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Comparative Metabolomic and Lipidomic Analysis of Phenotype Stratified Prostate Cells.

Burch TC, Isaac G, Booher CL, Rhim JS, Rainville P, Langridge J, Baker A, Nyalwidhe JO - PLoS ONE (2015)

Bottom Line: We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites.Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells.This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America; Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.

ABSTRACT
Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

No MeSH data available.


Related in: MedlinePlus

Validation of the expression profiles of ChoK-α and LPCAT3 genes.(A) Real time RT-PCR analysis of expression profiles of ChoK-α and LPCAT 3. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared non-malignant RC77N-E and the malignant RC77T-E cells. (B) Western blot analysis of expression of ChoK-α and GAPDH in prostate cell lines. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared to the normal RWPE-1, non-malignant RC77N-E and the malignant RC77T-E cells. GAPDH was used as a loading control and there are no significant differences in the expression of the protein. (C) Densitometry measurements of Western Blot detection of ChoK-α and GAPDH.
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pone.0134206.g006: Validation of the expression profiles of ChoK-α and LPCAT3 genes.(A) Real time RT-PCR analysis of expression profiles of ChoK-α and LPCAT 3. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared non-malignant RC77N-E and the malignant RC77T-E cells. (B) Western blot analysis of expression of ChoK-α and GAPDH in prostate cell lines. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared to the normal RWPE-1, non-malignant RC77N-E and the malignant RC77T-E cells. GAPDH was used as a loading control and there are no significant differences in the expression of the protein. (C) Densitometry measurements of Western Blot detection of ChoK-α and GAPDH.

Mentions: Our lipidomics data demonstrates the upregulation of specific phosphatidylcholine (PC) species in aggressive prostate cancer cells. PC accounts for between 40–60% of the phospholipids of eukaryotic membranes and they play a significant role in cellular structure and multiple biological functions [19]. PC, like other phospholipids, is synthesized de novo in eukaryotic cells through the Kennedy pathway [20]. The Lands cycle is responsible for acyl remodeling to modify fatty acid composition of phospholipids derived from the Kennedy pathway [21]. In this process, the fatty acyl composition at the sn-2-position of PC is then altered and the net result is the generation of a diverse population of PC species that have unique fatty acids with different carbon chain lengths and with different degrees of saturation [22–24]. The enzymes that control these two pathways play a central role in the regulation of the levels of PC and other phospholipids that are available in eukaryotic cells. In turn, the levels of the phospholipids species may control biochemical processes that might significantly affect processes and pathways that lead to aggressive tumorigenic phenotype. We have performed quantitative real time qRT-PCR gene expression analyses targeting choline kinase alpha (ChoK-α) the first enzyme in the Kennedy pathway and 3 Lands cycle enzymes, lysophosphatidylcholine acyltransferases 1, 2 and 3 (LPCAT1, LPCAT2 and LPCAT3) respectively. The primers that were used for real time qRT-PCR are shown in S1 Table. There is significant over expression of ChoK-α in the metastatic MDAPCa2b and LNCaP cells compared to the other cell lines (Fig 6A). The normal RWPE-1 cells showed the lowest expression levels of ChoK-α. The expression levels of LPCAT1, LPCAT2 and LPCAT3 are similar in the five cell lines (data not shown). As an example, the fold changes in the expression of LPCAT3 in the different cells which show non-significant differences are shown in Fig 6B. The differential gene expression of ChoK-α was validated at protein level by Western blot where both the LNCaP and MDAPCa2b cells show significantly higher levels of expression of ChoK-α protein compared to their non-metastatic counterparts Fig 6C. The quantitation of the western blot showing differential expression of ChoK-α was confirmed by densitometry (Fig 6D). GAPDH was used as loading control for the Western blot. Additional, comparative transcriptomic and biochemical analysis between the LNCAP cells and its aggressive sub-clone C4-2 further confirm a significant upregulation of ChoK-α in the aggressive cells (S3 Fig). Therefore, our genomic and biochemical data provide further supporting evidence that overexpression of ChoK-α may be involved in a potential role for the upregulation of different PC species which may be involved in the development aggressive prostate cancers.


Comparative Metabolomic and Lipidomic Analysis of Phenotype Stratified Prostate Cells.

Burch TC, Isaac G, Booher CL, Rhim JS, Rainville P, Langridge J, Baker A, Nyalwidhe JO - PLoS ONE (2015)

Validation of the expression profiles of ChoK-α and LPCAT3 genes.(A) Real time RT-PCR analysis of expression profiles of ChoK-α and LPCAT 3. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared non-malignant RC77N-E and the malignant RC77T-E cells. (B) Western blot analysis of expression of ChoK-α and GAPDH in prostate cell lines. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared to the normal RWPE-1, non-malignant RC77N-E and the malignant RC77T-E cells. GAPDH was used as a loading control and there are no significant differences in the expression of the protein. (C) Densitometry measurements of Western Blot detection of ChoK-α and GAPDH.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526693&req=5

pone.0134206.g006: Validation of the expression profiles of ChoK-α and LPCAT3 genes.(A) Real time RT-PCR analysis of expression profiles of ChoK-α and LPCAT 3. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared non-malignant RC77N-E and the malignant RC77T-E cells. (B) Western blot analysis of expression of ChoK-α and GAPDH in prostate cell lines. There is a significant upregulation in the expression of ChoK-α in the metastatic LNCAP and MDAPCa2b compared to the normal RWPE-1, non-malignant RC77N-E and the malignant RC77T-E cells. GAPDH was used as a loading control and there are no significant differences in the expression of the protein. (C) Densitometry measurements of Western Blot detection of ChoK-α and GAPDH.
Mentions: Our lipidomics data demonstrates the upregulation of specific phosphatidylcholine (PC) species in aggressive prostate cancer cells. PC accounts for between 40–60% of the phospholipids of eukaryotic membranes and they play a significant role in cellular structure and multiple biological functions [19]. PC, like other phospholipids, is synthesized de novo in eukaryotic cells through the Kennedy pathway [20]. The Lands cycle is responsible for acyl remodeling to modify fatty acid composition of phospholipids derived from the Kennedy pathway [21]. In this process, the fatty acyl composition at the sn-2-position of PC is then altered and the net result is the generation of a diverse population of PC species that have unique fatty acids with different carbon chain lengths and with different degrees of saturation [22–24]. The enzymes that control these two pathways play a central role in the regulation of the levels of PC and other phospholipids that are available in eukaryotic cells. In turn, the levels of the phospholipids species may control biochemical processes that might significantly affect processes and pathways that lead to aggressive tumorigenic phenotype. We have performed quantitative real time qRT-PCR gene expression analyses targeting choline kinase alpha (ChoK-α) the first enzyme in the Kennedy pathway and 3 Lands cycle enzymes, lysophosphatidylcholine acyltransferases 1, 2 and 3 (LPCAT1, LPCAT2 and LPCAT3) respectively. The primers that were used for real time qRT-PCR are shown in S1 Table. There is significant over expression of ChoK-α in the metastatic MDAPCa2b and LNCaP cells compared to the other cell lines (Fig 6A). The normal RWPE-1 cells showed the lowest expression levels of ChoK-α. The expression levels of LPCAT1, LPCAT2 and LPCAT3 are similar in the five cell lines (data not shown). As an example, the fold changes in the expression of LPCAT3 in the different cells which show non-significant differences are shown in Fig 6B. The differential gene expression of ChoK-α was validated at protein level by Western blot where both the LNCaP and MDAPCa2b cells show significantly higher levels of expression of ChoK-α protein compared to their non-metastatic counterparts Fig 6C. The quantitation of the western blot showing differential expression of ChoK-α was confirmed by densitometry (Fig 6D). GAPDH was used as loading control for the Western blot. Additional, comparative transcriptomic and biochemical analysis between the LNCAP cells and its aggressive sub-clone C4-2 further confirm a significant upregulation of ChoK-α in the aggressive cells (S3 Fig). Therefore, our genomic and biochemical data provide further supporting evidence that overexpression of ChoK-α may be involved in a potential role for the upregulation of different PC species which may be involved in the development aggressive prostate cancers.

Bottom Line: We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites.Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells.This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America; Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.

ABSTRACT
Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

No MeSH data available.


Related in: MedlinePlus