proteome analysis of hepatocellular carcinoma cell strains, mhcc97 … · 2015-08-07 · proteome...

Proteome analysis of hepatocellular carcinoma cellstrains, MHCC97-H and MHCC97-L, with differentmetastasis potentials

Shi-Jian Ding1, Yan Li2, Xiao-Xia Shao1, Hu Zhou1, Rong Zeng1, Zhao-You Tang2

and Qi-Chang Xia1

1Research Centre for Proteome Analysis, Key Laboratory of Proteomics, Institute ofBiochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, Shanghai, P. R. China

2Liver Cancer Institute and Zhongshan Hospital of Fudan University, Shanghai, P. R. China

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasisand to search for potential markers for HCC prognosis, differential proteome analysis on twoHCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, wasconducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laserdesorption/time of flight mass spectrometry and liquid chromatography ion trap mass spec-trometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed sig-nificant differential expression in MHCC97-H and MHCC97-L cells (Student’s t-test, P , 0.05)and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were fur-ther identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem massspectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolaseL1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulinprecursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 andcalumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of theseidentified proteins have been reported to be associated with tumor metastasis. The functionalimplications of alterations in the levels of these proteins are discussed.

Keywords: Hepatocellular carcinoma / Mass spectrometry / Metastasis / Proteome / Two-dimensional gelelectrophoresis

Received 15/7/03Revised 7/9/03Accepted 9/9/03

982 Proteomics 2004, 4, 982–994

1 Introduction

Hepatocellular carcinoma (HCC) is one of the most com-mon and aggressive malignant tumors with especiallyhigh prevalence in Asia and Africa and relatively low pre-

valence in Europe and North America [1]. Recent studiesindicate that the incidence of HCC in the US and UK hasincreased substantially over the last two decades [2, 3].Although a small subset of HCC patients qualify for surgi-cal intervention, many patients with advanced HCC havelittle chance of survival. The extremely poor prognosis ofHCC is largely the result of a high rate of recurrence andmetastasis [4]. This feature of HCC underscores the needto develop an accurate molecular profiling model toimprove diagnosis and identify therapeutic targets for thetreatment of HCC patients with metastases. Advances inthe fields of genomics and proteomics give rise of pro-mise for the discovery of new molecular targets for ther-apy, biomarkers for early detection and new endpoints fortherapeutic efficacy and low toxicity. Genomics-basedapproaches include the measurements of expression offull sets of mRNA, such as differential display, serial anal-ysis of gene expression, and large-scale gene expression

Correspondence: Prof. Qi-Chang Xia, Institute of Biochemistryand Cell Biology, Chinese Academy of Sciences, 320 Yue-yangRoad, Shanghai 200031, P. R. ChinaE-mail: [email protected]: 186-21-54920171

Abbreviations: CALUP, calumenin precursor; CD, cathepsin D;CHCA, a-cyano-4-hydroxy-trans-cinnamic; CK19, cytokeratin19; CRP55, calreticulin precursor; HCC, hepatocellular carci-noma; LDH-B, lactate dehydrogenase B; 67LR, 67 kDa lamininreceptor; Mn-SOD, managese superoxide dismutase; M2-PK,pyruvate kinase M2; nm23-H1, non-metastatic cell protein 1;PGP9.5, carboxy-terminal hydrolase L1; PMF, peptide mass fin-gerprinting; S100A4, S100 calcium-binding protein A4; Trx, thior-edoxin

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de

DOI 10.1002/pmic.200300653

Proteomics 2004, 4, 982–994 Differential proteome analysis and hepatocellular carcinoma metastasis 983

arrays. However, interpreting the data and adapting theresults to a particular application remains a challenge.Conflicting evidence also exists regarding the correlationbetween mRNA and protein abundance levels [5, 6].Comparatively, proteomics technologies allow for identifi-cation of the protein changes caused by the disease pro-cess in a relatively accurate manner. The inherent advan-tage of proteomics is that the identified protein is itself thebiological endpoint. Although new exciting approaches,such as isotope-coded affinity tags [7], surface enhancedlaser desorption/ionization-based protein chips [8] andmultidimensional protein identification technology [9] areavailable, currently, 2-DE is the mainstay of most prote-omic analysis because of its simplicity, reliability, highinformation content and ready accessibility to research-ers.

2-DE-based proteomic studies in liver cancer researchhave been conducted on cell lines, tissues, and seraat both the cellular and subcellular level to study carcino-genesis, liver cirrhosis and drugs toxicity [10–15]. 2-DEdatabases of HCC cell lines have also been establishedin these studies [16, 17]. However, to the best of ourknowledge, no proteomic studies have been reported onHCC metastasis, perhaps because of the lack of appro-priate materials. Recently, human HCC cell strains withhigh and low spontaneous metastasis potentials,MHCC97-H and MHCC97-L, have been established fromthe parental cell line MHCC97. They provide good modelsfor comparative proteome analysis of HCC metastasis-associated proteins [18, 19]. In our previous studies, weused 2-DE combined with MS to identify differentiallyexpressed proteins between HCC and normal liver celllines and investigated malignant growth-associated pro-teins by using antisense technology against the EGFreceptor [20, 21]. Here, the same strategy was used tostudy MHCC97-H and MHCC97-L cells. Fourteen differ-entially expressed protein spots were identified using2-DE followed by MALDI-TOF MS and LC-IT MS. Theimplications of the identified proteins in terms of theirinvolvement in the process of HCC metastasis are dis-cussed. These results provide the basis for searching forpotential markers for HCC prognosis and give some cluesto elucidate the mechanism of HCC metastasis.

2 Materials and methods

2.1 Chemicals

DTT, urea, agarose, glycerol, Bromophenol Blue, CHAPS,Bio-lytes1 (3–10), IPG Strips (3–10 L), mineral oil, acryl-amide, Bis, Tris base, glycine, SDS, ammonium persul-phate and TEMED were from Bio-Rad (Hercules, CA,

USA). Iodoacetamide (IAA), ammonium bicarbonate, for-mic acid and a-cyano-4-hydroxy-trans-cinnamic (CHCA)were from Sigma (St. Louis, MO, USA). ACN and metha-nol were from Fisher (Fair Lawn, New Jersey, USA). TFAwas from Merck (Schuchardt, Hohenbrunn, Germany).Trypsin (sequencing grade) was obtained from Boehrin-ger Mannheim (Mannheim, Germany). All buffers wereprepared with Milli-Q water (Millipore, Bedford, MA,USA).

2.2 Cell culture and sample preparation

MHCC97-H and MHCC97-L cells were established fromthe parent cell line MHCC97. Spontaneous pulmonarymetastasis occurred in 100% and 40% of recipient nudemice after orthotopic inoculation of MHCC97-H andMHCC97-L, respectively [18]. The cells were cultured at377C in 5% CO2 in DMEM medium (Gibco BRL, GrandIsland, NY, USA) supplemented with 10% fetal calf serum(Hyclone, Utah, USA). Sample preparation was per-formed as described previously [20]. Briefly, the cellswere grown to 90% confluency and harvested by treatingwith 0.25% trypsin and 0.02% EDTA. They were rinsedthree times in PBS and centrifuged in 1.5 mL microtubes.The cell pellets were dissolved in lysis buffer (8 M urea,4% CHAPS, 40 mM Tris, 65 mM DTT, 2% Bio-Lytes1) andcentrifuged at 25 0006g for 1 h at 47C. Protein concen-trations were determined using a modified Bradfordassay [22]. All samples were stored at 2807C prior toelectrophoresis.

2.3 2-DE and silver-staining

2-DE was performed as previously described [18], usingthe PROTEAN IEF and PROTEAN xi II systems (Bio-Rad).Total proteins (80 mg) were run in a IEF system using a17 cm pH 3–10 ReadyStrip (Bio-Rad). The total Vh was47 000,52 000. Following IEF separation, the gel stripwas equilibrated with buffer I (6 M urea, 30% glycerol,2% SDS, 1% DTT) and then buffer II (DTT was replacedwith 2.5% IAA) each for 15 min. The equilibrated gel stripwas placed on the top of a 12% Tslab gel and sealed with0.5% agarose. SDS-PAGE was performed for 30 min at aconstant current of 10 mA and then at 25 mA until the Bro-mophenol Blue reached the bottom of the gels. The sepa-rated proteins were visualized by silver diamine-stainingas described by Yu et al. [20]. For preparative 2-DE,400 mg of total proteins were separated as describedabove. The total Vh of the IEF was 90 000,120 000 Vh.Proteins were detected by a modified silver-stainingmethod compatible with MS analysis [23].


984 S.-J. Ding et al. Proteomics 2004, 4, 982–994

2.4 Image acquisition and statistical analysis

The silver-stained 2-D gels were scanned at an optical res-olution of 84.7 mm per pixel using a GS-710 imaging densi-tometer (Bio-Rad) in transmission mode. The digitizedimages were analyzed with the PDQuest 7.1 softwarepackage (Bio-Rad). Following spot detection, a matchsetincluding all three batches of MHCC97-H and MHCC97-Lgels was built. A reference gel was selected from one of theMHCC97-H gels and unmatched protein spots of themember gels were automatically added to the referencegel. The individual protein spot quantity was normalizedaccording to the following method: The raw quantity ofeach spot in a membergel was divided by the total quantityof the valid spots in the gel (i.e., all the spots in the gelminus cancelled spots) and expressed as ppm. Quantita-tive analysis was performed using the Student’s t-test be-tween MHCC97-H and MHCC97-L gels. The confidencelevel was 95%. The Mr and pI of each protein spot werecalibrated with six previously identified protein spots (PDI,57.5/4.76; Actin, 41.3/5.56; Aldose reductase, 36.3/7.12;Triosephosphate isomerase, 26.8/6.51; Cyclophilin A,18.1/7.12; Thioredoxin, 11.7/4.82). The results were ex-ported to Excel 2000 (Microsoft, Seattle, CA, USA) forfurther analysis.

2.5 In-gel digestion

Protein spots were excised from gels, destained for20 min in 30 mM potassium ferricyanide/100 mM sodiumthiosulfate (1:1 v/v) and washed in Milli-Q water until thegels became clear. The spots were kept in 0.2 M NH4HCO3

for 20 min, dried by lyophilization, and digested overnightin 12.5 ng/mL trypsin in 0.1 M NH4HCO3. The peptideswere extracted three times with 50% ACN, 0.1% TFAand dried in vacuo.

2.6 MALDI-TOF MS

The peptide mixtures were dissolved in 0.1% TFA anddesalted using a C18 ZipTip (Millipore). The eluted pep-tides in 0.1% TFA/50% ACN mixed with an equal volumeof 0.1% TFA/30% ACN saturated with CHCA solutionwere applied onto the target, air-dried and analyzed usinga Bruker REFLEX III MALDI-TOF mass spectrometer (Bru-ker-Franzen Analytik, Bremen, Germany) in reflectron-positive mode. The extraction voltage was set at 20 kVand the cut-off mass value was set at 500. Mass spectrawere recorded using 50–200 shots, depending on thesignal-to-noise ratio obtained for each sample. Thepeaks were externally calibrated with peptide standards(MH1: Angiotensin II, 1046.5420 Da; Angiotensin I,1296.6853 Da; Substance P, 1347.7361 Da; Bombesin,

1619.823 Da; ACTH clip 18–39, 2465.199 Da) from Brukeror internally calibrated with autodigest peaks of trypsin(MH1: 2163.057 Da, 2273.160 Da).

2.7 LC-IT MS

A Finnigan LCQ Deca XP ion-trap micro-electrospraymass spectrometer (ThermoQuest, San Jose, CA, USA)coupled with a Surveyor HPLC system (ThermoQuest)was used foron-line LC-MS analysis of the protein digests.For LC separation, a microbore RP column (C18,0.186150 mm id, 5 mm; ThermoHypersil, San Jose, CA,USA) was used. Solvent A was 0.1% v/v formic acid, andsolvent B was 0.1% v/v formic acid in 100% v/v ACN. Thegradient was held at 2% solvent B for 40 min, andincreased linearly to 98% solvent B in 110 min. The digestswere applied to the C18 column by an autosampler andeluted at a flow rate of 3 mL per min. The eluted peptideswere introduced on-line to the electrospray source. IT-MSexperiments were done with automatic gain control. Theion source instrumental parameter settings were as fol-lows: ESI voltage 3.2 kV, capillary voltage 5 V, capillarytemperature 1507C. In the full scan mode, ions were col-lected in three microscans with a maximum ion injectiontime of 200 ms, covering the mass range from m/z 400 to2000. MS/MS was performed in data-dependent mode.The MS/MS collision energy was 35%.

2.8 Protein identification and databasesearching

Protein identification using peptide mass fingerprinting(PMF) was performed by the MASCOT search engine(http://www.matrixscience.com/, MatrixSicence Ltd, UK)against the NCBI nonredundant protein database (http://www.ncbi.nlm.nih.gov/). The species is homo sapiens.The errors in peptide masses were in the range of 0.01–0.1%. One missed tryptic cleavage site per peptide wasallowed during the search. Proteins matching more thanfour peptides and with a MASCOT score higher than 63were considered significant (p , 0.05). Protein identifica-tion using MS/MS raw data was performed with theSEQUEST (University of Washington, licensed to ThermoFinnigan) searching program against the NCBI humanprotein database (version from May 16, 2003, 37 490 pro-tein entries). In both MASCOT and SEQUEST searching,carboamidomethylation of cysteine was selected as thestatic modification and oxidation of methionine as thedifferential modification. Both b ions and y ions wereincluded in the database search. Protein identificationresults were filtered with the Xcorr (11�1.9, 21�2.2,31�3.75) and DelCn (�0.1).



2.9 Western blot analysis

Total proteins (80 mg) were separated by 2-DE asdescribed in Section 2.3 and electroblotted to Immobi-lon-P membrane (Millipore). After blocking with 5% non-fat dried milk in 16TBST (25 mM Tris, pH 7.5, 150 mM

NaCl, 0.05% Tween 20, and 0.001% thimerosal) for 1 hat room temperature, membranes were incubated withmouse anti-human CK19 (DAKO, Glostrup, Denmark),goat anti-human Cathepsin D (Santa Cruz Biotechnology,Santa Cruz, CA, USA) and sheep anti-human managesesuperoxide dismutase (Calbiochem-Novabiochem, SanDiego, CA, USA) for 1 h at room temperature, followedby horseradish peroxidase-conjugated secondary anti-body for 1 h at room temperature. Target proteins weredetected by ECL (Amersham Life Science, Cleveland,OH, USA).

3 Results

3.1 Proteome differential expression betweenMHCC97-H and MHCC97-L cells

The experiments (from cell culture to 2-DE) were repeated3 times. The silver-stained 2-D gels of the proteomesexpressed by MHCC97-H and MHCC97-L cells are pre-sented in Fig. 1. We detected 1130 6 53 and 1107 6 47protein spots in gels of total protein prepared from

MHCC97-H and MHCC97-L cells, respectively. One ofthe MHCC97-H gels was selected as a reference gel and77.59% and 69.93% of the protein spots could bematched for MHCC97-H and MHCC97-L gels, respec-tively. Fifty-six protein spots were found to have theirvolumes changed significantly (Student’s t-test, p ,

0.05) in MHCC97-H gels in comparison with MHCC97-Lgels (Table 1). Among them, 22 protein spots hadincreased expression levels while 34 had reduced expres-sion levels in MHCC97-H cells. Four protein spots weredetected in MHCC97-H but not MHCC97-L cells. No pro-tein spots were exclusive to MHCC97-L cells. All differen-tially expressed protein spots are showed in Fig. 1.

3.2 Identification of differentially expressedproteins by PMF and MS/MS

The selected protein spots from the silver-stained gelswere excised and subjected to in-gel tryptic digestion.The extracted peptides were analyzed by MALDI-TOFMS to generate PMF. Fig. 2A shows a MALDI-TOF massspectrum of peptides derived from protein spot 5406.MASCOT search using the PMF data indicated that 14peptides matched with peptides from pyruvate kinaseM2 (M2-PK) (Fig. 2B), giving a sequence coverage of28%. The results showed that the mass error of eachmatched peptide was below 30 ppm. The experimentalmasses of 4 peptides exactly matched their theoretical

Figure 1. 2-DE patterns of whole-cell proteins from (A) MHCC97-H cells and (B) MHCC97-L cells.Red arrows indicate proteins that were up-regulated in MHCC97-H cells and blue arrows indicateproteins that were down-regulated. Protein spots detected only in MHCC97-H cells are circled in red.



Table 1. Protein spots whose expression levels were significantly changed between MHCC97-H andMHCC97-L cells

Proteinindexa)

Measuredb) Normalized volumec) P-value

Mr pI MHCC97-H MHCC97-L

0101 15.50 21 3275.38 6 579.46 2249.47 6 527.16 0.019810203 30.39 21 4989.92 6 472.27 6025.35 6 490.54 0.011380308 32.72 21 1675.12 6 266.60 2344.68 6 418.63 0.017830509d) 45.48 21 2962.19 6 403.46 8439.49 6 2414.87 0.002110701d) 64.27 21 5677.22 6 1939.59 10768.78 6 2845.82 0.012691012d) 11.70 4.82 27844.37 6 2656.43 21551.39 6 3185.64 0.011491202 26.43 21 1760.39 6 158.45 2357.67 6 256.09 0.003701203 28.25 21 80.77 6 63.54 649.23 6 371.47 0.015561302 37.17 21 260.44 6 201.72 187.32 6 68.66 0.020551409d) 41.03 4.76 1546.06 6 285.56 775.76 6 347.33 0.007021418 38.19 4.83 341.83 6 229.45 176.53 6 79.39 0.012191502 43.23 21 928.27 6 120.36 1339.98 6 299.00 0.021612008 10.92 5.14 533.48 6 387.32 1115.33 6 102.52 0.013592109 20.31 5.32 89.57 6 43.96 850.29 6 176.77 0.002172111 21.68 5.41 989.57 6 168.57 750.29 6 63.21 0.018812403d) 39.23 5.37 1837.42 6 438.56 114.72 6 73.95 0.000032501 45.66 5.01 91.17 6 107.18 335.03 6 51.62 0.003183005 12.70 5.54 1781.34 6 190.16 2372.85 6 709.05 0.018823006d) 10.78 5.62 8181.34 6 2164.68 2372.85 6 492.41 0.010733101d) 25.04 5.58 1247.76 6 137.93 802.02 6 122.66 0.001463106 22.94 5.77 1816.07 6 236.66 1219.37 6 416.97 0.023613306d) 30.72 5.83 823.47 6 218.38 1115.27 6 357.23 0.021603310d) 36.80 5.76 3279.73 6 675.50 9315.62 6 1409.87 0.001533411 37.97 5.73 4316.30 6 684.34 187.28 6 70.73 0.005603504 48.27 5.61 1877.15 6 321.83 1387.32 6 150.08 0.016454106d) 19.82 5.74 935.60 6 329.98 2374.10 6 470.79 0.010244310 32.21 6.24 154.38 6 35.21 876.29 6 63.73 0.001324406d) 38.11 5.98 5994.29 6 1167.46 2603.23 6 657.63 0.017924507 42.57 5.88 1055.87 6 280.21 145.60 6 195.95 0.000894516 48.61 6.02 55.63 6 40.19 142.01 6 60.64 0.001644611 58.87 5.98 187.83 6 102.37 254.60 6 205.63 0.023715009 12.61 6.51 1178.10 6 324.69 2077.63 6 241.86 0.012185102 19.48 6.31 943.17 6 193.76 163.29 6 83.24 0.002195108d) 19.02 6.25 1965.9 6 468.2 7960.1 6 2407.1 0.002625203 30.58 6.48 142.48 6 53.48 657.29 6 83.97 0.007455211 25.19 6.45 3319.23 6 251.05 4052.94 6 494.62 0.019135406 40.44 6.27 2705.42 6 551.43 17220.65 6 6286.74 0.001425410 38.74 6.39 3566.09 6 1523.03 429.62 6 499.40 0.003935608 56.91 6.28 1425.96 6 623.23 580.78 6 190.95 0.020525610 53.75 6.36 2715.19 6 1320.88 1540.44 6 905.69 0.014656001 12.10 6.46 881.03 6 611.07 1734.60 6 296.53 0.022856105d) 22.31 6.54 978.17 6 516.46 1753.57 6 468.12 0.021546107 14.73 6.72 149.52 6 175.98 437.68 6 84.03 0.012726111d) 22.30 6.86 4829.73 6 1137.47 9432.36 6 2127.01 0.002206113 46.15 6.54 815.99 6 283.91 3309.54 6 675.65 0.001166303 33.06 6.31 256.43 6 87.39 1170.29 6 389.2600 0.001276501 46.15 6.54 192.56 6 226.59 829.77 6 245.21 0.004406606 56.05 6.63 257.48 6 117.47 583.74 6 120.38 0.004096705 71.05 6.59 418.96 6 119.44 646.10 6 96.15 0.012607206 30.92 21 1665.28 6 147.04 1485.11 6 161.08 0.001717315 37.12 21 240.39 6 298.03 645.52 6 129.94 0.023517617 50.33 21 203.15 6 78.75 57.60 6 72.42 0.017347802 73.80 6.99 229.92 6 78.69 83.74 6 39.28 0.007968201 27.84 21 841.55 6 49.87 640.26 6 139.78 0.01749



Table 1. Continued

Proteinindexa)

Measuredb) Normalized volumec) P-value

Mr pI MHCC97-H MHCC97-L

8207 30.77 21 562.33 6 289.20 139.06 6 176.21 0.023278603 53.83 21 341.48 6 150.08 658.03 6 189.99 0.01993

a) Index in the reference gelb) Calculated from PDQuest software; – stands for no calibration made for pI and Mr

c) The individual protein spot volumes were normalized as a percentage of the total volume in all ofthe protein spots present in the gel, and are expressed as ppm

d) These spots were further identified by MS analysis

BStart–End Observed Mr(expt) Mr(calc) Delta Miss Sequence

32–42 1197.73 1196.72 1196.64 0.08 0 LDIDSPPITAR43–55 1359.67 1358.66 1358.70 20.04 0 NTGIICTIGPASR73–88 1883.89 1882.88 1882.90 20.01 0 LNFSHGTHEYHAETIK73–91 2253.19 2252.18 2252.11 0.07 1 LNFSHGTHEYHAETIKNVR92–114 2465.36 2464.35 2464.28 0.07 0 TATESFASDPILRPVAVALDTK92–119 3017.59 3016.58 3016.59 20.00 1 TATESFASDPILYRPVAVALDTKGPEIR

188–205 1779.96 1778.95 1778.87 0.08 0 GADFLVTEVENGGSLGSK188–206 1907.97 1906.96 1906.96 0.00 1 GADFLVTEVENGGSLGSKK266–277 1394.71 1393.70 1393.77 20.07 1 IISKIENHEGVR278–293 1821.92 1820.91 1820.91 0.00 1 RFDEILEASDGIMVAR278–293 1837.93 1836.92 1836.90 0.01 1 RFDEILEASDGIMVAR Oxidation (M)279–293 1665.78 1664.77 1664.81 20.04 0 FDEILEASDGIMVAR294–310 1828.02 1827.02 1827.01 0.00 1 GDLGIEIPAEKVFLAQK319–335 1893.03 1892.02 1891.96 0.06 0 AGKPVICATQMLESMIK Oxidation (M)

No match to: 1570.69, 2163.13, 2273.24, 2289.20, 2514.36

Figure 2. (A) MALDI-TOF mass spectrum obtained from spot 5406 after trypsin digestion. (B) Peptidesequences from M2-PK matched with peaks obtained from the MALDI-TOF mass spectrum.



masses without any deviation. The score given byMASCOT was as high as 115, indicating significant confi-dence (p , 0.05) in the identity.

In cases where no conclusive identification could beobtained by PMF, especially for low molecular massproteins, microspray-MS/MS was used to provide asequence tag for database searching. A database searchusing MS/MS raw data of peptides derived from spot4106 by SEQUEST revealed that the best match wasnon-metastatic cell protein 1 (nm23-H1) with a score of186. Eight peptides were derived from nm23-H1. Thebase peak chromatogram of in-gel digests derived fromspot 4106 is presented in Fig. 3A. Fig. 3B shows the CIDspectrum of peptide T2, which was doubly charged andhad an m/z of 743.24. The excellent spectrum reveals thatmost of the b-ions and y-ions were produced from thisprecursor ion with the exception of b1 and y1 ions. Theseresults suggest, with high confidence, that spot 4106 isnm23-H1.

Among the proteins identified by PMF, Cathepsin D (CD)had the lowest protein coverage but it was also identifiedby MS/MS. Except for spot 0509, all spots identified byMS/MS had at least two peptides consistently matchingthe same candidate protein.

A database search using MS/MS raw data of peptidesderived from spot 0509 by SEQUEST revealed that thebest match was calumenin precursor (CALUP). Althoughonly one peptide matched CALUP as the top candidate,all three scans of this peptide had Xcorr values above 2.0(3.19, 2.77, 3.15) and DelCn values above 0.1 (0.52, 0.43,0.46). To confirm that the peptide was derived from CALUP,their CID spectra were further examined. Figure 4 showsthe MS/MS spectrum obtained form the doubly chargedpeptide at m/z 790.78. The spectrum contained y2, y4-y5,y7-y11, b2-b6, and b8-b12 as expected from the candi-date peptide; thus a partial sequence, TFDQLTPEESKER,could be determined. Intriguingly, a proline effect wasobserved in the spectrum [25, 26]. Furthermore, a searchof Protein Information Resource (PIR) [27] using the partialsequence resulted in CALUP as the unique hit, and signifi-cant sequence alignment with CALUP was producedwhen SWISS-PROT was searched using the BLAST sys-tem [28]. Thus we conclude that this spot is CALUP.

In total fourteen differentially expressed protein spotswere identified using PMF or MS/MS. The identificationmethods, summary scores, protein coverage, p values ofthe spots from MHCC97-H and MHCC97-L cells, etc., arepresented in Table 2. Six spots increased in MHCC97-Hcells were identified as cytokeratin 19 (CK19), ubiquitincarboxy-terminal hydrolase L1 (PGP9.5), S100 calcium-binding protein A4 (S100A4), thioredoxin (Trx), 67 kDa

laminin receptor (67LR), and M2-PK. Seven spots de-creased in MHCC97-H were identified as managesesuperoxide dismutase (Mn-SOD), lactate dehydrolase B(LDH-B), cofilin 1, calreticulin precursor (CRP55), CALUP,nm23-H1 and CD. Two spots (6105 and 6111) were iden-tified as Mn-SOD, but MS data did not reveal any post-modifications.

3.3 Verification of differential expressedproteins by Western blot analysis

From the identified candidates, CK19, CD and Mn-SODwere selected for Western blot analysis as shown inFig. 5. The expression changes of the three selected pro-teins were consistent with 2-DE and silver-stainingresults. CK19 was up-regulated, while CD and Mn-SODwere down-regulated in MHCC97-H cells compared withMHCC97-L cells. Such results demonstrate that the pro-teomic analysis of MHCC97-H and MHCC97-L cells wasconvincing. Moreover, at least four spots were detectedas CK19 while MS only identified the most abundantone. Two spots were also detected as Mn-SOD inMHCC97-L cells in agreement with the MS results.

4 Discussion

4.1 Protein identification by MALDI-TOF MS andLC-IT MS

PMF is a sensitive and high-throughput method that hasbeen extensively used for rapid screening of proteins.In this study we employed it to identify differentiallyexpressed protein spots. Seven protein spots were un-ambiguously identified using this method. However,some protein spots, especially those expressed only inMHCC97-H cells, were not identified. For several ofthem, good MS data were collected but no identity couldbe assigned. This may be due to unknown genes or over-lapping spots. For other spots, the MS data were not suf-ficient for protein identification (usually because of a lowsignal-to-noise ratio of the mass spectra and a smallnumber of peptides derived from faint spots). For a verysmall proportion of the spots, no MS data could beacquired. In such cases peptide sequencing using MS/MS becomes the method of choice since one or a fewtryptic peptide sequences are usually sufficient to identifya protein reliably, even in spots containing more than oneprotein. In this study, CALUP was identified by MS/MSwith only one peptide that is unique to it.

Two parallel (22.31/6.54 and 22.30/6.86) spots were iden-tified as Mn-SOD. Western blots confirmed the MS resultsof Mn-SOD and also revealed at least four spots were



Figure 3. LC-MS analysis of tryptic digests from spot 4106. (A) Base peak chromatogram showingthe peptide peaks from which 8 peptides (T1-T8) were confidently matched to nm23-H1. (B) MS/MSspectrum of the doubly charged precursor ion with m/z 743.24 (T2) at retention time 86.13 min. Asequence was confirmed from the labeled b- and y-ions in the spectrum. Fragments observed inthe spectrum are underlined and assigned.



Figure 4. MS/MS spectrum of the [M12H]21 ion with m/z 790.78 from a tryptic peptide of spot 0509.The peptide was matched to the same sequence from CALUP by SEQUESTsearching. The sequenceis displayed with the predicted fragment ion masses, and those observed in the spectrum are under-lined.

Table 2. Identification results of proteins differentially expressed between MHCC97-H and MHCC97-L cells

Proteinindexa)

ProteinDescription

SWISS-PROT ID

TheoreticalMr/pIb)

MeasuredMr/pIc)

Summaryscored)

Proteincover-agee)

Proteinlevelf)

p-value Identifi-cationmethodg)

0509 Calumeninprecursor

CALU-HUMAN

41.04/4.51 45.48/- 30 5.73% ↓ 0.00211 MS/MS

0701 Calreticulinprecursor(CRP55)

CRTC-HUMAN

48.10/4.29 64.27/- 370 36.2% ↓ 0.01269 MS/MS

1012 Thioredoxin THI2-HUMAN

11.74/4.82 11.70/4.82 120 43% : 0.01149 MS/MS

1409 67 kDa lamininreceptor(67LR)

AAH34537 32.86/4.83 41.03/4.76 72 37% : 0.00702 PMF

2403 Cytokeratin19 (CK19)

K1CS-HUMAN

44.07/5.04 39.23/5.37 240 50% : 0.00003 PMF



Table 2. Continued

Proteinindexa)

ProteinDescription

SWISS-PROT ID

TheoreticalMr/pIb)

MeasuredMr/pIc)

Summaryscored)

Proteincover-agee)

Proteinlevelf)

p-value Identifi-cationmethodg)

3006 S100 calcium-bindingprotein A4(S100A4)

S104-HUMAN

11.73/5.85 10.78/5.62 140 46.5% : 0.01073 MS/MS

3101 ubiquitincarboxy-terminalhydrolase L1(PGP9.5)

UBL1-HUMAN

23.35/5.30 25.04/5.58 94 34% : 0.00146 PMF

3306 Cathepsin D CATD-HUMAN

45.04/ 6.10 30.72/5.83 64 17% ↓ 0.02160 PMF,MS/MS

3310 Lactatedehydrolase B(LDH-B)

LDHB-HUMAN

36.64/5.71 36.80/5.76 40 12% ↓ 0.00153 MS/MS

4106 Non-metastaticcell protein 1(nm 23-H1)

NDKA-HUMAN

17.15/5.83 19.82/5.74 186 51.3% ↓ 0.01024 MS/MS

4406 Pyruvatekinase M2

KPY2-HUMAN

58.45/7.58 38.11/5.98 115 28% : 0.01792 PMF

5108 cofilin 1 COF1-HUMAN

18.50/8.22 19.02/6.25 194 53% ↓ 0.00262 MS/MS

6105 Manganese super-oxide (Mn-SOD)

AAP34407 22.30/6.86 22.31/6.54 71 23% ↓ 0.02154 PMF

6111 Manganese super-oxide (Mn-SOD)

AAP34407 22.30/6.86 22.30/6.86 75 31% ↓ 0.00220 PMF

a) Index in the reference gelb) Calculated from the database entry without any processingc) Calculated from PDQuest software; – stands for no calibration made for pI and Mr

d) MASCOTscore for MALDI-TOF MS analysis, or Sequest score for LC-IT MS analysise) Protein coverage was calculated by amino acid countf) :, up-regulated in MHCC97-H cells; ↓ , down-regulated in MHCC97-L cellsg) PMF from MALDI-TOF data; database search based on LC-IT MS/MS data (MS/MS)

Figure 5. Western blot analysis of CK19, CD and Mn-SOD expression in MHCC97-H and MHCC97-L cells.

CK19. These spots might be isoforms of Mn-SOD orCK19 with differential phosphorylation. However, we didnot find phosphorylated peptides originating from Mn-SOD or CK19. Mn-SOD and CK19 may be modified bychemical modifications other than phosphorylation. Fur-ther investigation of possible modifications will be carriedout using different types of protease or alkaline phospha-tase treatments prior to MALDI-TOF MS.

4.2 Functional implication of differentiallyexpressed proteins

MHCC97-H and MHCC97-L cells which have high andlow metastatic potentials, respectively, were establishedfrom the same parental human HCC cell line (MHCC97)



[19]. They are significantly different in phenotype, cellcycle, and in vitro and in vivo metastasis ability [19]. Asthey have the same genetic background, these cells pro-vide desirable materials for investigating HCC metastasis-associated proteins. A model to interpret cancer cell inva-sion was proposed [29]. It consists of the cyclic repetitionof three major processes: (1) attachment of cancer cells tobasement components through specific cell surfacereceptors (2) degradation of basement following localsecretion of proteolytic enzymes and (3) migration of thecells into the adjacent tissue compartments [29]. In thisstudy, 14 spots with significant and reproducible changesbetween MHCC97-H and MHCC97-L cells were identi-fied. Interestingly, most of these proteins have beenreported to be involved in the three major processes oftumor metastasis listed above. According to their charac-teristics and functions, the identified proteins can be clas-sified into five groups as shown in Table 3 and describedas follows:

Table 3. Functional categories of proteins differentiallyexpressed between MHCC97-H and MHCC97-L cells

Functional categoriesa) Protein name

Cytoskeleton and its associatedproteins

CK19, nm23-H1 and cofilin 1

Extracellular matrix degradationassociated proteins

67LR, CD and Trx

Calcium-binding proteins S100A4, CRP55 and CALUP

Carbohydrate metabolism enzymes M2-PK, LDH-B

Malignant growth associatedenzymes

Mn-SOD, PGP9.5

a) Differentially expressed proteins (13) were classified bytheir characteristics and broad functional criteria

(1) Cytoskeleton and its associated proteins, includingCK19, nm23-H1 and cofilin 1. CK19 is the smallest inter-mediate filament member. Its significantly high expres-sion in highly metastatic MHCC97-H cells illustrated thepotential of CK19 to regulate tumor metastasis by pro-moting cell motility and destroying extracellular matrix inoral squamous cancer cells [30, 31]. However, resultswere conflicting as to whether CK19 was expressed inHCC cells [32, 33]. Our results clearly show its expressionin HCC and its possible association with metastasis. Ourfurther study on specimens and sera of HCC patients anda nude mouse model also confirm the function of CK19 inmetastasis (manuscript in preparation). Nm23 has highhomology to nucleotide diphosphate kinase, which mayaffect the state of tubulin polymerization [34]. Decreasedexpression of nm23-H1 in highly metastatic HCC cells is

consistent with the previous report of nm23 as a putativemetastasis suppressor protein in cancers [35]. Lizuka etal. [36] reported the expression level of nm23-H1, but notnm23-H2, in HCC and showed a significant inverse corre-lation as compared with intrahepatic metastasis. A recentstudy showed that nm23-H1 regulates Rho-familyGTPases, which are closely related to cell migration [37].Cofilin 1 was found to be decreased in highly metastaticMHCC97-H cells. Cofilin 1, an actin depolymerizing pro-tein, can control actin-based motility by reversible phos-phorylation [38], indicating its possible role in metastasis.

(2) Extracellular matrix degradation associated proteins,including 67LR, CD and Trx. Up-regulation of LR67 inhighly metastatic MHCC97-H cells is in agreement withprevious reports of its increased expression in a varietyof human carcinomas (colon, breast, stomach, liver, lung,and ovary) which directly correlated with a higher prolifer-ation rate and metastatic tendency [39]. 67LR (an integ-rin-accessory molecule) stabilizes the binding of lamininto cell surface integrins [40]. CD, a lysosomal acidic pro-tease, may degrade the extracellular matrix and thusfacilitate tumor metastasis. A significant positive correla-tion between increased CD expression and lymph nodemetastasis has been reported in oral cancer [41]. How-ever, in gastric carcinoma, no such positive correlationswere found [42]. Furthermore, Johnson et al. reportedthat a high level of intracellular and secreted CD is an indi-cator of a less aggressive phenotype in some breast can-cer cells examined [43]. Our finding of increased expres-sion of CD in low metastatic MHCC97-L cells is consistingwith these notions. Another protein up-regulated in highlymetastatic MHCC97-H cells was Trx. Trx has beenshowed to alter the matrix metalloproteinase/tissue inhib-itor of metalloproteinase balance and stimulate cancercell invasion in the presence of the Trx receptor [44].

(3) Calcium-binding proteins, including S100A4, CRP55and CALUP. In our study, a significant increase inS100A4 levels was found in highly metastatic MHCC97-H cells. There is increasing evidence that S100A4 is a mo-lecular marker of metastatic potential with high prognos-tic significance in different types of cancers [45, 46].S100A4 expression alters the adhesive properties of cells,possibly by remodelling the extracellular matrix and pro-moting a redeployment of adhesion-mediating macromo-lecules to the extracellular matrix [47]. Reduced levels ofCRP55 were observed in highly metastatic MHCC97-Hcells. This result is in accordance with the report that cal-reticulin and calreticulin fragments inhibit angiogenesisand suppress tumor growth [48]. Our previous study alsoshowed the down-regulation of CRP55 in hepatoma cells[20]. CALUP has been identified using a proteomicapproach as a metastasis-associated protein, which is



down-regulated in high metastatic head and neck cancercell lines [49]. We also detected decreased levels ofCALUP in MHCC97-H cells. However, the mechanism ofits involvement in tumor metastasis remains unknown.

(4) Carbohydrate metabolism enzymes, including M2-PKand LDH-B. Tumor formation is generally linked to in-creased activity of glycolytic enzymes, such as pyruvatekinase and lactate dehydrogenase. The isoenzymes ofpyruvate kinase and lactate dehydrogenase appear to bepromising tumor markers [50]. In addition, M2-PK alsoshowed good correlation to metastasis in pancreatic can-cer and renal carcinoma [51, 52]. In this study, M2-PKlevels were increased in MHCC97-H cells while LDH-Blevels dereased. In liver cancer, the serum LDH isoen-zyme 4/5 ratio discriminates between hepatocarcinomaand secondary liver neoplasia [53]. However, LDH-B wasnot reported to be associated with tumor metastasis.

(5) Malignant growth associated enzymes, including Mn-SOD and PGP9.5. Mn-SOD can reverse the process ofcarcinogenesis in some cancers and protects againstradiation-induced cell death by controlling the generationof mitochondrial reactive oxygen species and intracellularlipid peroxidation [54, 55]. Reduced levels of Mn-SOD inhighly metastatic MHCC97-H cells are in accordance witha previous report of its expression in esophageal carci-noma [56]. However, overexpression of Mn-SOD canalso stimulate the activation of MMP-2 in human breastcancer cells [57]. PGP9.5, an ubiquitin hydrolase, is up-regulated in highly metastatic MHCC97-H cells. Expres-sion of PGP9.5 significantly correlated with tumor weightand extent in colorectal cancer. PGP9.5 may be useful asa marker for invasive colorectal cancer [58]. PGP9.5 mayincrease deubiquitination of cyclins leading to uncon-trolled cell growth [59].

5 Concluding remarks

In summary, a proteomic approach was carried out onwell established HCC cell strains with high and low meta-static potentials. We identified 14 protein spots with sig-nificant alterations. Western blot analysis of selected pro-teins confirmed the proteomic analysis results. Most ofthe differentially expressed proteins have clear relation-ships with the process of tumor metastasis and theirchanges are consistent with the literature. Other experi-mental procedures, in addition to 2-DE techniques, areneeded to probe the biological functions of these proteinsin HCC metastasis. This study shows that proteomics canprovide new information regarding the complex eventsassociated with HCC metastasis and may identify usefulmarkers for HCC prognosis.

This work was supported by the State 863 High-Technol-ogy R&D Project of China (No. 2001AA233031) and aChina State Key Basic Research Program Grant(No. 2001CB5102). We thank Professor You-shan Zhangfor critical reading of the manuscript and Man-rong Jiangfor help with Western blot analysis.

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