Erastin   中文名称: Erastin

VDAC2; VDAC3

异位子宫内膜基质细胞 (EESC) 中的铁死亡由 erematin（10 μM；24 小时）触发，9 小时后，总 ROS 水平上升 [1]。在 EESC 细胞中，红十字蛋白可以缩短线粒体长度并提高其膜密度 [1]。当用红菊酯 (10 μM) 处理 9 小时时，EESC 中铁相关蛋白（包括 FPN（铁输出蛋白））的 mRNA 表达水平较低。另一方面，FPN 的过度表达可以显着预防 Erastin 诱导的 EESC 铁死亡[1]。在 HT-29 结直肠癌细胞中，erematin（10 μM；24 小时）会导致线粒体通透性转换孔 (mPTP) 打开 [2]。 eratin（30 μM；72 小时）可显着抑制 HT-29 结直肠癌细胞的增殖 [2]。控制铁代谢或线粒体脂肪酸代谢的基因参与促红细胞生成素引发铁死亡的生物学机制。包含四肽重复结构域 35、柠檬酸合酶、ATP 合酶 F0 复合体亚基 C3、核糖体蛋白 L8、铁反应元件结合蛋白 2 (IREB2) 和酰基辅酶 A 合成酶家族成员 2 (ACSF2)[3]。

可以用Erastin创建铁死亡诱导的动物模型。在子宫内膜异位症小鼠模型中，Erastin（40 mg/kg；腹腔注射；每 3 天一次，持续 2 周）抑制子宫内膜异位症着床，表明 Erastin 通过诱导铁死亡促进异位病变消退 [1]。在 SCID 小鼠中，eratin（10 mg/kg、30 mg/kg；腹腔注射；每天一次，持续 4 周）抑制 HT-29 异种移植物的生长，其中 30 mg/kg 显示出最大活性 [2]。

Erastin抑制电压依赖性阴离子通道（VDAC2/VDAC3）并加速氧化，导致内源性活性氧的积累。
我们在此评估了erastin（一种电压依赖性阴离子通道（VDAC）结合化合物）潜在的抗结肠癌症活性。我们的体外研究表明，erastin可能通过诱导氧化应激和胱天蛋白酶-9依赖性细胞凋亡，对多种人类结直肠癌癌症细胞系发挥了强大的细胞毒性作用。此外，在erastin处理的癌症细胞中观察到线粒体通透性转变孔（mPTP）开放，这通过VDAC-1和亲环蛋白-D（Cyp-D）结合、线粒体去极化和细胞色素C释放证明。胱天蛋白酶抑制剂、ROS清除剂MnTBAP和mPTP阻断剂（桑格列非林A、环孢菌素A和邦克雷酸），以及shRNA介导的VDAC-1敲低，都显著减弱了erastin诱导的结直肠癌癌症细胞的细胞毒性和凋亡。另一方面，VDAC-1的过度表达增加了erastin诱导的ROS产生、mPTP开放和结直肠癌癌症细胞凋亡。体内研究表明，在严重联合免疫缺陷（SCID）小鼠中，以耐受性良好的剂量腹腔注射erastin可显著抑制HT-29异种移植物的生长。总之，这些结果表明erastin对癌症细胞具有细胞毒性和促凋亡作用。Erastin可以作为一种新型的抗癌症药物被进一步研究。

细胞活力测定[1]
细胞类型：正常子宫内膜基质细胞 (NESC) 和子宫内膜基质细胞 (EESC)
测试浓度： 0、0.5、 0.8、1、1.5、2、2.5、5、10 μM
孵育时间： 24 小时
实验结果：诱导细胞脱离和明显EESC 死亡，但 NESC 不死亡。

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细胞凋亡分析[1]
细胞类型：感染表达 FPN cDNA 的腺病毒的 EESC（共孵育 24 小时）
测试浓度： 0、0.5、1.5、2.5、5 和 2.5 μM
孵育时间： 24 小时
实验结果：通过降低总 ROS 和脂质 ROS 水平。并通过腺病毒感染细胞中 FPN 的过度表达而逆转。

Animal/Disease Models: Mouse model of endometriosis[1]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip)injection; once every 3 days for 2 weeks
Experimental Results: demonstrated little impact on body weight of mice and hair of mice displayed neat and glossy. decreased the volume of ectopic lesions.

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Mouse model of endometriosis[1]
Ten C57BL/6 female mice (7–8 weeks, weight 20–22 g) were used. Endometriotic lesions were surgically induced by autotransplantation of uterine horns onto the peritoneal wall as previously described. Briefly, uterine horns were removed and opened longitudinally, cut into homogeneous fragments using a 3-mm dermal biopsy punch and then transplanted onto own peritoneal wall of mice by suturing. 17-β-Estradiol-3-benzoate (30 μg/kg) was administered to each postoperative mouse every 3 days for 28 days. At 14 day after operation, endometrial-like lesions were established, and it was time for intervention. They were randomly divided into two groups. In the experimental group, each mouse received erastin (40 mg/kg) by intraperitoneal injection over a 14-day period. In the control group, in place of erastin, soybean oil was used. At 28 days, the mice were sacrificed and we harvested the ectopic tissues. The volumes of ectopic lesions were measured and analyzed as previously described (Zhao et al., 2015).

[1]. Li Y, et al. Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis. Hum Reprod. 2021 Mar 18;36(4):951-964.
[2]. Huo H, et al. Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells. PLoS One. 2016 May 12;11(5):e0154605.
[3]. Xie Y, et al. Ferroptosis: process and function. Cell Death Differ. 2016 Mar;23(3):369-79.

Erastin is a member of the class of quinazolines that is quinazolin-4(3H)-one in which the hydrogens at positions 2 and 3 are replaced by 1-{4-[(4-chlorophenoxy)acetyl]piperazin-1-yl}ethyl and 2-ethoxyphenyl groups, respectively. It is an inhibitor of voltage-dependent anion-selective channels (VDAC2 and VDAC3) and a potent ferroptosis inducer. It has a role as a ferroptosis inducer, an antineoplastic agent and a voltage-dependent anion channel inhibitor. It is a member of quinazolines, a member of monochlorobenzenes, an aromatic ether, a N-acylpiperazine, a N-alkylpiperazine, a diether and a tertiary carboxamide.

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Study question: Could erastin activate ferroptosis to regress endometriotic lesions?[1]

Summary answer: Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis.[1]

What is known already: Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipid reactive oxygen species (ROS).[1]

Study design, size, duration: Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis.[1]

Participants/materials, setting, methods: The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs.[1]

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Main results and the role of chance: EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipid ROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration.[1]

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Current researches find that erastin-induced ferroptosis involves a unique constellation of features, including requirement for ROS accumulation and iron (Dixon et al., 2012). The levels of iron accumulation serve as a driving factor in the induction of ferroptosis (Zhang et al., 2020). In patients with endometriosis, iron overload has been proved in the peritoneal fluids and ectopic endometrium (Defrere et al., 2008). Thus, with the help of intrinsic iron overloading circumstances, activation of ferroptosis may be a novel therapeutic strategy for treating endometriosis. In the present work, we provided the evidence that erastin could regress ectopic lesions by trigging ferroptosis. We also identified that FPN participated in erastin-induced ferroptosis by regulating intracellular iron levels in EESCs.[1]

Erastin, a small molecule, selectively kills cells expressing oncogenic mutants of RAS via ferroptosis (Yang and Stockwell, 2008). Recent researches suggested that erastin triggered ferroptosis in kidney tubule cells, fibroblasts, human hepatocellular cells (HepG2) and human promyelocytic leukemia cells (HL60), but not in human prostate cancer cells (LNCaP) and human erythromyeloblastoid leukemia cell line (K562) (Yuan et al., 2016). In order to investigate the effect of erastin on endometriosis, we first identified that EESCs were more susceptible to erastin-induced ferroptosis, compared to NESCs. Given lipid peroxidation signaling is increasingly recognized as core mediators of ferroptosis (Yang and Stockwell, 2016), oxidative damage-related markers, such as total ROS, lipid ROS and MDA, were detected. The levels of oxidative products were elevated after erastin treatment in EESCs. These alterations also accompanied morphological changes of mitochondria. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. This is consistent with previous study (Dixon et al., 2012). In addition, the cytotoxicity of erastin could be attenuated by iron chelator, DFO, and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, but not apoptosis inhibitor ZVAD-FMK and necroptosis inhibitor. These findings together suggest that erastin is capable to induce EESCs death by activating ferroptosis (Fig. 1). In order to explore whether erastin could play a similar biological role in vivo, a mouse model of endometriosis was established and ectopic lesions in experiment group were regressed (Fig. 6). However, taking the limited sample size into consideration, the effectiveness and safety evaluation of erastin need to be further studied at multiple animal models of endometriosis. [1]

In the ectopic endometrium, which were shown abundant iron, the expression of FPN was lower (Fig. 3). In cultured EESCs treated with erastin, we observed that the expression of FPN further downregulated and iron continued to accumulate (Fig. 2). This encourages us to investigate the role of FPN and iron in the erastin-induced ferroptosis. It has been demonstrated that FPN is the sole recognized mammalian iron exporter and is thought to be essential for iron homeostasis (Donovan et al., 2005). In patients with hereditary hemochromatosis, associated with mutations of FPN, early Küpffer cell and hepatocyte were iron overloading (De Domenico et al., 2006). Ferroportin-deficient mice also showed accumulated iron in enterocytes, macrophages and hepatocytes (Donovan et al., 2005). Furthermore, there were no significant differences in the mRNA expression levels of TFR, DMT1, ferritin light chain (FTL) and ferritin heavy chain (FTH) (Fig. 2). So, we reasoned that erastin enriched the cellular iron pool mainly by downregulation of FPN expression. To further uncover the role of FPN in the process of erastin-induced ferroptosis, we overexpressed FPN and found that the effects of erastin-induced ferroptosis were ablated (Fig. 4). In addition, the deficiency of FPN accelerated cell death in EESCs (Fig. 5). Therefore, elevated intracellular iron level by FPN deregulation played an important role in the process. Recently, Chen et al. (2020) revealed that erastin could trigger ferroptosis by increased iron levels through regulating ferritin/FPN metabolism in human breast carcinoma cells (MDA-MB-231). These coordinated changes of ferritin/FPN relied largely on mutated in ataxia-telangiectasia (ATM) and metal regulatory transcription factor 1 (MTF1) activity. We are going to explore the biological mechanism of erastin-induced FPN deregulation in EESCs in the following research.[1]

Studies on the mechanism of iron are crucial for further identifying erastin-induced ferroptosis. The latest research demonstrated that iron could trigger ferroptosis in dopaminergic cells, murine primary hepatocytes and bone marrow-derived macrophage (Doll and Conrad, 2017; Wang et al., 2017; Zhang et al., 2020). In animal experiments of hemochromatosis, researchers found that ferroptosis occurred in severe iron overload mice but not in mild iron overload mice (Wang et al., 2017). This implies that the levels of iron accumulation serve as a driving factor in the ferroptosis process. Hydroxyl radical could be generated in the Fenton process with the help of iron (Fischbacher et al., 2017). As for mitochondrial respiratory function, the reserve respiratory capacity in EESCs was much lower than NESCs (Chen et al., 2019). It is the adaptative responses to oxidative stress by eliminating ROS. EESCs exhausted ROS-buffering capacity protecting from oxidative damage, thus tending to be more sensitive to oxidant (Chen et al., 2019). Thus, we presumed that EESCs could not eliminate more ROS producing from erastin-induced iron accumulation. This issue needs further investigations.[1]

In summary, we demonstrate that erastin is able to induce EESCs death via ferroptosis. In vivo, the administration of erastin reduced the volume of endometriotic-like lesions in mouse model with endometriosis. These findings indicate that erastin may serve as a promising novel therapeutic treatment for endometriosis. FPN, which regulates intracellular iron concentration, is a negative factor in erastin-induced ferroptosis. More work is still needed toward understanding the roles of FPN, so that optimal targets can be identified for future treatment efforts in endometriosis.[1]

C30H31CLN4O4

547.04

546.203

C, 65.87; H, 5.71; Cl, 6.48; N, 10.24; O, 11.70

571203-78-6

11214940

White to off-white solid

1.3±0.1 g/cm3

721.9±70.0 °C at 760 mmHg

390.4±35.7 °C

0.0±2.3 mmHg at 25°C

1.634

4.75

76.9

ClC1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C([H])(C([H])([H])[H])C1=NC2=C([H])C([H])=C([H])C([H])=C2C(N1C1=C([H])C([H])=C([H])C([H])=C1OC([H])([H])C([H])([H])[H])=O)=O

BKQFRNYHFIQEKN-UHFFFAOYSA-N

InChI=1S/C30H31ClN4O4/c1-3-38-27-11-7-6-10-26(27)35-29(32-25-9-5-4-8-24(25)30(35)37)21(2)33-16-18-34(19-17-33)28(36)20-39-23-14-12-22(31)13-15-23/h4-15,21H,3,16-20H2,1-2H3

2-(1-(4-(2-(4-chlorophenoxy)acetyl)piperazin-1-yl)ethyl)-3-(2-ethoxyphenyl)quinazolin-4(3H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.29 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 12.5 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 2 中的溶解度: ≥ 1 mg/mL (1.83 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 10.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 3 中的溶解度: 5% DMSO+corn oil: 2.5mg/mL

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配方 4 中的溶解度: 5 mg/mL (9.14 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；
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