A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA-1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2-DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA-1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA-1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2-DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100-A6 and S100-A11 suggested that apoptosis was induced by apigenin via the caspase-dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.
A nutraceutical is a food or a part of a food that provides medicinal and health benefits (
The higher incidence of cholangiocarcinoma (CCA), a malignant tumor derived from intrahepatic or extrahepatic biliary tracts, occurs in Southeast Asian countries such as Thailand (
Apoptosis, a process of programmed cell death in multicellular organisms, is one of the main types of cell death pathway and involves a series of biochemical events, which lead to cell morphology and mortality (
The use of apigenin as an anticancer agent
In the present study, MTT assays were performed to study the cytotoxicity of apigenin on a cholangiocarcinoma cell line, and flow cytometric analysis was employed to determine the induction of apoptosis. The proteomic analysis was also used to study the differential protein expression between apigenin-treated and untreated cells.
The HuCCA-1 cell line, derived from a bile duct tumor mass, was provided by Professor Stitaya Sirisinha, Faculty of Science, Mahidol University (Bangkok, Thailand) and grown as a monolayer culture in Ham's F12 culture medium (Gibco Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin, 100 mg/ml streptomycin and 125 ng/ml amphotericin B. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
Cells at 80% confluence were harvested by trypsinization from culture flasks and seeded in 96-well plates at 104 cells per 100 µl per well. After 24 h incubation, the cells were treated with apigenin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at various concentrations (1–250 µM) for 24, 48 and 72 h. Each well was then replaced with fresh medium containing 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) and incubated for 2 h. Finally, the medium was removed and 100 µl dimethyl sulfoxide was added to each well. The absorbance was measured at 550 nm with a microplate reader, subtracted with the absorbance at 650 nm. Data were expressed as % cell growth compared with the untreated cells as the control.
Apoptosis was detected by two different methods, flow cytometric analysis of phosphatidylserine externalization and a DNA fragmentation assay. For the flow cytometric analysis, the HuCCA-1 cells were seeded in 6-well plate at 4×105 cells per 2 ml per well. After 24 h incubation, the cells were treated with apigenin at concentrations of 20% inhibition of cell growth (IC20), 25 µM, IC50, 75 µM and IC90, 200 µM, respectively. After 48 h of compound treatment, floating cells in culture media were separated whilst adherent cells were harvested by trypsinization, then the two cell populations were pooled together and centrifuged at 778 × g for 10 min at 4°C. The supernatant was removed and the cell pellets were resuspended and adjusted to 1×106 cells/ml in culture media containing 1% FBS. Equal volumes of the cell suspension and reagent of Muse™ Annexin-V & Dead Cell kit (Merck KGaA) were mixed together in a tube and incubated at room temperature for 20 min, and analysis was performed using Muse™ Cell Analyzer (Merck KGaA) (
Tissue culture flasks measuring 75 cm2 were used for seeding HuCCA-1 cells and the cells were cultured at 37°C, 5% CO2 for 24 h. Apigenin was then added to the cells at final concentration of 200 µM, 90% inhibition, IC90, for 48 h. Since apigenin treatment causes cell detachment, the floating cells were collected from the medium by centrifugation at 778 × g for 10 min at 4°C and washed with 0.25 M sucrose-containing protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The adherent cells were harvested by trypsinization and washed twice with 0.25 M sucrose, scraped in the same sucrose solution and centrifuged at 778 × g for 10 min at 4°C. The two samples were resuspended in 100 µl lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% dithiothreitol (DTT), 2% ampholine pH 3–10 and a protease inhibitor cocktail, sonicated on ice and centrifuged at 13,800 × g for 10 min at 4°C. The supernatants were saved and the concentration of proteins was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
The samples were prepared by leaving them overnight in gel rehydration of nonlinear pH 3–10, 70-mm Immobiline DryStrip gels (IPG; GE Healthcare, Chalfont, UK). An Ettan IPGphor system (GE Healthcare) was used for running the first dimension isoelectric focusing at 6,500 Vh. The IPG strips were equilibrated in two steps of equilibration buffer as previously described (
The gels were scanned using ImageScanner II (GE Healthcare) and analyzed using ImageMaster 2D platinum software (version 7.0; GE Healthcare) for differential analysis.
The triplicate washing step was performed by adding 50 µl 0.1 M NH4HCO3 in 50% acetonitrile (ACN) in excised gel spots and incubating for 20 min at 30°C. The gel pieces were dried completely in SpeedVac (Labconco, Kansas City, MO, USA). The gel pieces were reduced and alkylated in 1X buffer solution, 0.1 M NH4HCO3, 10 mM DTT and 1 mM EDTA, and incubated at 60°C for 45 min. The buffer solution was replaced with freshly prepared 100 mM iodoacetamide in 0.1 M NH4HCO3 solution. The reaction mixture was incubated in the dark at room temperature for 30 min. The gel pieces were washed three times using 50% ACN in water and were dried completely. The trypsin (Promega Corporation, Madison, WI, USA) was aliquoted (1 µg trypsin/10 µl 1% acetic acid) and stored at −20°C. The digestion buffer, 0.05 M Tris-HCl, 10% ACN, 1 mM CaCl2, pH 8.5, was prepared. The tryptic digestion was performed by adding 50 µl digestion buffer and 1 µl prepared trypsin into the gel pieces. The reaction mixture was incubated at 37°C overnight. The digestion buffer was removed and saved. The gel pieces were then added to 60 µl 2% freshly prepared trifluoroacetic acid and incubated at 60°C for 30 min for peptide extraction. The saved digestion buffer and the final extract were then pooled and dried by SpeedVac.
The Q-TOF mass spectrometer (Micromass UK, Ltd., Manchester, UK) equipped with a Z-spray ion source operating in the nanoelectrospray mode was used. The analysis by LC was carried out using a capillary LC system (Waters Corporation, Milford, MA, USA). The instrument in MS/MS mode was calibrated by Glu-fibrinopeptide. The 75 mm id ×150 mm C18 PepMap column (LC Packings, Amsterdam, The Netherlands) was attached to the LC system. Eluents A and B were prepared as follows: Eluent A, 0.1% formic acid in 97% water and 3% ACN and eluent B, 0.1% formic acid in 97% ACN and 3% water. The gradient for peptide separation was 0 min 7% B, 35 min 50% B, 45 min 80% B, 49 min 80% B, 50 min 7% B and 60 min 7% B. ProteinLynx Global SERVER™ (version 2.2; Waters Corporation) screening Swiss-Prot and NCBI (
The untreated and apigenin treated HuCCA-1 cells were scraped separately and sonicated in an in-house 1X radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0 and 1 mM EDTA) to extract the proteins. Protein lysates (20 µg) were subsequently loaded in each lane. Proteins separation was performed by 12.5% SDS-PAGE and transferred onto FluoroTrans® polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). Subsequent to blocking with 5% nonfat dried milk in TBS-Tween 20 (TBST), 10 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20, at room temperature for 1 h, the membranes were washed with TBST and incubated with the following primary antibodies: Mouse monoclonal cytokeratin 7 (CK7; cat. no. MAB3554; dilution, 1:2,000; Merck KGaA); mouse monoclonal cytokeratin 8 (CK8; cat. no. MAB3414; dilution, 1:2,000; Merck KGaA); mouse monoclonal cytokeratin 18 (CK18; cat. no. MAB3236; dilution, 1:2,000; Merck KGaA); mouse monoclonal cytokeratin 19 (CK19; cat. no. MAB3238; dilution, 1:2,000; Merck KGaA); mouse monoclonal S100-A6 (cat. no. ab55680; dilution, 1:250; Abcam, Cambridge, UK); rabbit polyclonal S100-A11 (cat. no. 10237–1-AP; dilution, 1:250; Proteintech, Chicago, IL, USA); rabbit monoclonal S100-P (cat. no. ab133554; dilution, 1:1,000; Abcam); mouse monoclonal heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1; cat. no. ab6102; dilution, 1:2,000; Abcam); rabbit polyclonal heterogeneous nuclear ribonucleoprotein H (hnRNP H; cat. no. ab10374; dilution, 1:5,000; Abcam); mouse monoclonal annexin A1 (cat. no. MAB3773; dilution, 1:2,000; Merck KGaA); mouse monoclonal annexin A2 (cat. no. ab54771; dilution, 1:20,000; Abcam); rabbit polyclonal annexin A3 (cat. no. ab33068; dilution, 1:2,000; Abcam); mouse monoclonal peroxiredoxin-1 (cat. no. ab58252; dilution, 1:2,000; Abcam); mouse monoclonal prostaglandin E synthase 3 (PTGES3; cat. no. WH0010728M1; dilution, 1:500; Sigma-Aldrich; Merck KGaA); or rabbit monoclonal GAPDH (cat. no. ab75834; dilution, 1:10,000; Abcam) at 4°C overnight. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Dako; Agilent technologies, Inc., Santa Clara, CA, USA) at room temperature for 1 h. Following washing with TBST, membranes were visualized by using an enhanced chemiluminescence Western blotting detection kit (Advansta, Menlo Park, CA, USA) and an ImageQuant LAS 4000 mini (GE Healthcare). A total of three experiments were performed for each antibody.
The mean values and standard deviations of differential expression between treated and untreated cells were calculated. The significance of differences was analyzed by two-tailed unpaired Student's t tests, and P<0.05 was considered to indicate a statistically significant difference.
The MTT assay was performed to investigate the growth inhibition and assess the cytotoxic effect of apigenin. HuCCA-1 cells were treated with various concentrations of apigenin ranging from 1–250 µM at different times. The results in
To examine whether apigenin induced apoptotic cell death in HuCCA-1 cells, two different methods were used to detect apoptosis, flow cytometric analysis of phosphatidylserine (PS) externalization and DNA fragmentation assays. When cells undergo apoptosis, an early event is PS externalization on the cell surface, which are detected by Annexin-V staining. Subsequent to the progression to late stage of apoptosis, the membrane integrity of the cells is lost, allowing penetration of membrane-impermeant dyes such as 7-aminoactinomycin D (7-AAD) and propidium iodide. Mode of cell death in apigenin-treated HuCCA-1 cells was analyzed. The cells were treated with increasing concentration from IC20, 25 µM, IC50, 75 µM and IC90, 200 µM of apigenin compared with the untreated control for 48 h. Subsequent to the treatment, the cells that were positive for Annexin-V and 7-AAD staining with stages of early and late apoptosis were detected (
Additionally, another key feature of apoptosis, DNA fragmentation with a ladder pattern, was also investigated. The HuCCA-1 cells were treated with IC90, 200 µM, apigenin for 48 h, and subsequently analyzed for DNA fragmentation. As demonstrated in
From the DNA fragmentation of apigenin-treated HuCCA-1 cells, apoptosis was clearly demonstrated in the floating cells. Thus, the levels of protein expression in the untreated and apigenin-treated cells floating and adherent cells were compared using proteomic techniques. Only the floating cells demonstrated differential protein expression when compared with the untreated cells by 2-DE.
The expression levels of twelve proteins were not measured in HuCCA-1 cell lysate subsequent to apigenin treatment. These proteins are involved in metabolism, cytoskeletal/mobility, protein synthesis and degradation, chaperone/stress response, protection and detoxification and transport/binding. These proteins are phosphoenolpyruvate carboxykinase (GTP;
For proteins involved in cytoskeleton/mobility function, significant changes were revealed in expression in CK7, CK8, CK18, CK19, S100-A6 and S100-A11 (
Immunodetection was used to verify the presence of certain proteins from adherent and floating cells subsequent to treatment with apigenin compared with untreated cells, as demonstrated in
Cholangiocarcinoma is a malignant tumor derived from bile duct epithelium, with high incidence in northeast Thailand (
Apigenin is a promising nutraceutical, a food or part of a food that may elicit health benefits. Numerous reports of the effect of apigenin treatment on different human cancer cell lines have demonstrated an inhibition of cell growth via apoptosis and cell cycle arrest (
Apoptosis, or programmed cell death, leads to DNA fragmentation, cytoskeletal reorganization, plasma membrane blebbing, nuclear condensation and loss of cell adhesion. Induction of apoptosis by apigenin in HuCCA-1 cells was demonstrated by flow cytometric analysis and confirmed by DNA fragmentation assays. The cleavage of chromosomal DNA by cellular nucleases into oligonucleosomal size fragments is a characteristic feature of apoptosis. Agarose gel electrophoresis demonstrated that the DNA ladder pattern, indicating DNA fragmentation, was identified only with floating cells and not with attached cells. This confirms that the later stages of apoptosis are exhibited in HuCCA-1 cells treated with apigenin. Typically, apoptosis occurs through the activation of specific caspases including caspase-3 (
In the present study, the treatment of HuCCA-1 cells with apigenin demonstrated various types of protein alterations, namely changes in protein expression and/or cleavage. The proteins with roles in cytoskeleton/mobility revealed cleavage, and therefore this supports the presence of caspase activities during apoptosis.
The results of the present study revealed that subsequent to treatment with apigenin, the expression of S100-A6 and S100-A11 was higher in floating cells compared with attached HuCCA-1 cells during apoptosis. These two proteins are members of the S100 protein family and have been revealed to serve important roles in a number of tumorigenic processes, including apoptosis, cell differentiation, cell growth and cell cycle. The upregulation of S100-A6 has been demonstrated to enhance apoptosis and decrease cell viability by affecting caspase-3 activity in hepatocellular carcinoma cells (
Proteins involved in protein synthesis and degradation demonstrated low or no expression subsequent to apigenin treatment, including 26S protease regulatory subunit 10B, hnRNP H and hnRNP A2/B1. In particular, the 26S protease regulatory subunit 10B is a protein associated with apoptosis, and disappears in the floating cells. The decreased activity of the 26S proteasome and induction of cell death was observed only in breast cancer cells and not in normal cells, treated with
Chaperone/stress response proteins including prostaglandin E synthase 3, hypoxia upregulated protein 1 and 60 kDa heat shock protein, were revealed to be downregulated subsequent to treatment with apigenin. However, stress-induced-phosphoprotein 1 was upregulated. Molecular chaperones are involved in protein folding, transport and assembly (
In conclusion, apigenin, a nutraceutical present in several vegetables and fruits, demonstrated the cytotoxic effect toward HuCCA-1. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) with Annexin-V and dead cell assay kit, and DNA fragmentation confirmed the occurrence of early and late apoptosis. The proteins most significantly altered subsequent to treatment with apigenin were associated with apoptosis. The cleavage of cytokeratin 8, 18 and 19 and the high expression of S100-A6 and S100-A11 indicate that apoptosis was induced by apigenin via a caspase-dependent pathway. A marked reduction in the expression of hnRNP A2/B1 was also observed, possibly with changes of splicing forms, since it has been identified that the binding of apigenin to hnRNP A2/B1 resulted in changes of the splicing forms. The present study aimed to contribute to the understanding of the usefulness of dietary flavonoids such as apigenin.
The present study was supported by the Chulabhorn Research Institute (grant no. BC 2008-02).
Effect of apigenin on cell growth. (A) Treatment of HuCCA-1 cells by various concentration of apigenin (1–250 µM) for 24, 48 and 72 h. The number of surviving cell was calculated by MTT assay. The presented data are as mean ± standard deviation of three different experiments. (B) The cells were treated with apigenin at concentrations of 20, 50 and 90% growth inhibition, IC20, IC50 and IC90, respectively. The cells were observed by inverted phase-contrast microscopy (magnification, ×100).
Apigenin induces apoptosis in HuCCA-1 cells. (A and B) Flow cytometric analysis of apoptotic cells was performed by using Muse™ Cell Analyzer with Muse™ Annexin-V & Dead Cell kit. The treatment of HuCCA-1 cells was performed with 0.2% dimethyl sulfoxide (control) or different concentrations of apigenin (25–200 µM) for 48 h. Dot plots of flow cytometric results were from three independent experiments. Data are presented as mean ± standard deviation of three different experiments. (C) Detection of apigenin induced DNA fragmentation in HuCCA-1 cells. HuCCA-1 cells were treated with 200 µM apigenin, IC90, for 48 h. Genomic DNA samples were isolated from HuCCA-1 cells with or without apigenin, separation was performed on 2% agarose gels, and visualized using ethidium bromide staining. Lane M, DNA marker; lane 1, DNA from untreated cells; lane 2, DNA from the attached cells subsequent to treatment; lane 3, DNA from the floating cells subsequent to treatment; 7-ADD,
Comparison of 2-DE patterns between untreated and treated HuCCA-1 cells with apigenin. (A and B) Illustrate the 2-DE patterns of untreated and treated cells with 200 µM apigenin. NL pH 3–10 Immobiline DryStrip gels were used. Gels were stained with Coomassie Brilliant Blue R-250. Proteins with different expressions are marked by arrows with numbers. Data presented are the representative of three totally different experiments. 2-DE, two-dimensional gel electrophoresis; NL, non-linear.
Western blot analysis of proteins in untreated cells compared with adherent and floating cells subsequent to treatment with apigenin. C, control untreated cells; A, adherent cells; F, floating cells subsequent to treatment with apigenin, respectively. hnRNP A2/B1, heterogeneous nuclear ribonucleoprotein A2/B1; PTGES3, prostaglandin E synthase 3; hnRNP H, heterogeneous nuclear ribonucleoprotein H.
Differentially expressed proteins of HuCCA-1 following apigenin treatment.
Spot no. | Accession no. | Protein names | Theoretical pI/MW | Function | Fold Change |
---|---|---|---|---|---|
1 | Q9Y4L1 | Hypoxia up-regulated protein 1 | 5.21/111.3 | Chaperone/stress response | −2.04±0.17b |
2 | Q16822 | Phosphoenolpyruvate carboxykinase [GTP], mitochondrial | 7.40/70.6 | Metabolism | ND |
3 | P31939 | Bifunctional purine biosynthesis protein PURH | 6.26/64.6 | Metabolism | −16.21±0.35 |
4 | P10809 | 60 kDa heat shock protein, mitochondrial | 5.55/61.0 | Chaperone/stress response | −2.12±0.48b |
5 | P31948 | Stress-induced-phosphoprotein 1 | 6.76/62.6 | Chaperone/stress response | +1.58±0.03 |
6 | P07437 | Tubulin beta chain | 4.59/49.6 | Cytoskeleton/mobility | +2.19±0.39c |
7 | P08729 | Keratin, type II cytoskeletal 7 | 5.46/51.2 | Cytoskeleton/mobility | −2.51±0.13 |
8 | P08729 | Keratin, type II cytoskeletal 7 | 5.46/51.2 | Cytoskeleton/mobility | −5.33±0.41c |
9 | P31943 | Heterogeneous nuclear ribonucleoprotein H | 6.21/49.2 | Protein synthesis and degradation | −26.66±0.51 |
10 | Q16658 | Fascin | 7.28/54.4 | Cytoskeleton/mobility | −1.22±0.02c |
11 | P43490 | Nicotinamide phosphoribosyltransferase | 7.12/55.5 | Metabolism | −2.77±0.14 |
12 | P05787 | Keratin, type II cytoskeletal 8 | 5.52/53.5 | Cytoskeleton/mobility | −6.94±0.42d |
13 | P05787 | Keratin, type II cytoskeletal 8 | 5.52/53.5 | Cytoskeleton/mobility | −2.82±0.18 |
13 | P05783 | Keratin, type I cytoskeletal 18 | 5.42/47.9 | Cytoskeleton/mobility | −2.82±0.18b |
14 | P05787 | Keratin, type II cytoskeletal 8 | 5.52/53.5 | Cytoskeleton/mobility | −21.08±0.90 |
14 | P05783 | Keratin, type I cytoskeletal 18 | 5.42/47.9 | Cytoskeleton/mobility | −21.08±0.90c |
15 | P05787 | Keratin, type II cytoskeletal 8 | 5.58/53.5 | Cytoskeleton/mobility | −3.48±0.17 |
15 | P05783 | Keratin, type I cytoskeletal 18 | 5.42/47.9 | Cytoskeleton/mobility | −3.48±0.17c |
16 | P06733 | Alpha-enolase | 7.54/47.0 | Metabolism | −4.79±0.68 |
17 | P08727 | Keratin, type I cytoskeletal 19 | 5.09/44.1 | Cytoskeleton/mobility | ND |
18 | P08865 | 40S ribosomal protein SA | 4.59/32.8 | Protein synthesis and degradation | −1.81±0.04b |
19 | P07910 | Heterogeneous nuclear ribonucleoproteins C1/C2 | 4.99/33.6 | Protein synthesis and degradation | −2.01±0.12 |
20 | P06748 | Nucleophosmin | 4.71/30.9 | Protein synthesis and degradation | −1.63±0.16b |
21 | P07910 | Heterogeneous nuclear ribonucleoproteins C1/C2 | 4.99/33.6 | Protein synthesis and degradation | −4.64±0.49 |
22 | P05388 | 60S acidic ribosomal protein P0 | 5.60/34.3 | Protein synthesis and degradation | −1.42±0.03b |
23 | Q15365 | Poly(rC)-binding protein 1 | 7.11/37.5 | Protein synthesis and degradation | −5.59±0.14 |
24 | O00154 | Cytosolic acyl coenzyme A thioester hydrolase | 7.27/37.4 | Metabolism | −19.00±0.27d |
25 | P04083 | Annexin A1 | 7.00/38.6 | Signal transduction | −2.05±0.14 |
26 | P07355 | Annexin A2 | 8.04/38.4 | Signal transduction | +3.89±0.48b |
27 | P12429 | Annexin A3 | 5.82/36.4 | Signal transduction | +4.63±0.12 |
28 | Q06323 | Proteasome activator complex subunit 1 | 5.98/28.7 | Cell cycle | −5.84±0.10b |
29 | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 8.79/35.9 | Metabolism | ND |
30 | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 8.79/35.9 | Metabolism | −1.12±0.02 |
31 | P35232 | Prohibitin | 5.57/29.8 | DNA replication/gene regulation | −1.22±0.06b |
32 | P02545 | Lamin A/C | 6.13/53.2 | Cytoskeleton/mobility | D |
33 | P48556 | 26S proteasome non-ATPase regulatory subunit 8 | 7.16/30.0 | Protein synthesis and degradation | −3.17±0.05b |
34 | Q06830 | Peroxiredoxin-1 | 8.52/22.1 | Protection and detoxification | −2.41±0.09 |
35 | P21796 | Voltage-dependent anion-selective channel protein 1 | 8.85/30.6 | Ion channels | −1.35±0.09b |
36 | P43487 | Ran-specific GTPase-activating protein | 5.27/23.3 | Transport/binding proteins | ND |
37 | P28066 | Proteasome subunit alpha type-5 | 4.54/26.4 | Protein synthesis and degradation | +1.82±0.03 |
38 | Q9BV44 | THUMP domain-containing protein 3 | 5.97/57.0 | Protein synthesis and degradation | −2.45±0.03b |
39 | P05783 | Keratin, type I cytoskeletal 18 | 5.42/47.9 | Cytoskeleton/mobility | +9.17±0.15 |
40 | P25789 | Proteasome subunit alpha type-4 | 8.04/29.5 | Protein synthesis and degradation | −2.13±0.07c |
41 | P62333 | 26S protease regulatory subunit 10B | 7.09/44.2 | Protein synthesis and degradation | ND |
42 | Q15056 | Eukaryotic translation initiation factor 4H | 7.33/27.4 | Protein synthesis and degradation | −3.62±0.10 |
43 | Q06830 | Peroxiredoxin-1 | 8.52/22.1 | Protection and detoxification | −6.52±0.41c |
44 | P13693 | Translationally-controlled tumor protein | 4.84/19.6 | Transport/binding proteins | −2.11±0.07 |
45 | O75947 | ATP synthase subunit d, mitochondrial | 5.26/18.3 | Metabolism | −1.31±0.02b |
46 | P32119 | Peroxiredoxin-2 | 5.86/21.9 | Protection and detoxification | −3.20±0.11 |
47 | Q15185 | Prostaglandin E synthase 3 | 4.30/18.7 | Chaperone/stress response | ND |
48 | O00746 | Nucleoside diphosphate kinase, mitochondrial | 9.21/17.3 | Metabolism | +12.27±0.35b |
49 | P05783 | Keratin, type I cytoskeletal 18 | 5.42/47.9 | Cytoskeleton/mobility | D |
50 | P16949 | Stathmin | 5.96/17.2 | Cytoskeleton/mobility | ND |
51 | Q93020 | GTP-binding regulatory | 5.30/22.1 | Signal transduction protein Gi alpha-2 chain | −3.64±0.18 |
52 | P23528 | Cofilin-1 | 8.22/18.5 | Cytoskeleton/mobility | ND |
53 | P08727 | Keratin, type I cytoskeletal 19 | 5.09/44.1 | Cytoskeleton/mobility | D |
54 | P23528 | Cofilin-1 | 8.22/18.5 | Cytoskeleton/mobility | +2.46±0.13b |
55 | P62937 | Peptidyl-prolyl cis-trans isomerase A | 8.18/17.9 | Signal transduction | −4.24±0.66 |
56 | P62937 | Peptidyl-prolyl cis-trans isomerase A | 8.18/17.9 | Signal transduction | −10.17±0.46d |
57 | P22392 | Nucleoside diphosphate kinase B | 8.52/17.3 | Metabolism | −1.83±0.13 |
58 | P25398 | 40S ribosomal protein S12 | 6.30/14.5 | Protein synthesis and degradation | −2.35±0.10b |
59 | Q9UII2 | ATPase inhibitor, mitochondrial | 9.34/12.2 | Metabolism | ND |
60 | P06703 | Protein S100-A6 | 5.32/10.2 | Cytoskeleton/mobility | D |
61 | P31949 | Protein S100-A11 | 7.27/11.7 | Cytoskeleton/mobility | +4.13±0.12 |
62 | P04075 | Fructose-bisphosphate aldolase A | 8.06/39.4 | Metabolism | ND |
63 | Q04828 | Aldo-keto reductase family 1 member C1 | 8.25/36.8 | Protection and detoxification | ND |
64 | P22626 | Heterogeneous nuclear ribonucleoproteins A2/B1 | 9.12/37.4 | Protein synthesis and degradation | −4.56±0.75 |
65 | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 8.79/35.9 | Metabolism | −1.46±0.40b |
66 | P22626 | Heterogeneous nuclear ribonucleoproteins A2/B1 | 9.12/37.4 | Protein synthesis and degradation | ND |
67 | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 8.79/35.9 | Metabolism | −7.70±0.92 |
-, downregulated expression following treatment; +, upregulated expression following treatment; D, appearance of proteins upon treatment; ND, disappearance of proteins upon treatment; pI, isoelectric point; MW, molecular weight.
Fold change of protein expression was calculated from three independent experiments.
P<0.05
P<0.01
P<0.001.