CHM-1, Moscatilin及YC-1對人類肝癌細胞之抗癌作用及機轉研究

The Anti-Cancer Mechanisms of CHM-1, Moscatilin and YC-1 in Human Hepatocellular Carcinoma Cells

王士維(Shih-Wei Wang)

指導教授 ： 鄧哲明

國立臺灣大學/醫學院/藥理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02942

摘要

肝細胞癌是對現有化學治療藥物具有高度抗藥性的癌症，肝癌細胞周圍有許多間質細胞，這些間質細胞會釋放許多生長因子與蛋白溶解酵素協同肝癌細胞破壞細胞外基質，導致肝細胞癌具有高度侵襲的能力，造成肝細胞癌惡化時對於肝臟內部血管的侵潤及後續致命的轉移。在本論文中，將探索具有研發潛力抑制肝細胞癌生長及侵襲的抗癌藥物，並對其作用機轉進行探討。
第一部分主要討論2-phenyl-4-quinolone類化合物CHM-1在動物體內及體外模式抑制人類肝癌細胞生長的作用機轉。我們發現CHM-1具有強效且選擇性的抗癌活性，可經由對微小管聚合作用的抑制及Cdc2/cyclinB1 complex的活化而造成細胞週期之mitotic arrest，導致肝癌細胞的凋亡作用。此外，CHM-1可透過與caspase不相關的機制協同誘發細胞凋亡。在動物實驗中，CHM-1可以有效抑制動物腫瘤的生長，並延長實驗動物的生命週期，而且CHM-1與doxorubicin比較時具有較強的抗癌效果及較少的副作用。以上實驗證明CHM-1是個具有潛力的先導藥物，值得後續的研發與應用於肝細胞癌的治療。
第二部分主要討論CHM-1抑制肝細胞生長因子誘發人類肝癌細胞侵襲的作用與機轉。肝細胞生長因子可以活化c-Met受體誘發人類肝癌細胞SK-Hep-1的侵襲作用，我們發現CHM-1可以抑制c-Met受體tyrosine kinase的磷酸化，使Akt蛋白及NF-kB轉錄因子的活化被抑制，進而阻斷下游MMP-9的表現與活性，最後抑制肝癌細胞的侵襲作用。因此，CHM-1對肝細胞癌具有多方面的功能抑制，在未來的應用相當廣泛。
第三部分主要討論石斛萃取物moscatilin抑制人類肝癌細胞侵襲作用的機轉。我們發現moscatilin可以在細胞正常培養條件下抑制高侵襲性人類肝癌細胞SK-Hep-1的侵襲作用，moscatilin不影響細胞中MMP-2及MMP-9的活性，乃是透過破壞蛋白溶解酵素uPA的表現與活性來抑制肝癌細胞的侵襲，而此作用可能是阻斷PI3K/Akt訊息傳遞路徑所致。 Moscatilin是天然的萃取成分，可經由後續的結構修飾來加強抗癌活性及選擇性。我們認為moscatilin是相當值得未來開發及應用的成分。
第四部分主要討論benzyl-indazole類化合物YC-1抑制人類肝癌細胞增生的作用及機轉。實驗結果顯示，YC-1可以抑制肝癌細胞的生長，此作用乃是透過對於增生的抑制、而非對細胞的毒殺作用。研究發現，YC-1抑制增生的作用與cGMP訊息傳遞機制無關，乃是透過增加 p21蛋白的表現，促使細胞週期停留在G0/G1 期來調控對肝癌細胞增生的抑制作用。此外，YC-1可呈劑量相關性地抑制動物腫瘤的生長，顯示YC-1具有相當不錯的抗癌活性，有機會發展成為臨床肝細胞癌治療的藥物。

關鍵字

肝細胞癌 ； 微小管標的藥物 ； 細胞凋亡 ； 侵襲作用

並列摘要

Hepatocellular carcinoma (HCC) is highly chemoresistant to currently available chemotherapeutic agents. HCC cells are surrounded by stroma cells in liver, the tumor-stroma interactions can produce a lot of growth factors and proteases to degrade extracellular matrix in tumor microenvironment. Thus, invasion is a characteristic of HCC cells, which frequently show early invasion into blood vessels as well as intrahepatic metastasis and extrahepatic metastasis occurs subsequently. The present study is focused on the discovery of novel small-molecule agents to inhibit cell growth and invasion of human HCC cells.
The first part is to investigate the antitumor activity and mechanism of CHM-1, a 2-phenyl-4-quinolone derivative, in human HCC cells. CHM-1 induces mitotic arrest and apoptosis by binding to tubulin and inhibiting tubulin polymerization. CHM-1 causes mitotic arrest, at least partly, by modulating Cdc2/cyclin B1 complex activity and then apoptosis executed by a caspase-independent pathway. Importantly, CHM-1 inhibited tumor growth and prolonged the life span in mice inoculated with HA22T cells. CHM-1 is a promising chemotherapeutic lead compound worthy of further development into a drug candidate for treating cancer, especially HCC.
The second part is to evaluate the anti-invasive effects and mechanism of CHM-1 in hepatocyte growth factor (HGF)-induced invasion of SK-Hep-1 human HCC cells. We found that HGF stimulated invasion of SK-Hep-1 cells, together with increased expression of matrix metalloproteinase (MMP)-9. CHM-1 significantly inhibited tyrosine autophosphorylation of c-Met induced by HGF. CHM-1 also suppressed HGF-induced Akt phosphorylation, and NF-kB activation, the downstream regulators of HGF/c-Met signaling, resulting in the inhibition of MMP-9. Based on the findings herein, we suggest that CHM-1 could be effective candidate for prevention of HCC cell invasion associated with the HGF/c-Met system.
The third part is to study the anti-invasive mechanism of moscatilin in highly invasive human HCC cells SK-Hep-1. Moscatilin is a bibenzyl derivative extracted from the stems of a traditional Chinese medicine, Orchid Dendrobrium loddigesii. We found moscatilin significantly inhibited cell invasion in a concentration-dependent manner. Moscatilin did not affect the proteolytic activity of MMP-2 and MMP-9 in SK-Hep-1 cells. However, Moscatilin inhibited the enzyme activity and protein expression of urokinase-type plasminogen activator (uPA). We further demonstrated that anti-invasive activity of moscatilin may be through the selective suppression of uPA regulated PI3K/Akt signal transduction. Thus, we suggest that moscatilin is a potential therapeutic agent against tumor invasion.
The fourth part is to investigate the antiproliferative mechanism of YC-1 in human HCC cells. This study is the first to delineate that YC-1 directly inhibits proliferation of cancer cells. YC-1 inhibited the growth of human HCC cells in a concentration-dependent manner without significant cytotoxicity. We demonstrated that YC-1-induced antiproliferative effect is a cGMP-independent pathway. YC-1 induced Go/G1 arrest and inhibited tumor growth both in vitro and in vivo via the up-regulation of p21 expression. As a cytostatic agent, YC-1 slows the proliferation of cancer cells, while causing low toxicity to normal cells and allowing patients to “live with their cancer”. Used alone or in combination with other chemotherapeutic agents, YC-1 deserves further investigation in preclinical studies or clinical trial as a potential antitumor agent.

並列關鍵字

HCC, microtubule-targeted agent ； apoptosis ； invasion

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