text:
Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate
Journal of Translational Medicine volume 21, Article number: 575 (2023)
Benign prostatic hyperplasia (BPH) is a common disease in elderly men, mainly resulted from an imbalance between cell proliferation and death. Glutathione peroxidase 3 (GPX3) was one of the differentially expressed genes in BPH identified by transcriptome sequencing of 5 hyperplastic and 3 normal prostate specimens, which had not been elucidated in the prostate. This study aimed to ascertain the mechanism of GPX3 involved in cell proliferation, apoptosis, autophagy and ferroptosis in BPH.
Human prostate tissues, GPX3 silencing and overexpression prostate cell (BPH-1 and WPMY-1) models and testosterone-induced rat BPH (T-BPH) model were utilized. The qRT-PCR, CCK8 assay, flow cytometry, Western blotting, immunofluorescence, hematoxylin and eosin, masson’s trichrome, immunohistochemical staining and transmission electron microscopy analysis were performed during in vivo and in vitro experiments.
Our study indicated that GPX3 was localized both in the stroma and epithelium of prostate, and down-regulated in BPH samples. Overexpression of GPX3 inhibited AMPK and activated ERK1/2 pathway, thereby inducing mitochondria-dependent apoptosis and G0/G1 phase arrest, which could be significantly reversed by MEK1/2 inhibitor U0126 preconditioning. Moreover, overexpression of GPX3 further exerted anti-autophagy by inhibiting AMPK/m-TOR and up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4, mitochondrial GPX4 and cytoplasmic GPX4) to antagonize autophagy-related ferroptosis. Consistently, GPX3 deficiency generated opposite changes in both cell lines. Finally, T-BPH rat model was treated with GPX3 indirect agonist troglitazone (TRO) or GPX4 inhibitor RAS-selective lethal 3 (RSL3) or TRO plus RSL3. These treatments produced significant atrophy of the prostate and related molecular changes were similar to our in vitro observations.
Our novel data manifested that GPX3, which was capable of inducing apoptosis via AMPK/ERK1/2 pathway and antagonizing autophagy-related ferroptosis through AMPK/m-TOR signalling, was a promising therapeutic target for BPH in the future.
Benign prostatic hyperplasia (BPH) is the general non-malignant disease in elderly men and its prevalence rises with age, up to 50% in men over 50 years old . Lower urinary tract symptoms (LUTS), often secondary to BPH, are worrisome complication that place significant encumbrance on patients’ quality of life . The increase in the number of periurethral epithelial and stromal cells characterizes BPH histopathologically due to the maladjustment of cell proliferation and death . Although the molecular mechanism of BPH have been extensively studied, including a disequilibrium of sex hormone , stromal-epithelial interactions , cytokins , autoimmune  and oxidative stress (OS) , the exact pathogenesis remains unclear. In recent years, with the continuous progress of experimental technology, from PCR to a new generation of high-throughput sequencing to multi-omics, more and more molecular mechanisms related to BPH will be elucidated. Among them, the identification of differentially expressed genes (DEGs) through transcriptome sequencing (RNA-seq) to explore the molecular mechanism, the diagnostic markers and therapeutic targets have become the research directions of many diseases. So did with BPH. Indeed, we performed RNA-seq of 5 hyperplastic and 3 normal prostate specimens and found that there was a number of DEGs between hyperplastic prostate and normal prostate, of which the up-expressed matrix-remodeling associated 5 (MXRA5) , C-X-C motif chemokine ligand 13 (CXCL13) , bone morphogenetic protein 5 (BMP5)  and neural epidermal growth factor-like like 2 (NELL2) , and the down-expressed glutathione peroxidase 3 (GPX3) ranked at the top and most of them have been reported. However, the role and function of GPX3 gene in the development of BPH and its potential molecular mechanisms have not been elucidated.
The glutathione peroxidase family is composed of eight isozymes, and selenocysteine is present in five of them (GPX1–4, GPX6) . GPX3 encodes a 24 kDa 1homotetramer protein spanning five exons on chromosome 5q32 [14, 15]. In addition, plasma GPX3 is the dominant extracellular GPX isoform, and the kidneys are the major tissues that express it . Functionally, GPX3 exerts its antioxidant effect through the reduction of H2O2 and hydroperoxides by consuming glutathione (GSH) . And it has been observed involved a number of diseases, including cancers of the gastric , ovarian  and prostate , where it showed suppressive effect.
Mitochondria are not only the energy metabolism center of eukaryotic cells, but also the main source of intracellular reactive oxygen species (ROS) . Also, mitochondria regulates the type of cell death (e.g. apoptosis, autophagy and ferroptosis) through a variety of mechanisms [22, 23]. For example, the release of mitochondrial cytochrome c (Cyto-C) could initiate endogenous apoptosis with concomitant depolarization of the mitochondrial membrane potential (MMP) . However, the role of GPX3 related redox metabolism, especially its association with mitochondria on cell proliferation and cell death in BPH has not been well illustrated.
Ferroptosis was first reported in 2012 . And it’s a form of programmed cell death (PCD) that occurs as a result of iron-dependent oxidative reactions different in both biochemistry and morphology from others (apoptosis, necrosis and autophagy) . During ferroptosis, the lipids in the cyto-membranes undergo superfluous oxidative damage . This process is often accompanied by up-regulation of intracellular labile iron, transferrin and malondialdehyde (MDA), and down-regulation of GSH and anti-ferroptosis systems . It has been shown that the development of BPH is accompanied by abnormally elevated OS and ROS surplus , which can cause cellular dysfunction and tissue damage . There have been many studies on ferroptosis in several of tumors including prostate cancer . But whether and how ferroptosis affects BPH has not been reported.
Autophagy is a natural, regulated PCD behavior. The mechanism is through a variety of autophagy-related proteins (including Beclin1 and LC3B, which are the initial protein and structural components of autophagosomes, respectively) to form double membrane vesicles to engulf damaged proteins or organelles and bind to lysosomes . Functionally, autophagy exists in the life process as a survival mechanism of stress response . Although autophagy and ferroptosis are mechanistically and morphologically distinct cell death pathways, a growing number of recent studies have reported significant interactions between them [32,33,34]. In particular, elucidating the relationship between autophagy and ferroptosis in BPH, which has traditionally used apoptosis as the main PCD mode, will provide a deeper understanding of the regulation of cell death in BPH.
In our current study, we analysed the expression of GPX3 and PCD patterns (apoptosis, autophagy and ferroptosis) in human normal and hyperplastic prostates. Furthermore, we established GPX3 silencing and up-regulation prostate cell models to investigate the effects and mechanisms of GPX3 on cell proliferation, apoptosis, autophagy and ferroptosis level of prostate cells in vitro. Finally, we validated the role of GPX3 in vivo via testosterone-BPH (T-BPH) rat model.
Cell lines obtained and cultured, CCK-8 assay, total RNA extraction, RNA reverse transcription, qRT-PCR analysis, Western blot analysis, hematoxylin and eosin (H&E) staining and masson’s trichrome staining analysis were performed as we previously described [35, 36]. Primer sequences were summarized in Additional file 1: Table S1. The primary and secondary antibodies were summarized in Additional file 2: Table S2 and Additional file 3: Table S3. All experiments were repeated at least 3 times.
Cell immunofluorescence staining
For cell immunofluorescence microscopy, cells were seeded on 12 mm coverslips and washing by ice-cold (PBS, pH = 7.4). The coverslips were then fixed with 4% paraformaldehyde (PFA) for 30 min, followed by 0.1% Triton X-100 incubation, and then blocked in goat serum for 30 min at room temperature. Afterward, they were incubated with primary antibody (listed in Additional file 2: Table S2) at room temperature for 2 h, washed with PBS, and incubated with Cy3- or FITC-labeled secondary antibody (listed in Additional file 3: Table S3) for 1 h. Nuclei were labeled with DAPI (4ʹ,6-diamidino-2-phenylindole) (2 μg/ml). Visualization was done with a laser scanning confocal microscope (Olympus, Tokyo, Japan).
Tissue immunofluorescence staining
Human prostate tissues were sectioned in 10 μm-thick slices and thawed, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in PBS and incubated for 2 h in a mixture of PBS supplemented with 0.2% Triton X-100 and 0.1% bovine serum albumin, followed by incubation overnight with the primary antibodies (listed in Additional file 2: Table S2). The secondary antibodies (listed in Additional file 3: Table S3) employed to visualize the localization of primary antibodies were Cy3-conjugated goat anti-rabbit IgG (1:1000). DAPI was used for staining the nucleus. Visualization was done with a Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).
Human and rat prostate tissues fixed in 10% neutral buffered formalin for 48 h were routinely processed for paraffin embedding. Samples were sectioned at 5 μm and deparaffinized in xylene followed by descending grades of ethanol (100, 95, 70, 30%). Antigen retrieval was performed in 10 mM sodium citrate buffer at pH 6.0, heated to 96 °C, for 30 min, followed by proteinase K treatment for 10 min. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS for 15 min. Blocking was performed by incubating sections in 5% normal donkey serum with 2% BSA for 1 h. The sections were stained by routine immunohistochemical (IHC) methods, using horse radish peroxidase polymer conjugate (Invitrogen), to localize the antibody bound to antigen, with diaminobenzidine as the final chromogen. All immunostained sections were lightly counterstained with Hematoxylin. The primary antibodies (information listed in Additional file 2: Table S2) to target proteins were incubated for 1 h at room temperature. Slides were evaluated for immunostaining by light microscopy. All specimens were stained with IHC. For each field, integrated optical density (IOD) was calculated using Image-J software. The mean density was calculated by IOD/area and the average values were used for target protein expression quantitative analyses.
Public data acquiring
The gene expression profiling dataset GSE119195 was obtained from GEO (https://www.ncbi.nlm.nih.gov/geo/) of the NCBI and the mRNA expression profiles of GPX3 between human hyperplastic prostate and normal prostate specimens were analyzed.
Animals and tissues
In total, 40 male Sprague–Dawley rats (6 weeks old) were randomly allocated into five groups (n = 8 per group): control group, corn oil (MedChemExpress, China) injection [subcutaneously (s.c.)] + dimethyl sulfoxide (DMSO) (MedChemExpress, China) [intraperitoneally (i.p.)] + DMSO [intragastrically (i.g.)]; T group, T (testosterone propionate, Sigma-Aldrich, St. Louis, MO) (2 mg/day)/corn oil injection (s.c.) + DMSO (i.p.) + DMSO (i.g.); T + RSL3 group, T injection (s.c.) + RSL3 (RAS-selective lethal 3, induces ferroptosis by inhibiting GPX4, MedChemExpress, China) (10 mg/kg/day, i.p.) + DMSO (i.g.); T + TRO group, T injection (s.c.) + DMSO (i.p.) + TRO [troglitazone is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) as the GPX3 agonist, MedChemExpress, China] (15 mg/kg/day, i.g.); T + RSL3 + TRO group, T (s.c.) + RSL3 (i.p.) + TRO (i.g.). On day 28, ventral prostates and seminal vesicles were harvested and weighed. All surgical procedures were performed under anesthesia by i.p. injection of pentobarbital sodium (35 mg/kg; Abbott Laboratory, Chicago, IL). All animal protocols were approved by the Medical Ethics Committee for Experiments at Zhongnan Hospital of Wuhan University before the experiments were conducted.
Four prostate samples from young brain-dead men (mean age 25.2 ± 4.4 years old) undergoing organ donation were obtained as normal ones. Eight specimens of hyperplastic prostate (mean age 70.0 ± 7.5 years old) were came from the patients who underwent transurethral resection prostate. Postoperative prostate pathology examination revealed BPH. Human samples were collected after approval of Hospital Committee for Investigation in Humans and after receiving written informed consent from each patient or relative. All human studies were conducted in accordance with the principles of the Declaration of Helsinki.
Knockdown and overexpression of GPX3 in human prostate cells
GPX3-target specific small interfering RNA (siRNA) was synthesized by Genepharma Ltd. in Suzhou, China. When cells were 30–50% confluent in 6-well culture plates, the medium was replaced with fresh one 30 min before transfection. Following the instructions of Genepharma Ltd. in Suzhou, China, the transfection media were prepared and incubated for 10 min. Subsequently, we added 200 μL of lipofectamine (Genepharma Ltd. in Suzhou, China) complex solution to each well and then incubated for 6 h. After that the medium was replaced with fresh medium and incubated for 48–72 h. The sense sequences of si-GPX3 are listed in Additional file 4: Table S4.
GPX3 complementary DNA (cDNA) were PCR amplified from a cDNA library of human cell line and then a homologous recombination vector was constructed by cloning it into an empty FlagpcDNA3 vector. When cells were 40–60% confluent in 6-well culture plates, the medium was replaced with fresh one 30 min before transfection. Following the instructions of Genepharma Ltd. in Suzhou, China, the transfection media were prepared and incubated for 10 min. Subsequently, we added 200 μL of lipofectamine (Genepharma Ltd. in Suzhou, China) complex solution to each well and then incubated for 6 h. After that the medium was replaced with fresh medium and incubated for 48–72 h.
In order to deactivate ERK1/2, prostate cells were pretreated with 10 μM MEK1/2 inhibitor U0126 (MedChemExpress, China) for 24 h prior to plasmid transfection. And, prostate cells were pretreated with RSL3 (MedChemExpress, China) at 0.25 μM, 0.5 μM, 1 μM, 2 μM or 4 μM for 24 h to activate ferroptosis, isosilybin B (MedChemExpress, China) at 20 μM, 40 μM, 60 μM, 80 μM or 100 μM for 24 h to activate apoptosis, Rapamycin (Rapa, MedChemExpress, China) at 0.5 nM, 1 nM, 2 nM, 4 nM or 8 nM for 24 h to activate autophagy, Chloroquine (CQ, MedChemExpress, China) at 10 μM, 20 μM, 30 μM, 40 μM or 50 μM for 24 h to inhibit autophagy, and GSK621 (MedChemExpress, China) at 5 μM, 10 μM, 20 μM, 30 μM or 40 μM for 24 h to activate AMPK. Meanwhile, equivalent amount of DMSO without drugs were added to the cells served as a control.
Flow cytometry analysis
1 × 106 cells were collected for cell cycle analysis. After centrifugation, cells were resuspended in the dark in PBS containing 50 µg/mL propidium iodide (Sigma-Aldrich, USA) and 0.1 mg/mL RNaseA (20 µg/mL PBS). Detection after 30 min incubation at 37 °C. Cells were collected 48 h after transfection for analysis of apoptosis, stained with FITC Annexin V Apoptosis Detection Kit I ( BD biosciences, USA), and analyzed by flow cytometry. Intracellular ROS levels were assessed using a fluorescent probe 2ʹ, 7ʹ -dichlorofluorescein diacetate (DCFH-DA Sigma-Aldrich, USA). After transfection and growth for 48 h, 10 μM DCFH-DA was added into 1 mL medium and incubated at 37 °C for 30 min. To detect MMP, cells were collected and stained with JC-1 (10 μg/mL, Invitrogen, Rockford, IL, USA) at 37 °C for 10–20 min.
Isolation of cytosolic and mitochondrial fractions
A total of 2 × 107 cells were collected and then mitochondrial separation agent (Beyotime Biotechnology Co., Ltd. in Wuhan, China) was added. The cell suspension was centrifuged at 700 g for 10 min at 4 °C, then the supernatant was transferred to a new 2 mL tube and centrifuged at 12,000 g for 15 min at 4 °C. The supernatant (cytoplasmic portion) was transferred to a new tube, and the granules contained isolated mitochondria.
Measurement of intracellular iron, GSH and MDA
For the determination of iron levels, the collected cells and rat prostate tissue (10 mg) were homogenized in pre-cooled iron analysis buffer (Beyotime Biotechnology Co., Ltd. in Wuhan, China), and the supernatant was collected after centrifugation at 4 °C, 16,000 g for 10 min. The sample was incubated with an equal amount of analytical buffer at room temperature for 30 min. Then, 100 μL of the iron probe (Beyotime Biotechnology Co., Ltd. in Wuhan, China) was incubated in the dark at 25 °C for 60 min, and the absorbance was determined to be 593 nm.
Relative GSH concentration in cell and rat prostate tissue lysates was assessed using a Total Glutathione Quantification Kit (cat #T419; Dojingdo Laboratories). Samples were lysed using 5% 5‐sulfosalicylic acid solution. After centrifugation at 8000 g for 10 min, supernatants were collected and incubated with the substrate 5, 5′‐dithiobis (2‐nitrobenzoic acid) at 37 °C for 10 min, which produced yellow 5‐thio‐2‐nitrobenzoic acid. Subsequently, 20 μL substrate working solution was added and incubated for 10 min at room temperature. Absorbance was measured with a microplate reader at 412 nm.
Relative MDA concentration in cell and rat prostate tissue lysates was assessed using a MDA Quantification Kit (Beyotime Biotechnology Co., Ltd. in Wuhan, China) and MDA content was expressed as the relative absorbance of the sample at 532 nm.
Transmission electron microscopy analysis
Cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 1 h, followed by postfixation for 6 h in 1% OsO4. After dehydration with graded alcohol (50, 70, 80, 90, 100%) solutions for 15 min each time, each sample was embedded in epoxy resin (Sigma-Aldrich, 45,359). The cut-thin sample (70 nm) was mounted on a copper mesh (Sigma-Aldrich, TEM-74357) and post-stained with 2% uranyl acetate and 1% lead citrate, dried, and analyzed with a transmission electron microscope (JEOL).
Statistical analyzes were performed using SPSS statistical software, version 21.0 (IBM, Chicago, IL). The data were expressed as mean ± standard deviations from at least three independent experiments. Student’s two-tailed t-test was used to compare the means of two-group samples, and one-way ANOVA was applied for comparison of multiples groups. p-value < 0.05 was considered statistically significant. We set a single blind for the statisticians of the data in this study.
The expression and localization of GPX3 in human prostate tissues and cell lines
Our microarray dataset (serial number: GSE119195) was used to analyze the differential expression of GPX3 transcriptome in 5 hyperplastic and 3 normal prostate specimens, which showed GPX3 expression was significantly down-regulated at the transcriptional level in hyperplastic prostates (Fig. 1A and Additional file 5: Fig S1). Similarly, tissues harvested from our institute were observed that the expression of GPX3 was decreased at both mRNA (n = 4 control/8 BPH samples) (Fig. 1B) and protein (n = 4 control/4 BPH samples) levels (Fig. 1C, D). Meanwhile, immunofluorescence staining of prostate tissue demonstrated that GPX3 was localized both in epithelium and stroma (Fig. 1E, F). Consistently, cell immunofluorescence staining demonstrated GPX3 was present both in cultured human stromal cell WPMY-1 (Fig. 1G) and epithelial cell BPH-1 (Fig. 1H).
GPX3 silencing promoted the cell survival, shortened the cell G0/G1 phase, reduced mitochondria-mediated apoptosis and mitochondrial dysfunction of prostate cells via inhibiting ERK pathway activation
To investigate the functional activities of GPX3, the cell model of GPX3 silencing was constructed by three distinct si-GPX3s transfection. The mRNA level of GPX3 was more down-regulated by si-GPX3-1 and si-GPX3-3, which were selected for subsequent experiments (Fig. 2A). Meanwhile, GPX3 was silenced at the protein level after transfection of prostate cells with si-GPX3-1 and si-GPX3-3 (Fig. 2B and Additional file 6: Fig. S2A). Cell proliferation was detected with CCK-8 assay and it was found that downregulation of GPX3 accelerated cell proliferation at both 48 and 72 h (Fig. 2C) for both cell lines. Moreover, our flow cytometry assay showed that the ratio of cells in the G0/G1 phase was shortened in si-GPX3 transfected prostate cells with a corresponding increase of G2 phase but no significant change of S phase (Fig. 2D, E). Accordingly, G0/G1 phase cell cycle-related proteins (Cyclin D1, CDK4 and CDK6) underwent significant increase in prostate cells treated with si-GPX3, while G2 phase cell cycle-related proteins (Cyclin B1 and CDK1) were no significant difference (Fig. 2F and Additional file 6: Fig. S2B). Flow cytometry analysis also manifested that down-regulation of GPX3 restrained apoptosis of prostate cells by more than 50% (Fig. 2G, H). Meanwhile, we used 40 μM apoptosis-inducer isosilybin B (acting on the G0/G1 phase of the cell cycle. The IC50 of isosilybin B on cytotoxic of BPH-1 and WPMY-1 cells were 67.72 μM and 41.38 μM, respectively) for BPH-1 or 20 μM isosilybin B for WPMY-1 in combination with si-GPX3s, and the increase in apoptosis induced by isosilybin B treatment could be inhibited by the down-regulation of GPX3 (Additional file 6: Fig. S2C–E), which demonstrated that the down-regulation of GPX3 promoted cell survival by accelerating the G0/G1 phase. Furthermore, the active condition of mitochondria was determined via the detection of mitochondrial membrane potential (MMP). And the silence of GPX3 contributed to more agminated MMP [the ratio of JC-1 aggregates (orange) to JC-1 monomer (blue) increased] in prostate cells (Fig. 2I, J), indicating the initiation of apoptosis process was lessened by the more stable mitochondria. Since the release of Cytochrome C (Cyto-C) from mitochondria to cytoplasm is the committed step for concatenating mitochondrial damage to apoptosis, we performed mitochondrial and cytoplasmic compartments isolation on prostate cell lines. The knockdown of GPX3 contributed to the increase of mitochondrial Cyto-C (Cyto-Cmito) and the decrease of cytoplasmic Cyto-C (Cyto-Ccyto) (Fig. 2K and Additional file 6: Fig. S2F). Moreover, the release of Cyto-C is also affected by pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2. When receiving apoptotic signal, caspase 9 and caspase 3 are further activated step by step to initiate apoptosis. And immunoblotting analysis showed that GPX3 silencing promoted the expression of Bcl-2 and suppressed the expression of BAX. Notably, the expression of cleaved-caspase 9 and cleaved-caspase 3 were also prominently decreased, confirming that GPX3 silencing inhibited mitochondrial-mediated apoptosis in prostatic cells (Fig. 2L and Additional file 6: Fig. S2G). The MAPK family was also altered with phosphorylated ERK1/2 (p-ERK1/2) significantly weakened but no effect on p-JNK and p-p38 (Fig. 2M and Additional file 6: Fig. S2H) in si-GPX3 treated prostate cells. In addition, to verify the feasibility of the control group, we used the untreated control (con) group to replicate with the transfection control (si-con) group and the empty plasmid (vector) group. It was showed that there was no significant difference in GPX3 protein expression level (Additional file 7: Fig. S3A, B), cell proliferation activity (Additional file 7: Fig. S3C), cell cycle (Additional file 7: Fig. S3D, E), apoptosis level (Additional file 7: Fig. S3F, G), MMP level (Additional file 7: Fig. S3H, I) and MAPK pathway protein expression level (Additional file 7: Fig. S3J, K) among the above three groups.
Overexpression of GPX3 inhibited cell proliferation, induced G0/G1 phase arrest and triggered mitochondria-mediated apoptosis and mitochondrial dysfunction via activating ERK pathway in prostate cells
Both the mRNA and protein levels of GPX3 were up-regulated when we transfected the plasmid into prostate cells to overexpress GPX3 for 48 h or 72 h, respectively (Fig. 3A, B and Additional file 8: Fig. S4A). The CCK-8 assay manifested that GPX3 restrained cell growth (Fig. 3C). Furthermore, the flow cytometry assay observed that the proportion of cells in the G0/G1 phase was elevated with a decrease of G2 phase and no significant change of S phase (Fig. 3D, E), accompanied by a decreased expression of cycle-related proteins (Cyclin D1, CDK4 and CDK6) in GPX3 overexpressed prostate cells (Fig. 3F and Additional file 8: Fig. S4B). Meanwhile, overexpression of GPX3 facilitated cell apoptosis (Fig. 3G, H) and resulted in dissipative MMP [the ratio of JC-1 aggregates (orange) to JC-1 monomer (blue) decreased] in prostate cells (Fig. 3I, J). Moreover, it was observed GPX3 overexpression enhanced the translocation of Cyto-C from mitochondria to cytoplasm (Fig. 3K and Additional file 8: Fig. S4C) and led to the up-regulation of BAX, cleaved-caspase 9 and cleaved-caspase 3 with the expression of Bcl-2 suppressed (Fig. 3L and Additional file 8: Fig. S4D). Additionally, the upregulation of GPX3 significantly strengthened the phosphorylation of ERK1/2 in the MAPK family (Fig. 3M and Additional file 8: Fig. S4E).
U0126 reversed the effects of GPX3 overexpression on cell proliferation activity, cell cycle arrest and apoptosis
The prostate cells were treated with U0126 (MEK1/2 pathway inhibitor) and GPX3 overexpressed plasmid alone or together. As expected, U0126 significantly reversed attenuated cell proliferation viability (Fig. 4A), G0/G1 phase arrest (Fig. 4B, C) and increased apoptosis (Fig. 4D, E) caused by GPX3 overexpression. Furthermore, U0126 recovered the expression of apoptosis-related proteins (BAX, Bcl-2, cleaved-caspase 9 and cleaved-caspase 3) (Fig. 4F and Additional file 8: Fig. S4F), cycle-related proteins (Cyclin D1, CDK4 and CDK6) and p-ERK1/2 changed by GPX3 overexpression in BPH-1 and WPMY-1 cell lines to a certain extent (Fig. 4G and Additional file 8: Fig. S4G).
GPX3 silencing induced ferroptosis in prostate cells with the participation of GPX4
In order to explore the effect of GPX3 on ferroptosis in prostate cells, we detected the intracellular iron, lipid peroxidation product MDA and substrate GSH after GPX3 knockdown. It was observed that GPX3 silencing increased the content of iron (Fig. 5A) and MDA (Fig. 5B) while decreased the level of GSH (Fig. 5C) in prostate cells. At the same time, the level of intracellular ROS increased (Fig. 5D, E). Consistently, immunoblotting analysis indicated that si-GPX3 induced the down-regulation of nuclear factor erythroid 2-related factor-2 (Nrf2), glutathione peroxidase 4 (GPX4) and OS markers [superoxide dismutase 2 (SOD2) and catalase (CAT)] (Fig. 5F and Additional file 9: Fig. S5A). All these changes suggested GPX3 silence amplified ferroptosis. Since mitochondria is the important part of the regulation of ferroptosis, we performed mitochondrial and cytoplasmic compartments isolation on prostate cell lines. The knockdown of GPX3 contributed to the decrease of mitochondrial GPX4 (GPX4mito) and cytoplasmic GPX4 (GPX4cyto) and an increase of dihydroorotate dehydrogenase (DHODH, anti-ferroptosis system exists only in mitochondria) with no significant change of cytoplasmic ferroptosis suppressor protein 1 (FSP1, anti-ferroptosis system exists only in cytoplasm) (Fig. 5G and Additional file 9: Fig. S5B), indicating silenced GPX3 enhanced ferroptosis both in the cytoplasm and mitochondria with the involvement of dysfunctional GPX4mito and GPX4cyto, and was accompanied by upregulation of mitochondrial DHODH responsiveness.
Overexpression of GPX3 reversed the RSL3 induced ferroptosis
To further validate GPX3 mediated ferroptosis with the participation of GPX4, prostate cells were pretreated with RSL3 (GPX4 inhibitor/ferroptosis agonist) and transfected with GPX3 overexpressed plasmid alone or together. The viability of BPH-1 and WPMY-1 cells treated with RSL3 was detected by CCK-8 assay, and the half-maximal inhibitory concentration (IC50) of RSL3 was 0.71 μM and 0.75 μM, respectively (Additional file 9: Fig. S5C). Based on the IC50, 0.5 μM dosage of RSL3 was chosen. As shown in Fig. 6A–E, RSL3 treatment could induce the increase of iron content, MDA, intracellular ROS and the decrease of GSH in prostate cells, while GPX3 overexpression up-regulated the level of GSH, and down-regulated the content of iron, MDA and intracellular ROS in a RSL3-independent manner. In addition, in the detection of cell proliferation activity, both RSL3 treatment and GPX3 overexpression could attenuate the proliferation ability of prostate cells, and which was aggravated by RSL3 treatment and GPX3 overexpression combined (Fig. 6F). Meanwhile, ferroptosis-related proteins (GPX4 and Nrf2) and OS markers (SOD2 and CAT) were accordingly changed (Fig. 6G and Additional file 9: Fig. S5D). Moreover, we performed mitochondrial and cytoplasmic compartment isolation on GPX3 overexpressed BPH-1 and WPMY-1 cell lines. It was observed GPX3 overexpression enhanced the expression of GPX4cyto and GPX4mito, but FSP1 and DHODH did not change significantly (Fig. 6H and Additional file 9: Fig. S5E). More importantly, transmission electron microscopy (TEM) assays confirmed that RSL3 treatment substantially shanked mitochondria and decreased the number of ridges, whereas GPX3 plasmid largely abolished the typical changes of ferroptotic cells (Fig. 6I). Overall, these data indicated that GPX3 overexpressed exerted anti-ferroptosis effects both in the cytoplasm and mitochondria with the participation of GPX4.
GPX3 regulated ferroptosis in an autophagy-related manner
We also examined the levels of Beclin1 and LC3B, which are the initial protein and structural components of autophagosomes, respectively. Immunoblotting analysis showed that RSL3 could significantly up-regulate the expression of LC3B and Beclin1, while GPX3 overexpression could down-regulate the expression of LC3B and Beclin1 (Fig. 7A and Additional file 10: Fig. S6A). Meanwhile, TEM analysis further showed that RSL3 increased the autophagosome formation in prostate cells, which could be reversed by GPX3 overexpression (Fig. 7B). In order to further elucidate the relationship between ferroptosis and autophagy in prostate cells, we combined 2 nM Rapa (autophagy agonist. The IC50 of Rapa on cytotoxic of BPH-1 and WPMY-1 cells were 2.67 nM and 3.24 nM, respectively) with 20 μM CQ (autophagy inhibitor. The IC50 of CQ on cytotoxic of BPH-1 and WPMY-1 cells were 28.52 μM and 31.04 μM, respectively) (Additional file 10: Fig. S6B, C). Western blot indicated that under the premise of RSL3 pretreatment, GPX3 overexpression aggravated the down-regulation of LC3B and Beclin1 expression induced by CQ, and weakened the up-regulation of LC3B and Beclin1 expression triggered by Rapa (Fig. 7C and Additional file 10: Fig. S6D). Moreover, there were superimposed effects on CQ‐induced decrease of iron content, MDA, intracellular ROS and the increase of GSH, compared to prostate cells treated with GPX3 overexpression alone (Fig. 7D–H). Conversely, Rapa increased the levels of iron content, MDA and intracellular ROS, and decreased the GSH in prostate cells, which could be reversed by GPX3 overexpression (Fig. 7D–H). The above results indicated that GPX3 in prostate cells could inhibit autophagy-related ferroptosis by antagonizing autophagy.
GPX3 antagonized autophagy-related ferroptosis and activated ERK1/2 with the involvement of adenosine monophosphate-activated protein kinase (AMPK) pathway
AMPK is a positive regulator of autophagy via suppressing mammalian target of rapamycin (mTOR) . To investigate the role of AMPK/mTOR pathway in GPX3-mediated autophagy inhibition, we initially detected the activation levels of AMPK and mTOR and it was found that GPX3 overexpression inhibited the increase of AMPK phosphorylation and the decrease of mTOR phosphorylation triggered by RSL3 treatment (Fig. 8A and Additional file 10: Fig. S6E). We further used 20 μM GSK621 (AMPK agonist, the IC50 of GSK621 on cytotoxic of BPH-1 and WPMY-1 cells were 23.21 μM and 25.33 μM, respectively) (Additional file 10: Fig. S6F) pretreatment to determine whether AMPK was associated with GPX3-mediated autophagy. And the protein expression of p‐AMPK was significantly up-regulated in GSK621‐treated cells (Additional file 10: Fig. S6G). As shown in Fig. 8B and Additional file 10: Fig. S6H, GSK621 up-regulated the expression of LC3B and Beclin1, which could be partially rescued by GPX3 overexpression. In addition, GSK621 also mediated the upregulation of intracellular iron content, MDA and ROS, and the depletion of GSH, which were also reversed to some extent by GPX3 overexpression (Fig. 8C–G). Data suggested that GPX3 mediated autophagy-related ferroptosis of prostate cells with the involvement of AMPK/mTOR pathway. Moreover, AMPK acts as an upstream negative regulator of ERK1/2. Indeed, we observed GSK621 inhibited the expression of p-ERK1/2 protein, which could be reversed by GPX3 overexpression (Fig. 8H). It was suggested that GPX3 regulated ERK1/2 signaling with the involvement of AMPK pathway.
The level of apoptosis in hyperplastic prostate decreased, while OS, ferroptosis and autophagy increased
We further verified the expression of BAX, caspase 3 and Bcl-2 in tissue samples by IHC staining. The results revealed the expression of BAX and caspase 3 in the hyperplastic prostate tissues were significantly lower than that in normal tissues, while Bcl-2 was the opposite (Fig. 9A, B), which was consistent with the results of Western blotting (Fig. 9C). Moreover, to compare the levels of ferroptosis markers (GPX4 and Nrf2) and OS in normal and hyperplastic prostates, we performed IHC staining (Fig. 9D–G) and Western blotting (Fig. 9H) of GPX4, Nrf2 and antioxidant enzymes (CAT and SOD2) in normal and hyperplastic prostate tissues, and found that the expression of them declined in hyperplastic prostate tissues. Meanwhile, we verified the expression of autophagy-related factors (Beclin1 and LC3B) in tissue samples by Western blotting (Fig. 9I) and IHC staining (Fig. 9J, K). The results revealed that the expression of Beclin1 and LC3B in the hyperplastic prostate tissues were significantly higher than that in normal tissues. The above results indicated that the down-regulation of GPX3 (Fig. 1A–F) in hyperplastic prostate is accompanied by a low level of apoptosis and a high level of OS, ferroptosis and autophagy.
GPX3 inhibited BPH progress and cell proliferation, induced cell apoptosis and cell cycle arrest while antagonized ferroptosis and autophagy in vivo
Finally, we translated our in vitro study into in vivo experiment to further verify the function of GPX3. Rats supplemented with 4 week testosterone (T) showed significant increase in ventral prostate weight and prostate index (prostate weight/body weight) (Fig. 10A and (Table 1). In addition, compared with T group, RSL3 treatment significantly reduced the body weight (p < 0.01), ventral prostate weight (p < 0.001) and prostate index (p < 0.001). Furthermore, TRO (GPX3 agonist) treatment decreased the ventral prostate weight (p < 0.001) and prostate index (p < 0.001) while increased body weight (p < 0.01) when compared with T group. Moreover, compared with T + RSL3 group, T + RSL3 + TRO treatment had no significant effect on ventral prostate weight (p > 0.05), but decreased prostate index (p < 0.05) and increased body weight ( p < 0.001) (Table 1) In the prostate of T-BPH rats, the epithelium component was relatively increased. Nevertheless, RSL3, TRO and RSL3 + TRO effectively restrained the progression of BPH induced by T. The shrunk glands were lined with a single layer of columnar epithelium to low cuboidal cells, along with slight edema, which was most significant in the T + RSL3 + TRO group (Fig. 10B). Masson’s trichrome staining further indicated that the hyperplasia of the prostate occurred primarily in epithelium (p < 0.05) and secondarily in collagen fibers (p < 0.05), while smooth muscle (SM) had no difference in BPH rats. Moreover, when the T-BPH rat model treated with RSL3 or TRO, the components of epithelia and collagen fibers were relatively reduced, and the differences were statistically significant (p < 0.05). When compared with T + RSL3 group, T + RSL3 + TRO group only reduced the epithelial components (p < 0.05) with no effect on the stromal components (Fig. 10C, D). Furthermore, in order to quantify the proliferation level of rat prostates, we detected the nuclear antigen Ki-67, which is positively correlated with cell proliferation activity, and immunofluorescence staining of the rat prostate tissues showed that the positive rate of Ki-67 was significantly increased in BPH rats (p < 0.01) and was inhibited by RSL3 or TRO treatment (p < 0.05). When RSL3 and TRO treated simultaneously, the inhibition effect on the positive rate of Ki-67 was more significant than that of T + RSL3 group (p < 0.05) (Fig. 10E, F). Moreover IHC staining showed that the expression of GPX3 decreased in T-BPH rats (p < 0.05), and TRO treatment significantly upregulated the expression of GPX3 (p < 0.05) but there was no significant change with RSL3 treatment (p > 0.05) (Fig. 10G, H). The Western blot results were consistent to IHC (Fig. 10I, Additional file 11: Fig. S7A). Meanwhile, Western blot manifested that apoptosis-related proteins (BAX, cleaved-caspase 9 and cleaved-caspase 3) were down-regulated, and anti-apoptosis protein Bcl-2 and cycle-associated proteins (Cyclin D1, CDK4 and CDK6) were up-regulated in T-BPH rats, all of which were reversed by TRO intervention (Fig. 10J and Additional file 11: Fig. S7B). The above results indicated that the T-BPH rat model was successfully established, and the prostate of T-BPH rats showed accelerated proliferation activity, decreased apoptosis, shortened G0/G1 phase and down-regulated GPX3 expression, all of which could be reversed by TRO treatment.
Also, IHC staining and Western blot results showed that the expression of GPX4 (Fig. 11A, C, E and Additional file 11: Fig. S7C) and Nrf2 (Fig. 11B, D, E and Additional file 11: Fig. S7C) decreased in T-BPH rat prostate and further decreased after RSL3 treatment, while could be rescued by TRO treatment. Furthermore, T treatment significantly increased the levels of iron (Fig. 11F) and MDA (Fig. 11G), while decreased the levels of SOD2, CAT (Fig. 11E and Additional file 11: Fig. S7C) and GSH (Fig. 11H) in the rat prostate, indicating that the level of ferroptosis in the prostate of T-induced BPH rats was elevated, which could be further aggravated by RSL3 (Fig. 11E–H). In addition, TRO treatment antagonized ferroptosis caused by T or RSL3 (Fig. 11E–H). Meanwhile, IHC staining and Western blot experiments suggested that T treatment could up-regulate the levels of LC3B (Fig. 11I, K, M and Additional file 11: Fig. S7D) and Beclin1 (Fig. 11J, L, M, Additional file 11: Fig. S7D) in T-BPH rat prostates, which could be exacerbated by RSL3 treatment and partially reversed by TRO. This indicated that the autophagy level of T-BPH rat prostates was relatively elevated and could be further aggravated by ferroptosis inducer (RSL3), while the up-regulation of GPX3 expression (TRO treatment) could significantly antagonize autophagy induced by T or RSL3.
Our novel data demonstrated that GPX3 was localized in the stromal and epithelial compartments of prostate tissues and down-regulated in hyperplastic prostate tissues. Furthermore, we manifested that GPX3 not only had antioxidant properties, but also played roles in inhibiting AMPK pathway. Subsequently, it inhibited cell proliferation, promoted G0/G1 phase arrest and mitochondrial-dependent apoptosis via ERK1/2. On the other hand, it exerted anti-autophagy function by activating mTOR and antagonized autophagy-related ferroptosis mediated by Nrf2/GPX4 (Fig. 12).
GPX3 is an extracellular glycoprotein that accepts thioredoxin (Trx) and glutaredoxin (Grx) as substrates in addition to GSH . As an extracellular antioxidant device, plasma GPX3 is mostly produced by the kidneys, while other organs such as lung, breast, adipose, epididymis and prostate, express GPX3 locally in cell type dependent manner . It has been reported that GPX3 deficiency could enhance the cell proliferation of prostate cancer (PCa) tissues and reduce PCa cell apoptosis . On the other hand, Yu et al.  showed that overexpression of GPX3 could inhibit the proliferation of PCa cells and the size of xenograft tumors in vivo. Interestingly, GPX3 activity progressively decreased with age, which was more significant for people over 70 years old. It had been reported that the age-related decline of GPX3 may increase the risk of cardiovascular events in patients with atrial fibrillation . As we known, BPH is a common disease in aging male. And our previous microarray data did find the decrease of GPX3 in the aged prostate. Current study further validated that the expression of GPX3 in hyperplastic prostate was significantly lowered both at the transcriptional and translational levels. So did with the T-BPH rat model. However, the average age of healthy prostates and BPH prostates (25.2 ± 4.4 years vs. 70.0 ± 7.5 years) was significantly different, which seemed to be not conducive to distinguish the effect of aging on GPX3 expression. In fact, approximately 50% of men > 50 years of age will have pathological evidence of BPH, with this incidence increasing to > 80% as men reach their eighth decade of life or older , which means that we could hardly get healthy prostate samples from men over the age of 70 in existing clinical scenario. Comparing the down-regulated expression of GPX3 with age in healthy prostates and hyperplastic prostates around 70 years old will be of great significance for clarifying the expression pattern of GPX3 in prostate, which needs further study.
The imbalance between proliferation and apoptosis is one of the most important pathogenic mechanisms of BPH. In mammalian cells, Bcl-2 associated X protein (BAX) is recruited when various endogenous and exogenous stimuli induce apoptosis and form pores in the mitochondrial membrane , facilitating the release of Cyto-C (as a well conserved electron-transport protein and a part of the respiratory chain localized in mitochondrial intermembrane space.) into the cytoplasm. Cyto-C on the cytoplasm further activate caspase 9, which in turn triggers caspase 3 to initiate apoptosis . Bcl-2 acts as an anti-apoptotic protein, counteracting BAX activation to maintain this balance of programmed cell death . And the imbalance between proliferation and apoptosis is closely related to abnormal cell cycle regulation. Cyclin dependent kinases (CDKs) are a group of serine/threonine protein kinases. As the engine of cell cycle, CDKs and their regulatory factors exert an important effect in disease progression. CDK4 and CDK6 are important CDKs members that regulated the transition from G1 to S phase and bind to Cyclin Ds (D1, D2 and D3) to form CDK4/6-Cyclin complex after being activated by mitotic signals, while the over-activated CDK4/6 destabilizes the genome and chromosome, leading to uncontrolled proliferation and ultimately abnormal cell cycle regulation . Moreover, CDK1-Cyclin B1 complex is involved in the regulation of cell cycle as an important checkpoint of G2/M phase transition . In this study, we found GPX3 knocked-down shortened the G0/G1 phase, up-regulated the expression of Cyclin D1, CDK4 and CDK6, and arrested G2 phase, which seemed to contradict the accelerated cell proliferation and decreased apoptosis led by GPX3 silenced. Therefore, we combined si-GPX3s with isosilybin B, which plays a pro-apoptotic role by blocking the G1 phase , to detect the level of apoptosis. It was showed that the increase in apoptosis caused by isosilybin B could be significantly inhibited by GPX3 knockdown and GPX3 knockdown had no significant effect on the expression of Cyclin B1 and CDK1, which confirmed that the down-regulation of GPX3 regulated cell proliferation and apoptosis by shortening G0/G1 phase, while the prolongation of G2 phase seemed to be a compensatory change. Furthermore, we observed that overexpression of GPX3 could inhibit the proliferation of prostate cells, induce G0/G1 phase arrest, cell apoptosis, MMP dissipation and cytoplasmic transfer of Cyto-C. Specifically, overexpressed GPX3 induced mitochondrial dysfunction and cytoplasmic transfer of Cyto-C through up-regulation of BAX and down-regulation of Bcl-2, and ultimately induced mitochondria-dependent apoptosis via the caspase 9-caspase 3 cascade, and then cooperated with the blocking effect of GPX3 on G0/G1 phase to attenuate the proliferation activity of prostate cells. In addition, we also observed lower expression of BAX and caspase 3 and higher expression of Bcl-2 in hyperplastic prostate tissues compared with normal prostate tissues, which showed that with the down-regulation of GPX3, the apoptosis level of hyperplastic prostate was inhibited at the tissue level.
Three characteristic MAPK families (ERK, JNK and p38 kinases) have been identified in mammalian cells . There was evidence that all three MAPK cascades might be involved in the progression of BPH by regulating the local prostate environment [48, 49]. Although in most reports, the ERK1/2 pathway has been shown to promote cell survival, proliferation and motility, it was also reported inhibited cell apoptosis. In the study of cisplatin-induced apoptosis of renal epithelial cells, Kim et al.  found that activated ERK1/2 played a pro-apoptotic role by inducing BAX expression, MMP depolarization, mitochondrial Cyto-C release and caspase3 upregulation, which was in line with our findings. In this study, when GPX3 was knocked down in prostate cells, the phosphorylation level of ERK1/2 was significantly inhibited with no significant effect on JNK1/2 and p38. In current study, prostate cells were further pre-incubated with MEK1/2 inhibitor U0126 and then transfected with overexpressed GPX3. We found that GPX3 overexpression could significantly up-regulate the phosphorylation of ERK1/2 but had a weak effect on JNK1/2 and p38. Meanwhile, U0126 could largely offset the effects of GPX3 overexpression on cell proliferation, cell cycle and apoptosis. Our data suggested that GPX3 triggered G0/G1 arrest and mitochondrial-dependent apoptosis through ERK1/2 activation rather than JNK1/2 and p38 in hyperplastic prostate. Furthermore, we found that the activity of ERK1/2 was negatively regulated by AMPK in vitro. AMPK is an energy sensor that monitors the AMP: ADP: ATP ratio in eukaryotic cells and conclusive evidence showed that AMPK could directly phosphorylate the Raf proto-oncogene/Kinase suppressor of Ras (RAF/KSR) family kinases, the pivotal components of MAPK module, and alter their activities under variable conditions [51, 52]. In addition to mediating ERK1/2 signaling, we showed that AMPK pathway could also participate in autophagy-related ferroptosis of prostate cells.
Ferroptosis is a newly identified form of nonapoptotic programmed cell death characterized by iron-dependent accumulation of lipid peroxides, and has drawn wide attention since the term was put forward in 2012 . It is morphologically and biochemically different from known types of cell death. To be specific, the most notable morphological feature of ferroptotic cells under electron microscopy is the change in mitochondrial morphology; ferroptotic cells typically contain shrunken mitochondria with increased membrane density  and the main biochemical characteristics of ferroptosis consist of increased cellular labile iron, large amounts of ROS, decreased GPX4 activity, and accumulation of lipid metabolites . In this study, we were the first to examine the ferroptosis level in hyperplastic prostate. We found that the expression levels of Nrf2 and GPX4 were decreased in both human hyperplastic prostates and T-induced BPH rat prostates. Meanwhile, the expression of CAT and SOD2, which are antioxidant components, was also at a low level in hyperplastic prostate tissues. It is suggested that Nrf2/GPX4 dysregulation-associated ferroptosis and high levels of OS occur in GPX3 down-regulated hyperplastic prostates. Furthermore, when GPX3 was knocked down, which was regarded as an antioxidant, endogenous ROS were undeniably elevated, and accompanied by downregulation of antioxidant proteins SOD2 and CAT. Meanwhile, the levels of GPX4 and its cofactor GSH decreased, while the markers of labile iron content (Fe2+) and lipid peroxidation product MDA increased significantly. Moreover, GPX3 deficiency also led to the downregulation of Nrf2, a negative upstream regulator of ferroptosis, suggesting that the antagonistic effect of GPX3 on ferroptosis in prostate cells was related to Nrf2. We further pretreated prostate cells with RSL3 and then transfected with overexpressed GPX3. RSL3, a compound containing an electrophilic moiety and a chloroacetamide moiety, could reacted with the nucleophilic amino acid residue at the active site of GPX4. This binding directly led to the inactivation of GPX4. We observed that RSL3 treatment significantly increased the accumulation of labile iron, ROS and lipid peroxides and accompanied by the reduction or disappearance of mitochondrial cristae in prostate cells, while GPX4 decreased. Whereas, GPX3 overexpression could largely reverse the effect of RSL3 on activating ferroptosis via upregulating Nrf2 and GPX4. To escape ferroptosis, cells evolve to be equipped with defending systems that detoxify lipid peroxidation and suppress ferroptosis. GPX4 [including cytosol- and mitochondria-localized GPX4 (GPX4cyto and GPX4mito)] is the first reported anti-ferroptosis pathway . Moreover, FSP1, which locates in the plasma membrane, prevents lipid peroxidation and inhibits ferroptosis even in the absence of GPX4 . In addition, Mao et al.  reported that DHODH was an enzyme localized on the outer face of the mitochondrial inner membrane, which was functionally independent from GPX4mito, and both could detoxify lipid peroxides accumulated in mitochondria. In the present study we observed that when the expression level of GPX3 in prostate cells changed, both GPX4mito and GPX4cyto showed the consistent changes with GPX3, while FSP1 in the cytoplasm remained unchanged. However, when GPX4mito was down-regulated with GPX3 knockdown, the expression level of DHODH in mitochondria increased, whereas GPX3 overexpression did not affect it. This could be attributed to the parallel effect of DHODH and GPX4mito on inhibiting ferroptosis. When GPX4mito was inactivated, DHODH occurred an adaptive cellular response that attempted to antagonize ferroptosis. In addition, we found that RSL3 not only induced ferroptosis in prostate cells, but also further promoted the increase of autophagy levels.
Autophagy is a highly conserved evolutionary and complex cellular process in eukaryotic cells, where cytoplasmic long half-life proteins and organelles are sequestered within autophagosomes and delivered to lysosomes for degradation and recycling . Previous studies have revealed that autophagy was involved in BPH development [59, 60], and LC3B and Beclin1, as indicators of autophagy, were highly expressed in BPH stromal and epithelial tissues. Consistently, we found the high expression of LC3B and Beclin1 in hyperplastic prostate tissues and T-BPH rats, indicating that when the level of GPX3 in hyperplastic prostate tissues was down-regulated, it was accompanied by a high level of autophagy. Interestingly, there was a complex crosstalk between ferroptosis and autophagy through complex feedback loops [61, 62]. Ferroptosis induction was coupled to an increase in turnover of LC3B and autophagosome formation, consistent with the notion that lipid peroxidation as well as oxidized lipids could promote autophagy activation . Similar to the discovery that RSL3 could promote autophagy activation in glioma cells . In this study, RSL3 promoted autophagy activation in prostate cells, and this stimulation could be antagonized by GPX3 overexpression via down-regulating LC3B and Beclin1. However, functional contributions of GPX3 to autophagy-related ferroptosis have not been fully understood. The GPX3-mediated autophagic pathway could be modulated in a pharmacological approach. Ferroptosis in prostate cell lines could be aggravated by the autophagy activator Rapa, while the effects were reversed by the autophagy inhibitor CQ. Therefore, we inferred that GPX3-mediated autophagy may be an upstream mechanism that could adjust cellular OS and iron homeostasis during ferroptosis.
AMPK, as a key protein involved in various signal transduction pathways, activates autophagy through negative regulation of mTOR . As expected, our data suggested that increased GPX3 exerted antagonistic effects on autophagy by down-regulating AMPK phosphorylation and up-regulating mTOR. In addition, AMPK agonist GSK621 up-regulated the protein levels of LC3B and Beclin1, and further increased the level of ferroptosis, which could be antagonized by GPX3 overexpression. These data demonstrated that GPX3 antagonized autophagy and the associated ferroptosis by inhibiting AMPK/mTOR. The function of AMPK in mediation of ferroptosis was required for Beclin1 phosphorylation, with inhibition of system Xc − activity being reported . Perhaps AMPK could represented a mechanistic link between ferroptosis and autophagy. But its specific mechanism need be further studied.
Finally, we further translated our in vitro studies to in vivo. Since GPX3 recombinant protein or agonist was not commercially available, we used TRO gavage to mimic GPX3 overexpression  and intraperitoneal injection of RSL3 to initiate ferroptosis in vivo in rats. TRO is a ligand of peroxisome proliferator-activated receptor γ (PPARγ), which activates GPX3 as a direct upstream factor . Our T-induced BPH model was verified with the dramatically augmented weight of the prostate and seminal vesicle when compared with control group. In line with previous observations, T injection mainly led to a notable thickening of the epithelial layer and collagen fiber with no significant change in the percentage of the SM component, which were features of T-BPH rats . In addition, the prostate of T-BPH rats showed significantly accelerated proliferation activity, shortened cell cycle, decreased apoptosis, and up-regulated ferroptosis, OS and autophagy levels, which were consistent with what we observed in human hyperplastic prostate. Moreover, GPX3 overexpression (TRO treatment) could reverse the above T-induced phenotypic changes in rat prostate to a certain extent. In brief, all these changes were consistent with our in vitro findings. It should be noted that both RSL3 and TRO have inhibitory effects on the proliferation activity and epithelial thickness of the prostate in T-BPH rats (Fig. 10C–F), which seems unreasonable. In this in vivo study, the prostate of T-BPH rats treated with RSL3 showed significant increase in ferroptosis and autophagy, while TRO treatment showed a significant increase in apoptosis and G0/G1 phase arrest. This seems to indicate that the two have the same effect on the proliferation activity of rat prostates, but the mechanism is completely different, which needs further study.
Interestingly, our data observed that GPX3 had the pro-apoptosis but anti-autophagy-related ferroptosis effect in prostate cells. Study had shown that autophagy inhibition could enhance the apoptosis induced by androgen deprivation in human BPH-1 cells . In this study, both in human prostate tissues and in the prostate of T-BPH rats, there were inhibition of apoptosis and abnormal activation of autophagy, as well as increased ferroptosis levels (in an autophagy-related manner). This seemed to suggest the protective role of autophagy and autophagy-related ferroptosis in BPH. Their specific mechanism in the occurrence and development of BPH needs to be further studied.
There are still some shortcomings in this study. Even if GPX4 was inhibited by RSL3, the ferroptosis phenotype was still effective under the context of GPX3 overexpression. Whether GPX3-mediated ferroptosis regulation of prostate cells works only through GPX4 is still unknown. Furthermore, all rescue experiments in this study used chemical inhibitors or agonists, which may have the risk of off-target effects. The use of genetic technology to regulate specific genes would strengthen the evidence. And the transgenic mice will further corroborate the role of GPX3 in the pathogenesis of BPH. These will be supplemented in future study.
Our current study indicated that GPX3 protein was localized in both the stromal and epithelial compartments of prostate tissues with the transcription and translation levels of GPX3 were down-regulated in the hyperplastic prostate. Our novel data also demonstrated that GPX3 played double roles in the development of BPH in vivo and in vitro. On the one hand, GPX3 deficiency promoted cell proliferation, shortened G0/G1 phase and inhibited mitochondria-dependent apoptosis through AMPK/ERK1/2 pathway. On the other hand, GPX3 deficiency up-regulated autophagy-related ferroptosis via AMPK/mTOR pathway, and restrained Nrf2/GPX4 levels. Therefore, this study not only provided evidence for the future in-depth study of GPX3 in BPH, but also its multiple functional mechanisms were expected to become a new therapeutic target for BPH.
Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
Benign prostatic hyperplasia
Lower urinary tract symptoms
Differentially expressed genes
Matrix-remodeling associated 5
C-X-C Motif Chemokine Ligand 13
Bone morphogenetic protein 5
Neural epidermal growth factor-like like 2
Glutathione peroxidase 3
Reactive oxygen species
Mitochondrial membrane potential
Programmed cell death
Hematoxylin and eosin staining
RAS-selective lethal 3
Glutathione peroxidase 4
Peroxisome proliferator-activated receptor γ
Nuclear factor erythroid 2-related factor 2
Superoxide dismutase 2. CAT: catalase
Ferroptosis suppressor protein 1
Transmission electron microscopy
Adenosine monophosphate-activated protein kinase
Mammalian target of rapamycin
Kim EH, Larson JA, Andriole GL. Management of Benign prostatic hyperplasia. Annu Rev Med. 2016;67:137–51.
Woo HH, Huang CP, Huang WJ, Chang YH, Lien CS, Chkhotua A, et al. The EXPANDER-1 trial: introduction of the novel urocross expander system for treatment of lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH). Prostate Cancer Prostatic Dis. 2022;25(3):576–82.
Tamalunas A, Sauckel C, Ciotkowska A, Rutz B, Wang R, Huang R, et al. Inhibition of human prostate stromal cell growth and smooth muscle contraction by thalidomide: a novel remedy in LUTS? Prostate. 2021;81(7):377–89.
Joseph DB, Henry GH, Malewska A, Reese JC, Mauck RJ, Gahan JC, et al. 5-Alpha reductase inhibitors induce a prostate luminal to club cell transition in human benign prostatic hyperplasia. J Pathol. 2022;256(4):427–41.
McPherson SJ, Ellem SJ, Simpson ER, Patchev V, Fritzemeier KH, Risbridger GP. Essential role for estrogen receptor beta in stromal-epithelial regulation of prostatic hyperplasia. Endocrinology. 2007;148(2):566–74.
Fiard G, Stavrinides V, Chambers ES, Heavey S, Freeman A, Ball R, et al. Cellular senescence as a possible link between prostate diseases of the ageing male. Nat Rev Urol. 2021;18(10):597–610.
Vickman RE, Aaron-Brooks L, Zhang R, Lanman NA, Lapin B, Gil V, et al. TNF is a potential therapeutic target to suppress prostatic inflammation and hyperplasia in autoimmune disease. Nat Commun. 2022;13(1):2133.
Udensi UK, Tchounwou PB. Oxidative stress in prostate hyperplasia and carcinogenesis. J Exp Clin Cancer Res. 2016;35(1):139.
Xiao H, Jiang Y, He W, Xu D, Chen P, Liu D, et al. Identification and functional activity of matrix-remodeling associated 5 (MXRA5) in benign hyperplastic prostate. Aging (Albany NY). 2020;12(9):8605–21.
Liu D, Li M, Fu X, Yang S, Wang Z, Liu J, et al. Changes in the expression and functional activities of C-X-C motif chemokine ligand 13 (CXCL13) in hyperplastic prostate. Int J Mol Sci. 2022. https://doi.org/10.3390/ijms24010056.
Liu D, Liu J, Li Y, Liu H, Hassan HM, He W, et al. Upregulated bone morphogenetic protein 5 enhances proliferation and epithelial-mesenchymal transition process in benign prostatic hyperplasia via BMP/Smad signaling pathway. Prostate. 2021;81(16):1435–49.
Liu J, Liu D, Zhang X, Li Y, Fu X, He W, et al. NELL2 modulates cell proliferation and apoptosis via ERK pathway in the development of benign prostatic hyperplasia. Clin Sci. 2021;135(13):1591–608.
Brigelius-Flohe R, Flohe L. Regulatory phenomena in the glutathione peroxidase superfamily. Antioxid Redox Signal. 2020;33(7):498–516.
Maddipati KR, Marnett LJ. Characterization of the major hydroperoxide-reducing activity of human plasma Purification and properties of a selenium-dependent glutathione peroxidase. J Biol Chem. 1987;262(36):17398–403.
Bierl C, Voetsch B, Jin RC, Handy DE, Loscalzo J. Determinants of human plasma glutathione peroxidase (GPx-3) expression. J Biol Chem. 2004;279(26):26839–45.
Chang C, Worley BL, Phaeton R, Hempel N. Extracellular glutathione peroxidase GPx3 and Its role in cancer. Cancers. 2020. https://doi.org/10.3390/cancers12082197.
Brigelius-Flohe R, Maiorino M. Glutathione peroxidases. Biochim Biophys Acta. 2013;1830(5):3289–303.
Xie J, Fu L, Zhang J. Analysis of influencing factors on the occurrence and development of gastric cancer in high-incidence areas of digestive tract tumors based on high methylation of GPX3 gene. J Oncol. 2022. https://doi.org/10.1155/2022/3094881.
Worley BL, Kim YS, Mardini J, Zaman R, Leon KE, Vallur PG, et al. GPx3 supports ovarian cancer progression by manipulating the extracellular redox environment. Redox Biol. 2019;25:101051.
Yu YP, Yu G, Tseng G, Cieply K, Nelson J, Defrances M, et al. Glutathione peroxidase 3, deleted or methylated in prostate cancer, suppresses prostate cancer growth and metastasis. Cancer Res. 2007;67(17):8043–50.
Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal. 2012;24(5):981–90.
Tian C, Liu Y, Li Z, Zhu P, Zhao M. Mitochondria related cell death modalities and disease. Front Cell Dev Biol. 2022;10:832356.
Bock FJ, Tait S. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol. 2020;21(2):85–100.
Ow YP, Green DR, Hao Z, Mak TW. Cytochrome c: functions beyond respiration. Nat Rev Mol Cell Biol. 2008;9(7):532–42.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72.
Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ. 2018;25(3):486–541.
Kuang F, Liu J, Tang D, Kang R. oxidative damage and antioxidant defense in ferroptosis. Front Cell Dev Biol. 2020;8:586578.
Tang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021;31(2):107–25.
Vital P, Castro P, Ittmann M. Oxidative stress promotes benign prostatic hyperplasia. Prostate. 2016;76(1):58–67.
Ghoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD, et al. Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer. Cancer Res. 2021;81(6):1583–94.
Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147(4):728–41.
Li J, Liu J, Xu Y, Wu R, Chen X, Song X, et al. Tumor heterogeneity in autophagy-dependent ferroptosis. Autophagy. 2021;17(11):3361–74.
Hou W, Xie Y, Song X, Sun X, Lotze MT, Zeh HR, et al. Autophagy promotes ferroptosis by degradation of ferritin. Autophagy. 2016;12(8):1425–8.
Shen M, Li Y, Wang Y, Shao J, Zhang F, Yin G, et al. N(6)-methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells. Redox Biol. 2021;47:102151.
He W, Wang X, Zhan D, Li M, Wang Q, Liu J, et al. Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate. Clin Sci. 2021;135(1):167–83.
Liu J, Yin J, Chen P, Liu D, He W, Li Y, et al. Smoothened inhibition leads to decreased cell proliferation and suppressed tissue fibrosis in the development of benign prostatic hyperplasia. Cell Death Discov. 2021;7(1):115.
Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011;13(9):1016–23.
Chang SN, Lee JM, Oh H, Park JH. Glutathione peroxidase 3 inhibits prostate tumorigenesis in TRAMP mice. Prostate. 2016;76(15):1387–98.
Pastori D, Pignatelli P, Farcomeni A, Menichelli D, Nocella C, Carnevale R, et al. Aging-related decline of glutathione peroxidase 3 and risk of cardiovascular events in patients with atrial fibrillation. J Am Heart Assoc. 2016. https://doi.org/10.1161/JAHA.116.003682.
Chughtai B, Forde JC, Thomas DD, Laor L, Hossack T, Woo HH, et al. Benign prostatic hyperplasia. Nat Rev Dis Primers. 2016;2:16031.
Fleisher TA. Apoptosis. Ann Allergy Asthma Immunol. 1997;78(3):245–9.
Chen Q, Gong B, Almasan A. Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ. 2000;7(2):227–33.
Hata AN, Engelman JA, Faber AC. The BCL2 family: key mediators of the apoptotic response to targeted anticancer therapeutics. Cancer Discov. 2015;5(5):475–87.
O’Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4/6 inhibitors. Nat Rev Clin Oncol. 2016;13(7):417–30.
Wang Z, Fan M, Candas D, Zhang TQ, Qin L, Eldridge A, et al. Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression. Dev Cell. 2014;29(2):217–32.
Deep G, Oberlies NH, Kroll DJ, Agarwal R. Isosilybin B and isosilybin A inhibit growth, induce G1 arrest and cause apoptosis in human prostate cancer LNCaP and 22Rv1 cells. Carcinogenesis. 2007;28(7):1533–42.
Lee Y, Kim YJ, Kim MH, Kwak JM. MAPK cascades in guard cell signal transduction. Front Plant Sci. 2016. https://doi.org/10.3389/fpls.2016.00080.
Papatsoris AG, Papavassiliou AG. Molecular “palpation” of BPH: a tale of MAPK signalling? Trends Mol Med. 2001;7(7):288–92.
Gesmundo I, Di Blasio L, Banfi D, Villanova T, Fanciulli A, Favaro E, et al. Proton pump inhibitors promote the growth of androgen-sensitive prostate cancer cells through ErbB2, ERK1/2, PI3K/Akt, GSK-3beta signaling and inhibition of cellular prostatic acid phosphatase. Cancer Lett. 2019;449:252–62.
Kim YK, Kim HJ, Kwon CH, Kim JH, Woo JS, Jung JS, et al. Role of ERK activation in cisplatin-induced apoptosis in OK renal epithelial cells. J Appl Toxicol. 2005;25(5):374–82.
Freed E, Symons M, Macdonald SG, McCormick F, Ruggieri R. Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science. 1994;265(5179):1713–6.
Fu H, Xia K, Pallas DC, Cui C, Conroy K, Narsimhan RP, et al. Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science. 1994;266(5182):126–9.
Gan B. Mitochondrial regulation of ferroptosis. J Cell Biol. 2021. https://doi.org/10.1083/jcb.202105043.
Li J, Cao F, Yin HL, Huang ZJ, Lin ZT, Mao N, et al. Ferroptosis: past, present and future. Cell Death Dis. 2020;11(2):88.
Ingold I, Berndt C, Schmitt S, Doll S, Poschmann G, Buday K, et al. Selenium utilization by GPX4 Is required to prevent hydroperoxide-induced ferroptosis. Cell. 2018;172(3):409–22.
Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575(7784):688–92.
Mao C, Liu X, Zhang Y, Lei G, Yan Y, Lee H, et al. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature. 2021;593(7860):586–90.
Mizushima N, Levine B. Autophagy in human diseases. N Engl J Med. 2020;383(16):1564–76.
Liu C, Xu P, Chen D, Fan X, Xu Y, Li M, et al. Roles of autophagy-related genes Beclin-1 and LC3 in the development and progression of prostate cancer and benign prostatic hyperplasia. Biomed Rep. 2013;1(6):855–60.
Zhang N, Ji N, Jiang WM, Li ZY, Wang M, Wen JM, et al. Hypoxia-induced autophagy promotes human prostate stromal cells survival and ER-stress. Biochem Biophys Res Commun. 2015;464(4):1107–12.
Kang R, Tang D. Autophagy and ferroptosis—what’s the connection? Curr Pathobiol Rep. 2017;5(2):153–9.
Wu Z, Geng Y, Lu X, Shi Y, Wu G, Zhang M, et al. Chaperone-mediated autophagy is involved in the execution of ferroptosis. Proc Natl Acad Sci USA. 2019;116(8):2996–3005.
Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. Cell Res. 2019;29(5):347–64.
Wang X, Lu S, He C, Wang C, Wang L, Piao M, et al. RSL3 induced autophagic death in glioma cells via causing glycolysis dysfunction. Biochem Biophys Res Commun. 2019;518(3):590–7.
Song X, Zhu S, Chen P, Hou W, Wen Q, Liu J, et al. AMPK-mediated BECN1 phosphorylation promotes ferroptosis by directly blocking system X(c)(-) activity. Curr biol. 2018;28(15):2388–99.
Sekine Y, Osei-Hwedieh D, Matsuda K, Raghavachari N, Liu D, Furuya Y, et al. High fat diet reduces the expression of glutathione peroxidase 3 in mouse prostate. Prostate. 2011;71(14):1499–509.
Reddy AT, Lakshmi SP, Banno A, Reddy RC. Role of GPx3 in PPARgamma-induced protection against COPD-associated oxidative stress. Free Radic Biol Med. 2018;126:350–7.
Liu RF, Li J, Zhang J, Bai PD, Yang YF, Li W, et al. Crosstalk between apoptosis and autophagy in prostate epithelial cells under androgen deprivation. Exp Ther Med. 2018;15(3):2263–8.
We acknowledge the help of the staffs at Zhongnan Hospital of Wuhan University in completing the study.
This study was supported in part by National Natural Science Foundation of China N.82070780. The funding institutions did not provide assistance in designing research, collecting, analyzing and interpreting data and writing manuscripts.
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of Helsinki. Collection and treatment of all human specimens were carried out in accordance with the approved guidelines of the Ethics Committee at Zhongnan Hospital of Wuhan University (NO. 2021038). All animal protocols were approved by the Medical Ethics Committee for Experimental animals of Zhongnan Hospital of Wuhan University (No. ZN2021084).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Primer sequences used for qRT-PCR.
List of primary antibodies.
List of secondary antibodies.
Sense sequences of siRNA.
The heatmap plot of prostate DEGs (data from GSE119195 dataset). The heat map of 34 DEGs in 5 benign prostatic hyperplasia samples and 3 normal prostate samples. The legend color bar on the right side indicates the relation between scaled expression values and colors, and the red box highlights the GPX3 gene.
Effect of GPX3 knockdown on GPX3 protein expression, cell cycle-related proteins, cell apoptosis, apoptosis-related proteins and MAPK pathway-related proteins in BPH-1 and WPMY-1 cells A Immunoblot assay of GPX3 in BPH-1 and WPMY-1 after knockdown of GPX3. B Immunoblot assay of Cyclin D1, CDK4, CDK6, Cyclin B1 and CDK1 in BPH-1 and WPMY-1 after knockdown of GPX3. The results showed that GPX3 silencing only affected G0/G1 checkpoint proteins but not G2/M checkpoint proteins expression. C The inhibition of proliferation by isosilybin B from 0 to 100 μM, the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 67.72 and 41.38 μM, respectively. D Flow cytometry analysis of cell apoptosis. E Statistical analysis of apoptotic rate (%). Data showed that GPX3 silencing reversed the pro-apoptotic effect of isosilybin B, a G0/G1 phase apoptosis inducer, which confirmed GPX3 was involved in cell survival regulation by mediating G0/G1 phase. F The expression of Cyto-C in mitochondria or cytoplasm was detected after knockdown of GPX3. Tom 20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. G Immunoblot assay of apoptosis-related proteins (Cleaved-Caspase 9, Cleaved-Caspase 3, Bcl-2 and BAX) in BPH-1 and WPMY -1 after knockdown of GPX3. H Immunoblot assay of MAPK signaling pathway proteins in BPH-1 and WPMY -1 after knockdown of GPX3. GAPDH is used as loading control. *p < 0.05, **p < 0.01.
Effects of untreated group (con), transfected control (si-con) group and vector group on GPX3 expression, cell proliferation, cell cycle, apoptosis, MMP and MAPK pathway protein expression A Immunoblot assay of GPX3 in BPH-1 and WPMY-1 after different treatment. B Relative densitometric quantification of GPX3 protein in BPH-1 and WPMY-1 after different treatment. C The cell viability of BPH-1 and WPMY -1 after different treatment at different time points by CCK-8 assay; ns (red) conns (Biue) vs. si-con: con vs. vector. D Flow cytometry analysis of cell cycle. E Histogram showing percentage of cell populations at different stages of the cell cycle (%). F Flow cytometry analysis of cell apoptosis. G Statistical analysis of apoptotic rate (%). H The mitochondrial membrane potential level of BPH-1 and WPMY-1 cells was examined by JC-1 staining. The scatter plot of the flow cytometry analysis shows the distribution of JC-1 aggregates (Orange) and JC-1 monomer (Blue) cell population. I Histogram calculated the relative ratio of Orange against Blue fluorescence. J Immunoblot assay of MAPK signaling pathway proteins in BPH-1 and WPMY -1 after different treatment. K Relative densitometric quantification of MAPK signaling pathway proteins in BPH-1 and WPMY-1 after different treatment. GAPDH is used as loading control. ns means no significant difference.
Effect of GPX3 overexpression and U0126 on cell cycle, apoptosis and MAPK signaling pathway proteins in prostate cells A Immunoblot assay showed the efficiency of GPX3 overexpression in prostate cells at the protein level. B Immunoblot assay of proteins in relation to cell cycle in BPH-1 and WPMY-1 after overexpression of GPX3. C The expression of Cyto-C in mitochondria or cytoplasm was detected after overexpression of GPX3. Tom 20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. D Immunoblot assay of apoptosis-related proteins in BPH-1 and WPMY -1 after overexpression of GPX3. E Immunoblot assay of MAPK signaling pathway proteins in BPH-1 and WPMY -1 after overexpression of GPX3. F Immunoblot assay of apoptosis-related proteins (Cleaved-Caspase 9, Cleaved-Caspase 3, Bcl-2 and BAX) in BPH-1 and WPMY-1 after GPX3 overexpression or U0126 treatment. G Immunoblot assay of phosphorylated and total ERK1/2 as well as cell cycle related proteins (Cyclin D1, CDK4 and CDK6) in BPH-1 and WPMY-1 after GPX3 overexpression or U0126 treatment. GAPDH is used as loading control
Effect of GPX3 expression on oxidative stress and ferroptosis-related protein expression in prostate cells A Immunoblot assay of proteins in relation to ferroptosis (GPX4 and Nrf2) and OS (SOD2 and CAT) in BPH-1 and WPMY-1 after knockdown of GPX3. B The expression of GPX4, FSP1 and DHODH in mitochondria or cytoplasm were detected after knockdown of GPX3. Tom 20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. C The inhibition of proliferation by RSL3 from 0 to 4 μM, the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 0.71 and 0.75 μM, respectively. D Immunoblot assay of proteins in relation to ferroptosis (GPX4 and Nrf2) and OS (SOD2 and CAT) in BPH-1 and WPMY-1 cells by GPX3 overexpression or RSL3 treatment. E The expression of GPX4, FSP1 and DHODH in mitochondria or cytoplasm was detected after overexpression of GPX3. Tom 20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. GAPDH is used as loading control.
Effect of GPX3 overexpression on RSL3-mediated autophagy and AMPK-mTOR pathway proteins A Immunoblot assay of proteins in relation to autophagy (LC3B and Beclin1) in BPH-1 and WPMY-1 cells treated with GPX3 overexpression or RSL3. B The inhibition of proliferation by Rapa from 0 to 8 nM, and the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 2.67 and 3.24 nM, respectively. C The inhibition of proliferation by CQ from 0 to 50 μM, and the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 28.52 and 31.04 μM, respectively. D Immunoblot assay of autophagy associated proteins (LC3B and Beclin1) in RSL3-pretreated BPH-1 and WPMY-1 cells after GPX3 overexpression, CQ or Rapa treatment. E Immunoblot assay of proteins (AMPK, p-AMPK, mTOR and p-mTOR) in BPH-1 and WPMY-1 cells treated with RSL3 (0.5 μM) or GPX3 plasmid. F The inhibition of proliferation by GSK621 from 0 to 40 μM, and the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 23.21 and 25.33 μM, respectively. G Immunoblot assay of AMPK and p-AMPK in BPH-1 and WPMY-1 cells treated with 20 μM GSK621. H Immunoblot assay of proteins (LC3B and Beclin1) in RSL3-pretreated BPH-1 and WPMY-1 cells treated with GPX3 overexpression or GSK621 (20 μM). GAPDH is used as loading control.
Effect of GPX3 on cell cycle, apoptosis, ferroptosis and autophagy of prostate in vivo A Immunoblot assay of GPX3 protein in prostate of each treatment group. B Immunoblot assay of Cleaved-Caspase 9, Cleaved-Caspase 3, Bcl-2, BAX, Cyclin D1, CDK4 and CDK6 in prostate of Con, T, and T + TRO-treatment group. C Immunoblot assay of GPX4, Nrf2, SOD2 and CAT in prostate of each treatment group. D Immunoblot assay of LC3B and Beclin1 in prostate of each treatment group. GAPDH is used as loading control.
About this article
Cite this article
Li, Y., Zhou, Y., Liu, D. et al. Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate. J Transl Med 21, 575 (2023). https://doi.org/10.1186/s12967-023-04432-9
- Glutathione peroxidase 3
- Benign prostatic hyperplasia
- Mitochondria-mediated apoptosis
- Autophagy-related ferroptosis
- AMPK/ERK1/2 pathway
- AMPK/mTOR pathway


text:
Pemberton muslim girl personals
I married a muslim: katrina's incredible story the man was also a muslim at any time during your dating process did the spiritual roots of your life–ie. Sure enough, when you check out the forum on free muslim dating site, helahelcom, so what hope is there for the normal religious girls looking for love. Muslim porn videos members can watch all videos and we try to update the member area with new exclusive videos every month 18 year old muslim girl creampied wmv. This is what it's like to date as a young american muslim by don’t talk to the muslim girls, and muslim singles events a la speed dating are common. Watch muslim girls fucking porn videos for free, here on pornhubcom discover the growing collection of high quality most relevant xxx movies and clips no other sex tube is more popular.
Pemberton's best 100% free online dating site meet loads of available single women in pemberton with mingle2's pemberton dating services find a girlfriend or lover in pemberton, or just. That’s what you can expect when you date a muslim girl who carries her culture with her what men don’t get about dating muslim women. The challenges of young muslim girls to ignore the pressures of dating in the western world the muslim girl muslim girl problems: dating.
Watch free muslim girl porn videos on xhamster select from the best full length muslim girl xxx movies to play xhamstercom updates hourly. Muslim dating at muslimacom sign up today and browse profiles of women for dating for free. Albanian muslim matrimonial is a the best muslim matrimonial site where you can find muslim singles from around single albanian girl is seeking muslim. 8 things to expect when dating a muslim girl hesse a high proportion of muslim girls, the only thing one needs to know about “dating” a moslem women is.Live sex sex stories sex dating xxx rewards niqab covered muslim girl jungle fuck niqab covered muslim girl sexcom is made for adult by muslim porn. Islam russian women browse 1000s of russian dating profiles for free at russiancupidcom by joining that language of a body this art which each girl should own. The truth about dating muslim women muslim dating, muslim girls, muslim women dating skills required to date women from southeast asia. Can a non muslim guy date a muslim girl update cancel answer wiki 15 answers kenan Çelebi, sunni, hanafi, what are the perks of dating a muslim girl. Arab dating site with arab chat rooms arab women & men meet for muslim dating & arab matchmaking & muslim chat. Singlemuslimcom the world's leading islamic muslim singles, marriage and shaadi introduction service over 2 million members online register for free. In most of muslim countries number of girls are higher than boys so lots of girls who are not so pretty and how can i find muslim girls for dating update cancel. The guide to dating a muslim girl islam is in fashion while the word “terrorism” seems to be the first that comes to mind when you hear about muslims,. Black + muslim + woman i thought about how this would have played out totally differently if i was a white girl, dating ihssan tahir.
Besides, these women are as beautiful, as slavic girls check out any muslim dating site to see for yourself so why not try dating a muslim girl for a change. Single muslim women on dating: there was the muslim dating event at excel where, girls tend to find a middle path about their identity. Want to date a muslim girl and do not know how to start check our top tips of how to date a muslim woman. Are you looking for your thai muslim woman soulmate just browse loveawakecom dating site photo gallery of personals and find your girl within few minutes.
A white canadian atheist wants to get back together with his muslim girlfriend, but only through destroying her religion cappy shakes his head and explains. Best countries for dating arab muslim girls middle east may have some good looking women but we all know these most of the countries there are way too conservative and the girls are not. Don’t you also think that malaysian women are specialmeeting and dating these girls is learned from dating beautiful malaysian women muslim girl who.
Muslimfriends is an online muslim dating site for muslim men seeking muslim women and muslim boys seeking muslim girls 100% free register to view thousands profiles to date single muslim. Find the hottest muslim girls fucking porn videos on the planet at thumbzilla how do we know they're the hottest because the zilla is the fucking king.Send message

domain: sociology


text:
The government has announced that new legislation “The Parental Bereavement Leave and Pay Regulations” will be implemented in April 2020, giving parents two weeks paid leave upon the loss of a child.
The new regulations, known as “Jack’s Law”, come after 10 years of campaigning by his mother Lucy to improve care for parents who suffer the devastating loss of a child. The regulations apply to parents who suffer the death of a child under the age of 18, or a stillbirth from 26 weeks of pregnancy. Under the regulations, biological and adoptive parents will be entitled to two weeks paid leave irrespective of how long they have been employed. Parents who have been in employment for more than 26 weeks will also be eligible for statutory pay for this period.
The leave can be taken as a single block of two weeks or split into two separate weeks taken in the first 12 months after the child’s death, meaning parents can take it when they most need it.
The UK is the first country to introduce this measure; it’s just one of a set of reforms being introduced with the aim of making the UK the best place to work in the world.
The content of this article is correct as at the date of posting and is for information purposes only. Whilst we try and ensure it is accurate we do not warrant or guarantee that this is the case, nor do we accept any responsibility in the event that it is relied on. The information is not intended to be a substitute for legal advice which you are recommended to obtain.Back to News

domain: law


text:
Vote for one of four fiction works for our Twitter book club's book of the month.
Author Yiyun Li doesn't just study people on the subway—she studies her characters, unflinchingly imagining their gaze until she understands them fully.
The first published works from the creator of the best show on television were dark, philosophical pieces of fiction in The Atlantic.
Thirty Girls author Susan Minot says great writing—like T.S. Eliot's "The Love Song of J. Alfred Prufrock"—is a source of nourishment readers turn to again and again.
The Nobel Prize winner is a role model for writers looking to bridge the personal, domestic details of the short story with the global forces of history, author Kyle Minor says.
The Pulitzer Prize winner and former U.S. poet laureate, who died today at age 88, revealed to The Atlantic in 1992 and 2002 the ways education had influenced her work.
The lesson author Dorthe Nors took from Ingmar Bergman: It's not drugs, poverty, or wild lovers that make a great writer. It's discipline and time alone.
Discuss an acclaimed romance with our Twitter book club all this month.
Looking back on a troubled union, Jenny Offill's Dept. of Speculation nails the mundane intimacy and emotional complexity of married life after the "happily ever after."
Rebecca Mead, New Yorker staff writer and author of My Life in Middlemarch, shares what Eliot's Middlemarch taught her about love, marriage, and journalism.
Dismissing the classics' shortcomings as just a reflection of the era's norms mischaracterizes history and undermines the books' very relevance.
The Irish author's latest novel, The Guts, does what Doyle has done since he first published The Commitments: expertly weave sadness, tenderness, awkwardness, and pride into every page.
The actress's claims have horrified Orwell devotees, but if the book's romantic plot isn't convincing, it's only because the dystopian classic itself is flawed.
Author and journalist Jennifer Percy was a committed physics major until a Lawrence Sargent Hall story showed her a more satisfying way to approach life's complexities.
Author Ben Marcus says the beautiful but sorrowful strangeness of Kafka's "A Message from the Emperor" make it a perfect piece of writing.
The veteran author says Theodore Roethke's poetry is a reminder that sometimes you're hot, sometimes you're not.
Being a writer means knowing when to let go of bad ideas. And good ones.
Join our Twitter book club to read Eleanor Catton's astrological murder mystery.
New titles from Michael Cunningham, Maggie Shipstead, Hillary Clinton, and others
An imagined guide to successful self-promotion

domain: artistic


text:
For most of the past decade, February has been Audio Month at Paulson’s Audio & Video, Metro Detroit’s premier entertainment and smart home showroom.
This year, the celebration of sound is kicked up several notches to include better discounts on audio products throughout their showroom, and a special Music Matters event at the end of the month which will be predominantly virtual with some individual in-person elements for those who wish to visit them.
Audio Month – All February Long
The team at Paulson’s is knowledgeable and creative in finding the right fit for every client’s needs. Audio Month is an opportunity for anyone considering new audio for their home, office, home theater, and more to explore options, see and hear new products, and save. With discounts on speakers, components, audio electronics and accessories like wires, cables and power, it’s the best time of year to set up that perfect listening area or upgrade a home theater.
Music Matters – February 22-27
Audio Month will end with a bang when Paulson’s hosts Music Matters, a free event to showcase their diverse audio offerings for residential and commercial projects, indoor and outdoor spaces, from entry-level products to high-end.
Top audio vendors will be doing virtual keynote presentations via Zoom, including AudioQuest, Anthem, Bowers & Wilkins, Denon, GoldenEar, Marantz, McIntosh, Paradigm, Sonance and Sonos. Registration is required, and every attendee will receive an amenity box of gifts from Paulson’s and their brand partners.
For every virtual keynote attended, guests will receive one entry into a giveaway for a Sonos Move indoor/outdoor speaker, Sonance Patio Series outdoor audio system, Bowers & Wilkins vinyl collector’s box, and more.
Pick up your free ticket here: eventbrite.com/e/music-matters-week-2021-tickets-140130555263.

domain: entertainment


text:
You can raise your credit score, save enough cash for closing costs, and maintain accurate financial records. However, if you don’t have at least a 20 percent down payment when buying a house, you’ll have to pay private mortgage insurance.
Private mortgage insurance is required on all mortgage loans with a loan-to-value ratio greater than 80 percent. The annual premium for PMI — which is between 0.5 percent and one percent of the loan — is added to each mortgage payment. Therefore, if you get a mortgage for $100,000, and your mortgage insurance premium is one percent, you’ll pay $1,000 a year for PMI, or about $83 a month.
For the most part, private mortgage insurance is a win-win for both parties. It protects lenders against loss if a borrower defaults on the loan; and PMI puts homeownership within reach for borrowers with less than a 20 percent down payment.
Private mortgage insurance is costly for borrowers — but fortunately, it’s not permanent. Here’s a look at two ways to get rid of private mortgage insurance.
- Borrower-requested cancellation
Since PMI is only required by lenders when a homebuyer has less than a 20 percent down payment, you can ask your lender to cancel mortgage insurance once your property has 20 percent equity — but there are no guarantees.
There are several ways to build equity faster. For example, you can submit extra mortgage payments to reduce your principal balance. There’s the option of paying one half of your mortgage payment every two weeks, which is the equivalent of one extra mortgage payment a year. You can also pay a little more each month, and apply the extra payment to the principal only.
Additionally, home improvements can quickly raise your property’s value and eliminate mortgage insurance sooner. Home improvement projects that pay off include kitchen and bathroom remodels, room additions, and exterior improvements.
To request PMI cancellation, you’ll have to submit a request in writing and follow your lender’s guidelines. If your mortgage is current and your home’s value hasn’t declined, the bank may comply with your request. However, your lender will require a home appraisal before eliminating mortgage insurance. Appraisals cost between $300-$400, depending on the size of your home.
- Automatic PMI termination
The reality is that some borrowers can’t afford to pay down their mortgages early or spend money on costly home improvements. Don’t overly stress about mortgage insurance if you fall into this category. Be patient, and in a few years, your lender will automatically terminate PMI.
The Homeowner’s Protection Act (HOPA) makes it illegal for lenders to unnecessarily charge borrowers mortgage insurance. As a rule, lenders must automatically remove PMI on the date that your “mortgage balance is scheduled to reach 78 percent of the original value of the home” based on the initial amortization schedule, says Sara Millard, senior vice president and deputy general counsel at United Guaranty Corp.
So, even if your lender rejects your request to cancel mortgage insurance once your property has 20 percent equity, HOPA stops PMI from becoming a permanent or long-term expense.
Mortgage insurance isn’t cheap, and paying this premium every month will increase your mortgage payment. To avoid PMI, aim for a 20 percent down payment when purchasing a house. It’ll take longer to buy and you’ll have to make sacrifices, but it’s worth the effort. Besides, a 20 percent down payment not only eliminates mortgage insurance, it also helps you qualify for a better mortgage rate.
Filed Under: adjustable-mortgage-rates

domain: finance


text:
Local Life Krakow is about to select candidates for Best Places awards, dedicated to rewarding and encouraging excellence in products, service and ideas. You are helping us to make the best choice.
Pizzeria 00 has 2 votes. Click here to see the results.
You can vote once a day for your favourite place.
Pizzeria 00 is a cozy pizzeria with designer interior, serving the Neapolitan style pizza in casual atmosphere right in the centre of Old Podgórze district at Kalwaryjska. In Pizza 00, the dough for each pizza is prepared at least 48 hours in advance. Then, it slowly rises in cold temperature, for maximum flavour with a minimum use of fresh yeast. The slow rise makes the pizza dough easily digestible and very light for the stomach. When ready, the dough is garnished with simple, quality ingredients and baked in wood-fired oven for 90 seconds, according to the Pizza Napoletana regulations. Casual dining, good music and extraordinary pizza, the way it is done in Naples!
Editor & Krakow Local

domain: food


Engine OEM representatives discussed navigating challenges around support and spares when transitioning from old to new engine programs at Aviation Week Network’s Engine Leasing, Trading & Finance event in London.
GE Aviation, Pratt & Whitney and Rolls-Royce have expressed overall satisfaction with their new-generation engine programs despite some growing pains related to the entry-into-service phase and the transition from older programs to newer ones.
Despite Rolls-Royce’s satisfaction with the new engine types, there are some challenges to consider dependent on the size of the carrier awaiting new models to enter their fleets. “As engines and aircrafts have become more advanced technologically, the product is naturally more complex. This is especially daunting for smaller airlines rather than major ones as they tend to need a lot more support”, he says.
Collected and summarized from the source below by Ta Ngoc Diep:


text:
Sheet Pan Chicken and Mushrooms
The cooking coque et ecran samsung s8 and clean up are a breeze, and it’s amazingly flavorful!
What types of mushrooms do you recommendI like to use a variety of my favorite mushrooms, including shiitake, oyster, and sliced king trumpet coque s8 samsung mushrooms. Don’t coque paris samsung s6 edge worry: this dish still tastes great if you use only regular old white coque de telephone samsung note 2 and brown button mushrooms. enoki or brown coque note 10 samsung clamshell mushrooms), make sure you carefully coat them in the marinade and try not to break coque iphone 7 8 them up by accident. I normally add the sturdier mushroom varieties to the marinade first, and then add the more delicate ones at the end.
How do I cut the mushroomsMake sure you cut the mushrooms into roughly similar sizes so they finish cooking at the same time. I normally quarter large button mushrooms and adjust the size of the other coque samsung galaxy s7 walking dead mushrooms coque huawei nova accordingly.
Can I use another vegetable in place of coque silicone paillette samsung galaxy s6 edge mushroomsIf you have a mushroom allergy or you don’t quite have a pound of mushrooms, you can add some broccoli or cauliflower florets to the coque huawei p10 marinade and toss coque de samsung galaxie s7 them on the sheet pan. And if you absolutely samsung galaxy s8 plus coques 6 2 hate mushrooms might want to make some other recipe, ’cause this one’s all about the mushrooms!
Would chicken s6 edge coque samsung breast work in this recipeYes! Just make sure that you you don’t overcook them check that the coque 360 s7 samsung thickest part coque samsung galaxy s6 effet miroir of the chicken breast reaches between 150F and 160F and you’re good to go.
What do you serve with this sheet pan dinnerI like to make a big green salad, stir coque s7 samsung star wars fry some vegetables, heat up some cauliflower rice, or pressure cook a batch of Instant Pot Kale and Carrots. Pick a vegetable side dish that’s quick and easy enough to be finished by the time coques personnalisees iphone 5 5s se the chicken and mushrooms are ready.
Ready to make this super simple chicken and mushroom sheet pan dinnerServes 41 large shallot, thinly sliced3 tablespoons sherry vinegar or balsamic vinegar3 tablespoons extra virgin olive oil, avocado oil, melted ghee, or fat of choice1 tablespoon coconut aminos1 tablespoon coque samsung s7 amazon Red Boat fish sauce1 teaspoon Magic Mushroom coque samsung s8 Powder or Diamond Crystal kosher salt (if using fine grain salt or Morton’s kosher salt, use teaspoon)1 pounds boneless skinless chicken thighs1 pound assorted mushrooms, cut into the same size pieces cup minced fresh Italian parsley and/or chivesEquipment:Heat oven to 450F or 425F convection with the rack in the upper middle. As the oven heats up,combine the shallots, vinegar, extra virgin olive oil, coconut aminos, fish sauce, and coque samsung s5 mauve Magic Mushroom Powder (or salt) in a large bowl.
Stir well and then add the chicken to the marinade.
Shake off the excess marinade and transfer the chicken to a rimmed baking sheet. (Leave the marinade in the bowl, because you’re going to dump the mushrooms in next!)then transfer them to the rimmed sheet in a single layer.
Arrange the chicken, mushrooms, and shallots in a single layer on the sheet pan.
Pop the sheet pan dinner in the oven and roast for 25 to 35 minutes,rotating the coque samsung galaxy s8 plus fille tray 180 at the halfway point…


text:
Endwar Online Open Beta Trailer
After a long wait, Endwar Online officially launches into Open Beta.
Videos You May Enjoy
Articles You May Enjoy
- Mordheim: City of the Damned enters Early Access Phase 4
- Rogue Factor's turned based tactical RPG adaptation of Games Workshop's tabletop game, is receiving another content update for its E...
- Umbra Sets French Kickstarter Record
- We are absolutely flabbergasted at funding to such an incredible total on Kickstarter.
- Perfect World Entertainment Introduces CORE Connect
- Perfect World Entertainment has announced its new social networking platform, CORE Connect. CORE makes it easier for players to connect with o...

domain: news