Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding K_m values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent K_m (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).     

Distinct urinary metabolic profile of human colorectal cancer

A full spectrum of metabolic aberrations that are directly linked to colorectal cancer (CRC) at early curable stages is critical for developing and deploying molecular diagnostic and therapeutic approaches that will significantly improve patient survival. We have recently reported a urinary metabonomic profiling study on CRC subjects (n = 60) and health controls (n = 63), in which a panel of urinary metabolite markers was identified. Here, we report a second urinary metabonomic study on a larger cohort of CRC (n = 101) and healthy subjects (n = 103), using gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Consistent with our previous findings, we observed a number of dysregulated metabolic pathways, such as glycolysis, TCA cycle, urea cycle, pyrimidine metabolism, tryptophan metabolism, polyamine metabolism, as well as gut microbial-host co-metabolism in CRC subjects. Our findings confirm distinct urinary metabolic footprints of CRC patients characterized by altered levels of metabolites derived from gut microbial-host co-metabolism. A panel of metabolite markers composed of citrate, hippurate, p-cresol, 2-aminobutyrate, myristate, putrescine, and kynurenate was selected, which was able to discriminate CRC subjects from their healthy counterparts. A receiver operating characteristic curve (ROC) analysis of these markers resulted in an area under the receiver operating characteristic curve (AUC) of 0.993 and 0.998 for the training set and the testing set, respectively. These potential metabolite markers provide a novel and promising molecular diagnostic approach for the early detection of CRC.     

Metabolic control analysis of cellular respiration in situ in intraoperational samples of human breast cancer

The aim of this study was to analyze quantitatively cellular respiration in intraoperational tissue samples taken from human breast cancer (BC) patients. We used oxygraphy and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to measure a corresponding flux control coefficient (FCC). The activity of all components of ATP synthasome, and respiratory chain complexes was found to be significantly increased in human BC cells in situ as compared to the adjacent control tissue. FCC(s) were determined upon direct activation of respiration with exogenously-added ADP and by titrating the complexes with their specific inhibitors to stepwise decrease their activity. MCA showed very high sensitivity of all complexes and carriers studied in human BC cells to inhibition as compared to mitochondria in normal oxidative tissues. The sum of FCC(s) for all ATP synthasome and respiratory chain components was found to be around 4, and the value exceeded significantly that for normal tissue (close to 1). In BC cells, the key sites of the regulation of respiration are Complex IV (FCC?=?0.74), ATP synthase (FCC?=?0.61), and phosphate carrier (FCC?=?0.60); these FCC(s) exceed considerably (~10-fold) those for normal oxidative tissues. In human BC cells, the outer mitochondrial membrane is characterized by an increased permeability towards adenine nucleotides, the mean value of the apparent K(m) for ADP being equal to 114.8?±?13.6?μM. Our data support the two-compartment hypothesis of tumor metabolism, the high sum of FCC(s) showing structural and functional organization of mitochondrial respiratory chain and ATP synthasome as supercomplexes in human BC.     

Regulation of respiration in plants: a role for alternative metabolic pathways

Respiratory metabolism includes the reactions of glycolysis, the tricarboxylic acid cycle and the mitochondrial electron transport chain, but is also directly linked with many other metabolic pathways such as protein and lipid biosynthesis and photosynthesis via photorespiration. Furthermore, any change in respiratory activity can impact the redox status of the cell and the production of reactive oxygen species. In this review, it is discussed how respiration is regulated and what alternative pathways are known that increase the metabolic flexibility of this vital metabolic process. By looking at the adaptive responses of respiration to hypoxia or changes in the oxygen availability of a cell, the integration of regulatory responses of various pathways is illustrated.     

Proteomic analysis of colorectal cancer reveals alterations in metabolic pathways: mechanism of tumorigenesis

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7-10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.     

Application of fuzzy-logic models for metabolic control analysis

A priori information or valuable qualitative knowledge can be incorporated explicitly to describe enzyme kinetics making use of fuzzy-logic models. Although restricted to linear relationships, it is shown that fuzzy-logic augmented models are not only able to capture non-linear features of enzyme kinetics but also allow the proper mathematical treatment of metabolic control analysis. The explicit incorporation of valuable qualitative knowledge is crucial, particularly when handling data estimated from in vivo kinetics studies, since this experimental information is scarce and usually contains measurement errors. Therefore, data-driven techniques, such as the one presented in this work, form a serious alternative to established kinetics approaches.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.     

Regulation and control of metabolic fluxes in microbes

After about ten years of research renaissance in metabolism, the present challenge is to understand how metabolic fluxes are controlled by a complex interplay of overlapping regulatory mechanisms. Reconstruction of various regulatory network topologies is steaming, illustrating that we underestimated the broad importance of post-translational modifications such as enzyme phosphorylation or acetylation for microbial metabolism. With the growing topological knowledge, the functional relevance of these regulatory events becomes an even more pressing need. A major knowledge gap resides in the regulatory network of protein-metabolite interactions, simply because we lacked pertinent methods for systematic analyses - but a start has now been made. Perhaps most dramatic was the conceptual shift in our perception of metabolism from an engine of cellular operation to a generator of input and feedback signals for regulatory circuits that govern many important decisions on cell proliferation, differentiation, death, and naturally metabolism.     

Proteomic analysis of microvesicles derived from human colorectal cancer cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.     

Proteomic analysis of microvesicles derived from human colorectal cancer ascites

The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC.     

Optimization-based metabolic control analysis

In this work, a novel optimization-based metabolic control analysis (OMCA) method is introduced for reducing data requirement for metabolic control analysis (MCA). It is postulated that using the optimal control approach, the fluxes in a metabolic network are correlated to metabolite concentrations and enzyme activities as a state-feedback control system that is optimal with respect to a homeostasis objective. It is then shown that the optimal feedback gains are directly related to the elasticity coefficients (ECs) of MCA. This approach requires determination of the relative "importance" of metabolites and fluxes for the system, which is possible with significantly reduced experimental data, as compared with typical MCA requirements. The OMCA approach is applied to a top-down control model of glycolysis in hepatocytes. It is statistically demonstrated that the OMCA model is capable of predicting the ECs observed experimentally with few exceptions. Further, an OMCA-based model reconciliation study shows that the modification of four assumed stoichiometric coefficients in the model can explain most of the discrepancies, with the exception of elasticities with respect to the NADH/NAD ratio.     

Topological analysis of metabolic control

A topological approach is presented for the analysis of control and regulation in metabolic pathways. In this approach, the control structure of a metabolic pathway is represented by a weighted directed graph. From an inspection of the topology of the graph, the control coefficients of the enzymes are evaluated in a heuristic manner in terms of the enzyme elasticities. The major advantage of the topological approach is that it provides a visual framework for (1) calculating the control coefficients of the enzymes, (2) analyzing the cause-effect relationships of the individual enzymes, (3) assessing the relative importance of the enzymes in metabolic regulation, and (4) simplifying the structure of a given pathway, from a regulatory viewpoint. Results are obtained for (a) an unbranched pathway in the absence of feedback the feedforward regulation and (b) an unbranched pathway with feedback inhibition. Our formulation is based on the metabolic control theory of Kacser and Burns (1973) and Heinrich and Rapoport (1974).     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Application of electrical analogs to metabolic control analysis: Linear pathways with multiple feedback

• 1.1. Using electrical analogs, we have presented a systematic procedure for calculating the flux control coefficients of linear metabolic pathways with multiple feedback loops.
• 2.2. In this method, an electrical analog circuit is constructed first for the unregulated pathway.
• 3.3. This circuit is subsequently modified in a step-by-step fashion to take into account the effect of each feedback loop in the pathway.
• 4.4. An analog circuit consists of resistances which are connected in series (or parallel) with a voltage (or current) source.
• 5.5. The flux control coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's law.
• 6.6. We have investigated the possible patterns in linear pathways with two feedback loops.
• 7.7. This is followed by an analysis of a linear pathway with an arbitrary pattern of feedback inhibition.