Proteomic analysis of anti-tumor effects by tetrandrine treatment in HepG2 cells

Phytomedicine 17 (2010) 1000–1005

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Phytomedicine journal homepage: www.elsevier.de/phymed

Proteomic analysis of anti-tumor effects by tetrandrine treatment in HepG2 cells Zhixiang Cheng a,b , Keming Wang a , Jia Wei b , Xiang Lu c , Baorui Liu b,∗ a

Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China Department of Oncology, Drum Tower Hospital Affiliated to Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China c Department of Geriatrics, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China b

a r t i c l e

i n f o

Keywords: Tetrandrine Stephania tetrandra Mass spectrometry Peptide fingerprinting HepG2 cells

a b s t r a c t Tetrandrine (TET), a bis-benzylisoquinoline alkaloid isolated from the root of Hang-Fang-Chi (Stephenia tetrandra S Moore), exhibits broad pharmacological effects, including anti-tumor activity. Recently, the beneficial effects of TET on cytotoxicity towards tumor cells, radiosensitization, circumventing multidrug resistance, normal tissue radioprotection, and antiangiogenesis have been examined extensively. To explore the potential molecular mechanism of the anti-tumor effect of TET, we applied proteomic tools to profile the proteins in HepG2 cells subjected to TET treatment. The levels of 39 proteins in cells exposed to TET (IC50 = 5 ± 0.6 ␮g/ml) for 48 h were observed to undergo significant alterations. Six proteins were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS) using peptide fingerprinting from 10 protein spots (density difference >1.5-fold between the control and TET-treated group). Among them, 5 proteins were downregulated (proteasome activator complex subunit 3, 40S ribosomal protein S12, phosphoglycerate mutase 1, destrin, transaldolase) and 1 protein was upregulated (guanylate kinase 1) by TET treatment in HepG2 cells as determined by spot volume (P < 0.05). Most of the identified proteins were associated with tumor growth, migration, and anti-tumor drug resistance. These data will be helpful in elucidating the molecular mechanism of TET’s anti-tumor effect in HepG2 cells. © 2010 Elsevier GmbH. All rights reserved.

Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It usually causes death within a few weeks or months of detection (Bruix et al. 2004). Most patients diagnosed with HCC have low recovery rates. Moreover, the conventional and modified therapies currently available are rarely beneficial (Thomas and Zhu 2005). Therefore, the need to develop more effective agents for HCC is urgent. TET is a bis-benzylisoquinoline alkaloid isolated from the root of Hang-Fang-Chi (Stephania tetrandra S. Moore), which has been used in traditional Chinese medicine for several decades for treatment of arthritis, silicosis, and hypertension (Banks et al. 1993). Recently, the beneficial effects of TET on cytotoxicity towards tumor cells, radiosensitization, circumventing multidrug resistance, normal tissue radioprotection, and antiangiogenesis have been examined extensively (Chen 2002). TET shows striking anti-tumor efficacies

Abbreviations: TET, tetrandrine; HCC, hepatocellular carcinoma; 2DE, two-dimensional gel electrophoresis; MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; PA28gamma, proteasome activator complex subunit 3; RPS12, 40S ribosomal protein S12. ∗ Corresponding author. Tel.: +86 25 83107081; fax: +86 25 83107081. E-mail address: [email protected] (B. Liu). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.018

in a variety of malignant cell lines (Ng et al. 2006). Meanwhile, abundant evidence indicates the strong induction of apoptosis by TET in HepG2 cells (Ng et al. 2006). Besides its capability of reversing multidrug resistance (Chen et al. 2005; Dai et al. 2007), TET exhibits synergistic effects with chemotherapeutics in restricting tumor cell proliferation (Wei et al. 2007). However, the molecular mechanisms underlying TET’s anti-tumor efficacy remain poorly understood. In order to further investigate certain mechanisms, we applied a proteomic approach by utilizing two-dimensional gel electrophoresis (2DE) to profile the proteins in HepG2 cells treated by TET. HepG2 is a human hepatocellular liver carcinoma cell line isolated from a liver biopsy of a 15-year-old male Caucasian with a well-differentiated hepatocellular carcinoma.

Materials and methods Materials All chemicals used were of analytic grade. Tetrandrine (molecular formula C38 H42 N2 O6 ) was obtained as a powder with a purity of >98% from Beihai Sunshine Pharmaceutical Limited Company (Jiangxi, China). It was dissolved in 0.1N HCl and diluted with distilled water (pH value was adjusted to 6.6–6.8 with 1N NaOH) to achieve a TET concentration of 5 mg/ml.

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Cell culture and cytotoxicity assay

Mass spectrometry and database searching

Complete Dulbecco’s Modified Eagle’s Medium (DMEM) (100 U/ml penicillin, 100 mg/ml streptomycin, 12% fetal bovine serum, pH 7.4) was used to culture the HepG2 cells in standard incubator conditions (37 ◦ C, 5% CO2 ) until about 80% confluence. HepG2 cells (1 × 104 /well) were incubated with various concentrations of TET in at least five replicate wells for 48 h in 96-well plates. After 4 h incubation with MTT substrate (20 mg/ml), the culture medium was removed and DMSO added. Absorbance at 570 nm was measured with a multiwell spectrophotometer (BioTek, VT, USA). Growth inhibition was calculated as a percentage of the untreated controls, which were not exposed to drugs. The cytotoxicity was evaluated with reference to the IC50 value. The tests were performed at least three times.

Selected proteins were cut from the gels and digested by the method of Shevchenko et al. (1996) but with modified NH4 HCO3 concentration (from 100 mM to 25 mM). Proteins were reduced with 10 mM DTT/25 mM NH4 HCO3 at 56 ◦ C for 1 h and alkylated with 55 mM iodoacetamide/25 mM NH4 HCO3 in the dark at room temperature for 45 min in situ. Gel pieces were thoroughly washed with 25 mM NH4 HCO3 , 50% ACN, and 100% ACN, and dried in a Speedvac. Dried gel pieces were rehydrated with 2–3 ␮l of trypsin (Promega, Madison, WI, USA) solution (10 ng/␮l in 25 mM ammonium bicarbonate) at 4 ◦ C for 30 min. Excess liquid was discarded and gel plugs were incubated at 37 ◦ C for 12 h. TFA was added to a final concentration of 0.1% to stop the digestive reaction. Digests were immediately spotted onto 600 ␮m anchorchips (Bruker Daltonics, Bremen, Germany). Spotting was achieved by pipetting 1 ␮l of analyte onto the MALDI target plate in duplicate and then adding 0.05 ␮l of 2 mg/ml ␣-cyano-4hydroxycinnamic acid in 0.1% TFA/33% ACN which contained 2 mM ammonium phosphate. Bruker Peptide Calibration Mixture (containing Angiotensin II, MH+ 1046.542; Angiotensin I, MH+ 1296.685; Substance P, MH+ 1347.735; Bombesin, + MH 1619.822; Renin Substrate, MH+ 1758.932; ACTH 1–17 clip, MH+ 2093.086; ACTH 18–39 clip, MH+ 2465.198; Somatostatin 28 clip, MH+ 3147.471) was spotted down for external calibration. All samples were allowed to air dry at room temperature, and 0.1% TFA was used for on-target washing. All samples were analyzed in a time-of-flight Biflex IV mass spectrometer (Bruker Daltonics, Bremen, Germany). The spectrometer was run in positive ion reflectron mode with the accelerating voltage of 19 kV. Mass spectra (m/z range 700–4000, resolution 10,000–20,000) were processed using the FlexAnalysis software (version 2.4, Bruker Daltonics). The parameters used were Peak detection algorithm: SNAP (Sort Neaten Assign and Place), S/N threshold: 3.0, quality factor threshold: 50, and internal calibration: trypsin autodigestion peptides (trypsin [108–115], MH+ 842.509; trypsin [58–77], MH+ 2211.104). The masses detected frequently that arose from the matrix, trypsin, or known contaminants (e.g., keratins) were removed. The data were searched against NCBInr database by an in-house MASCOT (version 2.1, Matrix Science) search engine. For PMF, search conditions included: mass accuracy set as 100 ppm, one missed cleavage allowed, Homo sapiens (human) taxon, alkylation of cysteine by carbamidomethylation as a fixed modification, and oxidation of methionine as a variable modification. Proteins with a confidence of >95% and the number of peptides to match ≥4 were considered to be identified.

Sample preparation For the treated samples, complete DMEM was removed and the cells were washed twice with PBS. We diluted our stock of 5 mg/ml TET with media until a final concentration of 5 ␮g/ml TET was achieved. HepG2 cells were treated with 5 ␮g/ml TET and harvested by trypsinization after a 48-h incubation. HepG2 cells were lysed in lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 65 mM DTT, 2% (v/v) IPG buffer, pH 3–10, 1% (v/v) protease inhibitor cocktail), mixed well by vortexing, and ultrasonicated in an ice bath using a Soniprep instrument with 200 W 5 × 10 s pulses and short pauses in between, and then kept on ice for 1 h. The resulting lysate was centrifuged at 40,000 × g at 4 ◦ C for 1 h to remove cell debris. The supernatant was kept and its protein concentration estimated by the Bradford protein assay (Bradford 1976). 2DE and image analysis IPG strips (24 cm, pH 3–10, NL) were rehydrated with 80 ␮g protein in rehydration buffer containing 8 M urea, 2% CHAPS, 15 mM DTT, 0.5% (v/v) IPG buffer (pH 3–10, NL), and bromophenol blue, and focused with a maximum 8000 V to reach 55,000 Vh in the IPGphor isoelectric focus system (Amersham Pharmacia, San Francisco, CA, USA). After completion of focusing, the strips were equilibrated by two steps, first in a buffer containing 6 M urea, 50 mM Tris–Cl, pH 8.8, 30% (w/v) glycerol, 2% (w/v) SDS and 1% (w/v) DTT, and a second step replacing 1% DTT (w/v) with 2.5% (w/v) iodoacetamide. Each step lasted 15 min. The strips were loaded onto 12% SDS polyacrylamide gels and run in a buffer containing 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS. The system was run at 5 W per gel for the first 30 min followed by 12 W per gel for 6–7 h until the bromophenol blue line reached the bottom of the gels in the Ettan-Dalt II system (Amersham Pharmacia, San Francisco, CA, USA). Molecular weight markers were also separated to allow molecular mass determination of the resolved proteins. For analytic gels, the proteins were silver stained according to the published procedure (Shevchenko et al. 1996) except that glutaraldehyde was omitted in the sensitizing solution. The stained gels were scanned using an Umax image scanner (Umax, Taiwan, China). ImageMasterTM 2D platinum software (Version 5.0, GE Healthcare, San Francisco, CA) to detect, quantify, and match spots as well as to perform comparative and statistical analyses. The protein expression levels were determined using the relative volume of each spot in the gel and were expressed as %volume (%Vol = [spot volume/ volumes of all spots resolved in the gel]). Means and standard deviations were calculated from the data of three experiments, and analyzed by Student’s t-tests using ImageMasterTM 2D platinum software. Statistically significant differences were inferred at P values less than 0.05.

Western blot Samples containing 80 ␮g of protein from the control and TET-treated HepG2 cells were electrophoresed on a 12% SDS polyacrylamide gel and transferred to a nitrocellulose filter (Amersham Life Sciences). The filters were blocked in TBST containing 5% nonfat milk powder overnight and then incubated for 2 h with a 1:50diluted anti-human PA28gamma (Mid) rabbit polyclonal antibody (Zymed Laboratories, USA) in TBST containing 5% nonfat milk powder. They were washed with TBST three times for 10 min each. The filters were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Jingmei Co., China). Specific proteins were detected using a DAB kit (Pierce Co., USA). At the same time, a 1:2000-diluted anti-human actin rabbit polyclonal antibody (Abcam Biotechnology, USA) was used to verify the amount and integrity of the proteins. The blots were quantified by densitometry and densitometry analysis performed with Bandscan 5.0 software.

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Fig. 1. Dose-dependent inhibitory effect of TET on cell growth of HepG2 cells for 48 h measured by MTT assay.

Statistics Statistical analysis was performed using two-tailed Student’s ttest, and P < 0.05 was considered significant. Data were expressed as the mean ± SD of triplicate samples, and the reproducibility was confirmed in three separate experiments. Results Cytotoxic effects of TET in HepG2 cells To examine the cytotoxic effects of TET in HepG2 cells, cells were treated with increasing concentrations of TET (1, 3, 5, 10, 20 ␮g/ml) for 48 h and cell proliferation was evaluated by MTT assay. The results showed that the inhibition ratio increased significantly by TET in a dose-dependent manner (Fig. 1). The growth inhibition curve indicated the IC50 of TET for 48 h is 5 ± 0.6 ␮g/ml. The morphological changes of the HepG2 cells were assessed using light microscopy. HepG2 cells treated by 5 ␮g/ml TET for 48 h showed a distinct decreased cell population compared with the controls. Proteomic analysis to identify changes in protein expression stimulated by TET treatment in HepG2 cells To analyze the underlying mechanisms of TET’s anti-tumor effects, proteomic analysis was utilized to identify target-specific proteins differentially modulated by TET treatment. Three gels per sample were processed simultaneously and analyzed using

ImageMaster 2D Platinum Software. A total of 39 protein spots experienced differential expression before and after TET treatment (Fig. 2). Among them, 26 spots were upregulated and 13 spots downregulated. To further identify these targets, 10 differentially expressed spots were chosen according to their t-tests values and density difference >1.5-fold between the control and TET-treated group. These spots were then excised from one single gel, or from two to four gels and analyzed for protein identification by MALDITOF-MS. Seven peptide mass fingerprints (PMF) were successfully obtained. A selected PMF of protein spot 3217 is displayed in Fig. 3. All PMFs were searched with Mascot software in SWISS-PROT or NCBInr database to identify the protein spots. The result had high confidence if the protein was ranked as the best hit with a significant score and high sequence coverage. Finally, we identified 6 proteins in these spots (Fig. 2 and Table 1). Fig. 4 shows 6 differentially expressed proteins. To confirm the expression data derived from proteomic analysis after treatment of TET, Western blots were conducted for PA28gamma (Fig. 5). Results from the Western Blot showed the same trend as from proteomic analysis. Discussion TET has long been used for the treatment of arrhythmia, hypertension, inflammation, silicosis, and occlusive cardiovascular disorders (Banks et al. 1993). Many recent studies have reported the beneficial effects of TET on tumor cell cytotoxicity and radiosensitization, multidrug resistance, promotion of apoptosis, normal tissue radioprotection, and angiogenesis (Chen 2002). Earlier studies suggested that TET-induced anti-tumor actions displayed the following common features: (i) TET arrests cell cycle progression, mostly in the G1 phase. (ii) The antiproliferative action of TET is associated with increased p53 and/or p21 expression and decreased cyclin D1 expression. (iii) TET also increases several apoptotic signals including APO-1 (CD95, Fas) and caspase-3 activity, and causes the release of cytochrome c together with downregulation of Bcl-x (Kuo and Lin 2003; Meng et al. 2004). What is more, the pharmacokinetics and bioavailability of TET have been mentioned in a few researches (Huang et al. 1998; Ma et al. 2009; Song et al. 2008; Yang et al. 2007). Good linearity between peak heights and concentrations of TET was obtained in the certain concentration range (Huang et al. 1998; Song et al. 2008; Yang et al. 2007). The plasma peak concentration of TET was about 2 ␮mol/l and not less than 1 ␮mol/l until 18 h following TET administration (i.p.) at 30 mg/kg in mice (Dai et al. 2007). In vitro the release of TET from sustained release dosage forms went on a time of 12 h which fit-

Fig. 2. 2DE maps of HepG2 control and TET-treated HepG2 cells, silver stained. Protein spots marked on the maps are considered differentially expressed and identified by MS. These results are representative of at least three independent experiments.

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Fig. 3. Peptide mass fingerprint of protein spot 3217 (PA28gamma) obtained from MALDI-TOF-MS.

ted non-Fickian diffusion with matrix erosion significantly. In vivo the plasma concentration of TET extended preparation given in dogs reached Cmax 2.67 ± 0.69 ␮g/ml approximately at 5.67 ± 0.58 h after oral administration. The AUC0 → 24 and AUC0 → infinity were 24.64 ± 6.77 mg h/l and 29.75 ± 5.30 mg h/l, respectively. The elimination half-time was 9.6 ± 2.40 h (Ma et al. 2009). The TET concentrations in mouse lungs of TET microcapsules were significantly higher than those of TET injection, and the mean retained time of TET in lungs was prolonged from 157.1 h to 223.6 h after microencapsulation (Li et al. 2001). In the present study, we found that TET possessed an obvious cytotoxicity against HepG2 cells and the IC50 of TET for 48 h in HepG2 cells was 5 ± 0.6 ␮g/ml, which was very similar to the result of another research (Yoo et al. 2002). Proteomic changes accompanying the progression of 48-h TET treatment in HepG2 cells are of interest, as these may be related to the anti-tumor mechanisms of TET. Interestingly, we found some proteins related to tumor growth, migration, and anti-tumor drug resistance, such as PA28gamma, RPS12, transaldolase, PGAM1 and destrin, which varied greatly after TET treatment. Proteasome activator complex subunit 3 (PA28gamma) Proteasomes are responsible for the degradation of cellular proteins in eukaryotic cells, and they play an important role in cell cycle progression, apoptosis, angiogenesis and drug resistance. Since these pathways are fundamental for cell survival and proliferation, particularly in cancer cells, the inhibition of proteasomes is an attractive potential anti-cancer therapy (Milacic et al. 2006; Montagut et al. 2006). Many studies showed that proteasome inhibitors can inhibit growth, induce apoptosis, reverse drug resistance, and synergize with chemotherapeutic agents (Fujita et al. 2005; Leonard et al. 2006). PA28gamma is a member of the PA28

family, which has been shown to bind specifically to 20S proteasomes and greatly stimulate the hydrolysis of peptides (Rechsteiner and Hill 2005; Wilk et al. 2000). PA28gamma is an anti-apoptotic factor as it is an endogenous substrate for MEKK3, caspase-3 and caspase-7 (Barton et al. 2004; Hagemann et al. 2003). Thus, downregulation of PA28gamma may inhibit proteasome functions and induce apoptosis. Previous studies had demonstrated that TET could possess all the functions of proteasome inhibitors, such as inhibiting growth, inducing apoptosis, reversing drug resistance and synergizing effects with chemotherapeutic drugs. Our findings that TET can downregulate the expression of PA28gamma (Fig. 4) indicate that TET may indirectly inhibit proteasome functions and induce apoptosis through downregulating PA28gamma. Therefore, we propose that TET might be a novel proteasome inhibitor. Ongoing studies to further elucidate its anti-cancer mechanisms are under way in our laboratories. 40S ribosomal protein S12 (RPS12) RPS12, which is a highly conserved protein located at the functional center of the 30S subunit of the ribosome, is involved in the cellular processes of protein synthesis (Coukell and Polglase 1969). Many studies indicated that some ribosomal proteins are overexpressed in tumors (Woo et al. 2004) and appear to be involved in determining the fidelity of protein synthesis and drug resistance. Several ribosomal proteins, such as RPL36, RPL6, RPS13 and RPL23, are associated with drug resistance (Shen et al. 2006). These ribosomal proteins might promote MDR by suppressing drug-induced apoptosis or regulating the glutathione S-transferase-mediated drug-detoxifying system (Shi et al. 2004). RPS12 was demonstrated to be consistently over-expressed in human squamous cell cervical carcinoma, breast cancer and gastric cancer (Deng et al. 2006).

Table 1 MS identification of the differentially expressed protein spot in TET-treated HepG2 cells. Spot no.

Protein description

Accession no.

Theoretical mol. mass (kDa)/pI

Experimental mol. mass (kDa)/pI

Percentage coverage

3217

Proteasome activator complex subunit 3 (PA28gamma) (PSME3) 40S ribosomal protein S12 Phosphoglycerate mutase 1 (PGAM-B) Transaldolase (EC 2.2.1.2) Destrin (actin-depolymerizing factor) Guanylate kinase 1 (fragment)

P61289

29.6/5.69

31.0/5.95

46%

Q76M58 P18669 P37837 P60981 Q5T432

14.9/6.8 28.8/6.8 37.7/6.4 18.8/8.1 30.5/6.2

10.0/7.1 28.0/6.8 38.0/6.55 16.0/7.1 33.0/8.2

55% 69% 32% 44% 20%

4329 3385 2775 4111 3023

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Fig. 5. Western blot was performed to confirm proteomic results. As a result of Western blot, PA28gamma decreased. Comparisons of PA28gamma obtained from HepG2 (control) and TET-treated HepG2 cells. ␤-Actin was used to normalize protein loading. P < 0.01.

specific sensitivity to apoptotic signals (Banki et al. 1996). Inhibiting TAL expression will induce cell apoptosis. TET can downregulate the expression of TAL, so we suppose that TAL may play a role in TET-induced apoptosis. Phosphoglycerate mutase 1 (PGAM1)

Fig. 4. Differentially expressed protein of spot 3217, 4329, 3385, 2775, 4111 and 3023.

Genetic and biochemical analyses of ribosomes from streptomycinresistant mutants implicated RPS12 as the determinant of the various streptomycin phenotypes, including resistance, dependence, and pseudodependence (Maisnier-Patin et al. 2007). TET could reverse drug resistance through regulating MDR (Dai et al. 2007). Current results reported here imply that TET might reverse drug resistance through downregulating RPS12. Thus, RPS12 may be a novel target of TET in reversing drug resistance. Additional work is needed to prove this possible link.

PGAM1 is a glycolytic enzyme with postulated roles in cancer cell metabolism and proliferation (Kondoh et al. 2005). A peptide inhibitor of PGAM1 phosphorylation and activity promotes growth arrest in tumor cell lines (Engel et al. 2004), and over-expression of PGAM1 leads to the immortalization and indefinite proliferation of mouse embryonic fibroblasts (Kondoh et al. 2005). PGAM1 can serve as a potential anti-tumor therapeutic target (Evans et al. 2005). TET can downregulate PGAM1 and TET may be a inhibitor of PGAM1, as well. There is little literature about destrin and guanylate kinase 1 in tumors. In summary, our findings suggest that TET could inhibit HepG2 cells proliferation and might exert its effect on liver cancer at least partially through regulating these proteins’ expression directly or indirectly. Further study of these proteins will be helpful in the further elucidation of TET’s anti-tumor mechanism and the identification of new targets of drugs for chemotherapy. However, our present study still has some limitations (such as only one time point, one kind of cell lines, etc.). Preclinical and clinical studies will provide additional insights and assist in TET clinical use. Acknowledgments This work is supported in part by National Nature Science Foundation of China (30471701, 30670958) and Medical Technology Development Foundation of Nanjing (ZKX05015). The authors thank International Science Editing for the language editing of this manuscript.

Transaldolase (TAL)

References

TAL is a key enzyme of the reversible nonoxidative branch of the pentose phosphate pathway (PPP) that is responsible for the generation of NADPH to maintain glutathione at a reduced state (GSH). Thus, TAL is a critical determinant of tissue- and cell type-

Banki, K., Hutter, E., Colombo, E., Gonchoroff, N.J., Perl, A., 1996. Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J. Biol. Chem. 271, 32994–33001. Banks, D.E., Cheng, Y.H., Weber, S.L., Ma, J.K., 1993. Strategies for the treatment of pneumoconiosis. Occup. Med. 8, 205–232.

Z. Cheng et al. / Phytomedicine 17 (2010) 1000–1005 Barton, L.F., Runnels, H.A., Schell, T.D., Cho, Y., Gibbons, R., Tevethia, S.S., Deepe Jr., G.S., Monaco, J.J., 2004. Immune defects in 28-kDa proteasome activator gammadeficient mice. J. Immunol. 172, 3948–3954. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bruix, J., Sala, M., Llovet, J.M., 2004. Chemoembolization for hepatocellular carcinoma. Gastroenterology 127, S179–188. Chen, B.A., Du, J., Zhang, C.X., Cheng, J., Gao, F., Lu, Z.H., 2005. Using protein chips to study mechanism underlying reversion of drug resistance in leukemia cells in tetrandrime alone or in combination with droloxifen. Zhongguo shi yan xue ye xue za zhi 13, 999–1003. Chen, Y.J., 2002. Potential role of tetrandrine in cancer therapy. Acta Pharmacol. Sin. 23, 1102–1106. Coukell, M.B., Polglase, W.J., 1969. Relaxation of catabolite repression in streptomycin-dependent Escherichia coli. Biochem. J. 111, 279–286. Dai, C.L., Xiong, H.Y., Tang, L.F., Zhang, X., Liang, Y.J., Zeng, M.S., Chen, L.M., Wang, X.H., Fu, L.W., 2007. Tetrandrine achieved plasma concentrations capable of reversing MDR in vitro and had no apparent effect on doxorubicin pharmacokinetics in mice. Cancer Chemother. Pharmacol. 60, 741–750. Deng, S.S., Xing, T.Y., Zhou, H.Y., Xiong, R.H., Lu, Y.G., Wen, B., Liu, S.Q., Yang, H.J., 2006. Comparative proteome analysis of breast cancer and adjacent normal breast tissues in human. Genomics Proteomics Bioinformatics 4, 165–172. Engel, M., Mazurek, S., Eigenbrodt, E., Welter, C., 2004. Phosphoglycerate mutasederived polypeptide inhibits glycolytic flux and induces cell growth arrest in tumor cell lines. J. Biol. Chem. 279, 35803–35812. Evans, M.J., Saghatelian, A., Sorensen, E.J., Cravatt, B.F., 2005. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat. Biotechnol. 23, 1303–1307. Fujita, T., Washio, K., Takabatake, D., Takahashi, H., Yoshitomi, S., Tsukuda, K., Ishibe, Y., Ogasawara, Y., Doihara, H., Shimizu, N., 2005. Proteasome inhibitors can alter the signaling pathways and attenuate the P-glycoprotein-mediated multidrug resistance. Int. J. Cancer 117, 670–682. Hagemann, C., Patel, R., Blank, J.L., 2003. MEKK3 interacts with the PA28 gamma regulatory subunit of the proteasome. Biochem. J. 373, 71–79. Huang, J., Guo, J., Duan, G., 1998. Determination of 7 bio-active alkaloids in stephania plants by rp-hplc. Yao Xue Xue Bao 33, 528–533. Kondoh, H., Lleonart, M.E., Gil, J., Wang, J., Degan, P., Peters, G., Martinez, D., Carnero, A., Beach, D., 2005. Glycolytic enzymes can modulate cellular life span. Cancer Res. 65, 177–185. Kuo, P.L., Lin, C.C., 2003. Tetrandrine-induced cell cycle arrest and apoptosis in Hep G2 cells. Life Sci. 73, 243–252. Leonard, J.P., Furman, R.R., Coleman, M., 2006. Proteasome inhibition with bortezomib: a new therapeutic strategy for non-Hodgkin’s lymphoma. Int. J. Cancer 119, 971–979. Li, F.Q., Lu, B., Chen, W.B., Yang, H., 2001. Tetrandrine loaded sustained-release microcapsules for lung targeting. Yao Xue Xue Bao 36, 220–223. Ma, Y., Li, W.Z., Guan, S.X., Lai, X.P., Chen, D.W., 2009. Evaluation of tetrandrine sustained release calcium alginate gel beads in vitro and in vivo. Yakugaku Zasshi 129, 851–854.

1005

Maisnier-Patin, S., Paulander, W., Pennhag, A., Andersson, D.I., 2007. Compensatory evolution reveals functional interactions between ribosomal proteins S12, L14 and L19. J. Mol. Biol. 366, 207–215. Meng, L.H., Zhang, H., Hayward, L., Takemura, H., Shao, R.G., Pommier, Y., 2004. Tetrandrine induces early G1 arrest in human colon carcinoma cells by down-regulating the activity and inducing the degradation of G1-S-specific cyclin-dependent kinases and by inducing p53 and p21Cip1. Cancer Res. 64, 9086–9092. Milacic, V., Chen, D., Ronconi, L., Landis-Piwowar, K.R., Fregona, D., Dou, Q.P., 2006. A novel anticancer gold(III) dithiocarbamate compound inhibits the activity of a purified 20S proteasome and 26S proteasome in human breast cancer cell cultures and xenografts. Cancer Res. 66, 10478–10486. Montagut, C., Rovira, A., Albanell, J., 2006. The proteasome: a novel target for anticancer therapy. Clin. Transl. Oncol. 8, 313–317. Ng, L.T., Chiang, L.C., Lin, Y.T., Lin, C.C., 2006. Antiproliferative and apoptotic effects of tetrandrine on different human hepatoma cell lines. Am. J. Chin. Med. 34, 125–135. Rechsteiner, M., Hill, C.P., 2005. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol. 15, 27–33. Shen, D.W., Liang, X.J., Suzuki, T., Gottesman, M.M., 2006. Identification by functional cloning from a retroviral cDNA library of cDNAs for ribosomal protein L36 and the 10-kDa heat shock protein that confer cisplatin resistance. Mol. Pharmacol. 69, 1383–1388. Shevchenko, A., Wilm, M., Vorm, O., Mann, M., 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Biochem. 68, 850–858. Shi, Y., Zhai, H., Wang, X., Han, Z., Liu, C., Lan, M., Du, J., Guo, C., Zhang, Y., Wu, K., Fan, D., 2004. Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis. Exp. Cell Res. 296, 337–346. Song, N., Zhang, S., Li, Q., Liu, C., 2008. Establishment of a liquid chromatographic/mass spectrometry method for quantification of tetrandrine in rat plasma and its application to pharmacokinetic study. J. Pharm. Biomed. Anal. 48, 974–979. Thomas, M.B., Zhu, A.X., 2005. Hepatocellular carcinoma: the need for progress. J. Clin. Oncol. 23, 2892–2899. Wei, J., Liu, B., Wang, L., Qian, X., Ding, Y., Yu, L., 2007. Synergistic interaction between tetrandrine and chemotherapeutic agents and influence of tetrandrine on chemotherapeutic agent-associated genes in human gastric cancer cell lines. Cancer Chemother. Pharmacol. 60, 703–711. Wilk, S., Chen, W.E., Magnusson, R.P., 2000. Properties of the nuclear proteasome activator PA28gamma (REGgamma). Arch. Biochem. Biophys. 383, 265–271. Woo, M.S., Ohta, Y., Rabinovitz, I., Stossel, T.P., Blenis, J., 2004. Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site. Mol. Cell Biol. 24, 3025–3035. Yang, C., Guo, L., Liu, X., Zhang, H., Liu, M., 2007. Determination of tetrandrine and fangchinoline in plasma samples using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography. J. Chromatogr. A 1164, 56–64. Yoo, S.M., Oh, S.H., Lee, S.J., Lee, B.W., Ko, W.G., Moon, C.K., Lee, B.H., 2002. Inhibition of proliferation and induction of apoptosis by tetrandrine in hepg2 cells. J. Ethnopharmacol. 81, 225–229.