VDAC2; VDAC3

Ferroptosis in ectopic endometrial stromal cells (EESC) is triggered by erematin (10 μM; 24 hours), and at 9 hours, total ROS levels rise [1]. In EESC cells, erythrin can reduce the length of mitochondria and raise their membrane density [1]. Iron-related proteins, including FPN (iron export protein), have lower levels of mRNA expression in EESCs when treated with erythrin (10 μM) for nine hours. On the other hand, overexpression of FPN can considerably prevent Erastin-induced ferroptosis of EESCs [1]. In HT-29 colorectal cancer cells, erematin (10 μM; 24 hours) causes the opening of the mitochondrial permeability transition pore (mPTP) [2]. The proliferation of HT-29 colorectal cancer cells is greatly inhibited by eratin (30 μM; 72 hours) [2]. The genes that control iron metabolism or mitochondrial fatty acid metabolism are involved in the biological mechanism by which erythropoidin triggers ferroptosis. comprises tetrapeptide repeat domain 35, citrate synthase, ATP synthase F0 complex subunit C3, ribosomal protein L8, iron response element binding protein 2 (IREB2), and acyl-CoA synthetase family member 2 (ACSF2)[3].

Ferroptosis-induced animal models can be created with Erastin. In a mouse model of endometriosis, Erastin (40 mg/kg; i.p.; every 3 days for 2 weeks) inhibits endometriotic implantation, indicating that Erastin promotes regression of ectopic lesions by inducing ferroptosis [1]. In SCID mice, eratin (10 mg/kg, 30 mg/kg; intraperitoneally; once daily for 4 weeks) suppresses the growth of HT-29 xenografts, with 30 mg/kg showing the greatest activity [2].

Erastin inhibits voltage-dependent anion channels (VDAC2/VDAC3) and accelerates oxidation, leading to the accumulation of endogenous reactive oxygen species.
We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent.

Cell Viability Assay[1]
Cell Types: Normal endometrial stromal cells (NESCs) and endometrial stromal cells (EESCs)
Tested Concentrations: 0, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced cell detachment and overt death in EESCs, but not NESCs.

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Apoptosis Analysis[1]
Cell Types: EESCs infected with adenovirus expressing FPN cDNA (co-incubation for 24 hr)
Tested Concentrations: 0, 0.5, 1.5, 2.5, 5 and 2.5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced ferroptosis by decreasing the levels of total ROS and lipid ROS. And reversed by the overexpression of FPN in adenovirus-infected cells.

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]. Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis. Hum Reprod. 2021 Mar 18;36(4):951-964.

[2]. Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells. PLoS One. 2016 May 12;11(5):e0154605.

[3]. Ferroptosis: process and function. Cell Death Differ. 2016 Mar;23(3):369-79.

[4]. MT1DP loaded by folate-modified liposomes sensitizes erastin-induced ferroptosis via regulating miR-365a-3p/NRF2 axis in non-small cell lung cancer cells. Cell Death Dis. 2020 Sep 14;11(9):751.

[5]. Piperlongumine Inhibits Thioredoxin Reductase 1 by Targeting Selenocysteine Residues and Sensitizes Cancer Cells to Erastin. Antioxidants (Basel). 2022 Apr 4;11(4):710.

[6]. Activation of the reverse transsulfuration pathway through NRF2/CBS confers erastin-induced ferroptosis resistance. Br J Cancer. 2020 Jan;122(2):279-292.

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

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

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

[10]. The Tumor Suppressor p53 Limits Ferroptosis by Blocking DPP4 Activity. Cell Rep. 2017 Aug 15;20(7):1692-1704.

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?
Summary answer: Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis.
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).
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.
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.
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.
Large scale data: N/A.
Limitations, reasons for caution: This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated.
Wider implications of the findings: Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis.
Study funding/competing interest(s): This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.[1]

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We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent. [2]

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Piperlongumine, a natural alkaloid substance extracted from the fruit of the long pepper (Piper longum Linn.), is known to inhibit the cytosolic thioredoxin reductase (TXNRD1 or TrxR1) and selectively kill cancer cells. However, the details and mechanism of the inhibition by piperlongumine against TXNRD1 remain unclear. In this study, based on the classical DTNB reducing assay, irreversible inhibition of recombinant TXNRD1 by piperlongumine was found and showed an apparent kinact value of 0.206 × 10-3 µM-1 min-1. Meanwhile, compared with the wild-type TXNRD1 (-GCUG), the UGA-truncated form (-GC) of TXNRD1 was resistant to piperlongumine, suggesting the preferential target of piperlongumine is the selenol (-SeH) at the C-terminal redox motif of the enzyme. Interestingly, the high concentration of piperlongumine-inhibited TXNRD1 showed that its Sec-dependent activity is decayed but its intrinsic NADPH oxidase activity is retained. Furthermore, piperlongumine did not induce ferroptosis in HCT116 cells at 10 µM, whereas significantly promoted erastin-induced lipid oxidation, which could be alleviated by supplying glutathione (GSH) or N-acetyl L-cysteine (NAC). However, restricting GSH synthesis by inhibiting glutaminase (GLS) using the small molecule inhibitor CB-839 only slightly enhanced erastin-induced cell death. Taken together, this study elucidates the molecular mechanism of the antitumor capacity of piperlongumine by targeting TXNRD1 and reveals the potential possibility of inhibiting TXNRD1 to strengthen cancer cells' ferroptosis. [5]

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

0

6

8

39

871

0

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)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 1 mg/mL (1.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 5% DMSO+corn oil: 2.5mg/mL

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Solubility in Formulation 4: 5 mg/mL (9.14 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)