The Molecular Mechanism of Histone Deacetylase (HDAC) Inhibitor PCI-24781 as an Anti-tumor Agent in Neuroblastoma Cells

• Other Authors: Zhan, Qinglei (author.)
• Format: Others
• Language: English; Chinese
• Published: 2013
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292174

7－10％的兒科腫瘤是神經母細胞瘤, 並且它是最普遍的腦顱外的實體瘤。儘管使⽤用多個⾼高強度的治療⽅方案， 被診斷有⾼高危神經母細胞瘤病人的存活率仍然低於40％。 這更加突出了對於新的治療策略的迫切要求。組蛋⽩白去⼄乙酰化酶抑制劑是⼀一類新的有前景的抗腫瘤藥物， 它們在臨床上已經⽤用於許多類型的腫瘤的治療。PCI-24781 是⼀一種新型的屬於异羟肟酸家族的組蛋⽩白去⼄乙酰化酶抑制劑，對於癌症的治療俱有⾼高效率性和安全性。然⽽而，PCI-24781 對於神經母細胞瘤作⽤用的潛在機制仍然不是很清楚。 === 本文的研究显⽰示了以⾮非常低剂量的PCI-24781 的處理顯著地抑制神經母細胞瘤的細胞株SK-N-DZ 的⽣生⾧長， ⽽而對於正常的細胞株HS-68 沒有影響。 但是，PCI-24781 都能增加這兩個細胞株的乙酰化的組蛋白H3 的水平。PCI-24781 的處理能誘導SK-N-DZ 細胞的細胞週期停滯在G2/M 期，並且產⽣生⼤大量的sub-G1的凋亡細胞。對於凋亡信號通路的分析發現PCI-24781 能激活外在的和內在的凋亡信號通路。被上調的和凋亡相關的蛋⽩白質包括DR4, p21, p53, caspase3 以及斷裂的 caspase3。 === 進一步的蛋白質組學的分析顯示了在未處理和經過PCI-24781 處理的SK-N-DZ 細胞的不同的蛋⽩白表達譜。總共有42 個差異表達的蛋白被MALDI-TOF 質譜儀系統鑒定出來。功能性的歸類發現PCI-24781 影響了⼀一系列的細胞過程，包括細胞⾻骨架，能量代謝，DNA 修復， 細胞週期和轉錄的調控。免疫印跡的分析確證了五個候選蛋⽩白質在SK-N-DZ 細胞的表達，它們分別是prohibitin, hHR23a, RuvBL2, TRAP1 以及PDCD6IP。有趣的是，它們的表達譜在HS-68 細胞⾥里完全不同。 === PCI-24781 處理後，SK-N-DZ 的細胞保持高水平的hHR23a 和RuvBL2 蛋白質的表達，然而HS-68 的細胞抑制了它們的表達。這暗示了這兩個候選蛋白可能對於PCI-24781 誘導的細胞死亡中扮演着重要的角色。然⽽而，選擇性的沈默RuvBL2 基因，⽽而不是hHR23a 基因，減少了PCI-24781 引起的細胞死亡。總得來說，目前的結果強調了RuvBL2 基因在SK-N-DZ 細胞株中PCI-24781 抗腫瘤活性的重要性。這篇研究證實了組蛋⽩白去⼄乙酰化酶抑制劑在⾼高危神經母細胞瘤病⼈人中作為抗腫瘤藥物的價值，甚⾄至對於PCI-24781 在SK-N-DZ 細胞株中的潜在分子機制提供了一個新的思路。然而，要想深入理解正常細胞和癌細胞之間對於組蛋⽩白去⼄乙酰化酶抑制劑的不同敏感性的潛在機理，我們仍然需要大量的研究。 === Neuroblastoma accounts for 7-10% of all childhood cancers, and it is the most common extracranial solid tumor in childhood. The survival rate is less than 40% among children with high-risk neuroblastoma, despite of intensive multi-modality treatment, highlighting the urgent needs for new treatment strategies. Histone deacetylase (HDAC) inhibitors are a new class of promising anti-tumor agents that have been used in clinical trials for various tumor types. Among HDAC inhibitors, PCI-24781 is a novel hydroxamic acid that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated, especially in neuroblastoma cells. === In this study, I demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, the accumulation of acetylated histone H3 can be found both in SK-N-DZ and HS-68 cell line in response to PCI-24781. Treatment with PCI-24781 also induced cell cycle arrest in G2/M phase and large amounts of sub-G1 cells in SK-N-DZ cells. Analysis of apoptosis signaling pathways found that both extrinsic and intrinsic pathways were activated by PCI-24781. The increased apoptotic proteins included DR4, p21, p53, caspase3 and cleaved caspase3. === Different protein expression profiles were further revealed via proteomic analysis between SK-N-DZ cells with or without PCI-24781 exposure. MALDI-TOF MS system identified total 42 differentially expressed proteins. The functional classification revealed that PCI-24781 affected a series of cellular processes, including cytoskeleton organization, energy metabolism, DNA repair, cell cycle and transcriptional regulation. Western blotting confirmed the expression level of five candidate proteins in SK-N-DZ cells, including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Interestingly, expression patterns of the five proteins were totally different in HS-68 cells. === The high abundance of hHR23a and RuvBL2 was found in SK-N-DZ cells but not in HS-68 cells in response to PCI-24781, implying that these two proteins may be functionally important for PCI-24781-mediated cell death. However, selective knockdown of RuvBL2 not hHR23a rescued cells from PCI-24781-induced cell death. Collectively, my current results highlight the important role of RuvBL2 in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present study confirms the interest in HDAC inhibitors as anti-cancer agents for patients with high-risk neuroblastoma, and even provides a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line. However, many efforts are still needed to elucidate differential sensitivity between cancer and normal cells in response to HDACinhibitors treatment. === Zhan, Qinglei. === Thesis Ph.D. Chinese University of Hong Kong 2013. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.