Elsevier

Chemical Engineering Journal
化学工程杂志

Volume 495, 1 September 2024, 153364
第 495 卷，2024 年 9 月 1 日，153364
Chemical Engineering Journal

Organelle synergy unleashed: Modulating mitochondrial-endoplasmic reticulum contacts with a self-assembled prodrug amplifies ferroptosis for innovative cancer therapy
释放细胞器协同作用：用自组装前药调节线粒体-内质网接触，放大铁死亡，用于创新癌症治疗

https://doi.org/10.1016/j.cej.2024.153364 IF: 13.3 Q1
https://doi.org/10.1016/j.cej.2024.153364IF：13.3 第一季度 Get rights and content 获取权利和内容

Highlights 亮点

  • •
    LSA has excellent self-assembly capability.
    LSA具有优异的自组装能力。
  • •
    LSA induces MERC via Vdac-Ip3r interaction, driving calcium ion influx and lipid metablism.
    LSA 通过 Vdac-Ip3r 相互作用诱导 MERC，驱动钙离子流入和脂质代谢。
  • •
    LSA enhances ferroptosis via augmented lipid peroxidation through MERC.
    LSA通过 MERC 增强脂质过氧化作用，从而增强铁死亡。
  • •
    LSA has good in vivo antitumor capacity via ferroptosis.
    LSA通过铁死亡具有良好的体内抗肿瘤能力。

Abstract 抽象的

Mitochondrial-endoplasmic reticulum contacts (MERC), a crucial mediator in subcellular organelle communication, significantly influences the dynamics of tumor organelle. Despite the well-established correlation between MERC-associated enzymes and lipid peroxidation (LPO), the contribution of MERC to LPO-induced ferroptosis in tumor remains unclear. In this study, we unveil the role of MERC in amplifying ferroptosis within tumor cells by introducing a prodrug LSA (LA-SS-ART, Linoleic acid-disulfide bond-artesunate), which ingeniously utilizes fatty acids as MERC inducers and ferroptosis activators. LSA induces Vdac-Ip3r-mediated MERC, resulting in the influx of Ca2+ from ER into mitochondria. Consequently, mitochondrial dysfunction triggers lipid peroxidation, attributed to hindered β-oxidation. Furthermore, LSA upregulates the expression of Lpcat3 and Mfn2, promoting phospholipid synthesis and enhancing the production of polyunsaturated fatty acid-phospholipids, synergistically intensifying ferroptosis. In conclusion, this study leverages organelle interactions to finely modulate cellular metabolism and membrane function, ultimately amplifying the process of ferroptosis. These findings offer a perspective and direction for innovative cancer therapy.
线粒体-内质网接触（MERC）是亚细胞器通讯的关键介质，显着影响肿瘤细胞器的动态。尽管 MERC 相关酶和脂质过氧化 (LPO) 之间存在明确的相关性，但 MERC 对 LPO 诱导的肿瘤铁死亡的贡献仍不清楚。在这项研究中，我们通过引入一种前药LSA（LA-SS-ART，亚油酸-二硫键-青蒿琥酯）揭示了MERC在放大肿瘤细胞内铁死亡中的作用，该药物巧妙地利用脂肪酸作为MERC诱导剂和铁死亡激活剂。 LSA 诱导 Vdac-Ip3r 介导的 MERC，导致 Ca 2+从 ER 流入线粒体。因此，线粒体功能障碍会引发脂质过氧化，这归因于β-氧化受阻。此外，LSA上调Lpcat3和Mfn2的表达，促进磷脂合成并增强多不饱和脂肪酸磷脂的产生，协同加剧铁死亡。总之，本研究利用细胞器相互作用来精细调节细胞代谢和膜功能，最终放大铁死亡过程。这些发现为创新癌症治疗提供了视角和方向。

Keywords 关键词

Mitochondrial-endoplasmic reticulum contacts
Ferroptosis
Prodrug
Lipid peroxidation
Cancer therapy

线粒体-内质网接触
铁死亡
前药
脂质过氧化
癌症治疗

1. Introduction 一、简介

Subcellular organelles, such as mitochondria and the endoplasmic reticulum, are specialized microstructures residing within the cell’s cytoplasm [1], [2], [3]. They constitute the inner cellular environment, each playing distinct roles in maintaining cellular functions and metabolic processes [4], [5], [6]. Furthermore, they establish intricate connections through membrane contact sites, commonly known as organelle crosstalk [7], [8], [9]. These interactions significantly optimize material transport efficiency, fortify specific organelle functions, and influence cellular fate [10], [11], [12].
亚细胞器，例如线粒体和内质网，是存在于细胞质内的特殊微观结构[1] , [2] , [3] 。它们构成了细胞内部环境，各自在维持细胞功能和代谢过程中发挥着不同的作用[4] , [5] , [6] 。此外，它们通过膜接触位点建立复杂的连接，通常称为细胞器串扰[7] 、 [8] 、 [9] 。这些相互作用显着优化了物质运输效率，强化了特定的细胞器功能，并影响了细胞的命运[10] 、 [11] 、 [12] 。
Among various organelle interactions, the communication between the mitochondria and endoplasmic reticulum has recently gained substantial attention [12], [13], [14]. Research findings have illuminated the multifaceted roles of mitochondrial-endoplasmic reticulum contacts (MERC) in various cellular processes, including ion transport, programmed cell death, and lipid metabolism [15], [16], [17]. (1) The endoplasmic reticulum, acting as the primary reservoir for calcium ions, releases calcium ions into mitochondria via MERC, inducing endoplasmic reticulum stress and generating reactive oxygen species (ROS), ultimately causing cellular oxidative damage [18], [19], [20], [21]. (2) Additionally, calcium accumulation in the mitochondrial matrix can trigger programmed cell death [22], [23], [24]. (3) The regions of interaction between the mitochondria and endoplasmic reticulum, known as mitochondria-associated membranes (MAMs), are enriched in lipid synthesis enzymes, actively participating in numerous biochemical reactions within cancer cells by regulating phospholipid synthesis and lipid peroxidation (LPO) [11], [25], [26], [27]. Given the crucial role of MERC in cancer cell development, interfering these interactions presents novel possibilities for cancer treatment [28].
在各种细胞器相互作用中，线粒体和内质网之间的通讯最近引起了广泛关注[12] ， [13] ， [14] 。研究结果阐明了线粒体-内质网接触 (MERC) 在各种细胞过程中的多方面作用，包括离子转运、程序性细胞死亡和脂质代谢[15] 、 [16] 、 [17] 。 (1)内质网作为钙离子的主要储存库，通过MERC将钙离子释放到线粒体中，诱导内质网应激并产生活性氧(ROS)，最终引起细胞氧化损伤[18] 、 [19] 、 [20] 、 [21] 。 (2) 此外，线粒体基质中的钙积累可引发程序性细胞死亡[22] 、 [23] 、 [24] 。 （3）线粒体和内质网之间的相互作用区域，称为线粒体相关膜（MAM），富含脂质合成酶，通过调节磷脂合成和脂质过氧化（LPO）积极参与癌细胞内的众多生化反应[11] 、 [25] 、 [26] 、 [27] 。鉴于 MERC 在癌细胞发育中的关键作用，干扰这些相互作用为癌症治疗提供了新的可能性[28] 。
Several fatty acids, such as linoleic acid and oleic acid, have been unveiled to serve as pivotal inducers of MERC. For instance, linoleic acid reshapes phospholipid membrane structures through lipid metabolism reprogramming, elevating the frequency and area of MERC [29]. Furthermore, oleic acid can initiate MERC, leading to calcium accumulation in the mitochondrial matrix and, ultimately, causing mitochondrial dysfunction [30]. Additionally, polyunsaturated fatty acids (PUFA) can induce LPO by remodeling lipid metabolism [31], [32].
几种脂肪酸，例如亚油酸和油酸，已被发现可作为 MERC 的关键诱导剂。例如，亚油酸通过脂质代谢重编程重塑磷脂膜结构，提高MERC的频率和面积[29] 。此外，油酸可以启动 MERC，导致线粒体基质中钙积累，最终导致线粒体功能障碍[30] 。此外，多不饱和脂肪酸（PUFA）可以通过重塑脂质代谢来诱导LPO [31] , [32] 。
LPO is a unique characteristic of ferroptosis, an iron-dependent form of cell death [33], [34]. Numerous studies have documented the feasibility of inducing ferroptosis via enhanced lipid peroxidation [35], [36], [37], [38], [39]. On the basis of MERC researches mentioned above, our study hypothesizes that MERC can be strategically manipulated to remodel membrane lipid function, thereby amplifying ferroptosis.
LPO 是铁死亡的一个独特特征，铁死亡是一种铁依赖性细胞死亡形式[33] , [34] 。许多研究已经证明通过增强脂质过氧化诱导铁死亡的可行性[35] 、 [36] 、 [37] 、 [38] 、 [39] 。在上述MERC研究的基础上，我们的研究假设可以策略性地操纵MERC来重塑膜脂功能，从而放大铁死亡。
To investigate the induction of MERC by fatty acids to enhance ferroptosis, we designed and synthesized a series of GSH-responsive prodrugs employing fatty acids as MERC inducers and ferroptosis activators. Our results demonstrate the effectiveness of our prodrug LSA (LA-SS-ART, Linoleic acid-disulfide bond-artesunate) in stimulating MERC, consequently augmenting phospholipid (PL) synthesis and the generation of PUFA-PL. Meanwhile, MERC-induced calcium accumulation disturbs mitochondrial function, further blocking β-oxidation. This, in turn, intensifies the peroxidation of PUFA-PL, ultimately enhancing ferroptosis (Scheme 1). This innovative approach capitalizes on organelle interactions to reshape cellular metabolism and strategically integrate with ferroptosis, providing a novel perspective and direction for ferroptosis-dependent cancer therapy.
为了研究脂肪酸诱导MERC以增强铁死亡，我们设计并合成了一系列利用脂肪酸作为MERC诱导剂和铁死亡激活剂的GSH响应性前药。我们的结果证明了我们的前药 LSA（LA-SS-ART，亚油酸-二硫键-青蒿琥酯）在刺激 MERC 方面的有效性，从而增强磷脂 (PL) 合成和 PUFA-PL 的生成。同时，MERC 诱导的钙积累会扰乱线粒体功能，进一步阻断 β-氧化。这反过来又加剧了 PUFA-PL 的过氧化，最终增强铁死亡（方案 1 ）。这种创新方法利用细胞器相互作用来重塑细胞代谢并与铁死亡战略性整合，为铁死亡依赖性癌症治疗提供了新的视角和方向。
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Scheme 1. The structure and mechanism of prodrug LSA. Chemical structure of LA, ART, LSA. The schematic illustration of LSA promoted mitochondria-endoplasmic reticulum contacts, ultimately enhancing ferroptosis.
方案一。前药LSA的结构和机制。 LA、ART、LSA 的化学结构。 LSA 的示意图促进了线粒体与内质网的接触，最终增强了铁死亡。

2. Material and methods 2 材料与方法

2.1. Synthesis for prodrug LSA
2.1.前药LSA的合成

Bis(2-hydroxyethyl) dissulfide (0.12 mmol) was dissolved in 4 ml of dimethyl sulfoxide (DMSO) in a 50-ml round-bottom flask. Then, artesunate (ART) (0.24 mmol) and DMAP (0.48 mmol) were added dropwise sequentially and stirred in an ice bath for 30 min. Next, EDCI (0.36 mmol) was added, and the mixture was further stirred at room temperature for 24  h. The completion of the reaction was monitored by thin-layer chromatography. The target products were purified by column chromatography to give a Transparent oil like substance 1 (76 mg, Yield, 61 %). 1H NMR (500 MHz, CDCl3): δ 5.78 (d, J = 9.9 Hz, 1H), 5.44 (s, 1H), 4.38 (tt, J = 6.6, 2.0 Hz, 2H), 3.88 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.6 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.77 – 2.73 (m, 2H), 2.71 – 2.64 (m, 2H), 2.59 – 2.53 (m, 1H), 2.37 (ddd, J = 14.6, 13.4, 4.0 Hz, 2H), 2.03 (ddd, J = 14.5, 4.9, 2.9 Hz, 1H), 1.89 (ddt, J = 13.6, 6.7, 3.6 Hz, 1H), 1.81 – 1.69 (m, 2H), 1.66 – 1.59 (m, 1H), 1.53 – 1.42 (m, 4H), 1.40 – 1.25 (m, 3H), 1.06 – 0.94 (m, 4H), 0.86 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ 171.98, 171.19, 104.47, 92.26, 91.48, 80.10, 62.62, 60.36, 51.52, 45.19, 41.45, 37.22, 36.93, 36.19, 34.05, 31.76, 29.11, 28.81, 25.91, 24.55, 21.95, 20.20, 12.05.
将双(2-羟乙基)二硫化物(0.12mmol)溶解在50ml圆底烧瓶中的4ml二甲亚砜(DMSO)中。然后，依次滴加青蒿琥酯(ART)(0.24mmol)和DMAP(0.48mmol)并在冰浴中搅拌30分钟。接下来，加入EDCI(0.36mmol)，并将混合物在室温下进一步搅拌24小时。通过薄层色谱法监测反应的完成。目标产物经柱层析纯化，得到透明油状物质1 (76mg，收率61％)。 1 H NMR（500 MHz，CDCl 3 ）： δ 5.78（d， J = 9.9 Hz，1H），5.44（s，1H），4.38（tt， J = 6.6，2.0 Hz，2H），3.88（t， J） = 6.0 Hz, 2H), 2.94 (t, J = 6.6 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.77 – 2.73 (米, 2H), 2.71 – 2.64 (米, 2H), 2.59 – 2.53（米，1H）、2.37（ddd、 J = 14.6、13.4、4.0 Hz、2H）、2.03（ddd、 J = 14.5、4.9、2.9 Hz、1H）、1.89（ddt、 J = 13.6、6.7、 3.6 Hz, 1H), 1.81 – 1.69 (米, 2H), 1.66 – 1.59 (米, 1H), 1.53 – 1.42 (米, 4H), 1.40 – 1.25 (米, 3H), 1.06 – 0.94 (米, 4H) ，0.86（d， J = 7.2 Hz，3H）。 13 C NMR（126 MHz，CDCl 3 ）： δ 171.98、171.19、104.47、92.26、91.48、80.10、62.62、60.36、51.52、45.19、41.45、37.22、36.93、36.19、 34.05、31.76、29.11、28.81、25.91、 24.55、21.95、20.20、12.05。
Linoleic acid (LA) was dissolved in 4 ml of dimethyl sulfoxide (DMSO) in a 50-ml round-bottom flask. Then, intermediate 1 (0.24 mmol) and DMAP (0.48 mmol) were added dropwise sequentially and stirred in an ice bath for 30 min. Next, EDCI (0.36 mmol) was added, and the mixture as further stirred at room temperature for 24  h. The completion of the reaction was monitored by thin-layer chromatography. The target products were purified to give a Transparent oil like substance LSA (133 mg, Yield, 71 %). 1H NMR (500 MHz, CDCl3): δ 5.77 (d, J = 9.9 Hz, 1H), 5.44 – 5.26 (m, 5H), 4.32 (dt, J = 13.0, 6.6 Hz, 4H), 2.90 (t, J = 6.6 Hz, 4H), 2.78 – 2.50 (m, 7H), 2.40 – 2.27 (m, 3H), 2.08 – 1.94 (m, 5H), 1.87 (ddt, J = 13.5, 6.7, 3.5 Hz, 1H), 1.79 – 1.67 (m, 2H), 1.64 – 1.56 (m, 3H), 1.41 (s, 5H), 1.40 – 1.21 (m, 17H), 1.05 – 0.81 (m, 10H). 13C NMR (126 MHz, CDCl3): δ 173.55, 171.86, 171.04, 130.21, 130.04, 128.04, 127.91, 104.45, 92.20, 91.50, 80.10, 62.57, 62.05, 51.56, 45.24, 37.32, 37.26, 37.03, 36.22, 34.16, 34.10, 31.80, 31.52, 29.60, 29.34, 29.17, 29.14, 29.10, 28.80, 27.20, 25.96, 25.63, 24.88, 24.59, 22.57, 21.99, 20.22, 14.09, 12.07. HRMS (ESI, m/z): Calcd for C41H66O10S2 [M + Na]+: 805.4030, found: 805.3995.
将亚油酸 (LA) 溶解在 50 毫升圆底烧瓶中的 4 毫升二甲基亚砜(DMSO) 中。然后，依次滴加中间体1(0.24mmol)和DMAP(0.48mmol)，并在冰浴中搅拌30分钟。接下来，加入EDCI(0.36mmol)，并将混合物在室温下进一步搅拌24小时。通过薄层色谱法监测反应的完成。纯化目标产物，得到透明油状物质LSA (133mg，收率71％)。 1 H NMR（500 MHz，CDCl 3 ）： δ 5.77（d， J = 9.9 Hz，1H），5.44 – 5.26（m，5H），4.32（dt， J = 13.0，6.6 Hz，4H），2.90（t , J = 6.6 Hz, 4H), 2.78 – 2.50 (米, 7H), 2.40 – 2.27 (米, 3H), 2.08 – 1.94 (米, 5H), 1.87 (ddt, J = 13.5, 6.7, 3.5 Hz, 1H ), 1.79 – 1.67 (米, 2H), 1.64 – 1.56 (米, 3H), 1.41 (秒, 5H), 1.40 – 1.21 (米, 17H), 1.05 – 0.81 (米, 10H )。 13 C NMR（126 MHz，CDCl 3 ）： δ 173.55、171.86、171.04、130.21、130.04、128.04、127.91、104.45、92.20、91.50、80.10、62.57、62.05、 .56、45.24、37.32、37.26、37.03、36.22、 34.16、34.10、31.80、31.52、29.60、29.34、29.17、29.14、29.10、28.80、27.20、25.96、25.63、24.88、24.59、22.57、21.99、20。 22、14.09、12.07。 HRMS (ESI， m / z )：C 41 H 66 O 10 S 2 [M + Na] +计算值：805.4030，实测值：805.3995。

2.2. Preparation and characterization of nano-assemblies
2.2.纳米组件的制备和表征

Self-assembly of prodrugs was investigated in aqueous solution via the nanoprecipitation method. Briefly, the prodrugs were dissolved in DMSO and added dropwise into deionized water with vigorous stirring.The colloidal stability of ART prodrugs were investigated in PBS. The hydrodynamic diameters of prodrugs were characterized by Zetasizer (Nano ZS, Malvern Co.,UK). The morphologies of prodrugs were explored using transmission electron microscopy (TEM, HITACHI, HT7700, Japan).
通过纳米沉淀法研究了水溶液中前药的自组装。简而言之，将前药溶解在 DMSO 中，并在剧烈搅拌下滴加到去离子水中。研究 ART 前药在 PBS 中的胶体稳定性。前药的流体动力学直径通过 Zetasizer (Nano ZS, Malvern Co,UK) 进行表征。使用透射电子显微镜（TEM，HITACHI，HT7700，日本）探索前药的形态。

2.3. Subcellular localization
2.3.亚细胞定位

LA (2 μM), ART (2 μM), LSA (2 μM), LSA (2 μM) + nocodazole (Noc) (1 μM) dispersed in medium was incubated with cells over 24 h. Cells were incubated with Mito-Tracker Orange probe (Invitrogen, M7510) or ER-Tracker Green probe (Yeasen, Shanghai, 40763ES) for 15 min. Then cells were examined using Confocal Laser Scanning Microscope. Mito-Tracker and ER Tracker were respectively excited at the wavelength of 554, 488 nm.
LA (2 μM)、ART (2 μM)、LSA (2 μM)、LSA (2 μM) + 诺考达唑 (Noc) (1 μM) 分散在培养基中，与细胞一起孵育 24 小时以上。将细胞与 Mito-Tracker Orange 探针（Invitrogen，M7510）或 ER-Tracker Green 探针（Yeasen，上海，40763ES）孵育 15 分钟。然后使用共焦激光扫描显微镜检查细胞。 Mito-Tracker和ER Tracker分别在554、488 nm波长处激发。

2.4. Statistical analysis
2.4.统计分析

Data were expressed as means ± SD. For comparison between groups, statistical differences were analyzed by two-sided unpaired t tests. For multiple sample analyses, statistical differences were analyzed by one-way analysis of variance with Tukey’s multiple comparisons. All statistical analyses were performed using GraphPad Prism version 9 software. Statistical significance was defined as P < 0.05. Levels of significance were indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.
数据表示为平均值±SD。为了进行组间比较，通过两侧未配对 t 检验分析统计差异。对于多样本分析，通过Tukey多重比较的单向方差分析来分析统计差异。所有统计分析均使用 GraphPad Prism 版本 9 软件进行。统计显着性定义为 P< 0.05。显着性水平表示为*P < 0.05、**P < 0.01 和***P < 0.001。
Refer to Supplementary Section for reagents and apparatus used for this study.
有关本研究使用的试剂和仪器，请参阅补充部分。

3. Results 3. 结果

3.1. Self-Assembling potential and cytotoxicity of LSA
3.1. LSA的自组装潜力和细胞毒性

In our pursuit of potentiating ferroptosis through MERC, we innovatively designed and synthesized three compounds: LSA, OSA, and SSA (Fig. 1A and Scheme S1-S3). These compounds cleverly combine MERC enhancers with ferroptosis inducers, linked through a cleavable bond responsive to the tumor microenvironment’s GSH. MERC enhancers include linoleic acid (LA) representing PUFA, oleic acid (OA) symbolizing monounsaturated fatty acids (MUFA), and stearic acid (SA) signifying saturated fatty acids (SFA). The ferroptosis inducer is the widely recognized agent artesunate (ART). To validate our design’s integrity, we synthesized three control compounds (LCA, OCA, and SCA) (Scheme S4-S6), replacing the disulfide bond with a non-responsive carbon–carbon (CC) bond, rendering them unresponsive to GSH. The above six compounds were confirmed via rigorous structural analysis using 1H NMR, 13C NMR, and high-resolution mass spectrometry (Figs. S1-S22).
为了通过 MERC 增强铁死亡，我们创新地设计并合成了三种化合物：LSA、OSA 和 SSA（图 1 A 和方案 S1-S3 ）。这些化合物巧妙地将 MERC 增强剂与铁死亡诱导剂结合起来，通过响应肿瘤微环境 GSH 的可裂解键连接。 MERC增强剂包括代表PUFA的亚油酸(LA)、代表单不饱和脂肪酸(MUFA)的油酸(OA)和代表饱和脂肪酸(SFA)的硬脂酸(SA)。铁死亡诱导剂是广泛认可的药物青蒿琥酯（ART）。为了验证我们设计的完整性，我们合成了三种对照化合物（LCA、OCA 和 SCA）（方案 S4-S6 ），用非响应性碳碳 (CC) 键替换二硫键，使它们对 GSH 无响应。上述六种化合物通过使用1 H NMR、 13 C NMR 和高分辨率质谱的严格结构分析得到证实（图S1-S22 ）。
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Fig. 1. Self-assembly ability and cytotoxicity of prodrugs. (A) The structure of six compounds. (B) Characterization of nanoparticles with appearance, size distribution from DLS and TEM morphology, scale bars: 200 nm, Polydispersity index (PDI). (C, D) Stability analysis of nanoparticles by measuring average sizes and PDI of nanoparticles in aqueous environments by DLS for 7 days (n = 3). (E) TEM images depicting the morphological changes under GSH, scale bars: 200 nm. (F) IC50 values (μM) of free ART and nanoparticles against 4T1 and CT26 tumor cell lines for 48 h (n = 3). (G) Cell colony formation assay and quantitative result in the 4T1 and CT26 cells treated with LA, ART, LSA (n = 3). Scale bars = 400 μm. (H) Cell wound-healing assay and quantitative result in the 4T1 and CT26 cells treated with LA, ART, LSA (n = 3). (I) Calcein AM/PI staining assay of 4T1 and CT26 cells in the presence of LA, ART, LSA. Scale bars = 120 μm.
图1 .前药的自组装能力和细胞毒性。 (A) 六种化合物的结构。 (B)纳米粒子的外观表征、 DLS和TEM形态的尺寸分布、比例尺：200 nm、多分散指数(PDI)。 (C、D) 通过DLS测量水环境中纳米颗粒的平均尺寸和 PDI 7 天来进行纳米颗粒的稳定性分析 (n = 3)。 (E) TEM图像描绘 GSH 下的形态变化，比例尺：200 nm。 (F)游离ART和纳米颗粒针对 4T1 和 CT26 肿瘤细胞系 48 小时的IC 50值 (μM) (n = 3)。 (G) 用LA 、ART、LSA处理的 4T1 和 CT26 细胞的细胞集落形成测定和定量结果(n = 3)。比例尺 = 400 μm。 (H) 用LA 、ART、LSA 处理的 4T1 和 CT26 细胞的细胞伤口愈合测定和定量结果 (n = 3)。 (I)在 LA、ART、LSA 存在下对 4T1 和 CT26 细胞进行钙黄绿素AM/PI 染色测定。比例尺 = 120 μm。

Considering the presence of lipid chains in these compounds, we hypothesized their ability to spontaneously self-assemble into nanoscale particles in aqueous solutions. To validate this hypothesis, we conducted a thorough investigation, employing the Dahl effect, transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments. As anticipated, all six compounds demonstrated the ability to form spherical nanoparticles with an approximate 200-nanometer diameter (Fig. 1B). These nanoparticles exhibited remarkable stability in aqueous environments and PBS, maintaining their structural integrity for up to seven days (Fig. 1C, D and Fig. S23). Furthermore, the responsiveness of the nanoparticles to GSH was assessed. TEM images revealed substantial degradation of the disulfide bond within the nanoparticles after exposure to 0.1 mM GSH, leading to complete disappearance after 4 h. Conversely, under GSH-deficient conditions, the nanoparticles retained their spherical morphology, as evident in Fig. 1E.
考虑到这些化合物中存在脂质链，我们假设它们能够在水溶液中自发自组装成纳米级颗粒。为了验证这一假设，我们利用达尔效应、透射电子显微镜 (TEM) 和动态光散射(DLS) 实验进行了彻底的研究。正如预期的那样，所有六种化合物都表现出形成直径约为 200 纳米的球形纳米颗粒的能力（图 1 B）。这些纳米颗粒在水性环境和 PBS 中表现出显着的稳定性，可保持其结构完整性长达 7 天（图 1 C、D 和图 S23 ）。此外，还评估了纳米颗粒对 GSH 的响应性。 TEM 图像显示，暴露于 0.1 mM GSH 后，纳米粒子内的二硫键发生显着降解，导致 4 小时后完全消失。相反，在缺乏 GSH 的条件下，纳米颗粒保留了其球形形态，如图1 E 所示。
Next, MTT assays were conducted to evaluate their impact on cancer cell proliferation (Fig. 1F), encompassing mouse colorectal cancer CT26 and breast cancer 4T1 cells, both reliant on lipid metabolism (Fig. S24). Results demonstrated that LSA nanoparticles exhibited significantly heightened cellular antitumor efficacy, reducing the IC50 value to 0.3 μM, a more than 20-fold reduction compared to other nanoparticles in CT26 cells. A similar trend was consistently observed in the case of 4T1 cells. Our research reveals that compounds such as LCA and OSA exhibit lower cytotoxicity compared to free ART. On one hand, the diminished effectiveness be attributed to the incapacity of carbon–carbon bonds to react with GSH, thereby impeding their capacity to cleave within the tumor milieu to unleash LA and ART. On the other hand, the reduced efficacy of compounds such as OSA compared to ART might be ascribed to OA’s role as a monounsaturated fatty acid, which exhibits the capability to suppress ferroptosis [35]. To delve deeper into their cytotoxic effects, we employed colony formation and wound-healing assays. As illustrated in Fig. 1G and H, the results underscore the effective suppression of cell migration and colony formation by LSA nanoparticles. Furthermore, the fluorescence intensity of propidium iodide (PI), an indicator of cell death, was notably higher in the LSA nanoparticles group compared to the saline, LA, and ART groups (Fig. 1I).
接下来，进行MTT测定以评估它们对癌细胞增殖的影响（图1F ），包括小鼠结直肠癌CT26和乳腺癌4T1细胞，两者都依赖于脂质代谢（图S24 ）。结果表明，LSA纳米颗粒表现出显着增强的细胞抗肿瘤功效，将CT26细胞中的IC 50值降低至0.3 μM，与其他纳米颗粒相比降低了20倍以上。在 4T1 细胞中也一致观察到类似的趋势。我们的研究表明，与游离 ART 相比，LCA 和 OSA 等化合物的细胞毒性较低。一方面，有效性降低归因于碳-碳键无法与 GSH 发生反应，从而阻碍了它们在肿瘤环境中裂解释放 LA 和 ART 的能力。另一方面，与 ART 相比，OSA 等化合物的功效降低可能归因于 OA 作为单不饱和脂肪酸的作用，它具有抑制铁死亡的能力[35] 。为了更深入地研究它们的细胞毒性作用，我们采用了集落形成和伤口愈合测定。如图1G和H所示，结果强调了LSA纳米颗粒对细胞迁移和集落形成的有效抑制。 此外，与盐水组、LA 组和 ART 组相比，LSA 纳米颗粒组中碘化丙啶(PI)（细胞死亡指标）的荧光强度明显更高（图 1 I）。
These findings highlight the dual functionality of LSA nanoparticles in enhancing the elimination of cancer cells while concurrently restraining cell migration, setting them apart from LA and ART. Moreover, LSA nanoparticles exhibited superior cytotoxicity compared to other nanoparticle formulations.
这些发现强调了 LSA 纳米粒子的双重功能，即增强癌细胞的消除，同时抑制细胞迁移，使其与 LA 和 ART 区分开来。此外，与其他纳米颗粒制剂相比，LSA 纳米颗粒表现出优异的细胞毒性。

3.2. LSA-strengthened ferroptosis
3.2. LSA强化铁死亡

Ferroptosis is characterized by the accumulation of iron-dependent lipid peroxides and disturbances in redox balance [40]. To investigate LSA’s role in enhancing ferroptosis via LPO, we utilized confocal imaging and Western blot analysis. Initially, we employed C11 BODIPY 581/591 as a probe to gauge lipid peroxidation severity and DCFH-DA as a probe to estimate cellular ROS (Fig. 2A, Fig. S25). Notably, higher green fluorescence intensity in LSA group was exhibited compared to LA and ART, both indicative of peroxidized lipids and ROS. Subsequently, LSA treatment significantly elevated the LPO end-product MDA, contrasting with LA and ART (Fig. 2B and C). Western blot experiments highlighted a substantial upregulation of acyl-CoA synthetase long-chain family member 4 (Acsl4), a pivotal catalyst in PUFA peroxidation within the LSA group (Fig. 2D). Additionally, we employed the ferroptosis inhibitor Ferrostatin-1 (Fer-1) alongside an apoptosis inhibitor to authenticate the cell death mechanism. Our results indicate that Fer-1 effectively rescued cells post LSA treatment, while the apoptosis inhibitor M50054 and necroptosis inhibitor Necrostatin-1 (Nec-1) failed to do so (Fig. S26). These strongly suggest that ferroptosis is the predominant mechanism of cell death in this scenario. These findings underscore LSA’s ability to intensify ferroptosis through LPO.
铁死亡的特征是铁依赖性脂质过氧化物的积累和氧化还原平衡的紊乱[40] 。为了研究 LSA 通过 LPO 增强铁死亡的作用，我们利用共聚焦成像和蛋白质印迹分析。最初，我们使用C11 BODIPY 581/591作为探针来测量脂质过氧化的严重程度，并使用DCFH-DA作为探针来估计细胞ROS （图2A ，图S25 ）。值得注意的是，与 LA 和 ART 相比，LSA 组表现出更高的绿色荧光强度，均表明存在过氧化脂质和 ROS。随后，与 LA 和 ART 相比，LSA 处理显着提高了 LPO 最终产物 MDA（图 2 B 和 C）。蛋白质印迹实验强调了酰基辅酶A合成酶长链家族成员4（Acsl4）的显着上调，Acsl4是LSA组内PUFA过氧化的关键催化剂（图2D ）。此外，我们还使用铁死亡抑制剂 Ferrostatin-1 (Fer-1) 和凋亡抑制剂来验证细胞死亡机制。我们的结果表明，Fer-1在LSA处理后有效地拯救了细胞，而细胞凋亡抑制剂M50054和坏死性凋亡抑制剂Necrostatin-1（Nec-1）则未能做到这一点（图S26 ）。这些强烈表明铁死亡是这种情况下细胞死亡的主要机制。 这些发现强调了 LSA 通过 LPO 强化铁死亡的能力。
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Fig. 2. LSA-strengthened ferroptosis. (A) Measuring cellular lipid peroxidation by fluorescence microscopy using the C11 BODIPY 581/591 fluorescent probe. Total C11 BODIPY 581/591 (red), oxidized C11 BODIPY 581/591 (green), DAPI (blue) stained nucleus. Scale bars: 25 μm. (B, C) MDA level was measured in CT26 and 4T1 cells after treated with LA, ART or LSA (n = 3). (D) Western blot for ACSL4 was performed after treatment of LA, ART or LSA in CT26 and 4T1 cell. (E) FerroOrange staining for intracellular Fe2+ in CT26 and 4T1 cells treated in the presence or absence of LA, ART or LSA. Scale bars: 25 μm. (F) Western blot for TFR was performed, after treatment of LA, ART or LSA in CT26 and 4T1 cell. (G, H) The level of GSH was measured in CT26 and 4T1 cells after treatment of LA, ART or LSA (n = 3). (I, J) Western blot for Slc7a11 and Gpx4 was performed after treatment of LA, ART or LSA in CT26 and 4T1 cell.
图2 . LSA 强化铁死亡。 (A)使用C11 BODIPY 581/591荧光探针通过荧光显微镜测量细胞脂质过氧化。总 C11 BODIPY 581/591（红色）、氧化 C11 BODIPY 581/591（绿色）、 DAPI （蓝色）染色细胞核。比例尺：25 μm。 (B、C)在用LA 、 ART或 LSA 处理后测量 CT26 和 4T1 细胞的MDA水平 (n = 3)。 (D) CT26 和 4T1 细胞经LA 、ART 或 LSA 处理后进行ACSL4的蛋白质印迹。 (E)在存在或不存在LA、ART 或 LSA 的情况下处理的 CT26 和 4T1 细胞中细胞内 Fe 2+的铁橙染色。比例尺：25 μm。 (F)在 CT26 和 4T1 细胞中经 LA、ART 或 LSA 处理后，进行 TFR 的蛋白质印迹。 (G, H) 在 LA、ART 或 LSA 处理后测量 CT26 和 4T1 细胞中的 GSH 水平 (n = 3)。 (I，J) CT26 和 4T1 细胞经 LA、ART 或 LSA 处理后，进行 Slc7a11 和 Gpx4 的蛋白质印迹。

Subsequently, we probed whether the augmentation of LPO relies on iron. Confocal fluorescence analysis demonstrated that LSA-treated cells exhibited significantly higher orange fluorescence intensity of ferrous ions (Fig. 2E). The upregulation of Transferrin Receptor (Tfr) in Western blot assays suggested the transportation of iron to the cellular labile iron pool, where it becomes available for subsequent Fenton reactions (Fig. 2F). Previous literature has indicated a correlation between the expression levels of Tfr and ROS [40]. The results demonstrate a decrease in Tfr expression levels upon the addition of ROS scavenger N-Acetylcysteine (NAC) (Fig. S27). These further underscores the role of LSA-induced ROS in modulating Tfr and its associated iron metabolism. These results collectively indicate LSA’s capacity to enhance the abundance of iron ions, thereby intensifying Fe-dependent LPO.
随后，我们探讨了LPO的增强是否依赖于铁。共焦荧光分析表明，LSA处理的细胞表现出明显更高的亚铁离子橙色荧光强度（图2E ）。蛋白质印迹分析中转铁蛋白受体(Tfr) 的上调表明铁转运至细胞不稳定铁库，可用于随后的芬顿反应（图 2F ）。先前的文献已经表明Tfr和ROS的表达水平之间存在相关性[40] 。结果表明，添加ROS清除剂N-乙酰半胱氨酸（NAC）后Tfr表达水平降低（图S27 ）。这些进一步强调了 LSA 诱导的 ROS 在调节 Tfr 及其相关铁代谢中的作用。这些结果共同表明 LSA 具有增强铁离子丰度的能力，从而增强 Fe 依赖性 LPO。
Expanding upon the analysis of intracellular oxidized compounds, we investigated reductive components. The GSH-Gpx4 axis, crucial in cellular antioxidant defense, was examined to gauge cellular sensitivity to ferroptosis [41]. Measurement of GSH abundance in tumor cells revealed lower levels following LSA treatment compared to other groups (Fig. 2G, H). Simultaneously, Western blot experiments indicated a significant downregulation of Slc7a11, a component of the glutamate-cysteine antiporter (system Xc−), and Gpx4 within the LSA group (Fig. 2I, J). These results underscore LSA’s ability to disrupt the GSH-Gpx4 axis, diminishing intracellular antioxidant capability and enhancing cellular sensitivity to ferroptosis.
在细胞内氧化化合物分析的基础上，我们研究了还原成分。对细胞抗氧化防御至关重要的 GSH-Gpx4 轴进行了检查，以衡量细胞对铁死亡的敏感性[41] 。肿瘤细胞中 GSH 丰度的测量显示，与其他组相比，LSA 治疗后的 GSH 水平较低（图 2 G、H）。同时，Western blot 实验表明 LSA 组内谷氨酸半胱氨酸逆向转运蛋白（系统 Xc - ）的一个组成部分 Slc7a11 和 Gpx4 显着下调（图 2 I，J）。这些结果强调了 LSA 破坏 GSH-Gpx4 轴、降低细胞内抗氧化能力并增强细胞对铁死亡的敏感性的能力。
These consistent findings demonstrate that LSA amplifies iron-dependent lipid peroxidation in tumor cells and interferes with the intracellular reduction system, providing the mechanistic basis for enhanced ferroptosis. Furthermore, in comparison to ART, LSA significantly intensifies ferroptosis, potentially owing to the increased presence of LA within LSA.
这些一致的发现表明，LSA 放大了肿瘤细胞中铁依赖性脂质过氧化，并干扰细胞内还原系统，为增强铁死亡提供了机制基础。此外，与 ART 相比，LSA 显着加剧铁死亡，这可能是由于 LSA 中 LA 的存在增加。

3.3. LSA-induced MERC 3.3. LSA诱导的MERC

During our investigation, we have confirmed that the introduction of LA significantly enhances ART-induced ferroptosis. This prompts a critical question: does this augmentation originate from MERC?.
在我们的调查过程中，我们已经证实，LA 的引入显着增强了 ART 诱导的铁死亡。这就提出了一个关键问题：这种增强是否源自 MERC？
To address this, we conducted morphological assessments of mitochondria and endoplasmic reticulum through confocal imaging. Mito-Orange and ER-Tracker (green) labeled cells were utilized to distinguish mitochondria and endoplasmic reticulum. Notably, in cells treated with PBS or ART, there was no observable overlap between the red and green fluorescence in CT26 and 4T1 cells. In contrast, treatment with LA and LSA resulted in significant co-localization of red and green fluorescence (Fig. 3A), confirming LSA’s capacity to induce MERC.
为了解决这个问题，我们通过共聚焦成像对线粒体和内质网进行了形态学评估。 Mito-Orange 和 ER-Tracker（绿色）标记的细胞用于区分线粒体和内质网。值得注意的是，在用 PBS 或 ART 处理的细胞中，CT26 和 4T1 细胞的红色和绿色荧光之间没有观察到重叠。相反，LA和LSA处理导致红色和绿色荧光显着共定位（图3A ），证实了LSA诱导MERC的能力。
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Fig. 3. Mitochondrial and endoplasmic reticulum contacts were induced by LSA. (A) Representative confocal images of ER-Tracker (Green) and Mito-Tracker (Red) loaded 4T1 and CT26 cells treated with LA, ART, LSA. Scale bars: 10 μm. (B) Representative TEM images and confocal images of MERC in 4T1 and CT26 cells treated with LSA after 0, 8, 16, 24 h. TEM image: Red lines with arrow indicate the contact of Mito-ER. Scale bars: 200 nm. Confocal images: Scale bars: 10 μm. (C) The mode of localization of MERC in 4T1 and CT26 cells, green: Ip3r, red: Vdac, blue: DAPI. Scale bars: 10 μm. (D) Schematic diagram depicting the interplay between ER and mitochondria in tumor cell treated with LSA. (E) Representative confocal images of Rhod-2 (Red) and DAPI (Blue) loaded 4T1 and CT26 cells treated with LA, ART, LSA. Scale bars: 30 μm. Quantification of Ca2+ level (n = 3). (F) Western blot analysis of Chop, Bip, and p-EIF2a expression in 4T1 cells after 36 h of treatment with LA, ART, LSA and LSA + Noc. (G) Western blot analysis of Chop, Bip, and p-EIF2α expression in CT26 cells after treatment with LA, ART, LSA and LSA + Noc.
图3 . LSA 诱导线粒体和内质网接触。 (A) 加载经 LA、ART、LSA 处理的 4T1 和 CT26 细胞的 ER-Tracker（绿色）和 Mito-Tracker（红色）的代表性共焦图像。比例尺：10 μm。 (B) 在 0、8、16、24 小时后用 LSA 处理的 4T1 和 CT26 细胞中 MERC 的代表性 TEM 图像和共聚焦图像。 TEM 图像：带箭头的红线表示 Mito-ER 的接触点。比例尺：200 nm。共焦图像：比例尺：10 μm。 (C) MERC 在 4T1 和 CT26 细胞中的定位模式，绿色：Ip3r，红色：Vdac，蓝色：DAPI。比例尺：10 μm。 (D) 示意图描绘了经 LSA 处理的肿瘤细胞中 ER 和线粒体之间的相互作用。 (E) 加载经 LA、ART、LSA 处理的 4T1 和 CT26 细胞的 Rhod-2（红色）和 DAPI（蓝色）的代表性共焦图像。比例尺：30 μm。 Ca 2+水平的定量 (n = 3)。 (F)用 LA、ART、LSA 和 LSA + Noc处理 36 小时后 4T1 细胞中 Chop、Bip 和 p-EIF2a 表达的蛋白质印迹分析。 (G)用 LA、ART、LSA 和 LSA + Noc 处理后 CT26 细胞中 Chop、Bip 和 p-EIF2α 表达的蛋白质印迹分析。

To gain a deeper insight into the dynamics of LSA-induced MERC formation, we examined mitochondria and ER at various time points post-treatment (Fig. 3B). Notably, within the first 8 h following LSA treatment, substantial MERC features were not observed. However, a partial overlap of red and green fluorescence became evident during 8–16-hour mark and peaked at 24-hour mark. TEM corroborated these findings, highlighting the dynamic nature of LSA-induced MERC, with notable progress between 8 and 24 h. Next, the involvement of voltage-dependent anion channel (Vdac) and inositol trisphosphate receptor (Ip3r) in mediating MERC prompted immunofluorescence staining. The clear overlap between Vdac and Ip3r within the LSA group suggested that MERC is mediated through the binding of these two proteins (Fig. 3C), reinforcing the idea that LSA induces MERC through Vdac-Ip3r interaction (Fig. 3D).
为了更深入地了解 LSA 诱导的 MERC 形成的动态，我们在治疗后的不同时间点检查了线粒体和 ER（图 3 B）。值得注意的是，在 LSA 治疗后的前 8 小时内，没有观察到实质性的 MERC 特征。然而，红色和绿色荧光的部分重叠在 8-16 小时内变得明显，并在 24 小时内达到峰值。 TEM 证实了这些发现，突出了 LSA 诱导的 MERC 的动态性质，在 8 至 24 小时内取得了显着进展。接下来，电压依赖性阴离子通道 (Vdac) 和肌醇三磷酸受体 (Ip3r) 参与介导 MERC 促进了免疫荧光染色。 LSA组内Vdac和Ip3r之间的明显重叠表明MERC是通过这两种蛋白的结合介导的（图3C ），强化了LSA通过Vdac-Ip3r相互作用诱导MERC的观点（图3D ）。
The consequential increase in mitochondrial calcium levels and endoplasmic reticulum stress due to MERC formation through Vdac-Ip3r interaction were investigated [24], [28]. Using nocodazole (Noc) as a known MERC inhibitor [42], we assessed cellular status post-inhibition of MERC (Fig. S28). Rhod-2 probe analysis revealed notably higher red fluorescence intensity in the LSA group compared to the LA and ART groups (Fig. 3E). Co-treatment with LSA and Noc led to a significant reduction in red fluorescence levels, indicating that LSA-induced elevation of mitochondrial calcium ion levels depends on MERC. The accumulation of calcium ions resulted in decreased mitochondrial membrane potential, signifying damage to mitochondrial function (Fig. S29). Furthermore, Western blot analysis confirmed that in the LSA group, levels of endoplasmic reticulum stress-related proteins, including phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α), C/EBP homologous protein (Chop), and heavy-chain binding protein (Bip), were significantly higher compared to the ART group. Importantly, this increase was mitigated by Noc, indicating that LSA amplifies calcium influx through MERC, resulting in heightened endoplasmic reticulum stress (Fig. 3F-G).
研究了通过 Vdac-Ip3r 相互作用形成 MERC 导致线粒体钙水平和内质网应激随之增加[24] 、 [28] 。使用诺考达唑（Noc）作为已知的 MERC 抑制剂[42] ，我们评估了 MERC 抑制后的细胞状态（图 S28 ）。 Rhod-2探针分析显示，与LA和ART组相比，LSA组的红色荧光强度明显更高（图3E ）。 LSA 和 Noc 共同处理导致红色荧光水平显着降低，表明 LSA 诱导的线粒体钙离子水平升高取决于 MERC。钙离子的积累导致线粒体膜电位降低，表明线粒体功能受损（图S29 ）。此外，Western blot分析证实，在LSA组中，内质网应激相关蛋白的水平升高，包括真核起始因子2的磷酸化α亚基（ p-eIF2α）、C/EBP同源蛋白（Chop）和重链结合与 ART 组相比，蛋白质（Bip）显着升高。重要的是，Noc 减轻了这种增加，表明 LSA 放大了通过 MERC 的钙流入，导致内质网应激升高（图 3 FG）。
Our aboved experiments conclusively demonstrate that LSA induces MERC via Vdac-Ip3r interaction, subsequently resulting in an elevation of mitochondrial calcium levels and endoplasmic reticulum stress.
我们的上述实验最终证明，LSA 通过 Vdac-Ip3r 相互作用诱导 MERC，随后导致线粒体钙水平和内质网应激升高。

3.4. Ferroptosis and MERC validation through RNA-seq analysis
3.4.通过 RNA-seq 分析进行铁死亡和 MERC 验证

To further probe the mechanisms underlying LSA’s control over cellular fate, we performed RNA transcriptome sequencing on 4T1 cells. This analysis revealed notable differences in gene expression between the LSA-treated and control groups (Fig. 4A). In the LSA-treated group, 3,294 genes displayed significant differential expression, with 719 upregulated and 2,575 downregulated genes (Fig. 4B). High correlation coefficients exceeding 0.99 among samples within the same group underscored the experiment’s reproducibility (Fig. S30).
为了进一步探讨 LSA 控制细胞命运的机制，我们对 4T1 细胞进行了RNA转录组测序。该分析揭示了 LSA 处理组和对照组之间基因表达的显着差异（图 4 A）。在LSA处理组中，3,294个基因表现出显着差异表达，其中719个上调基因和2,575个下调基因（图4B ）。同一组内样本之间超过0.99的高相关系数强调了实验的再现性（图S30 ）。
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Fig. 4. The existence of ferroptosis and MERC was confirmed by RNA-seq. (A) Heatmap of differentially expressed genes after LSA treatment or untreatment (n = 3). (B) Volcano plot of differentially expressed genes. (C) KEGG enrichment analysis of differentially expressed genes after LSA treatment or untreatment. (D) GO enrichment analysis of differentially expressed genes after LSA treatment or untreatment. (E, F) Heatmap of main 20 genes in the ferroptosis (E), calcium signaling pathway (F) (n = 3). (G, H) GSEA analysis of genes in the protein processing in the endoplasmic reticulum pathway (G) and fatty acid beta oxidation (H) regulation identified in LSA for 24 h. (I) Mechanism of lipid degradation reprogramming from fatty acid oxidation to lipid peroxidation (n = 3). (J, K) Heatmap of main 20 genes in the UPR (J), ERAD (K).
图4 . RNA-seq证实了铁死亡和MERC的存在。 (A) LSA 处理或未处理后差异表达基因的热图 (n = 3)。 (B) 差异表达基因的火山图。 (C) LSA处理或未处理后差异表达基因的KEGG富集分析。 (D) LSA处理或未处理后差异表达基因的GO富集分析。 (E, F) 铁死亡 (E)、钙信号通路 (F) 中主要 20 个基因的热图 (n = 3)。 (G, H) GSEA 分析 LSA 中鉴定的内质网途径 (G) 蛋白质加工中的基因 (G) 和脂肪酸 β 氧化 (H) 调节 24 小时。 (I)从脂肪酸氧化到脂质过氧化的脂质降解重编程机制(n = 3)。 (J, K) UPR (J)、ERAD (K) 中主要 20 个基因的热图。

KEGG enrichment analysis emphasized LSA’s primary impact on ferroptosis and calcium signaling pathways, with calcium ions strongly associated with MERC (Fig. 4C). Gene Ontology (GO) enrichment analysis further highlighted that differentially expressed genes in the LSA-treated group primarily engage in the regulation of intracellular calcium ion transport and mitochondrial-endoplasmic reticulum physiological processes (Fig. 4D). These analyses reinforce our experimental observations, substantiating LSA’s enhancement of ferroptosis via MERC.
KEGG 富集分析强调了 LSA 对铁死亡和钙信号通路的主要影响，其中钙离子与 MERC 密切相关（图 4 C）。基因本体（GO）富集分析进一步强调，LSA处理组中差异表达的基因主要参与细胞内钙离子转运和线粒体内质网生理过程的调节（图4D ）。这些分析强化了我们的实验观察，证实了 LSA 通过 MERC 增强铁死亡。
A detailed examination of genes associated with ferroptosis and calcium signaling pathways (Fig. 4E, F) revealed LSA’s upregulation of pro-ferroptotic genes (Acsl4, Lpcat3, Trp53) and downregulation of anti-ferroptotic genes (Slc7a11, Acsl3, Pcbp1). Additionally, genes related to the calcium signaling pathway, including Cacna1i, Vdac2, Ryr2, Adcy2, exhibited a significant increase in response to LSA treatment. These findings underscore LSA’s pivotal role in modulating both ferroptosis and MERC.
对与铁死亡和钙信号通路相关的基因的详细检查（图4 E，F）揭示了LSA对促铁死亡基因（ Acsl4 ， Lpcat3 ， Trp53 ）的上调和抗铁死亡基因（ Slc7a11 ， Acsl3 ， Pcbp1 ）的下调。此外，与钙信号通路相关的基因，包括Cacna1i 、 Vdac2 、 Ryr2 、 Adcy2 ，对 LSA 治疗的反应表现出显着增加。这些发现强调了 LSA 在调节铁死亡和 MERC 中的关键作用。
Furthermore, GSEA indicated that the protein processing pathway in the endoplasmic reticulum was activated, while the fatty acid beta-oxidation pathway was suppressed within the LSA-treated group (Fig. 4G, H). This suggest LSA’s significant impact on endoplasmic reticulum function and lipid metabolism. A detailed analysis of the lipid degradation pathway revealed a significant reprogramming of lipid metabolism, with a shift in the degradation of PUFA from fatty acid oxidation to lipid peroxidation (Fig. 4I). This transition was associated with an increase in ROS generation and a decrease in ATP production (Fig. S31), possibly linked to mitochondrial dysfunction induced by calcium accumulation.
此外，GSEA 表明，LSA 治疗组内质网中的蛋白质加工途径被激活，而脂肪酸 β 氧化途径受到抑制（图 4 G、H）。这表明LSA对内质网功能和脂质代谢有显着影响。对脂质降解途径的详细分析揭示了脂质代谢的显着重编程，PUFA 的降解从脂肪酸氧化转变为脂质过氧化（图 4 I）。这种转变与 ROS 生成的增加和 ATP 生成的减少相关（图 S31 ），可能与钙积累引起的线粒体功能障碍有关。
Based on the GSEA results, we conducted a detailed examination of genes associated with endoplasmic reticulum function. The unfolded protein response (UPR) and endoplasmic reticulum-associated protein degradation (ERAD), two pivotal signaling pathways influencing endoplasmic reticulum function and highly related to endoplasmic reticulum stress [43], exhibited notable changes in their expression patterns in the LSA group compared to the control group (Fig. 4J, K). This further substantiates the occurrence of endoplasmic reticulum stress following LSA treatment, aligning with our previous experimental findings and underscoring the presence of MERC.
根据GSEA结果，我们对与内质网功能相关的基因进行了详细检查。未折叠蛋白反应（UPR）和内质网相关蛋白降解（ERAD）这两个影响内质网功能且与内质网应激高度相关的关键信号通路[43] ，与对照组相比，LSA组的表达模式出现显着变化。对照组（图4 J、K）。这进一步证实了 LSA 治疗后内质网应激的发生，与我们之前的实验结果一致，并强调了 MERC 的存在。
The above RNA-seq results confirm that following LSA treatment, both MERC and the ferroptosis pathway are triggered, accompanied by lipid metabolism reprogramming.
上述RNA-seq结果证实，LSA治疗后，MERC和铁死亡途径均被触发，并伴有脂质代谢重编程。

3.5. LSA enhances ferroptosis via augmented lipid peroxidation through MERC
3.5. LSA 通过 MERC 增强脂质过氧化作用，从而增强铁死亡

MAMs serve as repositories for numerous lipid synthesis enzymes, thereby fortifying lipid production [44]. Given that lipids are substrates crucial to lipid peroxidation and play a pivotal role in ferroptosis regulation [32], we postulated that MERC could potentially enhance lipid synthesis to provide the necessary substrates for LPO, thereby amplifying ferroptosis.
MAM 作为多种脂质合成酶的储存库，从而强化脂质生产[44] 。鉴于脂质是脂质过氧化的关键底物，并在铁死亡调节中发挥关键作用[32] ，我们假设 MERC 可能增强脂质合成，为 LPO 提供必要的底物，从而放大铁死亡。
To explore LSA’s impact on lipids, we conducted lipidomics analysis (Fig. 5A). Compared to the control group, LSA-treated cells exhibited significant alterations in 278 lipid metabolites, including fatty acids (FAs), triglycerides (TGs), phospholipids (PLs), and cholesterol esters (CEs). Notably, the most prominent changes were observed in phospholipids, particularly in phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and lysophosphatidylethanolamine (LPE). A reduction in monounsaturated phospholipids, indicating heightened cellular susceptibility to ferroptosis, was evident (Fig. S32A). Moreover, a decline in polyunsaturated phospholipids and PUFAs suggested active degradation (Fig. S32B, C), possibly through lipid peroxidation. In summary, these findings highlight LSA’s significant impact on phospholipids.
为了探讨 LSA 对脂质的影响，我们进行了脂质组学分析（图 5 A）。与对照组相比，LSA 处理的细胞在 278 种脂质代谢物中表现出显着变化，包括脂肪酸 (FA)、甘油三酯 (TG)、磷脂 (PL) 和胆固醇酯 (CE)。值得注意的是，最显着的变化是在磷脂中观察到的，特别是磷脂酰乙醇胺 (PE)、磷脂酰胆碱 (PC)、磷脂酰丝氨酸 (PS) 和溶血磷脂酰乙醇胺 (LPE)。单不饱和磷脂明显减少，表明细胞对铁死亡的敏感性增加（图 S32 A）。此外，多不饱和磷脂和 PUFA 的减少表明可能通过脂质过氧化进行主动降解（图 S32 B、C）。总之，这些发现强调了 LSA 对磷脂的显着影响。
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Fig. 5. LSA Enhances Ferroptosis via Augmented Lipid Peroxidation through MERC. (A) Heatmap of lipid metabolites. (B) Schematic illustration of phospholipid synthesis. (C) Relative expression of Gpat3, Lpin2, Cept1, Pemt, Ptdss1, Ptdss2, Lpcat3, Mfn2, Pisd in cytoplasm (CP), endoplasmic reticulum (ER), MAM and mitochondria (Mito) (n = 3). (D, E) Immunofluorescence of Mfn2 and Lpcat3. Scale bars: 25 μm. (F, G) Western blot for Mfn2 and Lpcat3 was performed after 36 h treated with LSA or LSA + Noc in CT26 and 4T1 cell. (H, I) MDA levels in CT26 and 4T1 cell treated with LSA or LSA + Noc (n = 3). (J, K) Measuring cellular lipid peroxidation in CT26 and 4T1 cell by fluorescence microscopy using the C11 BODIPY 581/591 fluorescent probe. Total C11 BODIPY 581/591 (red), oxidized C11 BODIPY 581/591 (green), DAPI (blue) stained nucleus. Scale bars: 25 μm.
图5 。 LSA 通过 MERC 增强脂质过氧化作用来增强铁死亡。 (A)脂质代谢物的热图。 (B)磷脂合成示意图。 (C) Gpat3 、 Lpin2 、 Cept1 、 Pemt 、 Ptdss1 、 Ptdss2 、 Lpcat3 、 Mfn2 、 Pisd在细胞质 (CP)、内质网 (ER)、MAM 和线粒体 (Mito) 中的相对表达(n = 3)。 (D, E) Mfn2和Lpcat3的免疫荧光。比例尺：25 μm。 (F, G) 在 CT26 和 4T1 细胞中用 LSA 或 LSA + Noc处理 36 小时后进行 Mfn2 和 Lpcat3 的蛋白质印迹。 (H, I)用 LSA 或 LSA + Noc处理的 CT26 和 4T1 细胞中的MDA水平 (n = 3)。 (J, K)使用C11 BODIPY 581/591荧光探针通过荧光显微镜测量 CT26 和 4T1 细胞中的细胞脂质过氧化。总 C11 BODIPY 581/591（红色）、氧化 C11 BODIPY 581/591（绿色）、DAPI（蓝色）染色细胞核。比例尺：25 μm。

Subsequently, RNA sequencing analysis of key genes in the phospholipid synthesis pathway was employed (Fig. 5B). Compared to the control group, a majority of genes linked to phospholipid synthesis exhibited pronounced upregulation. These included cytoplasmic genes (Cpat3, Lpin2, Cept1), endoplasmic reticulum genes (Pert, Ptdss1), mitochondria genes (Mfn2, Psid, Ptdss2), and MAMs genes (Lpcat3 and Lpcat4). Among these genes, Mfn2 and Lpcat3 displayed the most significant alterations (Fig. 5C), emphasizing LSA’s role in catalyzing phospholipid synthesis. Literature reports further suggest that Lpcat3 not only induces phospholipid synthesis but also facilitates the binding of PL and PUFA, promoting the formation of PUFA-PL, crucial substrates for lipid peroxidation [45]. RNA-seq analysis demonstrated substantial upregulation of Lpcat3 and Acsl4, indicating that lipid peroxidation is the degradation pathway for phospholipids and PUFAs. These results affirmed that LSA enhances phospholipid synthesis, augmenting the substrate pool for lipid peroxidation.
随后，对磷脂合成途径中的关键基因进行了RNA测序分析（图5B ）。与对照组相比，大多数与磷脂合成相关的基因表现出明显的上调。这些包括细胞质基因（ Cpat3 、 Lpin2 、 Cept1 ）、内质网基因（ Pert 、 Ptdss1 ）、线粒体基因（ Mfn2 、 Psid 、 Ptdss2 ）和 MAM 基因（ Lpcat3和Lpcat4 ）。在这些基因中， Mfn2和Lpcat3显示出最显着的改变（图 5 C），强调了 LSA 在催化磷脂合成中的作用。文献报道进一步表明， Lpcat3不仅诱导磷脂合成，而且促进PL和PUFA的结合，促进PUFA-PL的形成，PUFA-PL是脂质过氧化的关键底物[45] 。 RNA-seq 分析表明Lpcat3和Acsl4显着上调，表明脂质过氧化是磷脂和 PUFA 的降解途径。这些结果证实 LSA 增强磷脂合成，增加脂质过氧化的底物库。
Building upon these insights, we postulated that MERC intensifies ferroptosis by enhancing lipid peroxidation. To test this hypothesis, we examined the effect of MERC on Mfn2 and Lpcat3. Immunofluorescence showed that MERC inhibition significantly reduced Mfn2 and Lpcat3 expression in 4T1 and CT26 cells (Fig. 5D and E). These findings were corroborated by Western blot results, demonstrating a substantial downregulation of Mfn2 and Lpcat3 in the MERC-inhibited group (Fig. 5F and G). These outcomes suggest that MERC upregulates Mfn2 and Lpcat3.
基于这些见解，我们假设 MERC 通过增强脂质过氧化来加剧铁死亡。为了检验这一假设，我们检查了 MERC 对 Mfn2 和 Lpcat3 的影响。免疫荧光显示MERC抑制显着降低4T1和CT26细胞中Mfn2和Lpcat3的表达（图5D和E）。这些发现得到了蛋白质印迹结果的证实，表明 MERC 抑制组中 Mfn2 和 Lpcat3 显着下调（图 5 F 和 G）。这些结果表明 MERC 上调 Mfn2 和 Lpcat3。
To validate the influence of MERC inhibition on lipid peroxidation, we measured intracellular malondialdehyde (MDA) content (Fig. 5H and I), which significantly decreased following MERC inhibition. Using the C11-Bodipy probe to assess intracellular lipid peroxidation, we observed a considerable reduction in intracellular green fluorescence after MERC inhibition (Fig. 5J and K). These findings collectively support the conclusion that LSA augments ferroptosis by intensifying lipid peroxidation.
为了验证 MERC 抑制对脂质过氧化的影响，我们测量了细胞内丙二醛 (MDA) 含量（图 5 H 和 I），该含量在 MERC 抑制后显着下降。使用 C11-Bodipy 探针评估细胞内脂质过氧化，我们观察到 MERC 抑制后细胞内绿色荧光显着减少（图 5 J 和 K）。这些发现共同支持了 LSA 通过强化脂质过氧化而增强铁死亡的结论。
Our comprehensive experimental results confirm that LSA induces MERC, upregulates Mfn2 and Lpcat3, enhances phospholipid synthesis, increases PUFA-PL formation, and ultimately intensifies intracellular lipid peroxidation, culminating in amplified ferroptosis.
我们的综合实验结果证实，LSA 诱导 MERC，上调 Mfn2 和 Lpcat3，增强磷脂合成，增加 PUFA-PL 形成，并最终加剧细胞内脂质过氧化，最终导致放大的铁死亡。

3.6. In vivo evaluation of LSA
3.6. LSA 的体内评估

Building upon the significant in vitro anticancer efficacy of LSA and its ability to enhance ferroptosis, we conducted an evaluation of its potential antitumor effects in vivo. Fig. 6A and 6G depict the close monitoring of tumor volume changes in both the CT26 and 4T1 mouse xenograft models throughout the treatment period. Notably, while LA exhibited negligible inhibition of tumor growth and ART displayed moderate tumor suppression, LSA exerted the most substantial tumor-inhibitory effects (Fig. 6B-D and H-J). Upon concluding the experiments, tumor weights were assessed (Fig. 6E, K), indicating that LSA’s self-assembled nanoparticles enhance the in vivo stability of ART, effectively addressing concerns about its rapid degradation and limited bioavailability. Significantly, the therapeutic effect of the LSA + Noc group was less pronounced, suggesting that MERC inhibition can dampen LSA’s in vivo efficacy. In addition, we compared the in vivo efficacy of LSA with that of clinical chemotherapy agent (Cisplatin) and ferroptosis inducer (Erastin). The antitumor effect of LSA was significantly stronger than that of other groups (Fig. S33).
基于 LSA 显着的体外抗癌功效及其增强铁死亡的能力，我们对其潜在的体内抗肿瘤作用进行了评估。图6A和6G描绘了在整个治疗期间对CT26和4T1小鼠异种移植模型中肿瘤体积变化的密切监测。值得注意的是，虽然 LA 对肿瘤生长的抑制作用可以忽略不计，而 ART 显示出中等的肿瘤抑制作用，但 LSA 却发挥了最显着的肿瘤抑制作用（图 6 BD 和 HJ）。实验结束后，对肿瘤重量进行了评估（图6E 、K），表明LSA的自组装纳米颗粒增强了ART的体内稳定性，有效解决了对其快速降解和生物利用度有限的担忧。值得注意的是，LSA + Noc 组的治疗效果不太明显，表明 MERC 抑制可以抑制 LSA 的体内功效。此外，我们还比较了LSA与临床化疗药物（顺铂）和铁死亡诱导剂（Erastin）的体内疗效。 LSA的抗肿瘤作用明显强于其他组（图S33 ）。
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Fig. 6. In Vivo Evaluation of LSA. (A) Schematic illustration of the 4T1 tumor orthotopic establishment and therapeutic regimen. (B, C) Tumor volume of 4T1-tumor-bearing Balb/c mice after LA, ART, LSA and LSA + Noc treatments (n = 5). (D, E) Tumor photos (D) and weight (E) of mice from different groups at the 14th day (n = 5). (F) Western blot for Acsl4, Slc7a11, Lpcat3, Mfn2 was performed. (G) Schematic illustration of the CT26 tumor subcutaneous establishment and therapeutic regimen. (H, I) Tumor volume of CT26-tumor-bearing Balb/c mice after LA, ART, LSA and LSA + Noc treatments (n = 5). (J, K) Tumor photos (J) and weight (K) of mice from different groups at the 14th day (n = 5). (L) Western blot for Acsl4, Slc7a11, Lpcat3, Mfn2, Vdac, Ip3r was performed. (M, N) Acsl4, Slc7a11, Lpcat3, Mfn2 immunohistochemical staining of CT26 and 4T1 tumors. Scale bars: 100 μm.
图6 . LSA 的体内评估。 (A) 4T1肿瘤原位建立和治疗方案的示意图。 (B、C) LA、ART、LSA 和 LSA + Noc 治疗后 4T1 荷瘤 Balb/c 小鼠的肿瘤体积 (n = 5)。 (D,E)第14天时不同组小鼠的肿瘤照片(D)和体重(E)(n = 5)。 (F) 对 Acsl4、Slc7a11、Lpcat3、Mfn2 进行蛋白质印迹。 (G) CT26肿瘤皮下建立和治疗方案的示意图。 (H, I) LA、ART、LSA 和 LSA + Noc 治疗后 CT26 荷瘤 Balb/c 小鼠的肿瘤体积 (n = 5)。 (J,K)第14天时不同组小鼠的肿瘤照片(J)和体重(K)(n = 5)。 (L) 对 Acsl4、Slc7a11、Lpcat3、Mfn2、Vdac、Ip3r 进行蛋白质印迹。 (M，N) CT26 和 4T1 肿瘤的 Acsl4、Slc7a11、Lpcat3、Mfn2 免疫组织化学染色。比例尺：100 μm。

In-depth analysis of tumor tissues was performed to evaluate the expression levels of key proteins related to ferroptosis (Acsl4, Slc7a11), phospholipid synthesis (Lpcat3, Mfn2) and MERC (Vdac, Ip3r) (Fig. 6F, L and Fig. S34). Western blot results underscored the substantial increase in Acsl4, Lpcat3, Mfn2, Vdac and Ip3r expression in the LSA group, while Slc7a11 expression was significantly reduced compared to other groups.
对肿瘤组织进行深入分析，评估与铁死亡相关的关键蛋白（Acsl4、Slc7a11）、磷脂合成（Lpcat3、Mfn2）和MERC（Vdac、Ip3r）的表达水平（图6F、L和图6）。 S34 ）。 Western blot结果显示，与其他组相比，LSA组中Acsl4、Lpcat3、Mfn2、Vdac和Ip3r表达显着增加，而Slc7a11表达显着降低。
To further assess therapeutic outcomes, histological analyses were conducted, including HE staining for tumor sections and immunohistochemical staining for Acsl4, Slc7a11, Lpcat3 and Mfn2. Notably, the LSA treatment group exhibited the highest degree of tumor tissue necrosis in the HE-stained images (Fig. S35). Importantly, immunohistochemical images revealed a remarkable increase in ferroptosis markers (characterized by the upregulation of Acsl4 and downregulation of Slc7a11) and a substantial enhancement in phospholipid synthesis (indicated by the upregulation of Lpcat3 and Mfn2) (Fig. 6M, N). These findings are in agreement with the Western blot results, collectively affirming LSA’s robust anticancer effects. Additionally, through enhanced phospholipid synthesis, the LSA treatment group provided a significant pool of lipid peroxidation substrates, intensifying ferroptosis.
为了进一步评估治疗结果，进行了组织学分析，包括肿瘤切片的 HE 染色以及 Acsl4、Slc7a11、Lpcat3 和 Mfn2 的免疫组织化学染色。值得注意的是，LSA治疗组在HE染色图像中表现出最高程度的肿瘤组织坏死（图S35 ）。重要的是，免疫组织化学图像显示铁死亡标记物显着增加（以 Acsl4 上调和 Slc7a11 下调为特征），并且磷脂合成显着增强（以 Lpcat3 和 Mfn2 上调表示）（图 6 M，N）。这些发现与蛋白质印迹结果一致，共同证实了 LSA 强大的抗癌作用。此外，通过增强磷脂合成，LSA 治疗组提供了大量脂质过氧化底物，加剧了铁死亡。
Moreover, no significant differences in body weight were observed between the PBS and LSA treatment groups upon completing the treatment regimen (Fig. S36). Importantly, LSA did not induce any discernible adverse reactions in the mice during treatment, underscoring the reliable biological safety profile of LSA at the administered dosage. This robust safety profile can be attributed to LSA’s self-assembly stability in the in vivo milieu, facilitated by the presence of disulfide bonds that enable targeted release at the tumor site for therapeutic efficacy.
此外，在完成治疗方案后，在PBS和LSA治疗组之间没有观察到体重的显着差异(图S36 )。重要的是，LSA 在治疗期间没有在小鼠中引起任何明显的不良反应，这强调了 LSA 在给药剂量下具有可靠的生物安全性。这种强大的安全性可归因于 LSA 在体内环境中的自组装稳定性，二硫键的存在促进了 LSA 在肿瘤部位的靶向释放以达到治疗功效。
Subsequent to these observations, we conducted a comprehensive pathological assessment of major organs (including the heart, liver, spleen, lung and kidney) through tissue sectioning, supplemented by a panel of routine blood tests. As depicted in Figs. S37 and S38, the distinctions between the LSA group and the other positive control groups compared to the Saline group were marginal. Additionally, the majority of hematological parameters remained within the confines of normalcy, while hepatic function exhibited no discernible anomalies (Figs. S39, S40). Moreover, we conducted immunohistochemical evaluation of ferroptosis-associated proteins in liver tissue. The findings revealed unaltered expression levels of Acsl4 and Slc7a11, indicating the safety profile of LSA and its lack of causing normal cells ferroptosis in liver tissue (Fig. S41). In summary, the comprehensive results from these experiments collectively establish LSA as a potent anticancer agent characterized by robust efficacy and a distinguished safety profile.
在这些观察之后，我们通过组织切片，辅以一组常规血液检查，对主要器官（包括心脏、肝脏、脾脏、肺和肾）进行了全面的病理评估。如图所示。 S37和S38 ，与盐水组相比，LSA组和其他阳性对照组之间的差异是微乎其微的。此外，大多数血液学参数保持在正常范围内，而肝功能没有表现出明显的异常（图S39、S40 ）。此外，我们对肝组织中铁死亡相关蛋白进行了免疫组织化学评估。结果显示，Acsl4 和 Slc7a11 的表达水平没有改变，表明 LSA 的安全性及其不会引起肝组织中正常细胞铁死亡（图 S41 ）。总之，这些实验的综合结果共同证明 LSA 是一种有效的抗癌药物，具有强大的功效和卓越的安全性。

3.7. Enhancing ferroptosis via MERC: Clinical implications and therapeutic potential
3.7.通过 MERC 增强铁死亡：临床意义和治疗潜力

To assess the clinical applicability of the strategy aimed at reinforcing ferroptosis through MERC, a bioinformatics analysis of pivotal genes associated with ferroptosis (Acsl4 and Slc7a11) and phospholipid synthesis (Lpcat3 and Mfn2) in colorectal cancer patients was conducted.
为了评估旨在通过 MERC 强化铁死亡的策略的临床适用性，对结直肠癌患者中与铁死亡相关的关键基因（ Acsl4和Slc7a11 ）和磷脂合成（ Lpcat3和Mfn2 ）进行了生物信息学分析。
Commencing with Kaplan-Meier analysis to gauge the impact of Acsl4, Slc7a11, Lpcat3 and Mfn2 expression levels on the prognosis of colorectal cancer patients, important revelations emerged (Fig. 7A): patients with diminished expression levels of Acsl4, Lpcat3 and Mfn2 had lower survival rates compared to those with elevated expression levels of these genes. In contrast, colorectal cancer patients with low Slc7a11 expression exhibited more favorable survival rates than patients with heightened Slc7a11 expression. This dichotomy underscores the role of lower Acsl4, Lpcat3, and Mfn2 expression, as well as higher Slc7a11 expression, in driving colorectal cancer progression and adverse clinical outcomes.
从Kaplan-Meier分析开始，评估Acsl4 、 Slc7a11 、 Lpcat3和Mfn2表达水平对结直肠癌患者预后的影响，出现了重要的启示（图7A ）： Acsl4 、 Lpcat3和Mfn2表达水平降低的患者与这些基因表达水平升高的人相比，存活率较低。相比之下， Slc7a11低表达的结直肠癌患者比Slc7a11高表达的患者表现出更好的生存率。这种二分法强调了较低的Acsl4 、 Lpcat3和Mfn2表达以及较高的Slc7a11表达在驱动结直肠癌进展和不良临床结果中的作用。
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Fig. 7. Enhancing Ferroptosis via MERC: Clinical Implications and Therapeutic Potential. (A) The Kaplan-Meier survival analysis for Acsl4, Slc7a11, Lpcat3, Mfn2 genes along with the hazard ratio (HR) and log rank p value. (B, C) Correlation between Acsl4, Slc7a11, Lpcat3, Mfn2 in CRC. (D) The expression of Acsl4, Slc7a11, Lpcat3, Mfn2 in normal tissues and different grades tissue samples of CRC. (E) Immunohistochemical of Acsl4, Slc7a11, Lpcat3, Mfn2 in CRC patients tissue samples. Scale bars: 100 μm.
图7 .通过 MERC 增强铁死亡：临床意义和治疗潜力。 (A) Acsl4 、 Slc7a11 、 Lpcat3 、 Mfn2基因的 Kaplan-Meier 生存分析以及风险比 (HR) 和对数秩 p 值。 (B、C) CRC 中Acsl4 、 Slc7a11 、 Lpcat3 、 Mfn2之间的相关性。 (D) Acsl4 、 Slc7a11 、 Lpcat3 、 Mfn2在正常组织和CRC不同级别组织样本中的表达。 (E) CRC 患者组织样本中 Acsl4、Slc7a11、Lpcat3、Mfn2 的免疫组织化学。比例尺：100 μm。

A comprehensive exploration of the interplay among Acsl4, Slc7a11, Lpcat3 and Mfn2 revealed significant intercorrelations between these genes (Fig. 7B). Notably, a robust correlation was found between most phospholipid synthesis genes (Lpcat3 and Mfn2) and ferroptosis-related genes (Acsl4 and Slc7a11) (Fig. 7C). These findings affirm the clinical viability of enhancing phospholipid synthesis to potentiate ferroptosis.
对Acsl4 、 Slc7a11 、 Lpcat3和Mfn2之间相互作用的全面探索揭示了这些基因之间的显着相互相关性（图 7B ）。值得注意的是，大多数磷脂合成基因（ Lpcat3和Mfn2 ）与铁死亡相关基因（ Acsl4和Slc7a11 ）之间存在很强的相关性（图7C ）。这些发现证实了增强磷脂合成以增强铁死亡的临床可行性。
Next, we scrutinized the expression levels of Acsl4, Slc7a11, Lpcat3 and Mfn2 using clinical data from the TCGA database (Fig. 7D). Colorectal cancer samples were categorized based on the pTNM staging system into stages I-IV (N = 75, 177, 128, 64). A compelling observation emerged when comparing these gene expressions in tumor tissues with those in normal and adjacent tissues (N = 820). It became evident that Lpcat3 and Mfn2 were significantly downregulated within tumor tissues, while Slc7a11 displayed a marked increase in expression.
接下来，我们使用TCGA数据库的临床数据检查了Acsl4 、 Slc7a11 、 Lpcat3和Mfn2的表达水平（图7D ）。根据 pTNM 分期系统将结直肠癌样本分为 I-IV 期（N = 75、177、128、64）。当将肿瘤组织中的这些基因表达与正常组织和邻近组织中的基因表达进行比较时，出现了令人信服的观察结果（N = 820）。很明显， Lpcat3和Mfn2在肿瘤组织内显着下调，而Slc7a11的表达显着增加。
To corroborate this observation, immunohistochemical staining was performed on colorectal cancer tissue sections from clinical patients, further confirming the significant reduction of Acsl4, Lpcat3 and Mfn2 expression in tumor tissues and the pronounced elevation of Slc7a11 in comparison to adjacent tissues (Fig. 7E). These findings consistently highlight the potential of a therapeutic strategy involving MERC induction to bolster the expression of phospholipid synthesis genes (Lpcat3 and Mfn2), thus enhancing ferroptosis-related genes (Acsl4) and simultaneously curtailing the inhibition of ferroptosis genes (Slc7a11) for the treatment of colorectal cancer.
为了证实这一观察结果，对临床患者的结直肠癌组织切片进行了免疫组织化学染色，进一步证实了与癌旁组织相比，肿瘤组织中Acsl4、Lpcat3和Mfn2表达显着降低，而Slc7a11显着升高（图7E ） 。这些发现一致强调了涉及 MERC 诱导的治疗策略的潜力，以增强磷脂合成基因（ Lpcat3和Mfn2 ）的表达，从而增强铁死亡相关基因（ Acsl4 ）并同时减少铁死亡基因（ Slc7a11 ）的抑制。结直肠癌。
These results underscore the clinical significance of LSA in colorectal cancer treatment and emphasize the promise of MERC induction as a strategy to augment ferroptosis in clinical practice.
这些结果强调了 LSA 在结直肠癌治疗中的临床意义，并强调了 MERC 诱导作为临床实践中增强铁死亡策略的前景。

4. Discussion and conclusions
4 讨论与结论

The study underscores the pivotal role of MERC in cancer, particularly in contributing to metabolic reprogramming. To investigate the potential of MERC in potentiating ferroptosis, a novel prodrug LSA was conceived and synthesized. In addition, our experimental findings demonstrate the potent in vitro antitumor capabilities of LSA. Mechanistically, we demonstrate that MERC can enhance phospholipid synthesis and alter FFA degradation patterns, providing substrates for lipid peroxidation and ultimately enhancing ferroptosis. In vivo, experimental evidence validates LSA’s capacity to induce MERC, enhance ferroptosis in tumor cells, and exhibit outstanding antitumor potential. Bioinformatics analysis underscores the potential significance of strategies leveraging MERC induction to enhance ferroptosis in clinical cancer therapy.
该研究强调了 MERC 在癌症中的关键作用，特别是在促进代谢重编程方面。为了研究 MERC 在增强铁死亡方面的潜力，我们构思并合成了一种新型前药 LSA。此外，我们的实验结果证明了 LSA 强大的体外抗肿瘤能力。从机制上讲，我们证明 MERC 可以增强磷脂合成并改变 FFA 降解模式，为脂质过氧化提供底物并最终增强铁死亡。在体内，实验证据证实LSA具有诱导MERC、增强肿瘤细胞铁死亡的能力，并表现出出色的抗肿瘤潜力。生物信息学分析强调了利用 MERC 诱导来增强临床癌症治疗中铁死亡的策略的潜在意义。
In summary, the study’s design approach harnesses organelle interactions to modulate cellular metabolism and membrane function, ultimately amplifying ferroptosis. This presents a fresh perspective and orientation for cancer therapy.
总之，该研究的设计方法利用细胞器相互作用来调节细胞代谢和膜功能，最终放大铁死亡。这为癌症治疗提供了新的视角和方向。

CRediT authorship contribution statement
CRediT 作者贡献声明

Junyan Zhuang: Writing – review & editing, Writing – original draft, Methodology, Conceptualization. Renming Fan: Writing – review & editing, Methodology. Weike Liao: Writing – review & editing, Methodology. Ruizhuo Lin: Writing – review & editing, Methodology. Aohua Deng: Writing – review & editing, Methodology. Ting Zhao: Writing – review & editing, Methodology. Yongrui Hai: Writing – review & editing, Methodology. Heran Li: Writing – review & editing, Conceptualization. Lei Tang: Writing – review & editing, Conceptualization. Gaofei Wei: Writing – review & editing, Writing – original draft, Conceptualization.
庄俊彦：写作——审稿和编辑、写作——原稿、方法论、概念化。范仁明：写作——审稿与编辑，方法论。廖伟科：写作——评论和编辑，方法论。林瑞卓：写作——评论和编辑，方法论。邓敖华：写作-评论与编辑，方法论。赵婷：写作——评论和编辑，方法论。海永瑞：写作——评论和编辑，方法论。 Heran Li：写作——评论和编辑，概念化。唐雷：写作——评论和编辑，概念化。魏高飞：写作——审稿和编辑、写作——初稿、概念化。

Declaration of competing interest
竞争利益声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有已知的可能影响本文报告工作的相互竞争的经济利益或个人关系。

Acknowledgements 致谢

This work was supported by the National Natural Science Foundation of China (82173682), Shenzhen Science and Technology Program (JCYJ20210324133213037), Innovation Capability Support Program of Shaanxi (2021KJXX-92).
该工作得到国家自然科学基金（ 82173682 ）、深圳市科技计划（ JCYJ20210324133213037 ）、陕西省创新能力支撑计划（ 2021KJXX-92 ）的资助。

Appendix A. Supplementary data
附录 A 补充数据

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Data availability 数据可用性

Data will be made available on request.
数据将根据要求提供。

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