Mitochondrial alterations in human gastric carcinoma cell line

Hyoung Kyu Kim, Won Sun Park, Sung Hyun Kang, Mohamad Warda, Nari Kim, Jae-Hong Ko, Abd El-bary Prince and Jin Han
Am J Physiol Cell Physiol 293:C761-C771, 2007. First published 30 May 2007; doi: 10.1152/ajpcell.00043.2007 You might find this additional info useful... This article cites 80 articles, 20 of which you can access for free at: http://ajpcell.physiology.org/content/293/2/C761.full#ref-list-1
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Am J Physiol Cell Physiol 293: C761C771, 2007. First published May 30, 2007; doi:10.1152/ajpcell.00043.2007.

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
Mitochondrial Signaling Laboratory, Mitochondria Research Group, Department of Physiology and Biophysics, College of Medicine, 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 nal form 24 May 2007
1

Kim HK, Park WS, Kang SH, Warda M, Kim N, Ko J-H, Prince AE, Han J. Mitochondrial alterations in human gastric carcinoma cell line. Am J Physiol Cell Physiol 293: C761C771, 2007. First published May 30, 2007; doi:10.1152/ajpcell.00043.2007.We compared mitochondrial function, morphology, and proteome in the rat normal gastric cell line RGM-1 and the human gastric cancer cell line AGS. Total numbers and cross-sectional sizes of mitochondria were smaller in AGS cells. Mitochondria in AGS cells were deformed and consumed less oxygen. Confocal microscopy indicated that the mitochondrial inner membrane potential was hyperpolarized and the mitochondrial Ca2 concentration was elevated in AGS cells. Interestingly, two-dimensional electrophoresis proteomics on the mitochondria-enriched fraction revealed high expression of four mitochondrial proteins in AGS cells: ubiquinol-cytochrome c reductase, mitochondrial short-chain enoyl-coenzyme A hydratase-1, heat shock protein 60, and mitochondria elongation factor Tu. The results provide clues as to the mechanism of the mitochondrial changes in cancer at the protein level and may serve as potential cancer biomarkers in mitochondria. two-dimensional gel electrophoresis proteomics; biomarker; cancer
GASTRIC CANCER is the second most common malignant cancer after lung cancer worldwide (42, 43, 80), and advanced gastric cancer has a poor prognosis due to the limited efcacy of available therapies. Therefore, early diagnosis and better understanding of tumor progression and termination are essential for controlling this disease. Transcriptomics and proteomics afford a powerful and promising approach that allow for the comprehensive inspection of protein proling (20, 30, 82), which should disclose the suitability of various novel cancer biomarkers that contribute to an early diagnosis (6, 63). Consequently, proteomic studies on gastric 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 understand tumor development since Warburgs breakthrough discovery of mitochondrial injury in tumors (73). Consequently, studies have investigated the relationship between mitochondrial dysfunction and oncogenesis over the last 50 years to clarify the respiratory machinery impairment as a key event in carcinogenesis (73). Despite the increasing evidence implicating a mitochondrial defect or dysfunction as a key player in oncogenesis (2, 9, 13, 23), little is known of the alterations in mitochondrial structure and function that contribute to cellular

outcomes in gastric cell tumors. Therefore, we examined mitochondrial proteome alterations in gastric tumor cells and their correlation with mitochondrial function in the neo-homeostasis environment. This approach provides a better idea regarding the contribution of mitochondrial to neo-homeostasis in gastric cancer, and the impact of neo-homeostasis on mitochondrial proteome alterations may lead to the discovery of new biomarkers specic for mitochondrial involvement. Furthermore, the results suggest possible therapeutic interventions for gastric cancer that affect the mitochondrial membrane potential (m) and -oxidation pathway.
MATERIALS AND METHODS

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Cell Culture and Reagents Cells from the human gastric cancer cell line AGS were cultured in RPMI-1640 (Cambrex, Baltimore, MD) containing 10% FBS and 100 U/ml penicillin-streptomycin in a humidied 5% CO2 atmosphere. As countercells of AGS cells, normal rat gastric mucosal cells from cell line RGM-1 were cultured in DMEM-F-12 (Cambrex) under the same conditions (32). Solutions and Drugs Normal Tyrode solution contained (in mM) 143 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5.5 glucose, and 5 HEPES (pH 7.4) adjusted with NaOH. Fluorescent dyes [rhod-2 AM and tetramethylrhodamine ethyl ester (TMRE)] were purchased from Molecular Probes, prepared as 1 mM stock solutions in DMSO, and diluted into test solutions before each experiment. The nal concentration of DMSO in the bath solution (0.1%) has been previously proved not to affect recorded intensities. Acrylamide, methylene bis-acrylamide, glycine, Tris, urea, glycerol, CHAPS, thiourea, and bromophenol blue were purchased from Sigma (St. Louis, MO). N,N,N,N-tetramethylethylene diamine (TEMED), iodoacetamide, ammonium persulfate, DTT, and SDS were obtained from Merck (Darmstadt, Germany). IPG strips, IPG buffer, 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 membrane transfer kit were from Hoefer (San Francisco, CA). Immobilon-P membranes were purchased from Millipore (Bedford, MA). All other reagents used were of analytic grade. Mitochondrial Morphology Difference Comparisons by Electron Microscope Cells (1 106) were collected, washed twice with PBS, centrifuged for 5 min at 1,800 g, and glutaraldehyde xed for 2 4 h at 4C. Mitochondrial morphology differences, numbers, and shapes between

Address for reprint requests and other correspondence: J. Han, Mitochondrial Signaling Laboratory, Mitochondria Research Group, Dept. of Physiology and Biophysics, College of Medicine, Biohealth Products Research Center, Cardiovascular and Metabolic Disease Center, Inje Univ. 633-165 Gaegeum-Dong, Busanjin-Gu, Busan 613-735, Korea (e-mail: phyhanj@ijnc. inje.ac.kr). http://www.ajpcell.org

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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 uorescent indicators. Mitochondrial Ca2 was traced in both AGS and RGM-1 cells using the mitochondrial Ca2-sensitive uorescent dye rhod-2 AM. A cold/warm incubation protocol (59, 67) was used to exclusively load mitochondria with rhod-2 AM. Briey, cells (1 106 cells/sample) were washed using HEPES buffer [containing (in mmol/l) 130 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 10 HEPES, 11 glucose, and 0.2 CaCl2] at pH 7.4 and then stained using 5 mol/l rhod-2 AM for 120 min at 4C, followed by a 10-min incubation at 37C. To complete the deesterication of intracellular rhod-2 AM, cells were washed using HEPES buffer after being stained and stabilized for an additional 30 min. To monitor m, AGS and RGM-1 cells were incubated for 20 min with 200 nM of the cationic dye TMRE perchlorate (1). Cells were then washed twice and centrifuged. Confocal imaging. A confocal microscopy assay was performed with laser scanning confocal microscope (LSCM 510 META) connected to an inverted microscope (Axiovert 200M, Carl Zeiss, Jena, Germany) with a 40 water-immersion objective lens, optimal laser lines, and lter. The uorescence of TMRE was excited at 564 nm, and emitted signals were collected through a 600-nm long-pass lter. Rhod-2 AM was excited at 514 nm and emitted at 588 nm with a long-pass lter. Images were digitized at 8 bits and analyzed using LSCM 510 META software (Carl Zeiss). FACS analysis. For FACS tracing of m, TMRE-loaded cells were analyzed using FACScalibur (3,000 cells/sample, Becton Dickinson). After 5 min at a stable state, the mitochondrial uncoupler protein FCCP was added (1 M nal concentration), and m depolarization was then monitored. The uorescence intensity of TMRE was monitored at 582 nm (FL-2 channel). FACS data were analyzed using Cell Quest Pro version 5.2.1(Becton Dickinson) (1). Fluorimetric measurements. Fluorescence measurements were performed using a spectrouorometer (Ratio Master, Photon Technology) at an excitation constant of 550 nm and an emission contant of 560 nm. The mitochondrial Ca2 concentration ([Ca2]m) was determined as previously described (64). [Ca2]m (in nmol/l) was calculated using the following equation: [Ca2]m Kd [(R Rmin)/(Rmax R)], where Kd (570 nmol/l) is a dissociation constant, R is the measured uorescence intensity (after background subtraction), Rmin is the uorescence at 0 mM Ca2 and was derived by exposing cells to 25 M digitonin in Ca2-free HEPES buffer containing 10 M EGTA, and Rmax is the uorescence under saturating [Ca2] (32 M) and was derived by exposing cells to HEPES buffer containing 2.5 mmol/l CaCI2 without EGTA. Acquired data were analyzed using Felix version 1.43b (Photon Technology). Mitochondria Oxygen Consumption Measurement Respiration rates were measured in both cell types using 0.5-ml Clark oxygen electrodes (Rank Brothers, Cambridge, UK) thermostatted at 25C and connected to a Kipp and Zonen dual-channel chart recorder, assuming 250 nmol O2/ml at air saturation (53). Cell respiration rates were expressed as a function of mitochondria in cells. Mitochondria Protein Expression by 2-D Gel Electrophoresis Proteomics Preparation of mitochondria-enriched pellets. Collected AGS and RGM-1 cells were manually homogenized using a medium tting glass-Teon Potter-Elvehjem homogenizer in precooled mitochondrial isolation buffer, which contained 50 mM sucrose, 200 mM mannitol, 5 mM potassium phosphate, 1 mM EGTA, 5 mM MOPS, and 0.1% BSA plus protease inhibitor cocktail at pH 7.4. The resultant homogenate was centrifuged at 1,500 g for 5 min at 4C. Afterward,
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the supernatant was recentrifuged at 10,000 g for 10 min to obtain mitochondria-enriched pellets. 2-D gel electrophoresis proteomics of mitochondria proteins. The mitochondria-enriched pellet was dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris base, 1% DTT, 0.5% IPG buffer, 0.5% Triton X-114, and protease inhibitor cocktail) and kept at room temperature for 1 h. The protein content was assayed using a 2-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 (pH 310) IPG strip was rehydrated overnight at 30 V in 250 l of an isoelectric focusing solution that contained 50 g of the solubilized proteins. Isoelectric focusing was carried out at 60,000 V/h at 20C as follows: 500 V for 1 h, 1,000 V for 1 h, and nally 8,000 V incremented to 60,000 V/h. IPG strips were placed in 10 ml of an equilibration 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 rst equilibration step and 2.5% (vol/vol) iodoacetamide during the second equilibration step (15 min per equilibration step). The 2-D separation was performed using the SE 600 Ruby electrophoresis set (Amersham). IPG strips were loaded onto a 12.5% SDS-PAGE gel, and running buffer (25 mM Tris, 192 mM glycine, and 3.5 mM SDS at pH 8.3) was added. Low current (15 mA/gel) was applied to the gel for rst 15 min and increased up to 60 mA for 5 h. Gels were then stained with silver nitrate. Stained gels were scanned on a atbed scanner (UMAX power look 1100), and images were analyzed using commercially available software (Image Master 2D Platinum, Amersham Bioscience). Protein identication. For mass spectrometry ngerprinting, stained portions of the 2-D gel (see Fig. 5) were excised and then digested with trypsin as described by Rosenfeld et al. (57). Isolated protein spots were destained with 100 mM sodium thiosulfate and 30 mM potassium ferricyanide. After being washed with 50% acetonitrile (ACN), gel fragments were dried in a vacuum centrifuge. Dried gel fragments were rehydrated in 20 l of 25 mM NH4HCO3 that contained 0.5 g of sequencing grade trypsin (Promega). Fragments were then incubated overnight at 37C. Remaining peptides were extracted twice with 30 l of a 1:1 mixture of 50 mM NH4HCO3 and ACN. Extracts were evaporated in a vacuum centrifuge for further drying. Aliquots of the peptide-containing samples were applied to a target disk, and aliquots were left to evaporate. Spectra were obtained using a Voyager DE PRO MALDI-MS (Applied Biosystems, Foster City, CA). National Center for Biotechnology Information (NCBI) protein databases were searched with MASCOT PMF (http://www. matrixscience.com) using monoisotope peaks. A mass tolerance within 50 ppm was allowed initially, after which a recalibration was performed using the list of proteins that was obtained at 20 ppm. Proteins with Mowse scores over 63 were accepted as signicant. Statistical Analysis Results are expressed as means SE. Statistical signicance was estimated by Students t-test for comparison of two groups. Differences were classied as signicant at P 0.05.
RESULTS

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Mitochondria Shape and Numbers in AGS and RGM-1 Cells Electron microscopy showed differences in the shape and numbers of mitochondria in cells, where mitochondria appeared red. Figure 1 clearly shows that the region containing mitochondria in RGM-1 cells (number of mitochondria: 23.5 4, n 6) was much larger than in AGS cells (number of mitochondria: 16.3 3, n 6). Cancer cells contained 69% as many mitochondria as RGM-1 cells, and those mitochondrial size was smaller than in normal cells (RGM-1 cells: 3.5
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Fig. 1. Electron microscopic images of normal (RGM-1) and cancer (AGS) cells (A) and a histogram of mitochondrial sizes and numbers (B). Both the sizes and numbers of mitochondria were greater in RGM-1 cells (3.5 0.3 m, 23.5 4 mitochondria) than in AGS cells (1.3 0.5 m, 16.3 3 mitochondria). *P 0.01.

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 under laser scanning confocal microscopy with TMRE staining (Fig. 2A). The intensity of the uorescent TMRE probe was used as an indirect measure of m in AGS and RGM-1 cells (1). As shown in Fig. 2B, m of AGS cells (1,008.88 60.81, n 6) was higher than that of RGM-1 cells (834.93 68.57, n 6). To support the above data, we additionally carried out experiments using the FACS system. As shown in Fig. 2C, m of AGS cells (1,065.34 34.52) was proved to be hyperpolarized compared with that
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of RGM-1 cells (813.25 28.32), similar to results shown Fig. 2, A and B. Furthermore, the application of FCCP (1 M) induced more rapid depolarization of m in AGS cells than in RGM-1 cells (Fig. 2D). These results suggested that there were some changes of mitochondrial function in the cancer cell line. [Ca2]m [Ca2]m in both AGS and RGM-1 cells was traced using rhod-2 AM-loaded AGS and RGM-1 cells. The resulting intensity was an indirect measure of [Ca2]m (34). As shown in Fig. 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, uorimetric measurements revealed that
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Fig. 2. Mitochondrial membrane potential (m). A: confocal microscopic images of m in normal and cancer cells. B: summary of the experiments shown in A. C: FACS analysis results of tetramethylrhodamine ethyl ester (control) and FCCP-induced m depolarization of normal and cancer cells. D: summary of the experiments shown in C.

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 in the 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 and RGM-1 cells were used for 2DE proteomics analysis to compare the expression of different mitochondrial proteins.
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Using the automated spot-counting algorithm in Image Master 2D Platinum (Amersham Biosciences), 475 8.5 and 401 22.3 protein spots were detected from AGS and RGM-1 cells, respectively. Expression patterns of mitochondrial proteins are shown in Fig. 5A for RGM-1 cells and Fig. 5B for AGS cells. Mitochondrial proteins from RGM-1 and AGS cell lines were distributed in the region of pI 4-9 and had relative molecular masses between 30 and 150 kDa. 2-DE gel image analysis quantied the percent volume of each spot in both groups and revealed that the expression of 37 spots common to AGS and RGM-1 cells changed significantly by over 170%, including 20 spots that were downregulated and 17 spots that were upregulated in the AGS group. These 37 spots were sampled using a spot picker
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Fig. 3. Mitochondrial Ca2 concentration ([Ca2]m) measured by rhod-2 AM uorescence showing higher [Ca2]m in AGS cells than in RGM-1 cells. A: confocal microscopic images of [Ca2]m in normal and cancer cells. B: summary of the experiments shown in A. C: uoremetric measurements of [Ca2]m in AGS and RGM-1 cells. *P 0.05 vs. RGM-1 cells.

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General Discussion Understanding cancer progression is essential for the successful control of cancer. Since the common feature of malignancy is the ability of malignant cells to proliferate without restraint, apoptosis is considered to be the predominant pathway for the elimination of malignant cells. Despite a previous study (29) of proteomics proling in gastric tumors, the common link between proteome alterations and the role of mitochondria in apoptosis is still unclear. Mitochondria have dual functions in carcinogenesis, namely, cancer-associated changes in cellular metabolism: the Warburg effect (71, 73), which affects mitochondrial function, and the apoptosis-linked mitochondrial permeability transition pore (MPTP). Destabilizing mitochondria as a key determinant of the point of no return to apoptosis is still under debate (56). Moreover, the mechanisms responsible for the initiation and development of cancer, drug resistance, and disease progression remain to be elucidated. Therefore, understanding the changes in mitochondrial behav-

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

(One Touch Plus Spot Picker, The Gel Company) and subjected to matrix-assisted laser desorption/ionization-time of ight (MALDI-TOF) mass spectroscopy protein identication analysis. Protein Identication Note that although the mitochondrial fraction was isolated to enrich the mitochondrial yield as much as possible, the proteomics proling results still covered a range of expressed nonmitochondrial proteins. Thirty-seven selected spots were subjected to MALDI-TOF mass spectroscopy for protein identication. Peptide mass peaks were compared with those in the NCBI database (Fig. 6), and the protein identication data, including GenBank identication numbers, molecular masses, pI values, Mowse scores, numbers of matched peptides, and sequence coverage ratios (%), are shown in Table 1. The 14 identied protein spots were categorized into two groups based on their localization as mitochondrial or cytosolic proteins. The functions and expression 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 identied signicant changes of cancer-related proteins or cancer biomarkers in AGS cells compared with RGM-1 cells, including tumor rejection antigen (321%) (72), cytokeratin 8 protein (appear) (4, 25), cytokeratin 18 protein (716%) (4), enolase 1 (57%) (51), testis-specic bromodomain protein (64%) (60), transferrin (63%) (22), and annexin I (88%) (44, 77). Stress-related and chaperoning proteins were highly expressed in the gastric cancer cell line, including heat shock protein (HSP)70 (173%), HSP60 (appear), and mitochondrial translation elongation factor Tu protein (193%). The altered energy metabolism in the cancer cell line was reected in the expression of various related proteins, including aldehyde reductase 1 (70%), mitochondrial enoyl-CoA hydratase 1 (996%), and ubiquinol-cytochrome c reductase (appear).
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Fig. 5. Two-dimensional gel electrophoresis proteomic analysis of RGM-1 (A) and AGS (B) cells. Thirty-seven signicantly changed spots (labeled green in AGS cells) were detected from a total of 475 8.5 spots. Mw, molecular weight. www.ajpcell.org

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Fig. 6. Matrix-assisted laser desorption/ionization-time of ight mass spectroscopy spectra of ubiquinol-cytochrome c reductase. The 5 peptides (red) were matched with the National Center of Biotechnology Information database using the MASCOT search program. The sequence coverage was 11%, and the Mowse score was 86. m/z, mass-to-charge ratio.

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ior in gastric cancer is a promising approach for developing a cure. In this study, we compared the mitochondria of gastric cancer cells and control cells. On the basis of several previous studies (48, 74, 79), we compared a human gastric cancerderived cell line (AGS) with a control rat gastric epithelial cell line (RGM-1) to build our models. Our data suggest a possible correlation between differential proteomes and antiapoptotic cellular adaptations that lead gastric tumor cells toward apparent immortality. A State of Cellular Hypoxia It is well known that oxygen is the rst determinant substrate contributing to mitochondrial performance and, hence, cell function and survival. The rate of oxygen consumption is a direct function of mitochondrial performance. Therefore, tu-

mor oxygen status is strongly associated with the growth, malignant progression, and resistance of a cancer tumor to various therapies. A previous study (69) has suggested that hypoxia enhances malignant progression and increase aggressiveness. The reported decreased rate of oxygen consumption likely coincides with the decreased abundance and smaller size of mitochondria in the AGS cell line. Mitochondrial Response to Apparent Cellular Hypoxia Despite a decrease in the oxygen consumption rate and relative low abundance of mitochondria, a marked overexpression of ubiquinol-cytochrome c reductase in the gastric cancer cell line was observed. Ubiquinol cytochrome c reductase, the Rieske 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

Table 1. Identication of signicantly alternated proteins from AGS compared with RGM-1 cells
Spot No. GenBank Identication No. Protein Identication Mr, kDa pI Mowse Score Match Peptide Seqence Coverage Ratio, %

2 4 5 7 9 12 13 15 20 23 25 28 35 37

61656607 114326282 74143673 77702086 62913980 13325287 30311 899285 62020593 6978501 6978491 76642070 14286220 45768728

Tumor rejection antigen (gp96) Transferrin HSP70 Chaperonin-HSP60 KRT8 protein Enolase 1 KRT18 Elongation factor Tu (mitochondrial) BRDT protein Annexin I Aldehyde reductase 1 Nonmuscle myosin II Mitochondrial short-chain enoyl-CoA hydratase 1 Ubiquinol-cytochrome c reductase (Rieske iron-sulfur polypeptide 1)

91.3 79.8 72.4 61.1 41 47.4 47.3 49.8 53.1 39.1 36.2 22.8 31.8 29.9

4.75 6.75 5.07 5.7 4.94 7.01 5.27 7.26 9.34 6.97 6.26 5.52 8.34 8.55

121 72 128 190 163 124 109 126 73 73 62 72 76 86

11 7 12 14 15 12 8 8 6 6 6 13 6 5

18 12 20 34 42 30 25 23 16 22 14 7 29 11

Mr, relative mass; HSP, heat shock protein; KRT, cytokeratin; BRDT protein, testis-specic bromodomain protein. AJP-Cell Physiol VOL
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Fig. 7. Enlarged images with 3-dimensional spot expression of 8 protein spots upregulated (A) and 6 protein spots that were downregulated (B) in AGS cells compared with RGM-1 cells.

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coupled to ATP synthesis to transfer electrons from ubiquinol to cytochrome c. Logically, the observed upregulation of ubiquinol-cytochrome c reductase, which is in agreement with a report (40) suggesting amplication of the ubiquinol-cytochrome c reductase gene in different kinds of tumors, matched the elevation of m, as shown in the results of the present study. Whether this overexpression accompanies a defect in the mitochondrial energetic potential due to a possible complex III defect associated with mitochondrial DNA mutations (8) is still an open question. Nevertheless, the discrepancy between the cellular tendency toward local hypoxia (as demonstrated by the decreased respiration rate and reduced mitochondrial availabil-

ity) and the overexpression of the complex III component (ubiquinol-cytochrome c reductase) likely reects a decrease in ROS production as a cellular scavenger against apoptosis. Since a positive correlation between ROS production and apoptosis in other kinds of tumor has been demonstrated (41), this assumption needs further investigation to correlate the role of 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 inner membrane that plays a crucial role in cell death. A novel MPTP model of an intrinsic mitochondrial pathway of apoptosis has been recently proposed (3), which was regulated by a respira-

Table 2. Different protein expressions and their function in AGS compared with RGM-1 cells
Protein Identication (A/R) Known Function

Mitochondrial proteins Spot 7 Spot 15 Spot 35 Spot 37 Cytosolic proteins Spot 2 Spot 4 Spot 5 Spot 9 Spot 12 Spot 13 Spot 20 Spot 23 Spot 25 Spot 28

Chaperonin-HSP60 Elongation factor Tu (mitochondrial) Mitochondrial short-chain enoylCoA hydratase 1 Ubiquinol-cytochrome c reductase (Rieske iron-sulfur polypeptide 1) Tumor rejection antigen (gp96) Transferrin HSP70 KRT8 protein Enolase 1 KRT18 BRDT protein Annexin I Aldehyde reductase 1 Nonmuscle myosin 14

Appear 193 996 Appear 321 37 173 Appear 43 716 36 12 30 32

Mitochondrial matrix, apoptosis regulation Cell proliferation, mitochondria translation Fatty acid metabolism, fatty acid -oxidation enzyme Mitochondria electron transfer chain complex 3 Chaperone (HSP90), cancer related, response to hypoxia, antiapoptosis Glycoprotein, cancer-related cell proliferation, Fe3 transport Chaperone, antiapoptosis Cell proliferation, cancer-induced factor, antiapoptosis, structural molecule activity Carbohydrate metabolism, participates in glycolysis, cancer marker Cell proliferation, cancer-induced factor, antiapoptosis Cancer-related protein Ca2/phospholipid binding, lipid metabolism, antiapoptosis, cell motility, cancer-related marker NADPH-dependent oxidoreductase, glucose metabolism, aldehyde metabolism Cytoskeletal cell motility

(A/R), spot volume of AGS/RGM-1 cells 100 (%). AJP-Cell Physiol VOL
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tory supercomplex formed by NADH:ubiquininone dehydrogenase, cytochrome bc1, and adenine nucleotide translocator (ANT). The model suggested that complex III regulates two distinct pathways to the MPTP: one unregulated and involving mitochondrial ROS, and the other regulated and activated by Ca2. As recently addressed in gastric tumors, the possible oxidative damages to the redox centers of the respiratory chain that promoted the development of cell hypoxia (11) and the observed Ca2 overload clearly indicate the gastric tumor cells tolerance to Ca2 overload and possible ROS generation. Moreover, our data refute the role of Ca2 in triggering mitochondria-mediated apoptosis. It is also possible to speculate on the sensitivity of gastric cancer cells to ROS generation in our model. The Mitochondrial Interplay With Neo-Homeostasis The twofold increase in mitochondrial elongation factor Tu provides evidence of translational machinery remodeling in the AGS cell line to meet its primitive prokaryotic form (21). This assumed remodeling requires 1) an alternative energy source and 2) backing by sound chaperone systems that keep pace with the neo-homeostasis. The increased rate of fatty acid degradation can meet the rst requirement. Consistent with a recent study (78) postulating that mitochondrial short-chain enoyl-CoA hydratase 1 (EC 4.2.1.17) overexpression is a function of carcinogenesis, our data clearly conrm its higher expression in gastric cancer cells. The overexpression of this enzyme, which catalyzes the second step of the -oxidation pathway, suggests a novel biomarker for an aggressive type of gastric cell sarcoma and shows how cancer cells exploit an in-use energy source for their altered performance. We would expect the accelerated rate of -oxidation, together with the increased expression of a complex III component that does not meet with the consequent increase in oxygen consumption, to be related to the high coupling efciency of the cancer cell mitochondrial inner membrane. To conrm this assumption, however, further investigation is required. As biomarkers of carcinogenesis and components of cellular neo-homeostasis, HSPs are elevated during different kinds of stress (12, 16, 46), including gastric infection and tumors (36). HSP60, a chaperonin, is an evolutionarily conserved 60-kDa mitochondrial protein (27, 35) that affords protein chaperoning remodeling activity (31). Although the overexpression of HSP60 has been reported in other tumors (47), the mechanism of its upregulation in our model is still controversial. Previous studies (58, 76) have conrmed that unlike HSP70, which also showed increased expression, HSP60 helps in the induction of apoptosis by acting as a chaperone to pro-caspase-3 and aiding in its maturation into active caspase-3. HSP60 has both proand antiapoptotic roles in tumor cells (17, 19); therefore, it is tempting to speculate whether its overexpression could serve as a proapoptotic regulator rather than an antiapoptotic regulator. Moreover, our results provide additional evidence of neohomeostatic regulation via the decreased expression of aldehyde reductase 1, the rst limiting enzyme of the polyol pathway. Many known cascades that induce or exacerbate intracellular oxidative stress accelerate the polyol pathway, e.g., glutathione depletion, increased superoxide accumulation, increased JNK activation, and DNA damage (10). This nding
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strengthens the postulation that a tumor is susceptible to any adverse oxidative stress. Note that the disagreement between our nding and previous investigations suggests that the upregulation of glycolytic enzyme enolase 1 expression in gastric adenocarcinoma (29) is attributable to the difference in the samples examined, since all the tumors in the previous study were classied as intestinal type tumors. Cytoskeleton Remodeling as a Cellular Adaptation for Neo-Homeostasis
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The cytoskeleton remodeling via downregulation of both annexin I and nonmuscle myosin II supports a cellular adaptation for neo-homeostasis. Annexin I belongs to a family of cytosolic Ca2-binding proteins that plays an important role in actin remodeling and polymerization (24, 28). Its decreased expression, which suggests gastric transformation, is very similar to data for breast cancer (61), prostate cancer (44), lymphoma (70), and esophageal cancer (77). Moreover, deregulated annexin expression was recently observed in a highly invasive 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 cellular differentiation (45), which renders tumor cells more malleable at different possible surroundings. Additional Cellular Adaptations for Neo-Homeostasis Another observed adaptation was the increased expression of transferrin, a specic iron transfer protein that regulates the cellular free iron level (49, 54). Although iron is essential for the metabolism, viability, and proliferation of normal and cancer cells, it acts as a prooxidant that can damage biomolecules (22). Referring to the essential role of iron in cell metabolism and viability, the increased expression of transferrin is not surprising. Furthermore, the disruption of cellular iron homeostasis has been shown to inhibit the growth of different malignant cell types (7, 15, 75). Expectedly, there was a marked expression of tumor rejection antigen, which is very characteristic of gastric tumors (33). The overexpression of both tumor rejection antigen and cytokeratin 18 in gastric cell tumors provides insight into possible prognostic and diagnostic approachs for gastric cancer.
GRANTS This work was supported by the Korean Government through Korea Research 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-E00003 by and Korea Institute of Industrial Technology Evaluation and Planning through the Biohealth Products Research Center of Inje University. REFERENCES 1. Akao M, ORourke B, Teshima Y, Seharaseyon J, Marban E. Mechanistically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes. Circ Res 92: 186 194, 2003. 2. Alirol E, Martinou JC. Mitochondria and cancer: is there a morphological connection? Oncogene 25: 4706 4716, 2006. 3. Armstrong JS, Yang H, Duan W, Whiteman M. Cytochrome bc1 regulates the mitochondrial permeability transition by two distinct pathways. J Biol Chem 279: 50420 50428, 2004. 4. Athanassiadou P, Psyhoyiou H, Grapsa D, Gonidi M, Ketikoglou I, Patsouris E. Cytokeratin 8 and 18 expression in imprint smears of chronic www.ajpcell.org

293 AUGUST 2007

C770

MITOCHONDRIAL ALTERATIONS IN HUMAN GASTRIC CANCER CELL 30. He QY, Chiu JF. Proteomics in biomarker discovery and drug development. J Cell Biochem 89: 868 886, 2003. 31. Hemmingsen SM, Woolford C, van der Vies SM, Tilly K, Dennis DT, Georgopoulos CP, Hendrix RW, Ellis RJ. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333: 330 334, 1988. 32. Huang J, Zhou H, Mahavadi S, Sriwai W, Lyall V, Murthy KS. Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors. Am J Physiol Gastrointest Liver Physiol 288: G23G31, 2005. 33. Inoue H, Mori M, Honda M, Li J, Shibuta K, Mimori K, Ueo H, Akiyoshi T. The expression of tumor-rejection antigen MAGE genes in human gastric carcinoma. Gastroenterology 109: 15221525, 1995. 34. Kang SH, Park WS, Kim N, Youm JB, Warda M, Ko JH, Ko EA, Han J. Mitochondrial Ca2-activated K channels are more efcient at reducing mitochondrial Ca2 overload in rat ventricular myocytes. Am J Physiol Heart Circ Physiol. First published March 9, 2007; doi:10.1152/ajpheart.00789.2006. 35. Karlin S, Brocchieri L. Heat shock protein 60 sequence comparisons: duplications, lateral transfer, and mitochondrial evolution. Proc Natl Acad Sci USA 97: 11348 11353, 2000. 36. Kobayashi K, Yokota K, Yoshino T, Kawahara Y, Dey A, Hirai Y, Oguma K, Akagi T. Detection of Helicobacter pylori associated antigen and heat shock protein 60 on follicular dendritic cells in the germinal centres of low grade B cell lymphoma of gastric mucosa associated lymphoid tissue (MALT). J Clin Pathol 51: 396 398, 1998. 37. Li N, Guo R, Li W, Shao J, Li S, Zhao K, Chen X, Xu N, Liu S, Lu Y. A proteomic investigation into a human gastric cancer cell line BGC823 treated with diallyl trisulde. Carcinogenesis 27: 12221231, 2006. 38. Liu W, Liu B, Xin L, Zhang Y, Chen X, Zhu Z, Lin Y. Down-regulated expression of complement factor I: a potential suppressive protein for gastric cancer identied by serum proteome analysis. Clin Chim Acta 377: 119 126, 2007. 39. Melle C, Ernst G, Schimmel B, Bleul A, Kaufmann R, Hommann M, Richter KK, Daffner W, Settmacher U, Claussen U, von Eggeling F. Characterization of pepsinogen C as a potential biomarker for gastric cancer using a histo-proteomic approach. J Proteome Res 4: 1799 1804, 2005. 40. Ohashi Y, Kaneko SJ, Cupples TE, Young SR. Ubiquinol cytochrome c reductase (UQCRFS1) gene amplication in primary breast cancer core biopsy samples. Gynecol Oncol 93: 54 58, 2004. 41. Pan MH, Huang YT, Ho CT, Chang CI, Hsu PC, Sun Pan B. Induction of apoptosis by Meretrix lusoria through reactive oxygen species production, glutathione depletion, and caspase activation in human leukemia cells. Life Sci 79: 1140 1152, 2006. 42. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 55: 74 108, 2005. 43. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 94: 153156, 2001. 44. Paweletz CP, Ornstein DK, Roth MJ, Bichsel VE, Gillespie JW, Calvert VS, Vocke CD, Hewitt SM, Duray PH, Herring J, Wang QH, Hu N, Linehan WM, Taylor PR, Liotta LA, Emmert-Buck MR, Petricoin EF 3rd. Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma. Cancer Res 60: 6293 6297, 2000. 45. Pelham RJ Jr, Wang Y. Cell locomotion and focal adhesions are regulated by substrate exibility. Proc Natl Acad Sci USA 94: 13661 13665, 1997. 46. Pster G, Stroh CM, Perschinka H, Kind M, Knoach M, Hinterdorfer P, Wick G. Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy. J Cell Sci 118: 15871594, 2005. 47. Pignatelli D, Ferreira J, Soares P, Costa MJ, Magalhaes MC. Immunohistochemical study of heat shock proteins 27, 60 and 70 in the normal human adrenal and in adrenal tumors with suppressed ACTH production. Microsc Res Tech 61: 315323, 2003. 48. Pohle T, Becker JC, Markmann A, Lugering N, Pauels HG, Konturek JW, Domschke W. Aspirin effects on gastric epithelial cell proliferation and cytokine expression. Microsc Res Tech 53: 354 359, 2001. 49. Poon TC, Sung JJ, Chow SM, Ng EK, Yu AC, Chu ES, Hui AM, Leung WK. Diagnosis of gastric cancer by serum proteomic ngerprinting. Gastroenterology 130: 1858 1864, 2006. www.ajpcell.org

5. 6. 7. 8.

9. 10. 11. 12. 13. 14.

15.

16. 17. 18. 19.

20. 21. 22.

23. 24. 25. 26. 27. 28. 29.

viral hepatitis, autoimmune hepatitis and hepatocellular carcinoma. A preliminary study. Acta Cytol 51: 61 65, 2007. Berkelman T, Stenstedt T. 2-D electrophoresis, using immobilized pH gradients, principles and methods. Amersham Pharmacia Biotech, 1998, p. 14 36. Bichsel VE, Liotta LA, Petricoin EF 3rd. Cancer proteomics: from biomarker discovery to signal pathway proling. Cancer J 7: 69 78, 2001. Blatt J, Taylor SR, Kontoghiorghes GJ. Comparison of activity of deferoxamine with that of oral iron chelators against human neuroblastoma cell lines. Cancer Res 49: 29252927, 1989. Bonora E, Porcelli AM, Gasparre G, Biondi A, Ghelli A, Carelli V, Baracca A, Tallini G, Martinuzzi A, Lenaz G, Rugolo M, Romeo G. Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III. Cancer Res 66: 6087 6096, 2006. Brandon M, Baldi P, Wallace DC. Mitochondrial mutations in cancer. Oncogene 25: 4647 4662, 2006. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813 820, 2001. Burlaka AP, Sidorik EP, Ganusevich II, Osinsky SP. Effects of radical oxygen species and NO: formation of intracellular hypoxia and activation of matrix metalloproteinases in tumor tissues. Exp Oncol 28: 49 53, 2006. Cappello F, Zummo G. HSP60 expression during carcinogenesis: a molecular proteus of carcinogenesis? Cell Stress Chaperones 10: 263 264, 2005. Chatterjee A, Mambo E, Sidransky D. Mitochondrial DNA mutations in human cancer. Oncogene 25: 4663 4674, 2006. Chen J, Kahne T, Rocken C, Gotze T, Yu J, Sung JJ, Chen M, Hu P, Malfertheiner P, Ebert MP. Proteome analysis of gastric cancer metastasis by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identication of metastasisrelated proteins. J Proteome Res 3: 1009 1016, 2004. Chitambar CR, Matthaeus WG, Antholine WE, Graff K, OBrien WJ. Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. Blood 72: 1930 1936, 1988. Crouser ED, Julian MW, Huff JE, Mandich DV, Green-Church KB. A proteomic analysis of liver mitochondria during acute endotoxemia. Intensive Care Med 32: 12521262, 2006. Di Felice V, David S, Cappello F, Farina F, Zummo G. Is chlamydial heat shock protein 60 a risk factor for oncogenesis? Cell Mol Life Sci 62: 4 9, 2005. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specication. Cell 126: 677 689, 2006. Faried A, Sohda M, Nakajima M, Miyazaki T, Kato H, Kuwano H. Expression of heat-shock protein Hsp60 correlated with the apoptotic index and patient prognosis in human oesophageal squamous cell carcinoma. Eur J Cancer 40: 2804 2811, 2004. Feder ME, Walser JC. The biological limitations of transcriptomics in elucidating stress and stress responses. J Evol Biol 18: 901910, 2005. Furano AV. Content of elongation factor Tu in Escherichia coli. Proc Natl Acad Sci USA 72: 4780 4784, 1975. Gackowski D, Kruszewsk M, Banaszkiewicz Z, Jawien A, Olinski R. Lymphocyte labile iron pool, plasma iron, transferrin saturation and ferritin levels in colon cancer patients. Acta Biochim Pol 49: 269 272, 2002. Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 25: 4812 4830, 2006. Gerke V, Creutz CE, Moss SE. Annexins: linking Ca2 signalling to membrane dynamics. Nat Rev Mol Cell Biol 6: 449 461, 2005. Gires O, Mack B, Rauch J, Matthias C. CK8 correlates with malignancy in leukoplakia and carcinomas of the head and neck. Biochem Biophys Res Commun 343: 252259, 2006. Ha MW, Ma R, Shun LP, Gong YH, Yuan Y. Effects of allitridi on cell cycle arrest of human gastric cancer cells. World J Gastroenterol 11: 54335437, 2005. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 18521858, 2002. Hayes MJ, Rescher U, Gerke V, Moss SE. Annexin-actin interactions. Trafc 5: 571576, 2004. He QY, Cheung YH, Leung SY, Yuen ST, Chu KM, Chiu JF. Diverse proteomic alterations in gastric adenocarcinoma. Proteomics 4: 3276 3287, 2004. AJP-Cell Physiol VOL

Downloaded from http://ajpcell.physiology.org/ at Univ Cattolica Del Sacro Cuore-Bibl on June 4, 2013

293 AUGUST 2007

MITOCHONDRIAL ALTERATIONS IN HUMAN GASTRIC CANCER CELL 50. Qian HG, Shen J, Ma H, Ma HC, Su YH, Hao CY, Xing BC, Huang XF, Shou CC. Preliminary study on proteomics of gastric carcinoma and its clinical signicance. World J Gastroenterol 11: 6249 6253, 2005. 51. Racz A, Brass N, Hofer M, Sybrecht GW, Remberger K, Meese EU. Gene amplication at chromosome 1pter-p33 including the genes PAX7 and ENO1 in squamous cell lung carcinoma. Int J Oncol 17: 6773, 2000. 52. Ren H, Du N, Liu G, Hu HT, Tian W, Deng ZP, Shi JS. Analysis of variabilities of serum proteomic spectra in patients with gastric cancer before and after operation. World J Gastroenterol 12: 2789 2792, 2006. 53. Reynafarje B, Costa LE, Lehninger AL. O2 solubility in aqueous media determined by a kinetic method. Anal Biochem 145: 406 418, 1985. 54. Richardson DR, Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331: 1 40, 1997. 55. Rieske JS. Composition, structure, and function of complex III of the respiratory chain. Biochim Biophys Acta 456: 195247, 1976. 56. Riles WL, Erickson J, Nayyar S, Atten MJ, Attar BM, Holian O. Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells. World J Gastroenterol 12: 5628 5634, 2006. 57. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203: 173179, 1992. 58. Samali A, Cai J, Zhivotovsky B, Jones DP, Orrenius S. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells. EMBO J 18: 2040 2048, 1999. 59. Sato T, Saito T, Saegusa N, Nakaya H. Mitochondrial Ca2-activated K channels in cardiac myocytes: a mechanism of the cardioprotective effect and modulation by protein kinase A. Circulation 111: 198 203, 2005. 60. Scanlan MJ, Altorki NK, Gure AO, Williamson B, Jungbluth A, Chen YT, Old LJ. Expression of cancer-testis antigens in lung cancer: denition of bromodomain testis-specic gene (BRDT) as a new CT gene, CT9. Cancer Lett 150: 155164, 2000. 61. Shen D, Chang HR, Chen Z, He J, Lonsberry V, Elshimali Y, Chia D, Seligson D, Goodglick L, Nelson SF, Gornbein JA. Loss of annexin A1 expression in human breast cancer detected by multiple high-throughput analyses. Biochem Biophys Res Commun 326: 218 227, 2005. 62. Shi P, Huang Z. Proteomic detection of changes in protein synthesis induced by lanthanum in BGC-823 human gastric cancer cells. Biometals 18: 89 95, 2005. 63. Srinivas PR, Srivastava S, Hanash S, Wright GL Jr. Proteomics in early detection of cancer. Clin Chem 47: 19011911, 2001. 64. Sun H, Wang N, Halkos ME, Kerendi F, Kin H, Wang R, Guyton RA, Zhao Z. Involvement of Na/H exchanger in hypoxia/re-oxygenationinduced neonatal rat cardiomyocyte apoptosis. Eur J Pharmacol 486: 121131, 2004. 65. Takikawa M, Akiyama Y, Maruyama K, Suzuki A, Liu F, Tai S, Ohshita C, Kawaguchi Y, Bandou E, Yonemura Y, Yamaguchi K. Proteomic analysis of a highly metastatic gastric cancer cell line using two-dimensional differential gel electrophoresis. Oncol Rep 16: 705711, 2006. 66. Tomlinson AJ, Hincapie M, Morris GE, Chicz RM. Global proteome analysis of a human gastric carcinoma. Electrophoresis 23: 32333240, 2002.

C771

67. Trollinger DR, Cascio WE, Lemasters JJ. Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca2-indicating uorophores. Biophys J 79: 39 50, 2000. 68. Trumpower BL, Edwards CA. Purication of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate cytochrome c reductase complex of bovine heart mitochondria. J Biol Chem 254: 8697 8706, 1979. 69. Vaupel P, Kelleher DK, Hockel M. Oxygen status of malignant tumors: pathogenesis of hypoxia and signicance for tumor therapy. Semin Oncol 28: 29 35, 2001. 70. Vishwanatha JK, Salazar E, Gopalakrishnan VK. Absence of annexin I expression in B-cell non-Hodgkins lymphomas and cell lines. BMC Cancer 4: 8, 2004. 71. Wallace DC. Mitochondria and cancer: Warburg addressed. Cold Spring Harb Symp Quant Biol 70: 363374, 2005. 72. Wang XP, Qiu FR, Liu GZ, Chen RF. Correlation between clinicopathology and expression of heat shock protein 70 and glucose-regulated protein 94 in human colonic adenocarcinoma. World J Gastroenterol 11: 1056 1059, 2005. 73. Warburg O. On respiratory impairment in cancer cells. Science 124: 269 270, 1956. 74. Wei W, Wang G, Qi X, Englander EW, Greeley GH Jr. Characterization and regulation of the rat and human ghrelin promoters. Endocrinology 146: 16111625, 2005. 75. White S, Taetle R, Seligman PA, Rutherford M, Trowbridge IS. Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects. Cancer Res 50: 6295 6301, 1990. 76. Xanthoudakis S, Roy S, Rasper D, Hennessey T, Aubin Y, Cassady R, Tawa P, Ruel R, Rosen A, Nicholson DW. Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis. EMBO J 18: 2049 2056, 1999. 77. Xia SH, Hu LP, Hu H, Ying WT, Xu X, Cai Y, Han YL, Chen BS, Wei F, Qian XH, Cai YY, Shen Y, Wu M, Wang MR. Three isoforms of annexin I are preferentially expressed in normal esophageal epithelia but down-regulated in esophageal squamous cell carcinomas. Oncogene 21: 6641 6648, 2002. 78. Yeh CS, Wang JY, Cheng TL, Juan CH, Wu CH, Lin SR. Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by microarray-bioinformatics analysis. Cancer Lett 233: 297308, 2006. 79. Yeo M, Kim DK, Kim YB, Oh TY, Lee JE, Cho SW, Kim HC, Hahm KB. Selective induction of apoptosis with proton pump inhibitor in gastric cancer cells. Clin Cancer Res 10:8687 8696, 2004. 80. Zanghieri G, Di Gregorio C, Sacchetti C, Fante R, Sassatelli R, Cannizzo G, Carriero A, Ponz de Leon M. Familial occurrence of gastric cancer in the 2-year experience of a population-based registry. Cancer 66: 20472051, 1990. 81. Zhang G, Wang G, Wang S, Li Q, Ouyang G, Peng X. Applying proteomic methodologies to analyze the effect of hexamethylene bisacetamide (HMBA) on proliferation and differentiation of human gastric carcinoma BGC-823 cells. Int J Biochem Cell Biol 36: 16131623, 2004. 82. Zhu H, Bilgin M, Snyder M. Proteomics. Annu Rev Biochem 72: 783 812, 2003.

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