This article is mentioned in:

Introduction

In 2018, >1.27 million new cases and 35,800 deaths as a result of prostate cancer were recorded worldwide (1). Prostate cancer accounts for 13.5% of cancers diagnosed in men, which is only lower than lung cancer and is the fifth-leading cause of cancer-related mortality in men. Androgen deprivation therapy (ADT), the main treatment for locally advanced or metastatic androgen-dependent prostate cancer, can alleviate or stabilize symptoms in >80% of patients. However, most patients become non-responsive to this treatment and inevitably progress to castration-resistant prostate cancer (CRPC) following treatment for 12–18 months (2). The serum level of prostate-specific antigen (PSA), one of the most important target genes of androgen receptor (AR) and an important biomarker for the diagnosis and prognosis of prostate cancer, is elevated in patients with CRPC after ADT, suggesting that the AR signaling pathway is reactivated in CRPC cells (3–5). Two major mechanisms are involved in the reactivation of AR in CRPC, including AR upregulation and AR splice variant (AR-V) expression.

AR full-length (AR-FL) is a member of the steroid receptor subfamily that belongs to the nuclear receptor family and consists of four structural domains: Amino-terminal domain, DNA-binding domain, small hinge region and ligand-binding domain (LBD). Activation of the AR signaling pathway mainly depends on androgen, an AR ligand, in prostate cancer cells (5). After binding with its ligand, the AR protein is translocated from the cytoplasm to the nucleus. Two AR molecules are induced to homodimerize by the D-box in the nucleus, and the dimer binds to specific androgen response elements to promote target gene expression (6).

AR-Vs are named based on the majority that lack C-terminal LBD (7) and have constitutive transcriptional activity without androgen binding (3,8). Several drugs targeting AR reactivation, such as the FDA-approved abiraterone and enzalutamide (MDV3100), can prolong the overall survival of patients with CRPC. However, the majority of patients progress to drug-resistant disease partly because of AR-V expression, which is unaffected by these drugs; therefore, it is necessary to develop novel drugs targeting both AR-FL and AR-Vs to combat CRPC.

Erastin was first discovered to kill cancer cells overexpressing H-ras in engineered tumorigenic cells by synthetic lethal high-throughput screening of >20,000 compounds in 2003 (9). When erastin induces cell death, neither chromatin fragmentation nor caspase activation is detected, and the levels of intracellular reactive oxygen species (ROS) are upregulated and erastin-induced death can be prevented by iron chelation. In 2012, Dixon et al (10) proposed the term ferroptosis to describe this new death pattern. Erastin can induce ferroptosis via different mechanisms, such as directly targeting mitochondrial voltage-dependent anion channels (11), inhibiting the activity of the xCT light chain of the cystine/glutamate transporter (also known as system XC-) (12,13), playing a relevant role in the MAPK pathway (14) and increasing heme oxygenase-1 (15,16). Furthermore, Hasegawa et al (17) found that mucin 1 C-terminal subunit/xCT can downregulate erastin-induced ferroptosis in triple-negative breast cancer. As an inducer of ferroptosis, erastin has been demonstrated to inhibit cancer cell proliferation in acute myeloid leukemia, and hepatocellular, breast, ovarian, and head and neck cancer (16–18), although the mechanisms attributed to cell death differ among these cancers. Although a considerable number of studies have sought to determine whether erastin inhibits prostate cancer, it is necessary to investigate the specific mechanism of erastin, such as the activation of the AR signaling pathway in prostate cancer cells, specifically in CRPC.

Materials and methods

Prostate cancer cell lines and reagents

LNCaP, PC3, 22Rv1, C4-2, C4-2B, Du145 and 293T cells were obtained from the American Type Culture Collection. These cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium supplemented with 10% fetal bovine serum (FBS; Clark Bioscience) at 37°C in a humidified atmosphere with 5% CO2. LNCaP95 cells were provided by Dr Alan Meeker at the Johns Hopkins University (Baltimore, MD, USA) and were cultured in RPMI-1640 medium supplemented with 10% charcoal-stripped FBS (Gibco; Thermo Fisher Scientific, Inc.). Erastin, docetaxel (DTX), ferrostatin-1, liproxstatin-1, ZVAD-FMK, necrosulfonamide and chloroquine were purchased from Selleck Chemicals.

Sulforhodamine B (SRB) cell viability assay

After being treated with erastin at different concentrations (0, 2.5, 5.0, 10.0, 20.0 or 40.0 µM) for 24, 48 or 72 h, cell monolayers of 22Rv1 or LNCaP95 in 96-well plates were fixed using 20% (wt/vol) trichloroacetic acid (100 µl/well) at room temperature for at least 3 h, and then stained for 30 min using SRB (Shanghai YuanYe Biotechnology Co., Ltd.) at room temperature. Excess dye was removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution and then the OD was measured at 565 nm using a Multiscan Spectrum spectrophotometer. In order to obtain more samples in the subsequent experiments, the doses of erastin were determined based on the cell inhibition rate of 30%. Thus, 22Rv1 and LNCaP95 cells were treated with 10 or 20 µM erastin, while PC3 cells were treated with 1 µM erastin.

In order to verify whether the effect caused by erastin was ferroptosis, the cells were treated with erastin (20 µM). Meanwhile, the cells were added with or without ferroptosis inhibitors (1 µM ferrostatin-1 or 1 µM liproxstatin-1), apoptosis inhibitor (10 µM ZVAD-FMK), necroptosis inhibitor (1 µM necrosulfonamide), or autophagy inhibitor (25 µM chloroquine). After treatment for 48 h, the cell viability was detected by SRB as described above.

In order to calculate the combination index (CI) between DTX and erastin, 22Rv1 and LNCaP95 cells were treated with these two drugs at different concentrations (0, 2.5, 5.0 or 10.0 µM erastin and 0, 5, 10 nM DTX). After treatment for 48 h, cell viability was detected by SRB as described above.

Western blot analysis

Western blotting was conducted as previously described (19), and proteins were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences), following the manufacturer's protocols. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization of the densitometry signals. The following antibodies were used in this study: Anti-N-terminal AR antibody (cat. no. 5153; 1:1,000; Cell Signaling Technology, Inc.), anti-phospholipid hydroperoxide glutathione peroxidase (GPX4; cat. no. ab125066; 1:1,000; Abcam) and anti-GAPDH (cat. no. A00227; 1:1,000; Wuhan Boster Biological Technology, Ltd.). IRDye® 800CW goat anti-human IgG (H+L) (cat. no. 926-32232; 1:10,000; LI-COR Biosciences).

ROS assay

After treatment with erastin for 24 h, the cells were incubated with 1 µl DCFH-DA with 1 ml phosphate-buffered saline (PBS) for 1 h at 37°C in the dark to assess the cytosolic ROS levels. Samples were centrifuged at 860 × g at room temperature for 3 min, and the pellets were resuspended in 1 ml PBS. Measurements were performed on a FACSCalibur™ (BD Biosciences) flow cytometer using FlowJo software 7.6 (FlowJo LLC). All experimental results are reported as represented by three replicates.

Glutathione (GSH) and lipid peroxidation assays

The GSH and malondialdehyde (MDA) contents in cell lysates were assessed using GSH (cat. no. A061-1-2) and lipid peroxidation (cat. no. A003-4-1) assay kits, respectively, according to the manufacturer's instructions. Both kits were purchased from Nanjing Jiancheng Bioengineering Institute.

Dual-luciferase reporter gene assay

Three luciferase reporter plasmids were used in this research, and transfection was performed with TurboFect Transfection Reagent (cat. no. R0531; Thermo Fisher Scientific, Inc.). The androgen-responsive element-luciferase plasmid contains three repeat ARE regions ligated in tandem to a luciferase reporter (ARR3-Luc), which was provided by Dr Robert Matusik at Vanderbilt University School of Medicine (Nashville, TN, USA), and was used to reflect the AR-FL trans-activation activity. The ubiquitin-conjugating enzyme E2C-luciferase plasmid (UBE2C-luc) is driven by a minimal promoter and three repeats of an AR-V-specific promoter element. Thus, it was used to reflect AR-V7 trans-activation activity. pGL4-ARpro8.0 is driven by an 8.0 kb fragment of the 5′-flanking region of the human AR gene. The transfected cells, including 22Rv1, LNCaP95, PC3 and LNCaP cells, were divided equally into 24-well plates (1×105 cells/well) and cultivated in serum-free medium for 24 h. Subsequently, the cells were exposed to charcoal-stripped FBS with or without 1 nM R1881 (Sigma-Aldrich; Merck KGaA) at 37°C, which is a type of synthetic androgen, and erastin to detect AR-FL or AR-V7 activity. After 24 h, the cells were lysed with 100 µl reporter lysis buffer (Promega Corporation), and the luciferase activity was assayed using the Luciferase Assay System (Promega Corporation) and normalized based on the protein concentrations for each sample.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The total RNA of cells was extracted and collected using an E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Inc.) and quantified with a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription was performed with an RNA reverse transcription kit (Takara Bio, Inc.), and qPCR was performed using TransStart® Green qPCR SuperMix (TransGen Biotech Co., Ltd.), according to the manufacturer's instructions for 45 cycles of 5 sec at 94°C and 30 sec at 60°C. 36B4 served as the normalization control for these qPCR assays. The following primers were used: AR-FL sense, 5′-GTACAGCCAGTGTGTCCGAA-3′ and anti-sense, 5′-TTGGTGAGCTGGTAGAAGCG-3′; AR-V7 sense, 5′-AAAAGAGCCGCTGAAGGGAA-3′ and anti-sense, 5′-GCCAACCCGGAATTTTTCTCC-3′; PSA sense, 5′-CTCAGGCCAGGTGATGACTC-3′ and anti-sense, 5′-GTCCAGCACACAGCATGAAC-3′; transmembrane protease serine 2 (TMPRSS2) sense, 5′-ACACACCGATTCTCGTCCT-3′ and anti-sense, 5′-TGGCCTACTCTGGAAGTTCA-3′; UBE2C sense, 5′-TTCCCCAGTGGCTACCCTTA-3′ and anti-sense, 5′-CAGGGCAGACCACTTTTCCT-3′; transcription factor E2F7 (E2F7) sense, 5′-TTCTGTTGCTCAGACGGACC-3′ and anti-sense, 5′-ATCCCTCTCTGACCCTGACC-3′; and 36B4 sense, 5′-CGACCTGGAAGTCCAACTAC-3′ and anti-sense, 5′-ATCTGCTGCATCTGCTTG-3′. mRNA expression levels were quantified using the 2−ΔΔCq method (20).

Lentiviral packaging and lentivirus-infected cells

Three plasmids were used as lentiviral vectors: VSVG, ∆8.2, pLVX-AR-FL or pLVX-AR-V7. VSVG, ∆8.2, and oLVX were provided by the National Engineering Laboratory for AIDS Vaccine (Jilin University, Changchun, China). The preparation of the lentiviral particles and lentiviral infections were performed as previously described (21). When lentiviral particles (collected at 48 h after transfection) were used to infect 22Rv1 cells in 6-well plates (1×106 cells/well), polybrene was added at a final concentration of 6 µg/ml, fresh medium was replaced within 6 h, and the protein expression was determined by western blotting.

22Rv1 tumor xenograft model

A total number of 12 male BALB/c nude mice (age, 6–8 weeks old; weight, 15–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed at a constant temperature and constant humidity in a specific pathogen-free environment with free access to food and water. The present study was approved by the Animal Ethics Committee of Basic Medical College of Jilin University (approval no. 2016045). After 1 week of adaptation, 5×106 22Rv1 cells suspended in 50% Matrigel and PBS were injected subcutaneously into the right dorsal flank. Tumor formation was strictly monitored, and tumor volume was calculated by the modified ellipsoidal formula: Tumor volume = 0.52 × length × width2. When the tumor size reached ~50 mm3, the mice were randomly allocated into two groups (n=6/per group) and treated with or without erastin. The dose of erastin administered to the mice was 20 mg/kg, which was injected intraperitoneally twice every other day. After being treated for 2 weeks, all mice were injected with pentobarbital sodium at a dose of 20 mg/kg intraperitoneally. Then, 0.5 ml mouse blood was collected from the orbital vein to detect serum prostate-specific antigen (PSA) levels using a human KLK3 ELISA Kit (cat. no. KIT10771; Sino Biological, Inc.), tumors were removed for molecular analysis, and some organs were removed for a safety evaluation at the end of the experiment. Finally, all mice were sacrificed by intraperitoneal injection with pentobarbital sodium at a dose of 150 mg/kg. Erastin was dissolved in 3.3% dimethyl sulfoxide (DMSO) and 96.7% β-cyclodextrin (20%).

Histopathology assay

Hematoxylin and eosin (H&E) staining was performed as described in a previous study (22). Briefly, the tissues of the rats were fixed in 10% buffered formalin, and then decalcified in 10% ethylenediaminetetraacetic acid. The tissues were then treated with ethanol and xylene for dehydration. After being embedded in paraffin and sliced, several 4-µm thick histological slices were stained with H&E. The images were subsequently acquired using a light microscope (BX51; Olympus Corporation) at ×200 magnification.

Immunohistochemistry (IHC)

IHC staining was performed as described previously (23). Briefly, histological slices from tumor tissues were stained with anti-N-terminal AR antibody (1:200; cat. no. 5153; Cell Signaling Technology, Inc.). Histological images were captured by a microscope (BX51; Olympus Corporation) with an objective magnification of ×200.

Statistical analysis

The experiments were repeated three times independently. Statistical analysis was performed for multiple comparisons using one factor analysis of variance (ANOVA) followed by Dunnett's or Bonferroni's post hoc tests. All data were analyzed with the statistical software SPSS 11.0 (SPSS, Inc.), and the results are expressed as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference. SPSS software was also used to calculate the CI, a CI value of >1, 1 and <1 denotes antagonism, additivity and synergism, respectively.

Results

Erastin inhibits the growth of CRPC cells

Western blotting was employed to detect the expression of AR, AR-Vs and GPX4 in several prostate cancer cell lines including LNCaP95, LNCaP, Du145, C4-2B, 22Rv1, C4-2 and PC3 cells. The results showed that both 22Rv1 and LNCaP95 cell lines expressed AR, AR-Vs and GPX4 (Fig. 1A). Conversely, PC3 cells did not express AR or AR-Vs and only showed a low expression of GPX4. Thus, 22Rv1, LNCaP95 and PC3 cell lines were chosen for the subsequent experiments.

Figure 1. Erastin inhibits the proliferation of castration-resistant prostate cancer cells. (A) The protein expression of AR, AR-Vs and GPX4 in prostate cancer cell lines. (B and C) Sulforhodamine B assays showed that erastin inhibited the proliferation of 22Rv1 and LNCaP95 cells in a dose-dependent manner. (D-F) The proportion of sub-G1 cells was increased in 22Rv1 and LNCaP95 cells after treatment with erastin for 48 h. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. AR, androgen receptor; AR-V, AR splice variant; GPX4, phospholipid hydroperoxide glutathione peroxidase.

The effect of erastin on the proliferation of 22Rv1 and LNCaP95 cells was detected by the SRB assay. As presented in Fig. 1B and C, erastin inhibited the proliferation of these two CRPC cell lines in a dose-dependent manner. Then, PI flow cytometry was employed to examine the effect of erastin on the cell cycle. The results showed that erastin could increase the proportion of cells in sub-G1 phase and cause cycle arrest (Fig. 1D-F).

Erastin induces ferroptosis in CRPC cells

To verify that the effect caused by erastin is ferroptosis, the cells were treated with erastin with or without ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1), apoptosis inhibitor (ZVAD-FMK), necroptosis inhibitor (necrosulfonamide) and autophagy inhibitor (chloroquine). The SRB results showed that in both cell lines, ZVAD-FMK, necrosulfonamide and chloroquine had no effect on erastin-induced cell death (Fig. 2A and B). The death of the 22Rv1 cells induced by erastin was only reversed by ferrostatin-1 and liproxstatin-1.

Figure 2. Erastin induces ferroptosis in castration-resistant prostate cancer cells. (A and B) 22Rv1 and LNCaP95 cells were treated with 20 µM erastin with or without cell death inhibitor (1 µM ferrostatin-1, 1 µM liproxstatin-1, 10 µM ZVAD-FMK, 1 µM necrosulfonamide or 25 µM chloroquine) for 48 h. (C and D) The expression of GPX4 protein in 22Rv1 and LNCaP95 cells after treatment with erastin for 48 h. (E and F) ROS levels were assayed in the 22Rv1 and LNCaP95 cells treated with 10 or 20 µM erastin for 24 h. (G and H) GSH levels were assayed in the 22Rv1 and LNCaP95 cells treated with 10 or 20 µM erastin for 48 h. (I and J) MDA levels were assayed in the 22Rv1 and LNCaP95 cells treated with 10 and 20 µM erastin for 48 h. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. GPX4, phospholipid hydroperoxide glutathione peroxidase; ROS, reactive oxygen species; GSH, glutathione; MDA, malondialdehyde.

Then, the expression of the ferroptosis marker protein GPX4 was measured. The results showed that erastin downregulated the protein expression of GPX4 in both cell lines (Fig. 2C and D). Given that ferroptosis is characterized by lipid peroxidation, the level of GSH, a key regulator that maintains cellular redox homeostasis, was investigated. The results showed that ROS levels in both cell lines were increased with erastin treatment compared with the control group (Fig. 2E and F). Moreover, GSH levels were downregulated after erastin treatment in these two cell lines (Fig. 2G and H). MDA, an end product of lipid peroxidation, was notably increased following erastin treatment, as expected (Fig. 2I and J). According to the aforementioned results, it was hypothesized that ferroptosis was initiated in these two cell lines after erastin treatment.

Erastin downregulates AR protein expression by inhibiting the transcription of the AR gene

Given that AR-FL and AR-Vs play important roles in the development of CRPC, 22Rv1 and LNCaP95 CRPC cells that express AR-FL and AR-Vs were chosen in the present study to explore whether erastin can inhibit AR expression. In 22Rv1 cells, AR-Vs include AR-V7 (also called AR3), AR-V1 (also called AR4) and AR-V4 (also called AR5) (8,24), whereas LNCaP95 cells express only AR-V7 (25). Among these splice variants, AR-V7 has been found to be associated with the development of CRPC and is recognized as a biomarker of poor prognosis for patients with CRPC (26); thus, AR-V7 was selected as the representative of AR-Vs in the present study. The expression of AR-FL and AR-V proteins was detected by western blotting, and the results showed that erastin downregulated both AR-FL and AR-V protein expression levels (Fig. 3A and B).

Figure 3. Erastin downregulates AR protein expression by inhibiting the transcription of the AR gene. Erastin downregulated AR-FL and AR-V protein levels in (A) 22Rv1 and (B) LNCaP95 cells treated with 10 and 20 µM erastin for 48 h. Reverse transcription-quantitative PCR analysis showed that erastin at 10 and 20 µM decreased AR-FL and AR-V mRNA levels in (C) 22Rv1 and (D) LNCaP95 cells. Luciferase assays showed that erastin inhibited the activity of the 8.0 kb proximal AR promoter in (E) 22Rv1 and (F) LNCaP95 cells. Cells transfected with the pGL4-ARpro8.0 construct were treated with 10 or 20 µM erastin for 24 h. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. AR, androgen receptor; AR-V, AR splice variant; AR-FL, AR full-length.

To investigate how erastin decreases the protein levels of AR-FL and AR-V, AR-FL and AR-V7 mRNA expression levels were measured by RT-qPCR. As expected, erastin significantly reduced the levels of AR-FL and AR-V7 mRNA (Fig. 3C and D). Then, AR promoter activity was detected using dual-luciferase reporter plasmid pGL4-ARpro8.0. Erastin treatment resulted in the significant inhibition of AR promoter activity in both cell lines (Fig. 3E and F). Taken together, the data indicated that erastin had the ability to inhibit the transcription of the AR gene in CRPC cells.

Erastin downregulates AR-FL and AR-V transactivation

To evaluate the effects of erastin on AR transactivation, AR-FL and AR-V transcriptional activity and their target genes were measured by a reporter gene assay and RT-qPCR, respectively. First, 22Rv1 cells were transfected with the ARR3-luc luciferase construct, which contained three tandem repeats of androgen response elements. The results showed that erastin treatment induced a reduction in luciferase activity in 22Rv1 cells (Fig. 4A). Considering that the ARR3-luc construct can be regulated not only by AR-FL, but also by AR-Vs, the LNCaP cell line was selected for transfection with a ARR3-luc luciferase construct because these cells only express AR-FL. The results in LNCaP cells clearly showed that erastin inhibited AR-FL trans-activating activity (Fig. 4B). To specifically assess the effect of erastin on AR-V transcriptional activity, 22Rv1 and LNCaP95 cells were transfected with the UBE2C-luc construct in which the luciferase gene is driven by an AR-V-specific promoter element of the UBE2C gene (27). Most of the AR-Vs identified to date display constitutive activity, when AR-V transcriptional activity was measured (28); therefore, these cells were cultured with 10% charcoal-stripped FBS. As shown in Fig. 4C and D, erastin caused the effective inhibition of AR-V trans-activating activity.

Figure 4. Erastin downregulates AR-FL and AR-V transactivation. A luciferase assay showed that erastin inhibited endogenous (A and B) AR-FL and (C and D) AR-V transcriptional activity. Cells transfected with the ARR3-luc or UBE2C-luc construct were treated with erastin. (E) Sulforhodamine B assays showed that erastin inhibited the growth of PC3 cells in a dose-dependent manner. Then, erastin inhibited exogenous (F) AR-FL and (G and H) AR-V transcriptional activity in PC3 and LNCaP cells. Reverse transcription-quantitative PCR analysis showed that erastin decreased the levels of (I and K) AR-FL target genes PSA and TMPRSS2 and (J and L) AR-V target genes UBE2C and E2F7. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. AR, androgen receptor; AR-V, AR splice variant; AR-FL, AR full-length; PSA, prostate-specific antigen; TMPRSS2, transmembrane protease serine 2; UBE2C, ubiquitin-conjugating enzyme E2C; E2F7, transcription factor E2F7; luc, luciferase plasmid; ARR3, arrestin-C.

Considering that erastin had a significant inhibitory effect on endogenous AR-FL and AR-V transcriptional activity, it was speculated that erastin had an inhibitory effect on exogenous AR activity. To test this hypothesis, the effect of erastin on exogenously expressed AR-FL and AR-V7 trans-activating activity was evaluated in the PC-3 cell line (null-AR). The results showed that PC3 cells were significantly inhibited and the exogenous AR-FL and AR-V trans-activating activity was evidently decreased after the treatment of 1 µM erastin (Fig. 4E-G). The exogenous AR-V7 trans-activating activity was further tested in LNCaP cells (without AR-Vs), and the results were consistent with those of the PC3 cells (Fig. 4H).

Consistently, the mRNA levels of the AR-FL target genes PSA and TMPRSS2 and the AR-V-specific target genes UBE2C and E2F7 were significantly downregulated by erastin in both the 22Rv1 and LNCaP95 cell lines (Fig. 4I-L). Collectively, these findings indicated that erastin could downregulate AR-FL and AR-V trans-activation.

Upregulation of AR-FL and AR-V7 expression reverses the growth inhibition of erastin in 22Rv1 cells

To further test the importance of the roles of AR-FL and AR-V in the action of erastin in CRPC cells, AR-FL and AR-V7 were overexpressed in the 22Rv1 cell line using lentiviral infections that delivered AR-FL and AR-V7 RNA expression constructs, and growth inhibition was evaluated using the SRB method. As shown in Fig. 5A and B, the expression of AR-FL and AR-V7 proteins in the 22Rv1 cells was significantly upregulated, especially the AR-FL protein. The overexpression of AR-FL and AR-V7 significantly promoted the resistance to high concentrations of erastin after 48 and 72 h compared with controls in the 22Rv1 cells. However, there was no significant difference between the overexpression of AR-FL and AR-V7 protein (Fig. 5C and D).

Figure 5. Attenuation of erastin growth inhibition by overexpression of AR-FL or AR-V7 in 22Rv1 cells. (A and B) 22Rv1 cells were infected with a construct expressing AR-FL or AR-V7, and western blotting revealed successful infection. assay was used to detect growth inhibition in cells (C) 48 and (D) 72 h after treatment with 0–40 µM erastin. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. AR, androgen receptor; AR-V, AR splice variant; AR-FL, AR full-length.

Erastin inhibits the 22Rv1 ×enograft tumor growth rate

To investigate whether erastin inhibits the tumor growth in vivo, 22Rv1 cells were implanted into the subcutaneous space of immune-deficient nude mice. The tumor growth curve results are shown in Fig. 6A and revealed that erastin inhibited the growth of 22Rv1 tumors, with significance differences found on day 7 of the treatment. At the end of the experiments, the average tumor weight in the control group was 0.80±0.11 g, while that in the erastin-treated group was 0.55±0.17 g (Fig. 6B-D). The results of H&E staining of the tumor tissues showed that the nuclear/cytoplasmic ratio of tumor cells was notably decreased in the erastin group (Fig. 6E). Given that serum PSA concentration is one of most important clinical indexes for the diagnosis of prostate cancer, serum PSA levels were measured by ELISA, and a significant reduction in PSA serum levels in response to erastin treatment was observed (Fig. 6F). Then, AR protein levels were measured using IHC in tumor tissues. In erastin-treated tumor specimens, AR was obviously downregulated (Fig. 6G). In addition, the protein and mRNA levels of AR-FL and AR-V in the tumor tissues were measured by western blotting and RT-qPCR. Both the protein levels and mRNA levels of AR-FL and AR-V7 in the tumor tissues were decreased (Fig. 6H and I), which was consistent with the results obtained in vitro.

Figure 6. In vivo efficacy of erastin in the 22Rv1 ×enograft model. (A) Mean tumor volumes (n=6). The (B) general condition of the mice and (C) representative tumor images. (D) Mean tumor weights. (E) Histopathological examination of 22Rv1 tumors by H&E staining (mmagnification, ×200). (F) In vivo effect of erastin on serum PSA levels. (G) Representative immunohistochemistry images of AR in tumor tissues. (H) AR-FL and AR-V7 protein levels in the 22Rv1 ×enograft model. (I) AR-FL and AR-V7 mRNA levels in the 22Rv1 ×enograft model. (J) Mean body weights of the mice in each group. (K) H&E staining was used to evaluate the effect of erastin on the heart, liver, spleen and kidney of the mice (magnification, ×200). Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test. *P<0.05 and **P<0.01 vs. control. AR, androgen receptor; PSA, prostate-specific antigen; H&E, hematoxylin and eosin; AR-V, AR splice variant; AR-FL, AR full-length.

Evaluation of erastin safety in mice

To evaluate the toxicity of erastin in vivo, the body weights of mice were measured and mouse organs were collected, including the heart, liver, spleen and kidney, to observe changes in morphology by H&E staining. The mice appeared to tolerate erastin well, and neither a significant difference in body weight (Fig. 6J) nor noticeable organ damage (Fig. 6K) was detected between the treatment group and the control group.

Erastin synergistically enhances the growth inhibitory efficacy of docetaxel

Docetaxel, the standard therapy for CRPC, represents the only class of chemotherapy drugs that prolongs the survival of patients with CRPC. Previous studies have reported that docetaxel induces microtubule stabilization and abrogates AR nuclear translocation and transcriptional activity (29–31). However, microtubule stabilization has been found to have no effect on AR-Vs, especially AR-V7, with neither subcellular localization nor nuclear activity affected, indicating one of the mechanisms of docetaxel resistance (29,32). Given that erastin has the ability to downregulate AR-V expression and activity and that the underlying mechanism by which docetaxel inhibits AR-FL is different from that of erastin, it was hypothesized in the present study that erastin can enhance the efficacy of docetaxel in CRPC. To test this hypothesis, the growth of 22Rv1 and LNCaP95 cells was measured after treatment with erastin and docetaxel, and the combined index values were calculated. All the combinations produced a CI value <1, suggesting synergy between erastin and docetaxel in inhibiting cell growth (Tables I and II). When 5 µM erastin and 10 nM docetaxel were added to 22Rv1 and LNCaP95 cells alone or in combination, as shown in Fig. 7A and B, combination therapy inhibited tumor cell growth to a significantly greater extent than monotherapy. These data provided preliminary support for using erastin to enhance docetaxel efficacy in CRPC.

Figure 7. Erastin enhances docetaxel efficacy in prostate cancer cells. (A) 22Rv1 and (B) LNCaP95 cells were treated with 5 µM erastin with or without 10 nM DTX for 48 h, and cell growth was assessed using a sulforhodamine B assay. Statistical analysis was performed using ANOVA followed by Bonferroni's post hoc test. *P<0.05 vs. control; #P<0.05 vs. single-agent treatment groups. DTX, docetaxel.

Table I. Combination index values of DTX and erastin treatment in 22Rv1 cells.

Table I.

Combination index values of DTX and erastin treatment in 22Rv1 cells.

	Erastin, µM
DTX, nM	2.5	5	10
5	0.313	0.717	0.989
10	0.550	0.615	0.252
20	0.480	0.706	0.821

Table II. Combination index values of DTX and erastin treatment in LNCaP95 cells.

Table II.

Combination index values of DTX and erastin treatment in LNCaP95 cells.

	Erastin, µM
DTX, nM	2.5	5	10
5	0.765	0.837	0.961
10	0.545	0.617	0.868

Discussion

As the first ferroptosis inducer discovered, erastin has been found to have a significant antitumor effect in multiple types of tumors through different mechanisms (33). However, there has been relatively little research on elastin in prostate cancer. In the present study, erastin inhibited the proliferation of 22RV1 and LNCaP95 cells in a dose-dependent manner. Although the mechanism of erastin-induced ferroptosis in prostate cancer remains unknown, the ferroptosis marker GPX4 protein was downregulated in both cell lines after treatment with erastin, indicating the inherent ferroptosis. In addition, it was confirmed that erastin can inhibit the expression of AR-FL and AR-V proteins by reducing the transcriptional activity of AR-FL and AR-Vs and downregulating the transcription level of the AR gene. In addition, in vivo experiments confirmed that erastin inhibited the growth rate of tumors and downregulated the levels of AR protein and mRNA in tumors. There was no evident damage induced by erastin, as the weight of the body and organs, such as the heart, liver, spleen and kidney, was unaffected in the treated in mice.

While, previous studies have confirmed that erastin exerts an antitumor effect in multiple types of cancer, such as colorectal, breast and cervical cancer (33–35), the results of the present study revealed a novel underlying mechanism in prostate cancer in which erastin reduces AR-FL and AR-V protein expression by downregulating their mRNA levels. The increased expression of the full-length and splice variants of AR has been indicated as an important mechanism of resistance to traditional androgen deprivation therapy and newly developed androgen deprivation drugs, such as abiraterone and enzalutamide (5,36,37). However, none of the anti-androgens currently used in clinics can target AR-Vs directly to reduce their availability. In addition to erastin, the selective AR degradants UT-69, UT-155 and (R)-UT-155 bind to the AR transcriptional activation domain AF-1 in the amino terminus, and UT-69 and UT-155 can also bind to the carboxy terminal LBD, significantly reducing the activity of wild-type and splice mutants even in the presence of small amounts of AR (38). ASC-J9, an AR degradation enhancer, can degrade both AR-FL and AR-V7 in 22Rv1 cells and in C4-2 and C81 cells upon the addition of AR-V7 (39). These compounds may serve as effective antidotes for overcoming resistance to androgen deprivation therapy for the treatment of CRPC in the future.

Erastin can also enhance the efficacy of docetaxel chemotherapy in prostate cancer (Fig. 7A and B). It is important for AR-FL to translocate from the cytoplasm to the nucleus to form dimers, which have transcriptional activity (29,30,40,41). Docetaxel has been reported to attenuate the nuclear input of AR-FL by stabilizing microtubules, which play important roles in the process of AR translocation (29). However, the nuclear localization of AR-Vs, especially AR-V7, is not dependent on microtubules, and AR-V expression is proposed to be one of the mechanisms of docetaxel resistance. The results of the present study showed that erastin enhanced the growth inhibitory effect of docetaxel in CRPC cells, thus it is necessary to determine the optimal ratio for the two drugs in combination therapy in future studies.

One of the chemotherapeutic mechanisms of docetaxel is the promotion of the mitochondrial release of cytochrome C, interrupting the mitochondrial electron transport chain, and leading to the production of a large amount of ROS, which cause lipid peroxidation, DNA oxidation modification, protein oxidation and inactivation of various enzymes, ultimately causing apoptosis and necrosis (42,43). However, when CRPC cells become resistant to docetaxel, the levels of ROS in cells are quite low (44,45). In the process of erastin-induced ferroptosis, the xCT light chain of the cystine/glutamate transporter is blocked, thus depleting GSH and reducing GPX4 activity, and as a result ROS cannot be catalyzed by GPX4. Ultimately, ROS accumulate, which can reverse the decrease in ROS levels caused by the docetaxel resistance of CRPC cells.

In summary, the present study demonstrated that erastin can significantly downregulate the expression and activities of AR-FL and AR-Vs in prostate cancer in vitro and in vivo, and enhance the growth inhibitory efficacy of docetaxel in CRPC cells. These results indicated that erastin may be a promising therapeutic strategy for the treatment of human prostate cancer in the future, although further studies will be needed.

Acknowledgements

We would like to thank Dr Alan Meeker at the Johns Hopkins University (Baltimore, Maryland) for providing LNCaP95 cells, and to Dr Robert Matusik at Vanderbilt School of Medicine (Nashville, Tennessee) for providing the ARR3-luc construct.

Funding

This work was supported by the National Natural Science Foundation of China Project (grant nos. 81302206 and 81602228), Jilin Scientific and Technological Development Program (grant no. 20160101237JC), and Project of Jilin Provincial Department of Education (grant no. JJKH20170831KJ).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LZ, YY and TL designed and conducted the study. CH, HX, WeL, JC and SW performed the experiments. YY, TL and YJ performed the data analysis and interpretation. YX and WaL were responsible for data collection, validation and visualization. YY and TL drafted the manuscript with critical revision from LZ. LZ and YY confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Animal Ethics Committee of Basic Medical College of Jilin University (approval no. 2016045).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References