探討三環藥物Teroxirone抑制肝癌細胞生長的機制

Study of Mechanisms in Suppressing the Growth by Teroxirone in Hepatocellular Carcinoma Cells

王文興(Wang Wen-Hsing)

指導教授 ： 方剛

國立臺灣師範大學/理學院/生命科學系/碩士(2016年)

https://doi.org/10.6345/NTNU202204045

摘要

肝癌在全球癌症致死率排名第二，而肝內轉移途徑是造成肝癌復發與高死亡率的主要原因之一。但肝癌轉移機制仍不清楚。三環氧化物triptolide的衍生物teroxirone為一合成的抗癌試劑，過去研究發現teroxirone會使人類非小細胞肺癌細胞經內源性途徑細胞凋亡。本篇使用常見的肝癌細胞株Huh7 (具有突變p53)、HepG2 (野生型p53)與Hep3B (無p53)進行實驗，實驗顯示teroxirone會使肝癌細胞株Huh7產生外源性途徑細胞凋亡。進一步使用caspase-3抑制劑Z-DEVD-FMK來確認teroxirone所誘導Huh7產生細胞凋亡是否透過外源性途徑，發現細胞在加藥前經過caspase-3抑制劑處理，外源性細胞凋亡相關的蛋白質表現降低。另外，透過傷口癒合檢測 (wound-healing assay)與細胞遷移與侵入實驗 (transwell migration and invasion assay)都顯示藥物濃度低於IC50的teroxirone處理可以抑制Huh7產生細胞遷移與侵入。而利用酶譜分析 (zymography)與西方墨點法 (western blot)之結果可看出在低濃度的teroxirone處理後促進細胞轉移相關的蛋白基因表現皆有受到抑制。此外，證明了teroxirone會去降低Huh7 stem-like cells增生與其幹細胞指標。在動物模式中，異體移植的Huh7腫瘤teroxirone處理，會使腫瘤透過細胞凋亡的方式達到抑制腫瘤生長的目的。結論，teroxirone會透過活化外源性細胞凋亡途徑抑制人類肝癌細胞的生長，且低濃度的藥物處理能抑制肝癌細胞的遷移與轉移。另外，teroxirone能降低肝癌類幹細胞的幹細胞特性，並抑制動物模式中異體移植之腫瘤生長，顯示teroxirone在治療肝癌具有發展潛力。

關鍵字

肝癌細胞 ； teroxirone ； 外源性細胞凋亡 ； 轉移 ； 肝癌類幹細胞

並列摘要

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality, worldwide. The poor prognosis and high mortality rate is mainly the result of intra-hepatic metastases. The mechanisms underlying the metastasis of HCC remain unclear. The triepoxide derivative, teroxirone, is a synthetic antitumor agent. Previously, teroxirone was shown causing apoptotic cell death in human non-small cell lung cancer cells and the effects related to intrinsic pathway. In this study, human HCC cell lines HepG2 (wild type p53) , Huh7 (mutated p53), and Hep3B (p53 null) were used to study the effect of teroxirone on liver cancer cells. Teroxirone was shown inducing of apoptotic cell death of Huh7 cells through extrinsic pathway. Caspase-3 inhibitor, Z-DEVD-FMK, was used to confirm that teroxirone induced Huh7 apoptosis by inducing extrinsic pathway characters. Protein levels of extrinsic apoptosis pathway markers were decreased as a result caspase-3 inhibitor pretreatment. The data of wound-healing assay, transwell migration assay and transwell invasion assay, showed that low concentrations of teroxirone suppressed migration and invasion determination of Huh7 cells. The zymographic and western blot analysis indicated that the expression of metastasis promoting genes MMP-2, MMP-9, and VEGF were reduced after low concentrations teroxirone treatment. Furthermore, we also proved that teroxirone attenuated the cell viability and the stem cell markers intensity of Huh7 stem-like cells. In an in vivo model, subcutaneous injection of teroxirone suppressed the growth of xenograft Huh7 tumor cells by apoptosis. In conclusion, this research elucidates the effects and molecular mechanism for teroxirone on HCC cells. Thus, teroxirone is a new candidate in eradicating liver cancer cells.

並列關鍵字

liver cancer ； teroxirone ； extrinsic apoptosis ； metastasis ； liver cancer stem-like cell

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