分析植物對苯二酚衍生物HQ17(3)對帶有費城染色體之急性淋巴性白血病細胞株SUP-B15的抑制作用

Inhibitory effects of the botanical alkyl hydroquinone derivative HQ17(3)on acute lymphoblastic leukemia SUP-B15 cells harboring Philadelphia chromosome

張耀仁(Yao- Jen Chang)

指導教授 ： 胡忠怡

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01051

摘要

人類第9 號染色體與第22 號染色體發生轉位之後產生費城染色體(Philadelphia chromosome)，因而產生具有致癌性的融合蛋白BCR-ABL。BCR-ABL 為持續活化的酪胺酸激酶(tyrosine kinase)，會啟動下游許多關於細胞增生、存活、以及自我更新的訊號傳遞，而導致細胞惡性轉型。分子量為190 kD 的BCR-ABL 融合蛋白多發現於急性淋巴性白血病(ALL) (Ph+ ALL)，其疾病惡性度高，預後不佳；現階段以最高強度化學治療搭配tyrosine kinase inhibitors (TKI,如: Imatinib, Dasatinib)抑制BCR-ABL的活性，雖可使病人獲得暫時性緩解，但是仍有高比例病人血癌復發，並且發生抗藥性。因此，發展有別於化療的藥物或與tyrosine kinase inhibitor 合併搭配使用，是治療Ph+ ALL 的重要課題。10¢(Z),13¢(E),15¢(E)-heptadecatrienyl
hydroquinone (HQ17(3))是萃取自漆樹的天然小分子，對於多種腫瘤細胞具有毒殺能力，但對於人類正常周邊血液單核細胞以及實驗鼠無明顯毒性，HQ17(3)在腫瘤細胞中具有抑制DNA 拓樸異構酶II (DNA topoisomerase II)以及誘導產生reactiveoxygen species (ROS)能力，並可在肝癌細胞株中引起DNA 損傷及apoptotic cell death。我們在前驅測試中發現以低濃度HQ17(3)處理帶有BCR-ABL 的ALL 細胞株-SUP-B15 24 小時即有顯著毒殺作用。
本研究結果顯示HQ17(3)會誘導SUP-B15 細胞活性氧(ROS)上升、酸性胞器出
現、粒線體膜電位喪失、染色體斷裂及細胞自噬標記LC3-II 產生。抗氧化劑
(Antioxidantss, ROS scavenger)穀胱苷肽(GSH)與維生素C 可以減緩HQ17(3)所誘導的ROS 產生、減少粒線體膜電位損傷並降低細胞死亡。親脂性排鐵劑desferrioxamine mesylate (DFO)可阻止酸性胞器產生，並阻斷HQ17(3)所引起的粒線體損傷及細胞死亡；加入抑制細胞自噬抑制劑3methyl adenine (3-MA)與氯奎寧(Chloroquine)亦可以減少HQ17(3)誘發之細胞死亡。綜合本研究的實驗結果顯示HQ17(3)誘導ROS 啟動細胞自噬可能是造成SUP-B15 細胞死亡重要原因，並且此過程高度依賴鐵存在。因此，本研究結果指出，若可誘導癌細胞產生細胞自噬將有潛力發展輔助治療帶費城染色體急性淋巴性白血病的策略。

關鍵字

急性淋巴性白血病 ； SUP-B15 ； HQ17(3) ； 細胞自噬 ； 活性氧族群

並列摘要

Reciprocal t(9;22); BCR-ABL translocation gives rise to Philadelphia(Ph)chromosome and results in production of chimeric BCR-ABL fusion protein with constitutively active tyrosine kinase activity. The BCR-ABL protein activates a number of signaling pathways which promote cell proliferation, survival and self-renewal. Ph+ acute lymphoblastic leukemia (ALL) with 190kD BCR-ABL fusion protein presents very poor clinical outcomes. Although tyrosine kinase inhibitor (TKI) combined with multi-agent chemotherapy help to acquire a complete remission temporarily, still a high proportion of patients have leukemic relapse and develop drug resistance. Therefore,
developing drug(s) to act in different ways as in chemotherapies, or to be used in combination with the TKI in treatment of Ph+ ALL, is an alternative way to help the
patients affected by this very high risk disease. 10¢(Z),13¢(E),15¢(E)-heptadecatrienyl hydroquinone (HQ17(3)) is a small natural molecule extracted from the R. succedanea.
DNA topoisomerase IIα inhibition and oxidative stress were found to account for selective cytotoxicity caused by HQ17(3) in various types of tumor cells. In the preliminary data, we found that HQ17(3) has significant cytotoxic effect in a p190 BCR-ABL Ph+ ALL cell line, SUP-B15, within 24 hours in micromolar concentration.
We found in SUP-B15 cells, HQ17(3) induced reactive oxygen species (ROS) and acidic vesicle formation, mitochondrial membrane potential disturbance, chromosome breakage, and emergence of an autophagy marker, cleaved LC3-II. ROS scavengers(Glutathione, vitamin C) attenuated HQ17(3)-induced cell injury. Lysosomotropic iron chelator, desferrioxamine mesylate (DFO) abolished HQ17(3)-induced acidic vesicles formation, mitochondrial membrane potential loss and cell death. Inhibitors for autophagy (3-methyl adenine, chloroquine) partially rescued cells from
HQ17(3)-induced death. These results indicated that HQ17(3) may induce ROS production, and subsequently lead to autophagic cell death. In conclusion, HQ17(3) displayed a significant anti-leukemic activity in Ph+ ALL (SUP-B15) cells by ROS production and lysosomal iron-dependent events that contribute to autophagic cell death. These results suggest that agents selectively induce ROS in leukemic cells might induce autophagic cell death, and would potentially augment the treatment for Ph+ ALL.

並列關鍵字

acute lymphoblastic leukemia (ALL) ； SUP-B15 ； HQ17(3) ； autophagy ； reactive oxygen species (ROS)

參考文獻

1. 中華民國兒童癌症基金會: 兒童白血病, 2007

4. 行政院衛生署國民健康局: 中華民國 97 年癌症登記報告, 2010

3. A.V. Hoffbrand JEPaPAHM: Essential Haematology fourth edition

5. Thomas DA: Philadelphia chromosome positive acute lymphocytic leukemia: a new era of challenges, Hematology /the Education Program of the American Society of Hematology American Society of Hematology Education Program 2007, 435-443

7. Pui CH, Evans WE: Treatment of acute lymphoblastic leukemia, N Engl J Med 2006, 354:166-178