Proteomic identification of proteins involved in the anticancer activities of oridonin in HepG2 cells

Phytomedicine 18 (2011) 163–169

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Proteomic identification of proteins involved in the anticancer activities of oridonin in HepG2 cells Hui Wang a , Yan Ye a , Si-Yuan Pan b , Guo-Yuan Zhu a , Ying-Wei Li a , David W.F. Fong a , Zhi-Ling Yu a,∗ a b

Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China Department of Pharmacology, Beijing University of Chinese Medicine, Beijing, China

a r t i c l e Keywords: Apoptosis G2/M phase arrest Isodon rubescens Oridonin Proteomics

i n f o

a b s t r a c t Oridonin is the main bioactive constituent of the Chinese medicinal herb Isodon rubescens and has been shown to have anti-neoplastic effects against a number of cancers in vitro and in vivo. Here we report the proteomic identification of proteins involved in the anticancer properties of oridonin in hepatocarcinoma HepG2 cells. Cell viability assay showed that oridonin dose-dependently inhibited cell growth with an IC50 of 41.77 ␮M. Treatment with oridonin at 44 ␮M for 24 h induced apoptosis and G2/M cell cycle arrest, which were associated with nine differentially expressed proteins identified by proteomic analysis. The proteomic expression patterns of Hsp70.1, Sti1 and hnRNP-E1 were confirmed by quantitative real-time PCR and/or immunoblotting. Eight of the nine identified proteins are shown, for the first time, to be involved in the anticancer activities of oridonin. Up-regulation of Hsp70.1, STRAP, TCTP, Sti1 and PPase, as well as the down-regulation of hnRNP-E1 could be responsible for the apoptotic and G2/M-arresting effects of oridonin observed in this study. Up-regulation of HP1 beta and GlyRS might contribute to inhibitory effects of oridonin on telomerase and tyrosine kinase, respectively. These findings shed new insights into the molecular mechanisms underlying the anticancer properties of oridonin in liver cancer cells. © 2010 Elsevier GmbH. All rights reserved.

Introduction Isodon rubescens (Hemsl.) C.Y. Wu et Hsuan, native to the Yellow River valley in China, has been in use as a remedy for the treatment of respiratory and gastrointestinal bacterial infections as well as cancers (Sun et al., 2006). Oridonin (Fig. 1), a diterpenoid compound, is the major bioactive constituent of I. rubescens. In human patients oridonin exhibits curative effect for digestive system tumors such as esophageal and liver cancers (Wang, 1984; Wang and Wang, 1984). In animal experiments oridonin prolongs the lifespan of mice with Ehrlich ascites carcinoma, P388 lymphocytic leukemia or t (8;21) leukemia (Zhou et al., 2007). In various cancer cell lines, including HepG2, oridonin shows antiproliferative (Chen et al., 2007) and apoptotic effects (Ikezoe et al., 2003; Wang et al., 2008). Caspases, p53, reactive oxygen species, p38 MAPK pathway, and nuclear factor-kappa B (NF-␬B) have been implicated in the apoptosis-inducing activity of oridonin (Huang et al., 2008; Cheng et al., 2009). Oridonin can also induce G2/M or G0/G1 arrest depending on cell type (Liu et al., 2004; Wang et al., 2008). Other effects of oridonin such as inhibi-

tion of telomerase (Wang et al., 2008) and tyrosine kinase (Li et al., 2007), anti-angiogenesis (Meade-Tollin et al., 2004), and antimigration and differentiation induction (Ren et al., 2006) have also been reported. To further understand the molecular mechanism of oridonin in liver cancer therapy, in this study we identified differentially expressed proteins in oridonin-treated HepG2 cells by using two-dimensional gel electrophoresis (2-DE)-based proteomics. Materials and methods Oridonin Oridonin was purchased from Shanghai Shamrock Import & Export, Trading Co. Ltd., and the purity was determined to be 97% by HPLC. Stock solution of oridonin (274.5 mM) was prepared in dimethyl sulfoxide (DMSO, Sigma, France). Aliquots were stored at −20 ◦ C. Cell culture

∗ Corresponding author. Tel.: +852 3411 2465; fax: +852 3411 2461. E-mail address: [email protected] (Z.-L. Yu). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.06.011

HepG2 cells (ATCC, USA), grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, USA) and 1% peni-

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Fig. 1. The chemical structure of oridonin.

cillin/streptomycin (P/S, GIBCO, USA) were cultured at 37 ◦ C in an atmosphere containing 5% CO2 . Determination of cell viability, cell cycle distribution, and apoptosis Cytotoxicity was assessed using MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide) assay. HepG2 cells (7.0 × 103 cells per well) were seeded and grown overnight in 96-well plates and then treated with various concentrations of oridonin. Control cells were treated with DMSO (0.1%) in this experiment and in the following experiments. After 24-h incubation, 20 ␮l of MTT solution (5 mg/ml, USB, Austria) was added to each well and plates were incubated at 37 ◦ C for 4 h. Following medium removal, 100 ␮l of DMSO was added to each well and plates were gently shaken for 5 min. Optical absorbance was determined at 570 nm with a microplate spectrophotometer (BD Bioscience, USA). Each treatment was performed in triplicate and each experiment was repeated four times. The distribution of cells in various phases was determined from DNA content assessed by flow cytometry. Cells were seeded at a density of 45 × 104 per 60 mm dish and grown overnight. Oridonin was added to a final concentration of 44 ␮M and cells were incubated for 24 h. Both detached and adherent cells were collected and centrifuged at 1000 g for 5 min at 4 ◦ C. Pellets were rinsed with icecold phosphate-buffered saline (PBS) and fixed with 70% ethanol for 2 h. Cells were then stained with staining buffer (PBS containing 20 ␮g/ml of propidium iodide, 100 ␮g/ml RNase A, and 0.1% Triton X-100) for 15 min at 37 ◦ C in the dark. Samples were analyzed by a flow cytometer (BD Bioscience, USA) installed with the Modfit software version 3.1 (Verity Software House, USA). Apoptotic morphology was monitored in 4 , 6-diamidino-2phenylindole (DAPI)-stained cells. Cells (40 × 104 ) were grown for 24 h on cover slips in 35 mm dishes in the presence or in the absence of 44 ␮M oridonin. Cover slips were carefully washed with PBS, fixed with 4% paraformaldehyde for 10 min and incubated with 10 ␮g/ml DAPI for 10 min. Cells were washed with PBS and observed under a fluorescent microscope (Nikon, Japan). Cells with clear condensed nuclei were considered as apoptotic cells, and 500 cells in randomly selected fields were counted to calculate apoptosis rate. Proteomic sample preparation and 2-DE Cells were seeded in 100 mm culture dishes at 1.5 × 106 cells per dish, incubated overnight and then treated with 44 ␮M oridonin for 24 h. Cells were harvested by trypsinization, washed three times with isotonic buffer (10 mM Tris, 250 mM sucrose, pH 7.2) and lysed in a lysis buffer (8 M urea, 4% CHAPS, 40 mM dithiothreitol and 0.5% pH 3–10 NL IPG buffer) by gentle shaking for 60 min on ice. Extracts

were centrifuged at 25,000 g for 60 min at 4 ◦ C. Supernatants were purified with the 2D Clean Up Kit (GE Healthcare, USA) following manufacturer’s instructions. The protein concentration of each purified sample was determined using the 2D Quant kit (GE Healthcare, USA) and protein samples were stored in aliquots at −80 ◦ C until analyzed. First-dimension separation was performed using 13 cm IPG strip (pH 3–10 NL, GE Healthcare, USA). Samples were diluted in rehydration solution containing 8 M urea, 2% CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue, and 0.28% dithiothreitol (DTT, GE Healthcare, USA) to reach a final protein load of 100 ␮g (in 250 ␮l) per strip. IPG strips were actively rehydrated at 30 V for 12 h, focused at 500 V for 2.5 h, 1000 V for 0.5 h, and then the voltage was increased to 8000 V gradually over the subsequent 3 h and maintained at 8000 V for 40,000 Vh. Prior to the second-dimension separation, IPG strips were equilibrated in equilibration buffer (6 M urea, 75 mM Tris–HCl pH 8.8, 29.3% glycerol, 2% SDS) containing 1% (w/v) DTT for 15 min and then in the same equilibration buffer containing 2.5% iodoacetamide (IAA, Amersham Biosciences, USA) for a further 15 min. The second-dimension separation was carried out on 10% SDS-PAGE (20 mA/gel, 10 min; 25 mA/gel, 260 min). Silver staining and image analysis Protein spots were visualized by silver staining. Gels were fixed overnight in fixing solution (40% (v/v) methanol and 10% (v/v) acetic acid) and then treated for 30 min with sensitizing solution (30% ethanol, 4.05% (w/v) sodium acetate, and 0.2% sodium thiosulfate). Each gel was washed three times with MilliQ water for 5 min each, stained for 40 min in 0.1% silver nitrate (Sigma, USA) solution and then developed by incubation with a developing solution (2.5% (w/v) sodium carbonate and 0.02% formaldehyde) until protein spots appeared. The developing reaction was terminated by putting gels in the stopping solution (1.46% (w/v) EDTA, disodium salt) for 10 min. Finally gels were washed for 5 min with MilliQ water three times. The stained gels were scanned with an image scanner (GE Healthcare, USA) installed with LabScan 6.0 software, and data were analyzed using the Image Master 2D Platinum 6.0 software (GE Healthcare, USA). The intensity volume of each spot was processed by background subtraction and total spot volume normalization, and the resulting spot volume percentage was used for comparison. Only spots which were significantly (Student’s t-test, p < 0.05) and consistently up- or down-regulated (>2-folds) or spots which appeared or disappeared after treatment in three independent experiments were selected for in-gel digestion and mass spectrometry (MS) analysis. In-gel digestion and MALDI-TOF–MS/MS analysis Protein spots were excised from gels and each sample was transferred to a 1.5-ml Eppendorf tube and de-stained in a freshly prepared de-staining solution (15 mM potassium ferricyanate and 50 mM sodium thiosulfate) until the brownish color disappeared. Each de-stained sample was washed in 10 mM ammonium bicarbonate (NH4 HCO3 ) for 5 min, in 10 mM NH4 HCO3 containing 10 mM DTT for 15 min at 56 ◦ C, in 10 mM NH4 HCO3 containing 55 mM IAA for 20 min at room temperature in the dark, and finally in 10 mM NH4 HCO3 containing 50% ACN for 15 min. After drying in a Vacufuge concentrator (Eppendorf, Germany), each sample was incubated at 37 ◦ C overnight in 5 ␮l of 5 ␮g/ml trypsin gold solution (Promega, USA). The supernatant was collected and the gel was further extracted with 1% formic acid. The extracts were combined and dried in the Vacufuge concentrator and resuspended in formic acid for MS analysis. The mass spectra were obtained using the Bruker AutoflexIII MALDI-TOF/TOF Mass Spectrometer (Bruker Dal-

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tonics, USA). Protein identification was performed automatically by searching the Swiss-Prot 55.3 database using the MASCOT 2.04 search engine (Matrix Science, UK). Database searches were carried out using the following parameters: type of search, MS/MS ion search; enzyme, trypsin; and allowance of one missed cleavage. Carbamidomethylation was selected as a fixed modification and oxidation of methionine was allowed to be variable. The peptide and fragment mass tolerance were set at 50 ppm and 0.5 Da, respectively. The instrument was selected as MALDI-TOF-TOF. Proteins with probability-based MOWSE scores (p < 0.05) were considered to be positively identified. Western blot analysis Cells treated as described above were collected and proteins were extracted with RIPA lysis buffer [50 mM Tris-Cl, 1% (v/v) NP40, 0.35% (w/v) sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 1 mM Na3 VO4 ] containing a protease inhibitor cocktail (Roche, Germany). Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, USA). Twenty micrograms of individual protein samples were separated by SDS-PAGE and then electro-transferred onto the nitrocellulose membrane (Amersham Biosciences, USA). Membranes were blocked for 30 min with 5% skim milk in TBST buffer composed of 50 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20 and incubated with specific antibodies against human Hsp70.1 (Santa Cruz, USA; 1:2000 dilution) or hnRNP-E1 (Santa Cruz, USA; 1:500 dilution) overnight at 4 ◦ C. Beta-actin was used as a loading control and detected using an anti-actin polyclonal antibody (Santa Cruz, USA; 1:4000 dilution). After incubation with secondary antibodies, ECL detection reagents (Amersham Biosciences, USA) were used to detect signals. Real-time PCR Cells were treated as described above and total RNA was isolated using Trizol reagent (Invitrogen, USA) according to manufacturer’s protocol. Five micrograms of RNA was used for reverse transcription by oligo-dT using the SuperScriptII Reverse Transcription Kit (Invitrogen, USA). The real-time PCR primers were designed as follows: Hsp70.1 (sense 5 -cagaacaagcgagccgtgagg3 and anti-sense 5 -tcgtgaatctgggccttgtcc-3 ), Sti1 (sense 5 tgtaaggaggcggcagacgg-3 and anti-sense 5 -taaggcgcatggctgggtca3 ), hnRNP-E1 (sense 5 -aggtgaaaggctattgggcaagt-3 and anti-sense 5 -ggatcatgggagaacagcagaaa-3 ). To normalize the amounts of RNA in samples, a PCR reaction was also performed with primers of ␤-actin (sense 5 -gactacctcatgaagatc-3 and anti-sense 5 gatccacatctgctggaa-3 ). Real-time PCR was performed in a total volume of 20 ␮l, with 1× Power SYBR Green PCR Master Mixture (Applied Biosystems, UK) in the 7500 Fast Real-time PCR system (Applied Biosystems, USA). All the samples were run in triplicate. Data analysis All the results were expressed as the mean ± SEM, and statistical analyses were performed using the Student’s t test. Results Cytotoxicity of oridonin Initial MTT assay showed that treatment with oridonin for 24 h resulted in a marked decrease in cell viability in a dose-dependent manner (Fig. 2). The IC50 value was determined as 41.77 ␮M from

Fig. 2. Cytotoxicity of oridonin treatment in HepG2 cells. Cells were treated with various concentrations of oridonin or DMSO (control) for 24 h. Cell viability was measured by MTT assay. Data were expressed as mean ± SEM from four independent experiments (each treatment was performed in triplicate). *p < 0.01 vs the control.

the dose–response curve with GraphPad Prism 5.0 software. Thus, in subsequent assays 44 ␮M oridonin was used. Cell cycle arrest and apoptosis induced by oridonin In the cytotoxicity assay it was noted that oridonin treatment resulted in rounding up of cells when observed under the microscope (Fig. 3(A)). To verify whether oridonin caused cell cycle perturbation, DNA contents of HepG2 cells treated for 24 h with or without 44 ␮M oridonin were analyzed by flow cytometry. Fig. 3(B) shows that oridonin treatment caused a significant increase of G2/M phase cells from 19.39 ± 1.532% to 47.11 ± 2.090% (p < 0.01). Results also showed that sub-G1 cells significantly increased from 0.8357 ± 0.1799% to 2.499 ± 0.4642% after oridonin treatment (p < 0.01), suggesting an apoptotic effect. To verify the occurrence of apoptosis, cells were observed under a microscope after DAPI staining. Typical apoptotic nucleus alterations (chromatin condensation, nuclear fragmentation, appearance of apoptotic bodies) were observed after oridonin treatment (Fig. 3(C)). Apoptosis rate was 6.07 ± 0.581% in oridonintreated cells vs 1.80 ± 0.503% in control cells (p < 0.01) (Fig. 3(D)). 2-DE and MS/MS analysis results In an attempt to identify the molecular changes induced by oridonin, we monitored differential protein expression before and after oridonin treatment using 2-DE based proteomics. To ascertain reproducibility of results 2-DE was performed three times for each protein sample and each treatment was repeated three times. Fig. 4 shows representative gel images. Proteins within the range of 15–225 kDa and with isoelectric points (PI) between 3 and 10 were well separated. More than 1000 spots were detected on each gel. Twelve significantly (Student’s t-test, p < 0.05) and consistently up- or down-regulated protein spots with fold changes greater than 2 in terms of volume intensity in both triplicate gels and three independent experiments were cut from the gels and analyzed by MALDI-TOF–MS/MS after trypsin digestion. Eight up-regulated proteins, namely heat shock 70 kDa protein 1 (Hsp70.1), stress-induced phosphoprotein 1 (Sti1), serine–threonine kinase receptor-associated protein (STRAP), trifunctional purine biosynthetic protein adenosine-3, inorganic pyrophosphatase (PPase), chromobox protein homolog 1 (HP1 beta), translationally-controlled tumor protein (TCTP), glycyltRNA synthetase (GlyRS) and one down-regulated protein, namely poly(rC)-binding protein 1 (hnRNP-E1) were successfully identified. Table 1 lists the main properties of the identified proteins. The protein expression levels and identities of Hsp70.1 (the first up-regulated protein in Table 1) and hnRNP-E1 (the down-

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Fig. 3. Oridonin-induced cell cycle arrest and apoptosis in HepG2 cells. Cells were treated with oridonin (44 ␮M) or DMSO (control) for 24 h. (A) Cell morphological changes after oridonin treatment as observed under a light microscopy (100×). (B) Cellular DNA contents in oridonin-treated and control cells were monitored by flow cytometry and cell cycle distribution was determined using Modfit software version 3.1. The proportions of subG1 and G2/M phase cells were shown as indicated. These results are representative of three independent experiments. (C) Oridonin-treated and untreated control cells were stained with DAPI and visualized by fluorescent microscopy (400×). Representative DAPI-stained nuclei of cells are shown. Typical apoptotic changes of nucleus (chromatin condensation, nuclear fragmentation, appearance of apoptotic bodies) were indicated by arrows. (D) Cells with typical apoptotic changes were counted in randomly selected fields containing 500 cells. The percentage of apoptotic cells was calculated and presented as mean ± SEM of three independent experiments. *p < 0.01 vs the control.

Fig. 4. Representative 2-DE gels stained with silver nitrate. HepG2 cells treated with 44 ␮M oridonin or vehicle (DMSO) for 24 h. Eight protein spots with marked changes (see text) were identified and indicated. Their close-up spot images were shown on the right panel.

regulated) in response to oridonin treatment were confirmed by Western blotting (Fig. 5(A)). For the first two up-regulated proteins Hsp70.1 and Sti1 as well as the down-regulated protein hnRNP-E1, mRNA expression levels were assessed by quantitative real-time PCR. Results showed that, in oridonin-treated cells, mRNA expression levels of Hsp70.1 and Sti1 were 2.94 and 1.51-fold higher, and hnRNP-E1 0.59-fold lower

than in control cells (Fig. 5(B)); these level changes are comparable to their protein expression level changes. Discussion Oridonin has been shown to exhibit antiproliferative activity in various cancer cell lines. The concentrations needed to cause 50%

Fig. 5. Alterations in protein and mRNA expression of Hsp70.1, Sti1 and hnRNP-E1 in HepG2 cells treated with 44 ␮M oridonin for 24 h. (A) Western blot analysis of Hsp70.1 and hnRNP-E1. ␤-Actin was included as a protein-loading control. These results are representative of three independent experiments. (B) Real-time PCR analysis of mRNA expression levels of Hsp70.1, Sti1, and hnRNP-E1. Data were presented as mean ± SEM of three independent experiments. *p < 0.05 vs controls.

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Table 1 Summary of differentially expressed proteins in oridonin-treated HepG2 cells. Spot no.

Protein description

Function

MWa

PIb

MSc scores

Expression leveld

1 2 3 4 5 6 7e 7e 8

Heat shock 70 kDa protein 1 Stress-induced-phosphoprotein 1 Trifunctional purine biosynthetic protein adenosine-3 Serine–threonine kinase receptor-associated protein Poly(rC)-binding protein 1 Inorganic pyrophosphatase Chromobox protein homolog 1 Translationally-controlled tumor protein Glycyl-tRNA synthetase

Molecular chaperon Molecular chaperon Purine synthesis Signal transduction RNA binding Metabolism Chromatin MT stabiliztion Protein synthesis

70294 63227 108953 38756 37987 33095 21519 19697 83828

5.48 6.40 6.26 4.98 6.66 5.54 4.85 4.84 6.61

212 88 73 332 423 110 124 91 136

+ + + + − + + + +

a b c d e

Molecular weight. Isoelectric point. Mass spectrometry. Expression level in oridonin-treated HepG2 cells at the time point of 24 h compared with the control (+, increase; −, decrease). Two proteins were identified from a single spot.

inhibition of cell growth (IC50 s) are cell type-dependent, varying from several to tens of micromoles per liter (Zhang et al., 2009; Zhou et al., 2007; Ren et al., 2006; Chen et al., 2005). In HepG2 cells oridonin has been reported to have an IC50 value of about 30 ␮M (at 24 h) (Huang et al., 2008), while in the present report the IC50 value is 41.77 ␮M. The differences in IC50 values are probably due to the different culture conditions. In Huang’s study HepG2 cells were cultured in RPMI-1640 medium supplemented with 10% FCS, but we cultured the cells in DMEM medium supplemented with 10% heat-inactivated FBS. In addition, the different IC50 values may also be caused by different passage times of the cells. Whether the in vitro dosage has reference value for clinical application remains to be estimated. Early studies showed that oridonin at the dosage of 57–100 mg (every other day intravenous infusion for two months) exerted beneficial effects in patients with liver cancer (Gao et al., 1984; Wang, 1984). Animal studies have demonstrated that the bioavailability of oridonin is poor, and people have been trying to improve it by, for example, preparing it as nanoparticles (Zhang et al., 2005), or delivering it through self-microemulsifying drug delivery systems (Zhang et al., 2008). In several cancer cell lines oridonin has been reported to induce G2/M phase arrest (Chen et al., 2005; Han et al., 2007; Liu et al., 2004; Ren et al., 2006). Cell cycle arrest is often accompanied by apoptosis, and oridonin does have apoptotic effects in a variety of cancer cell lines (Huang et al., 2008; Ikezoe et al., 2003). It is generally believed that apoptosis appears in the phase in which the cell cycle is blocked (Hu et al., 2007). In oridonin-treated HepG2 cells we observed that apoptosis is associated with G2/M phase arrest. We are the first to report the G2/M phase-arresting activity of oridonin in HepG2 cells. Oridonin arrests different cancer cells at different phases. In human breast carcinoma MCF-7 (Hsieh et al., 2005) and prostate carcinoma LNCaP (Ikezoe et al., 2003; Chen et al., 2005) cells, which contain wild type p53, oridonin induces G1 arrest, while it blocks the cell cycle at the G2/M phase in human prostate carcinoma DU-145 cells with mutated p53 (Chen et al., 2005). It seems that p53 status plays a role in oridonininduced cell cycle arrest. Although HepG2 cells contain wild type p53, oridonin treatment induced G2/M arrest in this cell line. Our observation suggests that the G2/M arresting effect of oridonin may be not solely mediated by p53. Other molecules such as Sti1, Ppase, TCTP, STRAP and hnRNP-E1 identified in this study may also play roles in oridonin-induced G2/M cell cycle arrest (see Discussion). To further explore the molecular mechanisms underlying the anticancer effects of oridonin we applied proteomic analysis to identify differentially expressed proteins in oridonin-treated HepG2 cells. Nine proteins were successfully identified and expression patterns of Hsp70.1 and hnRNP-E1 were verified by Western blotting. In addition, mRNA expression levels determined by

quantitative real-time PCR for Hsp70.1, Sti1 and hnRNP-E1 were comparable to their protein expression levels. Sti1 is a co-chaperone and also functions in cell cycle progression. In oridonin-treated HepG2 cells, the up-regulation of Sti1 might inhibit the formation of 20S cyclosome complex (Yamashita et al., 1996), thus blocking separation of sister chromatids and consequently resulting in the retention of cells in the M phase. PPase has been shown to be involved in MAPK pathways. Oridonin can activate MAPK pathways (Huang et al., 2008; Wang et al., 2008). Oridonin may induce G2/M phase arrest through the activation of MAPK pathways and the consequent up-regulation of PPase (Zhang and Wang, 2007). TCTP overexpression in mammalian cells has been shown to cause a higher percentage of cells to be stalled in the G2/M phase (Gachet et al., 1999). The up-regulation of TCTP may be another mechanism for the G2/M-arresting effect of oridonin. Hsp70.1 has been shown to promote tumor necrosis factor (TNF)-mediated apoptosis by impairing nuclear factor-kappa B (NF␬B) survival signaling (Ran et al., 2004). The inhibitory effect of oridonin on TNF-␣-stimulated NF-␬B activity has been reported (Wang et al., 2008). Up-regulation of Hsp70.1 in oridonin-treated HepG2 cells may promote apoptosis by inhibiting NF-␬B activity. STRAP physically interacts with p53 and positively regulates p53 functions, including apoptosis induction and cell cycle arrest (Jung et al., 2007). Since the anticancer activity of oridonin has been shown to be p53-dependent (Huang et al., 2008; Wang et al., 2008), the up-regulation of STRAP may contribute to the apoptotic and G2/M arresting effects of oridonin. HnRNP-E1 has been shown to stimulate the translation of c-myc and Bag-1 (Carpenter et al., 2006). Suppression of c-Myc is associated with the induction of apoptosis in a variety of cells such as esophageal cancer cells (Thompson, 1998) and hepatoma cells (Hu et al., 2006; Qiao et al., 2008; Xu et al., 2004). Absence of c-Myc can prolong the G2 phase in c-myc (−/−) rat fibroblasts (Mateyak et al., 1999). G2/M arrest induced either by drugs or by overexpression of specific proteins is followed by a down-regulation of c-Myc expression in different cell types (Deng et al., 1999; Habel et al., 2005; Matsui et al., 2006). The down-regulation of hnRNP-E1 might be responsible for the apoptotic and G2/M arresting effects of oridonin in HepG2 cells by reducing the expression of c-Myc. Consistent with our hypothesis, it was shown that c-Myc expression is down-regulated after oridonin treatment (Wang et al., 2008; Xue et al., 2005). Furthermore, hnRNP-E1 has been reported to be an MPM-2 antigen. This also supports the role of hnRNP-E1 downregulation in the G2/M arresting effect of oridonin, because MPM-2 antigens are important mitotic regulators and effectors (Xiang et al., 2008). Bag-1 is identified as an anti-apoptotic protein based on its ability to bind Bcl-2 and augment Bcl-2 activity (Cato and Mink, 2001; Krajewski et al., 1999). The reduction of hnRNP-E1 protein

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expression might be responsible for the apoptotic effect of oridonin in HepG2 cells by reducing the expression of Bag-1 and subsequent reduction of Bcl-2 activity. Our suggestion is supported by previous reports that oridonin may cause a down-regulation of Bcl-2 protein expression (Deng et al., 1999; Liu et al., 2004). HP1 beta functions in telomere maintenance (Hayakawa et al., 2003). In human cells and a mouse xenograft model, overexpression of HP1 beta can lead to reduced telomere association of telomerase and growth potential (Sharma et al., 2003). The up-regulation of HP1 beta observed in this study might account for the telomerase inhibiting effects of oridonin (Wang et al., 2008). GlyRS gene can be up-regulated by a tyrosine kinase inhibitor, gefitinib, which has anti-tumor activity in a broad range of human cancers (Yano et al., 2006). Although it remains unclear as to the functional importance of up-regulation of GlyRS protein in oridonin-treated HepG2 cells, oridonin is reported to inhibit tyrosine kinase activity in human epidermoid carcinoma cells (Li et al., 2007). The up-regulation of GlyRS in response to oridonin treatment suggests that GlyRS might be a target for oridonin-mediated tyrosine kinase inhibition. Trifunctional purine biosynthetic protein adenosine-3 catalyzes purine de novo synthesis. Tumor cells generally have elevated activities of this de novo pathway and inhibitors of this protein are reported to cause a potent inhibition of purine synthesis and consequent cytotoxicity (Bronder and Moran, 2003). The cell growth promoting effect of trifunctional purine biosynthetic protein adenosine-3 might be one of the reasons of the weak apoptotic effect of oridonin observed in this study. In conclusion, this is the first report applying the proteomic approach to identify proteins involved in the biochemical activity of oridonin. Expression levels of nine proteins were differentially regulated in oridonin-treated HepG2 cells. The differential expressions of Hsp70.1, Sti1, STRAP, PPase, TCTP and hnRNP-E1 may contribute to the apoptotic and G2/M arresting effects of oridonin. Up-regulations of HP1 beta and GlyRS may contribute to the anticancer activities of oridonin through inhibiting the activities of telomerase and tyrosine kinase, respectively. The weak apoptotic effect of oridonin might be explained, at least in part, by the up-regulation of trifunctional purine biosynthetic protein adenosine-3. The results of this study advance our understanding of the molecular mechanisms responsible for the anticancer properties of oridonin in liver cancer cells. Acknowledgements We thank the Proteomic Laboratory for System Biology, School of Chinese Medicine, Hong Kong Baptist University, for proteomic analyses. This work was supported by a research grant (FRG/0809/I-08) from Hong Kong Baptist University. References Bronder, J.L., Moran, R.G., 2003. A defect in the p53 response pathway induced by de novo purine synthesis inhibition. J. Biol. Chem. 278, 48861–48871. Carpenter, B., MacKay, C., Alnabulsi, A., MacKay, M., Telfer, C., Melvin, W.T., Murray, G.I., 2006. The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression. Biochim. Biophys. Acta 1765, 85–100. Cato, A.C., Mink, S., 2001. BAG-1 family of cochaperones in the modulation of nuclear receptor action. J. Steroid Biochem. Mol. Biol. 78, 379–388. Chen, S., Gao, J., Halicka, H.D., Huang, X., Traganos, F., Darzynkiewicz, Z., 2005. The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage. Int. J. Oncol. 26, 579–588. Chen, J.H., Wang, S.B., Chen, D.Y., Chang, G.S., Xin, Q.F., Yuan, S.J., Shen, Z.Y., 2007. The inhibitory effect of oridonin on the growth of fifteen human cancer cell lines. Zhongguo Zhong Liu Lin Chuang 4, 16–20. Cheng, Y., Qiu, F., Ikejima, T., 2009. Molecular mechanisms of oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells. Autophagy 5, 430–431. Deng, Y., Lin, C., Zheng, J., Liang, X., Chen, J., Fu, M., Xiao, P., Wu, M., 1999. Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2. Sci. China C Life Sci. 42, 635–643.

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