RSL3

GPX4/glutathione peroxidase 4

RSL3（0-8 μM，72 小时）在 HN3-rslR 细胞中的 IC50 为 5.8 μM，在 HN3 细胞中为 0.48 μM，显着降低 HN3 细胞的活力 [1]。在 HN3-rslR 细胞中，RSL3（0-8 μM，24 小时）可使 Keap1 失活，提高 p62 和 Nrf2 的表达，并降低 GPX4 蛋白的表达 [1]。
RSL3或ML-162在不同程度上诱导了HNC细胞的铁凋亡。[1]
RSL3处理的HNC细胞对铁下垂的抗性与p62和Nrf2的表达有关。[1]
体外抑制Nrf2致敏的化疗耐药HNC细胞对RSL3治疗的反应[1]。

葫芦巴碱与RSL3联合使用（100 mg/kg，瘤内注射，每周两次，持续20天）可显着抑制HN3R因子细胞肿瘤生长[1]。

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在小鼠异种移植物模型中，所有小鼠在细胞植入和用载体、RSL3、胡芦巴碱或RSL3加胡芦巴林治疗期间和之后都存活良好。他们在治疗后20天被安乐死。与载体对照组相比，RSL3或胡芦巴碱单独使用并没有显著抑制体内肿瘤生长（P>0.1）（图6A和B）。然而，RSL3联合胡芦巴碱显著抑制了肿瘤生长（P<0.01）。对照组或治疗组的体重和每日食物摄入量没有显著变化（P<0.05）（图6C）。RSL3联合胡芦巴碱组体内肿瘤中测得的亚铁、脂质ROS和RPA水平明显高于对照组或其他治疗组（P<0.01）。对重要器官的组织学检查未发现两组之间有任何显著差异[1]。

ROS产生测量[1]
通过在37°C下加入10µM 2′，7′-二氯荧光素二乙酸酯（用于细胞质ROS）或2µM C11-BODIPY C11（用于脂质过氧化）30分钟，测量处理24小时的HNC细胞裂解物上清液中细胞ROS的产生。使用配备CellQuest Pro的FACSCalibur流式细胞仪分析ROS水平。

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不稳定铁池（LIP）和铁测定[1]
每个样品都接种在平板上，用指定的药物处理。4小时后，去除上清液，用Hank's平衡盐溶液（HBSS）洗涤板两次。通过在HBSS中加入8μg/ML钙黄绿素AM标记细胞，并在37°C的平板上孵育30分钟。30分钟后，去除标记溶液，用PBS洗涤细胞两次，然后用胰蛋白酶-EDTA进行胰蛋白酶消化。然后通过在HBSS中加入4%FBS中和它们，并在1500rpm下离心3分钟。收集的细胞在涡旋的同时用HBSS洗涤一次，并在1500rpm下离心3分钟。然后，在涡旋的同时将它们重新悬浮在250μL HBSS中，并进入FACS。使用配备CellQuest Pro的FACSCalibur流式细胞仪分析不稳定铁水平，并使用铁测定试剂盒测量细胞或组织提取物中的亚铁水平。

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Nrf2转录活性测定[1]
根据制造商的说明，使用Cignal抗氧化剂反应报告试剂盒测定Nrf2的转录活性。

细胞活力测定[1]
细胞类型： HN3 细胞、HN3-rslR 细胞
测试浓度： 0-8 μM
孵育持续时间：72小时
实验结果：HN3和HN3-rslR细胞中的IC50值分别为0.48 µM和5.8 µM[1]。

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蛋白质印迹分析 [1]
细胞类型： HN3-rslR 细胞
测试浓度： 0-8 μM
孵育持续时间：24 小时
实验结果：抑制 GPX4 表达，增加 p62 和 Nrf2 水平，并降低 Keap1 水平。

Animal/Disease Models: Tenweeks old athymic BALB/c male nude mice (nu/nu) carrying HN3R cells [1]
Doses: 100 mg/kg combined with trigonelline (50 mg/kg): intratumoral injection,
Experimental Results: Significant reduction in tumor volume in mice treated with trigonelline twice a week for 20 days.

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Tumor xenograft[1]
Ten-week-old athymic BALB/c male nude mice (nu/nu) were used and HN3R cells were injected subcutaneously into the flank of each. As soon as gross nodules from the tumor implants were detected, the mice were subjected to one of four treatments: vehicle; RSL3 (100 mg/kg intratumorally twice per week); trigonelline (50 mg/kg daily via oral administration); or RSL3 plus trigonelline. Each group included 10 mice. Tumor size and body weight were measured twice a week, and the tumor volume was calculated as (length × width2)/2. After scarification, the tumors were isolated and cellular lipid ROS and ferrous iron levels were measured.

[1]. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radic Biol Med. 2018 Dec;129:454-462.

[2]. Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance. Nat Cell Biol. 2022 Feb;24(2):168-180.

[3]. CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes. Nat Chem Biol. 2024 Jan 11.

[4]. Mitochondrial transplantation rescues neuronal cells from ferroptosis. Free Radic Biol Med. 2023 Nov 1;208:62-72.

[5]. Ibrutinib facilitates the sensitivity of colorectal cancer cells to ferroptosis through BTK/NRF2 pathway. Cell Death Dis. 2023 Feb 23;14(2):151.

[6]. A potent GPX4 degrader to induce ferroptosis in HT1080 cells. Eur J Med Chem. 2024 Feb 5;265:116110.

[7]. Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines. Cell Death Dis. 2021 Jul 13;12(7):698.

[8]. The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1. Redox Biol. 2023 Jun;62:102703.

[9]. Inhibition of cannabinoid receptor type 1 sensitizes triple-negative breast cancer cells to ferroptosis via regulating fatty acid metabolism. Cell Death Dis. 2022 Sep 21;13(9):808.

[10]. Prominin2 Drives Ferroptosis Resistance by Stimulating Iron Export. Dev Cell. 2019 Dec 2;51(5):575-586.e4.

Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (iron-dependent, non-apoptotic cell death); its inhibition can render therapy-resistant cancer cells susceptible to ferroptosis. However, some cancer cells develop mechanisms protective against ferroptosis; understanding these mechanisms could help overcome chemoresistance. In this study, we investigated the molecular mechanisms underlying resistance to ferroptosis induced by GPX4 inhibition in head and neck cancer (HNC). The effects of two GPX4 inhibitors, (1S, 3R)-RSL3 and ML-162, and of trigonelline were tested in HNC cell lines, including cisplatin-resistant (HN3R) and acquired RSL3-resistant (HN3-rslR) cells. The effects of the inhibitors and trigonelline, as well as of inhibition of the p62, Keap1, or Nrf2 genes, were assessed by cell viability, cell death, lipid ROS production, and protein expression, and in mouse tumor xenograft models. Treatment with RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees. RSL3 or ML-162 treatment increased the expression of p62 and Nrf2 in chemoresistant HN3R and HN3-rslR cells, inactivated Keap1, and increased expression of the phospho-PERK-ATF4-SESN2 pathway. Transcriptional activation of Nrf2 was associated with resistance to ferroptosis. Overexpression of Nrf2 by inhibiting Keap1 or Nrf2 gene transfection rendered chemosensitive HN3 cells resistant to RSL3. However, Nrf2 inhibition or p62 silencing sensitized HN3R cells to RSL3. Trigonelline sensitized chemoresistant HNC cells to RSL3 treatment in a mouse model transplanted with HN3R. Thus, activation of the Nrf2-ARE pathway contributed to the resistance of HNC cells to GPX4 inhibition, and inhibition of this pathway reversed the resistance to ferroptosis in HNC.[1]

C23H21CLN2O5

440.8762

440.113

C, 62.66; H, 4.80; Cl, 8.04; N, 6.35; O, 18.14

1219810-16-8

1750826

White to off-white solid powder

1.4±0.1 g/cm3

641.3±55.0 °C at 760 mmHg

341.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.636

3.07

88.7

1

5

6

31

696

2

COC(=O)[C@H]1CC2=C([C@@H](N1C(=O)CCl)C3=CC=C(C=C3)C(=O)OC)NC4=CC=CC=C24

TXJZRSRTYPUYRW-NQIIRXRSSA-N

InChI=1S/C23H21ClN2O5/c1-30-22(28)14-9-7-13(8-10-14)21-20-16(15-5-3-4-6-17(15)25-20)11-18(23(29)31-2)26(21)19(27)12-24/h3-10,18,21,25H,11-12H2,1-2H3/t18-,21+/m1/s1

methyl (1S,3R)-2-(2-chloroacetyl)-1-(4-methoxycarbonylphenyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylate

(1S,3R)-RSL3; 1S,3R-RSL3; RSL3 (1S,3R-); CHEMBL4747331; (1S,3R)-Methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate; RSL3 1S,3R-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~226.82 mM)

配方 1 中的溶解度: 5 mg/mL (11.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 50.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.67 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.72 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 0.56 mg/mL (1.27 mM) (饱和度未知) in 10% DMF 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。

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配方 5 中的溶解度: 20 mg/mL (45.36 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。