探討新組蛋白去乙醯酶抑制劑MPT0G009誘發細胞凋亡與合併TRAIL呈現協同作用於人類肝癌細胞之體外和體內的作用機轉

Novel synthetic histone deacetylase inhibitor MPT0G009 induces cell apoptosis and displays synergistic anticancer activity with TRAIL against human hepatocellular carcinoma in vitro and in vivo.

黃惠暄(Hui-Hsuan Huang)

指導教授 ： 鄧哲明 ； 潘秀玲

國立臺灣大學/醫學院/藥理學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10736

摘要

肝癌是造成國人癌症致死率居高不下的主要原因之一，雖然多激酶抑制劑 (multikinase inhibitor) sorafenib能有效地增加肝細胞癌病患的存活期，但目前還需要更多能有效治療此癌症的藥物。目前HDAC抑制劑被認為是具有潛力的抗癌藥物，其能促進細胞凋亡並使細胞週期停滯，因此本篇論文之目的為評估一個新合成的HDAC抑制劑 MPT0G009在肝細胞癌的抗癌效果。實驗結果顯示，MPT0G009作用在人類肝細胞癌細胞比起已核准上市的HDAC抑制劑 SAHA，有更強的細胞生長抑制作用、細胞毒殺作用和HDAC抑制作用。並透過fluorogenic HDAC assay kit得知MPT0G009為一個pan-HDAC抑制劑；我們發現MPT0G009在人類肝細胞癌Hep3B細胞中會增加組蛋白H3和α-tubulin乙醯化並且增加p21WAF1/CIP1蛋白表現，此現象證實MPT0G009在Hep3B細胞內的確具有HDAC抑制作用並引起下游的反應。進一步實驗顯示，MPT0G009會調控Hep3B細胞凋亡之訊息傳遞，包括其會增加subG1期的Hep3B細胞以及造成Hep3B細胞的粒線體膜電位下降，並且活化其caspase cascade、增加proapoptotic蛋白 Bim、減少 Bid proform以及減少antiapoptotic蛋白 Bcl-2、Bcl-xL和FLIPS/L，我們也在Hep3B細胞中，證實MPT0G009所引起的細胞凋亡與其抑制HDAC的作用有密不可分的關係。另外，我們還發現MPT0G009對Hep3B細胞短時間作用下，會透過加速FLIPS/L蛋白被分解和減少其轉錄來降低FLIPS/L蛋白表現。因此，MPT0G009合併使用TRAIL或TNF-α均能產生協同作用來抑制人類肝細胞癌細胞的生長，證實MPT0G009會使TRAIL-resistant的人類肝細胞癌細胞，如Hep3B和Huh7細胞對TRAIL轉而敏感化。最後，在腫瘤移植的動物模式中也看到，MPT0G009能有效地抑制Hep3B異種移植腫瘤的生長，MPT0G009會增加α-tubulin和組蛋白H3乙醯化、活化caspase 9、增加PARP cleavage的現象以及減少FLIPS/L，這些作用與in vitro結果相符。總結來說，新穎的HDAC抑制劑 MPT0G009在人類肝細胞癌細胞中，能有效地抑制HDAC並促進外生性和內生性的細胞凋亡路徑，迄今尚未有HDAC抑制劑被核准用來治療肝細胞癌，因此MPT0G009可望成為新一代的肝細胞癌治療藥物。

關鍵字

肝癌 ； 組蛋白去乙醯酶細胞凋亡

並列摘要

Hepatocellular carcinoma (HCC) is one of the common cause of cancer-related death in Taiwan. Although the multikinase inhibitor, sorafenib, significantly increases overall survival of HCC patients, more effective anti-cancer therapies for the treatment of HCC are needed. In addition, histone deacetylase (HDAC) inhibitors serve as promising anti-cancer drugs because of their ability to induce cell growth arrest and apoptosis. The purpose of this study is to evaluate the anti-cancer effects of a novel synthetic HDAC inhibitor, MPT0G009, in HCC. Our data showed that the growth-inhibitory, cytotoxicity and HDAC-inhibitory effects of MPT0G009 were more potent than suberoylanilide hydroxamic acid (SAHA), a FDA approved HDAC inhibitor, in HCC cells. By fluorogenic HDAC assay kit, we suggested that MPT0G009 is a pan-HDAC inhibitor. Moreover, MPT0G009 increased histone H3 acetylation, α-tubulin acetylation and p21WAF1/CIP1 protein, which indicated that MPT0G009 exhibits HDAC inhibitory activity in hepatocellular carcinoma Hep3B cells. Furthermore, MPT0G009- induced apoptosis of Hep3B cells was characterized by an increase in apoptotic (sub-G1) population of Hep3B cells, a loss of mitochondrial membrane potential, the activation of caspase cascade, an increase in proapoptotic protein Bim , a decrease in Bid proform and antiapoptotic proteins, such as Bcl-2, Bcl-xL and FLIPS/L. We also proved that MPT0G009-induced apoptosis was closely related to its activity of HDAC inhibition in Hep3B cells. Interestingly, we observed that the short-term treatment of Hep3B cells with MPT0G009 downregulated FLICE- inhibitory protein (FLIP) through proteasome-mediated degradation and transcriptional suppression. In addition, we observed the synergism of growth inhibition between MPT0G009 and TRAIL/TNF-α in HCC cells. It is proved that MPT0G009 sensitized Hep3B and Huh7 cells to TRAIL. Most importantly, MPT0G009 exhibited the tumor-inhibitory activity in Hep3B mouse xenograft models. MPT0G009 increased α-tubulin acetylation, increased histone H3 acetylation, activated caspase 9, increased PARP cleavage and decreased FLIPS/L in vivo. These results were consistent with in vitro results. In conclusion, MPT0G009, a new HDAC inhibitor, potently inhibits HDAC and triggers both the extrinsic and intrinsic apoptotic pathways in HCC cells. So far, there is no HDAC inhibitor approved for HCC. Thus, MPT0G009 may represent a new candidate for the treatment of HCC.

並列關鍵字

hepatocellular carcinoma ； histone deacetylase ； apoptosis

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