Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells

Chemico-Biological Interactions 256 (2016) 209e219

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Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells Zhan-Jun Ma, Xue-Xi Wang*, Gang Su, Jing-Jing Yang, Ya-Juan Zhu, You-Wei Wu, Jing Li, Li Lu, Long Zeng, Hai-Xia Pei School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu 730000, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 April 2016 Received in revised form 2 July 2016 Accepted 10 July 2016 Available online 12 July 2016

Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lariciresinol HepG2 cells Apoptosis Proteomics Bioinformatics

1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide and the third leading cause of cancer-related death, with an estimated incidence of 500,000 to 1 million new cases annually [1]. Systemic chemotherapy, administered mainly via oral, muscular, or venous routes, is one of the most common palliative methods. One of the main mechanisms of chemotherapeutic drug treatment is tumor cell apoptosis induction [2]. However, HCC is resistant to chemotherapy, and most current therapeutic strategies against HCC are unsatisfactory. In patients with advanced liver cancer due to liver dysfunction and

* Corresponding author. School of Basic Medical Sciences, Lanzhou University, School of Medicine, 205 Tianshui Rd South, Lanzhou, Gansu 730000, China. E-mail addresses: [email protected] (Z.-J. Ma), [email protected] (X.-X. Wang), [email protected] (G. Su), [email protected] (J.-J. Yang), [email protected] (Y.-J. Zhu), [email protected] (Y.-W. Wu), [email protected] (J. Li), [email protected] (L. Lu), [email protected] (L. Zeng), [email protected] (H.-X. Pei). http://dx.doi.org/10.1016/j.cbi.2016.07.011 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.

decompensation, the adverse effects of, and resistance to, chemotherapy drugs limit their efficacy [3]. Therefore, it is important to identify more effective and alternative therapeutic strategies benefitting HCC patients. Traditional Chinese medicine (TCM) offers a promising approach for the treatment of liver cancer. TCM has been applied in the diagnosis, treatment, and prevention of illnesses in China and other Asian countries for more than 3000 years. Many clinical studies have shown that TCM plays important roles in mitigating the adverse effects of chemotherapy, preventing tumor recurrence and metastasis, inhibiting tumor growth and tumor angiogenesis, and improving patient longevity, quality of life, and immune functioning [4]. Screening for effective anti-tumor components in TCM extracts has become an important approach to anticancer drug development [5]. Patrinia scabra Bunge (PSBE) is a valerianaceae patrinia species, also known as chicken grass and often confused with Patrinia heterophylla Bunge, commonly known as mutouhui. Previous studies have shown that various extracts, including polysaccharide, lignan, flavonoid, and saponin extracts, exhibit anti-tumor effects in vivo and can be used for the treatment of cervical cancer, leukemia, and colorectal cancer

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[6]. Our previous studies demonstrated that PSBE extract, together with lignans, polysaccharides, and saponins, exhibit anti-tumor effects in vivo [7]. The primary components of lignans include styraxlignolide D, styraxlignolide E, lariciresinol, and pinoresinol [8]. Specifically, lariciresinol (LA) shows significant inhibition of liver cancer cells in vivo. However, the precise mechanisms of action underlying the anticancer effects of LA remain unclear. TCM extracts comprise various chemical compositions and represent complex chemical systems; thus, effects on the body involve multiple organs, roles, and targets [9], but the action mechanisms of TCM are far from clear. Systems biology is regarded as the possible method that can bring breakthroughs in the study of TCM [10], because its advantage is consistent with the holistic philosophy of Chinese medicine. Meeting the urgent need for the systems biology, proteomics is a high-throughput protein screening approach; novel proteomics methods are available to evaluate complex chemical systems that are suitable for investigating anticancer mechanisms, identifying multi-drug targets, and aiding the development of novel drugs for TCM [11]. Proteomics approaches include two-dimensional electrophoresis (2-DE) and mass spectrometry. 2-DE provides a useful tool for rapid profiling of cellular factors differentially responding to a drug treatment [12]. In the study, we investigated LA-induced apoptosis induction of HepG2 cells using MTT assays, fluorescence microscopy, and flow cytometry. We then applied proteomic approaches combining 2-DE and mass spectrometry (MALDI-TOF-MS) to compare and identify differentially expressed proteins between untreated and LA-treated HepG2 cells. Finally, protein-protein interaction analysis revealed a potential signaling network affected by LA in HepG2 cells. These studies will increase our understanding of the anticancer mechanisms of LA and provide a solid basis for further development of LA as a novel anticancer agent.

2. Materials and methods 2.1. Plant extracts and chemicals Patrinia scabra Bunge was collected from Qingyang of Gansu province and identified according to the Chinese Pharmacopoeia (2005 edition) [13]. The plant name was verified using www. Theplantlist.org on 18-12-2015. Dried Patrinia scabra Bunge (30 kg) specimens were crushed and extracted by percolation using methanol (MeOH) at ambient temperature. The MeOH was evaporated, the residues were resuspended in water, and the contents were extracted using ether, chloroform (CHCl3), and watersaturated n-butanol. The extract was fractionated by normal phase silica gel column chromatography and eluted using various proportions of CHCl3eMeOH. This was followed by fractionation by reversed-phase silica gel column chromatography and repeated purification by Sephadex LH-20 column chromatography. The resulting Patrinia scabra Bunge lignan extract was 32.5% pure. This lignin extract was analyzed by thin-layer chromatography (TLC), in which the same or similar fractions were merged, ultimately obtaining LA (Fig. 1). The CAS Registry number for LA is 27003-73-2. LA was dissolved in dimethylsulfoxide (DMSO) and stored at 4
C. All chemicals and media for cell culture were purchased from Gibco BRL (Eggenstein, Germany), the chemicals for 2-DE were from Sigma Chemical Co (Milan, Italy), the 2-DE standard was from Bio-Rad (Munich, Germany), and the chemicals for silver staining were from CWBIO (Beijing, China). Antibodies specific for heat shock protein 27 (HSP27), ubiquitin, and ubiquitin-conjugating enzyme (UBC) were purchased from Proteintech (Wuhan, China).

Fig. 1. The chemical structure of lariciresinol.

2.2. Cell culture and treatments HepG2 cells were purchased from the cell bank of the Chinese Academy of Sciences, Shanghai, and maintained in RPMI-1640 medium containing 10% FBS (Gibco BRL, Grand Island, USA), penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37
C and 5% CO2. A 0.8 g/mL solution of LA was prepared in DMSO and stored in small aliquots at 4
C. Cells were seeded at a density of 5  104 per plate (25 cm2). When the cells reached 70% confluency, the culture medium was replaced with fresh medium containing different concentrations of LA. Control cells were cultured in medium containing an equal volume of DMSO without LA or in blank medium containing no more than 0.1% DMSO. 2.3. MTT analysis HepG2 cells (5  103/well) were seeded and grown in 96-well plates for 24 h and then treated with various concentrations of LA. Control cells were treated with 0.1% DMSO. After 24, 48, and 72 h, respectively, 10 mL 5 mg/mL MTT solution were added to each well, and the plates were incubated at 37
C for 4 h. Following removal of the medium, 150 mL DMSO were added to each well, and the plates were shaken gently for 5 min. Optical absorbance was measured at 570 nm using an ELISA reader (Bio-Rad, Hercules, CA, USA). Results are expressed as the fraction of surviving treated cells normalized to that of the control cells (the control was considered to contain 100% surviving cells). The data were representative of three independent experiments performed in triplicate. The IC50 (concentration inducing 50% inhibition) value was obtained from three independent trials. Statistical significance was assessed by ANOVA. A P value less than 0.05 was considered significant. 2.4. Fluorescence microscopic analysis of apoptosis HepG2 cells were seeded in 6-well culture plates and cultured for 24 h. After LA treatment for 48 h, cells were incubated in a methanol/acetic acid buffer (at a fixed 3:1 ratio) at room temperature for 10 min. The buffer was removed, and the cells were washed three times with ice-cold PBS and then incubated with Hoechst 33258 (5 mg/mL) at room temperature for 20 min in the dark. After washing with ice-cold PBS, morphological changes in the cells, including condensed chromatin in stained nuclei or

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smaller dense bodies fluorescing as bright blue, were observed and photographed under a fluorescence microscope (Nikon, Yokohama, Japan) at 200  magnification. 2.5. Measurement of apoptosis Flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining was performed to assess apoptosis. HepG2 cells were seeded at a density of 5  104 per plate (25 cm2). When the cells reached 70% confluency, the culture medium was replaced with fresh medium containing various concentrations of LA. After incubation for 48 h, the cells were washed twice with PBS and harvested by trypsinization. The harvested cells were rinsed twice with PBS and mixed with equal volumes of annexin V-FITC and PI supplemented with 50 mg/mL RNase and 0.1% (w/v) triton X-100 in sodium citrate (3.8 mM). Cells were analyzed by flow cytometry (BD Bioscience, Bedford, MA, USA). The results were analyzed using BD Bioscience Cell Quest Pro software. Three independent experiments were performed. 2.6. Cell cycle analysis For additional confirmation of apoptosis, flow cytometric analysis of cellular DNA was performed. HepG2 cells were seeded at a density of 5  104 per plate (25 cm2). When the cells reached 70% confluency, the culture medium was replaced with fresh medium containing various concentrations of LA for 48 h. After treatment, the cells were collected, washed twice with PBS, and fixed in 70% ice-cold ethanol at 4
C overnight. The fixed cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the pellets were washed twice with PBS. Before analysis, the cells were washed with PBS again and stained with 50 mg/mL PI for 30 min in the dark at room temperature. Finally, the cell cycle was analyzed by flow cytometry (BD Bioscience, Bedford, MA, USA). Three independent experiments were performed. 2.7. Protein extraction After treatment with 205 mg/mL LA for 48 h, the cultured cells were harvested, washed with PBS, and lysed in 400 mL lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 60 mM DTT, 1% (v/v) IPG buffer pH 3e10, and 1% (v/v) protease Inhibitor cocktail). The lysates were incubated at 4
C for 30 min, after which the insoluble material was pelleted by centrifugation at 12,000 g for 60 min at 4
C. The supernatant was measured for protein concentration using the Bradford protein assay and then stored at 20
C. 2.8. Two-dimensional gel electrophoresis For first-dimensional isoelectric focusing (IEF), IPG strips (BioRad, 17 cm, pH 3e10, NL) were rehydrated with 80 mg protein in rehydration buffer (7 M urea, 4% CHAPS, 50 mM DTT, 0.8% (v/v) IPG buffer, pH 3e10, and bromophenol blue) to obtain a final volume of 300 mL, followed by rehydration at 20
C and 50 V for 12 h. After rehydration, IEF was performed using the PROTEAN IEF Cell system (Bio-Rad, USA) and conducted by stepwise increases in voltage, as follows: the initial voltage was held at 250 V for 30 min, quickly increased from 250 to 1000 V within 60 min in the second step, linearly increased from 1000 to 10,000 V within 5 h in the third step, and then maintained at 500 V for 10 h in the last step. The plate temperature was maintained at 20
C. After IEF separation, the strips were incubated in equilibration buffer I (6 M urea, 0.375 M Tris-HCl, pH 8.8, 20% (w/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS) and 1% (w/v) DTT) for 15 min with gentle

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shaking, followed by incubation in buffer II (2.5% (w/v) iodoacetamide replacing 1% (w/v) DTT) for 15 min with gentle shaking. IPG strips were then rinsed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) running buffer and loaded directly onto 10% linear acrylamide gradient gels. Subsequently, the strips were overlayed with agarose solution (25 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.5% (w/v) agarose, and 0.01% (w/v) bromophenol blue). The second dimensional separation was performed by SDS-PAGE in two steps at 10
C: 50 V/gel for 30 min and 45 mA/gel until the bromophenol blue dye front reached the bottom of the gel. After SDS-PAGE, the gels were stained using silver nitrate (CWBIO, Beijing, China). Three replicates were run for each sample. 2.9. Image analysis The 2D gels were scanned using the GS-800 Calibrated Densitometer (Bio-Rad, USA). PDQuest™ 2-DE Analysis Software (Version 8.0.1, Bio-Rad, USA) was used for spot intensity calibration, spot detection, and background abstraction. Three separate gels were prepared for each group to minimize the influence of experimental variations. The gel spot pattern in each gel was summarized in a single gel after spot matching. Thus, we obtained one final gel per group. Means and standard deviations (SD) were calculated from three independent experiments, and paired Student’s t-tests were used to assess differences in the average protein abundance between gels. Protein spot densities with a more than two-fold significant change (P < 0.05) in a consistently increased or decreased pattern were considered differentially expressed and were selected for further identification. 2.10. Protein identification by mass spectrometry The protein spots of interest on the 2-DE gels were excised for MS fingerprinting. Briefly, isolated protein spots were destained with destaining solution and washed twice with double distilled water. Gel fragments were then dried in a vacuum centrifuge and rehydrated in 200 mL 200 mM NH4HCO3 for 20 min. Subsequently, the gel was washed with acetonitrile, after which the extracts were dried in a vacuum centrifuge. The remaining peptides were incubated with 50 mM NH4HCO3 (37
C for 24 h), and we obtained tryptic peptides from the gel using 10% formic acid. The spectral analysis was performed by Shanghai Sangon Biological Technology Co, Ltd, China, as described previously, using the ABI 5800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, Framingham, CA, USA). The obtained MS data were searched against the NCBI protein sequence database using an in-house MASCOT (version 2.1, Matrix Science, UK) search engine. The UniProt Knowledgebase was also used. 2.11. Interaction network of the identified proteins Functional interactions between proteins are fundamental for cell growth. Protein-protein interactions were analyzed using STRING software, version 9.1 (http://string-db.org), a Web-based bioinformatics search tool for interacting proteins [14]. The output includes a detailed network emphasizing several core proteins. 2.12. Western blot analysis To validate 2-DE and STRING results, following treatment with LA, whole cells were washed twice with ice-cold PBS, lysed in RIPA buffer, and quantified using the BAC protein assay kit (Beyotime, Hangzhou, China). Protein samples were separated on 15% SDS-

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PAGE gels, transferred to polyvinylidene fluoride membranes, and blocked for 1 h in blocking buffer (5% bovine serum albumin and 0.1% Tween 20 in tris-buffered saline (TBST)). After three washes in TBST, the membranes were incubated with the primary antibody overnight at 4
C. After incubating with an HRP-conjugated secondary antibody for 1 h at room temperature, the membranes were visualized using a chemiluminescence HRP substrate and the ChemiDoc XRS imaging system (Bio-Rad, USA). The expression level of GAPDH was also determined and used as an internal control.

of apoptosis (including marked nuclear condensation, improved brightness, cell shrinkage, and nuclear fragmentation) were obvious in HepG2 cells (Fig. 2B). 3.2. LA induces apoptosis of HepG2 cells

Quantitative results were expressed as means ± standard deviation. Two-sample t-tests assuming unequal variances were used for the statistical analyses (SPSS 18.0, IBM Corporation, Armonk, NY, USA). P < 0.05 was considered to indicate statistical significance.

To investigate the underlying mechanism of the antiproliferative activity induced by LA, LA-mediated HepG2 cell apoptosis was evaluated by flow cytometry using Annexin V-FITC/ PI staining. For this purpose, HepG2 cells were treated with various concentrations (0e800 mg/mL) of LA for 48 h. There was a significant increase in the percentage of apoptotic cells after LA treatment (Fig. 3A). Thus, flow cytometric analysis suggested that LA decreased the proliferation of HepG2 cells by inducing apoptosis in a dose-dependent manner (Fig. 3B). These data indicate that the decrease in cell viability in HepG2 cells potentially resulted from apoptosis induced by LA.

3. Results

3.3. LA affects cell cycle progression in HepG2 cells

3.1. LA inhibits proliferation of HepG2 cells

To confirm apoptosis, flow cytometric analysis of cellular DNA content was performed after staining with PI. Cells treated with LA showed an accumulation of cells in S phase, and the proportion of which increased to 52.02% after 48 h of treatment compared with 21.91% in the control (Fig. 4A). The accumulation of cells in S phase was dose-dependent (Fig. 4B). This result suggests that the growth inhibition induced by LA dose-dependently was due to blockade of the cell cycle at S phase, which can suppress tumor growth by preventing proper DNA replication.

2.13. Statistical analysis

To determine whether LA exhibits growth inhibitory activity, we investigated cell viability in the presence of LA using the MTT assay. HepG2 cells were treated with various concentrations from 0 to 800 mg/mL LA for 24, 48, and 72 h. Under these experimental conditions, HepG2 cell proliferation was inhibited after treatment with different concentrations of LA for different times. Furthermore, the inhibition was dose- and time-dependent with respect to control cells (Fig. 2A). The IC50 value of LA against HepG2 cells after 48 h was 205 mg/mL. Therefore, we selected a treatment concentration of 205 mg/mL for subsequent proteomic studies. After treatment with various concentrations of LA for 48 h, cell morphology was observed by Hoechst 33258 staining under a fluorescence microscope. In the control group, the cell nucleus was round and intact with faint staining, indicative of living cells. However, in HepG2 cells treated with LA, the characteristic features

3.4. 2-DE gel comparison To characterize the mechanism by which LA induces apoptosis in HepG2 cells, we analyzed the protein expression changes in treated HepG2 cells compared with control cells by proteomics using 80 mg protein rehydrated in rehydration buffer and separated by 2D-PAGE. Each protein sample was run in triplicate. The

Fig. 2. Effects of LA on the proliferation of HepG2 cells. A The cells were treated with various concentrations from 0 to 800 mg/mL LA for 24, 48, and 72 h, and the effects of LA on cell viability was examined by MTT test. After treatment, the cell viability significantly decreased dose- and time-dependent with respect to control cells (*P < 0.05). B Morphological change of HepG2 cells in response to various concentrations LA treatment for 48 h at magnification of 200  (Scale bar ¼ 50 mm). The arrows indicate several apoptotic cells with typical condensation of chromatin, cell shrinkage and nuclear fragmentation.

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Fig. 3. LA induced apoptosis of HepG2 cells. A Flow cytometric analysis of LA-induced apoptosis in HepG2 cells using Annexin V-FITC/PI staining. Cells in the lower right quadrant represent early apoptotic cells, and those in the upper right quadrant represent late apoptotic cells. B LA caused cell apoptosis in a dose-dependent manner, the apoptosis rate increased more rapid in HepG2 cells than control cells (*P < 0.05). Each figure represents three independent experiments.

Fig. 4. Effects of LA on the cell cycle distribution of HepG2 cells. The cells were treated with various concentrations of LA for 48 h a. LA induced S cell cycle arrest in cells after 48 h treatment. Showed were representative DNA histograms of cells obtained by flow cytometry analysis. b. LA caused a S cell cycle arrest in a dose-dependent manner, the arresting rate increased more rapid in HepG2 cells than control cells (*P < 0.05, **P < 0.01). Each figure represents three independent experiments.

proteomic maps of LA-treated and control cells were compared using the PDQuest program to identify protein spot variations (Fig. 5A, B). After LA treatment, significantly (P < 0.05) differentially expressed protein spots by two-fold or more were selected. Moreover, only well separated proteins with easily detectable spot intensity values were included. The red circles indicate the differentially expressed proteins after LA treatment, with a total of 165 spots. Of the 165 proteins, 8 were identified by MS analysis. The identified spots were marked with arrows and numbers.

3.5. Protein identification Protein identification was performed by MALDI-TOF-MS/MS analysis after in-gel digestion. MASCOT protein identification search software was used for the identification of differentially expressed protein spots. Eight differential proteins were identified from Spots 1 to 8. The identified proteins with their MASCOT score, MS/MS matched sequences, apparent and theoretical molecular weight, pI, coverage, and regulation values are shown in Table 1.

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Fig. 5. Comparison of 2-DE image of control HepG2 cells with that of LA-treated HepG2 cells. 80 mg protein were rehydrated with in rehydration buffer and separated by 2D-PAGE, then the gels were stained with silver nitrate and the synthetic gel images were generated using PDQUEST program. The red circle marked on behalf of differentially expressed proteins. A. The representative 2-DE image of cells treated with 205 mg/ml LA for 48 h. B. The representative 2-DE image of control cells for 48 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 MALDI-TOF-MS/MS identification results of differentially expressed protein spots in LA treated HepG2 cells. Spot no.

Protein name

Accession No.

MW (KD)

pI

Score

Sequence coverage(%)

Matches

Expression change

1 2 3 4 5 6 7 8

Heat shock protein 27 Haptoglobin Tropomodulin-2 Glyceraldehyde-3-phosphate dehydrogenase UDP-glucose 4-epimerase Annexin A1 Tubulin alpha-1A chain Brain acid soluble protein 1

P04792 P00738 Q9NZR1 O14556 Q14376 P04083 Q71U36 P80723

22.768 45.861 39.571 36.201 38.656 38.918 50.788 22.68

5.98 6.13 5.21 8.57 6.26 6.57 4.94 4.64

237 350 119 150 218 382 50 131

29 21 3 13 4 23 3 10

5(4) 10(8) 8(4) 8(6) 7(5) 7(7) 5(4) 9(8)

Decrease Decrease Decrease Increase Increase Increase Decrease Decrease

Among them, three proteins (glyceraldehyde-3-phosphate, UDPglucose 4-epimerase, and annexin A1) were upregulated and five proteins (heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1) were downregulated. The proteins were classified by the following functions: cell proliferation, metabolism, cytoskeletal organization, and movement. Representative MS/MS search results for Spots 1 and 6 are shown in Figs. 6 and 7. 3.6. Protein-protein interaction network analysis for the identified proteins To analyze protein-protein interactions among the proteins identified by MADLI-TOF/MS, we entered the protein information from Table 1 into the STRING database. Fig. 8 shows the proteinprotein interaction networks generated by the database and the web tool STRING, version 9.1 (http://string-db.org). As shown in Fig. 8, UBC is an important node within the entire network; thus, this enzyme may play an important role in the effects of LA. 3.7. Verification of the abnormally expressed proteins by Western blotting To validate the results of 2-DE analysis, Western blotting was used to analyze the expression of proteins in the LA treatment group and normal group. The expression level of HSP27 was determined by Western blot analysis. After treatment with various concentrations of LA for 48 h, HSP27 was downregulated in a dosedependent manner (Fig. 9A). When treated with 205 mg/mL LA for

24, 48, and 72 h, HSP27 was downregulated in a time-dependent manner (Fig. 9B). The Western blot results were consistent with those of 2-DE analysis for HSP27. To confirm the results of STRING analysis, the expression levels of ubiquitin and UBC in HepG2 cells with or without 48 h LA treatment were analyzed by Western blotting. LA treatment increased the levels of ubiquitin and UBC (Fig. 10). Western blotting further showed that ubiquitination plays an important role in apoptosis induction by LA in HepG2 cells. 4. Discussion Cancer is a major public health burden in many countries. Cancer treatments aim to block or reverse the initiation phase of carcinogenesis or to arrest its progression [15]. In this context, as a major component of comprehensive cancer treatment, TCM is considered to have a unique advantage [5]. Here, we performed a proteomic analysis of human HepG2 cells treated with LA to increase our understanding of the anticancer mechanisms of LA. In this study, human HepG2 cells were used as a model, and a 2DE-based proteomics approach was used to annotate the proteins altered by LA treatment in HepG2 cells. After LA treatment, the growth of HepG2 cells was inhibited, and typical morphological changes related to apoptosis were detected. Flow cytometry also revealed an increase in the rate of apoptosis in response to LA treatment. These data suggest that LA significantly inhibited cell growth and promoted apoptosis in HepG2 cells. Our results also showed that LA induced S-phase arrest in HepG2 cells, which may contribute to the growth inhibition. Subsequently, a proteomics

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Fig. 6. Identification of protein spot #1 from Fig. 4. A. Mass spectrum of tryptic peptide from spot #1. B. Output of the database searching by the MASCOT program using MS/MS data resulted in the identification of HSP27. C. Protein sequence of HSP27 is shown. The matched peptides are in bold red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

approach was introduced to investigate protein expression patterns in HepG2 cells in response to LA treatment. A total of eight differentially expressed proteins were identified by MALDI-TOF-MS followed by a database search. Among these proteins, five were downregulated (HSP27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1) and three upregulated (GAPDH, UDP-glucose 4-epimerase, and annexin A1). The Western blotting results for HSP27, ubiquitin, and UBC were consistent with those of 2-DE and STRING analyses. The functional roles of the proteins associated with LA-induced cell growth inhibition and apoptosis in HepG2 cells are briefly discussed below. Many anticancer agents arrest the cell cycle at G0/G1, S and G2/M phase and then induce apoptosis [16, 17] or directly induce apoptosis to kill cancer cell [18]. The effect of LA on cell cycle distribution of HepG2 cells was studied to verify the mechanism by which anticancer effect was achieved. We found that HepG2 cells exposed to LA were arrested at the S phase. The quality control of the cell cycle is regulated by restriction points and checkpoints. The restriction point (R) is defined as a point of no return in G1, following which the cell is committed to enter the cell cycle. Up to now, DNA damage checkpoints and spindle checkpoints have (partly) been elucidated. In response to DNA damage, checkpoints arrest the cell cycle in order to provide time for DNA repair. DNA damaging agents trigger checkpoints that produce arrest in G1 and G2 stages of the cell cycle. Cells can also arrest in S, which amounts to a prolonged S phase with slowed DNA synthesis [19]. Some studies demonstrated suppression of both the initiation and

elongation phases of DNA replication [20,21]. There is also evidence that ataxia-telangiectasia-mutated (ATM)-mediated phosphorylation of nijmegen breakage syndrome 1 (NBS1) is required to induce S phase arrest during the S phase checkpoint [22]. Therefor our results suggested that the growth inhibition induced by LA dosedependently was due to blockade of the cell cycle at S phase, which can suppress tumor growth by preventing proper DNA replication. HSP27 is a small protein expressed in the cytoplasm and is present in normal tissues. The expression of HSP27 was significantly upregulated in many tumor tissues compared with normal tissues and under stress conditions, and this was positively correlated with resistance to chemotherapeutic drugs [23]. HSP27 directly interacts with cytochrome c to inhibit the activation of caspase-3, and it regulates caspase-8 to inhibit the activation of Bid in the Bcl-2 family [24]. It also regulates the death domainassociated protein to inhibit the death receptor apoptotic pathway. In addition, HSP27 promotes phosphorylation of Akt, accelerates proteasomal degradation of NF-kB, and promotes activation of NF-kB to exert its anti-apoptotic effects [25]. In this study, the expression of HSP27 was downregulated in LA-treated HepG2 cells, as confirmed by Western blotting, thereby potentially reducing the anti-stress, anti-apoptotic, and anti-drug effects of HSP27 to inhibit proliferation and induce apoptosis. Annexin A1 is involved in the regulation of cell proliferation, differentiation, and apoptosis. The anti-proliferative effects of annexin A1 occur mainly in combination with various growth

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Fig. 7. Identification of protein spot #6 from Fig. 4. A. Mass spectrum of tryptic peptide from spot #6. B. Output of the database searching by the MASCOT program using MS/MS data resulted in the identification of Annexin A1. C. Protein sequence of Annexin A1 is shown. The matched peptides are in bold red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

factor proteins. Annexin A1, which can affect the formation and activity of protein complexes upstream of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway to activate ERK1/2MAPK signaling cascades, inhibits the expression of cyclin D1 and decreases cell production [26,27]. Annexin A1 also regulates caspase-3 activation and Ca2þ release, plays a role in the activation of p38 and JNK signaling transduction, and promotes cell apoptosis [28]. Exogenous annexin A1 can increase the concentration of intracellular Ca2þ and induce dephosphorylation of the Bcl-2/Bcl-xL-2eassociated death promoter to induce cell apoptosis [29]. Furthermore, annexin A1 regulates the activity of PLA2 and inhibits the production of antiapoptosis prostaglandin factor, thereby promoting apoptosis via hydrogen peroxide [30]. Therefore, Annexin A1 may be a tumor suppressor, and its increased expression in LA-treated cells may contribute to the anticancer activity of LA in liver cancer cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein involved in numerous subcellular processes, plays an integral role in glycolysis. However, further studies are required to establish the primary role of GAPDH in a variety of critical nuclear pathways apart from its already recognized role in apoptosis [31]. GAPDH can polymerize and dissolve microtubules,

has the function of the protein kinase [32]. Overexpression of GAPDH can induce apoptosis, including neuronal apoptosis [33]. Importantly, GAPDH seems to play dual role in the regulation of cell survival: it supports energy production, but when translocated into mitochondria, it can effectively induce proapoptotic mitochondrial membrane permeabilization (MMP) [34]; GAPDH is also a unique target of nitric oxide (NO) and is closely related to apoptotic signaling transduction [35]. Further studies are required to explore the functions of GAPDH in HepG2 cells. These proteins increase our understanding on the anticancer mechanisms of LA and may provide more potent molecular targets. STRING analysis was used to identify the interacting networks involving the proteins identified by MADLI-TOF/MS. In the interaction network, the proteins clustered in a tight interaction network centered on UBC. UBC is an important enzyme that affects the ubiquitin-proteasome pathway (UPP). UPP is the major pathway responsible for elimination of intracellular proteins, especially misfolded cellular proteins in eukaryotes including ubiquitin, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin ligases (E3), and the 26S proteasome [36]. Western blotting showed that LA treatment increased the levels of ubiquitin and UBC. This results further supports that UPP plays an

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Fig. 8. The protein-protein interaction network of the identified differentially expressed proteins. The network containing 8 identified differentially expressed proteins were mapped using the STRING system (http://string-db.org) based on evidence with different types. The signal pathways affected by LA were clustered in the network analysis.

Fig. 9. Western blot analysis to confirm the HSP27 in HepG2 cells LA-treatment. a. The cells were treated with various concentrations of LA for 48 h b. The cells were treated with 205 mg/mL LA for 24, 48, and 72 h. GAPDH was used as internal control.

important role in LA-induced apoptosis in HepG2 cells. This pathway is pivotal in cell cycle control, transcriptional regulation, signal transduction, antigen presentation, inflammation, apoptosis, and development [37e39]. UPP can influence and regulate apoptosis through multiple pathways [40]. First, degradation of the death domain receptor molecule Fas-like inhibitor protein (FLIP) is regulated by the proteasome pathway [41]. FLIP potentially interacts with tumor necrosis factor receptor activation factor-2

containing a RING finger domain with E3 activity, which promotes degradation of ubiquitinated FLIP. Second, UPP indirectly regulates the expression of pro-apoptotic protein Bax [42]. In nonapoptotic cells, the hydrophobic C-terminus of Bax includes the Bcl2 homology 3 (BH3) domain, thereby preventing BH3 from forming a dimer, since the monomer is sensitive to proteasomal degradation. Apoptotic signaling can induce a conformational change in the Bax monomer to prevent its degradation. Bax then translocates to

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Fig. 10. Effects of LA on the expression levels of proteins that are involved in UPP-mediated apoptosis. Dose-dependent effects of LA on the expression levels of ubiqitin and UBC. Protein expression in HepG2 cells treated with 0, 200 and 400 mg/mL LA for 48 h were analyzed by Western blotting. GAPDH was used as internal control.

the mitochondria, where it activates caspase-9 and promotes apoptosis. Third, inhibitor of apoptosis protein is an E3 containing a RING finger domain that binds to caspases directly and leads to degradation [43]. In this study, LA significantly inhibited cell growth and promoted apoptosis in HepG2 cells, and this antitumor mechanism may be associated with UPP, in that LA may eventually inhibit UPP activity and contribute to HepG2 apoptosis induction. In conclusion, LA is an important natural product that selectively induces HepG2 cell cycle arrest and apoptosis. Our study is the first to explore the molecular mechanism of LA by proteomic profiling. We also identified many novel protein targets of LA, never reported previously, and the protein-protein interaction networks involved in these effects. Although these results demonstrated that some of these differentially expressed proteins are potential inhibitors of tumor progression and therapy targets, further studies on the effects of LA on these proteins in cancer cells are required. Overall, these results provide novel insights into the anticancer mechanisms of LA and potential therapeutic strategies for liver cancer.

Conflict of interest The authors declare no conflict of interest.

Author contribution Zhanjun Ma: Experimental studies, Guarantor of integrity of entire study. Manuscript preparation. Xuexi Wang: Drafting the article or revising it critically. Gang Su: Analysis and interpretation of data. Jingjing Yang: Acquisition of data. Yajuan Zhu: Literature research. Youwei Wu: Data acquisition. Jing Li: Statistical analysis. Li Lu: Manuscript preparation. Long Zeng: Study concepts. Haixia Pei: Acquisition of data.

Acknowledgments This work was financially supported by the Lanzhou University National undergraduate innovation and entrepreneurship training program of China: No.201510730146; National Natural Science Foundation of China: 81000878.

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