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 belongs to the quinazoline class of compounds, with the structure quinazoline-4(3H)-one, where the hydrogen atoms at positions 2 and 3 are replaced by 1-{4-[(4-chlorophenoxy)acetyl]piperazin-1-yl}ethyl and 2-ethoxyphenyl, respectively. It is an inhibitor of voltage-dependent anion-selective channels (VDAC2 and VDAC3) and a potent inducer of ferroptosis. It possesses multiple functions, including ferroptosis induction, antitumor activity, and voltage-dependent anion channel inhibition. It belongs to the quinazoline class, monochlorobenzene class, aromatic ether class, N-acylpiperazine class, N-alkylpiperazine class, diether class, and tertiary amide class. Research Question: Can erastin activate ferroptosis to ablate endometriotic lesions? Summary Answer: Erastin can induce ferroptosis to ablate endometriotic lesions in endometriosis.
Known Information: Ectopic endometrial stromal cells (EESCs) exist in an iron-overloaded microenvironment, making them more susceptible to oxidative damage. Erastin-induced ferroptosis is characterized by the accumulation of iron-dependent lethal lipid reactive oxygen species (ROS).
Study Design, Sample Size, and Duration: This study included 11 patients without endometriosis and 21 patients with endometriosis. Primary normal and ectopic endometrial stromal cells were isolated, cultured, and subjected to various treatments. An endometriosis model was established using 10 C57BL/6 female mice in vivo.
Participants/Materials, Setup, and Methods: Ferroptosis markers were assessed by cell viability, lipid peroxidation levels, and morphological changes. Cell viability was measured by colorimetry, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. The expression of iron transporter (FPN) was detected by immunohistochemistry and Western blotting. Iron levels were semi-quantitatively analyzed using Prussian blue staining and immunofluorescence microscopy catalyzing ferrous iron. This study investigated the role of FPN in erastin-induced ferroptosis in endometrial stromal cells (EESCs) using adenovirus-mediated overexpression and siRNA-mediated gene knockdown.
Main Results and Incidental Factors: Compared with normal endometrial stromal cells (NESCs), EESCs were more sensitive to erastin treatment (P<0.05). Erastin treatment significantly increased total ROS levels (P<0.05, compared to the control group), lipid ROS levels (P<0.05, compared to NESCs), and intracellular iron levels (P<0.05, compared to NESCs). The iron chelator deferoxamine (DFO) and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 all attenuated the cytotoxicity of erastin to EESCs (P<0.05, compared to the erastin group). In erastin-treated EESCs, electron microscopy revealed shortened and condensed mitochondria. These results collectively indicate that erastin can induce EESC death through ferroptosis. However, erastin had a smaller effect on NESC. The process of erastin-induced EESC ferroptosis was accompanied by iron accumulation and decreased FPN expression. Overexpression of FPN eliminated erastin-induced EESC ferroptosis. In addition, knockdown of FPN accelerated erastin-induced EESC ferroptosis. In a mouse model of endometriosis, we found that ectopic lesions regressed after erastin administration.
Large-scale data: Not applicable.
Limitations and precautions: This study was conducted primarily in primary human endometrial stromal cells. Therefore, the function of FPN in vivo needs further investigation.
Broader implications of the results: Our results suggest that erastin may be a potential treatment for endometriosis.
Funding/conflict of interest: This study was not supported by any public, commercial or nonprofit organization. The authors declare no conflict of interest. [1]

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We evaluated the potential anti-colorectal cancer activity of erastin, a voltage-dependent anion channel (VDAC) binding compound. Our in vitro studies demonstrated that erastin exhibits significant cytotoxicity against various human colorectal cancer cell lines, likely through the induction of oxidative stress and caspase-9-dependent apoptosis. Furthermore, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, confirmed by VDAC-1 and cyclosporine D (Cyp-D) binding, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sangiferin A, cyclosporine A, and sangiferin), as well as shRNA-mediated VDAC-1 knockdown, significantly attenuated erastin-induced colorectal cancer cell cytotoxicity and apoptosis. Conversely, VDAC-1 overexpression enhanced erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of well-tolerated doses of erastin significantly inhibited the growth of HT-29 xenografts in severely ill combined immunodeficiency (SCID) mice. These results collectively indicate that erastin has cytotoxic and apoptotic effects on colorectal cancer cells. Erastin is expected to become a novel anti-colorectal cancer drug and can be further studied. [2] Piperlongumine is a natural alkaloid extracted from the fruit of long pepper (Piper longum Linn.), and is known to inhibit cytoplasmic thioredoxin reductase (TXNRD1 or TrxR1) and selectively kill cancer cells. However, the specific mechanism by which piperine inhibits TXNRD1 is unclear. Based on the classic DTNB reduction method, this study found that piperine has an irreversible inhibitory effect on recombinant TXNRD1, with an epikinase activity of 0.206 × 10⁻³ µM⁻¹ min⁻¹. Meanwhile, compared with wild-type TXNRD1 (-GCUG), UGA truncated TXNRD1 (-GC) is resistant to piperine, indicating that the preferred target of piperine is selenool (-SeH) on the C-terminal redox motif of the enzyme. Interestingly, high concentrations of piperine inhibited TXNRD1 and found that its selenium-dependent activity decreased, but its inherent NADPH oxidase activity was preserved. In addition, 10 µM piperidine did not induce ferroptosis in HCT116 cells, but significantly promoted erastin-induced lipid oxidation, and supplementation with glutathione (GSH) or N-acetylcysteine (NAC) could alleviate this promoting effect. However, using the small molecule inhibitor CB-839 to inhibit glutaminase (GLS) to limit GSH synthesis only slightly enhanced erastin-induced cell death. In summary, this study elucidates the molecular mechanism by which piperidine exerts its antitumor effect by targeting TXNRD1 and reveals the potential for inhibiting TXNRD1 to enhance ferroptosis in cancer cells. [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.)