Dioscin (Saponin)-Induced Generation of Reactive Oxygen Species through Mitochondria Dysfunction: A Proteomic-Based Study Ying Wang,† Chi-Ming Che,† Jen-Fu Chiu,*,‡ and Qing-Yu He*,§ Department of Chemistry and Open Laboratory of Chemical Biology and Department of Anatomy, The University of Hong Kong, Hong Kong SAR, People’s Republic of China, and Institutes of Life and Health Engineering, Jinan University, Guangzhou 510632, People’s Republic of China Received June 27, 2007
It is generally believed that traditional Chinese medicine such as saponins has great value as potent cancer prevention and chemotherapeutic agents; however, the molecular basis for their activities is for the most part lacking. In the present study, we used proteomics to examine the cytotoxic effect of dioscin, a glucoside saponin, on human myeloblast leukemia HL-60 cells. Dioscin induced apoptosis in HL-60 cells in a time-dependent manner. Protein profiling of the microsomal fraction with enriched plasma membrane proteins isolated from HL-60 cells revealed that proteins act as chaperones and/or mediators of protein folding and were substantially altered in expression cells upon dioscin stimuli. Further biochemical study indicated that mitochondria dysfunction caused generation of reactive oxygen species (ROS), leading to the changes in protein expression. The mitochondrial transmembrane potential (∆Ψm) inhibitor aristolochic acid (ArA) partially abrogated the dioscin-initiated death receptor apoptosis pathway and cell death. The current study provided detailed evidence to support that dioscin is capable of inducing apoptosis in mammalian cells, in which the mitochondria-initiated apoptosis pathway plays an important role. Keywords: anticancer drug • dioscin • mitochondria • reactive oxygen species • apoptosis
Introduction Saponins belong to a family of glycoconjugates with a broad range of biological and pharmacological activities, including immunomodulation and anticancer effects.1 Dioscin, a plant glucoside saponin extracted from the roots of Polygonatum zanlanscianense, was shown to induce apoptosis in a number of human carcinoma cell lines.2,3 Our previous study on dioscin-treated human myeloblast leukemia HL-60 cells suggested that dioscin exerted cytotoxicity through multiple apoptosis-inducing pathways.4 However, the detailed molecular basis of dioscin-triggered cell death remains largely unexplored. Programmed cell death or apoptosis is a tightly controlled multistep mechanism that is essential for elimination of unwanted cells in various biological systems. Diverse stimuli, such as DNA damage, death receptorsligand interaction, and increase of intracellular reactive oxygen species (ROS) level, may be responsible for initiating the apoptotic cascade.5 These pro-apoptotic signals induce several early events, jointly activating a common biochemical pathway, which then leads to the execution of apoptotic cell death. ROS are a group of reactive, short-lived, oxygen-containing species including superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), signlet oxygen (1O2•), and peroxyl radical (LOO).6 Under * To whom correspondence should be addressed. E-mail: tqyhe@ jnu.edu.cn for Q.Y.H.;
[email protected] for J.F.C. Fax: +86-20-8522-7039 for Q.Y.H.; +852-2817-1006 for J.F.C. † Chemistry, The University of Hong Kong. ‡ Anatomy, The University of Hong Kong. § Jinan University. 10.1021/pr070399r CCC: $37.00
2007 American Chemical Society
stress conditions, ROS are derived from at least two sources: the oxidative protein folding machinery in the endoplasmic reticulum (ER) and the mitochondria.7,8 In the ER, ROS are derived from the formation of disulfide bonds and the glutathione reduction of unstable and improper disulfide bonds through unfolded protein response (UPR).7 Meanwhile, mitochondria possess a total of nine potential sites of ROS generation that can leak single electrons to oxygen and convert it into superoxide anion, a progenitor ROS during the depletion of mitochondrial transmembrane potential (∆Ψm).8 Mitochondria also serve as a core component of the intrinsic cell death machinery.9 Attenuation of ∆Ψm causes release of cytochrome c from the mitochondrial intermembrane space and subsequently activates caspase 9 and caspase 3. These caspases are believed to cause cell death through the cleavage of poly(ADPribose) polymerase 1 (PARP-1), chromatin condensation, and DNA fragmentation.10 The extrinsic apoptosis pathway includes death receptor activation, which is initiated by death receptor-ligand interaction.11 Recruitment of death receptor-associated molecules, such as Fas-associated death domain (FADD), ensures the activation of initiator caspase 8, which can lead to the cleavage of the pro-apoptotic protein Bid. Truncated Bid then translocates to the mitochondria and subsequently activates caspase 9 and caspase 3, resulting in biochemical and morphologic changes that are characteristic of apoptotic cell death.11 In the present study, we focused on the microsomal fraction with enriched plasma membrane protein alterations by proteomic analysis and identified the major cellular pathways Journal of Proteome Research 2007, 6, 4703–4710 4703 Published on Web 11/02/2007
research articles responsible for the protein changes. Further functional study revealed that depletion of ∆Ψm induced ROS generation and partially activated the death receptor pathway. These findings support that dioscin is capable of inducing apoptosis through the mitochondrial death pathway.
Experimental Section Dioscin and Other Reagents. Dioscin was provided by Shanghai Institute of Organic Chemistry. All other chemicals, except as otherwise noted, were obtained from either SigmaAldrich Chemical Co. (St. Louis, MO) or Amersham Biosciences (Piscataway, NJ). Dioscin was dissolved in DMSO prior to use. Cell Line and Culture Conditions. Human myeloblast leukemia HL-60 cells were cultured in RPMI 1640 medium with 2.0 g/L sodium bicarbonate plus 10% fetal bovine serum, 1% L-glutamine, 1% penicillin, and streptomycin (100 units/mL), in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 °C. Drug Treatment. The cells were grown to about 80% confluence and were then either subcultured or treated with 7.6 µM dioscin (6.6 µg/mL). In some experiments, cells were pretreated with 50 µM aristolochic acid (ArA) and 100 µM caspase 8 inhibitor IETD-CHO (Ac-Ala-Ala-Val-Ala-Leu-LeuPro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr-AspCHO, CalBioChem, La Jolla, CA), respectively, 1 h prior to the addition of dioscin. Cytotoxicity Assay. The cytotoxicity of drug treatment was determined by MTT assay, in accordance with a previously reported procedure.5 Flow Cytometric Analysis of Apoptosis. Dioscin-induced apoptosis was determined by the Vybrant apoptosis assay kit (V-13241; Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Briefly, HL-60 cells were cultivated for 24 h before either left untreated or treated with 7.6 µM dioscin. At the end of each experiment, cells were harvested, resuspended in ice-cold PBS solution, stained by Alexa Fluor 488 Annexin V and/or propidium iodide (PI), and then analyzed with a FACStar Plus flow cytometer. One million cells were analyzed for each sample, providing a solid statistical basis for the determination of the percentage of apoptotic cells in each treatment using the WinMDI 2.8 software program. DNA Ladder. DNA laddering was assessed by a protocol developed by Fukuda et al.12 Equal quantities (5–30 µg) of DNA extracted from dioscin treatment or untreated control were run on 1.4% agarose gels in TBE buffer. Bands were detected by ethidium bromide staining. Preparation of Cell Microsomal Fraction Containing Enriched Plasma Membrane Proteins. Cell microsomal proteins were isolated from about 1 × 108 cells. To preserve intact surface glycoproteins, cells were not harvested with trypsin. After three washes with ice-cold PBS, the cells were scraped and incubated with 1.5 mL of sucrose buffer [10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 200 mM sucrose, 2 mM Na3VO4, 50 mM NaF, 10 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF)] for 30 min on ice to allow cell swelling. Cells were fragmented by 20 s of sonication, and then nuclei and cell debris were removed from the homogenates by centrifugation at 3000 rpm (Eppendorf Centrifuge 5415R) for 10 min at 4 °C. The supernatant was centrifuged at 35 000 rpm for 60 min at 4 °C (TLA 120.2 rotor, Beckman Coulter Optima LE-80K ultracentrifuge, Mississauga, Canada). The resulting pellet was solubilized in 2-DE lysis buffer [7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer, 1 mM PMSF] for a minimum of 1 h at 4704
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Dioscin-Induced ROS through Mitochondrial Pathway 4 °C. Amplification of the plasma membrane proteins was examined by Western blot analysis using integrin-beta 1 (BD Biosciences, San Jose, CA) as membrane fraction marker, FAK (Santa Cruz Biotechnology, Santa Cruz, CA) as cytoplasmic marker, and histone H1 (Santa Cruz Biotechnology) as nuclear marker. Two-Dimensional Gel Electrophoresis. Two-dimensional gel electrophoresis (2-DE) was carried out with Amersham Biosystems IPGphor IEF and Hoefer Tank (13 cm) units, in accordance with a previously described protocol.4 Protein samples (250 µg) of microsomal fraction extracted from untreated control and 7.6 µM dioscin treatment, respectively, were used for 2DE analysis. Triplicate electrophoresis was performed to ensure reproducibility. All gels were visualized by silver staining.4 Image Analysis and MS Peptide Sequencing. Image acquisition and analysis were performed with Image Scanner (Amersham Biosciences) and ImageMaster 2D Platinum software (Amersham Biosciences).4 Comparisons were made between gel images of cells treated with dioscin and untreated controls. Altered protein spots with consistent and significant volume changes (>2-fold difference) were selected for MALDI-TOF MS and tandem mass spectrometric analysis with a 4700 proteomics analyzer (“TOF/TOF”, Applied Biosystemes Inc., Foster City, CA). The resulting data were processed by using the 4700 Explorer software.13 Duplicate or triplicate runs were made to ensure the accuracy of the analysis. Detection of Oxidative Stress. Cells in HBSS were incubated for 15 min at 37 °C with 2 µM dihydroethidium (DHE, Molecular Probes) or 1 µM dichlorodihydrofluorescein diacetate (DCFHDA, Molecular Probes), respectively. After washing and resuspension in complete growth media, cells were treated with dioscin for various times as indicated in the figure. Dye oxidation (increase in fluorescence) was measured using a FACStar Plus flow cytometer with excitation and emission wavelengths at 488 and 530 nm, respectively.14 Glutathione Assay. Cellular glutathione (GSH) level was determined by a fluorometric glutathione detection kit (Oncogene, CN Biosciences Inc.), in accordance with the experimental protocol recommended by the supplier. Measurement of Mitochondrial Transmembrane Potential (∆Ψm). Changes in ∆Ψm were assayed by Rhodamine 123 (Rho123, Molecular Probes) staining in accordance with a procedure described previously.5 Western Blot Analysis. Western blot analysis was performed using primary antibodies against caspase 12 (Stressgen, Ann Arbor, MI), GRP78/ Bip (Santa Cruz Biotechnology), GRP94 (Santa Cruz Biotechnology), Fas (Laboratory Vision, Fremont, CA), FasL (Laboratory Vision), FADD (Laboratory Vision), caspase 8 (Laboratory Vision), Bid (Cell Signaling Technology, Beverly, MA), and alpha-tubulin (Sigma-Aldrich) at optimal dilution. Statistical Analysis. Statistical analysis was performed using a two-tailed Student’s t-test, and p < 0.05 was considered significant. Data were expressed as mean ( SD in triplicate, and reproducibility was confirmed in three separate experiments.
Results Dioscin Induces Apoptotic Cell Death in HL-60 Cells. Cytotoxicity of dioscin in HL-60 cells was first verified by cell proliferation assay in a time course treatment with 7.6 µM dioscin. This dose was the IC50 (half-maximal inhibitory concentration) value measured by dose response study in our
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Wang et al. 4
previous study. Microscopic results reported before showed that HL-60 cells treated with 7.6 µM dioscin exhibited a shrunken morphology compared with untreated control cells, indicating that dioscin adversely affects cell integrity.4 Figure 1A shows that dioscin inhibited HL-60 cell proliferation in a time-dependent manner. The effect of dioscin on cell death was further investigated by flow cytometric analysis using Vybrant apoptosis assay kit. This assay can distinguish apoptosis from necrosis, if cell death occurs. It can also quantitate three populations of cells with drug treatment: viable live cells, apoptotic cells, and populations of necrotic cells.15 Results in Figure 1B show that 24 h treatment of dioscin induced 16% increase in apoptotic cell population and 6% increase in necrotic cell population. Dioscin treatment dramatically reduced the cell number at 7.6 µM (50% at 24 h); however, this concentration caused only 22% apoptotic cell death (early and late) in comparison to untreated control, suggesting that dioscin may have a potent activity to inhibit cell proliferation besides induction of apoptotic cell death (manuscript in preparation). Dioscin-induced apoptosis was also confirmed by the oligonucleosomic degradation of cellular DNA, as this type of chromatin fragmentation is characteristic of apoptotic cell death.16 A DNA ladder was detectable after dioscin treatment for 24 h in HL-60 cells (Figure 1C). These observations together demonstrated that dioscin induced apoptosis in HL60 cells significantly after 24 h treatment. On the basis of these results, the treatment condition with 7.6 µM dioscin for 24 h was selected for the following analysis. Altered Microsomal Proteins Are Mainly Chaperones and Mediators of Protein Folding. Our previous results suggested that the F ring of the steroid and spirostanol structural unit formed by dioscin under physiological conditions played an important role in its cytotoxic effect toward cancer cells.17 This kind of structure was reported to interact preferentially with membrane proteins.18 We therefore applied 2-DE-based proteomic technology to further study the regulation of soluble microsomal membrane proteins that may play important roles in multiple signaling pathways by dioscin treatment. Amplification of plasma membrane proteins in the isolated microsomal fraction was determined by Western blot analysis using specific subcellular fraction markers (Figure 2A). The significantly enhanced quantity of integrin-beta 1 in the microsomal fractions of control and dioscin treatment samples indicated the enrichment of plasma membrane proteins compared to an equal amount of total proteins from whole cell lysate (Figure 2A). Four replica gels were used to compare the protein profiles and to calculate average changes by paired Student’s t-test for the detection of significant and consistent alterations. Separation of the microsomal fraction allowed the identification of 39 differentially expressed proteins after dioscin treatment for 24 h (Figure 2B and Table 1). These proteins are subsequently analyzed by MALDI-TOF and tandem mass spectrometry, and their identities as well as quantitative data are summarized in Table 1. These proteins can be classified into several groups on the basis of their major functions reported in the literature. One of the most noticeable groups is proteins that act as chaperones and/or mediators of protein folding (Figure 2C); the functions of these proteins depend on cellular redox state and the functions of mitochondria and endoplasmic reticulum
Figure 1. Dioscin-induced apoptosis in HL-60 cells. (A) Time course treatment of HL-60 cells with IC50 (half-maximal inhibition concentration) dose of dioscin (7.6 µM). Data were mean ( SD from three separate experiments. (B) Detection of apoptotic HL60 cells in the presence of dioscin for 24 h by annexin-V analysis. (C) DNA ladder was formed after dioscin treatment for 24 h. Results in (B) and (C) are representatives from three independent experiments.
(ER). We next investigated these possibilities to further determine the major cellular target of dioscin. Dioscin Stimulates Generation of Hydrogen Peroxide and Superoxide. Changes in cellular redox state and the presence of ROS can activate signaling events leading to Journal of Proteome Research • Vol. 6, No. 12, 2007 4705
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Figure 2. Dioscin treatment significantly altered microsomal proteins of HL-60 cells. (A) Western blot analysis showing the amplification of plasma membrane proteins in the microsomal fraction. (B) Representative overview of silver-stained 2-DE image of microsomal fraction from untreated control and dioscin-treated HL-60 cells. Arrows indicate those proteins whose expressions were altered after dioscin treatment and were unequivocally identified by PMF. Numbers are correlated with the spot no. listed in Table 1. (C) Detailed alteration patterns of proteins that act as chaperones and/or mediators of protein folding.
rebalance of protein folding capacity and folding demand or to cell death.7 Dioscin-stimulated ROS production in HL-60 cells was measured using the cell-permeable, oxidation-sensitive dyes DCFHDA and DHE, which can be oxidized to increase fluorescence by hydrogen peroxide and superoxide anions, respectively.19,20 Figures 3A and 3B show that dioscin induced increases in DCFHDA and DHE fluorescence within 1.5 h of treatment. Commonly, GSH is considered as the major thioldisulfide redox buffer of cells, and cellular GSH level is used to determine the redox state. Figure 3C reveals that exposure of HL-60 cells to 7.6 µM dioscin resulted in a drop in intracellular GSH level within 1 h and then a steady increase for 8 h. These results indicate that dioscin stimulated ROS generation in HL60 cells at an early time point. Dioscin-Induced ROS Generation Is Due to Mitochondria Dysfunction but Not ER Stress. Mitochondrial respiratory chain on the inner mitochondrial membrane and protein oxidation 4706
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Dioscin-Induced ROS through Mitochondrial Pathway in the ER are two major sources of intracellular ROS generation.5,21 We directly measured mitochondrial membrane integrity by Rho-123 staining. Flow cytometric results showed that ∆Ψm was depleted in a time-dependent manner soon after dioscin treatment (Figure 4A). Results from our previous study also showed that caspase 9 and caspase 3 were activated by dioscin in a time-dependent manner, indicating the correlation of the dioscin cytotoxicity with mitochondrial pathway.4 ER resident chaperones GRP78/Bip and GRP94 play an important role in protein quality control and act as a system of defense against any alteration in protein synthesis and proper folding. Apoptotic changes in ER generate ROS and then initiate signalregulating kinase 1 (ASK1) and inositol-requiring kinase 1 R (IRE1 R) activation and subsequently caspase 12 activation. However, no alteration in GRP78/Bip, GRP94, and caspase 12 levels was detected all the time during dioscin treatment (Figure 4B), ruling out the possibility of ER stress generating ROS by dioscin stimuli. Pretreatment with aristolochic acid (ArA), an uncoupling agent that inhibits mitochondrial permeability transition pore opening, partially abrogated dioscin-induced ROS generation (Figure 4C). These data indicate that loss of ∆Ψm leads to increased cellular ROS state. Death Receptor-Initiated Apoptosis Pathway Is Activated by Mitochondria Dysfunction under Dioscin Treatment. The death receptor apoptosis pathway involves binding of a deathinducing ligand (e.g., Fas ligand (FasL)) to a death receptor (e.g., Fas) and then the receptor clustering and transmission of signals via interactions of the cytoplasmic death domains of the receptors with adaptor proteins (e.g., FADD), which subsequently activate caspase 8.22 To determine whether dioscin triggers the death receptor signaling pathway, we directly measured the expression of Fas, FasL, FADD, caspase 8, and Bid by Western blot analysis. Figure 5A shows that dioscin treatment increased FasL, FADD, and caspase 8 expressions and decreased Bid levels in a time-dependent manner, but did not alter the surface expression of death receptor Fas. It has been demonstrated that caspase 8 can activate procaspase 3 and also cleave Bid into its truncated form (tBid), which translocates to the mitochondria and enhances cytochrome c release.23 To investigate the relationship of mitochondria dysfunction and death receptor activation, we pretreated HL-60 cells with caspase 8-specific inhibitor IETD-CHO and then measured ∆Ψm. Figure 5B shows that cotreatment with IETD-CHO did not inhibit dioscin-induced perturbation of ∆Ψm. On the other hand, differential expressions of FasL, FADD, caspase 8, and Bid under dioscin treatment were partially abrogated by ArA cotreatment (Figure 5C), and dioscin-induced cell death was partially inhibited by ArA (Figure 5D). These results suggested that depletion of ∆Ψm was responsible for the cytotoxicity of dioscin and the activation of the death receptor apoptosis pathway.
Discussion Apoptosis Is the Major Form of Cell Death Induced by Dioscin. Dioscin, one of the best characterized diosgenyl saponins, has been shown to exhibit potent cytotoxicity toward a number of human cancer cell lines.2,3,24 Our previous study demonstrated that multiple pathways leading to cell death were activated by dioscin treatment.4 However, the detailed molecular basis of dioscin cytotoxicity remained elusive. Apoptotic cell death can be characterized by a number of cellular and biochemical hallmarks, including externalization of phosphatidylserine to the outer cell membrane and DNA laddering.5,15
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Wang et al. Table 1. Dioscin (7.6 µM 24 h) Treatment-Induced Alterations in Membrane Fraction of HL-60 Cells
spot no.
245 246 249 299 419 486 1013 1017 1202 1224 1241 1366
687 693 825 999 1222 1268 1288
311 420 856 941 1178 1265 1266 1225 1322 1323 1387 218 220 231 281 303 877 892 1037 1038 a
NCBI access no.
protein ID
Proteins That Act HSP 70 kDa protein 8 isoform 1 HSP 70 kDa protein 8 isoform 2 HSP 70 kDa protein 8 isoform 1 ubiquilin 1 protein disulfide isomerase (PDI) chaperonin (HSP60) proteasome activator subunit 3 proteasome alpha 1 subunit isoform 2 proteasome beta 3 subunit peroxiredoxin 3 isoform, a precursor DJ-1 protein protein disulfide isomerase (PDI) NMR, 40 structures Proteins M2-type pyruvate kinase M2-type pyruvate kinase enolase 1 electron-transfer flavoprotein, alpha polypeptide lysophospholipase isoform ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunitprecursor (fragment)
MW (kDa)/pI
no. of peptides recovereda
as Chaperones/Mediators of Protein Folding 5729877 70.85/ 5.37 25 6 24234686 53.48/ 5.62 20 5 5729877 70.85/ 5.37 9 1 24659706 62.55/ 5.09 10 6 860986 56.76/ 6.10 17 5 306890 60.99/ 5.70 11 4 6755214 29.49/ 5.69 11 3 4506179 29.54/ 6.15 10 1 22538465 22.93/ 6.14 9 3 5802974 27.68/ 7.67 6 4 31543380 19.88/ 6.33 9 2 2098329 13.24/ 5.94 4 2
That Participate in Energy Production 189998 57.87/ 7.95 17 189998 57.87/ 7.95 21 4503571 47.13/ 7.01 5 4503607 35.06/ 8.62 9
sequence coverage %
fold difference (dioscin: ctrl)
59.0 45.2 20.9 31.2 21.8 7.1 8.7 10.3 11.7 14.2 5.8 10.3
7.26 ( 0.74 3.09 ( 0.25 1.94 ( 0.34 3.70 ( 0.49 2.53 ( 0.35 2.04 ( 0.25 0.51 ( 0.16 1.92 ( 0.33 2.02 ( 0.09 4.27 ( 0.15 0.61 ( 0.07 1.78 ( 0.36
4 5 2 1
19.0 30.4 8.9 5.3
0.62 ( 0.09 0.65 ( 0.08 2.60 ( 0.57 2.07 ( 0.24
4679012 23273230
22.86/ 6.05 18.48/ 5.21
6 10
3 4
12.5 11.8
1.21 ( 0.06 0.62 ( 0.12
179279
56.52/ 5.26
3
2
5.1
1.55 ( 0.01
4 2 7 4
32.2 10.3 35.3 19.0
0.36 ( 0.11 0.40 ( 0.03 1.66 ( 0.07 0.31 ( 0.15
3
39.7
0.57 ( 0.13
5
28.9
54.64 ( 7.66
2
14.7
3.52 ( 0.55
3
16.6
0.15 ( 0.11
2
12.5
1.76 ( 0.15
Proteins That Regulate Cell Death and Differentiation Ras-GTPase-activating protein (GAP) 5031703 52.12/ 5.36 17 adenylyl cyclase-associated protein 5453595 51.64/ 8.07 14 annexin I 4502101 38.69/ 6.57 17 voltage-dependent anion channel 2 42476281 31.55/ 7.40 10 (VDAC 2) Rho GDP dissociation inhibitor 14327952 22.97/ 5.10 10 (RhoGDI) beta Rho GDP dissociation inhibitor 14327952 22.97/ 5.10 9 (RhoGDI) beta (fragment) Rho GDP dissociation inhibitor 14327952 22.97/ 5.10 8 (RhoGDI) beta (fragment) translationally controlled tumor 4507669 19.58/ 4.84 7 protein 1 cofilin 1 5031635 18.49/ 8.22 7 Actin-related protein 2/3 complex, subunit 5-like sapiens Actin, gamma 1 propeptide
no. of peptides sequencedb
Cytoskeleton Proteins 13569956 16.93/ 6.15
5
3
24.5
0.50 ( 0.01
4501867
4
2
19.0
0.29 ( 0.11
7 6 1
43.8 32.8 8.5
0.28 ( 0.10 0.42 ( 0.17 0.15 ( 0.01
6
38.0
2.09 ( 0.31
5
13.6
3.34 ( 0.16
4 5 4
18.6 29.5 10.3
0.52 ( 0.13 2.66 ( 0.54 0.26 ( 0.14
3
9.1
0.36 ( 0.12
41.77/ 5.31
Transcription and mRNA Processing Proteins Guanine monophosphate synthetase 4504035 76.67/ 6.42 32 Guanine monophosphate synthetase 4504035 76.67/ 6.42 26 far upstream element-binding 16878077 68.56/ 6.85 14 protein (FUBP1 protein) heterogeneous nuclear 11527777 64.04/ 8.49 19 ribonucleoprotein L (hnRNP L) cleavage stimulation factor, 21619877 59.08/ 6.33 10 3′ pre-RNA subunit 2 ribosomal protein P0 4506667 34.25/ 5.71 13 ribosomal protein P0 4506667 34.25/ 5.70 12 eukaryotic translation 83376130 24.75/ 4.50 8 elongation factor 1 beta 2 eukaryotic translation 83376130 24.75/ 4.50 6 elongation factor 1 beta 2
Unique peptides identified by peptide mass fingerprinting.
b
Unique peptides identified by MS/MS sequencing.
Translocation of phosphatidylserine from the cytoplasmic surface to the outer leaflet of the plasma membrane occurs
under early cell death (apoptosis), while propidium iodide (PI) is only permeable to the late form of cell death (necrosis).15 In Journal of Proteome Research • Vol. 6, No. 12, 2007 4707
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Figure 3. Dioscin stimulated ROS generation in HL-60 cells. (A) Representative FACS profile for dioscin-induced DCFHDA oxidation in a time-dependent manner. (B) Representative FACS profile for dioscin-induced DHE oxidation in a time-dependent manner. (C) Measurement of cellular glutathione (GSH) level in HL-60 cells treated with dioscin; the values are shown as mean ( SD of triplicate experiments. Histogram markers in (A) and (B) were set at the same fluorescent intensity range; numbers on the marker indicate the percentage of cells in the gated region.
the present study, we showed that dioscin induced a significant increase of cell population in the annexin V positive and PI negative region (Figure 1B) and stimulated a DNA ladder (Figure 1C) after 24 h of treatment. Taken together with our previous finding that typical apoptotic changes in the nucleus were observed in HL-60 cells after dioscin treatment,4 these results suggest that the form of cell death induced by dioscin is mainly apoptosis. The dioscin-induced apoptosis can also be derived from the observation of the suppressed expression of voltage-dependent anion channel 2 (VDAC 2) and the significant increase of a fragmentation of Rho GDP dissociation inhibitor beta (RhoGDI beta) (Table 1). VDAC proteins constitute the major pathway governing the mitochondrial apoptotic death pathway, and three mammalian isoforms (VDAC 1, VDAC 2, and VDAC 3) have distinct functions.25 In most mammalian cells, Bak is mostly localized in the outer mitochondrial membrane and normally remains inactive by binding to VDAC 2.26 However, VDAC 2 expression is diminished upon death stimulation, thus allowing Bak to play a full-throttle role in mediating apoptosis.27 The RhoGDI is a cellular regulatory protein that functions primarily by controlling the cellular distribution and activity of Rho GTPases.28 The functions of Rho GTPases can be disrupted during Fas-induced apoptosis by caspase 3-mediated 4708
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cleavage of the RhoGDI protein.29 Proteomic results revealed the overexpression of Rho GDI in invasive ovarian cancers when compared with low malignant tumor or normal tissues.30 Further functional study indicated that RhoGDI can inhibit caspase-mediated apoptosis in part through protection of Rac1 GTPase from caspase cleavage.30 These findings are in accord with our current observation, suggesting that the altered proteins induced by dioscin are directly related to apoptosis. Protein Alterations Associated with Dioscin Cytotoxicity Are Related to ROS Generation. Saponins have been shown to primarily affect functions of membrane receptor proteins.31,32 We therefore used a comparative proteomic approach to examine the protein changes associated with dioscin-induced apoptosis in the cell microsomal fraction with enriched plasma membrane proteins. On the basis of the identified proteins, we revealed that several cellular pathways are potentially involved in dioscin-induced cell death. While substantial numbers of membrane proteins were identified here, some nonmembrane proteins were also found (e.g., HSP 70 and gamma Actin). These nonmembrane proteins may come from the endoplasmic reticulum and Golgi membranes. Additionally, the alteration of the nonmembrane proteins suggests the in vivo interactions of the intracellular domains of membrane proteins. There are also reports that some of the HSPs are
Wang et al.
Figure 4. Dioscin induced ROS generation from the mitochondria but not the endoplasmic reticulum. (A) Measurement of mitochondrial membrane potential (∆Ψm) depolarization in dioscintreated HL-60 cells by Rho-123 staining. (B) Western blot analysis of GRP78/ Bip, GRP94, and caspase 12 expression under dioscin treatment. (C) Effect of ArA on dioscin-induced DCFHDA and DHE oxidation. Data in (A) and (B) are representatives of three independent experiments. Data in (C) are mean ( SD from three separate experiments (*, p < 0.05 statistically significant difference compared with control; #, p < 0.05 statistically significant difference compared with dioscin treatment).
expressed and localized at the cell surface,33 implying that these nonmembrane proteins identified may be associated with heterogeneous cellular locations. Dioscin significantly altered the expression of chaperone proteins and proteasome subunits (Figure 2C and Table 1). Among them, heat shock proteins are known to be induced by ROS, and their expressions are related to several apoptosis pathways.34 Down regulation of DJ-1 expression by siRNA has been reported to enhance cell death by oxidative stress.35 Under stress conditions, increased cellular ROS can cause damage to cellular DNA, protein, and lipids, resulting in cellular senescence and then cell death.36 Proteasomes can recycle damaged and/or misfolded proteins under a high-level intracellular oxidative state.37 It has also been suggested that cells can regulate proteasome function in response to increased ROS level both by altering the total number of proteasomes and by altering the subunit components of the ubiquitin-proteasome.38 The identified elevated expression of several subunits of the proteasome complex in the present study further suggested that dioscin-induced microsomal protein alterations are associated with increased ROS level. Proteins that participate in cell growth, maintenance, and differentiation are also involved in dioscin-induced cell death
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Figure 5. Dioscin stimulated receptor-initiated apoptosis pathway through attenuation of ∆Ψm. (A) Western blot analysis of Fas, FasL, FADD, caspase 8, and Bid expressions under dioscin treatment. (B) Effect of caspase 8 inhibitor IETD-CHO on dioscininduced ∆Ψm depolarization after 6 h treatment. (C) Western blot analysis of dioscin-induced FasL, FADD, and Bid alteration in the presence of ArA. (D) Effect of ArA on dioscin-induced cell death by MTT assay after 24 h treatment. Results in (A) and (C) are representative data from three separate experiments. Data in (B) and (D) are mean ( SD from three separate experiments (*, p < 0.05 statistically significant difference compared with control; #, p < 0.05 statistically significant difference compared with dioscin treatment).
(Table 1). Additionally, dioscin treatment induced suppressed expression in proteins that act as signal transducers, transcription and mRNA processing proteins (Table 1). These results suggest that multiple pathways are associated with cytotoxicity of dioscin, and these pathways may work complementarily, resulting in dioscin-stimulated apoptotic cell death. Mitochondria Are the Primary Source of DioscinGenerated Oxidative Stress. Dioscin sensitizing HL-60 cells to apoptosis through a ROS-dependent mechanism is supported by direct measurement of ROS generation (Figure 3). Cells have developed effective mechanisms to reduce cellular ROS levels under normal conditions. However, ROS were found to play a pivotal role in activation of pro-apoptotic cell death signals under stress conditions such as chemotherapeutic agents treatment.39 ROS are generated from two major sources: the sustained UPR-regulated oxidative folding machinery in the ER and mitochondria.5,21 The regulated activation of GRP78/Bip and GRP94 provides a direct mechanism for all UPR transducers to sense the ER stress,40 and pro-apoptotic pathways emanating from the ER are mediated by caspase 12.41 In the present study, we failed to detect any changes in GRP78/Bip, GRP94, and caspase 12 expressions (Figure 4B), whereas depletion of ∆Ψm was observed in the early time of dioscin treatment by Rho 123 staining (Figure 4A). Furthermore, the generation of superoxide and hydrogen peroxide stimulated by dioscin can be blocked by the phospholipase A2 inhibitor ArA (Figure 4C). Together with previous reports that other saponins induced apoptosis through oxidative stress,42,43 the current Journal of Proteome Research • Vol. 6, No. 12, 2007 4709
research articles results collectively suggested that the mitochondria-derived ROS generation initiated dioscin-induced cell death. Attenuation of ∆Ψm Leads to Activation of the Death Receptor Signaling Pathway. Permeabilization of the outer mitochondrial membrane with consequent release of proapoptotic factors from the mitochondrial intermembrane space is an essential first step of the intrinsic apoptotic pathway and also serves as an amplification loop in the extrinsic pathway.44 In this study, we demonstrated that dioscin was able to induce expression of FasL soon after treatment (Figure 5A), which can promote apoptosis by binding to Fas. Addition of the caspase 8 inhibitor IETD-CHO cannot block ∆Ψm depletion (Figure 5B). On the other hand, dioscin-induced FasL and FADD expression, caspase 8 activation, and Bid truncation were partially restrained by the ∆Ψm inhibitor ArA (Figure 5C). Ahn and his colleagues have reported that platycodin D-induced NF-κB activity was related to a dose-dependent increase of FasL expression in HaCaT cells.45 Moreover, some studies indicated that mitochondria-derived apoptotic signals, such as ROS, JNK, and p38MAPK, are sufficient to induce FasL expression and receptor-ligand cross-linking initiated cell death.46,47 Taken together, dioscin treatment partially induced attenuation of ∆Ψm, leading to the activation of the death receptor signaling pathway and initiating apoptosis. However, the detailed mechanism of how dioscin-induced mitochondria dysfunction regulated FasL expression warrants further study.
Conclusions In the current study, dioscin inducing apoptosis in HL-60 cells was confirmed and further investigated. Proteomic analysis of microsomal fraction proteins revealed that dioscininduced microsomal protein alterations were mainly due to ROS generation. Further functional studies demonstrated that the major target responsible for dioscin cytotoxicity is mitochondria-derived oxidative stress. In addition, the death receptor initiated signaling pathway is partially activated by attenuation of ∆Ψm. Together with our previous findings, these experimental results support that mitochondria are the primary target of dioscin, raising the possibility that dioscin could be developed as a potential chemotherapeutic agent directed toward the mitochondria. Abbreviations: ∆Ψm, mitochondrial transmembrane potential; ER, endoplasmic reticulumn; GSH, glutathione; ROS, reactive oxygen species; UPR, unfolded protein response.
Acknowledgment. This work was partially supported by Hong Kong Research Grants Council Grants HKU 7512/ 05M (to Q.Y.H.), HKU 7218/02M and HKU 7395/03M (to J.F.C.). References (1) Saponins;Cambridge University Press: Cambridge, UK, 1995. (2) Liu, M. J.; Wang, Z.; Ju, Y.; Zhou, J. B.; Wang, Y.; Wong, R. N. Biol. Pharm. Bull. 2004, 27, 1059–1065. (3) Cai, J.; Liu, M.; Wang, Z.; Ju, Y. Biol. Pharm. Bull. 2002, 25, 193– 196. (4) Wang, Y.; Cheung, Y. H.; Yang, Z.; Chiu, J. F.; Che, C. M.; He, Q. Y. Proteomics 2006, 6, 2422–2432. (5) Wang, Y.; He, Q. Y.; Sun, R. W.; Che, C. M.; Chiu, J. F. Cancer Res. 2005, 65, 11553–11564. (6) Ueda, S.; Masutani, H.; Nakamura, H.; Tanaka, T.; Ueno, M.; Yodoi, J. Antioxid. Redox Signaling 2002, 4, 405–414. (7) Haynes, C. M.; Titus, E. A.; Cooper, A. A. Mol. Cell 2004, 15, 767– 776. (8) Andreyev, A. Y.; Kushnareva, Y. E.; Starkov, A. A. Biochemistry (Moscow) 2005, 70, 200–214.
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PR070399R
