Formosanin C 誘導之肝癌細胞自噬與鐵依賴型細胞死亡的關係

鐵依賴型細胞死亡（Ferroptosis）近來被指出是一種計畫性的細胞壞死。其特徵為依賴鐵產生的脂質過氧化（Lipid peroxidation），可能因細胞內自由鐵（Labile iron pool）增加導致。鐵是多種細胞生理現象所需的無機元素，並與細胞增殖及生長密切相關。腫瘤細胞，如肝癌細胞，對鐵的需求較高，且鐵相關蛋白基因會過度表現。此外，近期研究發現，細胞自噬中透過選擇性攜帶蛋白Nuclear coactivator 4（NCOA4）將鐵蛋白（Ferritin）降解的鐵蛋白自噬（Ferritinophagy），可促進鐵依賴型細胞死亡發生。本研究於SRB assay中發現隨植化素Formosanin C（FC）濃度上升，肝癌細胞Hep3B與HepG2生長顯著被抑制，而加入鐵依賴型細胞死亡抑制劑Ferrostatin-1、Liproxstatin-1及同時加Vitamin E與Vitamin C後，細胞生長有部分回復。處理FC後脂質過氧化物（Reactive oxygen species）的增加，及加入抑制劑後的部分減少，皆顯示FC有引發鐵依賴型細胞死亡的效果。FC同時會增加細胞中的酸性囊泡小體比例，顯示FC在兩個細胞中皆可誘導細胞自噬。西方墨點法中觀察到LC3-II產生，並在加入細胞自噬抑制劑Bafilomycin A1後累積，也證明了FC誘導細胞自噬的效果。在細胞自噬被抑制的情況下，處理FC 24小時後，NCOA4與鐵蛋白在兩細胞中皆會堆積。免疫螢光染色法中，LC3與NCOA4或LC3與鐵蛋白的Co-localization，也顯示FC誘導鐵蛋白自噬的潛力。值得注意的是，有較高NCOA4表現量的HepG2細胞，對FC誘導的鐵依賴型細胞死亡與細胞自噬，較鐵蛋白表現量較高的Hep3B細胞敏感，表明鐵依賴型細胞死亡誘導劑在鐵蛋白自噬相關的癌症治療中的作用。關鍵字：肝癌、皂素、鐵依賴型細胞死亡、細胞自噬、鐵蛋白自噬

關鍵字

肝癌；皂素；鐵依賴型細胞死亡；細胞自噬；鐵蛋白自噬

並列摘要

Ferroptosis is a recently described form of regulated necrotic cell death. A hallmark of ferroptosis is iron-dependent lipid peroxidation, which may caused by the increased level of labile iron pool. Iron is an essential inorganic element for various cellular events and is directly associated with cell proliferation and growth. Tumor cells, such as hepatocellular carcinoma (HCC) cells, necessitated higher concentrations of iron and the genes of iron uptake proteins were highly overexpressed. Moreover, recent studies indicated that autophagy contributes to ferroptosis through ferritinophagy, a degradation of the iron-storage protein, ferritin, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Here, SRB assay showed a significant dosage-related growth inhibition of HCC HepG2 and Hep3B cells by phytochemical Formosanin C (FC), which can be partially reversed by co-administration of ferroptosis inhibitor ferrostatin-1, liproxstatin-1 and vitamin E combined with vitamin C. Elevated production of lipid reactive oxygen species by FC and partially down regulation by co-administration of ferroptosis inhibitor indicating FC-induced ferroptosis. Percentage of acidic vesicular organelles-positive cells also increased after treated with FC, representing the induction of autophagic flux in both cell lines. In western blot analysis, the production of LC3-II by FC and a further elevation of LC3-II expression by addition of autophagy inhibitor bafilomycin A1 confirmed the autophagic effect of FC. In these autophagy inhibited conditions, NCOA4 and ferritin accumulation also observed in both cell lines after treated with FC for 24 h. Co-localization of LC3 with NCOA4 and LC3 with ferritin in immunoflurescence also showing the ferritinophagy potential induced by FC. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC-induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression, suggesting the role of ferroptosis inducer in ferritinophagy-related treatment of cancers. Keywords: hepatocellular carcinoma, saponin, ferroptosis, ferritinophagy, autophagy