Metabolite Processing - Essay Example

 Introduction Normally, cancer cells requires more energy and metabolites for the cell growth and proliferation. Unlike the normal cells, the growth rates of the cancer cells are very rapid and uncontrollable. They take up the nutrients excessively and shunt metabolite pathways supporting the growth. The alteration in the metabolic pathway and the mutation in the genes cause the rapid proliferation of the cancer cells. This type of variation in the growth is termed as metabolic reprogramming. (Zhou, Huang and Wei Y, 2010). Metabolic reprogramming enables the cells to synthesize specific metabolites in an uncontrollable manner. The scientists have identified many metabolites related to cancer. Some of the most important molecules are phosphocholine, glycine, glutamate, aspartate and essential aminoacids. Metabolic reprogramming alters the nutrient intake and excretion. The study of the metabolic pathways involved in the transformed cells is required for the study and treatment of cancer. There are about 60 human cancer cell lines producing nine types of tumors. (Zhou, Huang and Wei Y, 2010). The metabolic pathways present in these cell lines must be studied for the treatment of cancer in the humans. The quantitative analysis of the metabolites in the given cancer cell line are done using different biotechnological techniques. Some of the analysis methods like gas chromatography time- of – flight mass spectrophotometry ( GC- TOFMS), orthogonal partial least squares discriminate analysis ( OPLS- DA), and acquity ultra performance liquid chromatography – quadrupole time – of – flight mass spectrphotometry ( Acquity UPLC- QTOFMS), Liquid chromatography – tandem mass spectrometry ( LC – MS/MS) and micro array data analysis. (Yin, Qie and Sang, 2012). These methods are used for the analysis of the spent medium and the base line medium of the tissue. The concentration of the metabolites will vary from cell line to cell line. Hence the presence of the common metabolites in the medium is identified. The metabolites that show varying concentration were chosen for the analysis. The metabolite utilization will vary from cell line to cell line. If a metabolite concentration is same in all the cell lines, then it indicates that does not have any effect on cell proliferation and growth. (Semenza, 2010). A study was conducted by Jain et al. (2012) to study the effect of glycine on the rapid cancer cell proliferation. 60 human cancer cell lines from the nine well established tumor types were used for the study. 219 metabolites were analyzed from the cancer cell lines. Out of them, 111 metabolites were found in the spent medium and baseline medium. The major metabolites that are utilized from the medium are analyzed and the data were compared with the existing microarray pooled data. The studies have found that the rapid proliferation requires rapid utilization and synthesis of the metabolites in the medium. (Jain et al., 2012)). The studies were further extended for the analysis of the varying metabolites from the medium. Phosphocholine and glycine were found to be utilized during the cancer cell growth and proliferation. Since choline is essential for the phospholipid synthesis, the concentration became more on the glycine molecule. (Eliyahu, Kreizman and Degani, 2007). Glycine is a non-essential amino acid and they are not synthesized directly in the body. Glycine is produced by the conversion of L-serine into glycine by the mitochondrial serine hydroxymethyltransferase. The analysis of the glycine concentration for the rapid cell proliferation and the effect of glycine in cancer cell proliferation were analysed in this technique. The Gene silencing technique was used for the analysis of the enzyme mitochondrial serine hydroxymethyltransferase. It was observed that the glycine consumption was very less in the absence of this enzyme and the cell proliferation was very less in the glycine depriving medium. When glycine was supplied externally, the cell showed rapid cell proliferation.(DeBerardinis et al., 2008). Methodology Liquid chromatography and Tandem Mass spectrometry (LC- MS/MS) was used for the analysis of the metabolites released by the 60 human cancer cell lines. (Griffin and Shockcor, 2004). The LC- MS/MS technique requires less sampling time and the results are very accurate and all types of compound can be used for the analysis. Chemically similar metabolites can be easily separated and the concentration of the metabolite can be easily analysed from the tissue fluid using LC-MS/MS technique than the Gas chromatography ( GC) or Nuclear magnetic resonance ( NMR) techniques. (Griffin and Shockcor, 2004). The consumption and release profiling (CORE) provided a systematic and quantitative assessment of the metabolites in the spent medium and baseline medium. The flux rate of the metabolites is identified and measured in the cancer and other type of cells. CORE analysis enabled the researchers to identify 111 metabolites from the 219 metabolites present in the spent and baseline medium based on the variation in concentration on the 60 cell lines. (Griffin and Shockcor, 2004). The metabolic phenotype of the cancer cells and the link between each and every metabolite in the medium can be brought in using the core. The link between the metabolites and the cancer cell proliferation rates can be identified using the CORE analysis. Using this tool, phosphocholine and glycine were screened based on the cell proliferation rate across the 60 cell lines. (Griffin and Shockcor, 2004). The growth of the nontranformed cell in the presence of glycine was carried out and concluded that the rapid cell proliferation occurs only in the transformed cells. Microarray data analysis is a technique in which the expression level of the genes is monitored by comparing it with the existing data and the results are predicted to the accurate level. (Quackenbush, 2001). The gene expression of the metabolic enzymes of the 60 cell lines were analyzed using the microarray data analysis and the result showed that glycine biosynthesis enzymes were expressed more in the rapidly proliferating cancer cell lines. The glycine biosynthesis was found in two pathways at the mitochondria and cytosol. Of this, the mitochondrial pathway had the serine hydroxymethyltransferase enzyme (SHMT2). (Quackenbush, 2001). These data were revealed from the microarray data analysis technique. Gene silencing is the process of “switching off” the gene in the mechanism. In this process, either the precursor or the entire enzyme configuration is altered such that the function of the gene is lost. In this process, the SHMT2 enzyme was silenced and the effect of glycine concentration and SHMT2 effect was studied. It was found that the SHMT2 absence resulted in the reduction of the rapid growth and the growth was regained when the glycine was supplied externally. (Griffin and Shockcor, 2004). This confirms that glycine is very essential for the rapid proliferation of the cancer cells. After the confirmation at the lab level, the study was conducted in six large group of patients present in the initial stage of cancer. Since the mitochondrial enzymes are found to be important for the glycine synthesis, the enzymes SHMT2, Mitochondrial C1-tetrahydrofolate synthase ( MTHFD1L) and  methylene tetra hydrofolate dehydrogenase ( MTHFD2) concentration in the cancer cells were identified and the expression levels were analyzed using the microarray data analysis technique. (Quackenbush, 2001). The micro array data analysis was a Meta data analysis. These data confirmed that the SHMT2 enzyme was essential for the rapid cell proliferation of the cancer cells. The occurrence of the lymph node and the grade of tumor were found to be higher and worse for the patients with the higher concentration of the SHMT2 enzyme. (Griffin and Shockcor, 2004). The glycine and related metabolites pathway are found to be very important for the rapid cancer cell proliferation. Summary Cells produce the energy, complex substances such as proteins, carbohydrates and other fatty acid chains that are required for the growth and proliferation of the cells through the metabolic pathways. Metabolic pathways enable the cells to produce the required metabolites for the daughter cell proliferation. The metabolic pathways are altered based on requirements of the cells. The cancer cells alter the metabolic pathways to meet the demands of the proliferating cancer cells. The need for the identification of the major pathways and the metabolites involved in the cancer cell proliferation has increased. The mutations at the oncogenes increase the dependency of the tumor cells on specific nutrients. They also create an alteration in the metabolic pathways. This process is called as metabolite reprogramming. This process helps the daughter cells to obtain the required metabolites from the pathways. Glycine is one such amino acid that was found to be related to the cancer cell proliferation. (di Salvo et al., 2013). The rate of consumption of Glycine is directly proportional to the cancer cell proliferation. The Glycine produced by the mitochondria was found to play a major role in the cell proliferation. The enzyme that converts l – serine to glycine is mitochondrial serine hydroxyl methyltransferase. (di Salvo et al., 2013). The over expression of this enzyme increase the cancer cells. The metabolic phenotype of the cancer cells was explored and the relationship between the metabolites was identified using the CORE data. The data reveals that the cell lines of different types of cancer were found to be dependent on one or more metabolites for their growth and proliferation. Leukemia cells and melanoma cells released omithine and inosine which are unique for those cancers at a large concentration. Functionally related metabolites shared the same rate of consumption among the 60 cell lines. The 60 cell lines were derived from the major nine tumor types. Some of the major metabolites shared by the cell lines are choline, glucose, essential amino acids, polyamines and nucleotides. (di Salvo et al., 2013). The by-products of these metabolites were also produced at similar rates. Glutamine, the major amino acid consumed by the cancer cells released the same amount of glutamate and correlated with the input and output. Similarly the total carbon consumption was equal to the total carbon release. These data brings into account that all the cancer cell lines share some common metabolic phenotype. From the 111 metabolites identified as important from the cell lines, phosphocholine and glycine were identified to be related to the cancer cell proliferation. Phosphocholine was related to the major nutrient choline, which is the substrate for the phospholipid biosynthesis. Glycine is a non-essential amino acid and is not synthesized directly by the human body. The conversion of serine produces glycine. (Choi and Friso, 2006). Though not produced by our body, glycine acts as the precursor molecule for the synthesis of proteins. The studies have found that glycine is consumed largely by the rapidly proliferating cell and released by the slowly proliferating cells. This concludes that glycine is more utilized by the cancer cells than the human cells. Hence the need for the study of glycine production and consumption arises. The glycine consumption rate was measured in the primary human mammary epithelial cells ( HMECs) , human bronchial epithelial cells (HBE), human umbilical vein endothelial cells ( HUVECs) and human activated CD4+ T Lymphocytes (Jain et al., 2012). The doubling time for these cell lines varied from 8 to 18 hours. This is much similar to the cancer cell proliferation. The above cells were non- transformed cells and released the glycine. This indicates that glycine consumption is related to the rapidly proliferating cells. The metabolite CORE analysis also revealed that the highly expressed glycine biosynthesis enzymes are present in the rapidly proliferating cancer cells. Glycine is synthesized in two major metabolic pathways present in the cytosol and mitochondria. In the mitochondrial pathway, the enzyme serine hydroxymethyltransferase2 (SHMT2) is more active than the cytosolic enzyme. (di Salvo et al., 2013). Thus the mitochondrial glycine metabolic pathway is more important. The tracer analysis study for glycine also proved that about one-third of glycine is externally consumed and the remaining is produced by the cells itself. The conclusions from gene expression studies and metabolite core profiling are similar and they through a major impact on glycine for rapid cancer cell proliferation. The wet lab studies conducted with gene silencing of SHMT2 in Chinese hamster ovary cell strains proved that the proliferation of the cell was reduced because of the absence of the enzyme and the proliferation was optimized, only after the external supply of glycine. (Jain et al., 2012). The test was extended on the remaining 10 cell lines and the results were similar the Chinese hamster ovary cell line. The metabolic tracing of the glycine suggested that the glycine consumed was used for the synthesis of purine. This indicates that glycine is very important for the de novo synthesis pathway of the cells. The impact of glycine deprivation on the cell line was analyzed in the HeLa cancer cells. It was observed that silencing of the SHMT2 enzyme and lack of glycine in the medium slowed the proliferation of the glycine cells. The lack of glycine was found to increase the G 1 phase of the cell cycle, indicating that proliferation is very slow. The expression level of the mitochondrial glycine synthesizing enzymes such as SHMT2, Mitochondrial C1-tetrahydrofolate synthase ( MTHFD1L) and methylene tetrahydrofolate dehydrogenase (MTHFD2) were analyzed in the micro array data sets of six independent group of 1300 patients at the early cancer stage. (Jain et al., 2012). It was identified that SHMT2 was very significant among the patients and the glycine biosynthesis pathways was associated with the higher death rates among the patients. Thus the mitochondrial glycine synthesis and metabolism are much related to the human breast cancer. This article thus concludes that glycine is the key metabolite for the proliferation of the cancer cells. Conclusion and Critical analysis This study clearly indicates that glycine is one of the major metabolite for the proliferating cancer cells. This article is very clear in bringing out the importance of glycine in the purine synthesis through the De Novo pathway. The analysis of the data in relation to the gene expression and metabolic profiling are similar. The heme biosynthesis is not brought into consideration in this article. Heme synthesis is very essential for the cytochrome and oxidative phosphorylation complexes. Less importance is given for the cellular methylation reaction, which yields the one carbon molecule. Gene silencing is the major biotechnological concept used in this article. Similarly, micro array technique and Core analysis using the bioinformatics software play a role. References Choi, S. W. and Friso, S. (2006). Nutrient – Gene interactions in Cancer. CRC Press. DeBerardinis, RJ, Lum, JJ, Hatzivassiliou, G and Thompson, CB. (2008). The Biology of Cancer: Metabolic Reprogramming fuels Cell growth and Proliferation. Cell Metabolism, 7 (1): 11 – 20. di Salvo, M. L., Contestabile, R, Paiardini, A, and Maras, B. (2013). Glycine Consumption and Mitochondrial Serine Hydroxymethyltransferase in Cancer Cells: the Heme connection. Medical Hypotheses, 80 (5): 633-36. Eliyahu, G., Kreizman, T and Degani, H. (2007). Phosphocholine as a Biomarker of Breast Cancer: Molecular and Biochemical studies. International Journal of Cancer, 120 (8):1721 – 1730. Griffin, J. L, and Shockcor, J. P. (2004). Metabolic Profiles of Cancer cells. Nature Reviews Cancer, 4 (7): 551 – 561. Jain, M., Nilsson, R., Sharma, S., Madhusudhan, N., Kitami, T., Souza, A. L., Kafri, R., Kirschner, M. W., Clish C. B. and Mootha V. K. (2012). Metabolite Profiling identifies a key role for Glycine in Rapid Cancer Cell Proliferation. Science, 336(6084):1040-44 Quackenbush, J. (2001). Computational analysis of Microarray Data. Nature reviews Genetics, 2 (6): 418 -427. Semensa, G. L. (2010). HIF- 1: Upstream and Downstream of Cancer Metabolism. Current opinion in Genetics and Development, 20 (1): 51 – 56. Yin, C., Qie, S and Sang, N. (2012). Carbons Source Metabolism and its Regulation in Cancer Cells. 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