| 摘要: | 背景:肺癌不僅是全球死亡率最高的癌症，也影響超過 10% 的人口。因此，治 療肺癌的藥物數量正在穩定增加。然而，目前核准的藥物雖然最初有效，但往往 面臨隨著時間的推移而使疾病復發。這種復發歸因於癌細胞產生抗藥性，導致治 療效果減弱。因此，目前需要開發能夠使抗藥性癌細胞重新敏感、促進細胞死亡 的藥物。在我們先前的研究中，我們觀察到抗藥性癌細胞中 USP24 的表現增加， 導致細胞對治療的反應減弱。然而，在加入 USP24 抑制劑，NCI677397 後，細 胞恢復了對化療藥物的敏感性，進而促使細胞死亡。雖然我們發現了 NCI677397 會導致細胞死亡，但迄今為止 USP24 在肺癌細胞中的作用尚未完全闡明。
目的:本研究旨在探討 USP24 在調節抗藥性肺癌細胞中對鐵離子依賴性死亡和 粒線體功能中的作用。
材料與方法:我們分別使用 A549 和紫杉醇抗藥性 A549 (A549-T24)細胞作為 我們的藥物敏感和抗藥性細胞系，並使用 USP24 抑制劑 NCI677397 作為我們的 開發藥物。使用 LC-MS/MS 來分類脂質的類型。Seahorse XFe24 用於研究粒線體 功能。CCK8 測定用於研究細胞存活率。西方點墨法和免疫螢光分別檢測蛋白質 表現和定位。免疫沉澱用於研究 USP24 和電子鏈傳輸複合物之間的相互作用。
結果:NCI677397 誘導脂質增加，導致脂質過氧化和脂質活性氧(lipid ROS)的 累積，最終引發鐵死亡並導致細胞死亡。在加入 ROS 抑制劑(NAC)減少活性
氧後，脂質相關蛋白(如 HMGCS1)和鐵離子相關蛋白(如 NCOA4)顯著減少。 因此，細胞存活率顯著提高。除了脂質過氧化之外，我們還觀察到細胞內鐵離子 的升高，這是促進鐵死亡的標誌。添加鐵離子螯合劑(DFP)後，我們觀察到 HMGCS1 和 NCOA4 濃度下降，但細胞存活率沒有顯著提高。最後，我們闡明了 抗藥性肺癌細胞與藥物敏感肺癌細胞具有不同的代謝系統。我們也發現 NCI677397 透過增加粒線體 ROS(mtROS)、抑制電子傳遞鏈(ETC)複合物 I 和 降低粒線體膜電位(MMP)來中斷粒線體活性，阻礙細胞產生足夠能量的能力， 進而啟動粒線體自噬作用，最終癌細胞走入鐵離子依賴性死亡。
結論:NCI677397 是一種新型 USP24 抑制劑，透過觸發脂質過氧化和鐵離子過 載等過程誘導細胞死亡，最終導致鐵離子依賴性死亡。NCI677397 也透過抑制粒 線體電子傳遞鏈功能，導致粒線體功能障礙，最終引發粒線體自噬作用。
關鍵字:肺癌、鐵離子依賴性死亡、粒線體、粒線體自噬、電子傳遞鏈、泛素特 異性蛋白?二十四
Background: Lung cancer not only possesses the highest global mortality rate among all cancers, but also affects over 10% of the population. Consequently, the number of drugs for treating lung cancer is steadily increasing. However, currently approved drugs, while initially effective, often face the challenge of relapse over time. This recurrence is attributed to the development of drug resistance in cancer cells, leading to a diminished therapeutic effect. Therefore, there is an unmet medical need to develop drugs that can re-sensitize resistant cancer cells, promoting cancer cell death. In our previous study, we observed an increased expression of USP24 in drug-resistant cells, rendering the cells less responsive to treatment. However, upon the addition of the USP24 inhibitor, NCI677397, the cells regained sensitivity to the chemotherapeutic drug, then, promoting cell death. Although we found out that NCI677397 induced cell death, the role of USP24 in lung cancer cells has not been fully elucidated to date.
Objective: This study aims to investigate the role of USP24 in regulating ferroptosis and mitochondrial function on drug-resistant lung cancer cells.
Materials and Methods: We used A549 and Taxol-resistant A549 (A549-T24) cells as drug-sensitive and drug-resistant cell lines, respectively, and utilized the USP24 inhibitor, NCI677397, as a developmental drug. LC-MS/MS were used to clarify the
type of lipids. Seahorse XFe24 were used to study mitochondrial function. CCK8 assay was used to study cell viability. Western blot and immunofluorescence were used to detect the protein expression and localization, respectively. Immunoprecipitation was used to study the interaction between USP24 and electron chain transport complexes.
Results: NCI677397 induced an increase in lipids, leading to lipid peroxidation and the accumulation of Lipid Reactive Oxygen Species (Lipid ROS), ultimately triggering ferroptosis and resulting in cell death. After the addition of N-acetyl Cysteine (NAC) to reduce ROS, there was a significant decrease in lipid-related enzymes (such as HMGCS1) and iron-related proteins (such as NCOA4). Consequently, cell survival rates showed a remarkable increase. In addition to lipid peroxidation, we observed an elevation in intracellular irons, a hallmark in promoting ferroptosis. Upon adding an iron chelator, deferiprone (DFP), we observed a decrease in HMGCS1 and NCOA4 levels, though there was no significant improvement in cell survival rates. Finally, we clarified that drug-resistant lung cancer cells have different metabolic system compared to drug-sensitive lung cancer cells. We also found that NCI677397 interrupts mitochondrial activity through increasing mitochondrial ROS (mtROS), inhibiting complex I of electron transport chain (ETC) and decreasing mitochondrial membrane potential (MMP), hindering the ability of cells to generate sufficient energy, thus, initiating mitophagy.
Conclusion: NCI677397, a novel USP24 inhibitor, induces cell death by triggering processes such as lipid peroxidation and iron overload, ultimately leading to ferroptosis. NCI677397 also induces mitochondrial dysfunction by inhibiting ETC function, leading to mitophagy
Keywords: lung cancer, ferroptosis, mitochondria, mitophagy, electron transport chain, USP24 |
