrinsic pathways. Moreover, inhibitors of caspase-2, caspase-8 and caspase-9 drastically attenuated thiostrepton-mediated cell death; revealed that these caspases involved in thiostrepton-triggered apoptosis of HCC cells. Western blot analysis showed that the levels of t-Bid, Bax and DR4 were elevated upon treatment of thiostrepton; further supporting the possibility that thiostrepton indeed induced cell death via multiple apoptotic pathways. Results from Reactive oxygen species (ROS) determination and comet assay showed that thiostrepton caused an increase in intracellular H2O2 and OH content and induced DNA damage in HCC cells. Moreover, the administration of antioxidant N-acetyl-cysteine attenuated thiostrepton-induced the loss of mitochondrial membrane potential, the DNA damage and apoptosis, indicating that ROS might be a upstream event that primed the apoptosis of HCC cells when engaging with thiostrepton. Our observations provide evidence that thiostrepton effectively induces the apoptosis of HCC cells via both extrinsic and intrinsic pathways.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/12323
Appears in Collections:	[醫學研究所] 博碩士論文

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