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Comparative Proteomic Analysis of Indioside D-Triggered Cell Death in HeLa Cells Chi Chun Wong,† Ying Wang,‡ Ka-Wing Cheng,† Jen-Fu Chiu,‡ Qing-Yu He,*,§ and Feng Chen*,† School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China, Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China, and Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China Received January 11, 2008
Medicinal plants represent a rich source of cancer drug leads. Indioside D, a furostanol glycoside isolated from Solanum mammosum, was found to possess antiproliferative activity toward a panel of human cancer cell lines. Proteomic analysis of indioside D-treated HeLa cells revealed profound protein changes related to energy production and oxidative stress, suggesting that mitochondria dysfunction plays a role in indioside D-induced apoptosis. Indioside D caused a rapid dissipation of mitochondrial transmembrane potential (∆Ψm) and the generation of reactive oxygen species (ROS), leading to the activation of caspase-dependent apoptotic cell death. The Fas death receptor pathway was also activated following indioside D treatment, and triggered the activation of caspase-8 and cleavage of Bid, which also acted through the mitochondrial apoptosis pathway. These results suggest that indioside D induced apoptosis in HeLa cells via both intrinsic and extrinsic cell death pathways. Keywords: apoptosis • death receptors • indioside D • mitochondria • oxidative stress
Introduction Natural products derived from plants are an invaluable source of drug leads for the pharmaceutical industry over the past decades. Solanaceous plants such as Solanum lyratum and Solanum nigrum are widely used in traditional folk medicines for the treatment of cancers and herpes.1 Phytochemical investigations indicated that steroidal alkaloids and saponins are the major active components of Solanum mammosum.2 In the present study, we identified indioside D, a novel cytotoxic principle from S. mammosum fruits, as a potential chemotherapeutic drug lead. Indioside D is a furostanol saponin previously found in Solanum indicum and Solanum sodomaeum.3,4 Several studies have demonstrated that furostanol saponins such as protoneodiosin and protodioscin showed potent activity in NCI’s in vitro drug screen.5 Moreover, they did not produce toxic side-effects when administered to animals.5 The antiproliferative activity of these saponins is thought to be related to the induction of apoptosis;6 however, their intracellular targets and action mechanisms remained elusive. Apoptosis is a tightly regulated process of programmed cell death. Suppressing tumor growth by inducing apoptosis is an attractive option for cancer therapy as it does not provoke a pro-inflammatory response, thus, providing a better outcome of the treatment.7 Apoptotic cell death can proceed via mitochondria (intrinsic) and/or death receptors (extrinsic) path* All correspondence should be addressed to Prof. F. Chen or Prof. Q.-Y. He. E-mails: [email protected] (F.C.); [email protected] (Q.-Y.H.) Fax: +852-2299-0311 (F.C.); +86-20-8522-7039 (Q.-Y.H.). † School of Biological Sciences, The University of Hong Kong. ‡ Department of Anatomy, The University of Hong Kong. § Institute of Life and Health Engineering, Jinan University.
2050 Journal of Proteome Research 2008, 7, 2050–2058 Published on Web 04/01/2008
ways. Mitochondria, the ‘powerhouse’ of cells, are the focal point for a variety of pro- and antiapoptotic signals. A pivotal event in mitochondria-mediated apoptosis is the mitochondrial membrane permeabilization (MMP).8 Typical manifestation of MMP includes the dissipation of mitochondrial transmembrane potential (∆Ψm) and release of apoptotic proteins such as cytochrome c, followed by the activation of caspase-9 and caspase-3.9 It is also well-established that mitochondrialderived reactive oxygen species (ROS) such as O2–• and H2O2 are transient mediators of apoptosis following MMP.10 MMP leads to the overproduction of ROS, depletion of intracellular glutathione and sensitization to apoptotic stimuli.11 The extrinsic apoptosis pathway is initiated by the association of a death-inducing ligand with members of the tumor necrosis factor (TNF) receptor family.9 Subsequently, an adaptor protein called Fas-associated death domain (FADD) and pro-caspase-8 are recruited to the death receptor to form a death-inducing signaling complex (DISC).12 Pro-caspase-8 is proteolytically cleaved in DISC, and the activated caspase-8 would in turn initiate the effectors caspases cascades that mediates cell death in type I cells.9 In type II cells, formation of DISC is insufficient, and execution of apoptosis depends on an amplification loop involving mitochondria. Active caspase-8 cleaves pro-apoptotic Bid to truncated Bid (tBid), which translocates to the mitochondria and induces cytochrome c release and loss of ∆Ψm.13 In this study, we demonstrated that indioside D is a potent cytotoxic agent against several human cancer cells lines. The mechanisms of indioside D-induced apoptosis in HeLa cells were explored by proteomic approach. Proteomics is a powerful technique for the comprehensive analysis of protein complement within a cell line, tissue or organism.14,15 High-throughput 10.1021/pr800019k CCC: $40.75
2008 American Chemical Society
Indioside D-Induced Apoptosis
Figure 1. Schematic model of separation and purification of indioside D by column chromatography.
proteomic analysis can reveal multiple impacts of drug-target interaction in a global context, providing important insights for the assessment of drug efficacy and toxicity,16,17 and revealing potential proteins as novel drug targets involved in important biological processes, such as apoptosis.18,19 By comparative two-dimensional gel electrophoresis (2-DE) analysis of the untreated control and indioside D-treated cells, we identified the differentially expressed proteins by matrixassisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). These proteins are involved in energy generation, oxidative stress response, nucleic acid metabolism and calcium homeostasis. In concert with other biochemical studies, we revealed that indioside D induces apoptosis by activating both the intrinsic and extrinsic apoptosis pathways.
Experimental Section Chemical Reagents. MTT was purchased from Amersham Biosciences (Uppsala, Sweden). Caspase-8 inhibitor (ITEDCHO) and pan caspase inhibitor (z-VAD-FMK) were obtained from CalBiochem (San Diego, CA). All other reagents, except otherwise noted, were obtained from Sigma-Aldrich Chemical (St. Louis, MO) or Amersham Biosciences. Extraction and Isolation of Indioside D. The fruits of S. mammosum were collected in Hong Kong, China, in February 2006. The separation scheme is depicted in Figure 1. Briefly, fresh fruits (6 kg) were cut into slices and extracted at room temperature with 10 L of methanol for 48 h. The methanol extract was resuspended into water and partitioned with hexane and n-butanol. The n-butanol fraction containing saponins was fractionated on silica gel eluting with mixtures of chloroform, methanol and water (90:10:1; 60:10:1; 30:10:1; 10:10:1). Fraction III was further separated by a Sephadex LH20 (methanol) to give 20 fractions. Fractions 5–10 were combined and separated on an ODS gel with a mobile phase of acetonitrile and water (2:5). The 40 subfractions collected then were pooled according to their thin-layer chromatography patterns. Subfractions 11–20 showed two spots on the thinlayer chromatograph and they were resolved over silica gel to give indioside D (10 mg) and protodioscin (22 mg) with
corresponding yield of 0.00016 7% and 0.000367%, respectively. HPLC-UV analysis indicated that indioside D (95.4%) and protodioscin (97.2%) were obtained with good purity. Cell Lines and Culture Conditions. Human cervix epitheloid carcinoma (HeLa), human colon carcinoma (Caco2) and human hepatocellular carcinoma (HepG2) cells were cultured in DMEM (low glucose) medium with 2.0 g/L sodium bicarbonate, supplemented with 10% fetal bovine serum, 2 mmol/L Lglutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. Human nasopharyngeal carcinoma (HONE1 and CNE1) and human promyelocytic leukemia (HL-60) cells were cultured in RPMI-1640 medium with 2.0 g/L sodium bicarbonate, plus 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. All cells were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 °C. Drug Treatment. HeLa cells were treated with 15.0 µM indioside D for 24 h. In some experiments, cells were pretreated with 50 µM aristolochic acid (ARA), 1 µM trifluoperazine (TPZ), 100 µM pan Caspase Inhibitor (z-VAD-FMK), 100 µM Caspase-8 Inhibitor (ITED-CHO), separately, 1 h prior to the addition of indioside D. Cytotoxicity Assay. The cytotoxicity of indioside D was evaluated by MTT assay.15 Cells were suspended at 1 × 105 cells/mL, and 196 µL of suspension was plated onto a 96-well plate. After 24 h, 4 µL of media containing various concentrations of indioside D at 50, 30, 20, 10, 5, 2.5, and 1.25 µM was added. After 24 h- and 72 h-treatment, the medium was removed and replaced with 100 µL of RPMI medium with 0.5 mg/mL MTT. For nonadherent HL-60 cells, 25 µL of 5 mg/mL MTT in PBS was directly added to each well. Cells were then incubated at 37 °C for 4 h. Formazan was solubilized by adding 100 µL of DMSO and measured at 570 nm on a microplate reader. Morphological Assessment. Changes in cell morphology characteristic of apoptosis progression were detected by fluorescence microscopy (Olympus IX71 CTS Chinetek Scientific Microscope) after staining with 1 mg/mL 4,6-diamidino-2phenylindole (DAPI). Over 300 cells were counted for each well Journal of Proteome Research • Vol. 7, No. 5, 2008 2051
research articles and the results were obtained from three independent experiments. Flow Cytometry Analysis of Apoptosis. Indioside D-induced apoptosis was determined by propidium iodide (PI) staining. One million HeLa cells treated or untreated with 15 µM of indioside D were harvested, washed in PBS, stained with PI and analyzed with a FAC Plus flow cytometer. Percentage of apoptotic cells in each treatment was determined using the WinMDI 2.8 software program. Protein Extraction. After treatment with 15.0 µM indioside D for 24 h, the HeLa cells were washed with ice-cold washing buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.0). The cells were scraped in the buffer and spun down at 2000 rpm for 5 min. After two washes with 1 mL of washing buffer, the cell pellet was lysed in 50 µL of lysis buffer (8 M urea, 4% CHAPS, 2% IPG buffer, 0.2 mg/mL PMSF) and centrifuged at 13 500 rpm for 10 min at 4 °C. The supernatant were used directly for 2-DE analysis. Two-Dimensional Gel Electrophoresis (2-DE). 2-DE was performed with IPGphor IEF and electrophoresis units (GE healthcare). Whole cell protein lysates (100 µg) were mixed with rehydration solution (8 M urea, 4% CHAPS, 1 mM PMSF, 20 mM DTT and 0.5% IPG buffer) to a final volume of 250 µL. The precasted 13 cm IPG strips were rehydrated for 10 h at 30 V. IEF conditions were 500 and 1000 V, 1 h each; 8000 V to a total of 64 kVh. After IEF, strips were subjected to a two-step equilibration step in equilibration buffer (6 M urea, 30% glycerol, 2% SDS and 50 mM Tris-HCl, pH 6.8) with 1% dithiothreitol for the first step and 2.5% iodoacetamide for the second step. For SDS-PAGE, strips were transferred onto 1.5mm-thick 12.5% polyacrylamide gels at room temperature. All gels were visualized using silver staining. Stained gels were scanned with the Image Scanner and analyzed with Image Master 2D Elite software (GE Healthcare). Data were normalized, expressed as percentages of all valid spots to accounting for differences in protein loading and staining. The fold difference and standard deviation were calculated from normalized intensity volumes of individual spots between the control and drug-treated gels obtained in three independent experiments. A two-tailed Student’s t test was performed to determine if the relative change of protein spots was statistically significant (p < 0.05). Spots differed by >2-fold in silver stained gels or by >1.5-fold but confirmed by Western blot experiments were considered significant alternations. These spots were excised and analyzed by MALDI-TOF/TOF-MS.21 Tryptic In-Gel Digestion. Gel chips were destained in a 1:1 solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate and equilibrated in 50 mM ammonium bicarbonate to pH 8.0. After dehydrating with acetonitrile and drying in a SpeedVac, the gels were rehydrated in a minimal volume of trypsin solution (10 µg/mL in 25 mM ammonium bicarbonate) and incubated at 37 °C overnight. The supernatant was directly applied onto the sample plate with equal amounts of matrix. MALDI-TOF/TOF MS Analysis. Mass spectra were recorded on an Applied Biosystems 4700 Proteomics Analyzer (Framingham, MA). Instrument setting was reflector mode with 20 kV accelerating voltage. Laser shots at 5000 per spectrum were used to acquire the spectra with mass range from 600 to 3000 Da. The peptides of interest were further analyzed by MS/MS, using an energy adjustable collision cell filled with pure argon. MS and MS/MS spectra from the ABI 4700 Proteomics Analyzer were processed by using the 4700 Explorer software. MASCOT was used in database searching for protein identification by 2052
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Wong et al. incorporating MS and MS/MS data in the NCBI database. The search was restricted to one missed cleavage site, 50 ppm mass error tolerance for precursor ions and 0.1 Da for MS/MS fragments. Protein modifications including carboxyamidomethylation of cysteine and oxidation of methionine were also allowed. Duplicate or triplicate runs were made to ensure the accuracy of the analysis. Measurement of Mitochondria Transmembrane Potential (∆Ψm). The changes in ∆Ψm were assayed by measuring the uptake of Rhodamine-123 (Rho-123) (Molecular Probes).22 HeLa cells were incubated with 1 µM Rho-123 for 30 min at 37 °C, washed twice with HBSS, and treated with indioside D in complete medium. Rho-123 fluorescence was measured using a FACStar Plus flow cytometer with excitation and emission settings of 488 and 530 nm, respectively. Measurement of Oxidative Stress. Intracellular oxidative stress was determined by the increase in fluorescence due to DCFH-DA oxidation.23 Prior to drug addition, cells were incubated in HBSS containing 1 µmol/L DCFH-DA (Molecular Probes) for 15 min at 37 °C. At the end of incubation period with indioside D, cells were harvested and measured using a FACStar Plus flow cytometer with excitation and emission settings of 488 and 530 nm, respectively. Western Blotting. Western blotting was performed using primary antibodies against alpha-tubulin (Sigma-Aldrich), Bcl-2 (Santa Cruz Biotechnology), Bax (Santa Cruz Biotechnology), Bid (Cell Signaling), caspase-8 (Santa Cruz Biotechnology), caspase-9 (Cell Signaling), Enolase (Santa Cruz), GAPDH (Santa Cruz), FADD (Laboratory Vision), Fas (Laboratory Vision), FasL (Laboratory Vision), HSP60 (Santa Cruz), PARP-1 (Ab-2, Oncogene), procaspase-3 (Cell Signaling), pyruvate kinase (Abcam), and vimentin (Sigma) at optimized dilutions. Alphatubulin was taken as a marker for equal protein loading. Statistical Analysis. Statistical analysis was done using a twotailed Student’s t test, and p < 0.05 was considered significant. Data were expressed as mean ( SD of triplicate samples, and reproducibility was confirmed in at least two independent experiments.
Results Indioside D is Cytotoxic toward Several Human Cancer Cell Lines. Two furostanol saponins were isolated from nbutanol fraction of S. mammosum using a combination of silica gel, Sephadex and ODS gel chromatography (Figure 1). These compounds were identified as indioside D and protodioscin based on their 1H-NMR and 13C-NMR data.4 HPLC-UV analysis indicated that indioside D (95.4%) and protodioscin (97.2%) were obtained with good purity, with yields of 0.000167% and 0.000367%, respectively. We focused on the cytotoxic mechanisms of indioside D in the present study, since indioside D exhibited stronger cytotoxicity than protodioscin. By means of MTT assay, cytotoxicity profile of indioside D against six cancer cell lines was determined. As shown in Table 1, indioside D exhibited significant cytotoxic activity toward cell lines tested with IC50 values of 15.0–50.0 and 4.5–24.0 µM after 24 and 72 h of treatment, respectively. The most potent cytotoxic effects were observed in HeLa and Caco2 cells, while it has relatively lower cytotoxicity toward nasopharyngeal carcinoma cell lines (CNE1 and HONE1). IC50 of indioside D for HeLa cells was 15.0 µM after 24 h of treatment and this condition was used for the following experiments. Indioside D Induces Apoptosis in HeLa Cells. After treatment with 15 µM indioside D, typical morphological changes
Indioside D-Induced Apoptosis Table 1. Cytotoxicity (IC50, Half-Maximal Inhibitory Concentration) of Indioside D against Six Human Cancer Cell Lines IC50 (µM) cell line
associated with apoptosis were detected in HeLa cells by DAPI staining (Figure 2A). Chromatin condensation and fragmentation were evident after 12 h, and apoptotic bodies were clearly visible after 24 h-treatment (Figure 2A). The percentage of apoptotic cells increased from <5% for control to 12% and 35% after 12 and 24 h treatment, respectively. Indioside D-induced apoptosis was further investigated by flow cytometry. Analysis of PI-stained cells revealed that the percentage of sub-G1 peak (apoptotic phase) increased progressively after addition of indioside D, with 1.0 ( 0.5% for the control, 1.3 ( 0.6% at 12 h and 7.9 ( 1.2% at 24 h treatment (Figure 2B). The percentage apoptosis observed in DAPI staining is higher than that by PI flow cytometry. It is possibly because apoptotic fragments that constitute Sub-G1 peak do not form until the later stages of
Figure 2. Morphological features of indioside D-treated HeLa cells. (A) Typical morphological changes associated with apoptosis were identified after 12 and 24 h-treatment. Cells were stained by DAPI and then visualized by a fluorescent microscope (320×) (*, p < 0.05 statistically significant difference compared with control). (B) Percent cell cycle distribution obtained by flow cytometry analysis of PI-stained control and indioside D-treated cells. The data was representative of three independent experiments. Statistically significant difference were indicated (*p < 0.05).
apoptosis. Results of morphological and flow cytometric analyses suggested that the form of cell death induced by indioside D was apoptosis. Alternations in HeLa Cells Proteome in Response to Indioiside D Treatment. The proteomes of control and indioside D-treated cells were analyzed by 2-DE. Representative 2-D gel images are shown in Figure 3A and detailed alternations in Figure 3B. Table 2 lists the differentially expressed proteins identified by peptide mass fingerprinting (PMF). These proteins could be classified into several groups based on their functions. The first group was proteins involved in energy production. Expression of mitochondrial ATP synthase precursor was significant up-regulated, whereas several enzymes involved in glycolysis including enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase 3 (PK3) were suppressed by indioside D treatment. In addition, a PK3 fragment appeared under indioside D stimuli. The second group of proteins was related to stress response. Heat shock protein 60 (HSP60), a mitochondrial chaperone; glutathioneS-transferase (GST) and stress response cytoskeleton protein vimentin were increased; whereas the level of redox sensitive DJ-1 protein was decreased. Other protein alternations included under-expression of heterogeneous nuclear ribonucleoprotein L (hnRNP L), peptidyl-prolyl-cys-trans isomerase A (cyclophilin A), calreticulin, and translocase of outer mitochondrial membrane 40 (TOMM40). To confirm the altered expression, Western blot analysis was carried out on enolase, GAPDH, HSP60, PK3 and vimentin. Figure 3C shows that the altered intensity of the proteins matched well with the differences found in 2-DE analysis. Cytotoxicity of Indioside D Is Associated with Activation of Mmitochondria-Related Apoptosis Pathway. Alternations in multiple proteins related to energy production and stress response led us to further investigate the role of mitochondria in indioside D-induced apoptosis. ∆Ψm was monitored in HeLa cells using Rho-123 staining. Exposure to indioside D resulted in a decrease in ∆Ψm after 1 h, and the depletion was further intensified after 6 h-treatment (Figure 4A). Activation of intrinsic cell death pathway is regulated by the Bcl-2 family proteins. In particular, the ratio of antiapoptotic Bcl-2 versus pro-apoptotic Bax is critical for release of cytochrome c.24 Western blotting revealed that indioside D treatment resulted in Bax up-regulation with a concomitant decline in Bcl-2 expression level in a time-dependent manner (Figure 4B). We next examined whether the inhibition of mitochondrial permeability transition (MPT) could rescue indioside D-treated cells from apoptosis. As shown in Figure 4C, pretreatment with MPT inhibitors ARA or TFZ effectively attenuated indioside Dinduced cytotoxicity in HeLa cell, though ARA itself reduced ∼20% cell viability (Figure 4C). These data suggested that indioside D-induced apoptosis involved the activation of mitochondrial intrinsic apoptotic pathway. Indioside D Stimulates ROS Generation. Since ROS production is closely related to mitochondrial dysfunction, we assessed the intracellular ROS generation in HeLa cells using DCFHDA staining followed by flow cytometry analysis. Results showed that 15 µM indioside D caused a significant generation of ROS as early as 1 h-treatment (Figure 5). Intracellular ROS continued to accumulate after 3 h. Caspases Are Downstream Activators of Indioside DInduced Apoptosis. Caspases are a conserved family of enzymes that serve as the executioners of apoptosis from various cell death signals.9 We examined the proteolytic cleavJournal of Proteome Research • Vol. 7, No. 5, 2008 2053
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Figure 3. 2-D gels images of control and indioside D-treated cells. (A) Master gel images of whole cell proteome of control and indioside D-treated HeLa cells. Arrows indicate those proteins whose expressions were altered after indioside D-treatment and were unequivocally identified by PMF. Numbers are correlated with those spot no. listed in Table 2. (B) Detailed alternation patterns of identified protein spots. (C) Western blot analysis of the altered proteins in indioside D-treated HeLa cells.
age of initiator caspase-8 and -9 by Western blotting. After 24 h of indioside D treatment, a significant cleavage of caspase-3, -8 and -9 occurred (Figure 6A), as reflected by a decrease in intensity of the pro-enzyme and/or the emergence of cleaved forms. To further confirm the activation of caspase cascade, degradation of poly(ADP-ribose) polymerase (PARP-1) was analyzed with Western blotting (Figure 6A). PARP-1 (116 kDa), a substrate of caspase-3 and a hall marker of apoptosis,11 was found to be cleaved to produce an 89 kDa fragment after 24 h-treatment. Moreover, the addition of a pan-caspase inhibitor (z-VAD-FMK) significantly attenuated the cytotoxicity of indioside D by 39.3% (Figure 6B). These data indicated that indioside D induced cell death in a caspase-dependent manner. Indioside D Alters Expression of Proteins Related to Cell Death Receptor Pathway. Triggering of CD95/Fas death receptor by its ligand FasL leads to the recruitment of FADD and the proteolytic activation of pro-caspase-8.12 To determine whether indioside D affects the death receptor pathway, we analyzed expressions of Fas, FasL and FADD by Western blotting. As shown in Figure 7A, indioside D-treatment resulted 2054
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in a significantly enhanced level of FasL after 6 h-treatment, but the expression of Fas and FADD only increased slightly. Capase-8 cleavage was observed after 24 h-treatment (Figure 6A). Activated caspase-8 also cleaved Bid to form truncated Bid (tBid), which targets outer mitochondrial membrane and causes ∆Ψm dissipation.13 By Western blotting, we observed a decrease in the 22 kDa Bid after 24 h of treatment, indicating activation of death receptor apoptosis pathways. Furthermore, the addition of a caspase-8 specific inhibitor (IETD-CHO) resulted in a significant restoration of ∆Ψm after indioside D-treatment for 6 h (Figure 7B). Thus, the activation of Fas signaling pathway may have partly contributed to ∆Ψm dissipation and mitochondrial-mediated apoptosis pathway.
Discussion Saponins have been proposed as possible anticancer agents. Furostanol glycosides are a class of anticancer saponins found widely in Solanum plant species. They possess promising anticancer activity, exemplified by the potent antiproliferative activity in NCI 60 cell lines screen .5 Moreover, it has been
Indioside D-Induced Apoptosis a
Table 2. Protein Alterations after Treatment with Indioside D (15 µM for 24 h) spot no.
384 Glutathione-S-transferase 427 DJ-1 protein 32 Heterogeneous nuclear ribonucleoprotein L 193 Translocase of outer 5174723 37.9/6.8 Mitochondrial transport mitochondrial membrane 40 protein 501 Calreticulin precursor (fragment) 4757900 48.1/4.3 Calcium-binding chaperone 516 Peptidyl-prolyl-cys-trans 13543666 18.0/7.7 Protein folding isomerase A a The fold differences were calculated based on the image analysis of silver-stained gels. mass fingerprinting. c Peptides sequenced: unique peptides identified by MS/MS sequencing.
reported that saponins are significantly less cytotoxic toward normal cells.25–28 In this study we isolated indioside D from the fruits of S. mammosum and characterized its cytotoxic activity. Indioside D is cytotoxic toward several human cancer cell lines, in particular HeLa cell line (Table 1). Upon exposure to indioside D, HeLa cells exhibited changes in cell morphology distinctive of apoptosis (Figure 2A). FACS analysis also revealed an increase in the apoptotic cell populations (sub-G1 peak) in response to indioside D-treatment (Figure 2B). Therefore, indioside D-treatment resulted in apoptotic cell death. Mitochondria Dysfunction Is a Key Event Following Indioside D Treatment. Molecular basis of indioside D action was further elucidated using proteomic approach. The majority of protein alternations was related to energy generation, including an enhanced expression of ATP synthase precursor and suppression of several glycolytic enzymes (Figure 3B). Mitochondrial-mediated apoptosis is characterized by the loss of ∆Ψm and a reduced energy production.8 ATP synthase is crucial to ATP production. Transient augmentation of gene expression of ATP synthase subunits has been noted in the initial stages of apoptosis,29 and a dramatic rise in ATP synthase precursors was associated with saponin-induced and mitochondria-mediated apoptosis.26 Increased expression of ATP synthase precursor fragment indicates a probable failure in protein processing due to mitochondrial dysfunction. TOM40 is a subunit of the translocase of outer mitochondrial membrane (TOM complex) that mediates translocation of mitochondrial precursor proteins.30 TOM40 is essential for viability in eukaryotic cells. Altered TOM40 was shown to result in the defective import of mitochondrial precursor proteins into the mitochondria. 31 Our results revealed suppression of TOM40 (Figure 3B), which may compromise mitochondrial function. Energy production was further disturbed by the reduced levels of glycolytic enzymes enolase 1, GAPDH and PK3 (Figure 3B,C). Up-regulation of glycolysis has a remarkable prevalence among cancers, thus, providing a sound biochemical basis for selective killing of cancer cells by inhibition of glycolysis.32 The sup-
Peptides matched: unique peptides identified by peptide
pression of housekeeping genes such as GADPH, enolase and aldolase was reported to be important suicidal mechanisms in cell deaths induced by FasL and ROS stress.33,34 Altered expression of these proteins involved in energy production indicated that metabolic catastrophe represents an important mechanism of indioside D-induced growth inhibition, which may be initiated through mitochondrial and death receptor apoptosis pathways. Our results demonstrated that the intrinsic apoptosis pathway is involved in the indioside D-induced cytotoxicity. We observed an immediate and progressive ∆Ψm collapse after the addition of indioside D (Figure 4A), followed by the activation of caspase-9 (Figure 6A). The perturbation of outer mitochondrial membrane was directly controlled by Bcl-2 family proteins through formation of conducting pores.9 Bax is a pro-apoptotic Bcl-2 family member that forms multimeric pores on outer mitochondrial membrane and causes the release of cytochrome c upon MMP.8 Bcl-2, in contrast, promotes cell survival by abrogating oligomerization and activation of Bax.35 We found an increase in the level of Bax with suppression of Bcl-2, tipping the Bax/Bcl-2 ratio toward a pro-apoptotic attitude (Figure 4B). Another mechanism for MMP is the MPT, which involves opening of the permeability transition pores (PTP), rapid ∆Ψm collapse, mitochondrial swelling and rupture of outer mitochondrial membrane.36 ARA and TPZ, phospholipase inhibitors that attenuate MPT,37 partially countered apoptotic effects of indioside D (Figure 4C). Thus, PTP opening contributed, at least in part, to cytotoxicity caused by indioside D exposure. These results suggest that the cytotoxicity of indioside D involved the intrinsic apoptosis pathway. Generation of ROS Is Associated with Apoptosis Induction by Indioside D. ROS plays an important role in the induction of cell death via intrinsic apoptosis pathway.10 Mitochondria are both the source and target of oxidative stress. ∆Ψm dissipation is often accompanied by accumulation of ROS, which in turn causes further disruption of ∆Ψm.10 We found that treatment with indioside D resulted in a rapid elevation Journal of Proteome Research • Vol. 7, No. 5, 2008 2055
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Figure 6. (A) Activation of caspases during indioside D-treatment. Western blotting of caspase-8, caspase-9, procaspase-3 and PARP-1 demonstrated appearance of their cleavages forms after 24 h incubation with 15 µM indioside D. (B) The effect of a pan caspase inhibitor (z-VAD-FMK) on indioside D-induced cell death after 24 h. (*, p < 0.05 statistically significant difference compared to the control; #, p < 0.05 statistically significant difference compared to indioside D treatment.)
Figure 4. Indioside D-treatment induces apoptosis via intrinsic pathway of apoptosis. (A) Measurement of the ∆Ψm in HeLa cells after indioside D treatment. (B) Western blotting of Bcl-2 and Bax alternations. (C) Effect of mitochondrial permeability transition (MPT) inhibitors on cytotoxicity of indioside D after 24 htreatment measured by MTT assay. (*, p < 0.05 statistically significant difference compared to the control; #, p < 0.05 statistically significant difference compared to indioside D treatment.)
Figure 5. Indioside D induced generation of ROS in HeLa cells. HeLa cells were labeled with DCFH-DA and then treated with 15 µM indioside D for the indicated periods.
of ROS generation in HeLa cell after 1 h (Figure 5). This is in agreement with observations from our proteomic analysis, which revealed profound alternations of ROS stress response proteins (Figure 3B). Heat shock proteins (HSP) are induced in response to various stimuli including oxidative stress.38 Induction of HSP60 resulted in depression of mitochondrial 2056
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Figure 7. (A) Effects of indioside D on the expression of proteins related to Fas-signaling pathway by Western blotting. (B) The effect of a caspase-8 inhibitor (ITED-CHO) on indioside D-induced ∆Ψm dissipation after 6 h-treatment. (*, p < 0.05 statistically significant difference compared to the control; #, p < 0.05 statistically significant difference compared to indioside D treatment.)
apoptotic cascade by the sequestration of pro-apoptotic Bax and the inhibition of cytochrome c/caspase-3 cascades.39 GST plays a key role in detoxification and confers protection against ROS stress via the neutralization of peroxidized metabolites.40 GST expression was induced following ROS insults.41 Consistent with its role in cytoprotection, GST was reported to inhibit multiple steps of JNK-mediated apoptotic cascade.42 Overex-
Indioside D-Induced Apoptosis pression of HSP60 and GST thus represents a protective response against the ROS generation triggered by indioside D. Vimentin, a class III intermediate filament that is involved in apoptosis and cell proliferation, was up-regulated. Sapra and his colleagues reported that vimentin was overexpressed in ROS stress-induced cytotoxicity in macrophage.43 Additionally, DJ-1 (pI 6.2) protein was found to be suppressed. DJ-1 directly scavenges hydrogen peroxide via the oxidation of Cys-106, with a corresponding shift of isoelectric point from 6.2 to 5.8 in vitro.44 Reduced expression of the pI 6.2 isoform of DJ-1 protein in 2-DE analysis further suggested that indioside D-treatment enhanced intracellular ROS level. With previous studies showing ROS generation induced by other saponins,45 it appears that oxidative stress is an important factor for the commitment of apoptosis following indioside D treatment. Interplay between Mitochondria and Death Receptor Pathways. Death receptor–ligand cross-linking triggered apoptosis pathway is also responsible for chemotherapeutic agentsinduced cell death.46 Fas is activated upon binding to its ligand, FasL. Activated Fas forms DISC by recruitment of FADD, procaspase-8 and CAP3,12 resulting in the cleavage of procaspase-8, which mediates apoptosis by activating caspase-3. In some cells, activation of death receptor pathway also causes MMP, in which digestion of pro-apoptotic Bid by caspase-8 to truncated-Bid promotes MPT and executes cell death via intrinsic apoptosis pathway.9 Our results showed that indioside D induced the death receptor pathway by increasing FasL expression dramatically after 6 h-treatment (Figure 7A). Upregulation of FasL was also shown to be critical for cisplatininduced apoptosis in ovarian carcinoma cells.47 In addition, the caspase-8 inhibitor (IETD-CHO) strongly attenuated the depletion of mitochondrial potential (Figure 7B). Interplay of the intrinsic and extrinsic pathway was further evidenced by the decline in full-length Bid after 24 h-treatment, presumably due to Bid truncation (Figure 7A). tBid can translocate to the mitochondria and enhance release of cytochrome c.13 Taken together, these data suggest that there is an activation of extrinsic cell death pathway which contributed, at least in part, to the indioside D-induced mitochondrial dysfunction. Apart from proteins related to energy metabolism and stress response, our proteomic analysis suggests that additional mechanisms are also involved in indioside D cytotoxicity. Suppressed nucleic acid metabolism was evident from downregulation of hnRNP L. hnRNP L is a RNA binding protein implicated in the regulation of mRNAs stabilization, processing and translation. It has been reported that hnRNPs can serve as substrates of caspase-3 and other proteases during apoptosis,48 and their cleavage during indioside D-induced apoptosis may account for the reduction of its full-length form. Moreover, under-expression of calreticulin was observed. Calreticulin is a major calcium binding protein in endoplasmic reticulum (ER) lumen that regulates Ca2+ homeostasis.49 Ca2+ release from ER lumen is linked to mitochondria-induced apoptosis and the overexpression of calreticulin was found to block apoptosis.50 It is possible that, through lowering the level of calreticulin, indioside D mediates an enhanced loss of Ca2+ from ER that potentiates mitochondria apoptosis pathway.
Conclusions In this study, we isolated indioside D as a potent cytotoxic furostanol saponin from S. mammosum. Through proteomic analysis, we identified profound alternations in proteins related to energy production and oxidative stress response. Together
with biochemical studies, we demonstrated that mitochondria dysfunction occurred following indioside D treatment. Alternations in the Bax/Bcl-2 ratio and MPT contribute to the modulation of ∆Ψm in indioside D-induced cell death. The death receptor Fas may also play a role in apoptosis by acting through the mitochondrial cell death pathway. These experimental results support that mitochondria and death receptors are primary targets of indioside D, raising the possibility that indioside D could be developed as a potential anticancer drug directed toward the mitochondria. Expression proteomics is a powerful approach for elucidating mechanism of indioside D in apoptosis induction. However, with current 2-DE techniques, low-abundance proteins that may be crucial to the cellular signaling are difficult to be detected. Therefore, future studies should focus on the alternations of proteins enriched from the mitochondria, which is a major targeted organelle for indioside D.
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