ERMAs與protopine在癌細胞中引起細胞凋亡之機轉探討以及sorafenib與MPT0E028併用之抗癌效果

Abstract

癌症在全球來說，是已開發國家死亡原因之首，在開發中國家也高居死亡原因第二名。在台灣，惡性腫瘤自民國71年起已經連續29年高居台灣人民十大死因之首。因此，癌症對全世界人類的健康與生命無疑是一大威脅，也是科學家們亟欲解決的問題。本篇論文首先以前列腺癌為研究對象，分別針對細胞能量代謝與抗微管藥物探討其引起細胞凋亡的機轉。此外，也以肝癌細胞為對象，評估標靶藥物sorafenib與HDAC inhibitor-MPT0E028合併治療的效果。 本論文的第一部分以前列腺癌細胞株(LNCaP)為主要的研究模式，探討 energy restriction-mimetic agent (ERMA)引起細胞凋亡的機轉。我們發現新穎的ERMA-CG-12、2-dexoyglucose (2-DG)以及glucose starvation能夠經由epigenetic effect增加抑癌基因KLF6的轉錄；利用sh-RNA knockdown KLF6能夠反轉CG-12所引起的細胞凋亡。利用chromatin immunoprecipitation，我們證實KLF6轉錄增加的原因，主要與該基因啟動子附近分佈的acetyl-H3與histone H3K4Me3等histone markers增加有關。多重證據顯示，這個現象是因為Sp1的表現減少有關，進而抑制histone deacetylase (HDAC)與histone H3K4 demethylase的轉錄作用。在本篇研究中，我們釐清了能量限制與epigenetic調控抑癌基因KLF6之間的關係，此發現也提供了前列腺癌治療方面的新契機。 抗微管藥物(microtubule-targeting agent, MTA)在癌症病患的化學治療是很重要的，像是docetaxel目前被核准用來治療晚期前列腺癌的病人。然而這類的藥物在臨床上因為其抗藥性的產生，降低其廣泛的應用性，因此開發新的MTA有其急迫性。在本篇論文的第二部分，我們以人類的hormone-refractory prostate cancer (HRPC)細胞株為實驗材料，發現protopine具有抑制細胞生長以及促進細胞週期停滯在G2/M的能力，最後會引起細胞凋亡。透過 in vitro tubulin polymerization assay 以及 in situ labeling of β-tubulin的實驗，我們證實protopine為新穎的微管穩定劑(microtubule-stabilizing agent)。此外，protopine能夠活化Cdk1/cyclin B1 complex，進而增加Bcl-2的磷酸化與減少Mcl-1的表現，造成細胞凋亡。最後我們也排除了protopine是P-glycoprotein受質的可能性，且其結構較為簡單，因此protopine具有許多優點，值得當做一個先導藥物進一步開發。 Sorafenib 為一個口服的multiple tyrosine kinase inhibitor (TKI)， 目前用來治療晚期renal cell carcinoma (RCC)與hepatocellular carcinoma (HCC)的病人。過去的文獻指出histone deacetylase (HDAC) inhibitor像是trichostatin A、SAHA和MS-275等，能夠在HCC細胞中引起細胞凋亡；在動物實驗中也發現能夠抑制細胞生長與血管新生作用。在本篇論文的第三部分我們利用一個新穎的HDAC inhibitor-MPT0E028合併使用sorafenib，探討其在HCC的抗癌效果。實驗結果發現subG1-phase、nucleosome fragmentation、caspase 3 activation與PARP cleavage都有增加的現象，顯示兩者併用能夠增加細胞凋亡的現象。此外，我們也利用microarray來尋找受到藥物影響的基因，並且進行訊息傳導路徑的分析。在Hep 3B xenograft的動物實驗也發現兩者併用對於tumor growth delay有明顯的幫助。綜合以上發現，我們發現MPT0E028與sorafenib合併使用的確在HCC中有協同效果，值得進一步探討其抑制細胞生長的相關機轉。

Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Since 1982, cancer remains the top cause of death in Taiwan. Hence, cancer is undoubtedly the most life-threatening disease in the world and there is an urgeny to resolve the problem. In the first two parts of this thesis, we used prostate cancer cell lines as materials to investigate the mechanism of energy-restricted mimetic agents (ERMAs)- and protopine-induced apoptotic cell death. In the third part, we examined the synergistic effect of the targeted-therapy agent (sorafenib) and HDAC inhibitor (MPT0E028) in hepatocellular carcinoma (HCC). In the first part of this thesis, we investigated the underlying mechanisms of ERMA-induced apoptosis in LNCaP prostate cancer cells. By using a novel thiazolidinedione-derived ERMA, CG-12, 2-deoxyglucose (2-DG) and glucose starvation, we obtained the evidence that the tumor suppressor gene Kruppel-like factor 6 (KLF6) was elevated by epigenetic activation and shRNA-mediated KLF6 knockdown abrogated the ability of CG-12-induced apoptosis. Chromatin immunoprecipitation analysis indicated that this KLF6 transcriptional activation was associated with increased histone H3 acetylation and histone H3 lysine 4 trimethylation occupancy at the promoter region. Several lines of evidence demonstrate that the enhancing effect of ERMA on these active histone marks was mediated through transcriptional repression of histone deacetylases and H3 lysine 4 demethylases by down-regulating Sp1 expression. Together, these findings underscore the intricate relationship between energy restriction and epigenetic regulation of tumor suppressor gene expression, which has therapeutic relevance to foster novel strategies for prostate cancer therapy. Microtubule-targeting agents (MTAs) such as docetaxel, which is approved for treatment of metastatic castration-resistant prostate cancer, are important anti-cancer drugs. However, the development of drug resistance limited their extensive usefulness and there has been an impetus to develop novel agents to benefit patients with drug-resistant tumors. In the second part of this thesis, we identified that protopine exhibited an antiproliferative effect, causing cell-cycle arrest at the G2/M phase and leading to robust apoptosis in hormone refractory prostate cancer (HRPC) cells. From the in vitro tubulin polymerization assay and in situ labeling of β-tubulin in drug-treated cells, our data suggest that protopine is a novel microtubule-stabilizing agent. The data suggest that protopine increased the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex and that contributed to cell apoptosis by modulating mitochondria-mediated signaling pathways, such as Bcl-2 phosphorylation and Mcl-1 down-regulation. These findings underscore the novel effect of protopine as a microtubule-stabilizing agent with poor susceptibility to P-glycoprotein and simpler structure, which may foster novel strategies for prostate cancer therapy. Sorafenib is an orally active multiple tyrosine kinase inhibitor (TKI) approved for the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). It has been reported that histone deacetylase (HDAC) inhibitors, such as trichostatin A, SAHA and MS-275, have been shown to induce apoptosis in HCC cells and to inhibit growth of HCC by inhibiting proliferation and tumor-related angiogenesis in vivo. In the third part of this thesis, we examined the synergistic effect by the combination of a novel HDAC inhibitor MPT0E028 with sorafenib in HCC. We observed the induction of sub-G1 phase of HCC cells, nucleosome fragmentation, caspase-3 activation as well as poly (ADP-ribose) polymerase cleavage, indicating there is an increased apoptotic cell death. Moreover, we also evaluated the genes affected by drug combination through microarray and pathway analysis. In the Hep 3B xenograft model, we found that drug combination significantly delayed the tumor growth rate. Base on these findings, we demonstrated a synergistic effect of MPT0E028 in combination with sorafenib in HCC and it is worthy of further investigating the underlying mechanism of this anti-cancer effect.