第一型組織胺受體拮抗劑在人類雄性激素不依賴型前列腺癌細胞的抗癌作用機轉研究

Study of Anticancer Mechanism of H1 Receptor Antagonists in Human Androgen-Independent Prostate Cancer Cells

王韋婷(Wei-Ting Wang)

指導教授 ： 陳燕惠

共同指導教授 ： 顧記華(Jih-Hwa Guh)

國立臺灣大學/醫學院/藥學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03398

摘要

惡性腫瘤為世界死因排名的第一位，其中，前列腺癌為全球男性罹患率第一高且死亡機率為第六位。近年來，國人前列腺癌的發生率與死亡率有逐漸增加的趨勢。雖然目前有許多前列腺癌的治療方法，像是手術治療、放射線治療、荷爾蒙阻斷療法及化學療法；但是，一旦荷爾蒙阻斷後疾病仍持續惡化則稱為荷爾蒙抗性前列腺癌，針對此種類型的前列腺癌其治療方法仍然有限。因此，發展有效治療荷爾蒙抗性前列腺癌的藥物為當前之急。
在本研究中，我們探討四種不同的H1組織胺受體拮抗劑在人類荷爾蒙不依賴性前列腺癌中的抗癌機制。首先，發現在四種H1組織胺受體拮抗劑中 (包含Terfenadine、Loratadine、Fexofenadine及Diphenhydramine)，其中以Terfenadine抑制癌細胞生長的效果最佳，而且在PC-3和DU-145細胞都有抑制的作用，因此，選擇Terfenadine作後續更深入的抗癌機制探討。接著，由流式細胞儀的分析結果發現，細胞週期之subG1期的細胞數會增加，代表Terfenadine會造成細胞凋亡的現象。利用西方墨點法偵測與細胞凋亡相關的蛋白表現量，結果發現Terfenadine的作用下會迅速地造成促凋亡蛋白Bak表現量增加，及抗凋亡蛋白Mcl-1產生cleavage (28kDa)，而且此現象與時間呈正相關性；接著會造成後續PARP-1蛋白的斷裂及Caspase-9、-7和-3的活化。此外，藉由可以辨認Bak活化時之特殊構型的抗體進行偵測，結果發現具有功能性的Bak增加後會使得粒線體膜電位下降，進一步造成凋亡因子cytochrome C和apoptosis inducing factor (AIF) 釋出到細胞質中。另外，同時利用RT-PCR與Real-time PCR的技術偵測，發現在轉錄的調控上使Bak mRNA增加，進而蛋白表現量上升。當藥物處理到晚期的時間點，亦會影響到DNA損傷相關蛋白的表現量，導致DNA損傷的發生。總體而言，Terfenadine會先造成有功能性的Bak蛋白表現量增加及Mcl-1蛋白的降解，進一步影響粒線體的結構，使得粒線體膜電位下降後，造成caspases的活化及DNA損傷，最終導致細胞死亡。

關鍵字

前列腺癌 ； 組織胺受體拮抗劑 ； 內生性細胞凋亡

並列摘要

Cancer is the world's leading cause of death. Among these cancers, prostate cancer is the most frequently diagnosed cancer and the sixth leading cause of cancer death in men. In Taiwan, the occurrence and mortality rate of prostate cancer has increased over the past few years. There are several therapies for prostate cancer, such as surgery, radiation therapy, hormone deprivation therapy, and chemotherapy. However, the therapies for treating patients with hormone-refractory prostate cancers (HRPCs), which are become resistant to hormone therapy, are limite. Therefore, novel therapeutic agents for treating HRPC are urgently demanded.
In this study, we have demonstrated the anticancer mechanism of H1-receptor antagonists in human HRPCs. First, the effect of four H1-receptor antagonists, including Terfenadine, Loratadine, Fexofenadine, and Diphenhydramine on cell proliferation was examined. The data showed that Terfenadine was the most potent, displaying an antiproliferative activity in both human HRPC cell lines PC-3 and DU-145. We choosed Terfenadine to do further study in detail. The flow cytometric analysis showed that terfenadine induced an increase of subG1-phase population of the cell cycle. By using Western blot analysis, we found that Terfenadine rapidly induced Bak up-regulation and Mcl-1 degredation in time-dependent manner, leading PARP-1 cleavage and proteolytic activation of caspase -9, -7, and -3. Moreover, by using specific antibody to recognize activated Bak, it showed that the increase of functional Bak was correlated to the loss of mitochondria membrane potential, subsequently leading to the release of cytochrome C and apoptosis-inducing factor (AIF) to cytosol. We also found that Bak up-regulation in protein levels was due to increase of mRNA levels. The DNA damage was also induced by Terfenadine and was associated with the proteolytic activation of capsase cascades, which in turn caused further up-regulation of DNA-damage related proteins.
In summary, the data suggest that terfenadine induces up-regulation of functional Bak and Mcl-1 degradation, leading to the loss of mitochondrial membrane potential and the activation of caspase cascades that ultimately induce DNA damage and apoptotic cell death.

並列關鍵字

prostate cancer ； terfenadine ； bak

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