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. 2010 Dec;9(12):2617-28.
doi: 10.1074/mcp.M110.000661. Epub 2010 Aug 10.

A combined proteomics and metabolomics profiling of gastric cardia cancer reveals characteristic dysregulations in glucose metabolism

Zhen Cai  1 , Jiang-Sha Zhao, Jing-Jing Li, Dan-Ni Peng, Xiao-Yan Wang, Tian-Lu Chen, Yun-Ping Qiu, Ping-Ping Chen, Wen-Jie Li, Li-Yan Xu, En-Ming Li, Jason P M Tam, Robert Z Qi, Wei Jia, Dong Xie
Affiliations

A combined proteomics and metabolomics profiling of gastric cardia cancer reveals characteristic dysregulations in glucose metabolism

Zhen Cai et al. Mol Cell Proteomics. 2010 Dec.

Abstract

Gastric cardia cancer (GCC), which occurs at the gastric-esophageal boundary, is one of the most malignant tumors. Despite its high mortality and morbidity, the molecular mechanism of initiation and progression of this disease is largely unknown. In this study, using proteomics and metabolomics approaches, we found that the level of several enzymes and their related metabolic intermediates involved in glucose metabolism were deregulated in GCC. Among these enzymes, two subunits controlling pyruvic acid efflux, lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase B (PDHB), were further analyzed in vitro. Either down-regulation of LDH subunit LDHA or overexpression of PDH subunit PDHB could force pyruvic acid into the Krebs cycle rather than the glycolysis process in AGS gastric cancer cells, which inhibited cell growth and cell migration. Our results reflect an important glucose metabolic signature, especially the dysregulation of pyruvic acid efflux in the development of GCC. Forced transition from glycolysis to the Krebs cycle had an inhibitory effect on GCC progression, providing potential therapeutic _targets for this disease.

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Figures

Fig. 1.
Fig. 1.
An overview of two-dimensional gel images between GCC samples and corresponding normal samples. A–D, representative images of a two-dimensional gel for normal (left) versus cancer (right) tissues with poor differentiation (top) or with lymph node metastasis (bottom), respectively. Numbers indicated on the map highlight several of the protein spots that had differential expression. E, detailed alteration patterns of two glycolytic enzymes, ENO1 and LDHB, in different pooled samples.
Fig. 2.
Fig. 2.
Results from GC-TOF MS analysis. A, typical total ion current chromatograms of tissue samples obtained from a GCC specimen and its matched normal counterparts. The total ion current chromatogram for the normal tissues is shown below the GCC samples. B, OPLS-DA score plot discriminating the tissue samples from the GCC specimen and its matched normal counterpart using GC-TOF MS analysis.
Fig. 3.
Fig. 3.
Validation of glucose metabolic characteristics in GCC from RNA to protein level. A and B, relative mRNA expression of seven glycolysis- and anaerobic respiration-related genes and six Krebs cycle- and oxidative phosphorylation-related genes in 33 pairs of matched human GCC and corresponding normal tissues by quantitative real time PCR. The mRNA expression level was normalized to that of RP2; and each box plot shows the distribution of the relative expression level of the corresponding gene individually. Error bars, maximum and minimum values except for the outlier represented as an open circle in the figure. C, scatter plots demonstrating the concordance between the protein expression level from proteomics analysis and the RNA expression level given by real time PCR. D, protein expression level of ACO2, ENO1, LDHA, and LDHB in randomly selected eight pairs of matched human GCC and corresponding normal tissues using Western blot. Ponceau S staining was used as a loading control. 2D, two-dimensional gel; T, tumor; N, normal.
Fig. 4.
Fig. 4.
LDH and PDH influence cancer cell growth by controlling the fate of pyruvic acid. A, Western blot analysis showed RNAi-mediated knockdown of LDHA in AGS. Equal loading was ascertained using tubulin as an internal control. B, HPLC analysis indicated the decrease of lactic acid in both supernatant and cytoplasm of cells with LDHA knockdown. C, MTT assay showed the effect of LDHA gene silencing on cell growth of AGS. D, soft agar assay showed the effect of LDHA gene silencing on colony formation of AGS in vitro. E, Western blot analysis showed overexpression of PDHB in AGS. F, HPLC analysis indicated the decrease of lactic acid in both supernatant and cytoplasm of cells with PDHB overexpression. G, MTT assay showed the effect of PDHB overexpression on cell growth of AGS. H, soft agar assay showed the effect of PDHB overexpression on colony formation of AGS in vitro. All the experiments were performed in triplicate and expressed as mean ± S.E. *, p < 0.05; **, p < 0.01.
Fig. 5.
Fig. 5.
Unique glucose metabolic characteristics in GCC may have potential as _targets for cancer therapy. A, the fingerprint map of the glucose metabolic network in GCC highlighting those enzymes and metabolic intermediates that are dysregulated. B, normal cells preferred the Krebs cycle and oxidative phosphorylation (OXPHOS) compared with anaerobic respiration. This favored pathway is controlled by the high activity of PDH and relatively low activity of LDH (left). GCC cells not only favor glycolysis by up-regulating the enzymes involved but also strongly prefer anaerobic respiration with production of prominent lactic acids rather than the Krebs cycle and oxidative phosphorylation because of activating LDH and suppressing PDH (middle). This divergence between GCC and normal tissues will give us new therapeutic opportunities (right). GAP, glyceraldehyde 3-phosphate; Lac, lactic acid; Pyr, pyruvate.
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