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
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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
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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
24 h
72 h
Caco2 CNE1 HONE1 HeLa HepG2 HL-60
20.0 ( 1.3 48.5 ( 2.0 >50.0 15.0 ( 2.5 30.5 ( 1.8 38.4 ( 2.1
5.0 ( 0.3 23.9 ( 1.1 23.0 ( 1.1 4.5 ( 0.2 6.7 ( 0.7 8.7 ( 0.7
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
