內質網壓力抑制拓樸異構酵素抑制劑引發之細胞凋亡在人類肝癌細胞的機轉探討

Abstract

肝癌是常見的惡性腫瘤且常對於臨床化學治療的藥物產生抗藥性。Camptothecin與etoposide為臨床上已廣泛使用的藥物，其作用機轉分別為抑制拓樸異構酵素I和II。Tunicamycin能經由抑制蛋白質N端的醣化作用來引起大量未折疊蛋白的產生，因此可作為內質網壓力的誘導劑。實驗數據顯示合併使用tunicamycin時，可減少在肝癌細胞Hep3B中camptothecin/etoposide引起的細胞凋亡現象，而另一株肝癌細胞HepG2則無此現象，且僅可在Hep3B中觀察到caspase-3、-7及bcl-2家族蛋白的活化被抑制。因此本篇研究將探討tunicamycin對兩株細胞產生不同抑制作用的機轉。實驗中檢測了許多蛋白的表現量與功能，包括拓樸異構酵素(I, IIα and IIβ)、B型肝炎病毒的相關蛋白、p-glycoprotein及reactive oxygen species (ROS)的釋放程度。結果顯示上述蛋白、酵素或是ROS皆無法解釋tunicamycin主導的抗藥性為何僅出現在Hep3B而非HepG2，且使用p53 siRNA後亦可排除此腫瘤抑制基因參與抗藥性產生的機轉。合併處理tunicamycin與單獨處理camptothecin/etoposide相較時，僅在Hep3B中可觀察到許多細胞週期調控蛋白的表現會受影響。此外，由先給予camptothecin/etoposide而tunicamycin後加的實驗中，顯示當後加的時間點越晚，可觀察到tunicamycin在Hep3B對於camptothecin/etoposide的抗藥性貢獻會消失，此表示細胞週期的調控在此處的必要性。我們亦觀察到了內質網壓力的指標蛋白GRP78之表現量與Hep3B細胞的存活率有良好的相關性。進行GRP78 siRNA實驗後，可觀察到tunicmaycin導致的細胞凋亡現象，顯示了GRP78可促進細胞存活的角色，且同時tunicamycin主導的Hep3B抗藥性會因此消失。因此GRP78在Hep3B的抗藥性中扮演了重要的角色。由上述的實驗數據可知，tunicamycin在Hep3B而非HepG2造成對camptothecin/etoposide產生抗藥性的原因，是由於在Hep3B中tunicamycin的合併使用可經由改變細胞週期的調控，以及增加GRP78的表現來提供對抗藥性的貢獻。

Hepatocellular carcinoma is a very common malignancy and is chemoresistant to clinical chemotherapeutic agents. Camptothecin and etoposide are two well-known anticancer drugs, targeting topoisomerase I and II, respectively. Tunicamycin, an N-glycosylation inhibitor, can cause unfolded protein response (UPR) and be widely used as pharmacological inducer of endoplasmic reticulum (ER) stress. Our data demonstrated that the combined co-treatment with tunicamycin could prevent cell apoptosis induced by camptothecin or etoposide in hepatocellular carcinoma Hep3B other than HepG2 cells, and only inhibit the activation of caspase-3、-7 and bcl-2 family protein in Hep3B cell. The following assessments were conducted to characterize the varied responses to tunicamycin inhibition in these two cell lines. Several protein expressions and functions were examined in this study, including topoisomerases (I, IIα and IIβ), HBV-related protein, p-glycoprotein and the levels of reactive oxygen species (ROS).The data demonstrated that none of these proteins, enzymes or free radicals could explain tunicamycin-mediated resistance in Hep3B but not in HepG2 cells. Using siRNA to knockdown p53, the data also excluded the involvement of this tumor suppressor gene. During the co-treatment， the expression of several cell cycle regulator could be changed compare to alone treat TOP posions. Furthermore, in the design of post-treatment of tunicamycin after camptothecin/etoposide addition, the results showed that tunicamycin-reversed effect declined when the duration of post-treatment was increased in Hep3B cells, indicating the necessity of cell cycle regulation for the reduction of camptothecin/etoposide effect. GRP78 expression had a good correlation with survival percentage in Hep3B other than HepG2. By using siRNA approaches, the data demonstrated that the knockdown of GRP78 expression induced a moderate increase of tunicamycin-induced apoptosis, revealing the pro-survival properties of GRP78, and tunicamycin-mediated resistance to camptothecin/etoposide action in Hep3B could be declined, accompanying the change of cell cycle distribution. The data suggested that GRP78 played an important role in the resistant mechanism. Taken together, the data suggest that tunicamycin could cause resistance to TOP poisons through cell cycle regulation, and induce an increase of GRP78 expression contributing to resistance of TOP poisons in Hep3B other than HepG2.