• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Different Glucose Uptake and Glycolytic Mechanisms Between Hepatocellular Carcinoma and Intrahepatic Mass-Forming Cholangiocarcinoma With Increased ^Sup 18^F-FDG Uptake

Posted on: Sunday, 23 October 2005, 03:01 CDT

By Lee, Jong Doo; Yang, Woo Ick; Park, Young Nyun; Kim, Kyung Sik; Et al

^sup 18^F-FDG uptake in malignant tumors largely depends on the presence of facilitated glucose transporters, especially type 1 (Glut 1) and a rate-limiting glycolytic enzyme, hexokinase (HK) type II. Low expression of Glut 1 was reported in hepatocellular carcinoma (HCC), whereas high expression was found in cholangiocarcinoma. Immunohistochemistry and proteome analysis were performed to obtain a detailed evaluation of the mechanisms involved in glucose uptake and use in these tumors. Methods: Tumor tissues obtained from both HCC (n = 7) and mass-forming cholangiocarcinoma patients (n = 7) who showed increased ^sup 18^F-FDG uptake on PET were used, lmmunohistochemistry for Glut 1 and HK I-III was performed in all tumor tissues. To identify proteins that regulate carbohydrate metabolism, a proteome analysis with matrix-assisted laser desorption ionization-time of flight and enzymatic digestion in-gel were performed using 8 available tumor samples and 3 normal liver tissues. Of the 8 tumor samples, 4 were HCCs; one was an intermediate phenotype HCC, and 3 were cholangiocarcinomas. The spot intensity of the proteins was calculated using proteome data; the tissues then were divided into 2 groups on the basis of the protein expression pattern, because the protein expression pattern of the intermediate-phenotype HCC was close to that of the cholangiocarcinomas. Group A included the HCCs and group B included the intermediate-phenotype HCC as well as the cholangiocarcinomas. Results: Immunoreactivity for Glut 1 was positive in all cholangiocarcinomas, but was negative in all HCCs except the one intermediate phenotype. However, HK II was positive in HCCs but was negative in 6 of the 7 cholangiocarcinomas. A total of 331 protein spots with a P value of <0.05 were identified by proteome analysis. Thirteen of these proteins that regulate carbohydrate metabolism were selected. The pentose phosphate pathway was increased in both groups, but more significantly in group B. Gluconeogenesis enzymes were decreased in both groups, but the tricarboxylic acid cycle- regulating enzyme expression was variable. Conclusion: HCCs have different glucose-regulating mechanisms from those of cholangiocarcinomas, even though both tumors showed increased ^sup 18^F-FDG uptake on PET scans. Further studies are required with regard to energy metabolism and ^sup 18^F-FDG uptake patterns in association with various oncogenic alterations regulating multiple steps of the glycolytic pathways.

Key Words: ^sup 18^F-FDG; glucose metabolism; hepatocellular carcinoma; cholangiocarcinoma

J Nucl Med 2005; 46:1753-1759

An past years, ^sup 18^F-FDG PET has been widely used clinically in the field of oncology because of its high sensitivity and specificity, and it also has significant potential for assessing therapy response (1). Because cancer cell growth is heavily dependent on glucose metabolism as a major energy substrate, the ^sup 18^F-FDG uptake pattern on PET scans that reflects glycolytic activity could be an indicator of the prognosis and aggressiveness of the tumors. In addition, the association of neoplastic growth with increased aerobic glycolysis has been known as the Warburg effect for more than 7 decades (2). However, ^sup 18^F-FDG uptake patterns in primary hepatic tumors are variable. Increased uptake can be seen in only half of hepatocellular carcinoma (HCC) patients (J); mass-forming intrahepatic cholangiocarcinomas show intense ^sup 18^F-FDG uptake, whereas hilar cholangiocarcinomas show only mild uptake (4).

In our previous study, the ^sup 18^F-FDG uptake was closely correlated with the pathologic grading in HCC. HCCs with increased ^sup 18^F-FDG uptake showed molecular features of more aggressive biologic properties than those with a low ^sup 18^F-FDG uptake (5). However, the glucose uptake pattern and its clinical implications have not been evaluated in cholangiocarcinomas.

^sup 18^F-FDG uptake in malignant tumors depends largely on the presence of facilitated glucose transporters, including type 1 (Glut 1) and a rate-limiting glycolytic enzyme, hexokinase (HK) type II (6). In previous reports, it was demonstrated that Glut 1 is highly expressed in cholangiocarcinomas but rarely expressed in HCCs (7,8). In contrast, HK II expression is increased in HCCs (9) but unknown in cholangiocarcinomas. These findings suggest that glucose uptake and glycolytic mechanisms could differ between HCCs and cholangiocarcinomas, even though both tumors use glucose as a major energy source. To explore the differences of mechanisms involving glucose uptake and glycolysis in these tumors, immunohistochemistries for Glut 1 and HK I-III were performed as well as proteome analysis of the enzymes regulating the glucose metabolism using the tumor tissues obtained from HCC and mass- forming cholangiocarcinoma patients showing increased ^sup 18^F-FDG uptake on PET scans.

MATERIALS AND METHODS

Tissue Specimens

Tumor tissue specimens from 14 patients (7 HCCs and 7 cholangiocarcinomas) who had undergone partial hepatectomy were obtained and stored in a deep freezer (-70C) for proteomic analysis or embedded in paraffin for hematoxylin-eosin staining and immunohistochemistry. The age of the patients ranged from 40 to 73 y. All tumor tissues demonstrated increased ^sup 18^F-FDG uptake on a PET scan (standardized uptake values [SUVsJ of ^sup 18^F-FDG ranged from 3.2 to 17.4). Approval was obtained from the Ethical Committee in our institute for this study.

PET Procedure

Before surgery, all patients were examined with a whole-body PET camera (Advance; GE Healthcare). Before scanning, patients fasted for at least 6 h, and their plasma glucose concentrations were measured. The blood glucose level was <140 mg/dE. Approximately 370 MBq ^sup 18^F-FDG were injected intravenously, and the emission scan (5 min/bed) was started from the neck to the knee in 2-dimensional (2D) mode 50-60 min after the injection. Patients then underwent a transmission scan (3 min/bed) with rotating ^sup 68^Ge sources. The images were reconstructed with the attenuation-corrected ordered- subset expectation maximization algorithm (number of subsets, 16; number of iterations, 2). The PET scans were compared with the corresponding CT or MR images for an accurate localization of the tumors. The coronal, sagittal, and axial images of the patients were qualitatively evaluated to determine whether the ^sup 18^F-FDG uptake in the tumor was higher than that in the surrounding noncancerous hepatic tissue. For a semiquantitative evaluation, a small region of interest (8 pixels) was placed over the area of maximum ^sup 18^F-FDG uptake in the tumors and the maximum SUV (SUV^sub max^) normalized to body weight was calculated.

Immunohistochemistry

Hematoxylin-eosin staining of 5-m-thick paraffin sections was performed and sections were then deparaffinized and rehydrated for immunohistochemistry. After wax removal and dehydration, sections were pretreated in a 0.01 mol/L citrate buffer (pH 6.0) using a microwave oven (820 W, 15 min) for antigen retrieval. They were then incubated with primary antibodies at 4C overnight. These primary antibodies were polyclonal human anti-Glut 1, rat antihexokinase type II, monoclonal anti-HK type I and type III (Chemicon International Inc.), and monoclonal anticytokeratin (CK)-19 (Dako). The antibodies were diluted to 1:1,000, 1:5,000, 1:100, 1:200, and 1:50, respectively. Endogenous peroxidase activity was blocked by incubation in H^sub 2^O^sub 2^ for 10 min. After washing, signals were detected using an EnVision Kit (Dako) and diaminobenzidine. The presence of membrane-bound Glut 1 and intracytoplasmic HK I-III was examined for all tumor tissues, and CK-19 was evaluated in HCCs for further classification of HCCs into a primary HCC and an intermediate (hepatocyte-cholangiocyte) phenotype (10). sections incubated with preimmune serum served as negative controls. Red blood cells present in the tissues and rat skeletal muscle served as positive controls for Glut 1 and hexokinases, respectively,

Proteome Analysis

An adequate amount of protein was obtained from 8 tumor specimens (5 HCCs and 3 cholangiocarcinomas) and 3 adjacent normal tissues. Proteome analysis was undertaken using these samples.

Reagents. Urea, thiourea, 3-([3-cholamidopropy]dimethyammonio)-1- propanesulfonate (CHAPS), dithiothreitol (DTT), benzamidine, Bradford solution, acrylamide, iodoacetamide, bisacrylamide, sodium dodecylsulfate (SDS), acetonitrile, trifluoroacetic acid, and a- cyano-4-hydroxycinnamic acid were purchased from Sigma-Aldrich. Pharmalyte (pH 3.5-10) was from Amersham Biosciences and immobilized pH gradient (IPG) DryStrips (pH 4-10; nonlinear, 24 cm) were from Genomine Inc. Modified porcine trypsin (sequencing grade) was from Promega.

Protein Sample Preparation. Tissue samples were washed twice in ice-cold phosphate-buffered saline, blotted to dry, and directly homogenated using a motor-driven homogenizer (Fisher Scientific) with a sample buffer containing 7 mol/L urea, 2 mol/L thiourea (containing 4% [w/v] CHAPS, 1% [w/v] DTT, and 2% [v/v] pharmalyte), and 1 mmol/L benzamidine. Proteins were extracted for 1 h at room temperature. After being centrifuged at 15,000g for 1 h at \15C, the insoluble material was discarded and the soluble fraction was used for a 2D gel electrophoresis. In addition, a membrane fraction was obtained after centrifuging the sample at 1,400g. Protein loading was normalized using a Bradford Assay (11).

2D Polyacrylainide Gel Electrophoresis (PACE). IPG dry strips were equilibrated for 12-16 h with sample buffer containing 7 mol/L urea and 2 mol/L thiourea (containing 2% CHAPS, 1% DTT, and 1% pharmalyte) and loaded with 200 g of sample. Isoelectric focusing (IFF) was performed at 20C using a Multiphor II electrophoresis unit and an EPS 3500 XL power supply (Amersham Biosciences) following the manufacturer's instructions. For IEF, the voltage was increased linearly from 150 to 3.500 V for 3 h to allow for sample entry. This was followed by a constant 3.500 V. with focusing complete after 96 kVh. Before the second dimension, strips were incubated for 10 min in an equilibration buffer (50 mmol/L Tris-Cl. pH 6.8. containing 6 mol/L urea, 2% SDS, and 30% glycerol). first with 1% DTT and then with 2.5% iodoacetamide. The equilibrated strips were inserted into SDS-PAGE gels (20-24 cm, 10%-16%). SDS-PAGE was performed using the Hoefer DALT 2D system (Amersham Biosciences) following the manufacturer's instructions. Two-dimensional gels were run at 20C for 1.7 kVh and then silver stained us described by Oakley et al. (12).

linage Analysis. A quantitative analysis of digitized images was performed using PDQuest software, version 7.0 (BioRad). The quantity of each spot was normali/.ed by total valid spot intensity.

Enzvniatic Digestion of Protein In-CeL Protein spots were digested in-gel using modified porcine trypsin in a manner similar to that previously described by Shevchenko et al. (13). The gel pieces were washed with 50% acetonitrile to remove any remaining SDS, salt, and stain. Next, the pieces were dried to remove any solvent, then rehydrated with trypsin (8-10 ng/L) and incubated for 8-10 h at 37C. The proteolytic reaction was terminated by the addition of 5 L 0.5% trifluoroacetic acid. Peptides were recovered by combining the aqueous phases obtained from extracting pieces of gel with 50% aqueous acetonitrile. After concentration, the peptide mixture was desalted using C^sub 18^ZipTips (Millipore) and eluted in 1-5 L of acetonitrile. An aliquot of this solution was mixed with an equal volume of a saturated solution of α-cyano-4- hydroxycinnamic acid in 50% aqueous acetonitrile. and 1 L of the mixture was spotted onto a target plate.

Matrix-Assisted Laser Desorption Ionisation-Time-of-Flight (MALDI- TOF) Analysis and Database Search. A protein analysis was performed using MALDI-TOF (Amersham Biosciences). Peptides were evaporated with an N2 laser at 337 nm using a delayed extraction approach. They were accelerated with a 20-kV injection pulse to analyze the time of flight. Each spectrum was the cumulative average of 300 laser shots. The search program ProFound, developed by Rockefeller University (http://129.85.19.192/ profound_bin/WebProFound.exe), was used for protein identification using peptide mass fingerprinting. Spectra were calibrated with the trypsin autodigestion ion peaks m/z (842.510. 2211.1046) as internal standards.

FIGURE 1. (A) Two-dimensional gel electrophoresis map of human liver tumor tissue demonstrates numerous protein spots. (B) Proteome analysis enables grouping of tumors (normal, HCC, and cholangiocarcinoma groups) as seen on distance tree mapping of protein expression profiles. However, protein expression pattern of intermediate phenotype of HCC group (patient 7) is close to cholangiocarcinoma group.

Data Analysis. Tumor specimens were divided into 2 groups on the basis of their protein expression patterns. HCCs were part of group A. whereas group B included the intermediate-phenotype HCC and the cholangiocarcinomas, because the protein expression pattern of the intermediate-phenotype HCC was close to that of the cholangiocarcinoma group (Figs. 1A and 1B). Comparisons between the 2 characteristics (normal vs. group A. normal vs. group B. and group A vs. group B) were assessed using a Student t test, and relationships were considered statistically significant when P < 0.05. Relative spot intensity (tumor/normal) was calculated in each group for comparison.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was successfully isolated from 2 HCCs including the intermediate phenotype. 2 cholangiocarcinomas, and 3 adjacent normal liver tissues using a Trizol reagent (Introgen) as directed by the manufacturer's protocol. Messenger RNA (mRNA) expression of the first reaction enzyme in the pentose phosphate pathway (PPP)-that is, glucose-6-phosphate dehydrogenase (G6PDH)-was evaluated. First- strand complementary DNA was synthesi/ed from 3 L of the total RNA using random hexamer and Superscript II reverse transcriptase (Life Technologies). One microlitcr of the reverse transcription reaction mixture was amplified with primers specific for G6PDH. and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control in the total volume of 50 L. Linearity of the PCR was tested by amplification cycles between 25 and 30. Primers used in PCR were as follows:

TABLE 1

Clinical and Pathologic Data

* G6PDH-sense, 5'-ACCTGGCCAAGAAGAAGATC-3',

G6PDH-antisense, 5'-TAGGAGGCTGCATCATCGTA-3',

* GAPDH-sense, 5'-ACCAC AGTCC ATGCC ATCAC-3',

* GAPDH-antisense, 5'-TCCAC CACCC TGTTG CTGTA-3'.

RESULTS

lmmunohistochemistry

Immunoreactivity for Glut 1 was positive in all cholangiocarcinomas but was negative in all HCCs except one (Table 1). In contrast, HK II was positive in HCCs but was negative in 6 of 7 cholangiocarcinomas (Figs. 2 and 3). CK-19 was positive only in the HCC, which had positive reactions for both Glut 1 and HK II. This is consistent with the intermediate phenotype of HCC (Fig. 4).

HKI was weakly positive in only one HCC, and HK-III was weakly positive in one HCC and 2 cholangiocarcinomas.

Protein Expression Profiles

A total of 331 protein spots that had a P value of <0.05 were selected for identification. Thirteen proteins involved in the metabolism of carbohydrates were chosen from this total for comparison and were classified as shown in Table 2.

FIGURE 2. Nodular HCC (Edmondson grade H-III) in right lobe of liver, inferior segment, shows increased ^sup 18^F-FDG uptake on PET scan (patient 3). lmmunohistochemical staining shows a negative reaction for Glut 1 but a positive reaction for HK Il (400).

FIGURE 3. Mass-forming cholangiocarcinoma in right lobe of liver, inferior segment, shows intense ^sup 18^F-FDG uptake (patient 10). Pathologically, tumor shows a strong positive reaction for Glut 1 along cell membrane but a negative reaction for HK Il ( 400).

PPP. The expression of 6-phosphogluconolactonase, the second step enzyme in the PPP, was markedly increased in group B (Fig. 5). Although G6PD, the first step-regulating enzyme, was not identified on proteome analysis, mRNA expression on the RT-PCR significantly increased as the 6-phosphogluconolactonase protein expression pattern (Fig. 6).

Tricarboxylic Acid (TCA) Cycle. Six enzymes of 8 successive reaction steps in the TCA cycle were differentially expressed. Isocitrate dehydrogenase (3rd-step reaction enzyme) and β- succinyl CoA synthetase (5th-step reaction enzyme) were increased, whereas α-ketoglutarate dehydrogenase (4th-step reaction enzyme), succinate dehydrogenase (6th-step reaction enzyme), and fumarase (7th-step reaction enzyme) were decreased in both groups, more significantly in group B. Pyruvate carboxylase involving in anaplerotic reaction increased in group A but decreased in group B.

FIGURE 4. Mass with intense ^sup 18^F-FDG uptake as seen in Figures 2 and 3 shows a positive reaction for both Glut 1 and HK Il and, additionally, the biliary marker CK-19 (patient 7) (400). Morphologically, lesion is classified as an HCC; however, biologically, it has both HCC and cholangiocarcinoma phenotypes.

Gluconeogenesis. Glyoxalate reductase/hydroxypyruvate reductase was decreased in group B, and phosphoenolpyruν ate carboxykinase (PEPCK) was decreased in both groups, but was more pronounced in group A.

Others. Pyruvate kinase, mannosephosphate isomerase, citrate lyase, and ketohexokinase decreased in both groups. Glutathione peroxidase (GPO), which is essential in the normal detoxification of H^sub 2^O^sub 2^, was increased in both groups. This increase was higher in group B (Fig. 5).

Glut 1 and HK proteins could not be identified on proteome analysis, possibly due to technical difficulties encountered during the extraction of membranous proteins (14).

TABLE 2

Proteins Involved in Carbohydrate Metabolism

DISCUSSION

Hepatocyte origin HCCs and bile duct origin cholangiocarcinomas are the major primary hepatic carcinomas. Although both high-grade HCCs and intrahepatic mass-forming cholangiocarcinomas showed high rates of glycolysis-that is, increased ^sup 18^F-FDG uptake on PET scans-our results demonstrated that the mechanisms involved in glucose uptake and glycolytic metabolism appear to be different from each other.

FIGURE 5. Protein expression level of glutathione peroxidase (GPO) and especially 6-phosphogluconolactonase (6-PGL) is increased in both HCC and cholangiocarcinomas, more in cholangiocarcinoma group. (Group A includes HCCs; group B includes intermediate phenotype of HCC and cholangiocarcinomas.)

Although most malignant cells highly express glucose transporters, especially Glut 1 (6,15), it is not increased in HCCs (7), as was seen in our study. The exact mechanisms have not been fully evaluated, but previous reports have revealed that the growth of HCCs is glutamine dependent (16,17), and fatty acids are the major energy source in HCC cells (18). These findings might be possible explanations for the low incidence of Glut 1 expression and lower 18F-FDG uptake in HCCs compared with cholangiocarcin\omas.

In addition to Glut 1, HKs-the first rate-limiting enzymes of the glycolytic pathway-also play a pivotal role in ^sup 18^F-FDG uptake and glycolytic pathways in malignant cells. Among the 4 hexokinase isozymes in mammalian tissues (HK I-IV), HK II is markedly increased in rapidly growing, highly malignant tumors, including HCCs (19,20). Additionally, an isozyme shift from type IV (glucokinase) to HK II has been observed in HCCs (21,22). However, most of the cholangiocarcinoma tissues did not demonstrate increased expression of HK II despite high ^sup 18^F-FDG uptake. The mechanisms of low expression of HK II in cholangiocarcinoma were not investigated, but previous data demonstrated that glucose-6-phosphate production depends more on the phosphorylating enzyme activity of HK II than on the protein expression level (23) and hexokinase is inhibited by the increased concentration of intracellular glucose-6-phosphate, which suggests that ^sup 18^F-FDG uptake does not always parallel the HK protein expression level. However, in conditions in which carbohydrates are limiting, as is seen in HCCs (24), it is important to have an increased amount of HK II protein, especially the mitochondria! bond form for efficient glucose use in cancer cells. In fact, mitochondrialbound hexokinase accounts for as much as 70% of the total cellular hexokinase in hepatoma cells, whereas the amount found on the mitochondria in normal liver cells is negligible (25). HK II bound to porins on the outer mitochondrial membrane prevents apoptosis via Bax-induced inhibition of cytochrome c release (26), Bax conformation change, and BID cleavage in association with activated serine/threonine kinase Akt/PKB (Akt) (27,28).

Glucose-6-phosphate produced by HK II can be used for lactate production, glycogen synthesis, the PPP for lipid or nucleotide synthesis, and also reduced nicotinamide adenine dinucleotide phosphate (NADPH) generation to maintain the redox state of the tumor cells under oxidative stress. However, use of glucose-6- phosphate for oxidative phosphorylation via the TCA cycle is relatively lower than for glycolysis or the PPPs as described previously (29-31).

In our study, both tumors showed an increase in the expression of the second-step enzyme, 6-phosphogluconolactonase, in terms of the PPP. The increase was greater in group B. Although the first-step enzyme, G6PDH, was not identified by proteome analysis-probably due to failure in protein solubilization during the IEF step (14)-mRNA expression was markedly increased in group B on RT-PCR. These findings may support the fact that the PPP plays an important role in cancer cell survival in cholangiocarcinomas or intermediate- phenotype HCCs. NADPH generated by enhanced pentose phosphate shunt produces the reduced form glutathione (GSH) from the oxidized form (GSSH); therefore, it further activates 6-phosphogluconolactonase activity (32). Moreover, glutathione peroxidase, which renders the cells resistant to H^sub 2^O^sub 2^, was overexpressed in our study, particularly in group B. This appears to be an additional cellular protective mechanism against oxidative stress.

The enzyme expression pattern involved in the TCA cycle was variable. Fluctuation in the concentration of key metabolites such as glutamine and glutamate, which reflect the cellular balance between adenosine triphosphate production and consumption, could have caused these enzyme expression variations. The clinical implications of metabolic regulations in the TCA cycle were not fully investigated in these tumors. Further studies are required.

FIGURE 6. mRNA expression of glucose-6-phosphate dehydrogenase (G6PDH) is markedly increased in intermediatephenotype HCC and cholangiocarcinomas (Cholangio) compared with normal and HCC.

Gluconeogenic enzymes such PEPCK were diminished in both groups, which is concordant with a previous report (33). and activation of Akt is known to be sufficient to repress the glucocorticoid and cyclic adenosine monophosphate-stimulated increase in PEPCK gene transcription (34).

An intriguing rinding in our study was that the protein expression pattern in the intermediate phenotype of HCCs was quite close to that of cholangiocarcinomas. Detailed molecular and biologic analysis in this particular type of cancer is required for a better understanding of the tumor characteristics and behaviors.

CONCLUSION

HCCs have different glucose-regulating mechanisms and enzyme expression pattern than those of cholangiocarcinomas. even though both tumors show similarity in the enhanced glucose uptake pattern on PET scans. Further studies are required with regard to the energy metabolism and ^sup 18^F-FDG uptake patterns in association with various oncogenic alterations, because the tumor-suppressor genes p53. H-ras. HIF-α, c-myc. and Akt regulate glucose use in multiple steps of the glycolytic pathways (35).

ACKNOWLEDGMENTS

This study was supported by the Brain Korea 21 Project in Medical Science, a faculty research grant of Yonsei University College of Medicine for 2003 (no. n-2003-1033), the National Core Research Center for Nanomedical Technology (grant R15-2004-024-02002-0). and a nuclear research and development project. Ministry of Science and Technology, Korea

REFERENCES

1. Kelloff GJ. Huffman JM. Johnson B. et al. Progress and promise of FDG-PET imaging for cancer patient management and oncolugic drug development. Clin Cancer Res. 2005:11:2785-2808.

2. Warhurg O. On the origin of cancer cells. Science. 1956;123:309-314.

3. Hu CL. Yu CH. Yeung DWC. ^sup 11^C-Acetale PET imaging in hcpatocellular harcinoma and other liver masses. J Nuel Med. 2003;44:213-222.

4. Kim YJ. Yun M. Lee WJ. Kirn KS. Lee JD. Usefulness of ^sup 18^F-FDG PET in intraliepaiic cholangiocarcinoma. Fur J Nucl Med Mol Imaging. 2003;30:14671472.

5. Lee JD. Yun M. Lee JM. el al. Analysis of gene expression profiles of hepatoeellular carcinomas with regard to F-18-FDG uptake pattern on positron emission tomography. Eur J Nucl Med Mol Imaging. 2004;31:1621-1630.

6. Higashi T, Saga T, Nakamoto Y. et al. Relationship between retention index in dual-phase ^sup 18^F-FDG PET, and hexokinase-Il and glucose transporter-1 expression in pancreatic cancer. J Nucl Med. 2002l43:173-180.

7. Ron MS. Jeong JS. Kim YH. Kim MC. Hong SH. Diagnostic utility of GLUT 1 in the differential diagnosis of liver carcinomas. Hepatogastroenrology. 2004; 51:1315-1318.

8. Zimmerman RL. Fogt F, Burke M, Murakata LA. Assessment of Glut- 1 expression in cholangiocarcinoma. benign biliary lesions and hepatoeellular carcinoma. Ontol Rep. 2002;9:689-692.

9. Mathupala SP. Rempel A. Pedersen PL. Glucose cataholism in cancer cells: isolation, sequence, and activity of the promotor type Il hcxokinusc. J Biol Chem. 1995;270:16918-16925.

10. Kim H. Park C. Han KH. et 1. Primary liver carcinoma of intcrmcdiaic (hepatocyie-cholangiocyie) phcriolype. J Hepatol. 2004;40:298-304.

11. Bradford NM. A rapid and sensitive method for the quanlilation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.

12. Oakley BR. Kirsch DR. Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980;105:361-363.

13. Shevchenko A, Wi;m M. Vorm O. Mann M. Mass spectrometric sequencing of proteins in silver-stained polyacrylamide gels. Anal Client. 1996;68:850-858.

14. Santoni V. Molloy M. Rahilloud T. Membrane proteins and proleomics: Un amour impossible? Electrophoresis. 2000;21:1054- 1070.

15. Larson SM. Positron emission tomography-based molecular imaging in human cancer: exploring the link between hypoxia and accelerating glucose metabolism. Clin Cancer R1-S. 2004;10:2203- 2204.

16. Wong MS, Raab RM, Rigoutsos I, Stephanopoulos GN. Kelleher JK. Metabolic and transcriptional patterns accompanying glutamine depletion and repletion in mouse hepatoma cells: a model for physiological regulatory networks. Physiol Genomics. 2004;16:247- 255.

17. Bode BP, Fuchs BC, Hurley BP. et al. Molecular and functional analysis of glulamine uptake in human hepatoma and liver-derived cells. Am J Physiol Gastrointest Liver Physiol. 2002;283:01062- 01073.

18. Hansson PK, Asztely AK, Clapham JC. Schreyer SA. Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability. Biochiin Biophys Ada. 2004;1684:54-62.

19. Wilson JE. Isozymes of mammalian hexokinases: structure, subcellular localization and metabolic function. J Exp Biol. 2003;206:2049-2057.

20. Goel A. Mathupala SP, Pedersen PL. Glucose metabolism in cancer: evidence that mcthylalion events play a role in activating type II hexokinase gene expression. J Biol Cheat. 2003;278:15333- 15340.

21. Klimek F, Bannaseh P. Isozyme shift from glucokinase to hexokinase is not an early but a late event in hepatocarcinogenesis. Cnrcinogenesis. 1993;14:18571861.

22. Reneurel F, Munose-Alonso MJ, Girard J. Leturque A. An unusual high Kn, hexokinase is expressed in the mhAT3F hepatoma cell line. J Biol Chem. 1998;273:26187-26193.

23. Aloj L, Caraco C, Jagoda E, Eckelman WC, Neuman RD. Glut-1 and hexokinase expression: relationship with 2-fluoro-2-deoxy-D- glucose uptake in A431 and T47D cells in culture. Cancer Res. 1999;59:4709-4714.

24. Rempel A. Mathupala SP, Pedersen PL. Glucose caUibolism in cancer cells: regulation of the type Il hexokinase promoter by glucose and cyclic AMP. FEBS Lett. 1996;385:233-237.

25. Parry DM. Pedersen PL. Intracellular localization and properties of paniculate hexokinase in the Novikoff asciles tumor: evidence for an outer mitochondrial membrane location. J Biol Chem. 1983;258:10904-10912.

26. Paslorino JG, Shulga N. Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-indueed cylochrome e release and apoptosis. J Biol Chem. 2003: 277:7610-7618.

27. Ralhmell JC, Fox CJ. Plas DR. Hammerman PS, Cinalli RM, Thompson CB. Ak\l-dirccled glucose metabolism can prevent Bax conformation change and promote growth factor-independent survival. Mol Cell Biol. 2003;23:7315-7328.

28. Majewski N. Nogueira V. Robery RB. Hay N. Akt inhibits apoptosis downstream of BID cleavage via a glucose-dependent mechanism involving mitochondrial hexokinases. MoI Cell Biol. 2004;24:730-740.

29. Flstrom RL, Bauer DE. Buzzai M, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 2004;64:3892-3899.

30. Hacker HJ, Sreingerg P. Toshkov I, Oesch F. Bannaseh P. Persistence of cholangiocellular and hepatocellular lesions observed in rats fed a cholinedclicient/DL-ethionine-supplememed diet. Carcinogenesis. 1992;13:271-276.

31. Frederiks WM. Bosch KS. De Jong JSSG. Van Noorden CJF. Post- translational regulation of glucose-6-phosphate dehydrogenase activity in (pre)neoplastie lesions in the rat liver. J Hisiochem Cyiochem. 2003;51:105-112.

32. Fratelli M. Demol H. Puype M, et al. Identification by redox proteomics of glutaihionylated proteins in oxidative-stressed human T lymphocytes. Prot Nail Acad Sci USA. 2002;99:3505-3510.

33. Hammond KD. Balinsky D. Activities of key glueoneogenic enzymes and glycogen synthase in rat and human livers, hepatomas, and hepatoma cell cultures. Cancer Res. 1978;38:1317-1322.

34. Liao J, Barthel A, Nakatani K. RoIh RA. Activation of protein kinase B/Akt is sufficient to repress the glucoeorticoid and cAMP induction of the phosphoenolpyruvate carboxykinase gene. J Biol Cliem. 1998;273:27320-27324.

35. Dang CV, Semenza GL. Oncogenic alteration of metabolism. Trends Biochem Sci. 1999;24:68-72.

Jong Doo Lee, MD, PhD1; Woo Ick Yang. MD. PhD2; Young Nyun Park. MD, PhD2; Kyung Sik Kim, MD. PhD3; Jin Sub Choi, MD, PhD3; Mijin Yun, MD1; Dooheun Ko. MD1; Tae-Sung Kirn, MD1; Arthur E.H. Cho. MD1; Hye Mi Kim, MD, PhD1; Kwang-Hyub Han, MD, PhD4; Seung-Soon Im, MS5; Yong-Ho Ahn, MD, PhD5; Chang Woon Choi, MD, PhD6; and Jeon Han Park, MD, PhD7

1 Division of Nuclear Medicine, Department of Diagnostic Radiology, Yonsei University College of Medicine, Seoul, Korea; 2 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea; 3 Department of Surgery, Yonsei University College of Medicine, Seoul, Korea; 4 Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea; 5 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Korea; 6 Department of Nuclear Medicine, Korea Cancer Center Hospital, Seoul, Korea: and 7 Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea

Received Apr. 5, 2005; revision accepted Jun. 27, 2005.

For correspondence or reprints contact: Jong Doo Lee, MD, PhD, Division of Nuclear Medicine, Department of Diagnostic Radiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun- gu, Seoul, 120-752, Korea.

E-mail; jdlee@yumc.yonsei.ac.kr

Copyright Society of Nuclear Medicine Oct 2005

Source: Journal of Nuclear Medicine, The

More News in this Category