Ferroptosis Induction in Breast Cancer Cell Line MDA-MB-231 by Sulfasalazine and Ferric Ammonium Citrate

鐵對細胞具有雙面效應，是細胞生長所必需，但也可經由芬頓反應 (fenton reaction) 產生活性氧ROS而對細胞造成毒性與傷害。鐵依賴性死亡ferroptosis是2003年新發現的細胞死亡模式，屬於非凋亡性細胞死亡模式的一種。透過鐵代謝衍生的脂質過氧化 (lipid peroxidation, LROS) 累積而造成細胞死亡，並且受細胞內信號路徑嚴密的調控。有別於以往發現之死亡形態，ferroptosis 的形態、生化標記或是基因特徵皆不同於apoptosis、necrosis以及autophagic。Ferroptosis的路徑包括抑制還原態穀胱甘肽 (GSH) 的合成和利用，造成GSH耗竭，最終產生脂質過氧化效應和細胞死亡；以上任一節點受到干擾，皆可能影響ferroptosis的發生。已知乳癌細胞具有堆積鐵的能力，其代謝鐵池增大有利於乳癌細胞生長，而且惡性程度越高，堆積鐵的能力越強。因此本研究的目的是探討ferroptosis對乳癌細胞的作用。利用三陰性乳癌細胞株MDA-MB-231對柳氮磺口比口定水楊酸 (sulfasalazine, SAS) 敏感以及強積鐵能力兩種特性，實驗設計誘導MDA-MB-231細胞死亡，利用專一性抑制劑ferrostatin-1並檢測ferroptosis 分子特徵，以及鐵相關抗氧化蛋白質NRF2、HO-1及ferritin (FTH) 的表現，並評估抗氧化礦物質硒和鋅化合物之影響。實驗結果確認SAS與鐵共同處理可使MDA-MB-231細胞發生ferroptosis，並受ferrostatin-1之抑制，細胞死亡前有ROS以及LROS量增加的現象。隨著SAS與鐵的劑量增加，細胞的NRF2以及FTH表現增加。硒可以抑制此死亡作用，可能機制是透過增加GPX4蛋白表現量；鋅增加SAS與鐵複合組細胞死亡，Zn-MT的增加無助於複合組細胞存活。Iron plays dual roles in cells. It is essential for cell growth but also produce reactive oxygen species (ROS) via the Fenton reaction then inducing cell toxicity and damage. Iron-dependent cell death, ferroptosis, is a newly discovered non-apoptotic cell death mode in 2003. It is a regulated form of cell death driven by iron-induced lipid peroxide accumulation and regulation strictly by intracellular signaling pathway. The morphological, biochemical markers and genetic characteristics of ferroptosis are different from apoptosis, necrosis and autophagic. The mechanism of ferroptosis is inhibiting synthesis and utilization of reduced-glutathione (GSH) . Then, GSH depletion and lipid peroxide (LROS) accumulation lead to ferroptosis. Blockade of any steps is might prevent ferroptosis. Breast cancer cells have the ability of iron accumulation, which increases breast cancer cells growth. The higher malignancy degrees of breast cancer cells have the stronger ability of iron accumulation. Therefore, the purpose of this study is investigating the effect of ferroptosis on breast cancer cell. The study design is making use of the two characteristics of triple-negative breast cancer cell line MDA-MB-231 : sensitive to sulfasalazine (SAS) and strong ability of iron accumulation, to induce ferroptosis. We detect ferroptosis molecular characteristics, and the effects is blocked by specific inhibitors ferrostatin-1. Then, we analyze iron-related antioxidant proteins NRF2, HO-1 and ferritin (FTH) protein expression and assess the impact of the antioxidant mineral selenium and zinc compound in ferroptosis. The results show that combined group (SAS + iron) induce MDA-MB-231 cells ferroptosis, and ferrostatin-1 inhibit cell death. In addition, ROS and LROS are increased before cell death. The NRF2 and FTH protein expression levels are increased in a combined group dose-dependent manner. Finally, we found selenium can inhibit ferroptosis, and the mechanism may be through increasing the GPX4 protein expression. Zinc increase cell death in combined group and enhance Zn-MT protein expression, but Zn-MT does not contribute to prevent cell death