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  • 學位論文

AR42, QS-ZYX-1-61,及erolotinib和MPT0E028併用之抗腫瘤藥效與機制探討

Effects and action mechanisms of AR42, QS-ZYX-1-61, and combination of erlotinib-MPT0E028 in cancer cells

指導教授 : 鄧哲明
共同指導教授 : 陳慶士 潘秀玲(Shiow-Lin Pan)
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摘要


在台灣的十大癌症死亡率排行榜中,肺癌與肝癌分別為第一、二名。即使經過幾十年的研究,至今我們依然在找尋更好的治療策略。 Topo II在哺乳類細胞的分裂過程中是一個必須的酵素,並用來維持DNA的topology,在腫瘤細胞中也常見topo II過度表現的現象。作用在topo II的抗癌藥物可以導致DNA的斷裂以及引起致命的細胞凋亡。除了傳統的化學治療,作用在HDAC上的HDAC inhibitors以及作用在其他標靶的藥物(例如EGFR)都廣為被用來研究癌症的治療。在本篇論文當中,我們就以HDAC inhibitor,topo II-targeting drug以及合併治療為研究對象,探討在腫瘤細胞內所調控的訊息傳遞。 HDAC inhibitors可以干擾HDAC的活性,對於藥物開發針對廣泛的抗癌治療是很有希望的。第一部分,我們報導的HDAC inhibitor,AR42(也稱之為OSU-HDAC42),之前已經發現 AR42在in vivo可以有效的抑制肝癌細胞,且這現象是經由藥物作用在histone acetylation-dependent以及-independent的兩個路徑。在本篇我們則是報導AR42在人類肝癌細胞(hepatocellular carcinoma)中引起topo IIα降解的新作用機制。研究結果顯示此一HDAC inhibitor可以藉由增加acetylated histone H3至CK2α的promoter達到活化CK2α的轉錄,接著CK2α可以促進topo IIα磷酸化並藉著磷酸化的topo IIα和COP9其中的subunit Csn5相結合。我們更進一步發現一個會和Csn5有交互作用的F-box蛋白-Fbw7,為負責此topo IIα降解的E3-ligase。 本論文的第二部分則是探討一個新的etoposide衍生物,QS-ZYX-1-61,在A549細胞中的藥效強度(IC50)比etoposide高100倍且具備良好的活性造成DNA雙股斷裂,並啟動DNA damage response導致最後的細胞凋亡。藥物處理之下可以活化ATM、Chk2並造成細胞內p53的累積。為了研究QS-ZYX-1-61對於topoisomerase的作用,利用DNA relaxation assay以及band-depletion assay發現藥物可以抑制topoisomerase II的酵素活性並在細胞內形成可逆的cleavable complexes。我們若更進一步的減少細胞內p53, topo IIα, IIβ的表現,則可以減少QS-ZYX-1-61原本所活化的caspase 3、PARP、以及細胞生長的抑制,顯示藥物在細胞內造成細胞凋亡需要經由這些蛋白的作用。我們的研究更證實QS-ZYX-1-61為一個新的DNA damage agent並值得應用於非小型肺癌細胞的治療。 Erlotinib(Tarceva)為口服的小分子標靶抑制劑,以可逆的方式結合到EGFR細胞內的tyrosine kinase domain,用來為治療非小型細胞肺癌,如此一來就會阻斷EGFR的autophosphorylation以及下游的訊息傳遞。本論文的最後一個部分,則是利用測量細胞的生長抑制、細胞週期的分佈、以及細胞凋亡訊號的表現,專注於erlotinib-resistant的細胞A549來做抗癌效果的觀察學習。當細胞暴露在erlotinib和新的口服HDAC inhibitor MTP0E028時,可以在erlotinib-resistant的人類非小型細胞肺癌(A549, H1299以及NCI-H1975)內觀察到加成性的毒殺作用。在in vitro我們可以看到抑制細胞生長、acetylated histone H3以及acetylated tubulin的增加、PARP cleavage,γH2AX的增加,以及caspase-3的活化。同時也在癌症動物模式中確認到相同的細胞訊號反應,也證實此合併投予藥物在動物體內為可容忍劑量。根據這些發現,可以在非小型細胞肺癌內評估合併TKI (tyrosine kinase inhibitor)以及HDAC inhibitor的策略,並分析導致此加成性藥理作用的分子機制。

關鍵字

肝癌 肺癌 細胞凋亡 DNA損害 拓撲異構酶

並列摘要


According to recent reports on the top ten leading causes of cancer death, lung cancer and liver cancer still take first and second place in Taiwan. After decades of research, we are still trying to find better therapeutic strategies to cure cancers. Topoisomerase II is an essential enzyme to maintain DNA topology in mammalian cells and is often overexpressed in tumor cells. Targeting topoisomerase II in cancer treatment leads to DNA breaks and lethal apoptotic cell death. In addition to conventional chemotherapy, HDAC inhibitors, and other target theapies also have been studied and evaluated for cancer treatment strategy. In this thesis, we investigated HDAC inhibitor, topo II-targeting drug, and combination cancer therapy in regulating tumor cells signalings. HDAC inhibitors that interfere HDAC activity have been shown promising evidences in drug development for broad spectrum anti-cancer therapy. In the first chapter, we reported a novel phenylbutyrate-derived histone deacetylase (HDAC) inhibitor AR42 (formerly OSU-HDAC42) exhibiting high in vivo potency in suppressing HCC tumor growth, which was previously shown attributable to its ability to target both histone acetylation-dependent and -independent pathways. Here we reported a novel pathway by which HDAC inhibitor AR42 mediated topo IIα proteolysis in HCC cells. Our data indicate that this HDAC inhibitor (AR42) transcriptionally activated casein kinase (CK)2α expression through increased association of acetylated histone H3 with the CK2α gene promoter. In turn, CK2 facilitated the binding of topo IIα to COP9 signalosome subunit (Csn)5 by way of topo IIα phosphorylation. Furthermore, we identified Fbw7, a Csn5-interacting F-box protein, as the E3 ligase that targeted topoIIα for degradation. In the second chapter, we evaluated a novel etoposide analogue, QS-ZYX-1-61, which exhibits 100 folds more potency than etoposide in A549 cells. QS-ZYX-1-61 displays an outstanding activity by which induces DNA double-strand breaks and DNA damage response signaling. We showed activation of ATM (ataxia telangiectasia mutated), Chk2 (checkpoint kinase 2), and accumulation of p53 after QS-ZYX-1-61 treatment in A549 cells. To determine the effect of QS-ZYX-1-61 on topoisomerase, we used DNA relaxation assay and band-depletion assay to show that QS-ZYX-1-61 inhibits enzyme activity of topo IIα and induces reversible cleavable complexes in cells. Moreover, blocking of p53, topo IIα, and topo IIβ greatly protected against caspase-3 activation, poly-ADP-ribose polymerase cleavage and cell growth inhibition, indicating that QS-ZYX-1-61 acts through these proteins. Our data suggest that QS-ZYX-1-61 is a novel DNA damage agent that could be applied as targeted therapeutic drug for NSCLC. Tyrosine kinase inhibitor (TKI) elotinib (Tarceva) is an oral small molecule inhibitor which reversibly binds to the intracellular tyrosine kinase domain of epidermal growth factor receptor and is used to treat non-small cell lung cancer. This agent blocks autophosphorylation of EGFR with subsequent inhibition of downstream signaling pathways. In the last chapter, we focused on erlotinib-resistant cells (A549 cells) and studied the antitumor effect by measuring growth inhibition, cell cycle distribution, and expression of apoptotic signals by immunoblots. When cells were exposed to erlotinib and MPTOE028, a novel pan-HDAC inhibitor, cytotoxic synergism was observed in erlotinib-resistant human NSCLC cell lines (A549, H1299 and NCI-H1975). We showed the synergistic effect of MPTOE028 in combination with erlotinib, exhibiting EGFR downregulation, growth inhibition, increased level of acetylated histone H3 and acetylated tubulin, PARP cleavage, γH2AX increased, and caspase-3 activation in vitro. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. Based on these findings, the combination of tyrosine kinase inhibitor and HDAC inhibitor for relapsing NSCLC has to be evaluated and analyzed regarding the molecular basis for this synergistic effect.

參考文獻


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