Proteins involved in oxidative stress in leiomyoma tissues treated with ulipristal acetate
- Authors:
- Published online on: November 2, 2020 https://doi.org/10.3892/mmr.2020.11642
- Article Number: 4
Abstract
Introduction
Uterine leiomyoma is a benign tumor that affects 70–80% of women, which is the highest incidence amongst the benign tumors of the female reproductive tract (1,2). This neoplasm is responsible for serious health problems (3) and is characterized by abnormal extracellular matrix (4), altered phosphoproteins (5), dysregulated chaperones (6), and alterations in the proteins involved in cell migration (7).
The management of leiomyoma is an emerging clinical issue as the symptoms, which include poliabortivity, pain, and heavy uterine bleeding (3), can severely affect women and female fertility.
For the management of symptomatic leiomyoma, surgical techniques, such as myomectomy and hysterectomy, are traditionally used (8). However, due to the high morbidity of uterine leiomyoma, there is urgent need for a more effective therapeutic approach, capable of improving the quality of life of these patients (9).
For the treatment of symptomatic leiomyoma patients there are, in addition to surgery, pharmacological therapies based on gonadotropin-releasing hormone agonists and selective progesterone receptor modulators (10), that can effectively reduce the volume of the leiomyoma (11). Among them, ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) used preoperatively to control leiomyoma growth (12). It controls progesterone receptor activity and leads to apoptosis of leiomyoma cells. However, the exact mechanism of action of this drug is still unknown (13). Interestingly, Maruo et al (14) reported that treating leiomyoma cell cultures with UPA induced the expression of matrix metalloproteinase MMP1 and MMP8 and decreased the expression of TIMP, while treatment of myometrium cells with UPA did not lead to protein dysregulation.
The objective of our study was to identify dysregulated proteins in leiomyoma tissue treated with UPA and in untreated leiomyoma, to gain an insight into the mechanism of action of this drug.
Materials and methods
Uterine leiomyoma samples
Seven premenopausal patients treated with ulipristal acetate and seven untreated patients who underwent hysterectomy for symptomatic uterine leiomyomas were enrolled. To be eligible for this study, all patients signed a written informed consent form. The median age of the patients was 42 years, with a minimum of 35 and a maximum of 48 years.
All study procedures complied with the Declaration of Helsinki and were approved by the Ethical Review Board of the Institute for Maternal and Child Health-IRCCS ‘Burlo Garofolo’ (Trieste, Italy).
Oncologic patients, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) seropositive patients, and subjects with adenomyosis were excluded from the study. The patients were treated with UPA six months before surgery.
Tissue samples
All samples were collected from the central area of the leiomyomas, which were all benign and without atypical features. They were subserosal/intramural, with dimensions ranging from 4 to 6 cm. The samples were stored at −80°C until proteomic analysis was performed.
2-DE and image analysis
2-DE was performed as previously described (15). Briefly, clean samples of treated and untreated leiomyoma and myometrium (200 mg each) were homogenized in 1.2 ml of dissolution TUC buffer [7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 65 mM DTT and 0.24% Bio-Lyte (3–10)] with a protease inhibitor mix (2 mM PMSF, 1 mM benzamidine, 1 mM EDTA, 1 mM NaF). After vortex, the solutions were centrifuged at 10,000 × g at 4°C for 30 min, and the protein content of the supernatant was determined using the Bradford assay. For 2-DE analysis, 400 µg of proteins from each sample (leiomyomas treated with UPA and untreated leiomyomas) were used. ReadyStrip™ pH 4.0–7.0 and 3–10 NL 18-cm immobilized pH gradient (IPG) strips were rehydrated in a dissolution buffer at 50 V for 12 h at 20°C, and isoelectric focusing (IEF) was performed in a PROTEAN IEF Cell (Bio-Rad Laboratories). After IEF, serial incubations were performed: First, the IPG strips were equilibrated for 20 min in an equilibration buffer [6 M urea, 2% SDS, 50 mM Tris-HCl (pH 8.8), 30% glycerol and 1% DTT] and then in equilibration buffer containing 4% iodoacetamide instead of DTT. For the second dimension, the equilibrated IPG strips were transferred to a 12% polyacrylamide gel. After electrophoresis, the gels were fixed in 40% methanol and 10% acetic acid for 1 h, and then stained for 16 h with SYPRO Ruby. After SYPRO Ruby destaining, the gels were stained for 48 h with colloidal Coomassie Brilliant Blue. The 2-DE gels were scanned with a Molecular Imager PharosFX System. Two experimental replicates were performed for each sample. For all gels, molecular weights were determined by comparison with Precision Plus Protein Prestained Standards (Bio-Rad Laboratories, Inc.) covering a range from 10 to 250 kDa, and analyzed using the Proteomweaver 4.0 software (both from Bio-Rad Laboratories, Inc.).
Quantification of spot levels
2-DE image analysis was performed using the Proteomweaver 4.0 software, matching all gels from seven treated and seven untreated leiomyomas. to identify quantitative differences. Fold change was calculated as the ratio between the mean %V of the treated and untreated leiomyomas. Differences were considered significant when the ratio of the mean percentage relative volume (%V) (%V = V(single spot)/V(total spot)) was ±1.5-fold and satisfied the non-parametric Wilcoxon test (P<0.05).
Trypsin digestion and MS analysis
After excision from 2-DE gels, the spots were washed four times with 50 mM NH4HCO3 and acetonitrile (ACN; Sigma-Aldrich; Merck KGaA) and dried under vacuum in a SpeedVac system. Three microliters of 12.5 ng/µl sequencing grade modified trypsin (Promega Corporation) in 50 mM NH4HCO3 were added for gel spot digestion; samples were digested overnight at 37°C. Peptide extraction was performed with three changes of extraction solution of 50% ACN/0.1% formic acid (FA; Fluka), peptide mixtures were dried under vacuum and stored at −20°C until mass spectrometry (MS) analysis.
For MS analysis, samples were dissolved in 12 µl of 3% ACN/0.1% FA, and 4 µl of each sample were analyzed by LC-MS/MS on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Inc.) coupled with a nano-HPLC Ultimate 3000 (Dionex; Thermo Fisher Scientific, Inc.). Peptides were separated in a 10 cm pico-frit column (75 µm ID, 15 µm Tip; New Objective) packed in-house with C18 material (Aeris Peptide 3.6 µm XB-C18, Phenomenex). H2O/FA 0.1% and ACN/FA 0.1% were employed as eluents A and B, respectively, and peptides were analyzed at a flow rate of 0.25 µl/min using a linear gradient of eluent B from 3 to 40% in 20 min. A Data Dependent Acquisition (DDA) was used: A full scan between 300 and 1,700 Da was performed at high resolution (60,000) on the Orbitrap. The ten most intense ions were selected for CID fragmentation and acquisition of MS/MS data in low resolution in the linear ion trap.
Raw data files were analyzed with the Proteome Discoverer 1.4 software package (Dionex-Thermo Fisher Scientific, Inc.) and searched with the Mascot Search Engine (version 2.2.4, Matrix Science). Spectra were searched against the human section of the Uniprot database (version July 2018) using the following parameters: Enzyme specificity was set to trypsin with 1 missed cleavage allowed, precursor and fragment ions tolerance were 10 ppm and 0.6 Da, respectively. Carbamidomethylcysteine and oxidation of methionine were selected as fixed and variable modifications, respectively. Proteins were considered as positive hits if at least 3 unique peptides for each protein were identified with high confidence (FDR <0.01%).
Western blotting
Protein extracts (50 µg) used for 2-DE were separated by 12% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was blocked by treatment with 5% dry milk in TBS-Tween 20. After milk saturation, the membrane was incubated overnight at 4°C with 1:300 diluted primary rabbit polyclonal antibody against PDIA3, 1:300 diluted primary rabbit polyclonal antibody against HSPB1, 1:5,000 diluted primary rabbit polyclonal antibody against ACTG2. The membrane was washed three times in TBST for 10 min and then incubated for 90 min at 4°C with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Sigma-Aldrich; Merck KGaA) at 1:3,000 dilution. Protein expression was visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc.), and the intensity of the signals was quantified by VersaDoc Imaging System (Bio-Rad Laboratories, Inc.). No technical replicates were performed for each sample (5 cases and 5 controls were analyzed). The intensities of the immunostained bands were normalized with the protein intensities measured by Red Ponceau (Sigma-Aldrich; Merck KGaA) from the same blot.
Ingenuity pathway (IPA) analysis
Dysregulated proteins identified by MS in the treated and untreated leiomyomas, were analyzed by IPA (Qiagen GmbH) with a P-value <0.05 indicating a statistically significant value. Selected genes were used to generate bio-functions. For the filter summary, only associations with high (predicted) confidence or that had been experimentally observed, were considered.
Statistical analysis
Statistical analyses were carried out with the non-parametric Wilcoxon signed-rank test for matched samples for both 2-DE and western blotting data. P<0.05 was considered to indicate a statistically significant difference. All the analyses were conducted with Stata/IC 14.1 for Windows (StataCorp LP).
Results
Proteomic studies
In this study, we performed comparative proteomic analysis of leiomyoma tissues treated and untreated with ulipristal acetate with the aim of generating 2-DE reference maps and identifying dysregulated proteins. Gel-pairs correlation analysis highlighted an average matching efficiency of ~80%. Fig. 1 shows the 5,000 protein spots detected on the gel with ReadyStrip pH 4.0–7.0 and 6.0–10.0, for both types of proteomes. As reported in Table I, 13 protein spots were significantly upregulated (>1.5-fold), and 17 were significantly downregulated (<0.6-fold) in treated and untreated leiomyomas. Mass spectrometry analysis was applied to identify the 30 proteins modulated by the treatment, searching the MS/MS data against the human section of the UniProt database.
 | Table I.Dysregulated proteins identified by mass spectrometry in treated compared with untreated leiomyoma. |
Western blotting study of altered proteins
In this study, we validated the downregulation of PDIA3, HSPB1, and ACTG2. Following bioinformatic analysis, we selected HSPB1, an optimal candidate due to its involvement in the inhibition of the synthesis of reactive oxygen species, and PDIA3 and ACTG2, for which antibodies are commercially available. Fig. 2 shows a quantitative comparative western blot analysis of PDIA3, HSPB1, ACTG2 expression in a cohort of five treated and five untreated leiomyoma tissue samples (previously analyzed through 2-DE). To normalize the results of our WB analysis, we determined the total protein content of each sample by Red Ponceau. The reason for this was that, according to our previous results (16) the proteins that are usually selected as encoded by housekeeping genes (i.e., β-actin, tubulin or GAPDH) are upregulated in leiomyoma and, thus, they are not adequate to be used as controls for normalization. Because we could not establish which proteins should be considered as housekeeping in our samples, we decided to apply a total protein content normalization method, as reported in several other publications (17,18).
Functional analysis
The core analysis with IPA was performed on the proteins identified by mass spectrometry, comparing treated and untreated leiomyomas. Fig. 3 shows the networks in which these proteins are most involved, i.e. the degradation of hydrogen peroxide and the synthesis of reactive oxygen species. The regulatory network that controls the degradation of hydrogen peroxide includes three of the proteins identified: HSBA1, PRDX6, SOD1. In particular, PRDX6 and SOD1 are associated with the activation of the degradation of hydrogen peroxide, while eight other proteins participate in the network responsible for the synthesis of reactive oxygen species. Among these, TAGLN, C3, ALB, PDRX6 are involved in the activation of the synthesis of reactive oxygen species, while HBA1, APOA1, SOD1, HSPB1 are implicated in the inhibition of synthesis of reactive oxygen species. PRDX6 is linked both to the degradation of hydrogen peroxide and to the synthesis of reactive oxygen species.
Discussion
In response to the urgent need for an efficient therapeutic approach for leiomyomas, selective progesterone receptor modulators (SPRMs) might offer a valid option (19,20). This study represents a first attempt to characterize the proteomic profile of leiomyomas after treatment with UPA, in order to identify dysregulated proteins correlated with the drug's mechanisms of action.
Through a combination of 2-DE and mass spectrometry, we identified 30 proteins dysregulated by the pharmacological treatment. Subsequent western blot analysis confirmed the altered expression of three of these proteins: HSPB1, PDIA3, and ACTG2.
Peroxiredoxin 6 (PDRX6), an enzyme involved in the reduction of water and alcohol, exerts its enzymatic activity on H2O2, fatty acids and phospholipid hydroperoxides (21,22).
In our study, PDRX6 was up-regulated (in treated leiomyoma samples) suggesting that its detoxifying function in cancer cells might bear some relation with the mechanisms of action of the drug.
SOD1, which contributes to the destruction of toxic free radicals (23,24) activating the degradation of hydrogen peroxide, also appeared to be up-regulated in treated leiomyoma. Considering that SOD1, as well as PDRX6, are involved in the destruction of free radicals protecting the cell from oxidative stress.
APOA1 is involved in the mechanism of reverse transport of cholesterol from peripheral tissues back to the liver for excretion in the bile (25). Interestingly, it has been shown that APOA1 overexpression can improve abnormal lipid metabolism in non-alcoholic steatohepatitis (NASH). This protective effect is due to its ability to decrease the levels of reactive oxygen species and suppress COX-2-induced inflammation in hepatocytes (26,27). In our study, this protein was down-regulated and this effect could bear some connection with the activity of the drug on leiomyoma cell.
HSPB1 is a molecular chaperone capable of preventing the aggregation of misfolded proteins, an event which is easily triggered/induced in a variety of different types of cancer cells, following treatment with Erastin, a ferroptosis-inducing agent (28,29).
The phosphorylation of HSPB1 reduces the iron-mediated production of lipid reactive oxygen species, thus conferring cancer cells protection against ferroptosis (30,31).
The disruption of HSPB1 expression following UPA treatment, suggests that there may be a correlation between production of lipid reactive oxygen species, ferroptosis, and drug treatment.
TAGLN is a cytoskeletal protein of the calponin family that has been shown/suggested to contribute to replicative senescence (32,33). Downregulation of TAGLN in leiomyomas treated with UPA, induces the synthesis of reactive oxygen species, causing an increase of these reactive species inside the cell.
To our knowledge, this is the first study that applies proteomics to investigate the mechanisms underlying the effectiveness of the treatment of leiomyoma with ulipristal acetate. Using bioinformatic tools, we identified several proteins associated with oxidative stress, suggesting that the mechanisms of action of the drug may be related to its effect on oxidative stress The main limitation of the study is that it doesn't address the functional aspects of the mechanisms of action of UPA. Another limitation of the study is the lack of technical replicates of western blotting.
However, in terms of clinical application, our study offers the advantage of addressing directly the mechanisms involved not only in hormonal metabolisms but also in protein expression, with the aim of identifying new and more effective molecules.
We need to clarify that the aim of our study was to investigate the implication, in terms of oxidative stress, of using ulipristal acetate in leiomyoma from a biochemical point of view. We believe that our findings could direct further studies toward the research for new molecules (in addition to the ulipristal acetate, which has been currently removed from the market) with a specific target to reduce oxidative stress. The ulipristal acetate has been removed from market under EMA (European Medicines Agency) recommendation due to rare side effects to the liver.
In conclusion, by analyzing the leiomyoma tissue of patients treated with UPA and comparing their proteomic profiles with samples obtained from patients receiving no treatment, we found some markers of interest. Several proteins involved in oxidative stress were down- or up-regulated following UPA treatment. Since oxidative stress is critical in a variety of conditions, including cancer, the ability of UPA to modulate this event is highly promising. Therefore, we believe that additional studies would help better understand the role of UPA in the treatment of leiomyoma.
Acknowledgements
The authors would like to thank Dr Alessandra Knowles (Institute for Maternal and Child Health-IRCCS ‘Burlo Garofolo’, Trieste, Italy) and Dr Martina Bradaschia (Institute for Maternal and Child Health-IRCCS ‘Burlo Garofolo’, Trieste, Italy) for the English revision of the manuscript.
Funding
This research was funded by the Italian Ministry of Health (grant no. RC 27/17)-Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
BU, FS, FR, SB and GR conceived and designed the experiments. YCCDS, MA, BG, CF and EA performed the experiments. BU, DL, GDL, GS, GA and LM analyzed the data. BU, LM, FR, SB, FS and GR wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All study procedures complied with the Declaration of Helsinki and were approved by the Institutional Review Board of the Institute for Maternal and Child Health-IRCCS ‘Burlo Garofolo’ (Trieste, Italy) (RC 27/2017). All patients signed a written informed consent form.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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