- 學位論文
瑞香草醌誘導腎臟癌細胞細胞凋亡與自體吞噬之研究
Effects of Thymoquinone on Inducing Renal Cancer Cells Apoptosis and Autophagy
摘要
研究目的：瑞香草醌(thymoquinone)是一種從黑種草(Nigella sativa)種籽中分離出的化合物，許多研究顯示具有多種生物活性；包括抗發炎、抗氧化和抗癌作用。本篇研究中，我們進一步研究瑞香草醌對人類腎細胞癌細胞(786-O和786-O-SI3)與腎臟泌尿上皮癌細胞(BFTC-909)的抗癌作用及其相關的分子機制。 研究方法及資料：分別透過MTT測定、trypan blue exclusion assay和細胞colony形成等測定方法來分析細胞存活率、細胞數目和細胞colony的形成。以PI染色法、Annexin V/PI染色法和DAPI染色法等測定方法來檢測瑞香草醌作用下，腫瘤細胞的細胞週期分佈、腫瘤細胞凋亡比例及腫瘤細胞染色質的濃縮。進行acidic vesicular organelles染色，來研究瑞香草醌作用下，自體吞噬作用是否參與了瑞香草醌所引起的腫瘤細胞死亡。以西方點墨法分析瑞香草醌作用下，腫瘤細胞內細胞凋亡及細胞自體吞噬相關之蛋白表現。 研究結果：我們的研究結果證實，瑞香草醌作用下顯著降低了腫瘤細胞存活率，並抑制了腎癌細胞colony的形成，且顯著降低腫瘤細胞的粒線體膜電位，並誘導786-O-SI3細胞與BFTC-909細胞的細胞凋亡作用。瑞香草醌作用下，增加了腫瘤細胞內的活性氧物質(reactive oxygen species, ROS)和超氧化物(superoxide)的產生，以及腫瘤細胞凋亡和自體吞噬相關訊息反應路徑的激活。同時，ROS的抑制作用下，顯著降低了腫瘤細胞caspase-3的活性，並拮抗了瑞香草醌抑制腫瘤細胞存活率的作用。此外，儘管瑞香草醌在786-O-SI3細胞中誘導細胞自體吞噬並激活了自體吞噬作用反應路徑，但是自體吞噬作用的抑制並不能顯著拮抗瑞香草醌降低腫瘤細胞存活率的反應。這些結果證實，細胞凋亡在瑞香草醌誘導的786-O-SI3細胞和BFTC-909TC細胞的細胞死亡中扮演著重要的角色，而非自體吞噬作用。此外，瑞香草醌抑制了786-O-SI3細胞sphere的形成以及aldehyde dehydrogenase活性、Nanog、Nestin、CD44和Oct-4的表達，這證實瑞香草醌可抑制癌症幹細胞的特性。在免疫缺陷的小鼠實驗中，由小鼠皮下接種786-O-SI3細胞再餵食小鼠瑞香草醌，證實瑞香草醌誘導細胞凋亡及抑制786-O-SI3細胞腫瘤生長之能力。 結論與建議：綜上所述，這些發現證明瑞香草醌誘導786-O-SI3細胞和BFTC-909細胞死亡。這可能歸因於經由ROS/超氧化物的產生，進一步誘導腫瘤細胞走向細胞凋亡。以上研究證實，瑞香草醌未來或許可應用於輔助治療泌尿生殖系統癌症。
並列摘要
Objective: Thymoquinone, a phytochemical compound isolated from Nigella sativa, has been shown to have various biological effects including anti-inflammation, anti-oxidation, and anticancer. Here, we further investigated the anti-cancer effects of thymoquinone on human renal carcinoma cell 786-O and 786-O-SI3 and transitional carcinoma cell BFTC-909 and the associating molecular mechanism. Methods and Materials: The viability, number, and colony formation were determined by MTT assay, trypan blue exclusion assay, and colony formation assay, respectively. The cell cycle distribution, apoptotic cell death, and chromatin condensation of the thymoquinone-treated cells were detected by PI staining, Annexin V/PI staining, and DAPI staining. Acridine orange staining for autolysosome were performed to investigate whether autophagy participated in thymoquinone-provoked cell death. Results: Our results showed that thymoquinone significantly reduced the cell viability and inhibited the colony formation of renal cancer cells. We also observed that thymoquinone clearly induced cell apoptosis and mitochondrial membrane potential change in both cancer cells. Thymoquinone also triggered the production of reactive oxygen species (ROS) and superoxide and the activation of apoptotic and autophagic cascade. Meanwhile, inhibition of ROS suppressed the caspase-3 activation and restored the decreased cell viability of 786-O-SI3 in response to thymoquinone. Interestingly, inhibition of autophagy did not intensively restore the cell viability of 786-O-SI3 suppressed by thymoquinone. Moreover, thymoquinone suppressed the cell sphere formation and the expression of aldehyde dehydrogenase, Nanog, Nestin, CD44, and Oct-4 in 786-O-SI3 cells. These findings revealed that thymoquinone was able to suppress the induced stemness of 786-O-SI3 cell. Furthermore, the tumor-bearing model showed that thymoquinone in vivo inhibited the growth of implanted 786-O-SI3 cell. Conclusion and Suggestion: Taken together, these findings indicate that thymoquinone inhibits the proliferation of 786-O-SI3 and BFTC-909 cell, which may attribute to the induction of ROS/superoxide and the consequent apoptosis. It suggests that thymoquinone may be a potential anticancer supplement for genitourinary cancer.