天然物經由調控胞內訊息傳遞而抑制前列腺癌與肝癌之抗癌機轉探討

Identification of Anticancer Mechanism of Natural Products via Modulating Signaling Transduction in Human Prostate and Hepatocellular Carcinoma

江柏政(Po-Cheng Chiang)

指導教授 ： 顧記華

國立臺灣大學/醫學院/藥學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.03199

摘要

癌症是一種複雜且死亡率高的慢性疾病，於醫療上是個尚未被解決的棘手問題，雖然對於癌症的研究發展日新月異，然而對於癌症的治療，依然只能抱以「可以治療，無法治癒」的想法，抗癌藥物的研發，仍是一條漫漫長路。目前用於治療癌症的藥品中超過60%是來自天然物，如長春花生物鹼(Vinca alkaloids)、紫杉醇(Paclitaxel)與喜樹鹼(Camptothecin)等。新藥的開發往往是從天然物的活性成分尋找先導藥物，經由半合成修飾與活性測試探討其結構與活性之關係(structure- activity relationship (SAR))，以這些數據做基礎進而設計出更有潛力的抗癌藥物。以往對於天然物活性的研究，受限於對天然物資源的開發與應用，多侷限在陸生植物，直到近代科技發達後，海洋的生物資源才獲得較多的發展。本篇論文將針對數種天然物產物，探討其在人類前列腺癌與肝癌中的抗癌機轉。 第一篇研究主要在探討海洋羽珊瑚Clavularia viridis所萃取出之類前列腺素Bromovulone III，對荷爾蒙不依賴型前列腺癌所引發的細胞凋亡機轉。PGA1、PGA2與∆12-PGJ2等具有Cyclopentenone結構的前列腺素已在許多的研究中發現具有抑制前列腺癌細胞生長與促進細胞凋亡的能力。本篇研究證實，從海洋羽珊瑚Clavularia viridis中分離出的類前列腺素Bromovulone III相較於PGA1、PGA2與∆12-PGJ2，在荷爾蒙不依賴型前列腺癌細胞PC3中展現出30到100倍的抑制細胞生長活性。根據本實驗室之前的研究，Bromovulone III在人類肝癌細胞Hep3B，會藉由引發內質網迫力(endoplasmic reticulum (ER) stress)造成細胞凋亡。然而於人類荷爾蒙不依賴型前列腺癌PC3細胞中，卻發現Bromovulone III藉由不同於引發內質網迫力之途徑造成細胞凋亡。機轉方面，caspase-8的活化最早被觀測到，接著caspase-9、caspase-3與Bid的活化型隨處理時間增加也有明顯的表現， Mcl-1的裂解亦明顯產生。值得一提的是，Bromovulone III於不增加各死亡受器與其配體表現的情況下，利用共軛焦顯微鏡可以在短時間30分鐘開始觀測到Fas叢聚於脂筏(lipid raft)的現象，推測Bromovulone III是藉由活化Fas所調控之細胞凋亡。此外，caspase-2在Bromovulone III處理下也有活化的情形產生，利用csapase-2的抑制劑也證明caspase-2於caspase-8與caspase-3的活化有部分的貢獻。雖然許多研究已經指出DNA損傷會藉由caspse-2的活化啟動Fas/caspase-8調控的細胞凋亡機轉，然而經由Comet實驗與DNA裂解實驗證實Bromovulone III沒有造成DNA損傷的能力，對於topoisomerase IIα的活性也沒有影響。因此，Bromovulone III所引起的caspase-2活化與Fas/caspase-8細胞凋亡機轉應與DNA損傷無關。總結來說，Bromovulone III會藉由造成Fas重新分布叢聚活化外生性細胞凋亡(extrinsic apoptosis)機轉，再藉由Bid以及Mcl-1的裂解引發粒線體調控的內生性凋亡機轉，進而產生抗癌的活性。 內質網迫力的誘發被認為在肝癌生成的過程具有一定的影響力，然而對於其是在腫瘤新生還是死亡中有較重要的貢獻，依然是個混沌未清的難題，因此，於第二篇的研究中利用Tunicamycin抑制細胞內蛋白質的醣化(N-glycosylation)以造成內質網迫力，藉此探討內質網迫力之引發對於抑制人類肝癌細胞生長所扮演的角色。實驗結果顯示，Tunicamycin在人類肝癌細胞Hep3B與HepG2中抑制細胞生長的GI50分別為0.19與0.62 µg/ml，並可明顯的造成細胞週期於G1期累積以及細胞週期調控因子的減少，其中又以Cyclin D1與Cyclin A的表現最易受影響。此外p27與Cyclin D1/Cdk4複合物間的結合亦隨著Tunicamycin的處理而增加，對Tunicamycin引起的細胞週期G1期停滯提供了另一種解釋。於內質網迫力所引發的蛋白不完全摺疊反應方面，GRP78與CHOP這兩個標的蛋白表現亦明顯的增加，推測細胞週期調控因子的減少為內質網迫力造成細胞週期停滯與抑制肝癌細胞生長的主要原因。進一步利用antisense CHOP與GRP78 siRNA探討CHOP與GRP78的角色，結果顯示抑制GADD153的誘發無法回復Tunicamycin所造成的G1期停滯，對Cyclins與Cdks的表現下降亦沒有影響。抑制GRP78的表現，則可增加Tunicamycin在Hep3B中所引起的細胞凋亡，對於抑制細胞生長的能力亦有些微的促進。簡單來說，Tunicamycin造成細胞週期停滯在G1期的原因，推測是因為cyclin D1與cyclin A的表現量下降以及p27與Cyclin D1/Cdk4複合物之間的結合增加。內質網迫力所引發的兩個標的蛋白GRP78與GADD153對Tunicamycin抑制人類肝癌細胞Hep3B增生的能力沒有太大的貢獻，反倒是GRP78似乎還提供了點保護的角色。綜合來看，調控細胞週期的因子於內質網迫力造成之抑制癌細胞生長似乎扮演了最敏感的角色。 第三篇的研究是關於由牛樟芝分離出的antroquinonol於HepG2細胞所引起的抗癌機轉探討。本研究利用antroquinonol在多種肝癌細胞株以及人類肝臟胚胎細胞WRL-68中進行抑制細胞生長的活性測試，結果顯示，antroquinonol對於肝癌細胞與正常細胞有不錯的選擇性，於HepG2細胞中展現出最好的抑制細胞生長活性，活性之相對關係為HepG2> HepG2.2.15>Mahlavu> PLC/PRF/5>SK- Hep1>WRL-68。antroquinonol明顯的引發細胞週期停滯於G1期，並於沒有影響mRNA表現的情況下，造成多種調控G1期的細胞週期調控因子之表現與核內轉移的下降。進一步探討其成因，發現antroquinonol於不影響DNA轉錄與蛋白質降解的機制下，藉由抑制細胞內蛋白轉譯造成細胞週期調控因子的表現下降。Antroquinonol在短時間可以明顯的抑制p70S6K (Thr421、Ser424與 Thr389)與 4E-BP1 (Thr37、Thr46與Thr70)的磷酸化，進而阻斷蛋白質合成，此外TSC1與TSC2間的結合也在antroquinonol處理下有增加的現象。這些現象在併用AMPK抑制劑後，都可得到回復，推測AMPK在antroquinonol所引起的作用中扮演了重要的角色。此外粒線體的破裂與膜電位喪失也提供了AMPK活化的可能原因。簡單來說，antroquinonol利用活化AMPK阻斷mTOR訊息途徑，導致細胞內蛋白質生成受到抑制，最後造成細胞週期停滯進而產生抗癌效果。

關鍵字

類前列腺素 ； 死亡受器叢聚 ； 內質網迫力 ； 細胞週期停滯

並列摘要

Cancer is a high-risk malignant chronic disease worldwide. Although investigations of cancer progresses with each passing day, the treatment for cancer remains a tricky unsolved problem. There’s still a long way to go in the development of anticancer agents. Clinical therapeutic strategies are only made to treat but not cure cancer patients. There are good precedents for looking to nature for drug discovery. Over 60 percent of current anticancer drugs, such as vinca alkaloids, camptothecin and taxol, are derived in one way or another from natural sources. During new drug discovery, scientists usually aspire to get the lead compounds from natural sources and semi-synthesis, and use them as the templates to design derivatives with more potent activity base on the structure-activity relationship (SAR). Original investigations of natural products were limited to terrestrial resources through the exploitation and application of natural materials. However, with the improvement of technology, scientists paid more and more attentions to marine aquatic biological resources for the last several decades. In this thesis, we tried to identify the anticancer mechanism of several natural products in prostate cancer and human hepatocellular carcinoma. In the first study, we tried to identify the anticancer mechanism of Bromovulone III, isolated from soft coral Clavularia viridis, in hormone-independent prostate cancer. Cyclopentenone prostaglandins (PGs) such as PGA1, PGA2 and ∆12-PGJ2 have been shown to suppress tumor cell growth and to induce apoptosis in prostate cancer cells. Bromovulone III, which is isolated from the soft coral Clavularia viridis, is a cyclopentenone prostanoid. In this study, Bromovulone III displayed anti-tumor activity of 30 to 100 times more effective than PGA1, PGA2 and ∆12-PGJ2 in PC-3 cells. In our previous study, Bromovulone III triggered cell apoptosis in human hepatocellular carcinoma Hep3B cells via inducing ER stress. However, in this study,Bromovulone III displayed a totally different anticancer mechanism in prostate cancer PC3 cells. Several targets of caspases and Bcl-2 family of proteins were detected and the data demonstrated that Bromovulone III induced the activation of caspase-8, -9 and -3, and Bid cleavage in which the caspas-8 activation occurred first. Bromovulone III did not up-regulate the protein levels of death receptors and ligands. Of note, the Fas clustering in PC-3 cells responsive to bromovulone III was observed by confocal immunofluorescence microscopy suggesting the involvement of Fas-mediated pathway. Bromovulone III also induced the cleavage of Mcl-1 in this study. The cleavage fragments (24, 19 and 17 kDa) may partly share the apoptotic insult. Although it has been suggested that Fas-mediated signaling may contribute to the caspase-8 activation induced by DNAdamaging agents; however, bromovulone III did not induce any DNA breakage, suggesting that bromovulone III-induced Fas/caspase-8-dependent signaling is not through the direct target on DNA damage. In summary, the data suggest that bromovulone III causes a rapid redistribution and clustering of Fas in PC-3 cells. Subsequently, the Fas event causes the activation and interaction of caspase-8/Bid/caspase-9 signaling cascades, and the activation of executor caspase-3. The initiation of endoplasmic reticulum (ER) stress has been suggested to play potential roles in hepatocarcinogenesis. However, many obstacles remain as to whether ER stress plays a role in carcinogenesis or tumoricide. This secondary study sought to identify the signals that can serve as anticancer effectors in cells in response to ER stress. Tunicamycin (an N-glycosylation inhibitor) inhibited cell proliferation with IC50 values of 0.19 and 0.62 µg/ml in Hep3B and HepG2 cells, respectively. It induced G1 arrest of the cell cycle in both cell lines. The anticancer mechanism of tunicamycin was investigated in Hep3B cells. Tunicamycin induced a rapid decline of cyclin D1 and cyclin A expression and an early increase of glucose-related protein (GRP) 78 and growth arrest and DNA damage-inducible transcription factor (GADD) 153 levels. Cyclin D1 and was the most sensitive regulator to tunicamycin-triggered degradation mechanism. The association of p27 with cyclin D1/ Cdk 4 was also increased by tunicamycin. The inhibition of GADD153 expression by transfection of GADD153 antisense did not modify tunicamycin-induced G1 arrest and cyclin/Cdk expressions. The knockdown of GRP78 expression by the siRNA transfection technique moderately increased tunicamycin-induced apoptosis and the antiproliferative effect by sulforhodamine B assay. We suggest that tunicamycin induces G1 arrest through downregulation of cyclins and Cdks, in which cyclin A is more susceptible to ER stress-triggered degradation mechanism in Hep3B cells. The increased association of p27 with cyclin D1/Cdk4 may also contribute to tunicamycin-induced cell-cycle arrest. GADD153 and GRP78 play a minor role in tunicamycin-mediated antiproliferative effect, although GRP78 moderately inhibits apoptosis in Hep3B cells. These data provide evidence that cell-cycle regulators are susceptible factors in hepatocellular carcinoma (HCC) responsive to ER stress. In the third study, we tried to identify the anticancer mechanism of antroquinonol, isolated from Antrodia camphorate, In this study, antroquinonol presented great selectivity between HCCs and human liver embryonic cells, and induced anti-proliferative effect with the rank order of potency against HCCs being HepG2>HepG2.2.15>Mahlavu> PLC/PRF/5>SK-Hep1>WRL-68. Antroquinonol caused G1 arrest of the cell-cycle. The data were supported by down-regulation and reduced nuclear translocation of G1-cyclins and Cdks and further analysis showed the inhibition of translational but not transcriptional and protein degradational levels. Antroquinonol blocked cellular protein synthesis through inhibition of phosphorylation of p70S6K (Thr421/Ser424 and Thr389) and 4E-BP1 (Thr37/Thr46 and Thr70). The TSC1/TSC2 association was also increased by antroquinonol. These effects were significantly reversed by selective AMPK inhibitor, suggesting the central role of AMPK to antroquinonol action. The cellular stress on mitochondria may partly explain antroquinonol-mediated AMPK activation. Taken together, the data suggest that antroquinonol displays anticancer activity against HCCs through AMPK activation and inhibition of translational pathway that induce G1-arrest of the cell-cycle and subsequent apoptosis.

並列關鍵字

Prostanoid ； Fas clustering ； ER stress ； cell cycle arrest ； mTOR

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