Dehydroepiandrosterone-induced changes in mitochondrial proteins contribute to phenotypic alterations in hepatoma cells

Biochemical Pharmacology 117 (2016) 20–34

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Dehydroepiandrosterone-induced changes in mitochondrial proteins contribute to phenotypic alterations in hepatoma cells Mei-Ling Cheng a,b,c,d, Lang-Ming Chi e,f, Pei-Ru Wu a, Hung-Yao Ho b,d,g,⇑ a

Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan Healthy Aging Research Center, Chang Gung University, Tao-Yuan, Taiwan c Metabolomics Core Laboratory, Chang Gung University, Tao-Yuan, Taiwan d Clinical Phenome Center, Chang Gung Memorial Hospital, Linkou, Tao-Yuan, Taiwan e Clinical Proteomics Core Laboratory, Chang Gung Memorial Hospital, Linkou, Tao-Yuan, Taiwan f Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan g Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan b

a r t i c l e

i n f o

Article history: Received 14 May 2016 Accepted 4 August 2016 Available online 5 August 2016 Keywords: DHEA Mitochondria Proteomics FASTKD2

a b s t r a c t Dehydroepiandrosterone (DHEA)-induced growth arrest of hepatoma cells is associated with metabolic disturbance. Our previous study has suggested that DHEA may cause cellular energy drain. It is possible that mitochondrial dysfunction may be mechanistically implicated in DHEA-induced changes in cellular phenotype. Treatment of SK-Hep-1 cells with DHEA caused significant reduction in proliferation, colony formation, and growth in semi-solid medium. Such changes in cellular phenotype were associated with mitochondrial depolarization, increase in mitochondrial mass, and decrease in respiratory activity. Level of reactive oxygen species (ROS) increased in DHEA-treated cells. To explore the mechanistic aspect of DHEA-induced mitochondrial dysfunction, we employed SILAC approach to study the changes in the mitoproteome of SK-Hep-1 cells after DHEA treatment. Respiratory chain complex proteins such as NDUFB8 and SDHB were differentially expressed. Of mitochondrial proteins with altered expression, FAST kinase domain-containing protein 2 (FASTKD2) showed significantly reduced expression. Exogenous expression of FASTKD2 in SK-Hep-1 cells increased their resistance to growth-inhibitory effect of DHEA, though it alone did not affect cell growth. FASTKD2 expression partially reversed the effect of DHEA on mitochondria, and reduced DHEA-induced ROS generation. Our results suggest that DHEA induces changes in mitochondrial proteins and respiratory activity, and contributes to growth arrest. FASTKD2 may be an important regulator of mitochondrial physiology, and represent a downstream target for DHEA. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction DHEA is a steroid secreted by gonads, adrenal cortex, brain and gastrointestinal tract [1]. DHEA and its sulfated form are the most abundant steroid in circulation. The plasma level increases during adolescence; peaks at about 25 years of age; and decreases to about 10% of adolescent level by the age of 80 [2]. DHEA has multiple beneficial effects, including antiobesity [3,4], antiatherosclerosis [5,6], reduction of blood glucose level [7,8], and memory enhancement [9]. Decreased DHEA level correlates with an increased risk of carcinogenesis [10,11]. DHEA has been known ⇑ Corresponding author at: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, 259, Wen-Hua 1st Rd., Guishan Dist., Taoyuan City 33302, Taiwan. E-mail address: [email protected] (H.-Y. Ho). http://dx.doi.org/10.1016/j.bcp.2016.08.005 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

to inhibit chemically induced carcinogenesis in skin [12], breast [13], lung [14], liver [15–17], and colon [18], and spontaneous tumorigenesis in p53 nullizygous mice [19]. DHEA suppresses growth of numerous cell lines in vitro [20–22]. The doses of DHEA that inhibit cell growth and carcinogenesis are relatively high, when compared to those providing beneficial effects [22–26]. For growth inhibitory effect, DHEA is effective in a dose range of hundreds of lM [20,22]. DHEA deters the development of colorectal cancer, pancreatic cancer and mammary cancer at doses ranging from 1200 to 1800 mg/kg/day [25–27]. Several mechanisms have been proposed to account for the growth inhibitory effect of DHEA. DHEA has been shown to be an uncompetitive inhibitor of glucose-6-phosphate dehydrogenase (G6PD). Inhibition of G6PD was believed to reduce the flux of pentose phosphate pathway and the supply of ribose-5-phosphate and reduced nicotinamide adenine dinucleotide, both of which are

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essential to cell proliferation [28]. However, it remains dubious about whether G6PD accounts for DHEA-induced growth arrest [29–33]. We have found that DHEA treatment induced intracellular G6PD activity in hepatoma cells [34]. A similar finding has been observed in promyelocytic cells [32]. It has been suggested that inhibition of 3-hydroxy-3-methylglutaryl CoA reductase (HGMR) is related to growth inhibitory effect of DHEA. However, mevalonate treatment did not relieve cells from growth stagnation [34], ruling out the role of HGMR as primary target for growth inhibition. Our previous metabolomics study has shown that DHEA treatment induced anomalous changes in metabolism of such metabolites as glutathione, cardiolipin, phosphatidylcholine and carnitine [35]. Some of these metabolites are involved in energy metabolism, implying a defect in mitochondrial function in DHEA-treated cells. Indeed, mitochondrial membrane potential was lowered in these cells [35]. Mitochondria may represent a potential target organelle for DHEA in hepatoma cells. The inhibitory effect of DHEA has been observed in other cells. DHEA inhibited complex I activity in cerebellar granule cells and caused neurotoxic effect [36]. It reduced the respiratory control ratio of mitochondria isolated from rat brain, and protected them from the negative impact of anoxia-reoxygenation and uncoupling agent [37]. However, the mechanistic aspect of DHEA-induced mitochondrial dysfunction is not clear. In the present study, we studied the mechanism underlying DHEA-induced mitochondrial dysfunction and phenotypic changes in hepatic cells. DHEA treatment causes significant reduction in growth properties. Such phenotypic changes are accompanied by functional defects of mitochondria, and changes in expression of specific mitochondrial proteins. Expression of FASTKD2 is inhibited in the presence of DHEA. Conversely, exogenous FASTKD2 expression increases the resistance to DHEA-induced growth suppression, and partially reverses its inhibitory effect on respiratory activity.

2. Materials and methods 2.1. Chemicals and reagents Unless otherwise stated, DHEA and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thermo Pierce SILAC Protein Quantitation Kit-DMEM (PI89983), 13C6-L-ArginineHCl (PI88210) and 13C6-L-lysine-2HCl (PI88431) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal calf serum (FCS), penicillin, streptomycin and amphotericin were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mitotracker Red 580FM, 5,50 ,6,60 -t etrachloro-1,19,3,39-tetraethylbenz-imidazolocarbocyanine iodide (JC-1), 20 ,70 -di-chlorodihydrofluorescein diacetate (H2DCFDA), MitoSOX Red and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies to NDUFB8, UQCRC2, SDHB, MT-CO2 and ATP5A1 (ab110411; Abcam, Cambridge, MA, USA); to SDHA (ab110410; Abcam, Cambridge, MA, USA); to NDUFS4 (ab87399; Abcam, Cambridge, MA, USA); to porin (ab14734; Abcam, Cambridge, MA, USA); to FASTKD2 (AP9877c; Abgent, San Diego, CA, USA); and to actin (A5441; Sigma-Aldrich, St. Louis, MO, USA) were purchased from respective vendors.

2.2. Cell culture and cell biological techniques SK-Hep-1 cells (ATCC catalog number: HTB-52) were maintained in DMEM/10% FCS supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 lg/ml amphotericin at 37 °C in a humidified atmosphere of 5% CO2. Cell number was determined by neutral red assay [34].

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Colony formation assay was performed with 100 cells as previously described [38]. Soft agar assay was performed as described elsewhere [39]. Briefly stated, a bottom layer of 0.6% agar containing DMEM complete medium was set in a well of 6-well culture plate. About 2  104 cells were seeded in 0.3% agar containing complete DMEM with or without DHEA. The cell-containing agar solution was layered on the top of 0.6% agar layer. The plate was incubated at 37 °C in a humidified atmosphere of 5% CO2 for 2 weeks. The number and diameter of spheroid colony were determined microscopically. 2.3. Stable isotope labeling in culture for mass spectrometry (MS)based proteomic quantification Stable isotope-labeling by amino acids in cell culture (SILAC) was performed as previously described [40]. In brief, SK-Hep-1 cells were grown in SILAC DMEM/5% dialyzed FCS for at least 6 doublings prior to mitochondria fractionation and shotgun quantitative proteomic analysis. 2.4. Mitochondrial isolation and fractionation About 3  106 12C6-L-arginine and 12C6-L-lysine-labeled (lightlabeled) or 13C6-L-arginine and 13C6-L-lysine-labeled (heavylabeled) cells were seeded in a tissue culture dish with a diameter of 15 cm, and cultured respectively in normal and SILAC media to 80% confluence. Five cultures of either light- or heavy-labeled cells were treated without or with 200 lM DHEA. Mitochondria were isolated as described elsewhere with modifications [41,42]. Briefly stated, cells were washed 4 times with phosphate buffered saline (PBS), scraped in MIB buffer (25 mM Tris–HCl/1 mM EDTA/0.25 M sucrose), and centrifuged at 200g at 4 °C for 10 min. The cell pellet was resuspended in MTE (10 mM Tris– HCl/0.1 mM EDTA/270 mM mannitol). An aliquot of cell suspension was mixed with an equal volume of RIPA buffer, and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific; Waltham, MA, USA). An equal amount of light- and heavy-labeled cells, equivalent to 300 lg of cell lysate protein in each sample, were mixed. A 100 protease inhibitor solution (Qiagen, Germany) was added to the mixed cell suspension. The sample was immersed in ice bath, and sonicated with a sonicator tip for 10 s thrice. After centrifugation at 1250g at 4 °C for 10 min, the supernatant was retained. The pellet was resuspended in MTE buffer and centrifuged again. The supernatant was collected and combined with that from the previous step. It was centrifuged at 15,000g at 4 °C for 10 min. The pellet was washed thrice with MTE buffer, and resuspended in 5% iodixanol (OptiprepTM)/HM buffer (10 mM Tris–HCl, 4.4% mannitol). For isolation of highly enriched mitochondria, the mitochondrial preparation was loaded on a discontinuous gradient with 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, and 30% iodixanol. It was centrifuged at 150,000g at 4 °C for 16 h. Fractions were retrieved from the top of gradient. Each fraction was centrifuged, and washed twice with MTE buffer. The pellet was dissolved in denaturing buffer (20 mM HEPES/0.1% sodium dodecyl sulfate (SDS), pH 8.0). Samples from these fractions were subject to immunoblotting with anti-calreticulin, anti-actin and anti-porin antibodies. The fraction that is highly positive for porin expression but highly negative for actin and calreticulin represents the highly enriched mitochondria. 2.5. In solution digestion The mitochondrial suspension was extracted in denaturing buffer (20 mM HEPES/0.1% sodium dodecyl sulfate (SDS), pH 8.0). The protein extract was reduced with 5 mM dithiothreitol at 37 °C for

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1 h, and subsequently alkylated with 15 mM iodoacetamide at room temperature for 30 min. The sample was digested with sequencing grade modified trypsin (Promega, WI, USA) at an enzyme to substrate ratio of 1:30 (W/W) at 37 °C overnight. The reaction was stopped by addition of formic acid to a final concentration of 0.5%. 2.6. Shotgun quantitative proteomics analysis using LTQ-Orbitrap mass spectrometer Peptides prepared from in-solution digestion were separated, desalted by C18 tip-column, and analyzed with an online 2D LC–MS/MS technique using strong cation exchange (SCX) and reverse-phase 18 (RP18) nanoscale liquid chromatography coupled with LTQ-Orbitrap mass spectrometry (Thermo Electron, Bremen, Germany) [43]. Briefly stated, the mixture of light- and heavy-labeled peptides were injected into an in-house packed SCX column (Luna SCX, 5 lm, 0.5  150 mm; Phenomenex, Torrance, CA, USA), and fractionated into 30 fractions using a continuous ammonium chloride gradient in the presence of 30% acetonitrile and 0.1% formic acid. Each SCX-eluted fraction was diluted, trapped on a RP18 column (Source 15 RPC, 0.5  5 mm; GE Healthcare, Piscataway, NJ, USA), and then separated on BEH RP18 chromatographic column (1.7 lm, 0.1  120 mm; Waters Corp., Milford, MA, USA) using an acetonitrile gradient in 0.1% formic acid. Chromatographic separation was performed on Dionex UltiMate 3000 nanoLC system, and subsequent mass spectrometric analysis was performed using an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher, San Jose, CA, USA). Full scan spectra (m/z 430–2000) were acquired with resolution set to 60,000. The lock mass calibration feature was enabled. Automatic gain control (AGC, set to 106) was used to prevent over-filling of the ion trap. Up to ten most intense precursor ions with minimal signal intensity of 25,000 were sequentially isolated for MS/MS fragmentation using collision energy of 35%. 2.7. MS-based peptide/protein identification and quantitative analysis by Proteome Discoverer software and Mascot search engine, and subsequent analysis MS raw files are uploaded into Proteome Discoverer (version: 1.3; Thermo Fisher Scientific, Waltham, MA, USA) with default setting to generate a peak list for protein identification, which was performed by Mascot search (version 2.2.2; Matrix Science, Boston, MA, USA) against Swiss-Prot human protein database (released in 2010; containing 20,367 human entries). The search parameters were set as follows: Carbamidomethylation of Cys as fixed modification; oxidation of Met, pyro-Glu formation from peptide N-terminal Gln; acetylation of protein N-terminus; SILAC modification of Lys and Arg as variable modifications; 6 ppm for MS tolerance; 0.6 Da for MS/MS tolerance; 1 for missing cleavage. The identified peptides and proteins in all search results were integrated and further filtered by Proteome Discoverer with following criteria: 7 for minimum peptide length, 2 for minimum unique peptides for the assigned protein. The peptides shared (not unique for leading proteins) between multiple leading proteins were assigned to the one with highest protein score. Peptide and protein identifications with false discovery rate less than 1% were accepted. For protein quantification, the proteins with at least two ratio counts generated from unique peptides were analyzed. The median value of the spectrum ratios was calculated as protein abundance (H/L ratio). The peptides shared (not unique for leading proteins) between multiple leading proteins were assigned to the one with highest protein score. The global median normalization was applied to recalculate the protein abundance to generate the normalized protein ratio to

reduce the system error from sample preparation in each experiment. For pathway analysis, Ingenuity Pathway Analysis (IPA) (build version 346717M; content version 24390178) was used. The quantitative precision of an SILAC experiment is typically less than 20% RSD (our unpublished data) [44,45]. The proteins, expression of which were P30% higher in treatment group (or control group) than in control group (or treatment group), were selected for IPA analysis. These molecules had corresponding log2 (DHEA/Control) expression ratios P0.378512 or 60.378512. Proteins were categorized according to their ‘‘disease and biological functions”. 2.8. Quantitative analysis of DWm, mitochondrial mass and reactive oxygen species Mitochondrial membrane potential was determined using cationic, lipophilic dye JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetrae thylbenzimidazolocarbocyanine iodide) as described previously [46]. Cells receiving DHEA treatment were loaded with 0.5 lM JC-1 in DMEM at 37 °C for 60 min, and counterstained with 5 lg/ ml of Hoechst 33342 prior to examination with Leica TCS SP2 MP system (Leica Microsystems, Mannheim, Germany). Confocal fluorescence images of labeled cells were acquired using HCX PL-APO 1001.40 NA oil immersion objective. Image analysis was performed with Leica LCS software package. For cytometric analysis, DHEA-treated cells were loaded with JC-1 dye as described above, washed thrice with PBS, and trypsinized. Cell suspension was analyzed for JC-1 monomeric (FL1 channel) and J-aggregate (FL2 channel) with CELLQuest software. The ratio of the mean florescence intensity (MFI) of FL2 channel to that of FL1 channel was calculated. Mitochondrial mass was measured cytometrically using Mitotracker Red 580FM as previously described [46]. For quantification of superoxide anion production, DHEAtreated cells were loaded with 2 lM MitoSOX red at 37 °C for 20 min, washed twice with PBS, and trypsinized for cytometric analysis. Additionally, DHEA-treated cells were stained with 5 lM 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) for analysis in a similar way. The MFI of oxidized MitoSOX red or that of 20 ,70 -dichlorofluorescein (DCF) was quantified using CellQuest Pro software (Becton Dickinson, CA, USA). 2.9. Measurement of cellular and mitochondrial oxygen consumption Cellular oxygen consumption was measured as previously described [46]. Briefly stated, un- or DHEA-treated cells were washed twice with PBS, trypsinized, and resuspended in respiration buffer (20 mM NaKPO4 (pH 7.2), 65 mM KCl, 125 mM sucrose, 2 mM MgCl2). The cell suspension was placed in a Mitocell respiratory chamber equipped with a Clark-type electrode, which was connected to Strathkelvin 928 6-Channel Oxygen System (Strathkelvin Instruments, Glasgow, UK). Oxygen consumption rate was monitored. Data are expressed as lg O2 per 107 cells per min. Oxygen consumption rate can also be normalized to mitochondrial mass determined by Mitotracker Red 580FM staining. Data are expressed as lg O2 per mitochondrial mass unit (MMU) per min. Determination of mitochondrial oxygen consumption was performed as previously described [46]. In brief, DHEA-treated cells were washed twice with PBS, scrapped in ice-cold SHE buffer (0.25 M sucrose, 1 mM EGTA, 3 mM HEPES), and homogenized. The homogenate was centrifuged at 1000g at 4 °C for 10 min, and the supernatant was subject to further centrifugation at 10,000g at 4 °C for 10 min to pellet the mitochondria. The mitochondria were resuspended in a suitable volume of SHE buffer. An aliquot of mitochondria equivalent (0.5 mg) was added to 300 ll of assay buffer (20 mM K2HPO4/KH2PO4 (pH 7.2), 125 mM sucrose, 65 mM KCl, 2 mM MgCl2), and transferred to the Mitocell

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respiratory chamber equipped with Clark-type electrode. Malate and glutamate were added at the final concentration of 5 mM; ADP was added at the final concentration of 0.5 mM. Respiratory control ratio (RCR) is defined as rate of ADP-stimulated state 3 oxygen consumption divided by the rate of oxygen consumption (State 4o) determined in the presence of 2 lg/ml oligomycin [47]. 2.10. Molecular and biochemical techniques For transfection, cDNA encoding FASTKD2 (cDNA accession number: BC001544) was available as MGC premier human expression ready cDNA clone in pTCP vector (Transnomics, Huntsville, AL, USA). The expression vector was transfected into cells using Polyfect transfection reagent (Qiagen, Valencia, CA, USA) according to manufacturer’s instruction. SDS-PAGE and western blotting were performed as previously described [48]. ATP level was determined as previously described [34]. 2.11. Statistical analysis Results are presented as means ± SD. Data were analyzed by two way analysis of variance and t test where appropriate. A p value of less than 0.05 was considered significant.

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3. Results 3.1. DHEA induces growth arrest of SK-Hep-1 cells DHEA caused dose- and time-dependent reduction in growth of SK-Hep-1 cells, as assayed by neutral red assay (Fig. 1A). After treatment with 100 and 200 lM DHEA for 48 h, growth of SKHep-1 cells decreased, respectively, by 38.3 and 80.9%. Increase in treatment time to 96 h further reduced the growth of 200 lM DHEA-treated cells by about 86.6%. The growth stagnation was accompanied by reduction in clonogenicity (Fig. 1B). The total number of colonies formed by 100 lM DHEA-treated cells was significantly reduced by 46.2%, as compared with that of control (Fig. 1Bb). As DHEA concentration increased to 200 lM, the colony formation was nearly completely suppressed. Additionally, in the presence of 100 lM DHEA, the percentage of large colonies (with diameter greater than 1 mm) decreased to a greater extent than the small ones. The ability of SK-Hep-1 cells to form colonies in semi-solid medium was reduced in DHEA treatment group (Fig. 1C). The total number of spheroid colonies decreased by 47.1 and 75.7% in 100 and 200 lM DHEA-treatment groups, respectively. Again, there was greater reduction in large spheroid colonies (with diameter greater than 0.1 mm) than small one (with diameter of 0.03–1 mm) in the presence of DHEA. These findings

Fig. 1. DHEA inhibits anchorage dependent and independent growth of SK-Hep-1 cells. (A) SK-Hep-1 cells were seeded, and 24 h later (time = 0 h), were treated with indicated concentrations of DHEA for 24, 48, 72 and 96 h. Cell number at indicated time-points was determined by neutral red assay. The absorbance values were compared to cell number standard curve. The change in cell number with time is shown. Cell number is expressed as fold relative to that at time = 0 h, and data are expressed as mean ± SD, n = 6. *p < 0.05, §p < 0.01, vs. untreated cells. (B)(a) Cells were seeded in a culture plate, and treated without (plate on the left), or with 100 (panel in the middle) or 200 lM (panel on the right). Twelve days later, the plate was stained with 0.1% crystal violet, and the number of colonies with size P 1 mm or <1 mm was determined. Representative un- and treated cell plates are shown in (a). The distribution of colony size in control and treatment groups is shown in (b). Data are expressed as mean ± SD, n = 6. *p < 0.05, vs. untreated colonies with defined size. (C) Cells were seeded in top agar containing 0 (a0 ), 100 (b0 ), and 200 (c0 ) lM DHEA, and incubated for 2 weeks. Representative un- and treated soft agar plate are shown in (a0 –c0 ) (original magnification: 40). Spheroid colonies were counted. The distribution of the size of spheroid colonies in control and treatment groups is shown in (d0 ). Data are expressed as mean ± SD, n = 6. *p < 0.05, vs. untreated spheroid colonies with defined size.

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suggest that DHEA inhibits cellular proliferation, growth potential and anchorage-independent growth of SK-Hep-1 cells.

3.2. DHEA causes mitochondrial anomalies, ROS generation and dysfunction To address whether DHEA affects mitochondrial function and ROS generation, we treated SK-Hep-1 cells with DHEA for 48 h, and examined parameters related to mitochondrial function. DHEA treatment causes mitochondrial depolarization. As shown in Fig. 2A, the control cells that were stained for JC-1 dye displayed

strong red fluorescence (J aggregate of JC-1 dye) and less green fluorescence (monomeric form of JC-1). After treatment with 200 lM DHEA, mitochondrial depolarization in SK-Hep-1 cells was revealed by reduction in red fluorescence and significant enhancement of green fluorescence. We employed a flow cytometric method to quantify mitochondrial potential DWm. The FL2/FL1 ratio of SK-Hep-1 cells, indicative of DWm, decreased by 31.1 and 47.8% after treatment with 100 and 200 lM DHEA, respectively. Interestingly, DHEA appears to induce an increase mitochondrial mass. The mitochondrial mass, as measured by Mitotracker Red 580FM staining and cytometric analysis, increased by 27.3% in DHEA-treated cells.

Fig. 2. DHEA causes decline in DWm. (A) SK-Hep-1 cells were treated without (a–d) or with (e–h) 200 lM DHEA for 48 h; stained with JC-1 and Hoechst 33342 dyes, and examined by confocal microscopy. Intracellular distribution of aggregate (a, e) and monomer (b, f) of JC-1 dye is an indicator of DWm. Nuclei of are shown (c, g). The corresponding images are overlaid (d, h). The photographs shown here are representative of three experiments (scale bar, 10 lm). (B) SK-Hep-1 cells were treated with 0, 100 and 200 lM DHEA for 48 h, stained with JC-1 dye, and analyzed using flow cytometry. The ratio of the MFI of FL2 channel to that of FL1 channel (FL2/FL1) was calculated, and is expressed as the percentage of that of untreated cells. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (C) SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h, stained with Mitotracker red, and analyzed using flow cytometry. The MFI of the stained cells is expressed as the percentage of that of untreated cells. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells.

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Mitochondrial depolarization is accompanied by mitochondrial ROS generation. SK-Hep-1 cells were un- or treated with 200 lM DHEA for 48 h, and subsequently stained with ROS-sensitive dyes for flow cytometric analyses. DHEA treatment of SK-Hep-1 cells, which were stained with MitoSOX red dye, caused a 3.5-fold increase in fluorescence (Fig. 3A), indicating increased mitochondrial superoxide generation in these cells. Consistent with this, DCF fluorescence of H2DCF-DA-stained cells increased significantly (Fig. 3B). It is likely that mitochondrial depolarization and ROS generation are causally related to anomalous electron transport. We applied amperometric method to quantify the oxygen consumption rate, which indicates the function of electron transport chain. SK-Hep-1 cells were un- or treated with 200 lM DHEA for 48 h, and analyzed for their oxygen consumption rate. As shown in Fig. 4, the oxygen consumption rate of SK-Hep-1 cells decreased after DHEA. The rate normalized to cell number was reduced by 52.4% in DHEA-treated cells as compared with that of untreated cells (Fig. 4A). When normalized to mitochondrial mass unit, the rate was 62.6% lower in DHEA-treated cells than in control cells (Fig. 4B). We further measured state 3 and 4o respiration of mitochondria isolated from DHEA and untreated cells. The state 3 respiration refers to ADP-stimulated respiration, while state 4o refers to the respiratory state after termina-

Fig. 3. DHEA induces ROS generation. (A) SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h, stained with MitoSOX red, and analyzed using flow cytometry. The MFI of the stained cells is expressed as fold relative to that of untreated cells. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (B) SKHep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h, stained with H2DCFDA, and analyzed using flow cytometry. The DCF MFI of the stained cells is expressed as fold relative to that of untreated cells. Results are mean ± SD, n = 6. * p < 0.05, vs. untreated cells.

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tion of state 3 respiration in the presence of oligomycin. The state 3 respiration of mitochondria from DHEA-treated cells was inhibited by 58%, as compared with that of control cells (Fig. 4C). Accordingly, the RCR was 56.1% lower for mitochondria from DHEA-treated cells than for those from control cells (Fig. 4D). Decreases in electron transport and DWm are associated with diminution of ATP supply. Intracellular ATP level in DHEA-treated cells was reduced by nearly 25% (Fig. 4E). These findings suggest that DHEA induces functional alteration of mitochondria and defect in energy metabolism.

3.3. Identification of mitochondrial proteins differentially expressed in DHEA-treated SK-Hep-1 cells DHEA-induced mitochondrial dysfunction may be attributed to changes in mitochondrial proteins. To examine this possibility, we adopted SILAC approach, and isolated highly enriched mitochondrial preparations from un- and DHEA-treated cells to study changes in mitochondrial proteins. Two biological experiments were performed (Fig. 5A). The heavy isotopelabeled cells were treated with DHEA for 48 h, and the light isotope-labeled cells served as untreated control. In the replicate experiment, the light isotope-labeled cells were treated with DHEA for 48 h, and the heavy isotope-labeled cells were untreated. Differently labeled cells were mixed in 1:1 ratio, and mitochondria were isolated and fractionated using OptiprepTM density gradient centrifugation. The fractions highly enriched in mitochondria (porin-positive and calreticulinnegative) were collected for proteomic analysis (data not shown). 1728 and 2335 proteins were identified, respectively, in replicate experiments, experiment 1 and 2 (data not shown). They were characterized by definite ratios of their expression levels in DHEA-treated cells to those in control cells (i.e. DHEA/Control ratios). A total of 1662 duplicate proteins were identified and quantified (Fig. 5B; data not shown). The data for 488 proteins, expression of which is 30% higher in DHEAtreated cells (or control cells) than in control cells (or DHEAtreated cells), were submitted to IPA for pathway analysis. Top 15 categories of ‘‘disease and biological functions” are shown in Table 1. Two groups of proteins with biological functions entitled ‘‘mitochondrial respiratory chain deficiency” and ‘‘mitochondrial complex I deficiency” (within the categories entitled ‘‘developmental disorder”, ‘‘hereditary disorder”, and ‘‘metabolic disease” of the higher-rank category entitled ‘‘disease and disorder”) with 15 and 12 proteins were reduced in expression in DHEA-treated cells (Table 2). All 12 proteins within the latter category could be found in the former category. Fifteen proteins of the category entitled ‘‘mitochondrial respiratory chain deficiency” are shown (Fig. 5D), and are related to mitochondrial electron transport. The annotated IPA protein functions and Uniprot GO molecular functions of these proteins are listed (Table 3). Expression of proteins within this category was down-regulated in DHEA-treated cells. A number of mitochondrial proteins were picked from proteomic dataset for validation with western blotting (Fig. 5C). We treated SK-Hep-1 cells with or without DHEA for 48 h. Mitochondria were isolated from DHEA-treated and control cells for analysis of these proteins by western blotting. Expression of NDUFB8, NDUFS4, SDHB, UQCRC2, MT-CO2 and FASTKD2 decreased in DHEA-treated cells. On the contrary, the levels of SDHA and ATP5A1 increased and remained unchanged, respectively, in these cells. Trends in the changes in protein levels, revealed by western blotting, were consistent with the corresponding DHEA/Control ratios in proteomic dataset (data not shown).

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3.4. FASTKD2 as a downstream target for DHEA One of proteins differentially expressed in DHEA-treated cells is FASTKD2. It is a member of FAST kinase domain containing protein (FASTKD) family. Previous studies have shown that FASTKD3, another member of FASTKD family, is essential to mitochondrial respiration [49]. It is probable that the repression of FASTKD2 expression may partly mediate the cellular effect of DHEA. Decrease in FASTKD2 protein was consistent with reduction in 35% reduction in the level of FASTKD2 transcript in DHEAtreated cells (data not shown). To test whether down-regulation of FASTKD2 expression accounts for effect of DHEA, we transfected SK-Hep-1 cells with FASTKD2 expression vector for 48 h, and examined the consequence of DHEA treatment. Expression of FASTKD2 significantly

increased in whole cell lysate and mitochondrial lysate of transfected cells (Fig. 6A–D). However, the morphology of FASTKD2transfected was indistinguishable from that of control cells (Fig. 6E, F). When growth kinetics of these cells was examined, the growth rates of FASTKD2-transfected and control vectortransfected cells were nearly the same (Fig. 6G). Nevertheless, the FASTKD2-transfected cells were more resistant to DHEAinduced growth inhibition after 48 h of treatment, as compared with the control vector-transfected cells (Fig. 6H). Exogenous expression of FASTKD2 causes 32% increase in number of cells treated with 100 lM DHEA. The sensitivity of control vectortransfected cells to DHEA was similar to that of parental cells (data not shown). These findings suggest that the growth inhibitory effect of DHEA can be partly attributed to repression of FASTKD2 expression.

Fig. 4. DHEA suppresses cellular respiratory activity and energy production. (A) SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (lg O2/107 cells/min) was calculated. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (B) The oxygen consumption rate is normalized to mitochondrial mass unit (MMU), which was determined as in Fig. 2C. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (C) SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h. The mitochondria were isolated and assayed for oxygen consumption rate (102 lg O2/lg mitochondrial protein/min) during state 3 (solid bar) and 4o (open bar) respiration as described in Section 2. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (D) RCRs were calculated accordingly, and are shown. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells. (E) SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h, and ATP content was determined as described in Section 2. Results are mean ± SD, n = 6. *p < 0.05, vs. untreated cells.

M.-L. Cheng et al. / Biochemical Pharmacology 117 (2016) 20–34

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Fig. 5. Differential changes in the mitoproteome of hepatoma cells in response to DHEA. (A) The scheme of proteomic analysis of the highly enriched mitochondrial preparations from un- and DHEA-treated SK-Hep-1 cells is shown. Duplicate experiments were performed. In experiment 1, the heavy isotope-labeled cells were treated with DHEA; in experiment 2, the light isotope-labeled cells were treated with DHEA. (B) Venn diagram shows the number of proteins with quantitative expression ratios in duplicate experiments. (C) Validation of selected proteins from proteomic data. SK-Hep-1 cells were treated without () or with (+) 200 lM DHEA for 48 h, and harvested for isolation of mitochondria. The samples were subject to western blotting with antibodies to NDUFB8, NDUFS4, SDHA, SDHB, UQCRC2, MT-CO2, ATP5A1 and FASTKD2. Porin serves as control. A representative experiment out of six is shown (a0 ). The protein bands were quantified by densitometric scanning, and the expression levels of proteins are expressed as the percentages of those of untreated cells (b0 ). Results are mean ± SD, n = 6. *p < 0.05, §p < 0.01, vs. untreated cells. (D) One of significant IPA categories of biological functions, namely the mitochondrial respiratory chain deficiency, was determined by IPA analysis, and is shown.

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Table 1 Top disease and biological functions of mitochondrial proteins differentially abundant in control and DHEA-treated cells as revealed by Ingenuity pathway analysis. Diseases and disorders

p value

No. molecule

Developmental disorder

1.55  1002– 3.41  1012 1.55  1002– 3.41  1012 9.00  1003– 3.41  1012 1.54  1002– 3.15  1010 1.58  1002– 3.15  1010

49

Molecular and cellular functions

p value

No. molecule

Cellular movement

1.58  1002– 6.73  1009 1.58  1002– 3.52  1007 1.58  1002– 1.73  1006 1.58  1002– 2.07  1006 1.58  1002– 4.00  1006

81

Physiological system development and function

p value

No. molecule

Immune cell trafficking

1.58  1002– 6.73  1009 1.58  1002– 1.55  1007 1.51  1002– 4.48  1007 9.13  1003– 1.45  1005 1.51  1002– 2.74  1004

34

Hereditary disorder Metabolic disease Hematological disease Organismal injury and abnormalities

Cell-to-cell signaling and interaction Cellular assembly and organization Cell death and survival Cell morphology

Hematological system development and function Tissue development Hair and skin development and function Connective tissue development and function

60 49 41 155

60 71 105 64

38 36 15 14

3.5. Expression of FASTKD2 partially restores DHEA-induced inhibition of mitochondrial respiration and suppresses ROS generation It is possible that FASTKD2, like FASTKD3, may modulate mitochondrial respiration. DHEA-induced reduction in FASTKD2 expression may be related to decline in mitochondrial function. To test such possibility, we treated FASTKD2- and control vectortransfected cells without or with 200 lM DHEA for 48 h, and examined their oxygen consumption rates. As shown in Fig. 7A, expression of FASTKD2 partially reversed the inhibitory effect of DHEA on oxygen consumption. Under basal condition, the oxygen consumption rates of FASTKD2- and control vector-transfected cells were similar. Upon DHEA treatment, the oxygen consumption rate of FASTKD2-transfected cells was 46% higher than that of control vector-transfected cells. When normalized to mitochondrial mass unit, the rate of the former cells was 90% higher than that of the

latter cells (Fig. 7C). It was evident that expression of FASTKD2 significantly suppressed DHEA-induced increase in mitochondrial mass. The ability of FASTKD2 to partially restore mitochondrial respiration in DHEA-treated cells is associated with reduction in ROS generation. The FASTKD2- and control vector-transfected cells were treated without or with 200 lM DHEA for 48 h, and were subject to flow cytometric analyses of ROS. The level of mitochondrial superoxide in DHEA-treated FASTKD2-transfectant was 34% lower than that of similarly treated control vector-transfectant (Fig. 8A). In agreement with this, expression of FASTKD2 significantly suppresses ROS generation, as indicated by DCF fluorescence (Fig. 8B).

4. Discussion The cytostatic effect of DHEA is not completely understood. In the present study, we have shown that DHEA-induced growth inhibition was accompanied by inhibition of mitochondrial respiration and mitochondrial depolarization. Proteomic analysis of highly enriched mitochondrial fraction from DHEA- and untreated cells revealed that a number of proteins were differentially expressed in cells after DHEA treatment. Expression of several respiratory complex proteins was down-regulated. Moreover, another mitochondrial protein FASTKD2 had its expression repressed after DHEA treatment. Exogenous FASTKD2 expression reversed the inhibitory effect of DHEA on cellular oxygen consumption; reduced DHEA-induced increase in mitochondrial mass; and increased their resistance to growth inhibitory effect of DHEA. These findings suggest that FASTKD2 plays important roles in mitochondrial physiology, and represents a downstream target of DHEA. DHEA exerts antiproliferative effect on various cells [50]. We have previously shown that DHEA does not suppress growth of HepG2 cells via inhibition of glucose-6-phosphate dehydrogenase and 3-hydroxy-3-methylglutaryl CoA reductase [34]. Our further study of metabolic pathways indicated that DHEA disturbs Sadenosylmethionine metabolism and mitochondrial functions [35]. DHEA-induced declines in mitochondrial oxygen consumption and ATP level suggest that mitochondria are important target of DHEA action. Consistent with this, exogenous expression of FASTKD2 that enhances mitochondrial function in DHEA-treated cells confers an increased resistance to cytostatic effect of DHEA. Additionally, supplementation of DHEA-treated HepG2 cells with pyruvate alleviates the growth suppression by DHEA [34]. Apart from its effect on growth, DHEA appears to inhibit the ability of SK-Hep-1 cells to migrate and invade extracellular matrix. Using wound healing assay, we have found that the wound closure rate of DHEA-treated cells was significantly reduced. It was consistent with a reduction in the ability of DHEA-treated cells to migrate across uncoated and Matrigel-coated Transwell membranes (our unpublished data). Studies are under way to reveal the underlying mechanism.

Table 2 The number and identity of proteins in the categories of biological functions entitled ‘‘mitochondrial respiratory chain deficiency” and ‘‘mitochondrial complex I deficiency” as revealed by IPA analysis. Categories

Functions

Diseases or functions annotation

p-Value

Molecules

Molecule no.

Developmental Disorder, Hereditary Disorder, Metabolic Disease Developmental Disorder, Hereditary Disorder, Metabolic Disease

Mitochondrial respiratory chain deficiency Mitochondrial complex I deficiency

Mitochondrial respiratory chain deficiency Mitochondrial complex I deficiency

3.41E12

COX6B1, FASTKD2, MT-CO2, MT-ND1, NDUFA11, NDUFA12, NDUFA2, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2 MT-ND1, NDUFA11, NDUFA12, NDUFA2, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2

15

5.68E12

12

29

M.-L. Cheng et al. / Biochemical Pharmacology 117 (2016) 20–34 Table 3 Annotated functions of 15 proteins within the category entitled ‘‘mitochondrial respiratory chain deficiency”. UniProt/ SwissProt Accession

Symbol

Entrez Gene Name

Location

Type(s)

IPA Protein Functions

Uniprot GO – Molecular function

DHEA/ Control

Log2 Ratio

P14854

COX6B1

Cytoplasm (Mitochondria)

Enzyme

Cytochrome-c oxidase, enzyme

Cytochrome-c oxidase activity

0.726

0.462

Q9NYY8

FASTKD2

Poly(A) RNA binding, rRNA binding Cytochrome-c oxidase, enzyme

Poly(A) RNA binding, protein kinase activity, rRNA binding Copper ion binding, cytochrome-c oxidase activity

0.696

0.522

MT-CO2

Cytoplasm (Mitochondria) Cytoplasm (Mitochondria)

Other

P00403

0.725

0.463

P03886

MT-ND1

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase (ubiquinone)

NADH dehydrogenase (ubiquinone) activity

0.768

0.380

O43678

NDUFA2

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase (ubiquinone)

NADH dehydrogenase (ubiquinone) activity

0.631

0.663

Q86Y39

NDUFA11

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase (ubiquinone)

–

0.731

0.453

Q9UI09

NDUFA12

Cytoplasm (Mitochondria)

Enzyme

Enzyme

Electron carrier activity, NADH dehydrogenase (ubiquinone) activity

0.636

0.653

P28331

NDUFS1

Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) FAST kinase domains 2 Cytochrome c oxidase subunit II NADH dehydrogenase, subunit 1 (complex I) NADH: ubiquinone oxidoreductase subunit A2 NADH: ubiquinone oxidoreductase subunit A11 NADH: ubiquinone oxidoreductase subunit A12 NADH: ubiquinone oxidoreductase core subunit S1

Cytoplasm (Mitochondria)

Enzyme

2 iron, 2 sulfur cluster binding,4 iron, 4 sulfur cluster binding, electron carrier activity, ironsulfur cluster binding, metal ion binding, NADH dehydrogenase (ubiquinone) activity

0.608

0.718

O75306

NDUFS2

NADH: ubiquinone oxidoreductase core subunit S2

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase, NADH2 dehydrogenase (ubiquinone), protein binding Enzyme, protein binding, ubiquitin protein ligase binding

0.764

0.388

O43181

NDUFS4

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase (ubiquinone)

0.629

0.669

O75380

NDUFS6

Cytoplasm (Mitochondria)

Enzyme

Enzyme

Electron carrier activity, NADH dehydrogenase (ubiquinone) activity

0.728

0.458

O75251

NDUFS7

NADH: ubiquinone oxidoreductase subunit S4 NADH: ubiquinone oxidoreductase subunit S6 NADH: ubiquinone oxidoreductase core subunit S7

4 iron, 4 sulfur cluster binding, electron carrier activity, metal ion binding, NAD binding, NADH dehydrogenase (ubiquinone) activity, quinone binding, ubiquitin protein ligase binding NADH dehydrogenase (ubiquinone) activity

Cytoplasm (Mitochondria)

Enzyme

Enzyme, protease binding, protein binding

0.744

0.426

O00217

NDUFS8

Cytoplasm (Mitochondria)

Enzyme

Enzyme

0.760

0.396

P49821

NDUFV1

NADH: ubiquinone oxidoreductase core subunit S8 NADH: ubiquinone oxidoreductase core subunit V1

4 iron, 4 sulfur cluster binding, metal ion binding, NADH dehydrogenase (ubiquinone) activity, oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor, quinone binding 4 iron, 4 sulfur cluster binding, metal ion binding, NADH dehydrogenase (ubiquinone) activity

Cytoplasm (Mitochondria)

Enzyme

4 iron, 4 sulfur cluster binding, FMN binding, metal ion binding, NAD binding, NADH dehydrogenase (ubiquinone) activity

0.659

0.602

P19404

NDUFV2

NADH: ubiquinone oxidoreductase core subunit V2

Cytoplasm (Mitochondria)

Enzyme

Enzyme, NADH2 dehydrogenase, NADH2 dehydrogenase (ubiquinone), protein binding Enzyme, NADH2 dehydrogenase, NADH2 dehydrogenase (ubiquinone)

2 iron, 2 sulfur cluster binding, electron carrier activity, metal ion binding, NADH dehydrogenase (ubiquinone) activity

0.661

0.597

Enzyme

‘‘UniProt/Swiss-Prot Accession” represents Uniprot/Swiss-Prot accession number of the leading protein in the database; ‘‘Symbol” represents protein symbol; ‘‘Entrez Gene Name” represents the Entrez gene name corresponding to protein; ‘‘Location” represents the subcellular location as determined by IPA analysis (It should be noted that ‘‘cytoplasm” include such organelles as mitochondrion); ‘‘Type” represents the molecule type as determined by IPA analysis; ‘‘IPA Protein Functions” represents the protein function(s) assigned by IPA analysis; ‘‘Uniprot GO – Molecular function” represents the molecular function(s) annotated by Gene Ontology (GO) project; ‘‘DHEA/Control” represents the average of normalized ratios of the abundance (i.e. normalized protein ratios) of protein in DHEA-treated versus control samples in experiment 1 or 2 (rounded off to 3 decimal places); ‘‘Log2 Ratio” represents Log2 transformation of (Average DHEA/Control) (rounded off to 3 decimal places).

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Our finding that DHEA inhibits state 3 respiration and RCR of mitochondria of SK-Hep-1 cells is consistent with those of previous studies. A high concentration of DHEA inhibits mitochondrial respiration of different cell types [51]. For instance, DHEA suppresses respiratory chain activity at high concentration, and is neurotoxic under in vitro and in vivo conditions [36]. It is not completely understood how DHEA inhibits mitochondrial respiration. We have

found that lipophilic metabolites, such as phosphatidylcholine and cardiolipin, decrease in abundance in DHEA-treated cells [35]. Phosphatidylcholine amounts to 35–50% of mitochondrial lipids [52]. Cardiolipin plays essential roles in maintenance of the structure and activity of respiratory chain complex [53,54]; in complex III-mediated proton pumping [55,56]; and in respirasome assembly and mitochondrial biogenesis [57,58]. Apart from the changes

Fig. 6. FASTKD2 expression alleviates the growth inhibitory effect of DHEA. SK-Hep-1 cells were transfected with control vector (Vector) or FASTKD2 expression vector (FASTKD2) for 48 h, and whole cell lysate (A) and mitochondria (C) were prepared for western blotting with antibodies to FASTKD2 and to control proteins (actin for whole cell lysate, porin for mitochondrial preparation). A representative experiment out of three is shown. The protein bands were quantified by densitometric scanning. The expression levels of FASTKD2 are normalized to that of actin (for whole cell lysate) (B) and to that of porin (for mitochondrial preparation) (D), and are expressed as fold relative to FASTKD2 level of the control vector-transfected cells. Data are expressed as mean ± SD, n = 3. *p < 0.05, vs. control vector-transfected cells. The morphology of control vector- (E) and FASTKD2 expression vector- (F) transfected is shown (original magnification: 100). A representative experiment out of three is shown. (G) SK-Hep-1 cells were transfected with control vector (Vector) or FASTKD2 expression vector (FASTKD2) for 48 h. The transfected cells were re-plated, and cell number at indicated timepoints after re-plating was determined by neutral red assay. The absorbance values were compared to cell number standard curve. The temporal change in cell number is shown. Cell number is expressed as fold relative to that at the time of re-plating, and data are expressed as mean ± SD, n = 6. (H) SK-Hep-1 cells were similarly transfected. The transfected cells were re-plated, and 24 h later (time = 0 h), were treated with indicated concentrations of DHEA for 48 h. Cell number was determined by neutral red assay. The absorbance values were compared to cell number standard curve. Cell number is expressed as fold relative to that at time = 0 h. Data are expressed as mean ± SD, n = 6. * p < 0.05, vs. untreated cells.

M.-L. Cheng et al. / Biochemical Pharmacology 117 (2016) 20–34

in mitochondrial lipids, mitochondrial proteins are differentially expressed in cells after DHEA treatment. Expression of various subunits of complex I, including MT-ND1, NDUFA2, NDUFA11, NDUFA12, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, and NDUFV2, was specifically down-regulated in DHEA-

Fig. 7. Expression of FASTKD2 enhances respiratory activity in DHEA-treated cells. (A) SK-Hep-1 cells were transfected with control vector (Vector) or FASTKD2 expression vector (FASTKD2) for 48 h. The transfected cells were re-plated, and 24 h later, were further treated without () or with (+) 200 lM DHEA for 48 h. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (lg O2/107 cells/min) was calculated. Results are mean ± SD, n = 6. *p < 0.05, vs. control vector-transfected cells. (B) SK-Hep-1 cells were similarly transfected and treated without () or with (+) 200 lM DHEA for 48 h. They were stained with Mitotracker red, and analyzed using flow cytometry. The MFI of the stained cells is expressed as the percentage of that of untreated control vector-transfected cells. Results are mean ± SD, n = 6. *p < 0.05, vs. control vector-transfected cells. (C) The oxygen consumption rate of cells treated without () or with (+) 200 lM DHEA is normalized to the corresponding MMU, which was determined as in (B). Results are mean ± SD, n = 6. *p < 0.05, vs. control vector-transfected cells.

31

treated cells. Likewise, expression of SDHB was reduced in these cells, suggesting down-regulation of functional complex II. Additionally, two subunits of complex IV, namely COX6B1 and MTCO2, were under-expressed. Intriguingly, the complex IV subunit COX6A1 can bind cardiolipin, which stabilizes COX6A1 and COX6B1 [59]. Such interactions are essential to structural integrity of complex IV. It is likely that decreases in expression of complex IV subunit and in cardiolipin level diminish the formation of functional complex IV. Additionally, reduction in expression of SCO2, the metalloprotein essential to assembly of catalytic center of cytochrome c oxidase [60], is suggestive of reduced complex IV activity. These changes, together with reduced expression of complexes I and II, contribute to DHEA-induced suppression of mitochondrial respiration. Increased ROS generation by malfunctioning mitochondria accounts for DHEA-induced oxidative stress. Superoxide anion formation, as indicated by elevated MitoSox fluorescence, increases in DHEA-treated cells. This probably forms as a consequence of one electron transfer from the reduced redox centers in complexes of respiratory chain to molecular oxygen. Additionally, the translated products of the alternatively spliced transcripts of SHC1 gene may

Fig. 8. Expression of FASTKD2 reduces DHEA-induced ROS generation. (A) SK-Hep1 cells were transfected with control vector (Vector) or FASTKD2 expression vector (FASTKD2) for 48 h. The transfected cells were re-plated, and 24 h later, were further treated without () or with (+) 200 lM DHEA for 48 h. They were stained with MitoSOX red, and analyzed using flow cytometry. The MFI of the stained cells is expressed as fold relative to that of untreated control vector-transfected cells. Results are mean ± SD, n = 6. *p < 0.05, vs. control vector-transfected cells. (B) SKHep-1 cells were similarly transfected and treated with DHEA. They were stained with H2DCFDA, and analyzed using flow cytometry. The DCF MFI of the stained cells is expressed as fold relative to that of untreated control vector-transfected cells. Results are mean ± SD, n = 6. *p < 0.05, vs. control vector-transfected cells.

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be involved. p66Shc, an isoform of SHC1, can localize to mitochondria, and interact with cytochrome c to produce hydrogen peroxide [61,62]. Paradoxically, the level of SHC1 protein decreased in DHEA-treated cells (data not shown). As the peptides corresponding to SHC1 (listed in proteomics dataset) cannot uniquely identify different isoforms, the SHC1 entity may represent an assemblage of different isoforms. Upon further examination, p66Shc, p52Shc and p46Shc were present in mitochondria (our unpublished data). The p52Shc level was significantly reduced, while the p46Shc level remained largely unchanged in mitochondria of DHEA-treated cells. A form of p66Shc with a slightly decreased electrophoretic mobility, reminiscent of the Ser-phosphorylated form [63,64], was detected; and its level increased in mitochondria of treated cells. This modified form of p66Shc may contribute to DHEAinduced ROS. Consistent with an increased mitochondrial ROS generation, DHEA-treated cells have a robust increase in level of glutathione disulfide, but a relatively mild decrease in their glutathione content [35]. To cope with oxidative stress, these cells increase the synthesis of c-glutamylcysteine and glutathione. Some enzymes involved in glutathione metabolism and its antioxidative actions increased in expression modestly after DHEA treatment. These include microsomal glutathione transferase 1 (MGST1) and glutathione reductase (GSR). MGST1, which is localized to mitochondrial outer membrane, protects cells from reactive intermediates [65]. GSR can be targeted to mitochondria [66], and plays an important role in reduction of GSSG to GSH and maintenance of activity of iron-sulfur center-containing enzymes [67]. Up-regulation of the antioxidative system may represent an adaptive response to increased ROS generation. The DHEA-induced mitochondrial mass increase may represent a compensatory response of cells to reduction in respiratory activity. The compensatory mitochondrial mass increase is not unprecedented. Mitochondrial mass of fibroblasts undergoing replicative senescence or H2O2-induced senescence increase is higher than that of cells at early proliferating stage [68]. Increased ROS generation in the former cells is believed to promote mitochondrial biogenesis. It is plausible that DHEA-induced mitochondrial ROS generation contributes to mitochondrial mass increase. Consistent with this notion, the level of mitochondrial sirtuin SIRT3 increased modestly (by 10%) in DHEA-treated cells. SIRT3 can be induced by hydrogen peroxide to deacetylate its target proteins, and promotes mitochondrial biogenesis [69]. Still, other mechanism cannot be ruled out. DHEA may induce the expression of PGC-1a [70], which promotes mitochondrial biogenesis [71]. DHEA-induced mitochondrial biogenesis occurs concomitantly with repression of specific mitochondrial proteins. It follows that such compensatory change does not completely restore mitochondrial respiratory activity. It appears that the compensatory mitochondrial biogenesis can occur without effectually restoring mitochondrial functions. For instance, mitochondrial proliferation in human colon carcinoma cells is unable to make up for the functional loss caused by depolarization [72]. Likewise, the mitochondrial mass of qo cells devoid of mitochondrial DNA increases in response to H2O2 treatment [68]. Fas-activated serine/threonine phosphoprotein (FAST) and related proteins FASTKD1-5 constitute the FASTK family of proteins, which are characterized by the presence of an N-terminal mitochondrial targeting domain and three C-terminal domains, namely FAST_1, FAST_2 and RAP domains. FASTKD3 is required for mitochondrial respiration, and interacts with components of mitochondrial translational apparatus and metabolic pathways [49]. It has been recently found that FASTKD2 interacts with such mitochondrial transcripts as 16S RNA, and MT-ND6 and MT-CO2 mRNAs, and is involved in their processing and expression [73]. Apparently, it is required for mitochondrial ribosome biogenesis [74]. Down-regulation of FASTKD2 expression in DHEA-treated cells and the ability of exogenous FASTKD2 expression to alleviate

DHEA-induced growth inhibition suggest that FASTKD2 is a target for action of DHEA. DHEA may regulate FASTKD2 expression via intermediate regulatory molecules. Functionally, a decrease in FASTKD2 expression may reduce the assembly of respiratory complexes. Silencing of FASTKD2 expression has been shown to reduce the level of complex I and IV holoenzymes [74]. As mentioned earlier, FASTKD2 can bind MT-CO2 mRNA. We have found that the level of MT-CO2 mRNA decreased in DHEA-treated cells (data not shown). It is plausible that the reduced FASTKD2 and MT-CO2 expression, together with a decline in interaction between FASTKD2 and MT-CO2 mRNA, may hamper the assembly of functional complex IV (cytochrome c oxidase). Consistent with this, a nonsense mutation in FASTKD2 gene in an infant with mitochondrial encephalomyopathy was associated with cytochrome c oxidase deficiency [75]. DHEA may affect the signaling between mitochondria and cytosol/nucleus. Epidermal growth factor receptor (EGFR) and Lyn are mitochondrial targets for DHEA. EGFR can enter mitochondria, where it binds to MT-CO2 and regulates its activity [76,77]. Exogenous expression of mitochondria-targeting EGFR enhances ATP production and cell motility [78]. Diminished EGFR expression in mitochondria of DHEA-treated cells implies that the EGFR signaling to mitochondria and oxidative phosphorylation are dysregulated. Lyn, localized to mitochondrial intermembrane space, acts as a sensor for hydrogen peroxide generated by respiratory complexes. It can activate Syk, which is upstream of signaling molecules such as JNK and Akt [79]. Decrease in Lyn in mitochondria of DHEA-treated cells suggests that the mitochondrion-to-cyto sol/nucleus signaling may be affected.

Conflict of interest The authors declare no conflict of interest. Acknowledgments The research work was supported, in whole or in part, by grants from Chang Gung Memorial Hospital (BMRP819, BMRP564, CMRPD1C0753, CMRPD1E0421, CMRPD1E0422, CMRPD3D0192, CMRPD1C0443, CMRPD1C0763, CLRPD190014, and CLRPD190015), and Ministry of Science and Technology, Taiwan, R. O. C. (NMRPD1E0651 & NMRPD1E1061). References [1] C.R. Parker Jr., Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging, Steroids 64 (1999) 640–647. [2] S.S. Yen, G.A. Laughlin, Aging and the adrenal cortex, Exp. Gerontol. 33 (1998) 897–910. [3] M.P. Cleary, The antiobesity effect of dehydroepiandrosterone in rats, Proc. Soc. Exp. Biol. Med. 196 (1991) 8–16. [4] M.K. McIntosh, C.D. Berdanier, Antiobesity effects of dehydroepiandrosterone are mediated by futile substrate cycling in hepatocytes of BHE/cdb rats, J. Nutr. 121 (1991) 2037–2043. [5] G.B. Gordon, D.E. Bush, H.F. Weisman, Reduction of atherosclerosis by administration of dehydroepiandrosterone. A study in the hypercholesterolemic New Zealand white rabbit with aortic intimal injury, J. Clin. Invest. 82 (1988) 712–720. [6] A.N. Nafziger, D.M. Herrington, T.L. Bush, Dehydroepiandrosterone and dehydroepiandrosterone sulfate: their relation to cardiovascular disease, Epidemiol. Rev. 13 (1991) 267–293. [7] D.L. Coleman, E.H. Leiter, R.W. Schwizer, Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice, Diabetes 31 (1982) 830– 833. [8] D.L. Coleman, R.W. Schwizer, E.H. Leiter, Effect of genetic background on the therapeutic effects of dehydroepiandrosterone (DHEA) in diabetes-obesity mutants and in aged normal mice, Diabetes 33 (1984) 26–32. [9] K. Wojtal, M.K. Trojnar, S.J. Czuczwar, Endogenous neuroprotective factors: neurosteroids, Pharmacol. Rep. 58 (2006) 335–340.

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