Journal: Journal of Cellular Physiology

Article Title: Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

doi: 10.1002/jcp.25272

Figure Lengend Snippet: Effects of nandrolone on R2C cell viability. Effects of treatment of R2C cells with different nandrolone doses (0–500 μM) for 48 h evaluated by MTT cell viability tests. Vertical axis: percentage of cell viability. Horizontal axis: nandrolone concentration, in µM. Basal: untreated cells. Data are presented as the mean ± SD of triplicate experiments.

Article Snippet: When cells reached approximately 80% confluence, they were treated for 48 h with different concentrations of nandrolone prepared by first dissolving Nandrolone Vetranal analytical standard (Sigma–Aldrich, St. Louis, MO) in absolute ethanol to obtain a working solution, and then in the culture medium without horse serum to reach the desired concentrations (0–15.6 μM).

Techniques: Concentration Assay

Journal: Journal of Cellular Physiology

Article Title: Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

doi: 10.1002/jcp.25272

Figure Lengend Snippet: Testosterone production. Testosterone production in cells treated with 3.9 and 15.6 μM nandrolone concentrations for 48 h. Vertical axis: testosterone levels (ng/ml). Horizontal axis: nandrolone concentration, in µM. Basal: untreated cells. Data are presented as the mean ± SD of quadruplicate experiments. * = P < 0.001.

Article Snippet: When cells reached approximately 80% confluence, they were treated for 48 h with different concentrations of nandrolone prepared by first dissolving Nandrolone Vetranal analytical standard (Sigma–Aldrich, St. Louis, MO) in absolute ethanol to obtain a working solution, and then in the culture medium without horse serum to reach the desired concentrations (0–15.6 μM).

Techniques: Concentration Assay

Journal: Journal of Cellular Physiology

Article Title: Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

doi: 10.1002/jcp.25272

Figure Lengend Snippet: Effect of nandrolone stimulation on steroidogenic proteins. (A) Representative cropped blots for StAR (30 kDa), CYP11A1 (60 kDa), HSD3B1 (42 kDa), and CYP17A1 (55 kDa). The gels were run under the same experimental conditions and β‐actin was used as internal control. (B) Relative expression levels of StAR, CYP11A1, HSD3B1, and CYP17A1. Vertical axis: arbitrary units (AU). Horizontal axis: nandrolone concentration, in µM. Basal: untreated. Data are presented as the mean ± SD of triplicate experiments. * = P < 0.05.

Article Snippet: When cells reached approximately 80% confluence, they were treated for 48 h with different concentrations of nandrolone prepared by first dissolving Nandrolone Vetranal analytical standard (Sigma–Aldrich, St. Louis, MO) in absolute ethanol to obtain a working solution, and then in the culture medium without horse serum to reach the desired concentrations (0–15.6 μM).

Techniques: Expressing, Concentration Assay

Journal: Journal of Cellular Physiology

Article Title: Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

doi: 10.1002/jcp.25272

Figure Lengend Snippet: Effect of nandrolone supplementation on StAR and CYP17A1 levels: confocal analyses. (A and B) Representative microphotographs of immunofluorescence stain for StAR (A) and CYP17A1 (B) in cells treated with 3.9 and 15.6 μM of nandrolone. Basal: untreated cells. Bar = 25 μm. (C and D) Representative histograms of the immunofluorescence quantification of the staining intensity for StAR (C) and CYP17A1 (D). The stain intensity was expressed as the mean pixel intensity (PI) normalized to the cross‐sectional area (CSA) using the Leica application suite advanced fluorescence software. Vertical axis: PI/CSA *100. Horizontal axis: nandrolone concentration, in µM. Data are presented as the mean ± SD of quadruplicate experiments. * = P < 0.001.

Article Snippet: When cells reached approximately 80% confluence, they were treated for 48 h with different concentrations of nandrolone prepared by first dissolving Nandrolone Vetranal analytical standard (Sigma–Aldrich, St. Louis, MO) in absolute ethanol to obtain a working solution, and then in the culture medium without horse serum to reach the desired concentrations (0–15.6 μM).

Techniques: Immunofluorescence, Staining, Fluorescence, Software, Concentration Assay

Journal: Journal of Cellular Physiology

Article Title: Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

doi: 10.1002/jcp.25272

Figure Lengend Snippet: Effect of nandrolone on StAR and CYP17A1 gene expression. qRT‐PCR evaluation of StAR and CYP17A1 gene expression after nandrolone stimulation at 3.9 and 15.6 µM. The graphs show the normalization with the reference genes, according to the Livak Method (2 ‐ΔΔCT ). Vertical axis: 2 ‐ΔΔCT . Horizontal axis: nandrolone concentration, in µM. Basal: untreated cells. Data are presented as the mean ± SD of triplicate experiments. * = P < 0.05.

Article Snippet: When cells reached approximately 80% confluence, they were treated for 48 h with different concentrations of nandrolone prepared by first dissolving Nandrolone Vetranal analytical standard (Sigma–Aldrich, St. Louis, MO) in absolute ethanol to obtain a working solution, and then in the culture medium without horse serum to reach the desired concentrations (0–15.6 μM).

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Effect of nandrolone on cell viability. ( A ) Phase-contrast images of cultured HepG2 cells in ± 80 μM nandrolone for 72 h. Scale bars, 100 μm. The shown optical micro-photographs on the left are representative of several independent biological replicates yielding similar results; digital magnifications of selected areas are also shown on the right panel. ( B ) Cell growth curves of HepG2 seeded at the same density in presence or absence of nandrolone and counted every 24 h at the indicated times; the values shown are means of three independent ± SEM time-courses for each condition (where not visible the error bar is within the size of the symbol); * P < 0.05, ** P < 0.01, *** P < 0.005 vs relative CTRLs. ( C ) Effect of nandrolone on cell viability assessed by MTS assay, as described in Materials and Methods. Cell viability is expressed as the percentage (%) of untreated cells. The data shown are means ± SEM of at least three independent experiments; * P < 0.05. ( D ) Apoptosis measurement of untreated and ND - treated cells. HepG2 cells were incubated with annexin V and propidium iodine (PI) and the internal-sample percentage of early apoptotic, late apoptotic and necrotic cells assessed by flow cytometry as detailed in Materials and Methods. The superimposed bar graph shows the average values ± SEM of three independent experiments; * P < 0.05. CTRL: ethanol-treated Control cells, ND: nandrolone-treated cells.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Cell Culture, MTS Assay, Incubation, Flow Cytometry

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Effect of nandrolone on cell cycle. ( A ) Cell cycle analysis of HepG2 nandrolone treated cells (ND) using ModFit software (Verity Software House). Histogram plots were shown on the right. Data, expressed as percentage of total events analyzed, are the means ± SD of three independent experiments; ** P < 0.01; *** P < 0.005. PI: propidium iodide. ( B ) Protein expression levels of key regulators of cell cycle progression, Cyclin D1, Cyclin E, Cdk1/2, p21 and p53, assayed by Western blotting in untreated and 80 μM nandrolone-treated cells for 72 h. Upper panel: a representative cropped immunoblot. Full-length blots are presented in Supplementary Fig. . Bottom bar histogram: densitometry analysis normalized to β-actin as means ± SEM of three independent experiment; ** P < 0.01; *** P < 0.001. A.U.: arbitrary unit.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Cell Cycle Assay, Software, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Metabolic flux analysis of Nandrolone-treated HepG2 cells. Representative oxygen consumption rates (OCR) ( A ) and extra cellular acidification rates (ECAR) ( D ) profile of HepG2 cells treated with vehicle (CTRL) or 80 μM nandrolone (ND), assayed by the Seahorse XFe96 Analyzer as described under Materials and Methods. Oligomycin (Oligo), FCCP, and Rotenone (Rot) were added at the indicated points in A . Glucose (GLC), Oligo and 2-deoxyglucose (2DG) were added at the indicated points in D . The histograms in B , E , show OCR and ECAR normalized to the protein content of the cells removed from each well at the end of the assay. OCR in B Basal, resting OCR; Oligo, OCR measured after the addition of the ATP synthase inhibitor oligomycin also referred as proton leak; FCCP, OCR measured after the addition of the uncoupler FCCP eliciting the maximal respiratory capacity. The OCR were corrected for the residual OCR measured after the addition of the CxI inhibitor rotenone (not shown). ECAR in E Glycolysis, resting ECAR, measured after the addition of glucose and corrected for the 2DG-insensitive ECAR; Glycolytic Capacity, ECAR measured after the addition of oligomycin and refers to the maximal glycolytic activity with the OxPhos inhibited; Glycolytic Reserve, difference between ECAR measured in the presence of oligomycin and under resting conditions. ( C) The histograms show Reserve Capacity, ATP turnover and maximal capacity computed as described in Materials and Methods. ( F ) Energy map obtained plotting the basal and maximal OCR and basal and maximal ECAR measured in A , D The values are means ± SEM of three independent experiment carried out in 3 technical replicates under each condition; * P < 0.05; ** P < 0.01; # P < 0.001.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Effect of nandrolone on the mitochondrial respiratory chain complexes. ( A) Activity of mitochondrial respiratory chain complexes. Cells were lysed and assayed by spectrophotometric assays under condition of saturating substrates as detailed in Materials and Methods. CxI, NADH dehydrogenase; CxII, Succinate dehydrogenase; CxIII, Coenzyme Q-cytochrome c oxidoreductase; CxIV Cytochrome c oxidase. The results are expressed as nmoles of substrate transformed/min/10 6 cells and the bars indicate the means ± SEM of 4 independent biological replicates for each conditions; # P < 0.001. ( B) Expression of the OxPhos complexes. Left panel: representative cropped immunoblot of the OxPhos complexes protein expression in untreated (CTRL) and Nandrolone-stimulated (ND) cells using a cocktail of specific antibodies as detailed in Materials and Methods, full-length blots are presented in Supplementary Fig. ; β-actin was used as loading control. Graph bars on the right show the average (±SEM) of data resulting from densitometric analysis of three independent blots; * P < 0.01.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Activity Assay, Transformation Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Nandrolone treatment results in unbalance of the cellular redox state of HepG2 cells. Nandrolone elevates ROS production in HepG2 cells. ( A,B) Flow-cytometric analysis of ROS production assayed by the fluorescent peroxide probe DCF-DA ( A ) and the mitochondrial superoxide anion probe MitoSox ( B ) in untreated (grey areas) and Nandrolone-stimulated (colored areas) cells. Panel on the left: representative flow-cytometric histogram plots. The bar graph in the insets shows the mean intensity of the DCF-DA and MitoSox-related fluorescence (MFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. * P < 0.05; ** P < 0.01. ( C , D) Representative LS C M imaging of ROS production in living HepG2 cells treated with nandrolone and assessed by DCF-DA ( C ) and MitoSox ( D ) probes respectively. An enlarged detail of the optical field (square) and rendering of nandrolone-treated cells is shown on the right of each panel (and rendered in false colors). The images are representative of three different preparations yielding similar results. The histograms on the right of panels C and D show quantification of the fluorescence/cell elaborated by the freeware software ImageJ ( https://imagej.nih.gov/ij/ ); the values are means ± SEM of at least ten different optical fields, each containing 30–50 cells, from 3 independent experiments; * P < 0.05; ** P < 0.01.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Fluorescence, Imaging, Software

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Nandrolone causes a shift toward an immature state (stem cell-like phenotype) in hepatoma cells. ( A) Flow- cytometric analysis of cancer stem cell surface marker CD133 after 80 μM nandrolone treatment. Representative dot-plots of untreated (CTRL) and nandrolone-treated (ND) cells; Graph bars on the bottom show the average (±SEM) of data resulting from quantification of percentage of CD133 positive cells and from normalized mean fluorescence intensity (MFI) of six independent biological experiments; * P < 0.05; ** P < 0.01. FSC-H: forward scatter height. ( B) Expression of stemness transcription factors resulted up-regulated after nandrolone treatment. Quantitative real‐time‐polymerase chain reaction analysis of transcripts for Myc, Lin28, Nanog , and Kfl4 in HepG2 treated cells is shown as histograms. The values are means ± standard error of the mean (SEM) of normalized transcript levels of six independent biological experiments, * P < 0.05; ** P < 0.01; # P < 0.001 versus untreated. ( C) Effect of nandrolone on HepG2-derived spheroid. Cells were grown for 3 and 7 days under condition of 3D culturing (see Materials and Methods) without (CTRL) and with 80 μM nandrolone (ND treated). The micrographs are representative of two independent experiments; below the “ND treated” panels the averaged spheroid area normalized to the respective CTRL is indicated. ( D) qRT-PCR analysis of the Sox2 transcript level in untreated and nandrolone-treated HepG2-derived spheroids, normalized to CTRL sample at the lower timepoint (i.e. 3 days). Experimental conditions as in panel C. Bars are averaged normalized values ± SEM of three independent preparations; * P < 0.05.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Marker, Fluorescence, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Antimycin A treatment elevates ROS production and induces an increase of CD133 positive HepG2 cells. ( A) Dose-dependence effect of antimycin A (Ant. A) on cell endogenous respiratory activities. HepG2 cells were exposed to the indicated concentrations of Ant. A and the relative oxygen consumption rate (OCR) was determined. OCR is expressed as the percentage (%) of untreated cells. ( B) Superoxide anion production was evaluated through flow-cytometry assessment of MitoSox Red fluorescence. HepG2 cells incubated with 10 nM Ant. A for 48 hours presented elevated levels of superoxide production (middle panel), reduced by treatment with 10 mM of the ROS scavenger NAC added 4 h before the analysis (upper and lower panel); unstained controls (UN) are also shown. The bar histogram ( C ) shows the mean intensity of the MitoSox-related integrated fluorescence (iMFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. * P < 0.05; ** P < 0.01. ( D) Representative dot-plots of flow cytometric analysis of cancer stem cell surface marker CD133 expression. Cells were incubated for 48 h with 10 nM Ant. A and 80 μM Nandrolone ± 10 mM of the ROS scavenger NAC added 6 h before the analysis. The bar histogram on the right shows the percentage of CD133 positive cells expressed as fold-change of the untreated cells (indicated by dashed line) and are means ± SEM of three independent experiments. * P < 0.05; ** P < 0.01 vs untreated.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: Flow Cytometry, Fluorescence, Incubation, Marker, Expressing

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Nandrolone triggers the gain of stemness in several primary non-cancerous cells model. ( A) Effect of nandrolone on colony formation ability of CD34 + HS/PC in vitro . Left panel: representative images of CFU-GEMM, CFU-GM and BFU-E scored under an inverted microscope (magnification 40X) are shown. Right panel: quantitative analysis of CFU-GEMM, CFU-GM and BFU-E derived by CD34 + cells isolated form human umbelical cord blood with or without 80 μM nandrolone. ND was added to methylcellulose based medium at time of plating and colonies were detected and enumerated after 14 days of culture as described in Material and Methods. Results shown represent the mean of ± SD of two independent experiment, * P < 0.05 vs CTRL. ( B) Effect of nandrolone on calcific deposition during osteogenic differentiation of dental pulp mesenchymal stem cell (DPSCs). Mineral matrix deposition was assayed by Alizarin Red (red staining) in DPSCs incubated with vehicle (CTRL) cells and cells treated with several doses of nandrolone after 21 days in osteogenic conditions. The graph on the right shows quantitative analysis of alizarin red staining carried out by a densitometric analysis (Image J software). ( C) Nandrolone treatment induces stemness genes expression during osteogenic differentiation of dental pulp stem cell. Quantitative reverse transcriptase PCR analysis for Myc, Lin28, Nanog, Kfl4 in DPSCs incubated for 21 days in osteogenic medium with 80 μM nandrolone. Data are the mean ± SEM of normalized transcript levels of five independent experiment from 5 independent donors. Per each gene evaluated, fold change value of transcript level in nandrolone treated cells compared to untreated cells (indicated by dashed line) is shown, * P < 0.05; ** P < 0.01. ( D) Nandrolone elevates stem cells genes expression in vivo . Real time-PCR evaluation of stemness gene expression after nandrolone administration in mice model as describe in Material Methods. For each gene, relative expression levels of transcripts of nandrolone treated mice compared to animals treated with vehicle (dashed line) are shown. The graphs show normalization with the reference gene of Myc, Lin28, Nanog, Kfl4, Nestin expression in several tissues, as spleen, kidney and liver. Data are the mean ± SEM of normalized transcript levels of 3 independent biological experiment. * P < 0.05; ** P < 0.01; # P < 0.001.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: In Vitro, Inverted Microscopy, Derivative Assay, Isolation, Staining, Incubation, Software, Expressing, In Vivo, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity

doi: 10.1038/s41598-020-58871-1

Figure Lengend Snippet: Proposed mechanism of the differential action of nandrolone on normal/cancer stem and differentiated cells. The hypothesis is put forward that by inhibiting the mitochondrial respiratory chain CxIII, nandrolone induces a pro-oxidative setting (red arrowed lines) that depending on the cellular antioxidant supply (green arrowed lines) establishes a differential redox signalling. A low increase of reactive oxidant species would favour self-renewal/quiescence of normal or cancer stem cells and possibly retro-differentiation of early progenitors, all equipped with a robust antioxidant armoury. Conversely, a higher pro-oxidative state, such that caused by nandrolone in late progenitor or differentiated cells will induce cell cycle arrest and possibly cell death. See Discussion for further details.

Article Snippet: For in vitro experiments, cells at approximately 50% confluence were treated for 72 h with 80 µM nandrolone (Vetranal-analytical standard, Sigma–Aldrich, St. Louis, MO).

Techniques: