E2, phorbol myristate acetate (PMA; cat. no. PI585-1 mg), ionomycin calcium salt (cat. no. IO634), cisplatin (cat. no. 479306), sulfuric acid (cat. no. 7664-93-9), phosphotungstic acid hydrate (cat. no. 12501-23-4), 2-thio-barbituric acid (TBA; cat. no. 504-17-6) and metformin were purchased from Calbiochem (cat. no. D150959; Merck KGaA). 1-[4-(6-bromobenzo[1,3](8)dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G-1; cat. no. 10008933) was obtained from Cayman Chemical Company. DMEM, charcoal stripped fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Thermo Fisher Scientific, Inc.). mitoCapture™ (cat. no. K250) was acquired through BioVision, Inc., and 1-butanol and Baker Analyzed™ A.C.S. were from Avantor, Inc.

Cervical carcinoma cell lines were obtained from the American Type Culture Collection, including HeLa [positive to human papillomavirus (HPV) 18], SiHa (positive to HPV 16) and C33A (negative to HPV). Non-tumorigenic HaCaT was cultured as mock control; cells maintained normal properties of differentiation and were kindly provided by Dr. Petra Boukamp (German Cancer Research Center; DKFZ). Cell lines were tested and authenticated by the Multiplexion's Multiplex Cell Line Authentication tests in September 2018 (www.multiplexion.de).

Cells were cultured in DMEM containing 1% antibiotic [penicillin G (10,000 U/ml) and streptomycin (10,000 µg/ml)], amphotericin B (250 µg/ml) and 10% of charcoal-stripped FBS at 37°C in a humidified atmosphere with 5% CO₂. Cells were grown until reaching 70-80% confluence. Three to four passages were achieved before the experiments were performed.

Cell lines were cultured on 8-well slides (cat. no. 177402; Sigma-Aldrich; Merck KGaA) at 5×10³/well for HeLa and SiHa or at 10×10³/well for C33A and HaCaT. Cells were stimulated with E2 (10 nM), as previously determined (3) and incubated for 24 h. Staining was performed using the MitoCapture™ staining according to the manufacturer's instructions and examined under a fluorescence microscope coupled to a digital camera (magnification, ×10 and ×40). In healthy cells, dye aggregates and stains the mitochondria red; in abnormal cells, the mitochondrial membrane potential collapses and the dye remains in the cytoplasm in its green fluorescent monomeric form. As in recent years evidence suggested that low concentrations of chemotherapeutic agents, like cisplatin, facilitate malignancy of carcinoma cells (14,15), cisplatin (0.5 µg/ml) was used as control of cellular and mitochondrial damages and were compared with a basal control (no treatment). The evaluation of the mean optical density was determined using Image-pro Plus 6.0 (Media Cybernetics, Inc.) based on the red staining; data were normalized to the fluorescence of vehicle-treated cells. Each sample was tested by triplicate in three independent experiments.

The effect of E2 (10 nM) on intracellular Ca²⁺ concentration was measured using the Fluo-4 Calcium kit (cat. no. F36206; Molecular Probes; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The supernatant of the cells was obtained 48 h after applying E2. Ionomycin (750 ng/ml) and PMA (10 ng/ml) stimuli were applied for 5 min at 37°C before the analysis of the samples in the spectrophotometer. Excitation and emission wavelengths were 494 and 516 nm, respectively. Readings were recorded every minute over 16 min. G1 (1 µM) was used as a positive control alone or in combination with PMA (10 ng/ml) and ionomycin (750 ng/ml) and were compared with a basal control.

Cells (5×10³) were seeded in 96-well plates with DMEM containing 10% charcoal-stripped FBS. Cells were stimulated with E2 (10 nM) and/or metformin (10 mM) and were compared with a basal control. After 48 h, nitrite concentrations were measured using the Measure-iT™ High-Sensitivity Nitrite assay (Invitrogen; Thermo Fisher Scientific, Inc.). The amount of nitrite is extrapolated to NO concentrations following the manufacturer's instructions. Reagent (Measure-iT™ nitrite quantitation reagent; 2.0 ml of 100X in 0.62 M HCl; 1:100; 100 µl) was added to the wells and samples (10 µl) were added. After 10 min at room temperature, 5 µl developer solution was added and the fluorescence was measured using a spectrophotometer (excitation and emission wavelength, 360 and 460 nm, respectively).

The levels of lipid peroxides were calculated via determining the malondialdehyde (MDA) concentrations. Cells were stimulated with E2 (10 nM) and/or metformin (10 mM) and were compared with a basal control. After 48 h, 300 µl cell supernatant was mixed with 2 ml sulfuric acid (N/12), 0.3 ml 10% phosphotungstic acid and 1 ml 0.6% TBA. The mixture was placed in a boiling water bath at 95°C for 1 h, followed by cooling at room temperature. Then, 1.3 ml of n-butane was added, mixed vigorously and centrifuged at 1,400 × g for 15 min. The absorption of the organic phase was recorded at 534 nm.

Cell supernatants were used to determine iron concentrations and to evaluated ferritin levels. Cells were stimulated with E2 (10 nM) and incubated for 48 h and were compared with a basal control. The VITROS Fe slide method was performed using the VITROS Fe slides and the VITROS Calibrator 4 kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The difference in the density of the reflectance after 1-5 min is proportional to the concentration of iron in the samples. The determination of the ferritin levels was determined using the same samples with the IMMULITE^® kit (cat. no. L2KFE2; Siemens Healthineers) following the manufacturer's instructions.

Cells were stimulated with E2 (10 nM) and/or metformin (10 mM) and were compared with a basal control. The supernatant was obtained after 48 h. Glucose concentrations were measured in the BS-120 Clinical Chemistry analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) using the glucose oxidase method (COD11803; BioSystems S.A.) following the manufacturer's instructions. Glucose in the samples generated a colored complex that was quantified spectrophotometrically at 500 nm.

Cells were stimulated with E2 (10 nM) and/or metformin (10 mM) and were compared with a basal control. The supernatant was obtained after 48 h. The VITROS LAC Slide method was performed using the VITROS LAC slides and the VITROS Calibrator kit 1 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Lactate in samples is oxidized to pyruvate and hydrogen peroxide (H₂O₂). The generated H₂O₂ oxidizes 4-aminoantipy-rine, which forms a complex with 1,7-dihydroxynaphthalene. Absorbance at 540 nm was determined spectrophotometrically.

Cells were stimulated with E2 (10 nM) and incubated for 48 h and were compared with a basal control. Total RNA was extracted from SiHa and HeLa using the RNeasy Plus Mini kit (cat. no. 74136; Qiagen, Inc.), according to the manufacturer's instructions. RNA was quantified measuring the absorbance at 260/280 nm.

Transcriptome sequencing was performed using RNAseq technology. The samples (SiHa and HeLa cells) stimulated with E2 were sent to the Genome Institute (Novogene Corporation, Inc.), where the Illumina HiSeq 4000 system was used; the sequencing strategy included a 250-300 bp insert cDNA library, HiSeq platform, paired-end 150 and 50 bp single-end. The RNA quality was measured and selection criteria were: Amount ≥1 µg; volume ≥20 µl; concentration ≥50 ng/µl; RNA integrity number value ≥7; 28S/18S >1; no degradation or pollution. Subsequently, fragmentation of mRNA and synthesis of cDNA of the first and second chain was performed. For the mRNA isolation and fragmentation, 200 ng total RNA was purified using oligo-dT beads and poly (A)-containing mRNA were fragmented into small pieces with Fragment Buffer. For the cDNA synthesis, first-strand cDNA was generated using the First Strand Master Mix and Super Script II (Invitrogen; Thermo Fisher Scientific, Inc.) for 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. Then Second Strand Master Mix was added to synthesize the second-strand cDNA (16°C for 1 h).

cDNA fragments were purified, resolved for the final repair and the adenine single nucleotide addition was performed. cDNA fragments were ligated using adapters and fragments with a suitable size were selected for PCR amplification. The library construct was prepared for the quantification and qualification of libraries. For the end repair, the purified fragmented cDNA was combined with End Repair Mix and incubated at 30°C for 30 min. The end-repaired DNA was purified with Ampure XP beads (Thermo Fisher Scientific, Inc.) and incubated with A-Tailing Mix at 37°C for 30 min. For the adapter ligation, the adenylated 3'-DNAs were combined and RNA Index Adapter and Ligation Mix was added at 30°C for 10 min. The end-repaired DNA was purified with Ampure XP beads. Several rounds of PCR amplification with the PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments and products were purified with the Ampure XP beads.

For the validation of the library, quantitation was performed in two ways: (i) Determination of the mean molecule length using the Agilent 2100 bioanalyzer (Agilent Technologies, Inc.); (ii) reverse transcription-quantitative PCR (TaqMan Probe; Illumina, Inc.). Library sequencing was performed as follows: (i) Amplification within the flow cell on the Bot instrument for cluster generation (HiSeq^® 4000 PE Cluster Kit; Illumina, Inc.); and (ii) loading of the clustered flow cell onto the HiSeq 4000 Sequencer for paired-end sequencing (Illumina; Inc.) with recommended read lengths of 100 bp.

Downstream analysis was performed using a combination of programs, including FLEXBAR version 2.5 for filtration (16). Alignments of readings with index hg38 were parsed employing Kallisto version 0.44.0 (17). Differential expression analysis between groups (two biological replicates per group) was performed using the DESeq2 R package version 1.24.0 (18). The resulting P-values were adjusted applying the Benjamini-Hochberg approach to control the false discovery rate (FDR). Genes with an adjusted P<0.05 were considered as differentially expressed. An absolute fold change of 1 was considered in the significant differential expression. Gene Ontology (19) and KEGG enrichment (20,21) were implemented by the Cluster Profiler R package (https://www.R-project.org). A corrected P<0.05 was considered significantly enriched by differential expressed genes (DEGs).

Cell proliferation of HeLa, SiHa, C33A and HaCaT cells was assessed using the xCELLigence platform (Roche Applied Science). Cells were cultured at 0.25×10⁴/well in 96-well E-plates (cat. no. 058416; ACEA Biosciences, Inc.) and were introduced into the xCELLigence reading station. After 4 h, cells were stimulated with 10 nM E2, 10 mM metformin, 1 µg/ml cisplatin alone or in combination (E2+metformin, E2+cisplatin and E2+metformin+cisplatin), a vehicle-stimulated (DMEM+ethanol) cells and were compared with basal control. The proliferation rate was determined in real time every 30 min over 72 h. Cisplatin stimulation was used as a cell death control. Three independent experiments were performed with three replicates in each case.

Statistical analyses were made using SPSS 20 (IBM Corp.) and GraphPad Prism 6 (GraphPad Software, Inc.). Experiments were performed in triplicate and tested in three independent trials. One-way ANOVA with Bonferroni correction was used to determine significance following Levene's test for homogeneity of variances. Data are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

The effect of E2 on the mitochondrial permeability of cervical cancer and HaCaT cell lines was evaluated using a selective dye. In HeLa, SiHa and C33A, E2 significantly decreased the mitochondrial permeability compared with the basal control (P<0.0001; Fig. 1). The green color indicated that the dye accumulated in the cytoplasm. In HaCaT cells, the opposite effect was observed with increased mitochondrial permeability following E2 treatment as suggested by the red color (P<0.0001).

E2 had no significant effect on the calcium concentrations in cervical cancer cells or HaCaT compared with the basal control (P>0.05; Fig. 2). The use of ionomycin plus PMA significantly increased the ion concentration compared with the basal control (HeLa, P=0.003; SiHa, P=0.001; C33A, P=0.001). Exposure to E2, ionomycin and PMA significantly decreased the calcium concentration in SiHa and HeLa compared with the associated control (P<0.05), suggesting that E2 altered the calcium signaling pathway. In C33A and HaCaT, E2 did not significantly modify the cellular response to stimulation with ionomycin plus PMA (P>0.05). G1, a selective agonist of the ER in the membrane (GPR30) associated with non-genomic effects, such as calcium mobilization, was used as positive control. E2 and G1 exhibited similar behaviors in all cell lines.

To evaluate the participation of E2 in other mechanisms involved in the mitochondrial activity, we analyzed whether E2 modulated oxidative stress. First, we determined the effect of E2 on the concentration of NO in cervical cancer cells, as well as in HaCaT. E2 significantly decreased NO levels in HeLa, SiHa and C33A compared with the basal control (P=0.0002, P=0.0014 and P=0.0003; respectively; Fig. 3). This effect was significantly reversed when treating cells with metformin and E2 (P<0.05). In HaCaT cells, no differences were observed following treatment with E2, metformin or a combination of these compared with the control.

Furthermore, we analyzed the concentration of MDA, which is a final product of the lipid peroxidation and is used as a direct indicator of cell damage (22). E2 induced a significant decrease in lipid peroxidation in HeLa (P=0.0004) and SiHa (P=0.0005; Fig. 4) compared with the basal control. In C33A, the effect was the opposite and lipid peroxidation was significantly increased following E2 treatment (P=0.0002). Treatment with metformin significantly alleviated E2 effects in HeLa and C33A cells (P<0.05), but no significant effects were observed in SiHa (P>0.05). In HaCaT cells, no significant differences were observed following treatment with E2, metformin or a combination of these compared with the control.

The role of iron in lipid peroxidation has been associated with a form of cell death other than apoptosis or necrosis. Ferritin binds to iron and this complex is vital in controlling ROS generation (22). E2 stimulus did not significantly modify intracellular iron or ferritin levels in cervical cancer cells compared with the basal control (P>0.05; Fig. 5). In HaCaT, intracellular iron was significantly decreased following E2 treatment compared with the basal control (P=0.007).

The mitochondrial bioenergetics dysfunction favors the activation of the glycolytic pathway in different types of tumors (13). In HeLa, SiHa and C33A supernatants, there was a significant decrease in glucose levels following E2 stimulus (P=0.0001, P=0.002 and P=0.0003; respectively; Fig. 6), suggesting that the cells increased the uptake of glucose. A similar significant decrease was observed using metformin treatment (P<0.01). The combination of both treatments showed glucose levels similar to those observed in the basal control (P>0.05). It was hypothesized that the hypoglycemic effect of metformin was associated with E2. Furthermore, E2 significantly increased lactic acid levels in HeLa and SiHa compared with the basal control (P=0.0001 and P=0.004; respectively). The effects were more pronounced with metformin or a combination treatment with E2 (P<0.001).

Considering that E2 modified the metabolism of the cervical cancer cells, we analyzed the effects of E2 on the expression of molecules associated with metabolic pathways using the RNA sequencing technology. The total number of DEGs in SiHa following E2 treatment was ~3-fold higher than that in HeLa cells (Fig. 7). E2 stimulus had an impact on various metabolic pathways, including the upregulation of genes involved in the synthesis of proteins associated with the mitochondrial electron transport chain, which may affect OXPHOS and the energy metabolism (Table I). Additionally, upregulation of genes associated with glycolysis was observed (Table I and II), supporting the theory of the role played by E2 in the regulation of the Warburg effect associated with cervical cancer (Fig. 8). Defects in the metabolism of lipids, amino acids, proteoglycans, nucleotides, inositol phosphate, and choline have been reported in different types of cancer (23-25). E2 stimulation modulated various metabolic pathways (Table I). Table II summarized the DEGs shared by HeLa and SiHa.

E2 regulation of the differential expression of transcripts with functional roles in the processes of energy production was demonstrated by evaluating the impact on OXPHOS, glycolysis/gluconeogenesis, lipid processing, vitamins and cofactors, as well as on the metabolism of amino acids, nucleotides, phosphatidylinositol and choline (Table I). E2 regulated the expression of genes involved in mitochondrial complexes as follows: (i) Complex I, NDUFAB1, NDUFA4, and NDUFS5 upregulated; (ii) complex III, UQCRB and UQCRH upregulated; (iii) complex IV, COX6C, COX5B, COX7C, COX7B and COX6A1 upregulated; (iv) complex V, ATP5F1A, ATP5F1E, ATP5F1B, ATP5MC2, ATP5MG, ATP5PB and ATP5PO upregulated, and TCIRG1 downregulated.

Absolute changes in DEGs ranged between −0.4985 and 0.2496. A total of 5 genes associated with glycolysis were modified in both HeLa and SiHa by E2 stimulus, including LDHA, LDHB, PGK1, TPI1 and PFKP (absolute changes, −0.1893 to 0.2598). Other gene alterations modulated by E2 include: (i) Metabolism and synthesis of glycans (n=10), with GAA, NDST1, SGSH, PIGQ, MGAT5B, GPAA1 and MGAT1 downregulated and EXT1, PIGK and TUSC3 upregulated; (ii) lipid metabolism (n=10), with MGAT1, AGPS, FASN, ACADVL, CAD and SPHK1 upregulated and CPS1, PTGES3, PLA2G4A and PPT1 downregulated; (iii) metabolism of amino acids and nucleotides (n=9); (iv) metabolism of inositol phosphate (n=4); and (v) metabolism of choline, vitamins and cofactors (n=3; Table II and Fig. 8).

A total of 12 signaling pathways were affected by E2, including hypoxia-inducible factor (HIF)-1, Ras, mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF), phospholipase D, 5'-AMP-activated protein kinase (AMPK), cGMP-dependent protein kinase (PKG), calcium, appeline, phosphatidylinositol, PI3K-Akt and pathways mediated by sphingolipid (Table III). It should be noted that of these, genes that affected HIF-1, Ras, MAPK, VEGF and phospholipase D pathways that were upregulated included PGK1, LDHA and PLA2G4A (absolute changes, 0.1606, 0.2167 and 0.2494, respectively). Decreased genes following E2 stimulus included PFKP (−0.1893), FASN (−0.5459), CAD (−0.3228), SPHK1 (−0.3164), PLCD3 (−0.5829), INPPL1 (−0.7263) and LAMA5 (−0.7294), corresponding to the AMPK, cGMP-PKG, calcium, sphingolipid-mediated, appeline, phosphatidylinositol and PI3K-Akt signaling pathways (Table III).

Fig. 8 outlines important DEGs regulated by E2 and those directly involved in mitochondrial function, metabolism and associated signaling pathways. The impact of E2 in the pathophysiology of cervical cancer was suggested to be associated with the dysregulation of the respiratory mitochondrial electron chain, increasing OXPHOS, and activating glycolysis and the pentose phosphate pathway (Warburg effect). E2 was also suggested to inhibit the degradation of amino acids and provide metabolites for the synthesis of nucleotides, nucleic acids and coenzymes. Furthermore, it was hypothesized that E2 stimulates the synthesis of fatty acids associated with steroids, leukotrienes and prostaglandins metabolites, while it reduces the β-oxidation of lipids. It was suggested that E2 reduces oxidized glutathione, which regulates the oxidative state of tumor cells.

Metformin is proposed as an antitumor drug (26). The most relevant mechanism of its activity is the regulation of cellular metabolism (27). The potential role of E2 in metformin-induced cell death in cervical cancer was assessed in this study. In cervical cancer cells treated with metformin a significant decrease in proliferation was observed compared with the basal control at incubation times >20 h (P=0.0001; Fig. 9). In HeLa, the effect of metformin was similar to that induced by cisplatin, thus metformin was suggested to be a potential anti-carcinogen. E2 treatment did not markedly alleviate effects exerted by metformin.