A Morpholine Derivative N-(4-Morpholinomethylene)ethanesulfonamide Induces Ferroptosis in Tumor Cells by Targeting NRF2

Abstract

Small molecule drugs containing morpholine-based moieties have become crucial candidates in the tumor targeted therapy strategies, but the specific molecular mechanisms of these drugs causing tumor cell death require further investigation. The morpholine derivative N-(4-morpholinomethylene)ethanesulfonamide (MESA) was used to stimulate prostate and ovarian cancer cells and we focused on the ferroptosis effects, including the target molecule and signal pathways mediated by MESA. The results showed that MESA could induce ferroptosis to cause the proliferation inhibition and apoptosis effects of tumor cells according to the identification of ferroptosis inhibitor fer-1 and other cell death inhibitors. Further MESA could significantly increase the intracellular malondialdehyde (MDA), reactive oxygen species (ROS) and Fe²⁺ levels in tumor cells and mediate the dynamic changes of ferroptosis-relative molecules GPX4, nuclear factor erythroid2-related factor 2 (NRF2), ACSL4, SLC7A11 and P62-Kelch-like ECH-associated protein 1 (KEAP1)-NRF2-antioxidant response element (ARE) signal pathways. Further, NRF2 overexpression could reduce the tumor cell death and ROS levels exposure to MESA. Most importantly, it was confirmed that MESA could bind to NRF2 protein through molecular docking and thermal stability assays and NRF2 was a target molecule of MESA for inducing ferroptosis effects in tumor cells. Collectively, our findings indicated the ferroptosis effects of the morpholine derivative MESA in prostate and ovarian cancer cells and its function mechanism including targeted molecule and signal pathways, which would be helpful for developing MESA as a prospective small molecule drug for cancer therapy based on cell ferroptosis.

INTRODUCTION

Morpholine (C₄H₉NO, 1,4-tetrahydro-oxazine) is one of numerous nitrogen-containing heterocycles that are widely employed in medicinal and industrial preparations.¹⁾ It is a six-membered aromatic heterocyclic molecule with both amine and ether functional groups.²⁾ Compounds containing morpholine cores display a wide range of biological effects, including anti-cancer activity.³⁾ Small molecule drugs containing morpholine-based moieties have become the crucial candidates in tumor targeted therapy strategies. According to the literature, morpholine-containing small molecules have been developed into anti-cancer drugs due to its functions on tyrosine kinase, phosphatidylinositol 3 kinase, mitogen-activated protein kinase, poly ADP-ribose kinase, serine/threonine kinase, cysteine amidase, topoisomerase and so on.⁴⁾ However, the specific molecular mechanisms of these drug candidates require further investigation.

Ferroptosis is a unique form of regulated cell death and it is characterized by the iron-dependent lipid peroxidation, not consistent with the classical programmed cell death such as apoptosis, autophagy and necrosis.⁵⁾ Ferroptosis results from excessive lipid reactive oxygen species (ROS) accumulation which damages cell membranes.⁶⁾ More and more popular, it has been proved that ferroptosis plays a vital role in tumor progress and how to induce ferroptosis in tumor cells has been become a promising strategy in tumor precise therapy. But ferroptosis is a complicated physiological and biochemical development course, which is regulated by the dynamic changes of ferroptosis-relative genes and signal pathways. For tumor target therapy, global scientists are working on target and drug candidates that will drive the ferroptosis process to cause cell death of tumor cells.

Nuclear factor erythroid2-related factor 2 (NRF2) is a transcription factor that protects cells from oxidative stress by stimulating the gene expression of detoxification enzymes and antioxidant proteins.⁷⁾ It has been demonstrated that NRF2 is involved in the regulation of ferroptosis and this transcription factor is thought to be one of the most important coordinating components of the cellular antioxidant response.^8,9) Recent research has revealed that NRF2 has various activities other than redox regulation.^10–12) Moreover, NRF2 is highly expressed in a variety of tumors, including prostate cancer¹³⁾ and ovarian cancer,¹⁴⁾ and has emerged as a major target for cancer prevention and treatment research. The NRF2 signaling pathway is associated with tumor ferroptosis, which binds to antioxidant sites and activates downstream genes to mediate iron and ROS balance.¹⁵⁾ Thus, NRF2 is a pivotal gene to investigate ferroptosis during tumor progress and developing novel drugs to block NRF2 signal pathway may be a promising strategy for tumor targeted therapy based on ferroptosis.^16,17)

Prostate and ovarian cancer are common types of tumors in male and female reproductive system, while androgen deficiency is unique in prostate cancer.^18–20) There are at least four major therapeutic options depending on the type of tumor: resection, chemotherapy, radiation and targeted drugs.²¹⁾ And targeted therapy shows some better effects to boost patients’ chances of survival in both malignancies.^22,23) Thus, it is necessary to develop new approaches for prostate and ovarian cancer precise therapy.²⁴⁾ In our study, the morpholine derivative N-(4-morpholinomethylene)ethanesulfonamide (MESA) was a potential inducer of ferroptosis effects. Tumors cells of prostate and ovarian cancer would be treated with MESA in a dose-dependent manner. We were trying to confirm whether MESA could induce the cell death of tumor cells through ferroptosis-relative pathway and identify the target molecules and signal pathways involved. Ferrostatin-1 (fer-1), a ferroptosis inhibitor, would be employed to identify the cell death pathway mediated by MESA. Our study would present MESA as a promising small molecule drug and target candidate for cancer therapy based on cell ferroptosis.

MATERIALS AND METHODS

The Preparation of MESA

The morpholine derivative MESA was synthesized in our laboratory and the comprehensive synthesis procedure of MESA was developed based on the work of Yang et al.,²⁵⁾ chemical formula: C₇H₁₄N₂O₃S, molecular weight: 206. MESA was stocked at a concentration of 40 mM in dimethyl sulfoxide (DMSO) and diluted to a specific concentration and autoclaved prior to treatment.

To a solution of Morpholine (0.1 mmol) at room temperature was added Ethanesulfonamide (1.8 equivalent (equiv.)), 3-Butyn-2-one was added at room temperature carefully. The reaction mixture was stirred at room temperature for 2 min to acquire MESA as white solid (96% yield). Silicagel 60 (230–400 mesh, Merck, Germany), and TLC plates (Kieselgel 60F254, 0.25 mm, Merck, Germany) were used for column chromatography and analytical TLC, respectively.

The purity of MESA was determined using an HPLC system consisting of a Waters Alliance e2695 separation module with a Waters 2998 photo-diode array detector and a Waters Sunfire C18 reverse phase column. Methanol/H₂O (90/10, v/v) was used as the mobile phase, with a flow rate of 1.0 mL/min and a column temperature of 40 °C.

All NMR spectra, including ¹H-NMR and ¹³C-NMR spectra, were recorded on a Bruker Avance 400 MHz (Bruker, Fallanden, Switzerland). Chemical shifts were exhibited in parts per million (δ, ppm) and referenced to CDCl3 with 7.26 for ¹H and 77.16 for ¹³C.

Cell Culture

The prostate cancer cell lines 22RV1 (#CL-0004), LNCaP (#CL-0143) and C4-2 (#CL-0046), the ovarian cancer cell line SKOV3 (#CL-0215) were obtained from Procell Life (Wuhan, China) and the normal ovary cell line IOSE-80 (#IM-H366) were obtained from IMMOCELL (Xiamen, China). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (#8120365, Thermo Fisher Scientific, Waltham, MA, U.S.A.) supplemented with 10% fetal bovine serum (#2279604CP, Thermo Fisher Scientific, Waltham, MA, U.S.A.) and 1% penicillin-streptomycin liquid (#P1400, Solarbio, Beijing, China). All cells were routinely incubated at 37 °C in a humidified atmosphere of 5% CO₂.

Vector Construct and Transfection

The NRF2 overexpressed plasmid pEGFP-NRF2 and its negative control were synthesized by GenePharma (Shanghai, China). Advanced Lipofectamine 3000 Transfection Reagent (#L3000001, ThermoFisher, Shanghai, China) was used for transfection into cancer cells, the transfection operation was strictly in accordance with the reagent instructions, and cells was used for follow-up experiments 24 h after transfection.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

Cells (5 × 10³ cells/well) were inoculated into the 96-well plates and treated with either or both MESA and ferrostatin-1 (fer-1, an inhibitor of ferroptosis, # 347174-05-4, MedChem, Shanghai, China). The drug concentration and time were described in the figure legends below. MTT reagent of 5 mg/mL (#ST316, Beyotime, Jiangsu, China) was added in the dark and incubated for 4–6 h. The supernatant was discarded and the precipitate was dissolved with DMSO. The absorbance was measured at 490 nm using a microplate reader (BioTek, Winooski, VT, U.S.A.).

Colony Formation Assay

Cells (500 cells/well) were inoculated into the 6-well plates and treated with 0, 2.5, 5, 10, 20, and 40 µM MESA, respectively. The fresh medium was added every 3 d. After 7–10 d, cell colonies were fixed, stained, photographed and counted.

Apoptosis Assay

Cells (2 × 10⁵ cells/well) were inoculated into the 6-well plates and treated with either or both MESA and fer-1. The drug concentration and time were described in the figure legends below. The collected cells were stained with Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) (#C1062L, Beyotime) and detected by flow cytometry (Cytek^® DxP Athena™, Cytek Bioscience, Fremont, CA, U.S.A.).

Detection of Malondialdehyde (MDA)

Cells (2 × 10⁵ cells/well) were inoculated into the 6-well plates and treated with either or both MESA and fer-1. The drug concentration and time were described in the figure legends below. The collected cell lysate was subject to BCA Assay Kit (#P0011, Beyotime) and Lipid Peroxidation MDA Assay Kit (#S0131M, Beyotime) to the detect the protein concentration and MDA concentration in accordance with the manufacturers’ instructions. The relative MDA levels were indicated by the value of MDA concentration/protein concentration.

Detection of ROS

Cells (1 × 10⁵ cells/well) were inoculated into the 6-well plates and treated with either or both MESA and fer-1. The drug concentration and time were described in the figure legends below. Cells were incubated with 20 µM DCFH-DA (#S0033S, Beyotime, China) for 30 min at 37 °C. The collected cells were determined by flow cytometry for the ROS levels indicated by fluorescence intensity.

Detection of Fe²⁺ Detection

Cells (2 × 10⁵ cells/well) were inoculated into the 6-well plates and treated with either or both MESA and fer-1. The drug concentration and time were described in the figure legends below. The collected cells were washed and stained with 1 µM FerroOrange (#F374, Dojindo, Japan) for 30 min at 37 °C. Images were acquired using Imaging Multimode Reader (Biotek, Thorold, ON, Canada).

Western Blot

The experiments of protein extraction, electrophoresis and transfer were performed as per standard methods. Antibodies (SAB, Santa Monica, CA, U.S.A.) used were presented as follows: GPX4 (#32506), P62(#13222-2), ACSL4 (#36176), Kelch-like ECH-associated protein 1 (KEAP1) (#32450-2), SLC7A11 (#43437), NRF2 (#41255-2) and p-NRF2 (#C91434Bio). Signals were visualized using ECL reagent and analyzed via ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for a loading control.

Molecular Docking

The crystal structure of NRF2 was obtained from PDB database (PDB ID: 7K2A) and the protein and ligand molecules were prepared via DS 3.5 software. Molecular docking of MESA into the NRF2 protein binding sites was conducted via LibDock protocol and analyzed by LibDock Score.

Cellular Thermal Shift Assay

22RV1 cells (2 × 10⁶ cells/mL) were collected and they were treated with 10 µM MESA for 30 min at 37 °C. The collected cells were washed and resuspended in phosphate buffered saline (PBS) containing protease inhibitors. Cells (100 µL/tube) were incubated with gradient temperature from 40–60 °C (2 °C intervals) for 3 min in a thermal cycler. The samples were stored at room temperature for 3 min and then snap-freezed in liquid nitrogen for 3 min. The preceding cycles were performed for 3 times. The cell lysates were centrifuged and subject to Western blot assays to detect the levels of NRF2 and GAPDH proteins. The binding stability was indicated by melt curve of temperature.

Statistical Analysis

Data were shown as means  ±  standard deviation (S.D.). Differences between groups were analyzed via one- or two-way ANOVA. Asterisks (* p < 0.05, ** p < 0.01) were used to indicate differences between groups that were statistically significant.

RESULTS

MESA Inhibited the Proliferation of Prostate and Ovarian Cancer Cells

The synthesis approaches of morpholine derivative MESA were shown in Supplementary Fig. 1 and the chemical structure of MESA was presented in Fig. 1A (C₇H₁₄N₂O₃S, molecular weight: 206). The synthesis yield had reached to 96% and the product was white solid. The structure of MESA identified by NMR spectrum. The NMR data of MESA was showed as following: ¹H-NMR (400 MHz, CDCl3) δ: 8.05 (s, ¹H), 3.72 (s, 1H), 3.70–3.65 (m, ¹H), 3.47 (s, ¹H), 3.04–2.97 (m, ¹H), 1.28 (d, J = 3.7 Hz, ¹H); ¹³C-NMR (100 MHz, CDCl3) δ: 158.2, 66.9, 65.9, 50.2, 48.1, 44.1, 8.28 (Supplementary Fig. 2).

Fig. 1. MESA Inhibited the Proliferation of Tumor Cells

(A) The structure of MESA. (B) MTT assay. IOSE-80, LNCaP, SKOV3, C4-2 and 22RV1 cells were treated with 0, 2.5, 5, 10, 20, 40 µM MESA for 48 h, respectively. (C) Colony formation assay. 22RV1,¹⁾ SKOV3²⁾ and IOSE-80³⁾ cells were treated with 0, 2.5, 5, 10, 20, 40 µM MESA, respectively. Data were shown as mean  ±  S.D., ns: no statistical difference, ** p < 0.01.

To determine the effect of MESA on tumor cell viability, prostate cancer LNCaP, C4-2, and 22RV1 cells, as well as ovarian cancer SKOV3 cells, were stimulated with a gradient concentration of MESA and subject to MTT and cell colony assays. IOSE-80, ovarian epithelial cell line was used as normal control. The results showed that exposure to MESA significantly inhibited the proliferation of tumor cells in a dose-dependent manner in MTT and cell colony assays. In prostate cancer cells, MESA induced the strongest inhibitory effect on 22RV1 cells, which would be used for the following experiments together with ovarian cancer SKOV3 cells. Additionally, MESA had no discernible inhibitory effect on IOSE-80 cells, indicating that it was not harmful for the proliferation of normal cells (Figs. 1B, C, ** p < 0.01).

MESA Induced Ferroptosis in Prostate and Ovarian Cancer Cells

Different cell death inhibitors were tried alongside MESA to further explore the forms of tumor cell death induced by MESA. MESA-induced cell death of SKOV3 and 22RV1 cells were suppressed by ferroptosis inhibitors Fer-1, rather than Z-VAD-FMK (z-VAD, apoptosis inhibitor), 3-Methyladenine (3-MA, autophagy inhibitor) and necrostatin-1 (nec-1, necroptosis inhibitor). However, z-VAD could partially rescue the tumor cell death effects mediate by MESA, as indicated in Fig. 2A (** p < 0.01). As a result, the specific mechanism of MESA regulating ferroptosis in tumor cells would be investigated further in the subsequent study, but whether there was crosstalk between MESA-mediated apoptosis and ferroptosis in tumor cells needed to be further explored.

Fig. 2. MESA Induced Ferroptosis in Tumor Cells

(A) MTT assay. SKOV3 and 22RV1 cells were treated with either or both MESA/fer-1, MESA/z-VAD, MESA/3-MA, MESA/nec-1 for 12 h, respectively (MESA 10 µM, fer-1 1 µM, z-VAD 10 µM, 3-MA 1 µM, nec-1 10 µM). (B, C) MTT assay. SKOV3 and 22RV1 cells were treated with 0, 0.25, 0.5, 1 µM fer-1, respectively. MESA of 5 µM was added in (C). (D) Apoptosis assay. 22RV1¹⁾ and SKOV3²⁾ cells were treated with either or both 5 µM MESA and 0.5 µM fer-1 for 24 h. Data were shown as mean  ±  S.D., ns: no statistical difference, * p < 0.05, ** p < 0.01.

Next, we investigated whether MESA caused the tumor cell death via ferroptosis-relative pathway. Fer-1 (ferroptosis inhibitor) was employed to confirm the death effects of tumor cells treated with MESA. The results showed that fer-1 did not have detectable cytotoxic effects on the proliferation of SKOV3 and 22RV1 cells at the concentration less than 1 µM (Fig. 2B, p > 0.05). Meanwhile, MESA significantly hindered the proliferation of SKOV3 and 22RV1 cells and the inhibitory effects could be rescued with fer-1 (Fig. 2C, * p < 0.05, ** p < 0.01); MESA significantly induced the apoptosis of SKOV3 and 22RV1 cells and the apoptosis effects could be rescued with fer-1 (Fig. 2D, ** p < 0.01). Thus, exposure to MESA would cause the cell death of SKOV3 and 22RV1 cells and we supposed that the death effects might be related to ferroptosis pathway.

MESA Increased the Intracellular MDA, ROS and Fe²⁺ Levels in Prostate and Ovarian Cancer Cells

Since ferroptosis is iron ion dependent and distinguished by lipid peroxidation and MDA accumulation, we further detected the intracellular MDA, ROS and Fe²⁺ levels in 22RV1 and SKOV3 cells exposure to MESA. The results showed that compared to the control, the intracellular MDA and ROS levels were significantly increased 2-fold in 22RV1 and SKOV3 cells treated with MESA alone. Meanwhile, the intracellular Fe²⁺ signals indicated by FerroOrange were significantly increased in 22RV1 and SKOV3 cells with the individual stimulation of MESA. But all the increasing levels of intracellular MDA, Fe²⁺ and ROS levels in 22RV1 and SKOV3 cells induced by MESA could be dropped considerably after the addition of fer-1 (Figs. 3A–C, ** p < 0.01). Collectively, MESA increased the intracellular MDA, Fe²⁺ and ROS levels in 22RV1 and SKOV3 cells, and these results supported a disruptive role of MESA causing cell death in prostate and ovarian cancer cells via ferroptosis-relative pathway.

Fig. 3. MESA Up-Regulated the Intracellular MDA, ROS and Fe²⁺ Levels in Tumor Cells

(A) MDA detection with Lipid Peroxidation MDA Assay Kit. (B) Intracellular Fe²⁺ detection with FerroOrange. Scale bar, 100 µm. (C) ROS detection with DCFH-DA. 22RV1¹⁾ and SKOV3²⁾ cells were treated with either or both 5 µM MESA and 0.5 µM fer-1 for 24 h. Data were shown as mean  ±  S.D., ns: no statistical difference, ** p < 0.01.

MESA Mediated the Dynamic Changes of Ferroptosis-Relative Molecules and Signal Pathways in Prostate and Ovarian Cancer Cells

It had been demonstrated that MESA would cause proliferation inhibition and apoptosis effects of prostate and ovarian cancer cells via ferroptosis-relative pathway but the specific molecular mechanism involved needed further study. ACSL4, SLC7A11-GPX4, and P62-KEAP1-NRF2-ARE were essential molecules and signal pathways in the ferroptosis process of tumor cells. When exposure to MESA, the expression of ferroptosis suppressor molecules GPX4 and NRF2 were downregulated and the expression of ferroptosis promoting molecule ACSL4 was upregulated in 22RV1 and SKOV3 cells, which could be reversed by fer-1 in a dose-dependent manner (Figs. 4A, B, * p < 0.05, ** p < 0.01). Further, MESA stimulation downregulated the expression of SLC7A11, GPX4, NRF2, the phosphorylation level of NRF2 and upregulated the expression of ACSL4, P62, KEAP1 in 22RV1 and SKOV3 cells in a dose-dependent manner (Figs. 5A, B, * p < 0.05, ** p < 0.01). Thus, the results indicated that MESA might induce ferroptosis effects in 22RV1 and SKOV3 cells via suppressing the SLC7A11-GPX4, NRF2-ARE signal pathways and enhancing ACSL4 signal pathway. Interestingly, MESA not only dropped NRF2 protein levels but also repressed NRF2 phosphorylation, and the upregulation of P62, KEAP1 needed more attention and exploration.

Fig. 4. MESA Mediated the Dynamic Changes of Ferroptosis-Relative Genes

(A, B) Detection of protein levels of NRF2, GPX4 and ACSL4 by Western blotting assay. 22RV1 (A) and SKOV3 (B) cells were treated with either or both 5 µM MESA and fer-1 (0, 0.25, 0.5, 1 µM) for 24 h. Data were shown as mean  ±  S.D., ns: no statistical difference, * p < 0.05, ** p < 0.01.

Fig. 5. MESA Mediated the Dynamic Changes of Ferroptosis-Relative Signal Pathways

(A, B) Detection of protein levels of GPX4, ACSL4, SLC7A11, P62, KEAP-1 (A) and p-NRF2, NRF2 (B) by Western blotting assay. 22RV1¹⁾ and SKOV3²⁾ cells were treated with 0, 5, 10, 20 µM MESA for 24 h, respectively.

NRF2 Overexpression Antagonized MESA-Mediated Tumor Cell Death

NRF2, as previously mentioned, was involved in the process of MESA-induced tumor cell death. We first explored the NRF2 expression in IOSE-80, 22RV1, and SKOV3 cells. The gene was found to be significantly expressed in tumor cells, according to the findings (Fig. 6A). A NRF2 overexpression vector was constructed and transfected into 22RV1 and SKOV3 cells. The expression of the NRF2 gene were increased in tumor cells after 24-h transfection (Fig. 6B). Flow cytometry results demonstrated that NRF2 overexpression could drastically reduce MESA-induced tumor cell death (Fig. 6C). The results of intracellular ROS tests also revealed that NRF2 overexpression considerably downregulate tumor cell ROS levels following MESA exposure (Fig. 6D). Thus, MESA exposure might induce ferroptosis effects in tumor cells, which could be rescued by ferroptosis inhibitor and NRF2 overexpression. We supposed that the MESA-mediated cell death in tumor cells was related to the expression of NRF2 protein and the relationship between MESA and NRF2 protein was further explored.

Fig. 6. NRF2 Overexpression Antagonized MESA-Mediated Tumor Cell Death

(A) Western blotting assay of the expression level of NRF2 in IOSE-80, SKOV3 and 22RV1 cells. (B) Western blotting assay of the expression level of NRF2 in SKOV3 and 22RV1 cells after transfected with NRF2 overexpression vector. (C, D) 22RV1¹⁾ and SKOV3²⁾ cells were treated with either or both 5 µM MESA and transfected with NRF2 overexpression vector for 24 h. (C) Apoptosis assay. (D) ROS detection with DCFH-DA. Data were shown as mean  ±  S.D., ns: no statistical difference, ** p < 0.01.

NRF2 Was a Target Molecule of MESA Inducing Ferroptosis Effects in Tumor Cells

Molecular docking study was performed to investigate the interaction between MESA and NRF2 protein. As shown in Figs. 7A and B, the docked pose between MESA with NRF2 protein was applicable with a LibDockScore of 94. MESA formed one hydrogen bond to bind NRF2 protein at tyrosine (Tyr) 520 with a short distance of 2.41 Å. Since Tyr 520 belonged to the basic region-leucine zipper (bZIP) domain, we further found that MESA was able to strongly bind to the bZIP domain in the Neh1 region of NRF2 protein based on the analysis at the Uniprot Protein Data Bank (https://www.uniprot.org/, Fig. 7C).

Fig. 7. NRF2 Was the Target of MESA

(A, B) The molecular docking model of MESA and NRF2 protein ligands was conducted via DS 3.5 software. (A) The 3-dimensional protein model of NRF2 was shown by ribbon representation. MESA binding to a molecule-binding pocket in NRF2 protein was shown. (B) Putative binding sites between MESA and NRF2 protein. Prediction of binding sites was conducted by LibDock protocol and evaluated by LibDock Score. (C) MESA bound to the bZIP domain in the Neh1 region of NRF2 protein. (D) The band stability between MESA and NRF2 protein. 22RV1 cells (2 × 10⁶ cells/mL) were treated with 10 µM MESA for 30 min at 37 °C and analyzed by thermal shift assay (from 40 to 60 °C).

Thermal stability studies were conducted to confirm the binding between MESA and NRF2 protein in tumor cells. 22RV1 cells were treated with MESA and incubated with gradient temperature from 40–60 °C. The NRF2 protein in the control group was rapidly degraded with increasing temperature, however, the experimental group did not exhibit substantial degradation of NRF2 protein due to the addition of MESA. This outcome indicated that MESA was able to bind to NRF2 protein (Fig. 7D). Thus, NRF2 was identified as a target molecule of MESA in tumor cells and the binding sites were successfully predicted via molecular docking. MESA might bind to NRF2 protein and decreased its level in tumor cells, which mediated the ferroptosis effects and caused the cell death.

DISCUSSION

In this study, the morpholine derivative MESA was exposure to prostate and ovarian cancer cells to investigate the cell death effects it caused. Since the reversed effects of ferroptosis inhibitor fer-1 and the evaluation of increasing intracellular MDA, ROS and Fe²⁺ levels, we demonstrated that MESA was able to cause cell death in prostate and ovarian cancer cells via ferroptosis-relative pathway. NRF2 overexpression could rescue the ferroptosis effects in tumor cells exposure to MESA. Most importantly, it had been identified that NRF2 was a target molecule and SLC7A11-GPX4, ACSL4, P62-KEAP1-NRF2-ARE were signal pathways involved for MESA inducing ferroptosis effects in tumor cells.

Recent studies have revealed that NRF2 is involved in a number of physiological processes and a critical regulator of lipid peroxidation and intracellular oxidative homeostasis.^26,27) NRF2 binds to the downstream genes of antioxidant response elements (AREs) such as heme oxygenase-1 (HO-1) to regulate intracellular oxidative stress at transcriptional level.²⁸⁾ But in cytoplasm, the release and activation of NRF2 is negatively regulated by KEAP1, which mediates the ubiquitination degradation of NRF2 protein via the scaffolding protein P62-Cul3-E3 ubiquitin ligase pathway.²⁹⁾ We found that MESA exposure reduced the expression and phosphorylation of NRF2 and induced ferroptosis effects in tumor cells, while NRF2 overexpression could rescue the ferroptosis effects mediated by MESA. Based on molecular docking, bioinformatic analysis and thermal stability assays, it had been identified that NRF2 was a target molecule of MESA in response to inducing ferroptosis effects and cell death in tumor cells. The bZIP region of Nehl domain was the essential structure for NRF2 protein recognizing and binding AREs in nucleus.³⁰⁾ The binding sites between MESA and NRF2 protein was predicted to locate at Tyr 520 of bZIP domain and there was a strong binding due to the formation of hydrogen bond (2.41 Å). Thus, we supposed that MESA was able to block the bZIP region of NRF2 protein, suppressing the AREs at transcriptional level. Moreover, the expression of P62 and KEAP1 were upregulated in tumor cells treated with MESA. And we suspected that MESA and the P62-KEAP1 complex competed for binding to NRF2 protein, leaving a significant quantity of P62 and KEAP1 proteins in a free form.

A therapeutic target of NRF2 protein is anticipated to be a new treatment option for cancer patients due to its high expression status in tumor cells.³¹⁾ However, discovering and developing novel and efficient NRF2 inhibitors is very difficult and only a few NRF2 inhibitors have been reported to be ready for further preclinical testing. The primary issue is that these NRF2 inhibitors lack a precise and well-defined mechanism of causing cell death in tumor cells.³²⁾ The morpholine derivative MESA had anti-cancer efficacy in prostate and ovarian cancer cells. MESA targets the NRF2 protein and then regulates the P62-KEAP1-NRF2-ARE signaling pathway to cause ferroptosis effects in tumor cells.

Meanwhile, the morpholine derivative MESA had showed its latent function of causing ferroptosis effects via inactivating SLC7A11-GPX4 and activating ACSL4 signaling pathways, but the specific mechanism needed further investigation, which would contribute to developing MESA as a promising small molecule drug for cancer therapy based on cell ferroptosis in the future.

In summary, our findings indicated the ferroptosis effects of the morpholine derivative MESA in prostate and ovarian cancer cells and its function mechanism involving in targeted molecule (NRF2) and signal pathways (SLC7A11-GPX4/ACSL4/P62-KEAP1-NRF2-ARE), which would boost MESA as a prospective candidate of small molecule drug for tumor treatment based on cell ferroptosis. Further, whether there was crosstalk between MESA-mediated ferroptosis and other cell death effects (apoptosis) in tumor cells needed more study.

Funding

This work was supported by the Zhanjiang Science and Technology Research Plan Project (2020B01027, 2021B01031) and Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang, ZJW-2019-007).

Author Contributions

Conception and study design: BS, BW, XL. Data acquisition and Data analysis: BS, LZ, BW; Manuscript drafting and revising: BS, LZ, BW. All authors have read and approved the final version of this manuscript to be published.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data generated or analyzed during this research are included in this paper and available from corresponding author for reasonable need.

Supplementary Materials

This article contains supplementary materials.

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