新型HDAC抑制劑MPT0G157促進大腸直腸癌細胞株HCT116細胞凋亡與抑制血管新生之研究

Novel HDAC inhibitor MPT0G157 induced apoptosis and inhibited angiogenesis in colorectal cancer HCT116 cell line

黃彥嘉(Monica Huang)

指導教授 ： 楊家榮

國立臺灣大學/藥學專業學院/藥學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10655

摘要

實驗目的：Histone deacetylases (組蛋白去乙醯酶，HDACs)藉由減少組蛋白之乙醯化來調控基因表達，當HDACs作用使組蛋白去乙醯化時，抑癌基因會受到抑制，使得癌細胞不受控制而大量生長，因此透過抑制HDAC的作用可能可以抑制癌細胞增生。迄今雖然有四個HDAC抑制劑已被美國FDA允許上市，但尚未有用於治療固態瘤之HDAC抑制劑，此論文之研究目的為探討一新型HDAC抑制劑MPT0G157在大腸直腸癌中促使細胞凋亡及抑制癌細胞血管新生之效果與機轉。

實驗方法：本研究利用SRB assay評估MPT0G157抑制細胞增生之情形，並利用流式細胞儀觀察細胞週期變化；此外，利用HDAC assay kit測定MPT0G157對HDAC isoform的抑制效果及在大腸直腸癌細胞株HCT116中抑制HDAC活性之能力。蛋白質表現及mRNA變化以西方點墨法及即時定量聚合酶連鎖反應觀察。在血管新生方面，我們將細胞培養在低氧控制箱及使用氯化亞鈷誘導缺氧狀態，觀察MPT0G157 對缺氧誘導因子-1alpha(hypoxia-inducible factor-1alpha，HIF-1alpha)蛋白質之調控，並以免疫沉澱法觀察MPT0G157影響熱休克蛋白90 (heat shock protein 90，HSP90)乙醯化與HIF-1alpha之交互作用；藉由在細胞中過量表現HDAC1及HDAC6確認MPT0G157是否透過抑制HDAC活性影響細胞增生、細胞凋亡與抑制HIF-1alpha蛋白表現。利用異體移植(xenograft)腫瘤與matrigel plug assay動物實驗模型確認MPT0G157抑制腫瘤生成、血管新生及抗發炎的能力。

實驗結果：在四種不同癌症細胞株(乳癌、肺癌、大腸直腸癌、胰臟癌)中，MPT0G157在大腸直腸癌細胞株HCT116有最佳抑制細胞增生之效果，在低濃度(GI50 = 29.39 ± 2.63 nM)就能抑制細胞增生，但不影響正常細胞之增生情形。並且與已上市之兩種HDAC抑制劑 (Belinostat, PXD101; Vorinostat, SAHA)相比，MPT0G157在HCT116細胞株中抑制HDAC活性之效果最佳，並且有較好的誘發細胞凋亡的效果。MPT0G157同時也會使HSP90乙醯化增加，與其下游蛋白HIF-1alpha分離，失去保護的HIF-1alpha會經由蛋白酶體(proteasome)路徑被降解，減少血管內皮生長因子(vascular endothelial growth factor，VEGF)的mRNA表現，抑制血管新生。為了確認MPT0G157的抑癌效果是經由抑制HDAC活性而產生，我們在過量表現HDAC1及HDAC6後，抑制細胞增生、促進細胞凋亡及抑制HIF-1alpha蛋白表現的情形都有被回復，證實MPT0G157所產生之抗癌效果是經由抑制HDAC活性而產生。並且在動物實驗中MPT0G157可有效抑制腫瘤體積、減少血管新生及抗發炎，證實MPT0G157有良好的抗癌功效。

結論：實驗結果顯示，MPT0G157具有良好的抑癌效果，透過抑制HDAC活性可抑制大腸直腸癌細胞株HCT116之細胞增生，誘發細胞凋亡及抑制血管新生，在活體內實驗中也有明顯抑制腫瘤生成及血管新生的能力，因此MPT0G157有良好的發展潛力可成為固態瘤之治療藥物。

關鍵字

組蛋白去乙醯? ； 細胞凋亡 ； 血管新生 ； 缺氧誘導因子-1alpha ； 熱休克蛋白90

並列摘要

Obejective. Histone deacetylases (HDACs) regulate gene expression through chromatin remodeling by reducing histone acetylation. When histone acetylation decrease, tumor suppressor gene will be inhibited; therefore, HDACs are a promising target for cancer treatment. To date, four HDAC inhibitors have been approved, but no HDAC inhibitor has been accepted for solid tumor treatment. The aim of this study is to examine the anti-cancer and anti-angiogenesis effect of MPT0G157, a novel HDAC inhibitor, by in vitro and in vivo models.
Methods. Cell proliferation and HDAC activity were determined by SRB assay and HDAC assay kit, respectively. Cell cycle was studied by flow cytometry. Protein and mRNA expression was evaluated by western blot and real time PCR. In angiogenesis, we used cobalt chloride and hypoxia chamber to mimic hypoxia condition, and test HIF-1alpha protein expression. Immunoprecipitation was used to evaluate acetylation of HSP90 and interaction between HIF-1alpha and HSP90. Overexpressing HDAC 1, 6 to ensure the anti-cancer effect of MPT0G157 through inhibition of HDAC activity. In vivo study was determined by xenograft mice model and matrigel plug assay, then analyzed by H E stain, Masson’s trichrome stain, IHC stain, and western blot.
Results. MPT0G157 inhibited cell proliferation and was more potent in HCT116 cell line (GI50 = 29.39 ± 2.63 nM) but had no effect on normal cell line HS-5. Comparing with the marketed HDAC inhibitors (Belinostat, PXD101; Vorinostat, SAHA), MPT0G157 exhibited more potent inhibition of HDAC activity and induction of apoptosis. MPT0G157 inhibited angiogenesis through increasing acetylation of HSP90, promoting HIF-1alpha degradation by proteasome system and down-regulating the mRNA expression of VEGF. To further evaluate anti-cancer effect of MPT0G157 through inhibition of HDAC activity, we overexpressed HDAC1, 6 in HCT116 cell. After overexpression HDAC1, 6, the anti-cancer effects were all reversed, which indicated that the MPT0G157 had anti-cancer effect through inhibition of HDAC activity. In animal studies, MPT0G157 significantly inhibited tumor growth, angiogenesis, and down-regulated inflammation factors.
Conclusion. Our results demonstrate that MPT0G157 has potent effect in inhibition of cell proliferation, induction of apoptosis, and inhibition of HIF-1alpha expression through inhibition of HDAC activity. MPT0G157 also inhibits tumor growth, angiogenesis and inflammation in animal models. It suggests that MPT0G157 has potential as a new drug in solid tumor therapy.

並列關鍵字

Histone deacetylases ； apoptosis ； angiogenesis ； HIF-1alpha ； HSP90

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