- 學位論文
Gallic acid及WYC02-9在前列腺癌的應用
The application of gallic acid and WYC02-9 in prostate cancer
摘要
攝護腺癌好發於男性年長者，在西歐國家的發病率遠高於東方國家，對於年長者健康問題的影響日趨嚴重。目前醫學界尚未發現有效療法。傳統化療藥物對患者副作用大，往往病患於荷爾蒙阻斷治療一至兩年後易產生抗性，使其失去療效而復發。傳統上，新藥之開發不外乎來自天然物或化學合成，因此，由天然物尋找有效的成分，並能避免對病患引起副作用，以對抗荷爾蒙阻抗型的前列腺癌為一首要課題。本研究計畫主體gallic acid為香椿(Toona sinensis Roem.)嫩葉萃取純化合物及WYC02-9由台灣土生蕨類(Thelypteris torresiana Gaud. (T. torresiana))中提煉出來的新類黄酮類化合物之衍生物，研究其在前列腺癌相關作用機制之探討。本研究論文共分為二部分如下: 論文第一部分，以香椿葉萃取層之活性成分純化物gallic acid（GA）在前列腺癌細胞株的抗癌作用及分子機轉進行探討研究。在我們的研究結果發現，經GA處理後具有毒殺前列腺癌細胞株的效果。首先造成細胞型態的改變，DNA損害形成彗星拖曳及細胞凋亡現象。此乃經由細胞內活性氧化物(reactive oxygen species, ROS)生成，進而促使粒線體細胞膜膜電位改變；並且活化Chk1和Chk2及抑制cdc2、cdc25蛋白活性，細胞生長停滯在G2/M期。若癌細胞株預先處理NAC (N-acetylcysteine)或CAT (catalase)，再處理GA後，便可有效抑制GA所誘發的ROS導致的凋亡現象。當將GA和現有化療藥物Doxorubicin合併時，可以增強抗癌效果，減少抗癌藥物的劑量。由本研究結果推論，我們認為GA對於前列腺癌的治療具有相當大的開發潛力。 研究論文第二部分，在此篇研究中我們則以新合成類黃酮酚類化合物WYC02-9增加細胞內ROS生成，造成前列腺癌細胞株內DNA傷害和凋亡機制啟動。當前列腺癌細胞株以WYC02-9處理後可使癌細胞內產生大量ROS、造成DNA傷害，誘發ATM-p53-H2A.X途徑及活化Chk1和Chk2，使細胞週期停滯於S和G2/M期。此外， WYC02-9誘導細胞凋亡乃經由降低粒線體細胞膜膜電位，進而活化caspase-9、caspase-3和PARP裂解。當癌細胞株預先以NAC處理，再處以WYC02-9，則可有效抑制WYC02-9所誘發上述現象。再則，藉由接種前列腺癌細胞株腫瘤老鼠後，給於腹腔注射WYC02-9後可有效抑制腫瘤生長以驗證其抗癌效果。 總結來說，對於香椿葉粹取層之活性成分純化物gallic acid及新合成類黃酮酚類化合物WYC02-9在前列腺癌抗癌作用的機轉研究上，仍須再進一步研究探討，使之能應用於臨床癌症藥物治療之開發。
並列摘要
Prostate cancer, the most frequently diagnosed malignancy in elderly males of the Western countries, has also become a major malignancy in many Asian countries. It is a major public health issue in the United States and many other parts of the world. For treatment modalities remain very insufficient, despite the development of newer drugs supposed to offer novel therapeutic options. The traditional chemotherapy on patients suffers serious side effects. The hormone therapy for most patients with advanced prostate cancer develops resistance to after one to two years then loss of efficacy and relapse. With regard to prostate cancer chemotherapy, it is important to search for active compounds from natural products and synthetic chemicals, which already become a booming area for the chemotherapeutic application. This thesis study the molecular mechanisms and applications of gallic acid (Toona sinensis leaf extracts ) and WYC02-9 (the novel synthetic flavonoid from Thelypteris torresiana Gaud. extracts) on prostate cancer divided into two parts: First, we searched for active compounds, with cytotoxic activity against several cancer cells including prostate cancer cells, from leaf extracts of Toona sinensis Roem.. In this study, gallic acid was identified as the major anti-cancer compound in Toona sinensis leaf extracts. It was cytotoxic to DU145 prostate cancer cells, through generation of reactive oxygen species (ROS) and mitochondria-mediated apoptosis, which were reversed by antioxidants catalase and N-acetylcysteine. Furthermore, gallic acid was shown to enrich DU145 cells at G2/M phases by activating Chk1 and Chk2 and inhibiting Cdc25C and Cdc2 activities. In addition, gallic acid had a synergistic effect with doxorubicin in suppressing the growth of DU145 cells. Taken together, our results suggest that gallic acid has the potential to be developed into an anti-prostate cancer drug and is worthy of further studies. In the second part, we focused on the protoapigenone analogue WYC02-9, a novel synthetic flavonoid, which has been shown to act against a variety of cancer cells. However, its effects on prostate cancer and the underlying mechanism were unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells as well as the underlying mechanisms by which WYC02-9 induced DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage, activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased cell cycle in S and G2/M phases. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential (ΔΨm) decrease, and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation. Take together, these results warrant further investigation of the major anti-cancer compound in Toona sinensis leaf extracts, gallic acid, and the novel synthetic flavonoid, WYC02-9, on the anti-cancer activity mechanism of prostate cancer, toward the development of clinical applications in cancer therapy.