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doi: 10.1152/ajpcell.00043.2007 293:C761-C771, 2007. First published 30 May 2007; Am J Physiol Cell Physiol Jae-Hong Ko, Abd El-bary Prince and Jin Han Hyoung Kyu Kim, Won Sun Park, Sung Hyun Kang, Mohamad Warda, Nari Kim, cell line Mitochondrial alterations in human gastric carcinoma You might find this additional info useful... 80 articles, 20 of which you can access for free at: This article cites http://ajpcell.physiology.org/content/293/2/C761.full#ref-list-1 1 other HighWire-hosted articles: This article has been cited by http://ajpcell.physiology.org/content/293/2/C761#cited-by including high resolution figures, can be found at: Updated information and services http://ajpcell.physiology.org/content/293/2/C761.full found at: can be American Journal of Physiology - Cell Physiology about Additional material and information http://www.the-aps.org/publications/ajpcell This information is current as of June 4, 2013. 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/. Rockville Pike, Bethesda MD 20814-3991. Copyright © 2007 the American Physiological Society. ISSN: molecular physiology. It is published 12 times a year (monthly) by the American Physiological Society, 9650 is dedicated to innovative approaches to the study of cell and American Journal of Physiology - Cell Physiology at Univ Cattolica Del Sacro Cuore-Bibl on June 4, 2013 http://ajpcell.physiology.org/ Downloaded from

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Mitochondrial alterations in human gastric carcinoma

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doi: 10.1152/ajpcell.00043.2007293:C761-C771, 2007. First published 30 May 2007;Am J Physiol Cell Physiol 

Jae-Hong Ko, Abd El-bary Prince and Jin HanHyoung Kyu Kim, Won Sun Park, Sung Hyun Kang, Mohamad Warda, Nari Kim,cell lineMitochondrial alterations in human gastric carcinoma

You might find this additional info useful...

 80 articles, 20 of which you can access for free at: This article citeshttp://ajpcell.physiology.org/content/293/2/C761.full#ref-list-1

 1 other HighWire-hosted articles: This article has been cited by http://ajpcell.physiology.org/content/293/2/C761#cited-by

including high resolution figures, can be found at: Updated information and serviceshttp://ajpcell.physiology.org/content/293/2/C761.full

found at: can beAmerican Journal of Physiology - Cell Physiology about Additional material and information

http://www.the-aps.org/publications/ajpcell

This information is current as of June 4, 2013.

0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/. Rockville Pike, Bethesda MD 20814-3991. Copyright © 2007 the American Physiological Society. ISSN:molecular physiology. It is published 12 times a year (monthly) by the American Physiological Society, 9650

is dedicated to innovative approaches to the study of cell andAmerican Journal of Physiology - Cell Physiology

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Mitochondrial alterations in human gastric carcinoma cell line

Hyoung Kyu Kim,1 Won Sun Park,1 Sung Hyun Kang,1 Mohamad Warda,1,2 Nari Kim,1 Jae-Hong Ko,1

Abd El-bary Prince, and Jin Han1

1Mitochondrial Signaling Laboratory, Mitochondria Research Group, Department of Physiology and Biophysics, College ofMedicine, Biohealth Products Research Center, Cardiovascular and Metabolic Disease Research Center, Inje University,Busan, Korea; and 2Department of Biochemistry, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt

Submitted 31 January 2007; accepted in final form 24 May 2007

Kim HK, Park WS, Kang SH, Warda M, Kim N, Ko J-H,Prince AE, Han J. Mitochondrial alterations in human gastric carci-noma cell line. Am J Physiol Cell Physiol 293: C761–C771, 2007.First published May 30, 2007; doi:10.1152/ajpcell.00043.2007.—Wecompared mitochondrial function, morphology, and proteome in therat normal gastric cell line RGM-1 and the human gastric cancer cellline AGS. Total numbers and cross-sectional sizes of mitochondriawere smaller in AGS cells. Mitochondria in AGS cells were deformedand consumed less oxygen. Confocal microscopy indicated that themitochondrial inner membrane potential was hyperpolarized and themitochondrial Ca2� concentration was elevated in AGS cells. Inter-estingly, two-dimensional electrophoresis proteomics on the mito-chondria-enriched fraction revealed high expression of four mitochon-drial proteins in AGS cells: ubiquinol-cytochrome c reductase, mito-chondrial short-chain enoyl-coenzyme A hydratase-1, heat shockprotein 60, and mitochondria elongation factor Tu. The results provideclues as to the mechanism of the mitochondrial changes in cancer atthe protein level and may serve as potential cancer biomarkers inmitochondria.

two-dimensional gel electrophoresis proteomics; biomarker; cancer

GASTRIC CANCER is the second most common malignant cancerafter lung cancer worldwide (42, 43, 80), and advanced gastriccancer has a poor prognosis due to the limited efficacy ofavailable therapies. Therefore, early diagnosis and better un-derstanding of tumor progression and termination are essentialfor controlling this disease.

Transcriptomics and proteomics afford a powerful andpromising approach that allow for the comprehensive inspec-tion of protein profiling (20, 30, 82), which should disclose thesuitability of various novel cancer biomarkers that contribute toan early diagnosis (6, 63). Consequently, proteomic studies ongastric cancer have examined serum (38, 49, 50, 52) and tissue(14, 29, 39) specimens and cultured cell lines (37, 62, 66, 81).In addition, mitochondria have been targeted to better under-stand tumor development since Warburg’s breakthrough dis-covery of “mitochondrial injury” in tumors (73). Consequently,studies have investigated the relationship between mitochon-drial dysfunction and oncogenesis over the last 50 years toclarify the respiratory machinery impairment as a key event incarcinogenesis (73). Despite the increasing evidence implicat-ing a mitochondrial defect or dysfunction as a key player inoncogenesis (2, 9, 13, 23), little is known of the alterations inmitochondrial structure and function that contribute to cellular

outcomes in gastric cell tumors. Therefore, we examined mi-tochondrial proteome alterations in gastric tumor cells and theircorrelation with mitochondrial function in the neo-homeostasisenvironment. This approach provides a better idea regardingthe contribution of mitochondrial to neo-homeostasis in gastriccancer, and the impact of neo-homeostasis on mitochondrialproteome alterations may lead to the discovery of new biomar-kers specific for mitochondrial involvement. Furthermore, theresults suggest possible therapeutic interventions for gastriccancer that affect the mitochondrial membrane potential(��m) and �-oxidation pathway.

MATERIALS AND METHODS

Cell Culture and Reagents

Cells from the human gastric cancer cell line AGS were cultured inRPMI-1640 (Cambrex, Baltimore, MD) containing 10% FBS and 100U/ml penicillin-streptomycin in a humidified 5% CO2 atmosphere. Ascountercells of AGS cells, normal rat gastric mucosal cells from cellline RGM-1 were cultured in DMEM-F-12 (Cambrex) under the sameconditions (32).

Solutions and Drugs

Normal Tyrode solution contained (in mM) 143 NaCl, 5.4 KCl, 1.8CaCl2, 0.5 MgCl2, 5.5 glucose, and 5 HEPES (pH 7.4) adjusted withNaOH. Fluorescent dyes [rhod-2 AM and tetramethylrhodamine ethylester (TMRE)] were purchased from Molecular Probes, prepared as 1mM stock solutions in DMSO, and diluted into test solutions beforeeach experiment. The final concentration of DMSO in the bathsolution (�0.1%) has been previously proved not to affect recordedintensities. Acrylamide, methylene bis-acrylamide, glycine, Tris, urea,glycerol, CHAPS, thiourea, and bromophenol blue were purchasedfrom Sigma (St. Louis, MO). N,N,N�,N�-tetramethylethylene diamine(TEMED), iodoacetamide, ammonium persulfate, DTT, and SDSwere obtained from Merck (Darmstadt, Germany). IPG strips, IPGbuffer, Destreak rehydration solution, and the two-dimensional (2-D)Quant kit were purchased from Amersham Biosciences (Uppsala,Sweden). The one-dimensional gel electrophoresis kit and membranetransfer kit were from Hoefer (San Francisco, CA). Immobilon-Pmembranes were purchased from Millipore (Bedford, MA). All otherreagents used were of analytic grade.

Mitochondrial Morphology Difference Comparisons byElectron Microscope

Cells (1 � 106) were collected, washed twice with PBS, centri-fuged for 5 min at 1,800 g, and glutaraldehyde fixed for 2–4 h at 4°C.Mitochondrial morphology differences, numbers, and shapes between

Address for reprint requests and other correspondence: J. Han, Mitochon-drial Signaling Laboratory, Mitochondria Research Group, Dept. of Physiol-ogy and Biophysics, College of Medicine, Biohealth Products ResearchCenter, Cardiovascular and Metabolic Disease Center, Inje Univ. 633-165Gaegeum-Dong, Busanjin-Gu, Busan 613-735, Korea (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Cell Physiol 293: C761–C771, 2007.First published May 30, 2007; doi:10.1152/ajpcell.00043.2007.

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AGS and RGM-1 cells were observed under an electron microscope(JEOL 1200 EX2) (26).

Measurement of Mitochondrial Ca2� Concentration and ��m

Loading of cells with fluorescent indicators. Mitochondrial Ca2�

was traced in both AGS and RGM-1 cells using the mitochondrialCa2�-sensitive fluorescent dye rhod-2 AM. A cold/warm incubationprotocol (59, 67) was used to exclusively load mitochondria withrhod-2 AM. Briefly, cells (1 � 106 cells/sample) were washed usingHEPES buffer [containing (in mmol/l) 130 NaCl, 4.7 KCl, 1.2MgSO4, 1.2 KH2PO4, 10 HEPES, 11 glucose, and 0.2 CaCl2] at pH7.4 and then stained using 5 �mol/l rhod-2 AM for 120 min at 4°C,followed by a 10-min incubation at 37°C. To complete the deesteri-fication of intracellular rhod-2 AM, cells were washed using HEPESbuffer after being stained and stabilized for an additional 30 min.

To monitor ��m, AGS and RGM-1 cells were incubated for 20min with 200 nM of the cationic dye TMRE perchlorate (1). Cellswere then washed twice and centrifuged.

Confocal imaging. A confocal microscopy assay was performedwith laser scanning confocal microscope (LSCM 510 META) con-nected to an inverted microscope (Axiovert 200M, Carl Zeiss, Jena,Germany) with a �40 water-immersion objective lens, optimal laserlines, and filter. The fluorescence of TMRE was excited at 564 nm,and emitted signals were collected through a 600-nm long-pass filter.Rhod-2 AM was excited at 514 nm and emitted at 588 nm with along-pass filter. Images were digitized at 8 bits and analyzed usingLSCM 510 META software (Carl Zeiss).

FACS analysis. For FACS tracing of ��m, TMRE-loaded cellswere analyzed using FACScalibur (3,000 cells/sample, Becton Dick-inson). After 5 min at a stable state, the mitochondrial uncouplerprotein FCCP was added (1 �M final concentration), and ��m

depolarization was then monitored. The fluorescence intensity ofTMRE was monitored at 582 nm (FL-2 channel). FACS data wereanalyzed using Cell Quest Pro version 5.2.1(Becton Dickinson) (1).

Fluorimetric measurements. Fluorescence measurements were per-formed using a spectrofluorometer (Ratio Master, Photon Technology)at an excitation constant of 550 nm and an emission contant of 560nm. The mitochondrial Ca2� concentration ([Ca2�]m) was determinedas previously described (64). [Ca2�]m (in nmol/l) was calculatedusing the following equation: [Ca2�]m Kd � [(R Rmin)/(Rmax R)], where Kd (570 nmol/l) is a dissociation constant, R is themeasured fluorescence intensity (after background subtraction), Rmin

is the fluorescence at 0 mM Ca2� and was derived by exposing cellsto 25 �M digitonin in Ca2�-free HEPES buffer containing 10 �MEGTA, and Rmax is the fluorescence under saturating [Ca2�] (32 �M)and was derived by exposing cells to HEPES buffer containing 2.5mmol/l CaCI2 without EGTA. Acquired data were analyzed usingFelix version 1.43b (Photon Technology).

Mitochondria Oxygen Consumption Measurement

Respiration rates were measured in both cell types using 0.5-mlClark oxygen electrodes (Rank Brothers, Cambridge, UK) thermo-statted at 25°C and connected to a Kipp and Zonen dual-channel chartrecorder, assuming 250 nmol O2/ml at air saturation (53). Cellrespiration rates were expressed as a function of mitochondria in cells.

Mitochondria Protein Expression by 2-D GelElectrophoresis Proteomics

Preparation of mitochondria-enriched pellets. Collected AGS andRGM-1 cells were manually homogenized using a medium fittingglass-Teflon Potter-Elvehjem homogenizer in precooled mitochon-drial isolation buffer, which contained 50 mM sucrose, 200 mMmannitol, 5 mM potassium phosphate, 1 mM EGTA, 5 mM MOPS,and 0.1% BSA plus protease inhibitor cocktail at pH 7.4. The resultanthomogenate was centrifuged at 1,500 g for 5 min at 4°C. Afterward,

the supernatant was recentrifuged at 10,000 g for 10 min to obtainmitochondria-enriched pellets.

2-D gel electrophoresis proteomics of mitochondria proteins. Themitochondria-enriched pellet was dissolved in lysis buffer (7 M urea,2 M thiourea, 4% CHAPS, 40 mM Tris base, 1% DTT, 0.5% IPGbuffer, 0.5% Triton X-114, and protease inhibitor cocktail) and kept atroom temperature for 1 h. The protein content was assayed using a2-D Quant kit (GE Healthcare), and 2-D gel electrophoresis (2-DE)was performed as described by Berkelman and Stenstedt (5).

Each sample was run triplicate for a total of six gels. A 13-cm (pH3–10) IPG strip was rehydrated overnight at 30 V in 250 �l of anisoelectric focusing solution that contained �50 �g of the solubilizedproteins. Isoelectric focusing was carried out at 60,000 V/h at 20°C asfollows: 500 V for 1 h, 1,000 V for 1 h, and finally 8,000 Vincremented to 60,000 V/h. IPG strips were placed in 10 ml of anequilibration solution [50 mM Tris �HCl (pH 8.8) containing 6 M urea,30% glycerol, 2% SDS, and bromophenol blue] that contained 1%(vol/vol) DTT during the first equilibration step and 2.5% (vol/vol)iodoacetamide during the second equilibration step (15 min perequilibration step). The 2-D separation was performed using the SE600 Ruby electrophoresis set (Amersham). IPG strips were loadedonto a 12.5% SDS-PAGE gel, and running buffer (25 mM Tris, 192mM glycine, and 3.5 mM SDS at pH 8.3) was added. Low current (15mA/gel) was applied to the gel for first 15 min and increased up to 60mA for 5 h. Gels were then stained with silver nitrate. Stained gelswere scanned on a flatbed scanner (UMAX power look 1100), andimages were analyzed using commercially available software (ImageMaster 2D Platinum, Amersham Bioscience).

Protein identification. For mass spectrometry fingerprinting,stained portions of the 2-D gel (see Fig. 5) were excised and thendigested with trypsin as described by Rosenfeld et al. (57). Isolatedprotein spots were destained with 100 mM sodium thiosulfate and 30mM potassium ferricyanide. After being washed with 50% acetonitrile(ACN), gel fragments were dried in a vacuum centrifuge. Dried gelfragments were rehydrated in 20 �l of 25 mM NH4HCO3 thatcontained 0.5 �g of sequencing grade trypsin (Promega). Fragmentswere then incubated overnight at 37°C. Remaining peptides wereextracted twice with 30 �l of a 1:1 mixture of 50 mM NH4HCO3 andACN. Extracts were evaporated in a vacuum centrifuge for furtherdrying. Aliquots of the peptide-containing samples were applied to atarget disk, and aliquots were left to evaporate. Spectra were obtainedusing a Voyager DE PRO MALDI-MS (Applied Biosystems, FosterCity, CA). National Center for Biotechnology Information (NCBI)protein databases were searched with MASCOT PMF (http://www.matrixscience.com) using monoisotope peaks. A mass tolerancewithin 50 ppm was allowed initially, after which a recalibration wasperformed using the list of proteins that was obtained at 20 ppm.Proteins with Mowse scores over 63 were accepted as significant.

Statistical Analysis

Results are expressed as means � SE. Statistical significance wasestimated by Student’s t-test for comparison of two groups. Differ-ences were classified as significant at P � 0.05.

RESULTS

Mitochondria Shape and Numbers in AGS and RGM-1 Cells

Electron microscopy showed differences in the shape andnumbers of mitochondria in cells, where mitochondria ap-peared red. Figure 1 clearly shows that the region containingmitochondria in RGM-1 cells (number of mitochondria:23.5 � 4, n 6) was much larger than in AGS cells (numberof mitochondria: 16.3 � 3, n 6). Cancer cells contained 69%as many mitochondria as RGM-1 cells, and those mitochon-drial size was smaller than in normal cells (RGM-1 cells: 3.5 �

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0.3 �m2, n 6; and AGS cells: 1.3 � 0.5 �m2, n 6,respectively).

��m of AGS and RGM-1 Cells

We determined ��m in AGS and RGM-1 cells underlaser scanning confocal microscopy with TMRE staining(Fig. 2A). The intensity of the fluorescent TMRE probe wasused as an indirect measure of ��m in AGS and RGM-1cells (1). As shown in Fig. 2B, ��m of AGS cells(1,008.88 � 60.81, n 6) was higher than that of RGM-1cells (834.93 � 68.57, n 6). To support the above data, weadditionally carried out experiments using the FACS sys-tem. As shown in Fig. 2C, ��m of AGS cells (1,065.34 �34.52) was proved to be hyperpolarized compared with that

of RGM-1 cells (813.25 � 28.32), similar to results shownFig. 2, A and B. Furthermore, the application of FCCP (1�M) induced more rapid depolarization of ��m in AGScells than in RGM-1 cells (Fig. 2D). These results suggestedthat there were some changes of mitochondrial function inthe cancer cell line.

[Ca2�]m

[Ca2�]m in both AGS and RGM-1 cells was traced usingrhod-2 AM-loaded AGS and RGM-1 cells. The resulting in-tensity was an indirect measure of [Ca2�]m (34). As shown inFig. 3, A and B, the intensity of AGS cells (1,343.51 � 81.95,n 6) was higher than that in RGM-1 cells (1,054.62 � 43.28,n 6). Coincidently, fluorimetric measurements revealed that

Fig. 1. Electron microscopic images of normal(RGM-1) and cancer (AGS) cells (A) and a histo-gram of mitochondrial sizes and numbers (B). Boththe sizes and numbers of mitochondria were greaterin RGM-1 cells (3.5 � 0.3 �m, 23.5 � 4 mitochon-dria) than in AGS cells (1.3 � 0.5 �m, 16.3 � 3mitochondria). *P � 0.01.

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the calibrated [Ca2�]m of AGS cells (152.26 � 8.21 mM, n 6) was higher than that in RGM-1 cells (114.13 � 7.45 mM,n 6; Fig. 3C).

Mitochondria Oxygen Consumption

As shown in Fig. 4, the oxygen consumption was higher inthe normal cell line (2.6 � 0.2 nmol O2 �106 cells �min1)than in the cancer cell line (1.6 � 0.15 nmol O2 �106

cells �min1).

2-DE Proteomics Analysis

Mitochondrial-enriched fractions isolated from AGS andRGM-1 cells were used for 2DE proteomics analysis tocompare the expression of different mitochondrial proteins.

Using the automated spot-counting algorithm in Image Mas-ter 2D Platinum (Amersham Biosciences), 475 � 8.5 and401 � 22.3 protein spots were detected from AGS andRGM-1 cells, respectively. Expression patterns of mito-chondrial proteins are shown in Fig. 5A for RGM-1 cells andFig. 5B for AGS cells. Mitochondrial proteins from RGM-1and AGS cell lines were distributed in the region of pI 4-9and had relative molecular masses between 30 and 150 kDa.2-DE gel image analysis quantified the percent volume ofeach spot in both groups and revealed that the expression of37 spots common to AGS and RGM-1 cells changed signif-icantly by over 170%, including 20 spots that were down-regulated and 17 spots that were upregulated in the AGSgroup. These 37 spots were sampled using a spot picker

Fig. 2. Mitochondrial membrane potential(��m). A: confocal microscopic images of��m in normal and cancer cells. B: summaryof the experiments shown in A. C: FACSanalysis results of tetramethylrhodamineethyl ester (control) and FCCP-induced ��m

depolarization of normal and cancer cells.D: summary of the experiments shown in C.

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Fig. 3. Mitochondrial Ca2� concentration ([Ca2�]m)measured by rhod-2 AM fluorescence showing higher[Ca2�]m in AGS cells than in RGM-1 cells. A: confocalmicroscopic images of [Ca2�]m in normal and cancercells. B: summary of the experiments shown in A.C: fluoremetric measurements of [Ca2�]m in AGS andRGM-1 cells. *P � 0.05 vs. RGM-1 cells.

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(One Touch Plus Spot Picker, The Gel Company) andsubjected to matrix-assisted laser desorption/ionization-timeof flight (MALDI-TOF) mass spectroscopy protein identifi-cation analysis.

Protein Identification

Note that although the mitochondrial fraction was isolatedto enrich the mitochondrial yield as much as possible, theproteomics profiling results still covered a range of ex-pressed nonmitochondrial proteins. Thirty-seven selectedspots were subjected to MALDI-TOF mass spectroscopy forprotein identification. Peptide mass peaks were comparedwith those in the NCBI database (Fig. 6), and the proteinidentification data, including GenBank identification num-bers, molecular masses, pI values, Mowse scores, numbersof matched peptides, and sequence coverage ratios (%), areshown in Table 1. The 14 identified protein spots werecategorized into two groups based on their localization asmitochondrial or cytosolic proteins. The functions and ex-pression levels of the eight proteins that were upregulated(Fig. 7A) and the six proteins that were downregulated (Fig.7B) are shown in Table 2.

We identified significant changes of cancer-related pro-teins or cancer biomarkers in AGS cells compared withRGM-1 cells, including tumor rejection antigen (321%)(72), cytokeratin 8 protein (appear) (4, 25), cytokeratin 18protein (716%) (4), enolase 1 (57%) (51), testis-specificbromodomain protein (64%) (60), transferrin (63%)(22), and annexin I (88%) (44, 77). Stress-related andchaperoning proteins were highly expressed in the gastriccancer cell line, including heat shock protein (HSP)70(173%), HSP60 (appear), and mitochondrial translationelongation factor Tu protein (193%).

The altered energy metabolism in the cancer cell line wasreflected in the expression of various related proteins, includ-ing aldehyde reductase 1 (70%), mitochondrial enoyl-CoAhydratase 1 (996%), and ubiquinol-cytochrome c reductase(appear).

DISCUSSION

General Discussion

Understanding cancer progression is essential for the suc-cessful control of cancer. Since the common feature of malig-nancy is the ability of malignant cells to proliferate withoutrestraint, apoptosis is considered to be the predominant path-way for the elimination of malignant cells. Despite a previousstudy (29) of proteomics profiling in gastric tumors, the com-mon link between proteome alterations and the role of mito-chondria in apoptosis is still unclear. Mitochondria have dualfunctions in carcinogenesis, namely, cancer-associated changesin cellular metabolism: the Warburg effect (71, 73), whichaffects mitochondrial function, and the apoptosis-linked mito-chondrial permeability transition pore (MPTP). Destabilizingmitochondria as a key determinant of the point of no return toapoptosis is still under debate (56). Moreover, the mechanismsresponsible for the initiation and development of cancer, drugresistance, and disease progression remain to be elucidated.Therefore, understanding the changes in mitochondrial behav-

Fig. 5. Two-dimensional gel electrophoresis proteomic analysis of RGM-1 (A)and AGS (B) cells. Thirty-seven significantly changed spots (labeled green inAGS cells) were detected from a total of 475 � 8.5 spots. Mw, molecularweight.

Fig. 4. Oxygen consumption rates of AGS and RGM-1 cells. AGS cells had alower oxygen consumption rate than RGM-1 cells. *P � 0.01.

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ior in gastric cancer is a promising approach for developing acure.

In this study, we compared the mitochondria of gastriccancer cells and control cells. On the basis of several previousstudies (48, 74, 79), we compared a human gastric cancer-derived cell line (AGS) with a control rat gastric epithelial cellline (RGM-1) to build our models. Our data suggest a possiblecorrelation between differential proteomes and antiapoptoticcellular adaptations that lead gastric tumor cells toward appar-ent immortality.

A State of Cellular Hypoxia

It is well known that oxygen is the first determinant substratecontributing to mitochondrial performance and, hence, cellfunction and survival. The rate of oxygen consumption is adirect function of mitochondrial performance. Therefore, tu-

mor oxygen status is strongly associated with the growth,malignant progression, and resistance of a cancer tumor tovarious therapies. A previous study (69) has suggested thathypoxia enhances malignant progression and increase aggres-siveness. The reported decreased rate of oxygen consumptionlikely coincides with the decreased abundance and smaller sizeof mitochondria in the AGS cell line.

Mitochondrial Response to Apparent Cellular Hypoxia

Despite a decrease in the oxygen consumption rate andrelative low abundance of mitochondria, a marked overexpres-sion of ubiquinol-cytochrome c reductase in the gastric cancercell line was observed. Ubiquinol cytochrome c reductase, theRieske Fe-S protein, is a key subunit of the cytochrome bc1

complex (complex III) of the mitochondrial respiratory chain(55, 68). This complex generates an electrochemical potential

Fig. 6. Matrix-assisted laser desorption/ion-ization-time of flight mass spectroscopy spec-tra of ubiquinol-cytochrome c reductase. The 5peptides (red) were matched with the NationalCenter of Biotechnology Information databaseusing the MASCOT search program. The se-quence coverage was 11%, and the Mowsescore was 86. m/z, mass-to-charge ratio.

Table 1. Identification of significantly alternated proteins from AGS compared with RGM-1 cells

Spot No.GenBank

Identification No. Protein Identification Mr, kDa pI Mowse Score Match PeptideSeqence Coverage

Ratio, %

2 61656607 Tumor rejection antigen (gp96) 91.3 4.75 121 11 184 114326282 Transferrin 79.8 6.75 72 7 125 74143673 HSP70 72.4 5.07 128 12 207 77702086 Chaperonin-HSP60 61.1 5.7 190 14 349 62913980 KRT8 protein 41 4.94 163 15 42

12 13325287 Enolase 1 47.4 7.01 124 12 3013 30311 KRT18 47.3 5.27 109 8 2515 899285 Elongation factor Tu (mitochondrial) 49.8 7.26 126 8 2320 62020593 BRDT protein 53.1 9.34 73 6 1623 6978501 Annexin I 39.1 6.97 73 6 2225 6978491 Aldehyde reductase 1 36.2 6.26 62 6 1428 76642070 Nonmuscle myosin II 22.8 5.52 72 13 735 14286220 Mitochondrial short-chain enoyl-CoA hydratase 1 31.8 8.34 76 6 2937 45768728 Ubiquinol-cytochrome c reductase (Rieske

iron-sulfur polypeptide 1)29.9 8.55 86 5 11

Mr, relative mass; HSP, heat shock protein; KRT, cytokeratin; BRDT protein, testis-specific bromodomain protein.

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coupled to ATP synthesis to transfer electrons from ubiquinolto cytochrome c. Logically, the observed upregulation ofubiquinol-cytochrome c reductase, which is in agreement witha report (40) suggesting amplification of the ubiquinol-cyto-chrome c reductase gene in different kinds of tumors, matchedthe elevation of ��m, as shown in the results of the presentstudy. Whether this overexpression accompanies a defect in themitochondrial energetic potential due to a possible complex IIIdefect associated with mitochondrial DNA mutations (8) is stillan open question. Nevertheless, the discrepancy between thecellular tendency toward local hypoxia (as demonstrated by thedecreased respiration rate and reduced mitochondrial availabil-

ity) and the overexpression of the complex III component(ubiquinol-cytochrome c reductase) likely reflects a decrease inROS production as a cellular scavenger against apoptosis.Since a positive correlation between ROS production andapoptosis in other kinds of tumor has been demonstrated (41),this assumption needs further investigation to correlate the roleof ROS production with apoptosis in our AGS cell model.Apoptosis is strongly dependent on activation of the MPTP,which is a Ca2�-sensitive channel in the mitochondrial innermembrane that plays a crucial role in cell death. A novel MPTPmodel of an intrinsic mitochondrial pathway of apoptosis hasbeen recently proposed (3), which was regulated by a respira-

Fig. 7. Enlarged images with 3-dimensionalspot expression of 8 protein spots upregulated(A) and 6 protein spots that were downregu-lated (B) in AGS cells compared with RGM-1cells.

Table 2. Different protein expressions and their function in AGS compared with RGM-1 cells

Protein Identification �(A/R) Known Function

Mitochondrial proteinsSpot 7 Chaperonin-HSP60 Appear Mitochondrial matrix, apoptosis regulationSpot 15 Elongation factor Tu (mitochondrial) 193 Cell proliferation, mitochondria translationSpot 35 Mitochondrial short-chain enoyl-

CoA hydratase 1996 Fatty acid metabolism, fatty acid

�-oxidation enzymeSpot 37 Ubiquinol-cytochrome c reductase

(Rieske iron-sulfur polypeptide 1)Appear Mitochondria electron transfer chain complex 3

Cytosolic proteinsSpot 2 Tumor rejection antigen (gp96) 321 Chaperone (HSP90), cancer related, response to hypoxia,

antiapoptosisSpot 4 Transferrin 37 Glycoprotein, cancer-related cell proliferation, Fe3�

transportSpot 5 HSP70 173 Chaperone, antiapoptosisSpot 9 KRT8 protein Appear Cell proliferation, cancer-induced factor, antiapoptosis,

structural molecule activitySpot 12 Enolase 1 43 Carbohydrate metabolism, participates in glycolysis, cancer

markerSpot 13 KRT18 716 Cell proliferation, cancer-induced factor, antiapoptosisSpot 20 BRDT protein 36 Cancer-related proteinSpot 23 Annexin I 12 Ca2�/phospholipid binding, lipid metabolism, antiapopto-

sis, cell motility, cancer-related markerSpot 25 Aldehyde reductase 1 30 NADPH-dependent oxidoreductase, glucose metabolism,

aldehyde metabolismSpot 28 Nonmuscle myosin 14 32 Cytoskeletal cell motility

�(A/R), spot volume of AGS/RGM-1 cells � 100 (%).

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tory supercomplex formed by NADH:ubiquininone dehydro-genase, cytochrome bc1, and adenine nucleotide translocator(ANT). The model suggested that complex III regulates twodistinct pathways to the MPTP: one unregulated and involvingmitochondrial ROS, and the other regulated and activated byCa2�. As recently addressed in gastric tumors, the possibleoxidative damages to the redox centers of the respiratory chainthat promoted the development of cell hypoxia (11) and theobserved Ca2� overload clearly indicate the gastric tumorcells’ tolerance to Ca2� overload and possible ROS generation.Moreover, our data refute the role of Ca2� in triggeringmitochondria-mediated apoptosis. It is also possible to specu-late on the sensitivity of gastric cancer cells to ROS generationin our model.

The Mitochondrial Interplay With Neo-Homeostasis

The twofold increase in mitochondrial elongation factor Tuprovides evidence of translational machinery remodeling in theAGS cell line to meet its primitive prokaryotic form (21). Thisassumed remodeling requires 1) an alternative energy sourceand 2) backing by sound chaperone systems that keep pacewith the neo-homeostasis.

The increased rate of fatty acid degradation can meet the firstrequirement. Consistent with a recent study (78) postulatingthat mitochondrial short-chain enoyl-CoA hydratase 1 (EC4.2.1.17) overexpression is a function of carcinogenesis, ourdata clearly confirm its higher expression in gastric cancercells. The overexpression of this enzyme, which catalyzes thesecond step of the �-oxidation pathway, suggests a novelbiomarker for an aggressive type of gastric cell sarcoma andshows how cancer cells exploit an in-use energy source fortheir altered performance. We would expect the acceleratedrate of �-oxidation, together with the increased expression of acomplex III component that does not meet with the consequentincrease in oxygen consumption, to be related to the highcoupling efficiency of the cancer cell mitochondrial innermembrane. To confirm this assumption, however, further in-vestigation is required.

As biomarkers of carcinogenesis and components of cellularneo-homeostasis, HSPs are elevated during different kinds ofstress (12, 16, 46), including gastric infection and tumors (36).HSP60, a chaperonin, is an evolutionarily conserved 60-kDamitochondrial protein (27, 35) that affords protein chaperoningremodeling activity (31). Although the overexpression ofHSP60 has been reported in other tumors (47), the mechanismof its upregulation in our model is still controversial. Previousstudies (58, 76) have confirmed that unlike HSP70, which alsoshowed increased expression, HSP60 helps in the induction ofapoptosis by acting as a chaperone to pro-caspase-3 and aidingin its maturation into active caspase-3. HSP60 has both pro-and antiapoptotic roles in tumor cells (17, 19); therefore, it istempting to speculate whether its overexpression could serve asa proapoptotic regulator rather than an antiapoptotic regulator.

Moreover, our results provide additional evidence of neo-homeostatic regulation via the decreased expression of alde-hyde reductase 1, the first limiting enzyme of the polyolpathway. Many known cascades that induce or exacerbateintracellular oxidative stress accelerate the polyol pathway,e.g., glutathione depletion, increased superoxide accumulation,increased JNK activation, and DNA damage (10). This finding

strengthens the postulation that a tumor is susceptible to anyadverse oxidative stress.

Note that the disagreement between our finding and previousinvestigations suggests that the upregulation of glycolytic en-zyme enolase 1 expression in gastric adenocarcinoma (29) isattributable to the difference in the samples examined, since allthe tumors in the previous study were classified as intestinaltype tumors.

Cytoskeleton Remodeling as a Cellular Adaptation forNeo-Homeostasis

The cytoskeleton remodeling via downregulation of bothannexin I and nonmuscle myosin II supports a cellular adap-tation for neo-homeostasis. Annexin I belongs to a family ofcytosolic Ca2�-binding proteins that plays an important role inactin remodeling and polymerization (24, 28). Its decreasedexpression, which suggests gastric transformation, is very sim-ilar to data for breast cancer (61), prostate cancer (44), lym-phoma (70), and esophageal cancer (77). Moreover, deregu-lated annexin expression was recently observed in a highlyinvasive gastric cancer cell line (65). As recently noted (18),nonmuscle myosin II likely senses matrix elasticity. Therefore,its decreased expression is clearly indicative of reduced cellu-lar differentiation (45), which renders tumor cells more mal-leable at different possible surroundings.

Additional Cellular Adaptations for Neo-Homeostasis

Another observed adaptation was the increased expressionof transferrin, a specific iron transfer protein that regulates thecellular free iron level (49, 54). Although iron is essential forthe metabolism, viability, and proliferation of normal andcancer cells, it acts as a prooxidant that can damage biomol-ecules (22). Referring to the essential role of iron in cellmetabolism and viability, the increased expression of trans-ferrin is not surprising. Furthermore, the disruption of cellulariron homeostasis has been shown to inhibit the growth ofdifferent malignant cell types (7, 15, 75).

Expectedly, there was a marked expression of tumor rejec-tion antigen, which is very characteristic of gastric tumors (33).The overexpression of both tumor rejection antigen and cyto-keratin 18 in gastric cell tumors provides insight into possibleprognostic and diagnostic approachs for gastric cancer.

GRANTS

This work was supported by the Korean Government through KoreaResearch Foundation Grants KRF-2005-210-E00003, KRF-2005-211-E00006,KRF-2005-037-E00002, KRF-2005-042-E00010, KRF-2006-312-E00018,KRF-2006-312-C00601, KRF-2006-351-E00002, and KRF-2006-351-E00003by and Korea Institute of Industrial Technology Evaluation and Planningthrough the Biohealth Products Research Center of Inje University.

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