OVCAR3 and A2780 human ovarian cancer cell lines were acquired from the Chinese Type Culture Collection, Chinese Academy of Sciences. The cells were cultured at 37 °C and 5% CO${}_2$, in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin sodium, and 100 mg/mL streptomycin sulfate. All the cell lines were authenticated by short tandem repeat and were confirmed negative for mycoplasma contamination (Mycoalert Mycoplasma Detection Kit, Lonza, Monteggio, Switzerland).

The sequence of shRNA targeting ARHGAP10 (shARHGAP10) was GGTTCACAATTATCAGAAA. The shRNA was included in pLKO.1 lentiviral vector (Addgene, Watertown, MA, USA). Lentiviral constructs of pLKO.1-scramble shRNA (shNC), pLKO.1-shARHGAP10, pLVX-Puro empty vector (Clontech, Palo Alto, CA, USA), or pLVX-Puro-ARHGAP10 were co-transfected with viral packaging plasmids (psPAX2 and pMD2.G) into 293T cells. The viral supernatant was harvested after 48 h and filtered. A2780 cells were infected with pLVX-Puro-ARHGAP10 lentivirus while OVCAR3 cells were infected with pLKO.1-shARHGAP10 lentivirus with 8 µg/mL polybrene. Stable pools were obtained with 0.5 µg/mL puromycin (Sigma, St. Louis, MO, USA).

A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus were treated with or without 25 µM apoptosis inhibitor zVAD-fmk, 20 µM necroptosis inhibitor necrostatin-1 (Nec-1), or 5 µM ferroptosis inhibitor ferrostatin-1 (Fer; all from Selleck, Shanghai, China). Another set of A2780 cells was infected with pLVX-Puro-ARHGAP10 lentivirus and/or treated with 5 µM Fer-1.

OVCAR3 cells were infected with pLKO.1-shARHGAP10 lentivirus and/or treated with 1 µg/mL RSL3 (Selleck, Shanghai, China). Another set of OVCAR3 cells was treated with 0.5 mM sodium butyrate with or without pLKO.1-shARHGAP10 lentivirus infection. Cell Counting Kit-8 (CCK-8) Assay Kit (Dojindo Lab, Kumamoto, Japan) was then used to measure cell viability in 96-well plates (2 $\times$ 10${}^3$ cells per well). At 0, 12, 24, and 48 h, CCK-8 solution was added, and optical density values were measured using a microplate reader (Bio-Rad, Richmond, CA, USA) after 1 h.

A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus were treated with or without 25 µM zVAD-fmk, 20 µM Nec-1, or 5 µM Fer-1. Cell death rate was determined using the LDH cytotoxicity assay kit (Beyotime Biotechnology, Shanghai, China; C10016), as per the manufacturer’s instructions, and calculated as previously described [16].

Cells (3 $\times$ 10${}^5$ per well) were cultured overnight. A2780 cells were infected with pLVX-Puro-ARHGAP10 lentivirus and/or treated with 5 µM Fer-1. OVCAR3 cells were infected with pLKO.1-shARHGAP10 lentivirus and/or treated with 1 µg/mL RSL3. Another set of OVCAR3 cells was treated with 0.5 mM SB with or without pLKO.1-shARHGAP10 lentivirus infection. At 48 h, C11-BODIPY (10 µM; Thermo Fisher Scientific, Waltham, MA, USA) was added to the cell suspension and incubated for 30 min at 37 °C in the dark. After washing twice with PBS, the fluorescence intensity of the cells was measured by a CytoFLEX flow cytometer (Beckman Coulter, Miami, FL, USA). The Iron Assay Kit (Abcam, Waltham, MA, USA; ab83366) was used to detect Fe${}^{2+}$ content, as per the manufacturer’s instructions.

Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The mRNA levels of ARHGAP10 were determined by quantitative RT-PCR using GAPDH as a control. The primer sequences used were as follows: ARHGAP10, 5${}^{\prime }$-AAGGAGGACACCCCTACCAG-3${}^{\prime }$ and 5${}^{\prime }$-GGATGCCCAGTCTCCAAAG-3${}^{\prime }$; GAPDH, 5${}^{\prime }$-TCCCATCACCATCTTCCAGG-3${}^{\prime }$ and 5${}^{\prime }$-GATGACCCTTTTGGCTCCC-3${}^{\prime }$. Specific product amplification was verified by dissociation curve analysis. The foldchange for target genes normalized by internal control was determined by the 2${}^{-△△\text{CT}}$ formula.

Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate) containing protease inhibitors. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of cell lysates were separated on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes, and analyzed by Western blotting using an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). The antibodies used are listed in Table 1. Band intensities were analyzed using Image J (NIH, Bethesda, MD, USA) and normalized to $\beta$-actin.

OVCAR3 cells were treated with 0.5 mM sodium butyrate or left untreated for 48 h, and then fixed with 1% formaldehyde. The fixed cells were sonicated for chromatin fragmentation. The chromatin solutions were then incubated overnight with anti-H3K9ac (Cell Signaling Technology, Danvers, MA, USA; 9649) or control IgG antibody (Cell Signaling Technology; 3900) at 4 °C. H3K9ac binding in the ARHGAP10 promoter region was quantified by PCR using the following ARHGAP10 promoter primers: 5${}^{\prime }$-AAGAGCCCCCTGTGCGAGTT-3${}^{\prime }$ (Forward) and 5${}^{\prime }$-GGCGGGCGACATAACTCT-3${}^{\prime }$ (Reverse).

Athymic Balb/c nude mice (age: 5 weeks) were acquired from the Slac Laboratory Animal Co. Ltd. (Shanghai, China) and housed in a pathogen-free animal facility. Mice were randomly assigned to control or experimental groups (6 per group). A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus were injected intraperitoneally into the flank of each mouse (2 $\times$ 10${}^6$/0.1 mL). Injection of Fer-1 (5 mg/kg) was administered twice a week. Tumor volume was estimated once every 3 days: volume = 1/2 $\times$ length $\times$ width${}^2$. On day 35, the mice were sacrificed by cervical dislocation after an intraperitoneal injection of sodium pentobarbital (30 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). The animal study was carried out in accordance with the guidelines approved by the Animal Experimentation Ethics Committee of Shanghai Pudong Hospital, Fudan University Pudong Medical Center [(2021)No.(QKW-02)].

After fixation and permeabilization, the tissues collected from the xenograft tumor were incubated with 1% bovine serum albumin for 30 min for blocking and then with anti-Ki67 (Abcam; ab243878) and Alexa Fluor 488-labeled Goat Anti-Mouse IgG (H + L) (Beyotime Biotechnology; A0423). DAPI (Beyotime Biotechnology; C1002) labeling was used for nuclear staining. Then, positively stained cells were visualized using a confocal laser scanning microscope (Leica Microsystems, Inc., Wetzlar, Germany).

All experiments were conducted in triplicate. Data were expressed as mean $\pm$ standard deviation (SD). All statistical analyses were performed using GraphPad Prism 8.4.2 (GraphPad Software, San Diego, CA, USA). Differences between two groups were assessed for statistical significance using the Students t-test, while ANOVA was used for multi-group comparisons. p values 0.05 were considered indicative of statistical significance.

To understand the effects of ARHGAP10 on ovarian cancer, we first used pLVX-Puro-ARHGAP10 lentivirus to induce the overexpression of ARHGAP10. A2780 cell line was used as an in vitro cell model of human ovarian cancer. After infection with pLVX-Puro-ARHGAP10 lentivirus, the A2780 cells showed much higher mRNA and protein expression of ARHGAP10 compared with non-infected cells (Control) or cell infected with empty lentivirus (Vector) (Fig. 1A,B), confirming the success of the infection.

Fig. 1.

ARHGAP10 overexpression promotes A2780 cell ferroptosis. (A) mRNA and (B) protein levels of ARHGAP10 in A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus or control lentivirus (n = 3). A2780 cells were infected with pLVX-Puro-ARHGAP10 lentivirus and treated with 25 µM apoptosis inhibitor zVAD-fmk, 20 µM necroptosis inhibitor Nec-1, or 10 µM ferroptosis inhibitor Fer-1, and the (C) cell viability and (D) cell death were determined (n = 3). The (E) Fe${}^{2+}$ content, (F) lipid reactive oxygen species (ROS), and (G) expression of GPX4 and PTGS2 in A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus or control lentivirus (n = 3). ***p 0.001 vs. vector. ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ARHGAP10.

Consistent with a previous study [7], our results showed that ARHGAP10 overexpression caused a significant decrease in cell viability (Fig. 1C) and an increase in cell death (Fig. 1D). To determine the cause of ARHGAP10 overexpression-induced cell death, several cell death inhibitors were tested. Ferroptosis inhibitor Fer-1 markedly inhibited the ARHGAP10 overexpression-induced cell viability decrease and cell death. However, apoptosis inhibitor zVAD-fmk only slightly inhibited ARHGAP6 overexpression-induced cell viability decrease and cell death (Fig. 1C,D). To further examine the effects of ferroptosis on ARHGAP10 overexpression-mediated cell death, Fe${}^{2+}$ content, and lipid ROS levels were measured. Fe${}^{2+}$ content (Fig. 1E) and lipid ROS levels (Fig. 1F) were significantly increased in ARHGAP10-overexpressing A2780 cells, suggesting the intensification of ferroptosis, an iron-dependent PCD pathway distinctive from apoptosis, necrosis, and autophagy, characterized by the accumulation of lipid-based ROS [17]. Therefore, we focused on ferroptosis by measuring the expression level of two proteins indicative of ferroptosis, glutathione peroxidase 4 (GPX4), a protective protein that prevents lipid peroxidation and ferroptosis [18], and Prostaglandin-Endoperoxide Synthase 2 (PTGS2), a well-established biomarker of ferroptosis [19]. As shown by the Western blot assay, ARHGAP10 overexpression substantially decreased the expression of GPX4 and increased that of PTGS2 (Fig. 1G). These results consistently indicated that ARHGAP10 overexpression triggered ferroptosis in ovarian cancer cells.

To further confirm the triggering of ferroptosis by ARHGAP10 overexpression, we tested the effect of Fer-1. Both in the absence and presence of ARHGAP10 overexpression, treatment with Fer-1 significantly increased cell viability (Fig. 2A), and decreased the Fe${}^{2+}$ content (Fig. 2B) and lipid ROS levels (Fig. 2C,D), indicating protection from cell death. Importantly, Fer-1 upregulated the expression of GPX4 and down-regulated that of PTGS2 in uninfected cells; furthermore, it reversed ARHGAP10 overexpression-induced decrease of GPX4 expression and enhancement of PTGS2 expression, to the level approximating that in the uninfected untreated control cells (Fig. 2E). Collectively, these results indicated that ARHGAP10 overexpression-induced ferroptosis in A2780 cells, which was suppressed by Fer-1.

Fig. 2.

Fer-1 inhibits ARHGAP10 overexpression-induced ferroptosis in A2780 cells. (A) Cell viability, (B) Fe${}^{2+}$ content, (C,D) lipid ROS, and (E) expression of GPX4 and PTGS2 in A2780 cells infected with pLVX-Puro-ARHGAP10 lentivirus and treated with Fer-1 (n = 3). **p 0.01, ***p 0.001 vs. control. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ARHGAP10.

In a previous study, ARHGAP10 was found to suppress tumor growth [7], which was argued to be contributed by apoptosis. However, other molecular mechanisms may also be involved in contributing to this effect. Based on our above results which showed that ARHGAP10 triggers ferroptosis in cancer cells, we hypothesized that it also contributes to tumor suppression.

To test our hypothesis, mice were injected with A2780 cells with or without ARHGAP10 overexpression to induce tumor development. Some of the mice in both groups were then treated with Fer-1 for 35 days, while the rest were left untreated. The development of tumors in each mouse was monitored for 35 days. Compared to the control group, tumors in mice treated with Fer-1 were larger and heavier, suggesting that ferroptosis is indeed a regulator of ovarian tumor growth (Fig. 3A–C). Notably, whereas ARHGAP10 overexpression significantly decreased tumor size and weight, these effects were reversed by Fer-1 (Fig. 3A–C).

Fig. 3.

Fer-1 inhibits ARHGAP10 overexpression-induced suppression of tumor growth in vivo. Mice injected with A2780 cells were treated with Fer-1 for 35 days. Tumor (A) volume, (B,C) weight, and (D) Ki67 immunofluorescence staining (scale bar, 20 µm) were quantified (n = 6). **p 0.01, ***p 0.001 vs. control. ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. ARHGAP10.

We also looked at immunofluorescence staining of Ki67, a commonly used biomarker of cell proliferation [20], in the xenograft tumor tissue. While Fer-1 greatly increased the signal intensity of Ki67 in the tumor cells, suggesting higher viability and greater proliferation, the signal intensity was drastically decreased by ARHGAP10 overexpression, and was restored to a level comparable to the control group by the copresence of ARHGAP10 overexpression and Fer-1 (Fig. 3D). These results indicated that ARHGAP10-induced suppression of tumor growth was in part via the ferroptosis pathway, which can be inhibited by Fer-1.

ARHGAP10 silencing is another way to re-confirm the observed relation between ARHGAP10 and ferroptosis. A shRNA targeting ARHGAP10 (shARHGAP10) was developed for this purpose and used to construct the pLKO.1-shARHGAP10 lentivirus. Infection of OVCAR3 cells with the control lentivirus (shNC) had no effect on the mRNA and protein expression levels of ARHGAP10. However, OVCAR3 cells infected with pLKO.1-shARHGAP10 manifested much lower mRNA and protein expression levels of ARHGAP10, confirming the specificity of pLKO.1-shARHGAP10 in ARHGAP10 silencing (Fig. 4A,B). Silencing of ARHGAP10 significantly increased cell viability (Fig. 4C), Fe${}^{2+}$ content (Fig. 4D), and lowered lipid ROS level (Fig. 4E,F), as well as increased and decreased the expression of GPX4 and PTGS2, respectively (Fig. 4G). These findings are consistent with our above findings, indicating suppression of ferroptosis.

Fig. 4.

RSL3 inhibits ARHGAP10 silencing-induced suppression of ferroptosis in OVCAR3 cells. (A) mRNA and (B) protein levels of ARHGAP10 in OVCAR3 cells infected with pLKO.1-shARHGAP10 lentivirus or control lentivirus (n = 3). (C) Cell viability, (D) Fe${}^{2+}$ content, (E,F), lipid ROS, and (G) expression of GPX4 and PTGS2 in OVCAR3 cells infected with pLKO.1-shARHGAP10 lentivirus and/or treated with RSL3 (n = 3). *p 0.05, ***p 0.001 vs. control. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. shARHGAP10.

RSL3 is a facilitator of cell ferroptosis, which has been shown to inactivate GPX4 and stimulate ROS production in cancer cells [21, 22]. Here, we showed that RSL3 decreased the viability of OVCAR3 cells, increased the lipid ROS level, and decreased and increased the expression of GPX4 and PTGS2, respectively (Fig. 4C–G). Treatment of OVCAR3 cells with RSL3 reversed the effect of ARHGAP10 silencing so that the cell viability level, lipid ROS level as well as GPX4 and PTGS2 expression levels were at an intermediate level between the ARHGAP10-silenced group and RSL3-treated group (Fig. 4C–G).

Lastly, we investigated the potential relation of SB with ARHGAP10 in ferroptosis. Treatment of ovarian cancer cells with SB greatly increased the mRNA and protein expression levels of ARHGAP10 (Fig. 5A,B). SB also increased the expression level of H3K9ac, a marker of transcriptional activation of genes (Fig. 5B). This suggested that SB may enhance the expression of ARHGAP10 by regulating the transcription of its encoding gene. The results of the ChIP assay showed that SB can increase the binding of H3K9ac to the promoter region of ARHGAP10 (Fig. 5C,D).

Fig. 5.

Sodium butyrate promotes OVCAR3 cell ferroptosis by transcriptional regulation of ARHGAP10. (A) mRNA levels of ARHGAP10 and (B) protein levels of ARHGAP10 and H3K9ac in OVCAR3 cells treated with sodium butyrate (SB) (n = 3). (C) Chromatin from OVCAR3 cells was immunoprecipitated using control IgG or H3K9ac antibody, and the results were measured by quantitative PCR (n = 3). (D) Chromatin from OVCAR3 cells with or without SB treatment were immunoprecipitated using the H3K9ac antibody, and the results were measured by quantitative PCR (n = 3). (E) Cell viability, (F) Fe${}^{2+}$ content, (G) lipid ROS, and (H) expression of ARHGAP10, GPX4, and PTGS2 in OVCAR3 cells treated with SB were infected with or without pLKO.1-shARHGAP10 lentivirus (n = 3). **p 0.01, ***p 0.001 vs. control. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs. SB.

Consistent with the above finding, we further found that SB significantly decreased the viability of OVCAR3 cells (Fig. 5E), increased the Fe${}^{2+}$ content (Fig. 5F) and level of lipid ROS (Fig. 5G), as well as decreased and increased the expression level of GPX4 and PTGS2, respectively (Fig. 5H). All these findings indicate the role of SB in inducing ferroptosis. However, silencing the ARHGAP10 mRNA with shARHGAP10 abrogated all the above effects of SB, bringing the cell viability and lipid ROS, GPX4, and PTGS2 levels back to the levels comparable to the control condition (Fig. 5E–H). These results strongly suggested that transcriptional regulation of ARHGAP10 is the only mechanism by which SB promotes ferroptosis in ovarian cancer cells.