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Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-(11)C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-(11)C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-(11)C]-Met in HCC cells.A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-(3)H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively.Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na(+) dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-(3)H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of MAT2A expression (the duration of SAM treatment). The incorporated (3)H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector.The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of L-[methyl-(11)C]-Met detected by PET.

Radiolabeled methionine (Met) promises to be useful in the positron emission tomography (PET) imaging of hepatocellular carcinoma (HCC). However, its metabolic routes in HCC have not yet been fully understood. In this study, the metabolic pathway(s) of radiolabeled Met in HCC were investigated.To simulate the rapid blood clearance of radiolabeled Met, pulse-chase experiments were conducted. L-[methyl-(3)H]-Met or L-[1-(14)C]-Met was pulsed over control or cycloheximide-treated WCH17 cells and rat hepatocytes for 5 min and chased with cold media. The water-soluble, lipid-soluble, DNA, RNA, and protein phases were subsequently extracted and measured from the acid-precipitable and acid-soluble fractions of whole cells. The radioactive metabolites Met, S-adenosylmethionine (SAM), S-adenosylhomocysteine, Met sulfoxide, and Met sulfone were further separated by radio thin layer chromatography.(1) The uptake of L-[methyl-(3)H]-Met in both cell types was higher than that of L-[1-(14)C]-Met. In rat hepatocytes, the uptake of L-[methyl-(3)H]-Met was significantly higher than that of L-[1-(14)C]-Met, which may contribute to its physiologic accumulation in surrounding hepatic tissues seen in PET imaging of HCC using L-[methyl-(11)C]-Met. Compared to rat hepatocytes, WCH17 cells had significantly higher uptake of both radiotracers. (2) For L-[methyl-(3)H]-Met, the major intracellular uptake was found mostly in the protein phase and, to a lesser degree, in the phosphatidylethanolamine (PE) methylation pathway, which is fairly stabilized within the 55-min chase period (the main metabolites were SAM, Met, Met sulfoxide, and Met sulfone). In contrast, the uptake of Met in rat hepatocytes mainly points to phosphatidylcholine (PC) synthesis through the PE methylation pathway (the main metabolite was PC). (3) Both cell types incorporated L-[1-(14)C]-Met predominantly into protein synthesis. (4) Finally, when the protein synthesis pathway was inhibited, the incorporation of SAM derived from L-[methyl-(3)H]-Met to lipid class (PC was the main metabolite) occurred at a reduced rate in WCH17 cells, suggesting that the route may be impaired in HCC.This study demonstrated that different metabolic pathways of radiolabeled Met exist between HCC and surrounding hepatic tissue and contribute to the patterns of increased uptake of radiolabeled Met in HCC.

PET with [methyl-11C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG–avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Methods: Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-14C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio–high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. Results: PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89 ± 0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. Conclusion: HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5′-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

Background and Aim: We aimed to evaluate the transcriptional characteristics of viral infection-induced woodchuck hepatocellular carcinoma (HCC), to compare the use of human, rat and mouse gene arrays for cross-species hybridization, and to look into gene expression profiles in woodchuck HCC by the combined use of these arrays. Methods: Commercially available human, rat and mouse oligonucleotide microarrays were used to determine the gene expression profiles on the same woodchuck liver samples. Differentially expressed genes between HCC and the surrounding hepatic tissues found in the arrays were selected for quantitative reverse transcription polymerase chain reaction. Results: Despite the difference in the number of the probes from each array, the percentage of genes that were detectable was similar. Stringent microarray data analysis using both supervised and unsupervised methods identified 281 differentially expressed genes via the human array with a false discovery rate (FDR) of 0.99%, 107 genes via the rat array with an FDR of 1.85% and 78 genes via the mouse array with an FDR of 7.41%. Eleven genes were differentially changed in all three arrays that include the upregulation of NPM1, H2AFZ, EEF1G, HNRPAB, RPS18, EIF5, CKS2, ARIH1, RPS12 and RPS10, and the downregulation of EGR1. The quantitative reverse transcription polymerase chain reaction with woodchuck-specific primers confirmed the reliability of the microarray results. Conclusion: This study further demonstrated the utility of cross-species hybridization of microarrays on woodchuck HCC. A combined use of three types of arrays identified more differential genes in HCC than individual arrays with the human array providing the richest information among the three arrays used.

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorption at 580nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-μl sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-11C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-11C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. An significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P < 0.01). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P < 0.01) (total soluble fraction: 876.61 ± 34.64 vs. 361.62 ± 49.97 mU/g tissue; cytoplasmic fraction: 1122.02 ± 112.39 vs. 732.32 ± 84.44 mU/g tissue; organelle content: 815.79 ± 491.5 vs. 547.91 ± 97.05 mU/ g tissue; sedimentable fragment: 251.92 ± 143.7 vs. 90.94 ± 18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.

Altered choline (Cho) metabolism in cancerous cells can be used as a basis for molecular imaging with PET using radiolabeled Cho. In this study, the metabolism of tracer Cho was investigated in a woodchuck hepatocellular carcinoma (HCC) cell line (WCH17) and in freshly derived rat hepatocytes. The transporter responsible for [(11)C]-Cho uptake in HCC was also characterized in WCH17 cells. The study helped to define the specific mechanisms responsible for radio-Cho uptake seen on the PET images of primary liver cancer such as HCC. Cells were pulsed with [(14)C]-Cho for 5 min and chased for varying durations in cold media to simulate the rapid circulation and clearance of [(11)C]-Cho. Radioactive metabolites were extracted and analyzed by radio-HPLC and radio-TLC. The Cho transporter (ChoT) was characterized in WCH17 cells. WCH17 cells showed higher (14)C uptake than rat primary hepatocytes. [(14)C]-Phosphocholine (PC) was the major metabolite in WCH17. In contrast, the intracellular Cho in primary hepatocytes was found to be oxidized to betaine (partially released into media) and, to a lesser degree, phosphorylated to PC. [(14)C]-Cho uptake by WCH17 cells was found to have both facilitative transport and nonfacilitative diffusion components. The facilitative transport was characterized by Na(+) dependence and low affinity (K(m) = 28.59 ± 6.75 μM) with partial energy dependence. In contrast, ChoT in primary hepatocytes is Na(+) independent and low affinity. Our data suggest that transport and phosphorylation of Cho are responsible for the tracer accumulation during [(11)C]-Cho PET imaging of HCC. WCH17 cells incorporate [(14)C]-Cho preferentially into PC. Conversion of [(14)C]-PC into phosphatidylcholine occurred slowly in vitro. Basal oxidation and phosphorylation activities in surrounding hepatic tissue contribute to the background seen in [(11)C]-Cho PET images.

The lack of good molecular markers for diagnosis as well as treatment assessment has rendered the hepatocellular carcinoma (HCC) a major challenge in health care. In this study, woodchucks were used as an animal model for hepatitis virus-induced HCC, and gene expression studies were performed using a human oligonucleotide microarray. An analysis approach combing supervised significant analysis of microarray (SAM), prediction analysis of microarray (PAM), and unsupervised hierarchical cluster methodologies statistically determined 211 upregulated and 78 downregulated genes between liver cancer and non-cancer liver tissues, and demonstrated > or = 93% accuracy in classifying the tissue samples. RT-PCR results confirmed the differential expression of selected sequenced woodchuck genes (SAT, IDH3B, SCD) in the microarray. Our study showed that differentially expressed genes were involved in transcription, RNA splicing, translation, cell cycle, metabolism, protein folding and degradation, apoptosis, immune response, metal binding, etc. Interestingly, some genes were involved with signaling pathways such as Ras/MAPK (MAPKAP1), Src-dependent pathways (CSK), hedgehog signaling pathway (HHIP), while Wnt signaling pathway may not be dominant in woodchuck HCC as shown by the downregulation of beta-catenin (TNNB1) and the upregulation of CXXC4 and CSNK2B. Numerous genes found in this study were also differentially expressed in human HCC and many other human cancers including breast, prostate and lung cancers, etc., serving as tumor suppressors, promoters, prognostic markers or chemotherapy targets. In conclusion, this study has demonstrated the robustness of the data analysis and the potential of using human microarrays on woodchuck samples. In particular, some of the differentially expressed genes in the woodchuck HCC can be further explored for possible molecular imaging targets or biological markers in human HCC.