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Showing content from https://pubmed.ncbi.nlm.nih.gov/19558692/ below:

Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM)

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doi: 10.1186/1476-4598-8-41. Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM)

Affiliations

Affiliation

¹ Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, USA. twmfan@gmail.com

PMID: 19558692
PMCID: PMC2717907
DOI: 10.1186/1476-4598-8-41

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Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM)

Teresa W M Fan et al. Mol Cancer. 2009.

doi: 10.1186/1476-4598-8-41. Affiliation

¹ Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, USA. twmfan@gmail.com

PMID: 19558692
PMCID: PMC2717907
DOI: 10.1186/1476-4598-8-41

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Abstract

Background: Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma.

Results: Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein.

Conclusion: PC activation - revealed here for the first time in human subjects - may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

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Figures
Figure 1

Plasma samples were collected 0,…

Figure 1

Plasma samples were collected 0, 3 and 12 hr after [U- ¹³ C-Glc]…

Figure 1

Plasma samples were collected 0, 3 and 12 hr after [U-¹³C-Glc] infusion, as described in the Experimental Section. Unlabeled glucose and lactate concentrations were quantified by 1-D ¹H NMR using DSS as the calibration standard. Their % ¹³C enrichment was determined from the respective ¹³C satellite peaks in 1-D ¹H NMR spectra. Panel A illustrates the time course changes in glucose and lactate concentrations as well as their % ¹³C enrichment. Panel B shows the ¹³C satellite pattern of the 3-methyl group of lactate (¹³C-CH₃-lac) in the 1-D ¹H NMR spectrum of patient #6 after 3 hr of [U-¹³C-Glc] infusion. The chemical shifts of lactate and Ala reflected the acidic pH of the TCA extract.

Figure 2

2-D ¹ H TOCSY identification…

Figure 2

2-D ¹ H TOCSY identification of polar metabolites in the lung tumor tissue…

Figure 2

2-D ¹H TOCSY identification of polar metabolites in the lung tumor tissue of patient #6. The 2-D TOCSY contour map is displayed along with the corresponding 1-D high-resolution spectrum. Panels A, B and C, D show the 0.8–6.4 and 5.7–9.5 ppm regions of the spectra, respectively. The assignment of cystine residue of oxidized glutathione (GSSG) was illustrated, which was based on the ¹H covalent connectivity (traced by solid rectangles) and chemical shifts (traced by dashed blue lines). Lactate was discerned similarly and based on the peak splitting pattern (doublet for 3-methyl @ 1.32 ppm and quartet for 2-methine protons @ 4.11 ppm). In addition, the ¹³C satellite cross-peaks of 3-methyl and 2-methine protons of lactate (patterns 1 and 2) and Ala (patterns 3 and 4) were evident (traced by dashed green rectangles), and the peak pattern indicates that lactate and Ala were uniformly ¹³C labeled [19]. The ¹³C satellite cross-peak patterns for the protons of Glu (5, 6, 12, 20), Gln (9), glutamyl residue of oxidized glutathione (GSSG) (7, 10, 13) and Asp (16–19) were present and noted by vertical and horizontal dashed green lines. The ¹³C satellite cross-peaks 14 and 15 were contributed by a mixture of ¹³C-2-Glu, ¹³C-2-Gln, and ¹³C-2-Glu of reduced glutathione (GSH).

Figure 3

¹ H- ¹³ C 2-D…

Figure 3

¹ H- ¹³ C 2-D HSQC identification of ¹³ C-metabolites in the TCA…

Figure 3

¹H-¹³C 2-D HSQC identification of ¹³C-metabolites in the TCA extracts of lung tumor tissues of patient #6. Metabolites were identified based on ¹H-¹³C covalent linkages observed in the 2-D contour map (panel B) and from the TOCSY spectrum such as in Fig. 2B. Panel A is the 1-D projection spectrum of the 2-D data along the ¹³C dimension, which allows a better comparison of the peak intensity of different metabolites. Panel C displays the expanded spectral region of C3 and C4 resonances of Glu, Gln, and GSSG-Glu to illustrate the resolution of these resonances in the 2-D HSQC contour map.

Figure 4

Comparison of metabolite profiles in…

Figure 4

Comparison of metabolite profiles in TCA extracts of paired non-cancerous and cancerous lung…

Figure 4

Comparison of metabolite profiles in TCA extracts of paired non-cancerous and cancerous lung tissues of patient #6. Metabolites in the 1-D ¹H NMR (panel A) and ¹³C HSQC projection spectra (panel B) were assigned as in Fig. 2 and 3, respectively. The two sets of spectra were normalized to dry weight and spectral parameters such that the peak intensity of individual resonances is directly comparable. The dashed lines trace metabolites that differed in abundance between cancerous and non-cancerous lung tissues.

Figure 5

Relationships between Krebs cycle intermediates…

Figure 5

Relationships between Krebs cycle intermediates and glycolytic products in terms of ¹³ C-labeled…

Figure 5

Relationships between Krebs cycle intermediates and glycolytic products in terms of ¹³C-labeled and total concentrations for lung tumor and non-cancerous tissues resected from patients #6–10. Metabolite and ¹³C isotopomer concentrations ([metabolite]) were determined from GC-MS analysis as described in Methods. The R²of the linear fit (solid red lines for cancer and dash black lines for non-cancerous tissues) for these plots are listed in Table 4.

Figure 6

Expected ¹³ C labeling patterns…

Figure 6

Expected ¹³ C labeling patterns in mitochondrial Krebs cycle intermediates and byproducts with…

Figure 6

Expected ¹³C labeling patterns in mitochondrial Krebs cycle intermediates and byproducts with [U-¹³C]-Glc as tracer. The cycle reactions are depicted without (panel A) or with (panel B) anaplerotic pyruvate carboxylase (PC) reaction and the ¹³C positional isotopomer patterns illustrated are the result of one cycle turn. In the absence of pyruvate carboxylation, Glu is labeled at C4 and C5 positions via the forward cycle reactions while Glu is labeled at C2 and C3 when pyruvate carboxylation is active (panels A and B). The possibility that a separate pool of pyruvate derived from Ala for entry into the Krebs cycle via pyruvate carboxylation is depicted in panel B, along with the contribution of the non-oxidative branch of the pentose phosphate pathway (PPP) to the pyruvate pool. Isotopic scrambling occurs at the symmetric succinate, leading to the redistribution of ¹³C labels into its four carbons, two each at a time (blue and green carbons). Red or blue and green letter C's represent ¹³C labeled carbons before or after scrambling, respectively; blue pyruvate denotes a separate pool of pyruvate; solid and dashed arrows denote favorable single and multi-step reactions, respectively; open arrows in panel A delineate ¹³C-labeled OAA after one turn from unlabeled pre-existing OAA.

Figure 7

Western blot analysis of PC…

Figure 7

Western blot analysis of PC protein patterns of paired tumor and non-cancerous tissues…

Figure 7

Western blot analysis of PC protein patterns of paired tumor and non-cancerous tissues from patients #6–10. Western blotting (panel A) and image analysis (panel B) were performed as described in Methods. Normalized PC response represented PC image density normalized to α-tubulin image density. The non-cancerous tissue of patient #8 had a high interfering background with no discernable PC band in the blot image, which was not quantified. The cancer tissue of patient #10 had a very intense PC band, which along with the PC band of the non-cancerous tissue was quantified using the blot image with 2 min of film exposure, as was the case for the α-tubulin band of all tissues. PC band for the rest of tissues was quantified using the same blot but with 17 min of film exposure. N: non-cancerous; C: cancer; ND: not determined. The data shown is representative of two separate blot analyses.