分析植物對苯二酚衍生物HQ17(3)對費城染色體陽性之急性淋巴性白血病細胞株SUP-B15的毒殺機制

Cytotoxic Effects Imposed by the Botanical Alkyl Hydroquinone Derivative HQ17(3) on Philadelphia Chromosome Positive ALL SUP-B15 Cells

周映辰(Yin-Chen Chou)

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(2018年)

https://doi.org/10.6342/NTU201802853

摘要

人類第9與第22號染色體轉位產生的費城染色體(Philadelphia chromosome, Ph)會導致具致癌性的BCR-ABL融合蛋白產生; BCR-ABL為持續活化的酪氨酸激酶，會啟動與細胞增生、存活及自我更新相關的訊息傳遞路徑，導致細胞轉型癌化。帶有費城染色體的急性淋巴性白血病(Ph+ ALL)具高惡性度且不易治癒，即使以高強度化療搭配tyrosine kinase inhibitor (TKI)抑制BCR-ABL活性，仍有高比例的患者會對藥物產生抗性而導致疾病復發，因此針對這類高風險(high-risk)患者發展有效的替代輔助治療方針，仍是目前ALL研究上的重要課題之一。
10’(Z),13’(E),15’(E)-heptadecatrienyl hydroquinone (HQ17(3)) 是自台灣漆樹的漆液中所萃取出的天然小分子化合物，目前已知HQ17(3)在多種腫瘤細胞中會透過抑制DNA拓樸異構酶II (DNA topoisomerase II)的活性引起DNA斷裂，並對細胞造成氧化壓力導致依賴半胱胺酸蛋白酶介導的細胞凋亡，對人類周邊血單核球細胞則不具明顯毒性。我們之前研究發現，以低濃度(1-3 µM)的HQ17(3)處理帶有費城染色體的急性淋巴性白血病細胞株SUP-B15，在24小時之下即有顯著毒殺效果，因此希望藉由研究HQ17(3)在SUP-B15細胞的毒殺模式了解這類細胞可能帶有的特殊弱點，以發展輔助Ph+ ALL的治療策略。
我們先前的研究已知HQ17(3)處理下SUP-B15細胞發生不依賴半胱胺酸蛋白酶介導的細胞死亡，其中包含了活性氧分子堆積及粒線體膜電位喪失及DNA斷裂，另外也發現HQ17(3)會誘導細胞發生內質網壓力及細胞自嗜現象。在此研究中，我們發現HQ17(3)可能透過內質網壓力引發粒線體內鈣離子濃度上升，導致粒線體內膜蛋白apoptosis-inducing factor (AIF)切割並轉移至細胞核而造成不依賴半胱胺酸水解酶介導的細胞死亡。然而以PD150606抑制AIF的切割/核轉移僅小部分減緩HQ17(3)引起的細胞死亡，若以鈣離子螯合劑抑制粒線體內鈣離子濃度上升能更有效挽救細胞免於死亡，說明SUP-B15細胞在HQ17(3)處理下粒線體鈣離子的堆積可能誘發其他引起細胞死亡的機制。我們發現HQ17(3)除了引起粒線體內活性氧分子堆積、膜電位喪失等粒線體損傷，也會造成粒線體融合相關蛋白Opa1的切割與與Mfn1/2降解。利用粒線體螢光染色發現HQ17(3)引起的細胞死亡會伴隨粒線體出現片段化以及大量減少的現象，顯示HQ17(3)可能會促使粒線體進行分裂並啟動粒線體自嗜清除機制(mitophagy)，以免疫螢光染色看到自嗜體標誌LC3-II與粒線體complex IV訊號重疊的現象，可確定HQ17(3)增進粒線體自嗜的發生。HQ17(3)造成的粒線體損傷與清除現象可透過與鈣離子螯合劑共處理降低粒線體鈣離子堆積而得到緩解，顯示HQ17(3)所造成粒線體鈣離子增加導致粒線體損傷與自嗜。總合以上，我們認為HQ17(3)在帶有費城染色體的preB-急性淋巴性白血病 SUP-B15細胞會透過尚未明瞭的機制引發內質網壓力，導致粒線體內鈣離子的累積，除了引起AIF分子切割與核轉位，也會致使粒線體活性氧分子上升，導致粒線體損傷及引發粒線體自嗜，最終導致粒線體大量減少細胞死亡。透過實驗數據與過去文獻探討，也暗示Ph+ ALL易受內質網壓力及粒線體壓力影響而導致細胞死亡發生，並期望後續研究上得以提供未來在高風險ALL患者治療替代方針的一個新方向。

關鍵字

帶有費城染色體的急性淋巴性白血病 ； SUP-B15 ； HQ17(3) ； 粒線體損傷 ； 細胞死亡

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, which activates multiple signaling pathways that promote cell proliferation, survival and self-renewal. Ph+-acute lymphoblastic leukemia presents very poor clinical outcomes irrespective of the highest dose chemotherapy combined with tyrosine kinase inhibitors. Therefore, it’s important to explore new agents act as alternative/supplemental strategies in treatment of this high-risk disease. The 10’(Z),13’(E),15’(E) -heptadecatrienyl hydroquinone (HQ17(3)) is a small natural molecule extracted from the sup of Rhus succedanea. HQ17(3) was found to be a DNA topoisomerase II⍺ poison that cause DNA damage, induced oxidative stress and caspase-dependent apoptosis in various types of cancer cells, but didn’t affect normal peripheral blood leukocytes. We found HQ17(3) has significant cytotoxic effect on the Ph+ B-ALL SUP-B15 cells within 24 hours at micromolar concentration. HQ17(3) induced ROS production, loss of mitochondrial membrane potential, and DNA fragmentation in SUP-B15 cells, accompanied by signs of autophagy and ER stress, followed by caspase-independent cell death. Here, we found HQ17(3) induced mitochondrial Ca2+ ([Ca2+m]) elevation, leading to apoptosis-inducing factor (AIF) cleavage and nuclear translocation. However, inhibition of AIF cleavage by calpain-I inhibitor PD150606 only slightly attenuated the HQ17(3)-induced cell death. Furthermore, Ca2+ chelator Bapta-AM could prevent [Ca2+m] overload and rescue cell death more effectively, indicating [Ca2+m] participate in other mechanisms implicated in cell death. HQ17(3) treatment resulted in increased mitochondrial superoxide, and loss of mitochondrial membrane potential. Degradation of mitochondrial fusion protein Mfn1/2 and Opa1 was noted, while MitoView stain showed significant loss of mitochondrial mass preceded cell death, indicated mitochondria might undergo fission followed by mitophagy, the selective degradation of mitochondria by autophagy. Mitophagy was revealed by COX IV and LC3B co-localization in immune-fluorescence stain. Furthermore, Ca2+-chelator attenuated HQ17(3)-induced mitochondrial ROS elevation, Opa1 cleavage and mitophagy occurence, indicated HQ17(3)-induced mitochondrial damage features are consequences of [Ca2+m] overload and related to cell death. In conclusion, HQ17(3) induces ER stress by yet-defined mechanism in Ph+ preB-ALL SUP-B15 cells, leads to [Ca2+m] overload, elevated superoxide and mitochondrial damage, followed by mitophagy, and cell death ensue. Our finding also suggests that Ph+ ALL cells is vulnerable to ER stress or mitochondrial stress, may provide a new approach to study alternative strategies for high-risk ALL treatment in future.

並列關鍵字

Ph+ acute lymphoblastic leukemia ； SUP-B15 ； HQ17(3) ； mitochondria damage ； cell death

參考文獻

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