- 學位論文
植物對苯二酚衍生物HQ17(3)對急性淋巴性白血病細胞株Reh的毒殺機制
The Cytotoxic Effects of the Botanical Alkyl Hydroquinone Derivative HQ17(3) on Acute Lymphoblastic Leukemia Cell Line Reh
摘要
急性淋巴性白血病為兒童常見癌症，起因於淋巴系造血相關基因、致癌基因(Oncogene)、酪胺酸激酶(Tyrosine kinase)基因等變異或轉位導致細胞分化生長機制異常。雖然以現今治療策略可以幫助80~90% ALL兒童病患達到完全緩解，然而少部分兒童及高比例成人ALL病患會對治療藥物無反應或產生藥物抗性，或者病患本身攜有高風險基因變異，不一定有合適的標靶治療藥物因此造成預後不佳，這些情況都成為了目前ALL治療研究與開發的重點與方向。 天然小分子化合物10’(Z),13’(E),15’(E)heptadecatrienylhydroquinone (HQ17(3)) 萃取自台灣漆樹。在過去文獻中已知具有選擇性抑制癌細胞的能力，在多種實質腫瘤細胞具有良好毒殺效果，其中於Huh7細胞發現HQ17(3)會結合性修飾DNA拓樸異構酶導致DNA斷裂、半胱胺酸蛋白酶(caspase)活化及引起氧化壓力；正常人類周邊血單核球細胞(PBMCs)在HQ17(3)環境下生長情形不會受影響，且施打HQ17(3)不危害健康大鼠生理機能。我們過去研究發現HQ17(3)對ALL細胞株的毒殺效果更佳(IC50約1~3μM)，且短時間內即有作用(24小時)，HQ17(3)在T-ALL細胞株Jurkat造成caspase-dependent細胞死亡而在B-ALL細胞株RS4;11及SUP-B15則造成caspase-independent細胞死亡，顯示在不同基因背景或細胞種系之下HQ17(3)可能引導的促死亡訊號與機制不盡相同。 本論文研究以帶有ETV6-RUNX1 t(12;21) 基因轉位且對ALL治療一線用藥Dexamethasone具抗性之ALL細胞株Reh作為實驗細胞模型，我們發現HQ17(3)處理之下Reh出現死亡特徵: 內膜外翻、caspase活化/PARP切割、粒線體膜電位喪失及細胞毒殺。然而以pan-caspase inhibitor z-VAD與HQ17(3)共處理無法緩解細胞死亡說明HQ17(3)造成Reh caspase-independent 細胞死亡；我們在HQ17(3)處理5個小時觀察到錯誤蛋白摺疊反應相關蛋白表現提升，表示HQ17(3)誘發內質網壓力，為了解鈣離子由內質網釋出是否導致粒線體膜電位喪失與損傷促進ROS壓力累積引發細胞死亡，利用鈣離子螯合劑BAPTA-AM或抗氧化劑vitamin C, glutathione可以減輕HQ17(3)誘導粒線體膜電位下降及細胞死亡，顯示鈣離子失衡及氧化壓力為促成細胞死亡之重要因素。HQ17(3)也會引發細胞自噬，利用自噬抑制劑chloroquine及bafilomycin A1可以顯著挽救粒線體膜電位喪失及細胞死亡，顯示過度自噬機制的發生可能為HQ17(3)引起細胞死亡當中之重要的角色。此外，我們發現HQ17(3)處理5小時細胞內粒線體總量即發生下降情形，雖然鈣離子螯合劑、抗氧化劑與細胞自噬抑制劑皆可削弱HQ17(3)造成之細胞死亡，但粒線體總量下降情形並未緩解。而HQ17(3)如何引發Reh細胞內質網壓力、細胞自噬與粒線體總量下降機制之間關連仍待深入探討。 在preB-ALL細胞模型中，HQ17(3)常可同時誘發內質網壓力、氧化壓力及細胞自噬，達到快速抑殺白血病細胞的效果。除探討HQ17(3)引發Reh細胞內機制外，我們也測試HQ17(3)是否可以與ALL治療一線用藥合併使用之可行性，結果顯示以低於HQ17(3) IC50濃度處理可以增加細胞對Dexamethasone的感受性。本論文研究期望透過了解HQ17(3)誘發細胞死亡詳細訊息傳遞或機轉，在未來能對高風險ALL治療藥物與合併用藥策略的發展有所助益。
並列摘要
Acute lymphoblastic leukemia (ALL) is the most common cancer among children. The pathogenesis of ALL involves chromosomal translocation and genetic alterations which disrupt genes that regulate hemopoiesis, activate oncogenes or tyrosine kinases, and characterized by abnormal proliferation and differentiation of lymphoid precursor cells. Despite improvements in treatment protocols resulting in complete remission of around 80%~90% of pediatric ALL patients, prognosis remains poor in ALL patients who are of high-risk cytogenetic changes (both in children and adults) or those who are resistant to therapy. New strategies should be explored to improve ALL treatment outcome. The 10’(Z),13’(E),15’(E) -heptadecatrienylhydroquinone (HQ17(3)) is a small natural molecule extracted from the sup of Rhus succedanea. Previous studies reported that HQ17(3) was cytotoxic to various types of cancer cell lines while it exerted no effect on PBMCs and the rat model. In Huh7 cells, HQ17(3) was found to be a topoisomerase IIα poison that caused DNA breakage, induced caspase activation and oxidative stress. We previously found that ALL cell lines were more sensitive to HQ17(3) (IC50: 1~3μM)than other malignant cell lines reported before and that HQ17(3) induced caspase-dependent cell death in T-ALL Jurkat cells and caspase-independent cell death in B-ALL RS4;11 and SUP-B15 cells, suggesting that HQ17(3) exerts potent anticancer activity on various ALL cells through different molecular pathways. Here we investigated the cytotoxic effect of HQ17(3) on ALL cell line Reh, which harbors ETV6-RUNX1 t(12;21) chromosome translocation and is resistant to ALL first-line drug Dexamethasone. Externalization of phosphatidylserine, caspases activation, and PARP cleavage in accompany with mitochondrial membrane potential loss and cell demise were observed in Reh cells upon HQ17(3) treatment. HQ17(3) cotreatment with pan-caspase inhibitor z-VAD could not restore cell viability, indicating that HQ17(3) induced caspase-independent cell death in Reh cells. In addition, we found HQ17(3) induced ER stress as revealed by upregulation of the unfolded protein response associated protein. To investigate whether calcium outflow following ER stress and subsequent oxidant stress might account for further mitochondria damage and cell death , we found that cotreatment of cells with calcium chelator BAPTA-AM or ROS scavengers (vitamin C, glutathione) partially attenuated HQ17(3)-induced MMP loss and cell death, indicating that perturbation of intracellular calcium homeostasis and ROS stress participated in the pro-death signaling. Furthermore, autophagy might also implicate in HQ17(3)-induced cell death as cotreatment cells with lysosome-targeting inhibitor Chloroquine and bafilomycin A1 could alleviate MMP loss and cell death. We also found that mitochondrial mass declined upon HQ17(3) treatment for 5 hours but could not be restored after treatment with calcium chelator, ROS scavenger or autophagy inhibitor. How ER stress, mitochondrial mass loss and autophagy signaling are integrated into each other remained to be defined. In preB-ALL cell models, HQ17(3) presented rapid cytotoxicity via inducing ER stress, ROS and extensive autophagy simultaneously. To investigate the clinical potential of HQ17(3), we tested its ability to sensitize Reh cells to ALL chemotherapeutic Dexamethasone. Synergy was observed between HQ17(3) and Dexamethasone combined treatment, indicating that HQ17(3) may be a promising chemosensitizer. However, further experiments and studies are required to obtain more insights into the cytotoxic mechanism, providing new therapeutic strategies for high risk ALL.