Tubeimoside-1 Exerts Cytotoxicity in HeLa Cells through

Feb 12, 2009 - Copyright © 2009 American Chemical Society. * To whom correspondence should be addressed. For Prof. Q.-Y. He: e-mail, [email protected]...
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Tubeimoside-1 Exerts Cytotoxicity in HeLa Cells through Mitochondrial Dysfunction and Endoplasmic Reticulum Stress Pathways Yang Xu,† Jen-Fu Chiu,‡ Qing-Yu He,*,§ and Feng Chen*,† School of Biological Sciences, and Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China, and Institute of Life and Health Engineering and Guangdong Provincial Key Laboratory of Bioengineering Pharmaceutics/National Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China Received November 19, 2008

Traditional Chinese herbal medicines are a great source of cancer chemotherapeutic agents. Tubeimoside-1 (TBMS1) is a triterpenoid saponin extracted from Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae), a Chinese herb with anticancer potential named as “Tu Bei Mu”. In the present study, we used proteomics to examine the cytotoxic effects of TBMS1 on HeLa cells. Protein profiling of TBMS1-treated HeLa cells revealed profound protein alterations related to energy metabolism and protein synthesis and folding, suggesting that mitochondria and endoplasmic reticulum (ER) play a role in TBMS1-initiated apoptosis. TBMS1 induced the depletion of mitochondrial transmembrane potential (∆Ψm), leading to the activation of caspase-dependent apoptotic cell death. Unfolded Protein Response (UPR) signaling pathways are also activated after TBMS1 treatment and these changes were accompanied by increased expression of GADD153/CHOP, a transcription factor associated with growth arrest and apoptosis in the event of prolonged ER stress. Salubrinal (Sal), a selective inhibitor for ER stress, partially abrogated the TBMS1-related cell death. These results suggest that TBMS1 exerts cytotoxicity in HeLa cells through both mitochondrial dysfunction and ER stress cell death pathways. Keywords: Tubeimoside-1 • Mitochondrial Dysfunction • Endoplasmic Reticulum Stress

Introduction The tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae) is a Chinese herb named as “Tu Bei Mu” (TBM). It was compiled in the Supplement of the Compendium of Materia Medica in 1765 (the Qing Dynasty).1 TBM has been widely used in traditional Chinese medicine for the treatment of tumor, inflammation and snake venoms. Especially, it was an important element of the anticancer formula in the treatments of breast cancer, nasopharyngeal carcinoma and esophageal carcinoma. Tubeimoside-1 (TBMS1) (Figure 1), a triterpenoid saponin extracted from TBM, is the main component with anticancer potential. It was shown to induce apoptosis in a number of human carcinoma cell lines.1-3 However, the detailed mechanisms and intracellular targets of TBMS1induced cell death remain elusive. Apoptosis is an essential way to regulate cell death process during tissue development, homeostasis and it works as a defense system against many toxic chemicals.4 It is well-known that anticancer chemotherapies mainly exert their elimination of cancer cells by apoptotic process and this process can be * To whom correspondence should be addressed. For Prof. Q.-Y. He: e-mail, [email protected]; fax, +86-20-8522-7039. For Prof. F. Chen: e-mail, [email protected]; fax, +852-2299-0311. † School of Biological Sciences, The University of Hong Kong. ‡ Department of Anatomy, The University of Hong Kong. § Jinan University. 10.1021/pr801001j CCC: $40.75

 2009 American Chemical Society

Figure 1. Chemical structure of tubeimoside-1 (TBMS1).

triggered either at the plasma membrane (extrinsic pathways) and/or within cells (intrinsic pathways).5,6 Recent studies have suggested that the intrinsic pathways are initiated by the biochemical events, affecting on the organelles inside the cells causing intracellular stresses. Among these organelles, mitochondria and endoplasmic reticulum (ER) play important roles involved in the intrinsic pathways to execute apoptosis.7,8 Journal of Proteome Research 2009, 8, 1585–1593 1585 Published on Web 02/12/2009

research articles Dysfunction of mitochondria has been found to be one of the major events during apoptosis. In this process, Bax, one of a pro-apoptotic Bcl-2 family, leads to membrane permeabilization by oligomerizing, translocating and consequently causing pores at the outer mitochondrial membrane.9 Upon the depletion of the mitochondria transmembrane potential, cytochrome c releases from the mitochondrial intermembrane spaces to initiate subsequent activation of caspase-9 further activating the downstream effector caspase-3. Caspase-3 is demonstrated to cleave its substrate poly (ADP-ribose) polymerase 1 (PARP-1), inducing characteristic apoptotic changes, such as chromatin condensation and DNA chromatin fragmentation.10 ER serves as a critical site responsible for regulating protein synthesis, protein folding and intracellular calcium (Ca2+) levels.11 The abnormalities in the ER function can cause ER stress, activating a series of signal transduction termed as “unfolded protein response” (UPR) and ultimately leading to apoptosis if the ER stress becomes prolonged and severe.12 This response is mediated by three ER proximal sensors: PKR-like ER-associated kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring enzyme-1 (IRE1). Among the UPR downstream cell death signaling pathway, GADD153/CHOP, a transcription factor, is a well-known ER stress-mediated apoptotic executor.11,13-15 In the present study, we applied a comparative proteomics approach to delineate the possible molecular basis of TBMS1induced cancer cell death. Altered proteins related to energy metabolism and protein synthesis were identified by twodimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). In combination with other biochemical characterizations, we demonstrated that TBMS1 induces apoptotic cell death through at least mitochondria and ER-stress pathways.

Experimental Section Reagents. TBMS1 was purchased from National Institute for the Control of Pharmaceutical and Biological Products. 4′,6Diamidino-2-phenylindole (DAPI) was purchased from Roche (Mannheim, Germany). Salubrinal was obtained from CalBiochem (San Diego, CA). All other chemicals, except otherwise noted, were purchased from either Amersham Biosciences (Uppsala, Sweden) or Sigma-Aldrich Chemical. TBMS1 was dissolved in PBS and stored at -20 °C. Cell Lines and Culture Conditions. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (high glucose) with 2.0 g/L sodium bicarbonate plus 10% fetal bovine serum (JRH Bioscience, Lenexa, KS), 1% L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin (GIBCO-BRL, Grand Isle, NY), and maintained in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 °C. When the cells grew to about 80% confluence, they were subcultured or treated with drug. Drug Treatment. Cells were treated with 25 µM TBMS1 based on the IC50 value measured (see below). After treatment, cells were washed three times with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) and transferred to a clean 1.5 mL Eppendorf tube, spun down at 2500 rpm for 5 min, and then washed with washing buffer (10 mM Tris-HCl and 250 mM sucrose, pH 7.0), 1 mL each. Cell pellet was lysed by adding 80 µL of lysis solution (8 M urea, 4% CHAPS, 2% IPG buffer, and 0.2 mg/mL PMSF). After centrifugation at 14 000 rpm for 10 min at 37 °C to clarify the 1586

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Xu et al. cell lysate, the lysis supernatant was used for 2-DE. Protein concentration was determined by protein assay (BioRad). To determine whether TBMS1 induced cell cytotoxicity via mitochondria transmembrane potential depletion or ER stress, the mitochondrial permeability transition inhibitors, 25 µM aristolochic acid, 0.5 µM cyclosporine A, 0.5 µM trifluoperazine and ER stress inhibitor, 5 µM salubrinal were used to pretreat cells 1 h before TBMS1 treatment. Imaging of Morphological Changes. Morphological changes of apoptosis were detected by fluorescence microscopy (Olympus IX71 CTS Chinetek Scientific Microscope) after staining with 1 µg/mL DAPI. More than 300 cells were counted for each well by three independent experiments. Cytotoxicity Assay. The cytotoxicity of TBMS1 was evaluated by MTT assay according to a method reported previously.16 HeLa cells were plated in 96-well plates at 2 × 104/well in the complete medium 200 µL/well. After 24 h incubation, the medium was replaced with the medium containing various amounts of TBMS1. At the end of experiments, 20 µL of 5 µg/ mL MTT was directly added to each well. Cells were then incubated at 37 °C for 4 h. Formazan was solubilized by 100 µL of DMSO and measured at 570 nm on the Model E1 310 Autoplate reader (Bio-Tek Instruments, Winooski,VT). Flow Cytometric Assessment of Apoptosis. TBMS1-induced apoptosis was determined by two-color analysis of Annexin V-FITC/PI kit (BD Biosciences) according to the introductions of the manufacturer. Cells were cultivated for indicated time before either left untreated or treated with different concentration of TBMS1. At the end of each experiment, cells were harvested, resuspended in PBS solution, stained by Annexin V-FITC/PI, and analyzed with a FACStar Plus flow cytometer. For each sample, 1 × 106 cells were analyzed, and percentage of apoptotic cells in each treatment was determined by the WinMDI 2.9 software program. DNA Laddering. DNA laddering was analyzed by a protocol developed by Fukuda et al.17 Equal quantities (5-30 µg) of DNA extracted from treated or untreated HeLa cells were run on 1% agarose gels in TBE buffer. Bands were detected by ethidium bromide staining. 2-DE. 2-DE was performed with Amersham Biosciences IPGphor IEF and Ettan Dalt Six electrophoresis units using the protocol described previously.16 Two 10 cm plates of cells from each treatment were used to prepare a single sample. Proteins (100 µg) extracted from whole cell were loaded on precast 13 cm IPG strips for IEF and then were separated on SDS-PAGE (12.5%). Silver Staining, Image Analysis and Tryptic In-Gel Digestion. With the use of the procedures previously described,16 the 2-DE gels were stained with silver staining and then subjected to image analysis with the ImageMaster 2-D Elite software (GE Healthcare). Protein spots were detected, matched and manually edited. The spot intensities were normalized and compared. The fold difference and standard deviation were calculated from the normalized data that was obtained in three independent experiments. Only those significantly and consistently altered protein spots (over 1.5-fold up- or downregulation) were selected for digestion analysis. The protein spots of interest were excised, destained and then subjected to tryptic in-gel digestion.16 The digest was then applied onto the sample plate and coated with matrix for MALDI-TOF/TOF MS analysis. MALDI-TOF/TOF MS and Protein Identification. MS analysis was performed using a Voyager 4700 mass spectrometer

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Figure 2. The cytotoxicity of TBMS1 was determined by MTT assay. (A) Cytotoxicity (IC50, half-maximal inhibitory concentration) of TBMS1 toward four human cancer cell lines. TBMS1 has the greatest cytotoxicity effect on HeLa cells. (B) Concentration dependence of TBMS1 cytotoxicity on HeLa cells. The IC50 of HeLa cells was 25 µM for 24 h TBMS1 treatment.

(Applied Biosystem). MALDI-TOF mass spectra were acquired in reflector positive ion mode with average 1500 laser shots per spectrum. Peptide ion masses were internally calibrated using trypsin autolytic peptides at m/z 842.51 and 2211.10. TOF/TOF tandem MS fragmentation spectra were acquired in a data-dependent fashion based on the MALDI-TOF peptide mass map for each protein, and 10 most abundant ions present were selected in each sample (excluding trypsin autolytic peptides and other known background ions). All these data were processed by using 4700/explorer software. MASCOT was used in searching for protein identification by NCBInr protein database. Species search was limited to Human. Western Blot Analysis. The prepared cell lysates were loaded onto SDS-PAGE. Semidry transfer unit (Hoefer TE77, GE Medical Systems) was applied for electroblotting and proteins were detected by ECL detection reagents (Amersham). The primary antibodies were used against HSP60 (Santa Cruz), HSP70 (Santa Cruz), GAPDH (Santa Cruz), enolase (Santa Cruz), β-actin (Sigma-Aldrich), cytochrome c (Oncogene), Bcl-2 (Santa Cruz), Bax (Santa Cruz), pro-caspase-9 (Calciochem), procaspase-3 (Cell Signaling), PARP-1 (Ab-2, Oncogene), GRP78 (Santa Cruz), GRP94 (Stressgen), calnexin (Santa Cruz), PERK (Santa Cruz), phospho-PERK (Cell Signaling), eIF2 (Santa Cruz) and phospho-eIF2 (Cell Signaling) at optimized dilutions. Mitochondrial Transmembrane Potential (∆Ψm). Changes in ∆Ψm were detected by 1 µM Rho-123 (Molecular Probes, Eugene, OR) for 30 min at 37 °C according to the procedure of Huigsloot et al.18 Rho-123 fluorescence was measured by a FACStar Plus flow cytometer with excitation and emission wavelengths of 488 and 530 nm. Statistical Analysis. Statistical analysis was done using a twotailed Student’s t-test and p < 0.05 was considered significant. Data are expressed as the mean ( SD of triplicate samples, and the reproducibility was confirmed in three separate experiments.

Results TBMS1 Is Cytotoxic toward Several Human Cancer Cell Lines. By means of MTT assay, cytotoxicity of TBMS1 in several cancer cell lines including A549, HeLa, HepG2, and EC109 was determined in time- and dose-course treatment. The IC50 values varied from 25 to 40 µM and 10.5-19 µM after TBMS1 treatment for 24 and 72 h, respectively (Figure 2A). As shown

in Figure 2A, HeLa cells were more sensitive to the TBMS1 exposure with the IC50 25 µM for 24 h treatment (Figure 2B). On the basis of these data, the condition of IC50 value 25 µM and 24 h treatment of TBMS1 was applied for the following experiments. HeLa Cells Show Apoptotic Characteristics with TBMS1 Treatment. After treatment with TBMS1 25 µM for indicated time, typical apoptotic morphological changes of HeLa cells were detected by DAPI staining (Figure 3A). Figure 3A clearly shows that TBMS1 induced chromatin condensation after 8 h treatment and generous apoptotic bodies appeared after 24 h. The percentage of apoptotic cells increased by about 7% and 30% over untreated HeLa cells, after TBMS1 treatment for 8 and 24 h, respectively. Flow cytometric analysis was applied to the further study of TBMS1-induced cytotoxicity. By using double staining of Annexin V-FITC/PI, this assay can classify the cells into three different populations: apoptotic cells (Annexin V-FITC positive, PI negative), necrotic cells (Annexin V-FITC positive, PI positive) and viable live cells (Annexin V-FITC negative, PI negative).16 Figure 3B shows that administration of TBMS1 for 24 h resulted in 18.7% increase in the apoptotic cell population and 0.8% increase in the necrotic cell population. In addition, DNA laddering was detectable after TBMS1 treatment for 12 and 24 h at the concentration of 12 and 25 µM in HeLa cells, and the degradation of cellular DNA increased in a dose- and time-dependent manner (Figure 3C). All these data demonstrated that TBMS1 obviously induced apoptosis in HeLa cells. Cellular Proteins Alter in Response to TBMS1 Administration. Figure 4A shows the representative 2-DE images for total proteins extracted from HeLa cells treated and untreated with TBMS1 for 24 h. More than 1100 protein spots were separated on the gel ranging from MW 6-200 kDa and pI 3-10. Detailed protein alterations in expression are shown in Figure 4B,C. The spots with great differences (up- or down-regulation over 1.5-fold) were selected for protein identification. Table 1 lists the identified proteins with their spot number, accession number, MW/pI values, fold difference and scores. These altered proteins can be classified into three categories according to their main functions and locations in cells (Table 1). The first group is involved in energy production. Several enzymes related to glycolysis, including enolase1, GAPDH, pyruvate kinase and aldolase A, were suppressed by TBMS1 treatment, Journal of Proteome Research • Vol. 8, No. 3, 2009 1587

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Figure 3. Morphological changes induced by TBMS1 in HeLa cells. (A) The cells were stained by DAPI and visualized by fluorescent microscope at indicated time; the values are shown as mean ( SD of triplicate experiments (×320 magnification) (*, p < 0.05 statistically significant difference compared with untreated HeLa cells). (B) Detection of apoptotic HeLa cells in the presence of TBMS1 for 24 h by annexin V-FITC/PI analysis. (C) DNA ladder was formed after TBMS1 treatment for 12 and 24 h at indicated concentrations. Results in panels B and C are representatives from three independent experiments.

whereas mitochondria chaperonins HSP60 and MTHSP75 were significantly up-regulated. Especially, MTHSP75 increased in its precursor form. The second group is related to protein synthesis and folding, involving chaperonins GRP78/Bip, calnexin and HSP70, and GAPDH. GRP78/Bip and calnexin locate at the ER and retain unfold and unassembled forms in the ER.19 GAPDH is related to protein transport between the ER and the Golgi apparatus.20 Other altered proteins include cytoskeleton proteins tubulin and keratin 8, transcription factor heterogeneous nuclear ribonucleoprotein K and neuroblast differentiation associated protein AHNAK. To confirm the 2-DE results, Western blotting was used to detect enolase, GAPDH, GRP78/ Bip, HSP60 and HSP70. The data matched well with the differences exhibited in the 2-DE (Figure 4D). TBMS1 Causes Mitochondrial Dysfunction. Corresponding to the proteomic changes of glycolytic enzymes and mitochondrial chaperonins, mitochondrial transmembrane potential (∆Ψm) was measured to detect the mitochondrial integrity by Rho-123 staining.16 The depletion of ∆Ψm was observed upon TBMS1 treatment for 4 h, and was further intensified in 8 h exposure of TBMS1 (Figure 5A). The mitochondrial cell death pathway is regulated by the Bcl-2 family. Particularly, the ratio of Bax (pro-apoptotic member) to Bcl-2 (antiapoptotic member) is critical for cytochrome c release and the following caspase activation.21-23 Western blot analysis revealed that Bax expression increased with the suppression of Bcl-2 in a time1588

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dependent manner which was accompanied with cytochrome c increasing in the cytoplasmic fraction (Figure 5C). Pretreatment with the inhibitors of the mitochondrial permeability transition, ArA, CyA and TFZ alone or in combination, increased the cell viability (Figure 5B). These data suggest that TBMS1-induced apoptosis involved the activation of mitochondrial-related pathway. Caspase-Dependent Pathway Is Activated by TBMS1 Treatment. Caspases, a conserved enzymatic family, serve as the executioners of apoptosis. In particular, casepase-9 and caspase-3 are activated toward mitochondrial cell death signals.23 In this investigation, we detected a time-dependent down-regulation of pro-caspase-9 and pro-caspase-3 by Western blot analysis (Figure 5D). The decreased intensity of the pro-enzymes reflected the emergence of the cleaved form. To further identify the activation of the caspase cascade, PARP-1 (116 kDa), one of caspase downstream effectors, was examined by Western blot analysis. Figure 5D shows that the cleaved fragment of PARP-1 (89 kDa) was observed after 24 h treatment of TBMS1. TBMS1 Induces the Activation of ER Stress Pathway. Protein alterations related to protein synthesis and folding and chaperones within ER promoted us to further study the role of ER in the TBMS1-induced apoptosis. It is well-established that there are three ER sensors (PERK, Ire1 and ATF6) that can activate the UPR response under ER dysfunction. PhosphoPERK can phosphorylate eIF2R to decrease the gene expression;

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Figure 4. Protein alterations in HeLa cells upon TBMS1 treatment. (A) 2-DE images of total cellular proteins extracted from TBMS1treated HeLa cells and control. (B and C) Altered protein spots and their intensity subjected to protein identification by MS. (D) Western blot confirmation for the identified proteins. Table 1. Protein Alterations in Response to TBMS1 Treatment (25 µM for 24 h) spots

216 105 325 1173 1168 1063 1081

protein ID

Chaperonin (HSP60) MTHSP 75 Enolase 1 GAPDH Aldolase A Pyruvate kinase 3 isoform 1 Pyruvate kinase

733 503 107

BiP protein Calnexin precursor Heat shock 70 kDa protein 9B precursor 1167 GAPDH

198 Tubulin, beta 213 Keratin 8 1096 Heterogeneous nuclear ribonucleoprotein K 127 Neuroblast differentiation associated protein AHNAK a

NCBI acc. no.

protein score C.I.%

no. of peptides matcheda

no. of peptides sequencedb

fold difference (TBMS1: Ctrl)

100 100 100 100 100 100 100

13 25 11 12 9 16 12

1 3 2 1 2 1 1

2.84 ( 0.37 1.95 ( 0.41 -1.75 ( 0.27 -2.13 ( 0.42 -1.91 ( 0.21 -1.78 ( 0.33 -2.26 ( 0.29

Endoplasmic Reticulum 6470150 70.89/5.23 147 10716563 67.52/4.47 186 24234688 73.67/5.83 522

100 100 100

12 9 26

1 1 3

2.59 ( 0.63 1.88 ( 0.32 2.65 ( 0.67

31645

306890 292059 4503571 31645 4557305 33286418 35505

protein score

MW (kDa)/pI

Mitochondria 60.98/5.7 190 73.73/5.97 580 47.14/7.01 187 36.03/8.26 233 39.4/8.3 181 57.9/7.96 231 57.84/7.58 156

235

100

16

1

-4.14 ( 0.78

Cytoskeleton and 18088719 49.64/4.75 4504919 53.67/5.52 1338462 50.94/5.39

Others 252 502 264

100 100 100

13 28 17

3 1 2

-1.65 ( 0.23 2.19 ( 0.40 3.24 ( 0.47

39932549 132.42/8.24

333

100

15

2

-4.48 ( 0.82

36.03/8.26

Peptides matched: unique peptides identified by peptide mass fingerprinting.

Ire1 and ATF6 pathway promotes the expression of ER chaperones.8,24-27 In the current detection, the ER chaperones GRP78/Bip, GRP94 and calnexin were evaluated by Western blot analysis (Figure 6A). In response to the TBMS1 treatment,

b

Peptides sequenced: unique peptides identified by MS/MS sequencing.

the levels of GRP78/Bip and GRP94 increased all the way up to 24 h, while the expression of calnexin reached its peak level at 8 h and then decreased to the basal level by 24 h. p-PERK, p-eIF2R and ATF6 fragments increased at the early period (after Journal of Proteome Research • Vol. 8, No. 3, 2009 1589

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Figure 5. TBMS1 induced apoptosis via mitochondria-related pathway. (A) Measurement of the ∆Ψm in HeLa cells after TBMS1 treatment. (B) Effect of mitochondrial permeability transition (MPT) inhibitors on cytotoxicity of TBMS1 after 24 h treatment as measured by MTT assay. (*, p < 0.05 statistically significant difference compared with the control; #, p < 0.05 statistically significant difference compared with TBMS1 treatment). (C) Western blot analysis of the expression of Bcl-2, Bax and Cyt c. (D) Activation of caspases and their downstream effecter PARP-1 as detected by Western blotting.

4 h treatment), whereas their normal forms did not change (Figure 6B), meaning that ER stress was induced on the TBMS1treated HeLa cells. At the same time, GADD153/CHOP, a hallmark of the ER stress-mediated apoptosis, was also activated (Figure 6B). We then investigated whether the inhibitor of ER stress could rescue TBMS1-treated cells from apoptosis. Salubrinal (Sal), an established ER stress inhibitor, can inhibit eIF2R dephosphorylation and protect cells against ER stressmediated apoptosis. As shown in Figure 6C by MTT assay, the cell viability of the TBMS1-treated cells increased from 40% to 60% after pretreatment with 5 µM Sal (Figure 6C). These results demonstrated that ER stress was partially involved in the TBMS1-induced apoptosis.

Discussion TBMS1 Induces Apoptosis in Cancer Cells. Anticancer chemotherapeutic drugs with different anticancer actions are badly needed in clinical medicine, particularly for the patients resistant to the standard treatment. Traditional Chinese herbal medicines are important sources of drugs for the development of novel chemotherapeutics. Among them, triterpenoid saponins extracted from traditional Chinese herbal medicines have been shown to have the anticancer potential with very low cytotoxicity toward normal cells.28-30 Especially, TBMS1 has been tested for their antitumor potential in vitro and in vivo and demonstrated to have low toxicity.3 TBMS1, as a triterpenoid saponin extracted from TBM, has been explored for many years, exhibiting potent cytotoxicity to a number of human cancer cell lines.1,3,31-33 However, the exact molecular mechanism of TBMS1 drug action remains largely unknown. 1590

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Evoked by the anticancer chemotherapeutic drugs, apoptosis is an essential cell death, characteristic by a number of cellular and biochemical hallmarks, including DNA chromatin fragmentation, nucleus condensation and externalization of phosphatidylserine.16,23 Apoptosis is the major phenomenon of the TBMS1 cytotoxicity as revealed in the present study, in which great changes in apoptotic morphological characters were observed in HeLa cells (Figure 3A). This apoptotic effect was further validated through the significant increase of the apoptotic cell population (Figure 3B) and the stimulation of the DNA laddering (Figure 3C) after TBMS1 treatment. Mitochondria and ER Are Involved in the TBMS1Induced Cytotoxicity. Given the fact that proteins are the key elements for cellular activities, the alterations of cellular protein expression related to drug stimulation can provide valuable information to help understanding the mechanism of drug actions. A comparative proteomic approach was therefore applied to examine the protein alterations associated with TBMS1-induced cell death. The substantial changes of proteins were related to energy generation and mitochondrial protein chaperonins. We showed that the energy production was disturbed by the reduced expression of glycolytic enzymes including enolase1, GAPDH, pyruvate kinase and aldolase A (Table 1). Considering that the mitochondria are described as “cellular power plants” and cellular energy production is often down-regulated by mitochondrial dysfunction under anticancer drug treatment,34 these results are indicative of mitochondria dysfunction with TBMS1 exposure. Because of mitochondria damage by TBMS1, the mitochondrial-related proteins’ expression decreased, including tubulin whose maturation depends

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Figure 6. TBMS1 stimulated ER stress-mediated apoptotic pathway. (A) Western blot analysis of ER stress related chaperones GRP78, GRP94 and calnexin. (B) Western blot analysis of ER stress sensors PERK, p-PERK, eIF2R, p-eIF2R, ATF 6 fragment and CHOP. (C) Effect of ER stress inhibitor, salubrinal (Sal), on TBMS1-induced cell death by MTT assay. (*, p < 0.05 statistically significant difference compared with the control; #, p < 0.05 statistically significant difference compared with TBMS1 treatment).

on mitochondrial chaperone HSP60.35 Located in the mitochondrial matrix, HSP60 and MTHSP75 help the proteins inside the mitochondria to fold into their proper conformation.36,37 Under stress conditions, a rise of HSP60 and MTHSP75 in expression was associated with TBMS1-induced and mitochondria-mediated cytotoxicity (Figure 4A,B). Other proteins participated in the process of protein folding, secretion and synthesis, including GRP78/Bip, HSP70 and calnexin, were also significantly up-regulated in response to the mitochondrial dysfunction (Figure 4A,C). Especially, GRP78/ Bip is a key element in maintaining the normal ER function and protecting the ER from dangerous stimulation. It can control the activation of transmembrane ER stress sensors through a binding-release mechanism and activate the downstream signaling pathways.26 The expression alteration of GRP78/Bip and calnexin also suggest that ER system was under challenge upon TBMS1 treatment. Regulation of intracellular calcium (Ca2+) levels is one of main functions of ER and calnexin is an integral protein of the ER related to Ca2+ storage and signaling.38 Under ER stress condition by TBMS1 exposure, calnexin may be released as precursor from ER before being converted into functional mature form, and the overexpression of calnexin precursor indicated the less expression of the functional calnexin. In addition, as a multifunctional protein except for the traditional metabolic function, GAPDH combined with cytoskeleton proteins to regulate the microtubule activity and ultimately took part in the membrane transport between the ER and the Golgi apparatus.20,39,40 As a fact that ER is an

essential site for protein secretion and proper folding and Ca2+ regulation, it may be another important organelle mediating the TBMS1-induced apoptosis. In a word, these observations suggest that the mitochondria and ER are deserved to be further studied as the potential targets of TBMS1-induced apoptosis. Mitochondria Dysfunction Is an Important Event in TBMS1-Related Cytotoxicity. Recent studies suggested that mitochondria play an important role in apoptosis, characterizing with the loss of ∆Ψm and the following caspase activation.41 The mitochondria-related apoptotic pathway was obviously involved in the TBMS1-induced cytotoxicity. In fact, the attenuation of ∆Ψm was observed within 4 h under TBMS1 treatment in the present experiment (Figure 5A). To further investigate whether mitochondrial permeabilization is involved in the TBMS1-induced apoptosis, we used phospholipase inhibitors ArA, CyA and TFZ to detect the effect on TBMS1related cytotoxicity. We found that using these inhibitors alone or in combination could partially prevent the cytotoxic effects of TBMS1 on the HeLa cells. In addition, TBMS1 treatment affected the expression of Bcl-2 family proteins that usually reside in the mitochondrial outer membrane serving as the central regulators of mitochondrial integrity. In normal condition, Bcl-2, an antiapoptotic member in Bcl-2 family, forms heterodimers with pro-apoptotic element Bax. Under stress, Bax inserts into the mitochondrial outer membrane, increasing membrane permeability and then leading to cytochrome c release by forming pores on the outer mitochondrial membrane.42 The balance of Bax and Bcl-2 could Journal of Proteome Research • Vol. 8, No. 3, 2009 1591

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42

on ER stress. Our current findings also indicated that TBMS1induced cytotoxicity on HeLa cells is through multiple apoptosis-inducing pathways, which suggests that TBMS1 could be a potent anticancer drug for cancer treatment. Additional functional studies to identify the specific targets of TBMS1 are undergoing.

thus decide cell destiny. The increased ratio of Bax to Bcl-2 shown in Figure 5C suggests the potential of the release of proapoptotic mitochondrial factors such as cytochrome c, which activate caspase-9 to form apoptosome complex, and finally activating caspase-3 and cleaving PARP-1 to execute apoptosis (Figure 5D). These results imply that mitochondria dysfunction, at least in part, contributed to the cytotoxicity of TBMS1. ER Stress Partially Initiates TBMS1-Induced Cytotoxicity. Recent studies pointed out that ER is a subcellular compartment involved in the intrinsic apoptotic pathway.11 Besides the mitochondrial-related protein alterations, proteomic changes also implicated that ER may be another target of TBMS1-induced cytotoxicity. GRP78/GRP94 and calnexin/ calreticulin, respectively, belong to two different chaperone systems in the ER: GRP78/Bip and GRP94 are the chaperones of heat shock protein family and the calnexin belongs to the chaperone lectins.43 These two systems have distinct functions, GRP78/GRP94 are responsible for the folding and maturation of nonglycosylated proteins and calnexin/calreticulin play an important role in the newly synthesized glycoproteins.44 The difference in the protein expression of GRP78/GRP94 and calnexin implied that the two systems have different response to the TBMS1-induced ER stress. In addition, during ER stress, when protein aggregation persists and the stress cannot be resolved, the UPR signaling switches from prosurvival to proapoptotic. The increased calnexin precursor (from the 2DMS results) and the time-dependent effects of calnexin reflected the switches of the UPR signaling. In the current experiment, ER stress sensors were also assayed to validate the involvement of ER stress in the apoptotic process. After TBMS1 exposure, PERK and eIF 2R were phosphorylated at the early stage, but the total protein level of PERK and eIF2R was not affected, as shown in Figure 6B. At the same time, ATF6 was cleaved to the activated form ATF6 fragmentation (Figure 6B).26 These signaling transduction pathways are the adaptation responses to ER stress which could suppress the protein synthesis to relief the burden of ER and increase transcription of ER related-chaperones genes.12 If ER stress prolongs and becomes severe, these responses can trigger apoptosis, however. GADD153/CHOP, a proapoptotic transcription factor, is minimally expressed under physiological conditions, but it is strongly induced in response to ER stress. The overexpression of CHOP could lead to growth arrest and apoptosis as revealed from the study of traditional ER stress inducers (tunicamycin and thapsigargin).45,46 Sal is a selective phosphatase inhibitor of eIF2R for ER stress inhibition47 and it partially abrogated the TBMS1-induced cell death (Figure 6C). This observation indicated that the ER dysfunction partially contributed to the TBMS1-mediated apoptosis. ER stress has been studied for a long time, but mainly in the field of neuropathology; the information about the correlation between ER stress and cancer was available only in recent years. Further investigation to identify the downstream effectors of ER stressrelated cytotoxicity will be interesting and informative.

Conclusions In the present study, TBMS1-induced apoptosis in HeLa cells was validated and further investigated. Proteomic analysis of the total proteins profiles indicated that energy metabolismand protein synthesis-related proteins were involved in the apoptotic progress. Further functional studies demonstrated that TBMS1 induced the dysfunction of mitochondria and ER system. This is the first report describing the effect of TBMS1 1592

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Acknowledgment. This work was partially supported by the 2007 Chang-Jiang Scholars Program and “211” Projects (to Q.-Y.H.), and HKU Genomics, Proteomics and Bioinformatics Strategic Research Theme, Faculty of Science Development Fund, the Outstanding Young Researcher and Outstanding Research Student Supervisor Awards of the University of Hong Kong (to F.C.). References (1) Wang, F.; Ma, R.; Yu, L. Role of mitochondria and mitochondrial cytochrome c in tubeimoside I-mediated apoptosis of human cervical carcinoma HeLa cell line. Cancer Chemother. Pharmacol. 2006, 57 (3), 389–99. (2) Liu, W. Y.; Zhang, W. D.; Chen, H. S.; Gu, Z. B.; Li, T. Z.; Chen, W. S. New triterpenoid saponins from bulbs of Bolbostemma paniculatum. Planta Med. 2004, 70 (5), 458–64. (3) Yu, L.; Ma, R.; Wang, Y.; Nishino, H. Potent anti-tumor activity and low toxicity of tubeimoside 1 isolated from Bolbostemma paniculatum. Planta Med. 1994, 60 (3), 204–8. (4) Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 1995, 267 (5203), 1456–62. (5) Wajant, H. The Fas signaling pathway: more than a paradigm. Science 2002, 296 (5573), 1635–6. (6) Denicourt, C.; Dowdy, S. F. Medicine. Targeting apoptotic pathways in cancer cells. Science 2004, 305 (5689), 1411–3. (7) Liao, P. C.; Tan, S. K.; Lieu, C. H.; Jung, H. K. Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis. J Cell Biochem 2008, 104 (4), 1509–23. (8) Yokouchi, M.; Hiramatsu, N.; Hayakawa, K.; Kasai, A.; Takano, Y.; Yao, J.; Kitamura, M. Atypical, bidirectional regulation of cadmiuminduced apoptosis via distinct signaling of unfolded protein response. Cell Death Differ. 2007, 14 (8), 1467–74. (9) Green, D. R.; Reed, J. C. Mitochondria and apoptosis. Science 1998, 281 (5381), 1309–12. (10) Nicholson, D. W.; Thornberry, N. A. Apoptosis. Life and death decisions. Science 2003, 299 (5604), 214–5. (11) Rao, R. V.; Ellerby, H. M.; Bredesen, D. E. Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 2004, 11 (4), 372–80. (12) Boyce, M.; Yuan, J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ. 2006, 13 (3), 363–73. (13) Moenner, M.; Pluquet, O.; Bouchecareilh, M.; Chevet, E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 2007, 67 (22), 10631–4. (14) Xu, C.; Bailly-Maitre, B.; Reed, J. C. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 2005, 115 (10), 2656– 64. (15) Oyadomari, S.; Mori, M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004, 11 (4), 381–9. (16) Wang, Y.; Che, C. M.; Chiu, J. F.; He, Q. Y. Dioscin (saponin)induced generation of reactive oxygen species through mitochondria dysfunction: a proteomic-based study. J. Proteome Res. 2007, 6 (12), 4703–10. (17) Fukuda, K.; Kojiro, M.; Chiu, J. F. Induction of apoptosis by transforming growth factor-beta 1 in the rat hepatoma cell line McA-RH7777: a possible association with tissue transglutaminase expression. Hepatology 1993, 18 (4), 945–53. (18) Huigsloot, M.; Tijdens, I. B.; Mulder, G. J.; van de Water, B. Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells. J. Biol. Chem. 2002, 277 (39), 35869–79. (19) Kleizen, B.; Braakman, I. Protein folding and quality control in the endoplasmic reticulum. Curr. Opin. Cell Biol. 2004, 16 (4), 343–9. (20) Tisdale, E. J.; Kelly, C.; Artalejo, C. R. Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity. J. Biol. Chem. 2004, 279 (52), 54046–52.

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