沙利竇邁及降膽固醇藥物抗癌新機轉之研究

Abstract

本論文探討沙利竇邁(thalidomide)以及降膽固醇藥物(statins)之抗癌新機轉。Thalidomide早期用於緩解懷孕婦女嘔吐之鎮定劑，但造成畸形兒的產生，因而勒令停用。近幾年發現thalidomide具有抗發炎、抑制血管新生、調節免疫之作用，因而重新使用於癌症治療。細胞黏著分子(Intercellular adhesion molecule-1, ICAM-1)在癌症轉移扮演重要的角色。本論文發現臨床肺癌病患其肺癌組織ICAM-1表現量高低和疾病惡化的分期成正相關性。過度表現ICAM-1之癌細胞會增強細胞之侵襲能力，使得癌細胞有機會轉移到其他組織。Thalidomide阻斷ICAM-1表現或使其細胞侵襲能力失效，則癌細胞轉移的程度會大幅降低。進一歩探討thalidomide之分子機轉，其抑制tumor nacrosis factor-alpha (TNF-alpha)所活化之nuclear factor-kappaB (NF-kappaB)及其結合到ICAM-1 promoter的能力，因而抑制ICAM-1 之表現，這些作用是透過抑制IkappaB kinase (IKK)及p65之磷酸化。 降膽固醇藥物statins擁有長鏈型之酸性官能基，結構類似histone deacetylase (HDAC) 抑制劑tricostatin (TSA)，並且可以增加組織蛋白乙醯化，因此進一步研究statins是否為新型HDAC抑制劑。利用電腦模擬HDAC2和lovastatin之分子間結合，證實lovastatin其酸性官能基可以鑲嵌到HDAC2之酵素活性部位，並且和鋅離子結合，抑制HDAC2之活性。利用細胞外HDAC活性試驗證實statins抑制HDAC活性，為一種競爭型HDAC抑制劑。Statins抗癌之分子機轉是促進HDAC1/2離開p21的promoter，CREB-binding protein (CBP)接近最後造成p21 promoter組織蛋白乙醯化，增加p21基因表現，達到抑制癌細胞生長之功能。 細胞自我吞噬(autophagy)是指細胞透過吞噬細胞質的物質產生能量來度過外界壓力的保護機制。但是，過度的細胞吞噬會導致細胞死亡，稱為autophagic cell death。本論文發現statins在癌細胞中引發autophagy，其分子機轉透過活化AMP-activated protein kinase (AMPK)，造成mammalian target of rapamycin (mTOR)訊息傳遞抑制和p21表現增加。Statins引起autophagy之現象在HCT116 p21-/-細胞消失，過度表現p21增加endoplasmic rediculum (ER) stresss signaling之eukaryonic translation initiation factor 2alpha (eIF2alpha)造成autophagy。進一步分析大腸癌病人腫瘤組織與正常組織發現p21表現和autophagy呈正相關。評估statins之抗癌效果發現，statins可以抑制癌細胞生長並且在azoxymethane (AOM)引發之小鼠大腸癌之動物模式或是肝癌裸鼠實驗，statins也具有抑制效果。總括這些研究結果，本論文研究成果提供老藥新用對抗癌細胞，並且提出分子機轉作為藥物研發之參考。

The novel anticancer mechanisms of thalidomide and HMG-CoA reductase inhibitors (statins) were investigated in this study. Thalidomide has been reported to have anti-angiogenic and anti-metastatic effects. Intercellular adhesion molecule-1 (ICAM-1) was demonstrated to be involved in cancer cell invasion. We investigated the role of ICAM-1 in tumorigenesis. High expression of ICAM-1 in human lung cancer specimens was correlated with a greater risk of advanced cancers (stage III and IV). A549/ICAM-1 cells were demonstrated to induce in vitro cell invasion and in vivo tumor metastasis. Anti-ICAM-1 antibody and thalidomide had inhibitory effect on these events. Thalidomide also suppressed tumor nacrosis factor-alpha(TNF-alpha-induced ICAM-1 expression through inhibition of nuclear factor-kappaB (NF-kappaB) binding to the ICAM-1 promoter. The in vivo xenograft model showed the effectiveness of thalidomide on tumor formation. We show that statins inhibited histone deacetylase (HDAC) activity, increased the accumulation of acetylated histone-H3 and the expression of p21 in human cancer cells. Computational modeling demonstrated the direct interaction of the carboxylic acid moiety of statins with the catalytic site of HDAC2. Transcription factor Sp1 but not p53 sites was found to be the statins-responsive element demonstrated by p21 luciferase-promoter assays. DNA-affinity protein binding assay (DAPA) and chromatin immunoprecipitation assay (ChIP) assays showed the dissociation of HDAC1/2 and association of CREB-binding protein (CBP), leading to the histone-H3 acetylation on the Sp1 sites of p21 promoter. Autophagy is a process describing the degradation and recycling of proteins or intracellular organelles. In this study, we found that statins induced autophagy through a mechanism relying on AMP-activated protein kinase (AMPK) activation. Statins activate AMPK, leading to inhibition of mTOR and expression of p21. AMPK inhibitor and AMPK siRNA abrogated atorvastatin-induced autophagy, which was also attenuated in p21-/- HCT116 cells. Positive correlation between the reduced expression of p21 and beclin 1 was seen in human colon cancer specimens. Overexpression of p21 induced not only LC3-II expression but also phosphorylation of eIF2alpha suggesting that p21 induces autophagy through ER stress signaling. Taken together, all the results provide a mechanistic insight into the crucial role of thalidomide and statins in anti-cancer therapy.