Article

Molecular Causes of the Aberrant Choline Phospholipid Metabolism in Breast Cancer

June 2004
Cancer Research 64(12):4270-6

June 2004
64(12):4270-6

Source
PubMed

Authors:

Proton magnetic resonance spectroscopy ((1)H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-(13)C]choline, using (1)H and (13)C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-(13)C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased (13)C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.

Mapping of exogenous choline uptake and metabolism in rat glioblastoma using deuterium metabolic imaging (DMI)

Article

Full-text available

Apr 2023

Introduction: There is a lack of robust metabolic imaging techniques that can be routinely applied to characterize lesions in patients with brain tumors. Here we explore in an animal model of glioblastoma the feasibility to detect uptake and metabolism of deuterated choline and describe the tumor-to-brain image contrast. Methods: RG2 cells were incubated with choline and the level of intracellular choline and its metabolites measured in cell extracts using high resolution 1H NMR. In rats with orthotopically implanted RG2 tumors deuterium metabolic imaging (DMI) was applied in vivo during, as well as 1 day after, intravenous infusion of 2H9-choline. In parallel experiments, RG2-bearing rats were infused with [1,1',2,2'-2H4]-choline and tissue metabolite extracts analyzed with high resolution 2H NMR to identify molecule-specific 2H-labeling in choline and its metabolites. Results: In vitro experiments indicated high uptake and fast phosphorylation of exogenous choline in RG2 cells. In vivo DMI studies revealed a high signal from the 2H-labeled pool of choline + metabolites (total choline, 2H-tCho) in the tumor lesion but not in normal brain. Quantitative DMI-based metabolic maps of 2H-tCho showed high tumor-to-brain image contrast in maps acquired both during, and 24 h after deuterated choline infusion. High resolution 2H NMR revealed that DMI data acquired during 2H-choline infusion consists of free choline and phosphocholine, while the data acquired 24 h later represent phosphocholine and glycerophosphocholine. Discussion: Uptake and metabolism of exogenous choline was high in RG2 tumors compared to normal brain, resulting in high tumor-to-brain image contrast on DMI-based metabolic maps. By varying the timing of DMI data acquisition relative to the start of the deuterated choline infusion, the metabolic maps can be weighted toward detection of choline uptake or choline metabolism. These proof-of-principle experiments highlight the potential of using deuterated choline combined with DMI to metabolically characterize brain tumors.

Novel approach reveals lipid metabolite reduction in nails of breast cancer patients as potential biomarker

Preprint

Full-text available

Apr 2020

BACKGROUND: Molecular adaptations in intracellular and extracellular microenvironment of breast cancer cells promote pro-tumor metabolic reprogramming. Hence, metabolic reprogramming is seen as a crucial factor in various tumor hallmarks including drug resistance, invasiveness and metastasis. Among well-known metabolic features of breast carcinoma including Warburg effects, altered amino acid metabolism, lipid remodeling is considered as key factors in achieving pro-tumor microenvironment. Therefore, a better understanding on molecular aspects of lipid remodeling is highly appreciated that may contribute towards future therapeutics and diagnostics purpose including the need of potential biomarkers. The identification and validation of lipid biomarkers are reported in the literature, but evidence on lipid metabolites as biomarkers in nails of breast cancer patients is completely unexplored. METHODS: This study reported a novel and specifically designed vertical tube gel electrophoresis (VTGE) system to assist in the purification of metabolites in the range of (~100-1000 Da) from nail samples. Fingernail clippings of breast cancer patients (N=10), and healthy subjects (N-12) were used for extraction and purification of metabolites. The VTGE system uses 15% polyacrylamide under non-denaturing and non-reducing conditions that makes eluted metabolites directly compatible with LC-HRMS and other analytical techniques. The characterization of lipid metabolites in nail lysates was done by positive ESI mode of Agilent LC-HRMS platform. RESULTS: Data suggest a novel observation that healthy and breast cancer patients show distinct accumulation of lipid metabolites specifically choline-based lipids. This is a first report that suggests that levels of choline, phosphorylcholine and lyso-PC are highly reduced and undetectable in nails of breast cancer patients over healthy subject. Furthermore, the potential use of reduced level of choline, phosphorylcholine and lyso-PC in nails of breast cancer patients is in line with current notion that these lipids are diverted to meet the pro-tumor activities in the tumor microenvironment. CONCLUSION: Data strongly provide a proof of concept for the potential use of lipid metabolites including choline, phosphorylcholine and lyso-PC as a set of biomarkers in nails of breast cancer patients. However, the authors propose that validity of these lipid biomarkers may be extended to large population size of breast cancer patients for future applications in early detection, grading, staging, predicting prognosis and therapeutic targeting of breast carcinoma.

Roles and Mechanisms of Choline Metabolism in Nonalcoholic Fatty Liver Disease and Cancers

Article

May 2024

Magnetic Resonance Imaging (MRI) and MR Spectroscopic Methods in Understanding Breast Cancer Biology and Metabolism

Article

Full-text available

Mar 2022

A common malignancy that affects women is breast cancer. It is the second leading cause of cancer-related death among women. Metabolic reprogramming occurs during cancer growth, invasion, and metastases. Functional magnetic resonance (MR) methods comprising an array of techniques have shown potential for illustrating physiological and molecular processes changes before anatomical manifestations on conventional MR imaging. Among these, in vivo proton (1H) MR spectroscopy (MRS) is widely used for differentiating breast malignancy from benign diseases by measuring elevated choline-containing compounds. Further, the use of hyperpolarized 13C and 31P MRS enhanced the understanding of glucose and phospholipid metabolism. The metabolic profiling of an array of biological specimens (intact tissues, tissue extracts, and various biofluids such as blood, urine, nipple aspirates, and fine needle aspirates) can also be investigated through in vitro high-resolution NMR spectroscopy and high-resolution magic angle spectroscopy (HRMAS). Such studies can provide information on more metabolites than what is seen by in vivo MRS, thus providing a deeper insight into cancer biology and metabolism. The analysis of a large number of NMR spectral data sets through multivariate statistical methods classified the tumor sub-types. It showed enormous potential in the development of new therapeutic approaches. Recently, multiparametric MRI approaches were found to be helpful in elucidating the pathophysiology of cancer by quantifying structural, vasculature, diffusion, perfusion, and metabolic abnormalities in vivo. This review focuses on the applications of NMR, MRS, and MRI methods in understanding breast cancer biology and in the diagnosis and therapeutic monitoring of breast cancer.

Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression

Article

Full-text available

Jul 2022
NAT BIOTECHNOL

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes (‘reporter genes’) that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides (‘reporter probes’). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.

Image-guided metabolomics and transcriptomics reveal tumour heterogeneity in luminal A and B human breast cancer beyond glucose tracer uptake

Article

Full-text available

Feb 2024

Background Breast cancer is a metabolically heterogeneous disease, and although the concept of heterogeneous cancer metabolism is known, its precise role in human breast cancer is yet to be fully elucidated. Methods We investigated in an explorative approach a cohort of 42 primary mamma carcinoma patients with positron emission tomography/magnetic resonance imaging (PET/MR) prior to surgery, followed by histopathology and molecular diagnosis. From a subset of patients, which showed high metabolic heterogeneity based on tracer uptake and pathology classification, tumour centre and periphery specimen tissue samples were further investigated by a targeted breast cancer gene expression panel and quantitative metabolomics by nuclear magnetic resonance (NMR) spectroscopy. All data were analysed in a combinatory approach. Results [ ¹⁸ F]FDG (2‐deoxy‐2‐[fluorine‐18]fluoro‐ d ‐glucose) tracer uptake confirmed dominance of glucose metabolism in the breast tumour centre, with lower levels in the periphery. Additionally, we observed differences in lipid and proliferation related genes between luminal A and B subtypes in the centre and periphery. Tumour periphery showed elevated acetate levels and enrichment in lipid metabolic pathways genes especially in luminal B. Furthermore, serine was increased in the periphery and higher expression of thymidylate synthase (TYMS) indicated one‐carbon metabolism increased in tumour periphery. The overall metabolic activity based on [ ¹⁸ F]FDG uptake of luminal B subtype was higher than that of luminal A and the difference between the periphery and centre increased with tumour grade. Conclusion Our analysis indicates variations in metabolism among different breast cancer subtypes and sampling locations which details the heterogeneity of the breast tumours. Correlation analysis of [ ¹⁸ F]FDG tracer uptake, transcriptome and tumour metabolites like acetate and serine facilitate the search for new candidates for metabolic tracers and permit distinguishing luminal A and B. This knowledge may help to differentiate subtypes preclinically or to provide patients guide for neoadjuvant therapy and optimised surgical protocols based on individual tumour metabolism.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer

Article

Full-text available

Nov 2023

Background Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. Methods We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. Results Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3β and MAPK signaling. Conclusions Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+ breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth

Article

Full-text available

Jan 2023
J EXP CLIN CANC RES

Background Intrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 ( GPCPD1 ), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored. Methods EDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets ( n = 540) and with immunohistochemistry on a tissue microarray ( n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo. Results In the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3β, and transcription factors, including HIF1α, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo. Conclusions Our results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.

Harmonization of Rapid Evaporative Ionization Mass Spectrometry Workflows across Four Sites and Testing Using Reference Material and Local Food-Grade Meats

Article

Full-text available

Nov 2022

Rapid evaporative ionization mass spectrometry (REIMS) is a direct tissue metabolic profiling technique used to accurately classify tissues using pre-built mass spectral databases. The reproducibility of the analytical equipment, methodology and tissue classification algorithms has yet to be evaluated over multiple sites, which is an essential step for developing this technique for future clinical applications. In this study, we harmonized REIMS methodology using single-source reference material across four sites with identical equipment: Imperial College London (UK); Waters Research Centre (Hungary); Maastricht University (The Netherlands); and Queen’s University (Canada). We observed that method harmonization resulted in reduced spectral variability across sites. Each site then analyzed four different types of locally-sourced food-grade animal tissue. Tissue recognition models were created at each site using multivariate statistical analysis based on the different metabolic profiles observed in the m/z range of 600–1000, and these models were tested against data obtained at the other sites. Cross-validation by site resulted in 100% correct classification of two reference tissues and 69–100% correct classification for food-grade meat samples. While we were able to successfully minimize between-site variability in REIMS signals, differences in animal tissue from local sources led to significant variability in the accuracy of an individual site’s model. Our results inform future multi-site REIMS studies applied to clinical samples and emphasize the importance of carefully-annotated samples that encompass sufficient population diversity.

Salivary biomarkers in cancer

Chapter

Jul 2022
ADV CLIN CHEM

In recent years, the comprehensive analysis of saliva, i.e., salivaomics, has played an increasing role in biomarker discovery for disease detection in general and cancer specifically. Saliva is a readily accessible, non-invasive and low-cost specimen that can be used to detect biomarkers of clinical relevance. Saliva-based “omics” technologies, which include proteomics, transcriptomics, metabolomics and microbiomics, have rapidly evolved and may be applicable in point-of-care detection, liquid biopsy and nanoscience. Advances in analytical methods has increased the scope and application of salivaomics from solely the oral cavity to the entire physiologic system, and accordingly to personalized medicine. In this chapter, we highlight recent advances in analytical approaches to identify and detect biomarkers in saliva and their potential use as diagnostic, prognostic and therapeutic markers with a focus on cancer.

Pathophysiological Integration of Metabolic Reprogramming in Breast Cancer

Article

Full-text available

Jan 2022

Simple Summary Tumors exhibit metabolic changes that differentiate them from the normal tissues from which they derive. These metabolic changes favor tumor growth, are primarily induced by cancer cells, and produce metabolic and functional changes in the surrounding stromal cells. There is a close functional connection between the metabolic changes in tumor cells and those that appear in the surrounding stroma. A better understanding of intratumoral metabolic interactions may help identify new vulnerabilities that will facilitate new, more individualized treatment strategies against cancer. We review the metabolic changes described in tumor and stromal cells and their functional changes and then consider, in depth, the metabolic interactions between the cells of the two compartments. Although these changes are generic, we illustrate them mainly with reference to examples in breast cancer. Abstract Metabolic changes that facilitate tumor growth are one of the hallmarks of cancer. The triggers of these metabolic changes are located in the tumor parenchymal cells, where oncogenic mutations induce an imperative need to proliferate and cause tumor initiation and progression. Cancer cells undergo significant metabolic reorganization during disease progression that is tailored to their energy demands and fluctuating environmental conditions. Oxidative stress plays an essential role as a trigger under such conditions. These metabolic changes are the consequence of the interaction between tumor cells and stromal myofibroblasts. The metabolic changes in tumor cells include protein anabolism and the synthesis of cell membranes and nucleic acids, which all facilitate cell proliferation. They are linked to catabolism and autophagy in stromal myofibroblasts, causing the release of nutrients for the cells of the tumor parenchyma. Metabolic changes lead to an interstitium deficient in nutrients, such as glucose and amino acids, and acidification by lactic acid. Together with hypoxia, they produce functional changes in other cells of the tumor stroma, such as many immune subpopulations and endothelial cells, which lead to tumor growth. Thus, immune cells favor tissue growth through changes in immunosuppression. This review considers some of the metabolic changes described in breast cancer.

Crosstalk of FGFR1 signaling and choline metabolism promotes cell proliferation and survival in prostate cancer cells

Article

Full-text available

Jan 2022
INT J CANCER

The acquisition of ectopic type I fibroblast growth factor receptor (FGFR1) is a common feature of prostate cancer (PCa), the most frequently diagnostic cancer in men. However, how ectopic FGFR1 contributes to PCa progression is not well understood. In our study we showed that ablation of FGFR1 in DU145 human PCa cells changed the cell metabolite profile. Among the changes, the choline metabolism profile was the most significantly altered by FGFR1 ablation. Detailed characterization revealed that ablation of FGFR1 altered expression of multiple choline metabolism enzymes. Among the changes of FGFR1‐regulated choline metabolic enzymes, downregulation of choline kinase α (CHKA) is the most prominent changes, which phosphorylates free choline to phosphocholine. Ablation of FGFR1 blunted the activity of choline to promote cell proliferation and survival. Furthermore, depletion of CHKA compromised FGF signaling activity in DU145 cells. We also first time demonstrated that FGFR1 formed complex with CHKA, suggesting that FGFR1 regulated CHKA at the posttranslational level. Together with the previous report that ectopic FGFR1 contributes to PCa progression and metastasis, our results here unravel a novel mechanism by which FGFR1 promotes PCa progression by dysregulating choline metabolism, and that the crosstalk between FGFR1‐choline metabolism can be a potential target for managing PCa progression.

Overexpression of Cystine/Glutamate Antiporter xCT Correlates with Nutrient Flexibility and ZEB1 Expression in Highly Clonogenic Glioblastoma Stem-like Cells (GSCs)

Article

Full-text available

Nov 2021

Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/GluHigh GSCs were found to be resistant to glutamine deprivation, whereas Gln/GluLow GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.

Consideration of Metabolite Efflux in Radiolabelled Choline Kinetics

Article

Full-text available

Aug 2021

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore, may confound the uptake of choline radiotracer [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). The aim of this study was to investigate how hypoxia affects the choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [18F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and the efflux of [18F]-D4-FCH and its phosphorylated product [18F]-D4-FCHP. The effects of hypoxia on [18F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer MDA-MB-231 cells. The mechanisms of radiotracer efflux were assessed by the cell uptake and immunofluorescence in vitro and examined in vivo (n = 24). The mathematical modelling methodology was further developed to verify the efflux hypothesis using [18F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (n = 17). We report a novel finding involving the export of phosphorylated [18F]-D4-FCH and [18F]-D4-FCHP via HIF-1α-responsive efflux transporters, including ABCB4, when the HIF-1α level is augmented. This is supported by a graphical analysis of human data with a compartmental model (M2T6k + k5) that accounts for the efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting the consideration of efflux in the modelling of radiotracer dynamics.

Elucidating the chemical and structural composition of breast cancer using Raman micro-spectroscopy

Article

Full-text available

Jul 2021

The current gold standard for breast cancer (BC) diagnosis is the histopathological assessment of biopsy samples. However, this approach limits the understanding of the disease in terms of biochemical changes. Raman spectroscopy has demonstrated its potential to provide diagnostic information and facilitate the prediction of the biochemical progression for different diseases in a rapid non-destructive manner. Raman micro-spectroscopy was used to characterize and differentiate breast cancer and normal breast samples. In this study, tissue microarrays of breast cancer biopsy samples (n=499) and normal breast (n=79) were analyzed using Raman micro-spectroscopy, and principal component analysis (PCA) was used for feature extraction. Linear discriminant analysis (LDA) was used for feature validation. Normal breast and breast cancer were successfully differentiated with a sensitivity of 90 % and specificity of 78 %. Dominance of lipids, specifically fatty acids, was identified in the normal tissue whereas proteins dominated the malignant spectra. Higher intensities of carotenoids, β-carotenoids, and cholesterol were identified in the normal breast while ceramide related peaks were mostly visible in the BC spectra. The biochemical characterization achieved with Raman micro-spectroscopy showed that this technique is a powerful and reliable tool for the monitoring and diagnosis of BC, regardless of the cohort heterogeneity. Raman spectroscopy also provided a powerful insight into the biochemical changes associated with the BC progression and evolution.

Polymerization as a Strategy to Improve Small Organic Matrices for Low-Molecular-Weight Compound Analytics with MALDI MS and MALDI MS Imaging

Article

Jul 2021

Metabolomics and cancer preventive behaviors in the BC Generations Project

Article

Full-text available

Jun 2021

Metabolomics can detect metabolic shifts resulting from lifestyle behaviors and may provide insight on the relevance of changes to carcinogenesis. We used non-targeted nuclear magnetic resonance to examine associations between metabolic measures and cancer preventive behaviors in 1319 participants (50% male, mean age 54 years) from the BC Generations Project. Behaviors were dichotomized: BMI < 25 kg/m2, ≥ 5 servings of fruits or vegetables/day, ≤ 2 alcoholic drinks/day for men or 1 drink/day for women and ≥ 30 min of moderate or vigorous physical activity/day. Linear regression was used to estimate coefficients and 95% confidence intervals with a false discovery rate (FDR) of 0.10. Of the 218 metabolic measures, 173, 103, 71 and 6 were associated with BMI, fruits and vegetables, alcohol consumption and physical activity. Notable findings included negative associations between glycoprotein acetyls, an inflammation-related metabolite with lower BMI and greater fruit and vegetable consumption, a positive association between polyunsaturated fatty acids and fruit and vegetable consumption and positive associations between high-density lipoprotein subclasses with lower BMI. These findings provide insight into metabolic alterations in the context of cancer prevention and the diverse biological pathways they are involved in. In particular, behaviors related to BMI, fruit and vegetable and alcohol consumption had a large metabolic impact.

MR spectroscopy in breast cancer metabolomics

Article

Full-text available

May 2021

Breast cancer poses a significant health care challenge worldwide requiring early detection and effective treatment strategies for better patient outcome. A deeper understanding of the breast cancer biology and metabolism may help developing better diagnostic and therapeutic approaches. Metabolomic studies give a comprehensive analysis of small molecule metabolites present in human tissues in vivo. The changes in the level of these metabolites provide information on the complex mechanism of the development of the disease and its progression. Metabolomic approach using analytical techniques such as magnetic resonance spectroscopy (MRS) has evolved as an important tool for identifying clinically relevant metabolic biomarkers. The metabolic characterization of breast lesions using in‐vivo MRS has shown that malignant breast tissues contain elevated levels of choline containing compounds (tCho), suggesting rapid proliferation of cancer cells and alterations in membrane metabolism. Also, tCho has been identified as one of the important biomarkers that help to enhance the diagnostic accuracy of dynamic contrast enhanced magnetic resonance imaging and also for monitoring treatment response. Further, metabolome of malignant tissues can be studied using ex vivo and in vitro MRS at high magnetic fields. This provided the advantage of detection of a large number of compounds that facilitated more comprehensive insight into the altered metabolic pathways associated with the cancer development and progression and also in identification of several metabolites as potential biomarkers. This article briefly reviews the role of MRS based metabolic profiling in the discovery of biomarkers and understanding of the altered metabolism in breast cancer.

Metabolomic characterization of colorectal cancer cell lines highlighting stage-specific alterations during cancer progression

Article

Full-text available

Mar 2021

Introduction: Metabolomic studies on various colorectal cancer (CRC) cell lines have improved our understanding of the biochemical events underlying the disease. However, the metabolic profile dynamics associated with different stages of CRC progression is still lacking. Such information can provide further insights into the pathophysiology and progression of the disease that will prove useful in identifying specific targets for drug designing and therapeutics. Thus, our study aims to characterize the metabolite profiles in the established cell lines corresponding to different stages of CRC. Methods: Metabolite profiling of normal colon cell lines (CCD 841 CoN) and CRC cell lines corresponding to different stages, i.e., SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D), was carried out using liquid chromatography-mass spectrometry (LC-MS). Mass Profiler Professional and Metaboanalyst 4.0 software were used for statistical and pathway analysis. METLIN database was used for the identification of metabolites. Results: We identified 72 differential metabolites compared between CRC cell lines of all the stages and normal colon cells. Principle component analysis and partial least squares discriminant analysis score plot were used to segregate normal and CRC cells, as well as CRC cells in different stages of the disease. Variable importance in projection score identified unique differential metabolites in CRC cells of the different stages. We identified 7 differential metabolites unique to stage A, 3 in stage B, 5 in stage C, and 5 in stage D. Conclusion: This study highlights the differential metabolite profiling in CRC cell lines corresponding to different stages. The identification of the differential metabolites in CRC cells at individual stages will lead to a better understanding of the pathophysiology of CRC development and progression and, hence, its application in treatment strategies.

Lipid remodeling in response to methionine stress in MDA-MBA-468 triple-negative breast cancer cells

Article

Full-text available

Feb 2021

Methionine is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike non-transformed cells, cancer cells have a unique metabolic requirement for methionine and are unable to proliferate in growth media where methionine is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as “methionine dependence”, “methionine stress sensitivity”, or the Hoffman effect. The response of lipids to methionine stress, however, is not well-understood. Using mass spectroscopy, label-free vibrational microscopy, and next-generation sequencing, we characterize the response of lipids to methionine stress in the triple-negative breast cancer cell line MDA-MB-468 and its methionine stress insensitive derivative, MDA-MB-468res-R8. Lipidome analysis identified an immediate, global decrease in lipid abundances with the exception of triglycerides and an increase in lipid droplets in response to methionine stress specifically in MDA-MB-468 cells. Furthermore, specific gene expression changes were observed as a secondary response to methionine stress in MDA-MB-468, resulting in a downregulation of fatty acid metabolic genes and an upregulation of genes in the unfolded protein response pathway. We conclude that the extensive changes in lipid abundance during methionine stress is a direct consequence of the modified metabolic profile previously described in methionine stress sensitive cells. The changes in lipid abundance likely results in changes in membrane composition inducing the unfolded protein response we observe.

Surface receptor-mediated targeted drug delivery systems for enhanced cancer treatment: A state-of-the-art review

Article

Nov 2020

Enhanced cancer treatment remains as one of the focused areas for researchers around the world. Hence, the progress in this direction will be a challenge and an opportunity in, inter-disciplinary field to mitigate the suffering of millions in the upcoming decades. As we see, cancer death rate has also progressively increased despite the current impressive treatment regimens but also due to the nonavailability of vaccines and the re-occurring of cancer in substantially recovered patients. Currently, numerous treatment strategies like surgical removal of solid tumors followed by radiation with a combination of immunotherapy/chemotherapy by the researchers and clinicians are routinely being followed. However, recurrence and distant metastasis often occur following radiation therapy, commonly due to the generation of radio-resistance through deregulation of the cell cycle, cell death, and inhibition of DNA damage repair mechanisms. Thus, chemotherapeutic/immunotherapeutic treatment systems have progressed remarkably in the latest years owing to destroying tumors, noninvasive, and affordable charge of therapy. But, traditional chemotherapeutic approaches target the DNA of mutated and normal healthy cells, resulting in a significantly increased risk of toxicity and drug resistance. Thus, many receptors targeted therapies are in the developmental phase of discovery. Cancer cells have a specialized set of surface receptors that provide potential targets for cancer therapeutics. Cell surface receptor-dependent endocytosis is well a known major mechanism for the internalization of macromolecular drugs. This review emphasizes the recent development of several surface receptors mediated cancer-targeting approaches for the effective delivery of various therapeutic formulations.

Aberrant activation of super enhancer and choline metabolism drive antiandrogen therapy resistance in prostate cancer

Article

Full-text available

Oct 2020

Next generation antiandrogens such as enzalutamide (Enz) are effective initially for the treatment of castration-resistant prostate cancer (CRPC). However, the disease often relapses and the underlying mechanisms remain elusive. By performing H3-lysine-27 acetylation (H3K27ac) ChIP-seq in Enz-resistant CRPC cells, we identified a group of super enhancers (SEs) that are abnormally activated in Enz-resistant CRPC cells and associated with enhanced transcription of a subset of tumor promoting genes such as CHPT1, which catalyzes phosphatidylcholine (PtdCho) synthesis and regulates choline metabolism. Increased CHPT1 conferred CRPC resistance to Enz in vitro and in mice. While androgen receptor (AR) primarily binds to a putative CHPT1 enhancer and mediates androgen-dependent expression of CHPT1 gene in Enz-sensitive prostate cancer cells, AR binds to a different enhancer within the CHPT1 SE locus and facilities androgen-independent expression of CHPT1 in Enz-resistant cells. We further identified a long-non coding RNA transcribed at CHPT1 enhancer (also known as enhancer RNA) that binds to the H3K27ac reader BRD4 and participates in regulating CHPT1 SE activity and CHPT1 gene expression. Our findings demonstrate that aberrantly activated SE upregulates CHPT1 expression and confers Enz resistance in CRPC, suggesting that SE-mediated expression of downstream effectors such as CHPT1 can be viable targets to overcome Enz resistance in PCa.

Distribution and clinical relevance of phospholipids in hepatocellular carcinoma

Article

Full-text available

Jun 2020

Background Hepatocellular carcinoma (HCC) is the most common liver cancer and featured with prominent disparity in incidence and mortality rate between male and female. It remains unclear whether alterations of phospholipids (PL) in hepatic tissues contribute to the pathogenesis, progression, and disparity of HCC.Methods Using electrospray ionization mass spectrometry (ESI–MS), PL profiles including 320 individual phospholipid species in 13 PL classes were determined in paired samples from HCC and adjacent benign hepatic tissues (BHT).Results(1) Concentrations of PLs in most of individual species, in subgroups and in total were decreased in HCC than in BHT in all studied population; (2) the number of individual PL species significantly different between HCC and BHT, and the number of PLs in six subgroups and in total decreased in HCC were more in male population than in female population; (3) panels of PL parameters (more in male population than in female population) were identified as biomarkers in differentiation of HCC from BHT, and in the prediction of pathological grade and clinical stage of HCC with high sensitivity, specificity, and accuracy.Conclusion It is concluded that alterations of PLs in hepatic tissues play important roles in pathogenesis, progression, and gender disparity of HCC.

Current and Emerging Magnetic Resonance-Based Techniques for Breast Cancer

Article

Full-text available

May 2020

Breast cancer is the most commonly diagnosed cancer among women worldwide, and early detection remains a principal factor for improved patient outcomes and reduced mortality. Clinically, magnetic resonance imaging (MRI) techniques are routinely used in determining benign and malignant tumor phenotypes and for monitoring treatment outcomes. Static MRI techniques enable superior structural contrast between adipose and fibroglandular tissues, while dynamic MRI techniques can elucidate functional characteristics of malignant tumors. The preferred clinical procedure—dynamic contrast-enhanced MRI—illuminates the hypervascularity of breast tumors through a gadolinium-based contrast agent; however, accumulation of the potentially toxic contrast agent remains a major limitation of the technique, propelling MRI research toward finding an alternative, noninvasive method. Three such techniques are magnetic resonance spectroscopy, chemical exchange saturation transfer, and non-contrast diffusion weighted imaging. These methods shed light on underlying chemical composition, provide snapshots of tissue metabolism, and more pronouncedly characterize microstructural heterogeneity. This review article outlines the present state of clinical MRI for breast cancer and examines several research techniques that demonstrate capacity for clinical translation. Ultimately, multi-parametric MRI—incorporating one or more of these emerging methods—presently holds the best potential to afford improved specificity and deliver excellent accuracy to clinics for the prediction, detection, and monitoring of breast cancer.

A comparative pharmaco-metabolomic study of glutaminase inhibitors in glioma stem-like cells confirms biological effectiveness but reveals differences in target-specificity

Article

Full-text available

Apr 2020

Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment.

17β‑estradiol‑induced mitochondrial dysfunction and Warburg effect in cervical cancer cells allow cell survival under metabolic stress

Article

Full-text available

Nov 2019

Mitochondria from different types of cancer show bioenergetics and dysfunction that favor cell proliferation. The mechanistic understanding of estrogen in cervical cancer is poorly understood. Therefore, the objective of this study was to determine how 17β‑estradiol (E2) affects mitochondrial function and the Warburg effect in SiHa, HeLa and C33A cervical cancer cells. Mitochondrial compromise was evaluated measuring changes in the membrane permeability by immunofluorescence, calcium concentration, redox status, iron and ferritin reserves. Glucose consumption and lactic acid assays were used to detect the metabolic activity. Results were confirmed at molecular level by analysis of the differential gene expression using RNA sequencing. E2 modified the mitochondrial permeability and produced an alteration in the calcium signaling pathway. In HeLa and SiHa, there was a significant decrease in nitric oxide levels and lipid peroxidation, and an increase in glucose consumption and lactic acid levels when stimulated with E2. Intracellular iron or ferritin reserves were not affected by the E2 treatment. Genes differentially modulated by E2 were involved in the mitochondrial electron transport chain, oxidative phosphorylation system, glycolysis, pentose phosphate pathway and the regulation of metabolic signaling pathways. Herein, we provide evidence for a primary effect of estrogen on mitochondrial function and the Warburg effect, favoring the metabolic adaptation of the cervical cancer cell lines and their survival.

Metabolic crosstalk in the breast cancer microenvironment

Article

Sep 2019

During tumorigenesis, breast tumour cells undergo metabolic reprogramming, which generally includes enhanced glycolysis, tricarboxylic acid cycle activity, glutaminolysis and fatty acid biosynthesis. However, the extension and functional importance of these metabolic alterations may diverge not only according to breast cancer subtypes, but also depending on the interaction of cancer cells with the complex surrounding microenvironment. This microenvironment comprises a variety of non-cancerous cells, such as immune cells (e.g. macrophages, lymphocytes, natural killer cells), fibroblasts, adipocytes and endothelial cells, together with extracellular matrix components and soluble factors, which influence cancer progression and are predictive of clinical outcome. The continuous interaction between cancer and stromal cells results in metabolic competition and symbiosis, with oncogenic-driven metabolic reprogramming of cancer cells shaping the metabolism of neighbouring cells and vice versa. This review addresses current knowledge on this metabolic crosstalk within the breast tumour microenvironment (TME). Improved understanding of how metabolism in the TME modulates cancer development and evasion of tumour-suppressive mechanisms may provide clues for novel anticancer therapeutics directed to metabolic targets.

Low SAR 31P (multi-echo) spectroscopic imaging using an integrated whole-body transmit coil at 7T

Article

Full-text available

Aug 2019

Catalina Arteaga de Castro

Phosphorus (31 P) MRSI provides opportunities to monitor potential biomarkers. However, current applications of 31 P MRS are generally restricted to relatively small volumes as small coils are used. Conventional surface coils require high energy adiabatic RF pulses to achieve flip angle homogeneity, leading to high specific absorption rates (SARs), and occupy space within the MRI bore. A birdcage coil behind the bore cover can potentially reduce the SAR constraints massively by use of conventional amplitude modulated pulses without sacrificing patient space. Here, we demonstrate that the integrated 31 P birdcage coil setup with a high power RF amplifier at 7 T allows for low flip angle excitations with short repetition time (TR ) for fast 3D chemical shift imaging (CSI) and 3D T1 -weighted CSI as well as high flip angle multi-refocusing pulses, enabling multi-echo CSI that can measure metabolite T2 , over a large field of view in the body. B1+ calibration showed a variation of only 30% in maximum B1 in four volunteers. High signal-to-noise ratio (SNR) MRSI was obtained in the gluteal muscle using two fast in vivo 3D spectroscopic imaging protocols, with low and high flip angles, and with multi-echo MRSI without exceeding SAR levels. In addition, full liver MRSI was achieved within SAR constraints. The integrated 31 P body coil allowed for fast spectroscopic imaging and successful implementation of the multi-echo method in the body at 7 T. Moreover, no additional enclosing hardware was needed for 31 P excitation, paving the way to include larger subjects and more space for receiver arrays. The increase in possible number of RF excitations per scan time, due to the improved B1+ homogeneity and low SAR, allows SNR to be exchanged for spatial resolution in CSI and/or T1 weighting by simply manipulating TR and/or flip angle to detect and quantify ratios from different molecular species.

In vivo MR spectroscopy for breast cancer diagnosis

Article

Full-text available

Mar 2019

Breast cancer is a significant health concern in females, worldwide. In vivo proton ( ¹ H) MR spectroscopy (MRS) has evolved as a non-invasive tool for diagnosis and for biochemical characterization of breast cancer. Water-to-fat ratio, fat and water fractions and choline containing compounds (tCho) have been identified as diagnostic biomarkers of malignancy. Detection of tCho in normal breast tissue of volunteers and in lactating females limits the use of tCho as a diagnostic marker. Technological developments like high-field scanners, multi channel coils, pulse sequences with water and fat suppression facilitated easy detection of tCho. Also, quantification of tCho and its cut-off for objective assessment of malignancy have been reported. Meta-analysis of in vivo ¹ H MRS studies have documented the pooled sensitivities and the specificities in the range of 71–74% and 78–88%, respectively. Inclusion of MRS has been shown to enhance the diagnostic specificity of MRI, however, detection of tCho in small sized lesions (≤1 cm) is challenging even at high magnetic fields. Potential of MRS in monitoring the effect of chemotherapy in breast cancer has also been reported. This review briefly presents the potential clinical role of in vivo ¹ H MRS in the diagnosis of breast cancer, its current status and future developments.

Context-dependent roles for ubiquitous mitochondrial creatine kinase CKMT1 in breast cancer progression

Article

Apr 2024

Glutamine transporter SLC38A3 promotes breast cancer metastasis via Gsk3β/β-catenin/EMT pathway

Article

Feb 2024

The role of phospholipase D in breast cancer

Chapter

Jan 2023

Warren Thomas

Systems biology approaches to unveiling the expression of phospholipases in various types of cancer—Transcriptomics and protein-protein interaction networks

Chapter

Aug 2023

Systems biology is an interdisciplinary field that aims to understand biological systems and processes in their entirety, by taking a holistic and integrated approach. In several ways, systems biology has revolutionized the study of diseases by taking a holistic approach in bringing together several scientific disciplines to get a deeper understanding into fundamental cellular processes underling human diseases and health. It draws on a range of biological and mathematical disciplines, including genetics, molecular biology, biochemistry, and computational biology. The goal of systems biology is to develop a comprehensive understanding of biological systems and how they interact, so that we can better understand the causes of disease and develop new treatments. Phospholipases, being one of the key enzymes involved in cellular response to hormones/growth factors and stress, hydrolyze glycerophospholipids to produce lipid mediators, which transduce signal relays into the cells to control a variety of cellular physiological functions. Traditional methods have provided understanding of the mechanisms and processes controlled by the enzyme. However, the contributions of these enzymes are beyond the scope of traditional methods and, hence, advanced methods are used to obtain data, and computational biology analysis is needed to obtain meaningful breakthroughs in molecular diagnostics and medicine.

Rapidly identifying and quantifying of unsaturated lipids with carbon-carbon double bond isomers by photoepoxidation

Article

Apr 2023

Unsaturated lipids play an essential role in life activities. Identifying and quantifying their carbon-carbon double bond (CC) isomers have become a hot topic in recent years. In lipidomics, the analysis of unsaturated lipids in complex biological samples usually requires high-throughput methods, which puts forward the requirements of rapid response and simple operation for identification. In this paper, we proposed a photoepoxidation strategy, which uses benzoin to open the double bonds of unsaturated lipids to form epoxides under ultraviolet light and aerobic conditions. Photoepoxidation is controlled by light and has a fast response. After 5 min, the derivatization yield can reach 80% with no side reaction products. Besides, the method has the advantages of high quantitation accuracy and a high yield of diagnostic ions. It was successfully applied to rapidly identify the double bond locations of various unsaturated lipids in both positive and negative ion modes, and to rapidly identify and quantitatively analyze the various isomers of unsaturated lipids in mouse tissue extract. So the method has the potential for large-scale analysis of unsaturated lipids in complex biological samples.

Assignment Strategies for Nuclear Magnetic Resonances in Metabolomic Research

Chapter

Jan 2013

Metabolomics, the global characterisation of the small molecule complement involved in metabolism, has evolved into a powerful suite of approaches for understanding the global physiological and pathological processes occurring in biological organisms. The diversity of metabolites, the wide range of metabolic pathways and their divergent biological contexts require a range of methodological strategies and techniques. Methodologies for Metabolomics provides a comprehensive description of the newest methodological approaches in metabolomic research. The most important technologies used to identify and quantify metabolites, including nuclear magnetic resonance and mass spectrometry, are highlighted. The integration of these techniques with classical biological methods is also addressed. Furthermore, the book presents statistical and chemometric methods for evaluation of the resultant data. The broad spectrum of topics includes a vast variety of organisms, samples and diseases, ranging from in vivo metabolomics in humans and animals to in vitro analysis of tissue samples, cultured cells and biofluids.

Hyperpolarized Nuclear Magnetic Resonance Spectroscopy: A New Method for Metabolomic Research

Chapter

Jan 2013

Metabolomic Magnetic Resonance Spectroscopy of Human Tissues: Comparison of In Vivo and High-Resolution Magic Angle Spinning Ex Vivo Techniques

Chapter

Jan 2013

Ginsenoside CK induces apoptosis in triple-negative breast cancer cells by targeting glutamine metabolism

Article

May 2022
BIOCHEM PHARMACOL

Breast cancer (BC) has replaced lung cancer as the most common cancer worldwide. Ginsenoside CK (CK) can effectively inhibit triple-negative breast cancer (TNBC), the occurrence and development of which are associated with glutamine addiction. However, the connection between CK and glutamine metabolism in TNBC proliferation and the mechanism of cell death induction remains unclear. Here, we found that high glutamine-addicted TNBC cells were particularly sensitive to CK treatment. CK exerted antitumour activity against TNBC by suppressing glutamine consumption and glutamate production via downregulation of glutaminase 1 (GLS1) expression. CK treatment further decreased cellular ATP production, reduced the utilisation of amino acids associated with glutamine metabolism, and induced glutathione (GSH) depletion and reactive oxygen species (ROS) accumulation, consequently triggering apoptosis in TNBC. Furthermore, CK decreased GLS1 expression in SUM159 xenograft mouse mammary tumours and significantly inhibited tumour growth with few side effects. Together, our data provide a powerful theoretical basis for the application of CK as a glutamine metabolic inhibitor in TNBC treatment.

A multimodal pipeline using NMR spectroscopy and MALDI‐TOF mass spectrometry imaging from the same tissue sample

Article

Full-text available

May 2022

Nuclear magnetic resonance (NMR) spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple‐negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several metabolites phospholipid species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy – MALDI imaging to advance metabolomic and lipidomic studies.

Insights into the hepatotoxicity of pyrene and 1-chloropyrene using an integrated approach of metabolomics and transcriptomics

Article

Mar 2022
SCI TOTAL ENVIRON

The toxicity of pyrene (Pyr) and its chlorinated species have not be comprehensively and clearly elucidated. In this study, an integrated approach of metabolomics and transcriptomics were applied to evaluate the hepatotoxicity of Pyr and 1-chloropyrene (1-Cl-Pyr) at human exposure level, using human L02 hepatocytes. After 24 h exposure to Pyr and 1-Cl-Pyr at 5–500 nM, cell viability was not significantly changed. Transcriptomics results showed that exposure to Pyr and 1-Cl-Pyr at 5 and 50 nM obviously altered the gene expression profiles, but did not significantly induce the expression of genes strongly related to the activation of aryl hydrocarbon receptor (AhR), such as CYP1A1, CYP1B1, AHR, ARNT. Pyr and 1-Cl-Pyr both induced a notable metabolic perturbation to L02 cells. Glycerophospholipid metabolism was found to be the most significantly perturbed pathway after exposure to Pyr and 1-Cl-Pyr, indicating their potential damage to the cell membrane. The other significantly perturbed pathways were identified to be oxidative phosphorylation (OXPHOS), glycolysis, and fatty acid β oxidation, all of which are related to energy production. Exposure to Pyr at 5 and 50 nM induced the up-regulation of fatty acid β oxidation and OXPHOS. The similar result was observed after exposure to 5 nM 1-Cl-Pyr. In contrast, exposure to 50 nM 1-Cl-Pyr induced the down-regulation of OXPHOS by inhibiting the activity of complex I. The obtained results suggested that the modes of action of Pyr and 1-Cl-Pyr on energy production remarkably varied not only with molecular structure change but also with exposure concentration.

Cancer Insights from MR Spectroscopy of Cells and Excised Tumors

Article

Mar 2022

Multinuclear ex vivo MRS of cancer cells, xenografts, human cancer tissue and biofluids is a rapidly expanding field that is providing unique insights into cancer. Starting from the 1970’s, the field has continued to evolve as a stand‐alone technology or as a complement to in vivo MRS to characterize the metabolome of cancer cells, cancer associated stromal cells, immune cells, tumors, biofluids and, more recently, changes in the metabolome of organs induced by cancers. Here we have reviewed some of the insights into cancer obtained with ex vivo MRS and have provided a perspective of future directions. Ex vivo MRS of cells and tumors provides opportunities to understand the role of metabolism in cancer immune surveillance and immunotherapy. With advances in computational capabilities, the integration of artificial intelligence to identify differences in multinuclear spectral patterns, especially in easily accessible biofluids, is providing exciting advances in detection and monitoring response to treatment. Metabolotheranostics to target cancers and to normalize metabolic changes in organs induced by cancers to prevent cancer‐induced morbidity are other areas of future development.

NIR Fluorescent Imaging and Photodynamic Therapy with a Novel Theranostic Phospholipid Probe for Triple-Negative Breast Cancer Cells

Article

Jun 2021

1 H MR Spectroscopy of Fine-Needle Aspiration Biopsy Specimens for the Discrimination of Breast Cancer

Article

Full-text available

Nov 2020

Purpose: To determine whether MR spectroscopic assessment of fine-needle aspiration (FNA) biopsy specimens from suspicious breast lesions could be used to improve the diagnostic utility of FNA biopsies for the characterization of breast lesions. Materials and methods: In this prospective study, a previously reported technique using high-spatial-resolution proton MR spectroscopy was modified and used to examine the utility of FNA biopsies in the evaluation of suspicious breast lesions. Tissue samples from 115 lesions (from 102 women; average age, 54 years) were excised by using FNA and core biopsies and were collected between September 7, 2012, and April 11, 2014. Histologic results from core biopsy specimens determined the lesions to be benign (n = 55), invasive ductal carcinoma (n = 51), invasive lobular carcinoma (n = 5), or ductal carcinoma in situ (n = 4). Measures of phosphocholine (PC), glycerophosphocholine, and choline relative to each other and to total creatine (tCr) were obtained from usable spectra. Planned comparisons among lesion groups were carried out using t test contrasts, and differences of each contrast level from zero were judged significant when the two-tailed P value was less than .05. Results: Of the 115 samples, 69 (60%) yielded no usable MR spectra. Analysis of the 46 with usable spectra found that only the difference in PC/tCr between benign and cancer lesions was statistically significant (P = .028). Conclusion: Given that 60% of FNA biopsy specimens yielded no usable spectra and that results were largely inconclusive when derived from usable spectra, the combined MR and FNA technique, as modified and implemented in this study, is of little value for detection and diagnosis of breast cancer.Keywords: Breast, MR-Spectroscopy, Neoplasms-Primary© RSNA, 2020.

Comparative Study of Alterations in Phospholipid Profiles upon Liver Cancer in Humans and Mice

Article

Aug 2020

Comparative studies of molecular alterations upon cancer between mice and humans are of great importance in order to determine the relevance of research involving mouse cancer models to the development of diagnostic and therapy approaches in clinical practice as well as for the mechanistic studies of pathology in humans. Herein, using molecular fingerprinting by internal extractive electrospray ionization mass spectrometry (iEESI-MS), we identified 50 differential signals in mouse liver tissue and 62 differential signals in human liver tissue that undergo significant intensity alterations (Variable Importance in the Project (VIP) > 1.0) upon liver cancer, out of which only 27 were common in both mouse and human tissues. Out of the 27 common differential signals, six types of phospholipids were also identified to undergo significant alterations in human serum upon liver cancer, including PC(34:2), PC(36:4), PC(38:6), PC(36:2), PC(38:4) and PC(42:9). Statistical analysis of the relative intensity distribution of these six identified phospholipids in serum allowed confident determination of liver cancer in humans (sensitivity 91.0 %, specificity 88.0 %, and accuracy 90.0 %). Our results indicate that, despite the significant difference in the overall alterations of phospholipid profiles upon liver cancer between humans and mice, the identified six ‘core’ differential phospholipids of liver cancer found in the liver tissues of both humans and mice as well as in human serum show high potential as a minimal panel for the rapid targeted diagnosis of liver cancer with high accuracy, sensitivity and specificity using direct mass spectrometry (MS) analysis.

Bioactive Lipid (BAL)-Based Therapeutic Approach to Cancer That Enhances Antitumor Action and Ameliorates Cytokine Release Syndrome of Immune Checkpoint Inhibitors

Chapter

May 2020

Undurti Das

It is the aim of cancer therapy to selectively kill tumor cells with few or no side effects. But current therapeutic approaches such as surgery, radiotherapy, and chemotherapy including immune checkpoint inhibitors (ICI) are associated with mild to moderate to severe side effects that are considered to be responsible for significant morbidity and mortality to patients with cancer. The recently discovered and employed ICI therapy though produced significant remission in not more than 20–30% of the patients is capable of inducing severe side effects due to excess production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). It is paradoxical that appropriate amounts of IL-6 and TNF-α are needed to induce apoptosis of tumor cells. Hence, it is important to device newer methods of eliminating rather selectively tumor cells but at the same time prevent or eliminate or reduce cytokine storm-induced side effects. IL-6 and TNF-α induce the release of polyunsaturated fatty acids (PUFAs), especially GLA, DGLA, AA, EPA, and DHA, from the cell membrane phospholipids by activating phospholipases. Previously we and others showed that PUFAs induce apoptosis/ferroptosis/necrosis and other forms of tumor cell death and at the same time are capable of suppressing the release of excess IL-6 and TNF-α. NK cells, TILs (tumor-infiltrating cells), and γδ T cells release toxic granules (also called as cytolytic granules) that contain unsaturated fatty acids. Thus, PUFAs seem to be a universal component of cytolytic granules and are responsible for the cytotoxic action of NK cells, TILs, and γδ T cells especially against tumor cells. Hence, it is hypothesized that a combination of ICI/TILs/NK cells/γδ T cells and PUFAs is likely to form a novel, reliable, and robust method of inducing apoptosis of tumor cells with few side effects.

Identified metabolic related genes to construct a prognostic model of Breast cancer patients

Preprint

Full-text available

Jan 2020

Background: More and more evidence confirms that there are many metabolic disorders in the tumor. The occurrence and development of breast cancer (BC) is closely related to metabolism. Methods: A metabolic related genes table was obtained by the Kyoto Encyclopedia of Genes and Genomes (KEGG) related metabolic pathway. The edgeR package was used to identify differentially expressed genes (DEGs) of The Cancer Genome Atlas (TCGA) breast cancer. We established a prognostic model by univariate Cox regression analysis and lasso-penalized Cox regression. The validation prognostic model was built through the Group on Earth Observations (GEO) database. Use the nomogram and Receiver Operating Characteristic (ROC) curve to verify the accuracy of models. Result: We identified 178 DEGs and 14 prognostic-related genes to construct a prognostic model. In the TCGA prognostic model and the GEO validation prognostic model, patients were divided into high riskscore group and low riskscore group, the high riskscore group had worse prognosis. Conclusion: We constructed a prognostic model of metabolic related genes and verified the feasibility and accuracy of the model. It is hoped that the model can provide a basis and biomarker for breast cancer related metabolic therapy and prognosis.

Magnetic resonance imaging of neuroinflammation in chronic pain: a role for astrogliosis?

Article

Jan 2020

Non-invasive measures of neuroinflammatory processes in humans could substantially aid diagnosis and therapeutic development for many disorders, including chronic pain. Several proton Magnetic Resonance Spectroscopy (H-MRS) metabolites have been linked with glial activity (i.e. choline and myo-inositol) and found to be altered in chronic pain patients, but their role in the neuroinflammatory cascade is not well known. Our multimodal study evaluated resting fMRI connectivity and H-MRS metabolite concentration in insula cortex in 43 patients suffering from fibromyalgia, a chronic centralized pain disorder previously demonstrated to include a neuroinflammatory component, and 16 healthy controls. Patients demonstrated elevated choline (but not myo-inositol) in anterior insula (p=0.03), with greater choline levels linked with worse pain interference (r=0.41, p=0.01). In addition, reduced resting functional connectivity between anterior insula and putamen was associated with both pain interference (whole brain analysis, pcorrected<0.01) and elevated anterior insula choline (r=-0.37, p=0.03). In fact, anterior insula/putamen connectivity statistically mediated the link between anterior insula choline and pain interference (p<0.01), highlighting the pathway by which neuroinflammation can impact clinical pain dysfunction. In order to further elucidate the molecular substrates of the effects observed, we investigated how putative neuroinflammatory H-MRS metabolites are linked with ex-vivo tissue inflammatory markers in a nonhuman primate model of neuroinflammation. Results demonstrated that cortical choline levels were correlated with glial fibrillary acidic protein, a known marker for astrogliosis (Spearman r=0.49, p=0.03). Choline, a putative neuroinflammatory H-MRS assessed metabolite elevated in fibromyalgia and associated with pain interference, may be linked with astrogliosis in these patients.

Mass spectrometry imaging to detect lipid biomarkers and disease signatures in cancer

Article

Full-text available

Jan 2020

Background Current methods to identify, classify, and predict tumor behavior mostly rely on histology, immunohistochemistry, and molecular determinants. However, better predictive markers are required for tumor diagnosis and evaluation. Due, in part, to recent technological advancements, metabolomics and lipid biomarkers have become a promising area in cancer research. Therefore, there is a necessity for novel and complementary techniques to identify and visualize these molecular markers within tumors and surrounding tissue. Recent Findings Since its introduction, mass spectrometry imaging (MSI) has proven to be a powerful tool for mapping analytes in biological tissues. By adding the label‐free specificity of mass spectrometry to the detailed spatial information of traditional histology, hundreds of lipids can be imaged simultaneously within a tumor. MSI provides highly detailed lipid maps for comparing intra‐tumor, tumor margin, and healthy regions to identify biomarkers, patterns of disease, and potential therapeutic targets. In this manuscript, recent advancement in sample preparation and MSI technologies are discussed with special emphasis on cancer lipid research to identify tumor biomarkers. Conclusion MSI offers a unique approach for biomolecular characterization of tumor tissues and provides valuable complementary information to histology for lipid biomarker discovery and tumor classification in clinical and research cancer applications.

Feasibility of 31 P spectroscopic imaging at 7 T in lung carcinoma patients

Article

Full-text available

Sep 2019

Currently, it is difficult to predict effective therapy response to molecular therapies for the treatment of lung cancer based solely on anatomical images. 31P MR spectroscopic imaging could provide as a noninvasive method to monitor potential biomarkers for early therapy evaluation, a necessity to improve personalized care and reduce cost. However, surface coils limit the imaging volume in conventional 31P MRSI. High-energetic adiabatic RF pulses are required to achieve flip angle homogeneity but lead to high SAR. Birdcage coils permit use of conventional amplitude modulated pulses, even over large FOV, potentially decreasing overall SAR massively. Here, we investigate the feasibility of 3D 31P MRSI at 7T in lung carcinoma patients using an integrated 31P birdcage body coil in combination with either a dual-coil or a 16-channel receiver. Simulations showed a maximum decrease in SNR per unit of time of 8% for flip angle deviations in short TR low flip-angle excitation 3D CSI. The minimal SNR loss allowed for fast 3D CSI without time-consuming calibration steps (>10:00 min.). 31P spectra from four lung carcinoma patients were acquired within 29:00 minutes and with high SNR using one of both receivers. The latter allowed discrimination of individual phosphodiesters, inorganic phosphate, phosphocreatine and ATP. The receiver array allowed for an increased FOV compared to the dual-coil receiver. 3D 31P-CSI were acquired successfully in four lung carcinoma patients using the integrated 31P body coil at ultra-high field. The increased spectral resolution at 7T allowed differentiation of multiple 31P metabolites related to phospholipid and energy metabolism. Simulations provide motivation to exclude 31P B1 calibrations, potentially decreasing total scan duration. Employing large receiver arrays improves the field of view allowing for full organ coverage. 31P MRSI is feasible in lung carcinoma patients and has potential as a non-invasive method for monitoring personalized therapy response in lung tumors.

Can Bioactive Lipid(s) Augment Anti-cancer Action of Immunotherapy and Prevent Cytokine Storm?

Article

Aug 2019
ARCH MED RES

Undurti Das

It is desired to selectively kill tumor cells with little or no action on normal cells. Current treatment options are associated with significant side effects including therapy with immune check point inhibitors (ICI). ICI therapy induced side effects are due to excess production of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). At the same time, production of appropriate amounts of IL-6 and TNF-α are needed to eliminate tumor cells. Hence, methods are needed that can selectively eliminate tumor cells and tone down the side effects of cytokine storm. Studies showed that IL-6 and TNF-α activate phospholipases to induce the release of polyunsaturated fatty acids (PUFAs) from the cell membrane phospholipid pool. PUFAs form precursors to pro- and anti-inflammatory eicosanoids and are capable of suppressing IL-6 and TNF-α excess production. PUFAs are endowed with capacity to selectively kill tumor cells by augmenting free radical generation and accumulation of toxic lipid peroxides in tumor but not normal cells. NK cells, TILs (tumor infiltrating cells) and γδ T cells release toxic granules (also called as cytolytic granules) that contain unsaturated fatty acids localized between the granule delimiting membrane and the granule core. Thus, lipids are a universal component of cytolytic granules and play an important role in their cytotoxic actions. Based on this evidence, it is suggested that a combination of ICI/TILs and PUFAs may form a novel method of eliminating cancer with few side effects.

Overexpression of group II phospholipase A2 in human breast cancer tissues is closely associated with their malignant potency

Article

Full-text available

Jul 1994

Membrane-associated phospholipase A2 (M-PLA2) is an enzyme that hydrolyses the sn-2 fatty acyl ester bond of phosphoglycerides. We measured M-PLA2 concentration in tissue extracts from 325 human breast cancers using a specific radioimmunoassay recently developed. Correlation analyses between the tissue concentration of M-PLA2 and clinicopathological factors showed that the enzyme level was significantly higher in patients with distant metastasis than in those without. In addition, M-PLA2 concentration was significantly higher in scirrhous carcinoma than in other histological types. No significant association was found between M-PLA2 concentration and age, menstrual status, tumour size, histological grade, vessel involvement or oestrogen receptor (ER) and progesterone receptor (PR) status. The expression of M-PLA2 mRNA was examined in a fibroadenoma, a stage IV breast cancer and its metastatic site of skin. Northern blot analysis showed a clear hybridisation band corresponding to M-PLA2 mRNA in both primary breast cancer and its metastatic site, while the fibroadenoma expressed a faint band corresponding to M-PLA2 mRNA. Breast cancer patients with high M-PLA2 concentrations exhibited significantly shorter disease-free and overall survival than those with low M-PLA2 concentration at the cut-off point of 5 ng 100 mg-1 protein, which was determined in a separate study. In multivariate analysis, M-PLA2 was found to be an independent prognostic factor for disease recurrence and death in human breast cancer. The possible significance of M-PLA2 expression in human breast cancer tissue is discussed. Images Figure 2

Three-Dimensional Magnetic Resonance Spectroscopic Imaging of Brain and Prostate Cancer 1

Article

Full-text available

Apr 2000

Clinical applications of magnetic resonance spectroscopic imaging (MRSI) for the study of brain and prostate cancer have expanded significantly over the past 10 years. Proton MRSI studies of the brain and prostate have demonstrated the feasibility of noninvasively assessing human cancers based on metabolite levels before and after therapy in a clinically reasonable amount of time. MRSI provides a unique biochemical "window" to study cellular metabolism noninvasively. MRSI studies have demonstrated dramatic spectral differences between normal brain tissue (low choline and high N-acetyl aspartate, NAA) and prostate (low choline and high citrate) compared to brain (low NAA, high choline) and prostate (low citrate, high choline) tumors. The presence of edema and necrosis in both the prostate and brain was reflected by a reduction of the intensity of all resonances due to reduced cell density. MRSI was able to discriminate necrosis (absence of all metabolites, except lipids and lactate) from viable normal tissue and cancer following therapy. The results of current MRSI studies also provide evidence that the magnitude of metabolic changes in regions of cancer before therapy as well as the magnitude and time course of metabolic changes after therapy can improve our understanding of cancer aggressiveness and mechanisms of therapeutic response. Clinically, combined MRI/MRSI has already demonstrated the potential for improved diagnosis, staging and treatment planning of brain and prostate cancer. Additionally, studies are under way to determine the accuracy of anatomic and metabolic parameters in providing an objective quantitative basis for assessing disease progression and response to therapy.

Magnetic resonance detect changes in phosphocholine associated with RAS activation and inhibition in NIH-3T3 cells

Article

Full-text available

Apr 2001

Ras is frequently mutated in cancer, and novel therapies are being developed to target Ras signalling. To identify non-invasive surrogate markers of Ras activation and inhibition, we used(31)P magnetic resonance spectroscopy (MRS) and investigated NIH 3T3 cells compared to a mutant ras transfected counterpart. The MR spectra indicated that phosphocholine (PC) levels increased significantly from 3 +/- 2 fmol cell(-1)in NIH 3T3 cells to 13 +/- 4 fmol cell(-1)in the transfected cells. The PC/NTP ratio increased significantly from 0.3 +/- 0.1 to 0.7 +/- 0.3. This could not be explained by either a faster proliferation rate or by alterations in cell cycle distribution. Both cell lines were treated with simvastatin, 17-AAG and R115777, agents which inhibit Ras signalling. Cell proliferation was inhibited in both cell lines. The spectrum of NIH 3T3 cells was not affected by treatment. In contrast, in the ras transfected cells growth inhibition was associated with an average 35 +/- 5% drop in PC levels and a comparable drop in PC/NTP. Thus the MRS visible increase in phosphocholine is associated with Ras activation, and response to treatment is associated with partial reversal of phosphocholine increase in ras transfected cells. MRS might therefore be a useful tool in detecting Ras activation and its inhibition following targeted therapies.

Increased choline kinase activity in human breast carcinomas: Clinical evidence for a potential novel antitumor strategy

Article

Full-text available

Jun 2002
ONCOGENE

Choline kinase (ChoK) and its product, phosphocholine (PCho), have been implicated in human carcinogenesis. Inhibition of this enzyme has been shown to be an efficient antitumor strategy in vivo. The aim of this study was to assess the relationship between the regulation of ChoK and clinical features in patients with breast cancer. To that end, normal and tumoral tissues from 53 patients with breast carcinomas were analysed for ChoK activity and expression, and compared to some clinical parameters. We report a relevant increase in ChoK activity in 38.5% of the tumoral tissues analysed when compared to the normal levels in healthy tissues. Furthermore, some clinical features were found significant versus ChoK status. First, an association of choline enzymatic activity with histological tumor grade was observed (P<0.001). In addition, increased ChoK activity was also associated with ER-negative breast carcinomas (P=0.037). A significant association between ChoK overexpression and both high histologic tumor grade (P=0.017) and ER-negative tumors (P=0.003) was found. Finally, ChoK overexpression was found in 17% of the samples and all corresponded with those that display the highest increase in ChoK activity (P<0.001). Here we provide evidence that ChoK may be related to the development of human breast cancer, suggesting that this finding may constitute the basis for the development of a novel antitumoral strategy for these patients.

Exposure of Human Breast Cancer Cells to the Anti-inflammatory Agent Indomethacin Alters Choline Phospholipid Metabolites and Nm23 Expression

Article

Full-text available

Dec 2002

We previously observed that changes in choline phospholipids of two malignant human mammary epithelial cells (HMECs) following treatment with a high dose of the cyclooxygenase (COX) inhibitor, indomethacin, mimicked changes following transfection with a metastasis suppressor gene, nm23. The similarity between response to indomethacin and nm23 transfection led us to 1) expand our (1)H NMR spectroscopy study of indomethacin treatment by determining the response at two doses for two nonmalignant and three malignant HMECs, 2) investigate COX-1 and COX-2 levels in HMECs and their relationship with choline phosholipid metabolites, and 3) determine changes in Nm23 expression following treatment with indomethacin. All HMECs exhibited a significant change in choline phospholipids following treatment with 300 microM indomethacin. At the lower dose of 50 microM, only nonmalignant HMECs and the estrogen-dependent malignant cell line, MCF-7, responded. COX-1 levels were significantly higher in malignant HMECs than in nonmalignant HMECs. A significant increase in Nm23 expression following 300 microM indomethacin was detected in MCF-12A and MCF-7 cells but not in MDA-MB-231 and MDA-MB-435 cells. These results suggest that COX-1 expression and its inhibition play a role in the choline phospholipid metabolism of HMECs, and the effect of indomethacin on HMECs may be mediated, in part, through upregulation of nm23.

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP : phosphocholine cytidylyltransferase by glucosylceramide

Article

Full-text available

Dec 2002
FASEB J

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site-directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDP-choline levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild-type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post-translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200-fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.

Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells

Article

Full-text available

Apr 2003

Phospholipase D (PLD) activity is elevated in response to mitogenic and oncogenic signals. PLD also cooperates with overexpressed tyrosine kinases to transform rat fibroblasts. 3Y1 rat fibroblasts overexpressing the tyrosine kinase c-Src undergo apoptosis in response to serum withdrawal. We report here that elevated expression of either PLD1 or PLD2 in these cells prevents apoptosis induced by serum withdrawal. 3Y1 cells transformed by the activated tyrosine kinase v-Src have elevated PLD activity and are resistant to apoptosis induced by serum withdrawal. However, if PLD activity is blocked, the v-Src-transformed cells underwent apoptosis. MDA-MB-231 cells are a human breast cancer cell line with substantially elevated levels of PLD activity. Inhibiting PLD activity in these cells similarly rendered them sensitive to the apoptotic insult of serum withdrawal. These data indicate that elevated PLD activity generates a survival signal(s) allowing cells to overcome default apoptosis programs.

Inhibition of choline kinase as a specific cytotoxic strategy in oncogne-transformed cells

Article

Full-text available

Jan 2004
ONCOGENE

Cancer treatment is in the need of selective drugs that can interfere specifically with signalling pathways affected during the carcinogenic process. Identification of new potential molecular targets is the key event in the design of new anticancer strategies. Once identified, attempts for the generation of specific molecules to regulate their function can be achieved. The relevance of deregulation of choline kinase (ChoK, E.C. 2.7.1.32) in oncogene-driven cell transformation has been previously demonstrated. Here we provide strong evidence that MN58b, a selective inhibitor of ChoK, is rather specific to this enzyme, with no effect on a variety of oncogene-activated signalling pathways involved in the regulation of cell proliferation. MN58b does not affect MAPKs, PI3K, and other enzymes involved in the regulation of phospholipid metabolism such as phospholipases C, D, and A2, CTP:phosphocholine cytidylyltransferase, or diacylglycerol choline-phosphotransferase. Consistent with this specificity, ectopic expression of ChoK resulted in resistance to its inhibitor. Finally, nontransformed cells were able to resume cell proliferation after removal of the drug, while transformed cells were irreversibly affected. These results indicate that inhibition of ChoK is a rather specific strategy for the cytotoxic treatment of transformed cells.

Nm23-transfected MDA-MB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: A (31)P nuclear magnetic resonance study in vivo and in vitro

Article

May 1999

Nm23 genes are involved in the control of the metastatic potential of breast carcinoma cells. To understand the impact of nm23 genes on tumor physiology and metabolism, a 31P nuclear magnetic resonance (NMR) spectroscopic study was performed on tumors formed in the mammary fat pad of severe combined immunodeficiency mice by MDA-MB-435 human breast carcinoma cells transfected with cDNA encoding wild type nm23-H1 and nm23-H2 proteins. Tumors formed by MDA-MB-435 cells transfected with vector alone were used as controls. All transgene tumors exhibited significantly higher levels of phosphodiester (PDE) compounds relative to phosphomonoester (PME) compounds in vivo compared with control tumors. Similar differences in PDE and PME also were observed for spectra obtained from cells growing in culture. Intracellular pH was significantly lower and extracellular pH was significantly higher for transgene tumors compared with control tumors. Histologic analysis of lung sections confirmed reductions in incidence, number, and size of metastatic nodules for animals bearing transgene tumors. These results suggest that nm23 genes may affect suppression of metastasis through phospholipid-mediated signaling and cellular pH regulation. Magn Reson Med 41:897–903, 1999.

Clinical Utility of Proton Magnetic Resonance Spectroscopy in Characterizing Breast Lesions

Article

Aug 2002
J NATL CANCER I

Rachel Katz-Brull

Proton magnetic resonance spectroscopy (1H MRS) of the breast has been proposed as an adjunct to the magnetic resonance imaging (MRI) examination to improve the specificity of distinguishing malignant breast tumors from benign breast tumors. In this review, we carry out a pooled analysis of the clinical breast 1H MRS studies undertaken to date to determine the factors that influence the diagnostic performance of this method. In total, five studies of breast 1H MRS from four independent centers around the world have been published to date. Altogether, 153 tumors were examined, 100 of which were confirmed histologically to be malignant and 53 of which were benign. The lesions presenting a detectable composite choline signal in their corresponding 1H MR spectra were diagnosed as malignant, whereas the lesions with no choline signal were diagnosed as benign. The sensitivity and specificity of breast 1H MRS for detecting breast cancer were 83% (95% confidence interval [CI] = 73% to 89%) and 85% (95% CI = 71% to 93%), respectively, and both values could be as high as 92% after technical exclusions. In a subgroup of 20 young women, the sensitivity and the specificity of the method approached 100%. The factors limiting the sensitivity of the examination were mainly technical. The use of the composite choline signal as a marker for malignancy in breast 1H MRS is a robust method with highly reliable interpretation, because it is based on the appearance of a single peak. The method is likely to provide even better results with technologic advances in breast MRS that lead to the improved detection of the composite choline signal.

Corrigendum to: Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN-γ/TNF-α in HT-29 cells (FEBS 27292)

Article

Jun 2003

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-α for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-γ for 7 h. Perchloric acid extracts were analyzed by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5’-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-γ/TNF-α treatment, but not in cells which were TNF-α-treated without IFN-γ preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors.

The application of13C NMR to the characterization of phospholipid metabolism in cells

Article

Jun 1992
MAGN RESON MED

31P and 31C NMR spectroscopy of lipid extracts of T47D human breast cancer spheroids and the use of 13C-labeled lipid precursors [3-13C ] serine, [1,2-13C] ethanolamine, and [1,2-13C]choline enabled us to determine the rate of 13C incorporation into the major phos-pholipids and to show that the synthesis of phosphatidylethanolarnine in T47D cells is via both the CDP-ethanolamine pathway and serine decarboxylation, with the extent of each depending on the concentration of ethanolamine in the medium. In the presence of low ethanolamine (3.4 μ M), both pathways contribute in equal proportions, while in the presence of high ethanolamine, the CDP-ethanolamine pathway predominates. © 1992 Academic Press, Inc.

Phospholipid metabolism as indicators of cancer cell function

Article

Sep 1992
NMR BIOMED

NMR methods are being applied to study phospholipid metabolism of cancer cells by monitoring the resonances which appear in the 31P spectrum. This review, aside from considering the applicability of NMR to this specific pathway, raises the question of whether the phospholipid metabolite peaks observed by MR are indicators of cancer cell function or tumor response to treatment. After assessing the results from many investigations, it is concluded that there is no clear correlation and that a combination of techniques, including in vitro and extract studies, will be necessary for a more comprehensive evaluation of the in vivo data.

Li X, Lu Y, Pirzkall A, McKnight T, Nelson SJ.. Analysis of the spatial characteristics of metabolic abnormalities in newly diagnosed glioma patients. J Magn Reson Imaging 16: 229-237

Article

Sep 2002
J MAGN RESON IMAGING

PurposeTo evaluate the role of 3D MR spectroscopic imaging (MRSI) as a tool for characterizing heterogeneity within a lesion in glioma patients.Materials and Methods Forty-nine patients with newly diagnosed glioma were studied with 3D water-suppressed proton (1-H) MRSI. Signal intensities from choline (Cho), creatine (Cr), N-acytel aspartate (NAA), and lactate/lipid (LL) were estimated from the spectra. Regions of interest (ROIs) corresponding to the metabolic abnormalities were defined and compared with the anatomic lesions.ResultsThis study showed that the tumor burden measured with either the volumes of the metabolic abnormalities or the metabolic levels in the most abnormal voxels was correlated with the degree of malignancy of the tumor. The volumes of elevated Cho and decreased NAA were useful for distinguishing low-grade from high-grade lesions. The volume of abnormal LL was correlated with the existence of necrosis and with the volume of contrast-enhancing lesions in high-grade lesions. The differences in the volume of abnormal LL were also statistically significant between patients in each grade.Conclusion These 3D-MRSI data provide important additional information to conventional MRI for evaluating and characterizing gliomas. J. Magn. Reson. Imaging 2002;16:229–237. © 2002 Wiley-Liss, Inc.

Measurements of human breast cancer using MR spectroscopy: a review of clinical measurements and a report of localized 31P measurements of response to treatment (Review Article)

Article

Jan 1998

MRTE; ; ;

The application of 13C NMR to the characterization of phospholipid metabolism in cells

Article

Jul 1992

31P and 13C NMR spectroscopy of lipid extracts of T47D human breast cancer spheroids and the use of 13C-labeled lipid precursors [3-13C]serine,[1,2-13C]ethanolamine, and [1,2-13C]choline enabled us to determine the rate of 13C incorporation into the major phospholipids and to show that the synthesis of phosphatidylethanolamine in T47D cells is via both the CDP-ethanolamine pathway and serine decarboxylation, with the extent of each depending on the concentration of ethanolamine in the medium. In the presence of low ethanolamine (3.4 microM), both pathways contribute in equal proportions, while in the presence of high ethanolamine, the CDP-ethanolamine pathway predominates.

Characterization of epithelial phenotypes in mortal and immortal human breast cells

Article

Feb 1992

We have previously described the mortal human breast epithelial culture MCF-10M, that was derived from fibrocystic breast tissue, was cultivated in medium with low calcium content for over 2 years, and spontaneously gave rise to the immortal MCF-10 cell line. The emergence of immortalized cells, characterized by growth in conventional calcium levels, from mortal cells has proven to be a reproducible event. Here we report the establishment of a second immortal line from MCF-10M, designated MCF-10-2, and establishment of the MCF-12 immortal line after long-term cultivation of MCF-12M mortal cells from reduction mammoplasty tissue. DNA fingerprinting demonstrated the independent, human origin and lineage of the MCF-10-2 and MCF-12 cell lines. Both lines require cortisol and EGF for maximal growth. The expression in these cultures of in vivo breast epithelial phenotypes was analyzed using 2-dimensional gel Western blots and immunoperoxidase staining with antibodies to cytokeratins and polymorphic epithelial mucin. MCF-10M and MCF-12M retain the cytokeratin profile of the luminal cell (7, 8, 18, 19), and also express cytokeratin 14, found predominantly in basal cells. The immortal lines express a similar profile, except that cytokeratin 19, a component of the fully differentiated luminal cell, is not expressed in the more uniform population seen in MCF-10 and MCF-12, but is retained in the morphologically mixed, less-selected population of MCF-10-2. Epitopes on the polymorphic epithelial mucin, recognized by antibodies HMFG 1, HMFG 2 and SM-3, were detected in the mortal cultures and in the immortal lines, indicating the occurrence of both normal and abnormal mucin processing. MCF-10, MCF-10-2 and MCF-12 cells do not form tumors in nude mice, but appear to organize as duct-like structures before regressing in the 5th week post injection.

Phospholipid metabolites as indicators of cancer cell function

Article

Nov 1992

Phospholipases A2 in ras-transformed and immortalized human mammary epithelial cells

Article

Nov 1994
CANCER LETT

Phospholipase A2 (PLA2) activities of non-tumorigenic and tumorigenic human mammary epithelial cells, 184B5 cells (immortalized cell line from a reduction mammoplasty) and B5KTu cells (cells from a tumor induced by ras-transformed 184B5 cells), are characterized, with emphasis on lipid biomediator-related phospholipases A2. Phospholipases A2 associated with regulation of arachidonic acid metabolism include the high molecular mass cytosolic PLA2 (cPLA2) and group II PLA2. The major PLA2 activity in the mammary epithelial cells has the characteristics of cPLA2; this activity is higher in the B5KTu cells. In contrast, the 184B5 and B5KTu cells have similar levels of a Ca(2+)-independent, cytosolic PLA2 activity and low levels of a particulate fraction PLA2 activity, which does not have the properties of group II PLA2. Thus, cPLA2 activity is selectively elevated in the tumorigenic human mammary epithelial cells and this may result in increased generation of lipid biomediators such as arachidonic acid metabolites.

Simultaneous Extraction of Cellular Lipids and Water-Soluble Metabolites: Evaluation by NMR Spectroscopy

Article

Feb 1996

A method for simultaneous extraction of lipids and water-soluble metabolites from a single cell sample was developed and optimized for NMR spectroscopy. Intermediary metabolites in cultured M2R mouse melanoma cells and changes therein in response to challenge with melanotropin were studied by 31P and 13C NMR. Cells were extracted with methanol, chloroform, and water (1:1:1, v/v/v). The contents of the chloroform and methanol-water phases were separated and quantitatively recovered. The contents of the upper and lower phases compared well with the homologous fractions obtained by perchloric acid and Folch's lipid extraction methods. The pH of the extracts remained within the physiologic range, eliminating potential deleterious effect on cellular metabolites. The water phase contained minimal amounts of salts, making these extracts amenable to subsequent analytical procedures. Obtaining lipid- and water-soluble metabolites from the same sample enables characterization of metabolic pathways that bridge the two cellular components in a quantitative manner.

Variations in energy and phospholipid metabolism in normal and cancer human mammary epithelial cells

Article

May 1996

By comparing the metabolism of human mammary epithelial cells and human breast cancer cells (MCF7 and T47D), proliferating at approximately the same rate, it was possible to isolate the effect of malignancy alone on the energetics and phospholipid metabolism of cancer cells. 31p NMR of perfused mammary cells and of water soluble extracts of these cells showed that the levels of phosphocholine, phosphoethanolamine, and glycerol derivatives of these metabolites were very low and significantly less than that in the cancer cells, suggesting an association of malignancy with induction of phospholipid biosynthesis and breakdown. The level of the high energy phosphates and the rates of glucose consumption and aerobic glycolysis did not reveal distinct differences between normal and cancer cells. The comparable energetic appear to be related to the similarity of proliferating capacity in culture of the normal and cancer cells.

Kinetics of choline transport and phosphorylation in human breast cancer cells; NMR application of the zero trans method

Article

Nov 1995

The mechanism and kinetics of choline transport and phosphorylation in MCF7 human breast cancer cells was studied by 31P, 13C and 2H NMR, applying the zero trans method. Choline was transported by a Michaelis-Menten like mechanism with a maximum transport rate T(max) = 13.5 +/- 2.6 nmol/hour/mg protein (3.06 +/- 0.6 fmol/cell/hour) and choline concentration at half maximal transport rate of Kt = 46.5 +/- 2.8 microM. The rate of choline phosphorylation was more than two orders of magnitude faster than the rate of its transport (T(max)) maintaining the ratio [phosphocholine]/[choline] higher than 100. The results demonstrated enhanced choline transport and choline kinase activity in breast cancer cells.

Magnetic Resonance Spectroscopic Changes in Alzheimer's Disease

Article

Oct 1997

In vitro and in vivo 31P magnetic resonance (MR) spectroscopy studies of Alzheimer's disease (AD) brain have revealed alterations in membrane phospholipid metabolism and high-energy phosphate metabolism. Mildly demented AD patients compared with control subjects have increased levels of phosphomonoesters, decreased levels of phosphocreatine and probably adenosine diphosphate and an increased oxidative metabolic rate. As the dementia worsens, levels of phosphomonoesters decrease and levels of phosphocreatine and adenosine di-phosphate increase. The changes in oxidative metabolic rate suggest that the AD brain is under energetic stress. The phosphomonoester findings support our in vitro findings and implicate basic defects in membrane metabolism in AD brain. MR spectroscopy provides new diagnostic insights and a noninvasive method to follow the progression of the disease and the metabolic response to therapeutic interventions.

Evaluating Human Breast Ductal Carcinomas with High-Resolution Magic-Angle Spinning Proton Magnetic Resonance Spectroscopy

Article

Dec 1998

We report the results of a study of human breast ductal carcinomas, conducted by using high resolution magic angle spinning proton magnetic resonance spectroscopy (HRMAS 1HMRS). This recently developed spectroscopic technique can measure tissue metabolism from intact pathological specimens and identify tissue biochemical changes, which closely correspond to tumor in vivo state. This procedure objectively indicates diagnostic parameters, independent of the skill and experience of the investigator, and has the potential to reduce the sampling errors inherently associated with procedures of conventional histopathology. In this study, we measured 19 cases of female ductal carcinomas. Our results demonstrate that: (1) highly resolved spectra of intact specimens of human breast ductal carcinomas can be obtained; (2) carcinoma-free tissues and carcinomas are distinguishable by alterations in the intensities and the spin-spin relaxation time T2 of cellular metabolites; and (3) tumor metabolic markers, such as phosphocholine, lactate, and lipids, may correlate with the histopathological grade determined from evaluation of the adjacent specimen. Our results suggest that biochemical markers thus measured may function as a valuable adjunct to histopathology to improve the accuracy of and reduce the time frame required for the diagnosis of human breast cancer.

Malignant Transformation Alters Membrane Choline Phospholipid Metabolism of Human Mammary Epithelial Cells1

Article

Feb 1999

Transduction of mitogenic signals in cells can be mediated by molecules derived from the synthesis and breakdown of the major membrane phospholipid, phosphotidylcholine. Studies were performed on human mammary epithelial cells in culture to understand the impact of malignant transformation and progression on membrane phospholipid metabolism. In the model system used here, phosphocholine levels and total choline-containing phospholipid metabolite levels increased with progression from normal to immortalized to oncogene-transformed to tumor-derived cells. These changes occurred independently of cell doubling time. A "glycerophosphocholine to phosphocholine switch" was apparent with immortalization. This alteration in phenotype of increased phosphocholine relative to glycerophosphocholine was observed in oncogene-transformed and for all human breast tumor cell lines analyzed. The results demonstrate that progression of human mammary epithelial cells from normal to malignant phenotype is associated with altered membrane choline phospholipid metabolism.

Nm23-transfected MDA-MB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: A 31P nuclear magnetic resonance study in vivo and vitro

Article

Jun 1999

Noh DY, Ahn SJ & Lee RA. et al Overexpression of phospholipase D1 in human breast cancer tissues. Cancer Lett161: 207-214

Article

Jan 2001
CANCER LETT

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. PLD is a major enzyme implicated in important cellular processes, such as cell proliferation. We designed this study to investigate the expression of PLD in human breast carcinomas and non-malignant tissues using RT-PCR, Western blot analysis, immunohistochemistry and an Arf-dependent PLD activity assay. We examined about 550 bp of PCR product and 120 kDa of PLD protein. Our results showed that PLD protein and mRNA levels were overexpressed in 14 of 17 breast cancer tissues. We also observed increased expression by immunohistochemistry and Arf-dependent PLD activity in microsomes of human breast tumors, which correlated well with PLD expression. PLD expression was elevated in human breast tumors compared with normal breast tissues. These results implicate a possible role of PLD in human breast tumorigenesis and suggest that PLD may be useful as a marker for malignant disease in the breast.

Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection

Article

Feb 2001

Recent advances in cDNA and oligonucleotide DNA arrays have made it possible to measure the abundance of mRNA transcripts for many genes simultaneously. The analysis of such experiments is nontrivial because of large data size and many levels of variation introduced at different stages of the experiments. The analysis is further complicated by the large differences that may exist among different probes used to interrogate the same gene. However, an attractive feature of high-density oligonucleotide arrays such as those produced by photolithography and inkjet technology is the standardization of chip manufacturing and hybridization process. As a result, probe-specific biases, although significant, are highly reproducible and predictable, and their adverse effect can be reduced by proper modeling and analysis methods. Here, we propose a statistical model for the probe-level data, and develop model-based estimates for gene expression indexes. We also present model-based methods for identifying and handling cross-hybridizing probes and contaminating array regions. Applications of these results will be presented elsewhere.

Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application

Article

Feb 2001

A model-based analysis of oligonucleotide expression arrays we developed previously uses a probe-sensitivity index to capture the response characteristic of a specific probe pair and calculates model-based expression indexes (MBEI). MBEI has standard error attached to it as a measure of accuracy. Here we investigate the stability of the probe-sensitivity index across different tissue types, the reproducibility of results in replicate experiments, and the use of MBEI in perfect match (PM)-only arrays. Probe-sensitivity indexes are stable across tissue types. The target gene's presence in many arrays of an array set allows the probe-sensitivity index to be estimated accurately. We extended the model to obtain expression values for PM-only arrays, and found that the 20-probe PM-only model is comparable to the 10-probe PM/MM difference model, in terms of the expression correlations with the original 20-probe PM/MM difference model. MBEI method is able to extend the reliable detection limit of expression to a lower mRNA concentration. The standard errors of MBEI can be used to construct confidence intervals of fold changes, and the lower confidence bound of fold change is a better ranking statistic for filtering genes. We can assign reliability indexes for genes in a specific cluster of interest in hierarchical clustering by resampling clustering trees. A software dChip implementing many of these analysis methods is made available. The model-based approach reduces the variability of low expression estimates, and provides a natural method of calculating expression values for PM-only arrays. The standard errors attached to expression values can be used to assess the reliability of downstream analysis.

Levels of phospholipid metabolites in breast cancer cells treated with antimitotic drugs: A P-31-magnetic resonance spectroscopy study

Article

Nov 2001

Magnetic resonance spectroscopy (MRS) methods have provided valuable information on cancer cell metabolism. In this study, we characterized the 31P-MR spectra of breast cancer cell lines exhibiting differences in hormonal response, estrogen receptors (positive/negative), and metastatic potential. A correlation was made between the cytotoxic effect of antimitotic drugs and changes in cell metabolism pattern. Because most anticancer drugs are more effective on proliferating cells, our study attempted to elucidate the metabolic profile and specific metabolic changes associated with the effect of anticancer drugs on proliferating breast cancer cell lines. Accordingly, for the 31P-MRS experiments, cells were embedded in Matrigel to preserve their proliferation profile and ability to absorb drugs. The MRS studies of untreated cells indicated that the levels of phosphodiesters and uridine diphosphosugar metabolites were significantly higher in estrogen receptor-positive and low metastatic potential cell lines. 31P-MRS observations revealed a correlation between the mode of action of anticancer drugs and the observed changes in cell metabolic profiles. When cells were treated with antimicrotubule drugs (paclitaxel, vincristine, colchicine, nocodazole), but not with methotrexate and doxorubicin, a profound elevation of intracellular glycerophosphorylcholine (GPC) was recorded that was not associated with changes in phospholipid composition of cell membrane. Remarkably, the rate of elevation of intracellular GPC was much faster in cell population synchronized at G2-M compared with the unsynchronized cells. The steady-state level of GPC for paclitaxel-treated cells was reached after approximately 4 h for synchronized cells and after approximately 24 h (approximate duration of one cell cycle) for the unsynchronized ones. These observations may indicate a correlation between microtubule status and cellular phospholipid metabolism. This study demonstrates that 31P-MRS may have diagnostic value for treatment decisions of breast cancer and reveals new aspects of the mechanism of action of antimicrotubule drugs.

Metabolic markers of breast cancer: Enhanced choline metabolism and reduced choline-ether-phospholipid synthesis

Article

May 2002

Specific genetic alterations during malignant transformation may induce the synthesis and breakdown of choline phospholipids, mediating transduction of mitogenic signals. The high level of water-soluble choline metabolites in cancerous breast tumors, relative to benign lesions and normal breast tissue, has been used as a diagnostic marker of malignancy. To unravel the biochemical pathways underlying this phenomenon, we used tracer kinetics and 13C and 31P magnetic resonance spectroscopy to compare choline transport, routing, and metabolism to phospholipids in primary cultures of human mammary epithelial cells and in MCF7 human breast cancer cells. The rate of choline transport under physiological choline concentrations was 2-fold higher in the cancer cells. The phosphorylation of choline to phosphocholine and oxidation of choline to betaine yielded 10-fold higher levels of these metabolites in the cancer cells. However, additional incorporation of choline to phosphatidylcholine was similar in both cell types. Thus, enhanced choline transport and augmented synthesis of phosphocholine and betaine are dominant pathways responsible for the elevated presence of choline metabolites in cancerous breast tumors. Uniquely, reduced levels and synthesis of a choline-ether-phospholipid may also serve as a metabolic marker of breast cancer.

Eliminating spurious sidebands in 1H MRS of breast lesions

Article

Aug 2002

Detecting metabolites in breast lesions by in vivo (1)H MR spectroscopy can be difficult due to the abundance of mobile lipids in the breast which can produce spurious sidebands that interfere with the metabolite signals. Two-dimensional J-resolved spectroscopy has been demonstrated in the brain as a means to eliminate these artifacts from a large water signal; coherent sidebands are resolved at their natural frequencies, leaving the noncoupled metabolite resonances in the zero-frequency trace of the 2D spectrum. This work demonstrates that using the zero-frequency trace-or equivalently the average of spectra acquired with different echo times-can be used to separate noncoupled metabolite signals from the lipid-induced sidebands. This technique is demonstrated with simulations, phantom studies, and in several breast lesions. Compared to the conventional approach using a single echo time, echo time averaging provides increased sensitivity for the study of small and irregularly shaped lesions.

Real-time changes in 1H and 31P NMR spectra of malignant human mammary epithelial cells during treatment with the anti-inflammatory agent indomethacin

Article

Nov 2002

Choline metabolites in malignant human mammary epithelial cells (HMECs) are significantly altered compared to normal HMECs. (1)H NMR studies of cell extracts have shown that treatment of malignant HMECs with a nonsteroidal anti-inflammatory agent, indomethacin, results in a distribution of choline compounds more typical of nonmalignant HMECs. To follow the time course of these changes, in this study real-time monitoring of choline compounds of malignant MDA-MB-231 cells was performed during treatment with indomethacin. The contribution of changes in intra- and extracellular pH to changes in choline compounds was also examined. Changes in water-soluble choline phospholipid metabolites, such as phosphocholine (PC), glycerophosphocholine (GPC), and total choline, as well as intracellular pH, were monitored by (31)P and diffusion-weighted (1)H NMR spectroscopy of living cells using an NMR-compatible perfusion system. An accumulation of GPC and a decrease of PC, resulting in an increased [GPC]/[PC] ratio, were detected within 2 hr of treatment with 200 microM indomethacin. Since a decreased [GPC]/[PC] ratio is associated with increased malignancy, these data demonstrate that nonspecific cyclooxygenase inhibition by indomethacin alters the choline metabolite profile of malignant cells towards a less malignant phenotype. These changes were not related to alterations of intra- or extracellular pH.

Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN-gamma/TNF-alpha in HT-29 cells

Article

Jul 2003

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-alpha for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-gamma for 7 h. Perchloric acid extracts were analyzed by (1)H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5'-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-gamma/TNF-alpha treatment, but not in cells which were TNF-alpha-treated without IFN-gamma preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors.

Diffusely elevated cerebral choline and creatine in relapsing-remitting multiple sclerosis

Article

Jul 2003

It is well known that multiple sclerosis (MS) pathogenesis continues even during periods of clinical silence. To quantify the metabolic characteristics of this activity we compared the absolute levels of N-acetylaspartate (NAA), creatine (Cr), and choline (Cho) in the normal-appearing white matter (NAWM) between relapsing-remitting (RR) MS patients and controls. Metabolite concentrations were obtained with 3D proton MR spectroscopy at 1.5 T in a 480 cm(3) volume-of-interest (VOI), centered on the corpus callosum of 11 MS patients and 9 matched controls. Gray/white-matter/cerebral-spinal-fluid (CSF) volumes were obtained from MRI segmentation. Patients' average VOI tissue volume (V(T)), 410.8 +/- 24.0 cm(3), and metabolite levels, NAA = 6.33 +/- 0.70, Cr = 4.67 +/- 0.52, Cho = 1.40 +/- 0.17 mM, were different from the controls by -8%, -9%, +22% and +32%. The Cho level was the only single metric differentiating patients from controls at 100% specificity and >90% sensitivity. Diffusely elevated Cho and Cr probably reflect widespread microscopic inflammation, gliosis, or de- and remyelination in the NAWM. Both metabolites are potential prognostic indicators of current disease activity, preceding NAA decline and atrophy.

Choline Phospholipid Metabolites of Human Vascular Endothelial Cells Altered by Cyclooxygenase Inhibition, Growth Factor Depletion, and Paracrine Factors Secreted by Cancer Cells

Article

May 2003

Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.

Molecular Causes of the Aberrant Choline Phospholipid Metabolism in Breast Cancer

Abstract

No full-text available

Recommended publications

Platelet-Activating Factor Mediates Phosphatidylcholine Hydrolysis by Phospholipase D in Human Endom...

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endom...