抗癌活性成分在胰臟癌和前列腺癌的抗癌機轉探討

Abstract

惡性腫瘤連續31年蟬聯國人十大死因的第一名，根據衛生署統計平均每5分鐘48秒就有1人罹患癌症，其惡性腫瘤的特性為生長速度不受到細胞的調控而快速的增生，其中胰臟癌是所有惡性腫瘤中為最具侵襲性的癌症，通常於末期才有顯著的臨床症狀，常常因為預後不佳的因素，其5年存活率不到5%，並且對於化學療法具有抵抗性，胰臟癌因此有「沉默殺手」的稱號；然而，前列腺癌則是生長的速度緩慢，甚至經年不會引起症狀或問題，導致許多老年男性患者不知因患有前列腺癌而致死，其中罹患前列腺癌的機率為六分之一，而因轉移性的前列腺癌致死率為三十分之一，因此，前列腺癌轉移侵入至其他器官如：淋巴結、骨骼、肺臟和肝臟，導致極高的致死率發生。癌症的治療是當今臨床上最重要的課題之一，也因此，抗癌藥物的研發也更顯得重要。 第一部分我們將探討天然物antroquinonol對人類胰臟癌細胞的抗癌機制，antroquinonol為牛樟芝(Antrodia camphorata)中萃取分離而得到ubiquinone類的化合物，在人類胰臟癌細胞株PANC-1和AsPC-1細胞中，具有濃度相關的生長抑制活性，antroquinonol經由抑制PI3-Kinase/Akt/mTOR訊息傳遞路徑，進而使得人類胰臟癌細胞之細胞生長週期停滯在G1期和伴隨著細胞凋亡(apoptosis)的現象，並藉由p21蛋白和K-ras的表現量增加，而加速人類胰臟癌細胞細胞自噬(autophagy)與細胞老化(senescence)的現象，最後造成人類胰臟癌細胞的生長受到抑制，導致細胞凋亡的現象，顯示antroquinonol對於人類胰臟癌細胞具有抗腫瘤的活性作用。 第二部分我們探討了天然物ardisianone對人類荷爾蒙不依賴型前列腺癌細胞的抗癌作用機轉，ardisianone為黑星紫金牛(Ardisia virens Kurz.)植物莖部中萃取分離而得到benzoquinone類的化合物，在人類荷爾蒙不依賴型前列腺癌細胞株PC-3和DU-145細胞中，具有抑制細胞生長的能力和促進細胞凋亡的活性，並誘發粒線體損傷的產生，對於Akt/mTOR/p70S6K調控蛋白質轉譯的抑制，促使Bcl-2家族蛋白和survivin表現量下降，導致caspases活化的訊息傳導路徑，除此之外，也會造成caspase-independent pathway的AIF自粒線體釋出而活化細胞凋亡途徑，顯示出ardisianone對於人類前列腺癌細胞透過粒線體損傷更具有抗腫瘤的活性作用。 第三部分我們將探討經由循理性設計的合成化合物KUD773，對人類荷爾蒙不依賴型前列腺癌細胞的抗癌作用機轉。KUD773化合物是依據thiazole和imidazole雜環做循理性設計的衍生物，在人類荷爾蒙不依賴型前列腺癌細胞株PC-3和DU-145細胞中，具有抑制細胞生長的能力和subG1期細胞凋亡的增加，並且使細胞週期停滯在有絲分裂期prometaphase到metaphase之間，其為有絲分裂相關蛋白PLK1(Thr210)的磷酸化表現量上升所調控，利用微管聚合實驗偵測KUD773可抑制微管的聚合能力，此外，藉由冷光偵測Aurora A kinase的活性受到抑制，導致細胞單極紡錘體（monopolar spindle）的形成。KUD773會調控cyclin A蛋白的下降，伴隨著cyclin B1蛋白的表現量上升，並且藉由CDK1(Thr161)的磷酸化表現量上升和CDK1(Tyr15)的磷酸化表現量下降而活化CDK1；除此之外，KUD773會促進Bcl-2蛋白的高度磷酸化和粒線體損傷，皆會造成caspases訊息傳導路徑的活化，並且對於細胞轉移調控相關因子MMP-2和MMP-9的蛋白表現量有抑制作用，顯示KUD773對於癌症的治療藥物發展上展露出一道曙光。 第四部分我們將探討經由循理性設計的合成化合物DS-435對人類荷爾蒙不依賴型前列腺癌細胞的抗癌作用機轉。DS-435利用組合式化學方式，依據拓樸異構酶（topoisomerase）抑制劑DACA和組織蛋白去乙醯酶（HDAC）抑制劑SAHA合成一系列循理性設計的衍生物，透過抑制拓樸異構酶和組織蛋白去乙醯酶的活性，來抑制人類荷爾蒙不依賴型前列腺癌細胞PC-3的細胞生長，DS-435會使細胞週期停滯在G1期，並隨著DS-435的濃度增加，使得細胞週期停滯在G2期。利用HDAC activity assay發現DS-435為非選擇性的HDAC抑制劑，並且會促進topoisomerase I-DNA斷裂複合物的結合，而抑制topoisomerase I的活性，更進一步地，DS-435會誘發DNA損傷反應使相關蛋白Chk1(Ser345)和Chk2(Thr68)的磷酸化表現量上升，並且也會造成組蛋白histone H3的乙醯化和磷酸化表現量顯著性地上升，其為HDAC活性受到抑制所調控。此外，利用合併療法觀察DS-435和蛋白質水解酶抑制劑MG-132，對於細胞凋亡的訊息傳導路徑具有協同性地作用。總結研究成果得知DS-435藉由同時抑制topoisomerase和HDAC的活性，造成DNA損傷相關蛋白的訊息途徑活化，最後導致細胞走向caspase-dependent細胞凋亡的路徑。

Cancer has been the first leading cause of death in Taiwan for the past 31 years. In 2011, one patient in Taiwan was diagnosed with cancer every 5 minutes and 48 seconds according to the Department of Health (DOH). Cancer is a class of diseases characterized by out-of-control cell growth. Pancreatic adenocarcinoma is among the most aggressive of all cancers, which signs and symptoms frequently occur when the cancer is at the advanced stage. Pancreatic cancer has a poor prognosis with the 5-year survival less than 5%, which is largely resistant to chemotherapy with a name called 'silent killer'. However, prostate cancer is mostly a very slow progressing disease. In fact, many men die of old age without ever knowing they had prostate cancer. Moreover, the risk of prostate cancer is 1 in 6 and the risk of death due to metastatic prostate cancer is 1 in 30. Therefore, prostate cancer can metastasize to lymph nodes, bone, lung, and liver with advanced stage, leading to a high mortality. Cancer therapy is one of the top issues in clinical therapy. Accordingly, the development of anticancer agents is getting important. On the part one of the thesis, we have identified the anticancer mechanism of natural product antroquinonol, a ubiquinone derivative isolated from a camphor tree mushroom Antrodia camphorata. Antroquinonol induced a concentration-dependent inhibition of cell proliferation in PANC-1 and AsPC-1 pancreatic cancer cells. The data suggest that antroquinonol induces anticancer activity in human pancreatic cancer cells through an inhibitory effect on PI3-kinase/Akt/mTOR pathways which induced G1 arrest of the cell-cycle and a subsequent apoptosis. Antroquinonol also induced the crosstalk between apoptosis, autophagic cell death and accelerated senescence, which was, at least partly, explained by the up-regulation of p21Waf1/Cip1 and K-ras. On the part two, we have identified the anticancer mechanism of natural product ardisianone, a natural benzoquinone derivative isolated from Ardisia virens. Ardisianone displayed anti-proliferative and apoptotic activities against human hormone-refractory prostate cancer (HRPC) PC-3 and DU-145 cell lines. The data suggest that ardisianone induces apoptosis in human prostate cancers through mitochondrial damage stress, leading to the inhibition of Akt/mTOR/p70S6K translational pathway, down-regulation of Bcl-2 family members, degradation of survivin and activation of caspase cascades. The data support further development of ardisianone as an anticancer agent through mitochondrial damage stress. On the part three, we have investigated the anticancer mechanism of rational designed based on thiazole and imidazole, KUD773, which induced anti-proliferative effect and a subsequent apoptosis in PC-3 and DU-145, two HRPC cell lines. Further detection of mitotic phosphoproteins and PLK1 phosphorylation at Thr210 indicated that KUD773 induced mitotic arrest, in particular, at prometaphase to metaphase of the cell cycle. The microtubule assembly assay showed that KUD773 inhibited tubulin polymerization. Moreover, KUD773 inhibited Aurora A activity by in vitro kinase assay and by the detection of monopolar spindle formation, a character of Aurora A inhibition. KUD773 triggered the up-regulation of cyclin B1 associated with the concomitant down-regulation of cyclin A. Accordingly, KUD773 induced an increase of Cdk1 phosphorylation at Thr161 and a decrease of phosphorylation at Tyr15 suggested Cdk1 activation. Moreover, the phosphorylation and subsequent down-regulation of Bcl-2 explain KUD773-mediated mitochondrial dysfunction and activation of caspase cascades. KUD773 also induced a significant decrease of protein levels of matrix metalloproteinase (MMP)-2 and -9, two central MMPs for cancer cell metastasis. In summary, the data suggest that KUD773 is a dual inhibitor against tubulin polymerization and Aurora A activity with the anti-cancer activity in HRPCs. On the part four, we have investigated the mechanism of rational designed compound DS-435, which displayed anticancer activity through both topoisomerase and HDAC activity. DS-435 was effective against the cell growth of prostate cancer PC-3 cells, and induced an arrest of the cell cycle at both G1 (at lower concentrations) and G2 phase (at higher concentration). The HDAC activity assay demonstrated that DS-435 was a pan HDAC inhibitor. Additionaly, DS-435 induced the formation of topoisomerase I-DNA cleavage complex, suggesting an inhibition on topoisomerase I activity. Furthermore, DS-435 induced the phosphorylation of Chk1 and Chk2 at Ser345 and Thr68, respectively, indicating the activation of both kinases and the occurrence of DNA damage response. DS-435 also dramatically increased the levels of histone H3 acetylation and phosphorylation, confirming the inhibitory effect on HDAC activity. Moreover, the combination between DS-435 and the proteasome inhibitor MG-132 caused a synergistic effect on cell apoptosis. In summary, the data suggest that DS-435 is a dual inhibitor against topoisomerase I and HDAC activity. It induces DNA-damage response signaling, leading to caspase-dependent apoptotic cell death in PC-3 cells.