探討 vincristine-vorinostat 合併使用與微管抑制劑 MPT0B392 之抗癌機轉及eIF4E binding protein 1 在癌症中的重要性

Abstract

目前最新的世界衛生組織統計，癌症是世界上致死率與發病率的主要原因之一，在 2012 年有八百二十萬的人口死於癌症相關疾病，且預測在未來的 20年裡，新的案例會以每年成長 57% 的速率增加。而且根據台灣衛生福利部的統計結果，在過去的三十年裡，癌症在台灣的十大死因中始終位於首位。尋求理想的合併療法以降低抗癌藥物的毒性和減少抗藥性的產生，發展新的小分子藥物來改善目前臨床上抗癌藥物使用的瓶頸，以及建立重要的癌症預後因子並根據其研發新穎的分子標拔藥物一直是治療癌症的最終目標。在此篇論文中，我們將研究 (1) vincristine 和 vorinostat 合併使用在人類急性淋巴性白血病的作用機轉，(2) 探討 MPT0B392 在人類急性白血病中的抗癌機轉，和 (3) 評估 eIF4E binding protein 1 (4E-BP1) 對於人類大腸直腸癌在臨床上的重要性。 Vincristine 是一種傳統的微管抑制劑，在臨床上常用於治療急性淋巴性白血病的合併療法中；vorinostat，俗稱SAHA，是第一個被 FDA 認可使用在 T 細胞淋巴瘤的廣泛型組織蛋白去乙醯酶抑制劑 (HDAC inhibitor)。在此論文的第一部分中，我們將著重 vincristine 和 vorinostat 合併使用之抗白血病機轉探討，由實驗結果發現當合併使用 vincristine 和 vorinostat後使得白血病細胞加成性的停留在 G2M 期，並隨之明顯地累積在 subG1 期；低濃度的 vincristine 會影響微管的動態平衡，在加入 vorinostat 之後會增加此現象，主要是因為 vorinostat 會藉由抑制 HDAC6 的活性因而促進微管蛋白的乙醯化進而使得微管的動態平衡改變；且在當我們併用 vincristine 和 HDAC6 專一性抑制劑 (tubastatin A 和 Acy1215) 也觀察到此加成的現象。除此之外， vincristine-vorinostat 的合併使用在小鼠體內異種移植的動物實驗中也具有較佳的療效且耐受性良好。根據這些結果，合併使用微管抑制劑和組織蛋白去乙醯酶抑制劑可以做為臨床上治療急性淋巴性白血病的基礎概念。 在第二部分中，我們探討一個全新可口服的 quinoline 衍生物-MPT0B392 (B392) 在急性白血病的抗癌機轉。B392 會使得人類急性白血球細胞滯留在 G2M 期，但最終還是會進行細胞凋亡，從小鼠異體移植的切片染色得知，投與 B392 會增加 cleavage-caspase 3的表現。B392 也會抑制微管的聚合作用，並且不會影響 P-gp 的活性，同時會活化 mitotic spindle checkpoint 以及 c-Jun-N-terminal-kinase (JNK)，而 JNK 的磷酸化會進一步流失粒線體的膜電位以及各種 caspases 的活化和 PARP cleavage。另外，我們也發現 B392 在對 sirolimus (mTOR 抑制劑) 具有抗性的細胞中會增強sirolimus 的細胞毒性。因此，由以上結果可知，不論是單獨使用或是合併療法，B392 是個具有發展潛力的抗白血病藥物。 4E-BP1 (eIF4E binding protein 1) 在 cap-dependent 以及 cap-independent 轉譯的路徑中扮演著關鍵的調控角色，也因此控制了蛋白質的生合成。在第三部分，我們認為 4E-BP1 在大腸直腸癌中是個重要的預後因子。在人類大腸直腸癌細胞株與癌化的組織中4E-BP1 蛋白質的表現量遠高於正常的大腸細胞株和組織，且藉由分析臨床病人的病理參數發現 4E-BP1 蛋白的表現量與疾病進程具有統計上的相關性。在缺氧的情況下，4E-BP1 在 cap-independent 轉譯過程中更顯重要；此外，cryptopleurine 衍生物 YXM110 也已被證實會藉由抑制 4E-BP1 的表現來達到腫瘤抑制作用，因此，我們更進一步去研究在缺氧情況下 YXM110 抑制蛋白質生合成的情況。由結果得知，YXM110 可以經由抑制 HIF-1

Cancer is the leading causes of mortality and morbidity worldwide with approximately 8.2 million cancer related deaths in 2012 and is expected that annual new cases will rise 57% within the next two decades as reported by World Health Worldwide. According to the statistics from Taiwan’s Ministry of Health and Welfare, cancer has taken the first position in causes of death in Taiwan in the past three decades. Searching for the optimal combination therapeutic approaches to decrease drugs toxicities and resistance, developing novel small molecular agents to improve current treatments and identifying the important prognostic factors to design the targeted therapies are our ultimate goals of eliminating cancers. In this thesis, we investigated (1) the effect of vincristine-vorinostat combination on human acute lymphoblastic leukemia (ALL), (2) the mechanism underlying anticancer activity of MPT0B392 on human acute leukemia and (3) the clinical importance of eIF4E binding protein 1 (4E-BP1) on human colorectal cancer (CRC). Vincristine, a traditional microtubule-depolymerizing agent, is one of combination chemotherapy drugs used in treatment of ALL; vorinostat, also called SAHA, was the first approved pan-HDAC inhibitor for T cell lymphoma. In the first study, we investigated the vincristine and vorinostat combined effect and the underlying antileukemic mechanism. Our data showed that combination of vincristine and vorinostat induced cells arrest synergistically in the G2M phase, following by obviously accumulation in the subG1 phase. Low concentration of vincristine caused microtubule dynamic changes, and this phenomenon was enhanced by co-incubated with vorinostat, which inhibited HDAC6, causing tubulin acetylation, also leading to microtubule instabilities. This result was consistent with vincristine in combination with HDAC6 inhibitors, tubastatin A and Acy1215. In vivo xenografting experiments corroborated the in vitro activity and tolerability of the combination. Based on these findings, the combination of microtubule depolymerizing agents and HDAC inhibitors can provide the basis of clinical treatment for ALL. In the second study, we elucidated the underlying mechanisms of a novel quinoline derivative, MTP0B392 (B392), which was specially designed for oral administration. B392 induced acute leukemic cells arrest in the G2M phase and ultimately led to apoptosis, which was corroborated by the observation of cleavaged-caspase 3 by immunohistochemistry staining in B392-treated groups of xenograft experiments. B392 also suppressed microtubule polymerization with less susceptibility to P-gp activity, activated mitotic spindle checkpoint as well as caused activation of c-Jun-N-terminal-kinase (JNK), which further contributing to mitochondrial membrane potential loss, caspases and PARP cleavages. Furthermore, B392 had an ability to enhance the cyototoxicity of sirolimus (mTOR inhibitor) in the sirolimus-resistant cells. Our results suggest that B392 is a potential antileukemic drug that could be applied in mono- and combination therapy. 4E-BP1 (eIF4E binding protein 1) is a key regulator in cap-dependent and independent translation, thereby controlling protein synthesis. In the third study, we identified 4E-BP1 as an important prognostic factor in CRC. We found that the protein expression level of 4E-BP1 was more abundant in colorectal cancer cell lines and patients’ tissues when compared to normal colon cell line and tissues. By the analysis of clinical patients’ pathological parameters, 4E-BP1 expression has statically correlation with disease progression. Under hypoxia, 4E-BP1 is thought to be a crucial factor for cap-independent translation. Additionally, YMX-110, a cryptopleurine derivative, led to tumor suppression by deletion of 4E-BP1. Thus, the inhibition of protein synthesis by YXM110 in hypoxic condition was further evaluated. Our data show that YXM110 decreased hypoxia-inducible factor 1