探討LYF-5與anthracenedione衍生物在人類荷爾蒙不依賴型前列腺癌細胞的抗癌作用機轉

Investigation of anticancer mechanism of LYF-5 and anthracenedione analogues in hormone- resistant prostate cancer PC-3 Cells

詹雅玲(Ya-Ling Chan)

指導教授 ： 顧記華

國立臺灣大學/醫學院/藥學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10081

摘要

在歐美等西方國家，前列腺癌為男性好發的癌症之一，在美國每年約有三萬人死於前列腺癌；在台灣，前列腺癌也晉升為十大癌症死亡因素。但是就目前為止，對於前列腺癌的治療尚未有一個有效的藥物，有鑑於此，發展一個有效對抗前列腺癌的抗癌藥物，對於癌症的治療是必須的。於是在本篇論文的第一部分中，我們使用sulforhodamine B (SRB)的分析方法，以男性素不依賴型的前列腺癌細胞株PC-3為細胞模式，針對多種化合物進行抗癌活性篩選。我們發現化合物LYF-5(3’-methoxyl- 2-phenyl-6-methyl-1,8-naphthyridine-4-one)能有效的抑制癌細胞的生長，其達到50%的抑制濃度(IC50)為0.12 μM。因此，我們便進一步探討其抗癌作用機轉。我們使用TUNEL做螢光染色，證實LYF-5的確會造成細胞的凋亡作用(apoptosis)。我們也以propidium iodide結合到細胞DNA，利用流式細胞儀來分析探討LYF-5對於細胞週期的影響。結果發現在LYF-5的作用下，能促使細胞的週期停止在G2/M期，並在作用24小時之後明顯的引起細胞subG1期(Hypodiploid phase，代表細胞凋亡)的增加。使用共軛焦顯微鏡觀察LYF-5對細胞微小管(microtubule)的作用，發現LYF-5能直接影響微小管的聚合作用。接下來，我們利用西方墨點法來探討Bcl-2家族蛋白、caspases以及其他與細胞凋亡有關的蛋白在LYF-5作用下的表現情形。結果顯示，LYF-5能造成Bcl-2與Bcl-xL的磷酸化，並使得Mcl-l的表現下降；同時也發現在LYF-5作用之下會造成Bcl-2與Mcl-1的斷裂。在caspases方面，caspase-3、caspase-8與caspase-9皆受到明顯的活化；另外LYF-5也會抑制XIAP(X-type inhibitor of apoptosis protein)與cIAP2的表現。 最後，我們使用共軛焦顯微鏡進行免疫螢光染色的實驗，以觀察在LYF-5的作用之下是否會造成粒線體中cytochrome c的釋放作用；由實驗的結果顯示LYF-5的確會造成細胞cytochrome c釋放至細胞質中。綜合以上的結果，我們可以認為LYF-5造成癌細胞進行細胞凋亡作用，首先是藉由直接影響微小管而造成細胞週期停止在G2/M期，之後磷酸化Bcl-2以及Bcl-xL這兩個抑制細胞進行凋亡的蛋白，並造成Mcl-1蛋白的裂解作用，接下來活化一系列的caspases蛋白並促使cytochrome c的釋放，最後導致細胞進行凋亡作用。
而在本論文的第二部份中，主要是針對一系列anthracenedione衍生物在前列腺癌細胞PC-3的抗癌作用，對其結構與作用的相關性以及可能的作用機轉做探討。藉由SRB assay的篩選，我們找到其中抗癌活性最強的化合物為WRC-76，其IC50是0.28 μM。為了了解這一系列化合物的作用機轉，我們以WRC-76為代表，觀察其對於細胞週期的影響。由實驗結果得知WRC-76能造成細胞的週期停止於S期，在長時間作用下能造成細胞的週期停止於G2/M期；並且高濃度的WRC-76會使細胞進行凋亡作用。接下來我們使用螢光染色的方式觀察WRC-76對於細胞微小管的形成是否造成影響，由結果發現WRC-76並不會影響微小管的形成作用。為了更進一步了解WRC-76的作用機轉，我們使用西方墨點法觀察WRC-76對於細胞內蛋白的影響，由實驗結果得知在藥物作用之下，Bcl-2蛋白家族只有Mcl-1的表現受到抑制。另外，WRC-76能藉由活化caspase-8、caspase-9與caspase-3來導致細胞凋亡。Death Receptorreceptor包括DR4、DR5以及Fas，以及他們的Ligandligand包括TRAIL以及FasL在WRC-76作用之下，皆不受到影響。另外我們使用螢光顯微鏡觀察細胞內Fas聚集(clustering)的情形，由實驗結果發現WRC-76並不會造成Fas的聚集作用。XIAP與survivin主要的功能為抑制caspases的活化，在WRC-76的作用之下，XIAP的表現量減少，這與細胞凋亡作用的結果是一致的，但是survivin卻不受影響。Caspase-12、CHOP以及CRP78是內質網受到壓力(Endoplasmic reticulum stress)會被活化的蛋白。在WRC-76作用下可以觀察到casapase-12以及CHOP的活化。並且在WRC-76的作用之下，能夠導致JNK以及p38這兩個MAP kinase的活化。綜合以上結果得知，WRC-76除了藉由活化caspase-8、caspase-9以及caspase-3的路徑導致細胞凋亡外，WRC-76另一方面還會造成內質網壓力，並活化caspase-12造成細胞凋亡。而在WRC-76作用之下可以看到JNK與p38這兩個MAP kinase的活化，而這兩個MAP kinase也同樣使細胞走向凋亡。

關鍵字

細胞凋亡 ； 前列腺癌 ； 微小管 ； 染色體

並列摘要

Prostate cancer is the most frequently diagnosed malignancy in males in the United States and most other industrialized Western countries. It kills more than 30,000 American men yearly and it is one of the top 10 deadliest cancers in Taiwan. There have been no therapeutic agents that provide a survival advantage in prostate cancer. Therefore, it is necessary to develop novel agents for the treatment of hormone-refractory prostate cancer.
In the first part of this study, we used human hormone-resistant prostate cancer cell line PC-3 to investigate the anti-tumor effect and mechanism of compound LYF-5 (3’-methoxyl-2-phenyl-6-methyl-1,8-naphthyridine-4-one). LYF-5 inhibited cell growth in a dose-dependent manner with an IC50 of 0.12 μM by sulforhodamine B (SRB) assays. Using FACScan flow cytometric analysis, the data showed that LYF-5 induced the arrest of cells in G2/M phase of the cell cycle and a subsequent increase of hypodiploid sub-G1 cells. The apoptotic cell death was also identified by TUNEL reaction technique. Immunofluoresence microscopic examination indicated that LYF-5 had an action target on tubulin/microtubule. Several apoptosis-related proteins were examined in this study. After the detection of expression of Bcl-2 family members by Western blotting, the data showed that LYF-5 induced phosphorylation of Bcl-2 and Bcl-xL, and Mcl-1 down-regulation. In addition, the cleaved forms of Mcl-1 was also apparent. Moreover, LYF-5-induced apoptosis was associated with processing of pro-caspase-8, pro-caspase-3 and pro-caspase-9, indicating the involvement of caspase activation. The expression of XIAP and cIAP2 were decreased after treating with LYF-5. Furthermore, we used immunofluoresence microscopic examination to examine the release reaction of cytochrome c. The data showed that LYF-5 significantly induced the release of cytochrome c from mitochondria. Taken together, it is suggested that LYF-5 induces apoptotic cell death through the target on tubulin/microtubule, the induction of mitotic arrest of the cell cycle, phosphorylation and cleavage of anti-apoptotic Bcl-2 family protein members, the activation of caspase and the release of cytochrome c.
In the second part of this study, the structure-activity relationship of a series of anthracenedione derivatives on anticancer activity and their action mechanisms in human hormone-resistant prostate cancer PC-3 cells were investigated. By using SRB assays, we found that several compounds displayed anticancer activity and amongst, WRC-76 was the most effective one with an IC50 of 0.28 μM. Accordingly, WRC-76 was selected for the mechanism study. The data showed that WRC-76 induced the arrest of the cell cycle in S and G2/M phase and a subsequent increase of hypodiploid sub-G1 phase. Immunofluoresence microscopic examination indicated that WRC-76 had no effect on tubulin/microtubule function. Several apoptosis-related proteins were examined in this study. After the detection of expression of Bcl-2 family members by Western blotting, the data showed that the expression of Mcl-1 was decreased after theatment with WRC-76. Moreover, WRC-76-induced cell apoptosis was associated with processing of pro-caspase-8, pro-caspase-3 and pro-caspase-9. The expression of XIAP but not survivin was inhibited by WRC-76. Caspase-12, CHOP and GRP78 are three ER stress markers and the expression of caspase-12 and CHOP were significantly increased by WRC76. Furthermore, JNK and p38 MAP kinases involved pathways were also activated in cells responsive to WRC-76. Taken together, it is suggested that WRC-76 induces apoptotic cell death through the activation of caspase-8, -9 and -3. WRC-76 also induces the ER stress of increase of CHOP expression and caspase-12 activity. Furthermore, the activation of JNK and p38 MAPK may, at least partly, explain WRC-76-induced apoptotic cell death.

並列關鍵字

apoptosis ； prostate cancer ； microtubule ； DNA

參考文獻

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