Plasma Metabolomics for Discovery of Early Metabolic Markers of Prostate Cancer Based on Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry.

BACKGROUND: The prevention and early screening of PCa is highly dependent on the identification of new biomarkers. In this study, we investigated whether plasma metabolic profiles from healthy males provide novel early biomarkers associated with future risk of PCa. METHODS: Using the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, we identified plasma samples collected from 146 PCa cases up to 13 years prior to diagnosis and 272 matched controls. Plasma metabolic profiles were characterized using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). RESULTS: Orthogonal partial least squares discriminant analysis (OPLS-DA) discriminated PCa cases from controls, with a median area under the receiver operating characteristic curve (AU-ROC) of 0.92 using a 1000-time repeated random sub-sampling validation. Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) identified the top 10 most important metabolites (p < 0.001) discriminating PCa cases from controls. Among them, phosphate, ethyl oleate, eicosadienoic acid were higher in individuals that developed PCa than in the controls during the follow-up. In contrast, 2-hydroxyadenine, sphinganine, L-glutamic acid, serotonin, 7-keto cholesterol, tiglyl carnitine, and sphingosine were lower. CONCLUSION: Our results support the dysregulation of amino acids and sphingolipid metabolism during the development of PCa. After validation in an independent cohort, these signatures may promote the development of new prevention and screening strategies to identify males at future risk of PCa.

NMR metabolomic profiles associated with long-term risk of prostate cancer.

INTRODUCTION: Prostate cancer is a multifactorial disease whose aetiology is still not fully understood. Metabolomics, by measuring several hundred metabolites simultaneously, could enhance knowledge on the metabolic changes involved and the potential impact of external factors. OBJECTIVES: The aim of the present study was to investigate whether pre-diagnostic plasma metabolomic profiles were associated with the risk of developing a prostate cancer within the following decade. METHODS: A prospective nested case-control study was set up among the 5141 men participant of the SU.VI.MAX cohort, including 171 prostate cancer cases, diagnosed between 1994 and 2007, and 171 matched controls. Nuclear magnetic resonance (NMR) metabolomic profiles were established from baseline plasma samples using NOESY1D and CPMG sequences. Multivariable conditional logistic regression models were computed for each individual NMR signal and for metabolomic patterns derived using principal component analysis. RESULTS: Men with higher fasting plasma levels of valine (odds ratio (OR) = 1.37 [1.07-1.76], p = .01), glutamine (OR = 1.30 [1.00-1.70], p = .047), creatine (OR = 1.37 [1.04-1.80], p = .02), albumin lysyl (OR = 1.48 [1.12-1.95], p = .006 and OR = 1.51 [1.13-2.02], p = .005), tyrosine (OR = 1.40 [1.06-1.85], p = .02), phenylalanine (OR = 1.39 [1.08-1.79], p = .01), histidine (OR = 1.46 [1.12-1.88], p = .004), 3-methylhistidine (OR = 1.37 [1.05-1.80], p = .02) and lower plasma level of urea (OR = .70 [.54-.92], p = .009) had a higher risk of developing a prostate cancer during the 13 years of follow-up. CONCLUSIONS: This exploratory study highlighted associations between baseline plasma metabolomic profiles and long-term risk of developing prostate cancer. If replicated in independent cohort studies, such signatures may improve the identification of men at risk for prostate cancer well before diagnosis and the understanding of this disease.

Two Metabolomics Phenotypes of Human Hepatocellular Carcinoma in Non-Alcoholic Fatty Liver Disease According to Fibrosis Severity.

Non-Alcoholic Fatty Liver Disease (NAFLD) is considered as the forthcoming predominant cause for hepatocellular carcinoma (HCC). NAFLD-HCC may rise in non-cirrhotic livers in 40 to 50% of patients. The aim of this study was to identify different metabolic pathways of HCC according to fibrosis level (F0F1 vs. F3F4). A non-targeted metabolomics strategy was applied. We analyzed 52 pairs of human HCC and adjacent non-tumoral tissues which included 26 HCC developed in severe fibrosis or cirrhosis (F3F4) and 26 in no or mild fibrosis (F0F1). Tissue extracts were analyzed using 1H-Nuclear Magnetic Resonance spectroscopy. An optimization evolutionary method based on genetic algorithm was used to identify discriminant metabolites. We identified 34 metabolites differentiating the two groups of NAFLD-HCC according to fibrosis level, allowing us to propose two metabolomics phenotypes of NAFLD-HCC. We showed that HCC-F0F1 mainly overexpressed choline derivatives and glutamine, whereas HCC-F3F4 were characterized by a decreased content of monounsaturated fatty acids (FA), an increase of saturated FA and an accumulation of branched amino acids. Comparing HCC-F0F1 and HCC-F3F4, differential expression levels of glucose, choline derivatives and phosphoethanolamine, monounsaturated FA, triacylglycerides were identified as specific signatures. Our metabolomics analysis of HCC tissues revealed for the first time two phenotypes of HCC developed in NAFLD according to fibrosis level. This study highlighted the impact of the underlying liver disease on metabolic reprogramming of the tumor.

Untargeted plasma metabolomic profiles associated with overall diet in women from the SU.VI.MAX cohort.

PURPOSE: Dietary intakes are reflected in plasma by the presence of hundreds of exogenous metabolites and variations in endogenous metabolites. The exploration of diet-related plasma metabolic profiles could help to better understand the impact of overall diet on health. Our aim was to identify metabolomic signatures reflecting overall diet in women from the French general population. METHODS: This cross-sectional study included 160 women in the SU.VI.MAX cohort with detailed dietary data (≥ 10 24-h dietary records) selected according to their level of adherence to the French dietary recommendations, represented by the validated score mPNNS-GS; 80 women from the 10th decile of the score were matched with 80 women from the 1st decile. Plasma metabolomic profiles were acquired using untargeted UPLC-QToF mass spectrometry analysis. The associations between metabolomic profiles and the mPNNG-GS, its components and Principal Component Analyses-derived dietary patterns were investigated using multivariable conditional logistic regression models and partial correlations. RESULTS: Adherence to the dietary recommendations was positively associated with 3-indolepropionic acid and pipecolic acid (also positively associated with fruit and vegetable intake and a healthy diet)-2 metabolites linked to microbiota and inversely associated with lysophosphatidylcholine (LysoPC(17:1)), acylcarnitine C9:1 (also inversely associated with a healthy diet), acylcarnitine C11:1 and 2-deoxy-D-glucose. Increased plasma levels of piperine and Dihydro4mercapto-3(2H) furanone were observed in women who consumed a Western diet and a healthy diet, respectively. Ethyl-ß-D-glucopyranoside was positively associated with alcohol intake. Plasma levels of LysoPC(17:1), cholic acid, phenylalanine-phenylalanine and phenylalanine and carnitine C9:1 decreased with the consumption of vegetable added fat, sweetened food, milk and dairy products and fruit and vegetable intakes, respectively. CONCLUSION: This study highlighted several metabolites from both host and microbial metabolism reflecting the long-term impact of the overall diet. TRIAL REGISTRATION: SU.VI.MAX, clinicaltrials.gov NCT00272428. Registered 3 January 2006, https://clinicaltrials.gov/show/NCT00272428.

Diet-Related Metabolomic Signature of Long-Term Breast Cancer Risk Using Penalized Regression: An Exploratory Study in the SU.VI.MAX Cohort.

BACKGROUND: Diet has been recognized as a modifiable risk factor for breast cancer. Highlighting predictive diet-related biomarkers would be of great public health relevance to identify at-risk subjects. The aim of this exploratory study was to select diet-related metabolites discriminating women at higher risk of breast cancer using untargeted metabolomics. METHODS: Baseline plasma samples of 200 incident breast cancer cases and matched controls, from a nested case-control study within the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, were analyzed by untargeted LC-MS. Diet-related metabolites were identified by partial correlation with dietary exposures, and best predictors of breast cancer risk were then selected by Elastic Net penalized regression. The selection stability was assessed using bootstrap resampling. RESULTS: 595 ions were selected as candidate diet-related metabolites. Fourteen of them were selected by Elastic Net regression as breast cancer risk discriminant ions. A lower level of piperine (a compound from pepper) and higher levels of acetyltributylcitrate (an alternative plasticizer to phthalates), pregnene-triol sulfate (a steroid sulfate), and 2-amino-4-cyano butanoic acid (a metabolite linked to microbiota metabolism) were observed in plasma from women who subsequently developed breast cancer. This metabolomic signature was related to several dietary exposures such as a "Western" dietary pattern and higher alcohol and coffee intakes. CONCLUSIONS: Our study suggested a diet-related plasma metabolic signature involving exogenous, steroid metabolites, and microbiota-related compounds associated with long-term breast cancer risk that should be confirmed in large-scale independent studies. IMPACT: These results could help to identify healthy women at higher risk of breast cancer and improve the understanding of nutrition and health relationship.

Plasma Metabolomic Signatures Associated with Long-term Breast Cancer Risk in the SU.VI.MAX Prospective Cohort.

BACKGROUND: Breast cancer is a major cause of death in occidental women. The role of metabolism in breast cancer etiology remains unclear. Metabolomics may help to elucidate novel biological pathways and identify new biomarkers to predict breast cancer long before symptoms appear. The aim of this study was to investigate whether untargeted metabolomic signatures from blood draws of healthy women could contribute to better understand and predict the long-term risk of developing breast cancer. METHODS: A nested case-control study was conducted within the SU.VI.MAX prospective cohort (13 years of follow-up) to analyze baseline plasma samples of 211 incident breast cancer cases and 211 matched controls by LC/MS. Multivariable conditional logistic regression models were computed. RESULTS: A total of 3,565 ions were detected and 1,221 were retained for statistical analysis. A total of 73 ions were associated with breast cancer risk (P < 0.01; FDR ≤ 0.2). Notably, we observed that a lower plasma level of O-succinyl-homoserine (OR = 0.70, 95%CI = [0.55-0.89]) and higher plasma levels of valine/norvaline [1.45 (1.15-1.83)], glutamine/isoglutamine [1.33 (1.07-1.66)], 5-aminovaleric acid [1.46 (1.14-1.87)], phenylalanine [1.43 (1.14-1.78)], tryptophan [1.40 (1.10-1.79)], γ-glutamyl-threonine [1.39 (1.09-1.77)], ATBC [1.41 (1.10-1.79)], and pregnene-triol sulfate [1.38 (1.08-1.77)] were associated with an increased risk of developing breast cancer during follow-up.Conclusion: Several prediagnostic plasmatic metabolites were associated with long-term breast cancer risk and suggested a role of microbiota metabolism and environmental exposure. IMPACT: After confirmation in other independent cohort studies, these results could help to identify healthy women at higher risk of developing breast cancer in the subsequent decade and to propose a better understanding of the complex mechanisms involved in its etiology.

NMR metabolomics of fibroblasts with inherited mitochondrial Complex I mutation reveals treatment-reversible lipid and amino acid metabolism alterations.

INTRODUCTION: Elucidating molecular alterations due to mitochondrial Complex I (CI) mutations may help to understand CI deficiency (CID), not only in mitochondriopathies but also as it is caused by drugs or associated to many diseases. OBJECTIVES: CID metabolic expression was investigated in Leber's hereditary optic neuropathy (LHON) caused by an inherited mutation of CI. METHODS: NMR-based metabolomics analysis was performed in intact skin fibroblasts from LHON patients. It used several datasets: one-dimensional 1H-NMR spectra, two-dimensional 1H-NMR spectra and quantified metabolites. Spectra were analysed using orthogonal partial least squares-discriminant analysis (OPLS-DA), and quantified metabolites using univariate statistics. The response to idebenone (IDE) and resveratrol (RSV), two agents improving CI activity and mitochondrial functions was evaluated. RESULTS: LHON fibroblasts had decreased CI activity (- 43%, p < 0.01). Metabolomics revealed prominent alterations in LHON including the increase of fatty acids (FA), polyunsaturated FA and phosphatidylcholine with a variable importance in the prediction (VIP) > 1 in OPLS-DA, p < 0.01 in univariate statistics, and the decrease of amino acids (AA), predominantly glycine, glutamate, glutamine (VIP > 1) and alanine (VIP > 1, p < 0.05). In LHON, treatment with IDE and RSV increased CI activity (+ 40 and + 44%, p < 0.05). IDE decreased FA, polyunsaturated FA and phosphatidylcholine (p < 0.05), but did not modified AA levels. RSV decreased polyunsaturated FA, and increased several AA (VIP > 1 and/or p < 0.05). CONCLUSION: LHON fibroblasts display lipid and amino acid metabolism alterations that are reversed by mitochondria-targeted treatments, and can be related to adaptive changes. Findings bring insights into molecular changes induced by CI mutation and, beyond, CID of other origins.

NMR metabolomic signatures reveal predictive plasma metabolites associated with long-term risk of developing breast cancer.

Background: Combination of metabolomics and epidemiological approaches opens new perspectives for ground-breaking discoveries. The aim of the present study was to investigate for the first time whether plasma untargeted metabolomic profiles, established from a simple blood draw from healthy women, could contribute to predict the risk of developing breast cancer within the following decade and to better understand the aetiology of this complex disease. Methods: A prospective nested case-control study was set up in the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, including 206 breast cancer cases diagnosed during a 13-year follow-up and 396 matched controls. Untargeted nuclear magnetic resonance (NMR) metabolomic profiles were established from baseline plasma samples. Multivariable conditional logistic regression models were computed for each individual NMR variable and for combinations of variables derived by principal component analysis. Results: Several metabolomic variables from 1D NMR spectroscopy were associated with breast cancer risk. Women characterized by higher fasting plasma levels of valine, lysine, arginine, glutamine, creatine, creatinine and glucose, and lower plasma levels of lipoproteins, lipids, glycoproteins, acetone, glycerol-derived compounds and unsaturated lipids had a higher risk of developing breast cancer. P-values ranged from 0.00007 [odds ratio (OR)T3vsT1=0.37 (0.23-0.61) for glycerol-derived compounds] to 0.04 [ORT3vsT1=1.61 (1.02-2.55) for glutamine]. Conclusion: This study highlighted associations between baseline NMR plasma metabolomic signatures and long-term breast cancer risk. These results provide interesting insights to better understand complex mechanisms involved in breast carcinogenesis and evoke plasma metabolic disorders favourable for carcinogenesis initiation. This study may contribute to develop screening strategies for the identification of at-risk women for breast cancer well before symptoms appear.

Specificities of Human Hepatocellular Carcinoma Developed on Non-Alcoholic Fatty Liver Disease in Absence of Cirrhosis Revealed by Tissue Extracts ¹H-NMR Spectroscopy.

There is a rising incidence of non-alcoholic fatty liver disease (NAFLD) as well as of the frequency of Hepato-Cellular Carcinoma (HCC) associated with NAFLD. To seek for putative metabolic pathways specific of the NAFLD etiology, we performed comparative metabolomics between HCC associated with NAFLD and HCC associated with cirrhosis. The study included 28 pairs of HCC tissue versus distant Non-Tumoral Tissue (NTT) collected from patients undergoing hepatectomy. HCC was associated with cirrhosis (n = 9), normal liver (n = 6) and NAFLD (n = 13). Metabolomics was performed using 1H-NMR Spectroscopy on tissue extracts and combined to multivariate statistical analysis. In HCC compared to NTT, statistical models showed high levels of lactate and phosphocholine, and low level of glucose. Shared and Unique Structures (SUS) plots were performed to remove the impact of underlying disease on the metabolic profile of HCC. HCC-cirrhosis was characterized by high levels of ß-hydroxybutyrate, tyrosine, phenylalanine and histidine whereas HCC-NAFLD was characterized by high levels of glutamine/glutamate. In addition, the overexpression glutamine/glutamate on HCC-NAFLD was confirmed by both Glutamine Synthetase (GS) immuno-staining and NMR-spectroscopy glutamine quantification. This study provides evidence of metabolic specificities of HCC associated with non-cirrhotic NAFLD versus HCC associated with cirrhosis. These alterations could suggest activation of glutamine synthetase pathway in HCC-NAFLD and mitochondrial dysfunction in HCC-cirrhosis, that may be part of specific carcinogenic processes.

Metabolomics and transcriptomics demonstrate severe oxidative stress in both localized chemotherapy-treated and bystander tumors.

BACKGROUND: Localized radiotherapy is long known to cause damages to not only targeted but also non-targeted cells, the so-called bystander (BS) effect. Recently, BS effect was demonstrated in response to chemotherapy. To get further insight into the mechanism of chemotherapy-induced BS effect in vivo, we investigated the response of normal tissues and untreated BS melanomas, at distance from localized chemotherapy-treated melanomas. METHODS: B16 melanoma cells were inoculated sc in one flank, in mice. Chemotherapy was administered intratumorally. After 3 weeks, untreated melanomas were implanted into the other flank. Tumors were analyzed morphologically, and using metabolomics and transcriptomics. RESULTS: Locally-treated melanomas showed growth inhibition and pleiotropic metabolic and transcriptional alterations. Tumors recovered slow proliferation while exhibiting prominent oxidative stress response (decreased glutathione level, and increased expression of genes including Mt1, Gpx3, Sod3, and Hmox1). Plasma contained increased levels of oxidative stress products. However, liver and soleus muscle displayed unaltered morphological characteristics. In contrast, untreated BS melanomas induced from naive B16 cells showed reduced growth, marked oxidative stress response (decreased glutathione level, and increased expression of genes including Sod2, Gpx1 and Gsr), and ras oncogene expression alterations. Furthermore, metabolomics and transcriptomics enabled to estimate the proportion of cells undergoing the BS effect within treated tumors. CONCLUSION: Treatment of tumors with chemotherapy induces BS effects, underpinned by oxidative stress, in abnormal proliferating tissues in vivo, not in normal tissue, that significantly contribute to overall tumor response. General significance BS effect significantly contributes to response to chemotherapy, and may be exploited to improve overall response to cancer treatment.

Normal human melanocytes exposed to chronic insulin and glucose supplementation undergo oncogenic changes and methyl group metabolism cellular redistribution.

Recent epidemiological studies have suggested a link between cancer and pathophysiological conditions associated with hyperinsulinemia. In this report, we address the possible role of insulin exposure in melanocyte transformation. To this aim, normal melanocytes were exposed to chronic insulin and glucose supplementation (twice the standard medium concentration) for at least 3 wk. After 3-wk treatment, melanocytes increased proliferation (doubling time: 2.7 vs. 5.6 days, P < 0.01). After 3-wk treatment or after 3-wk treatment followed by 4-wk reculture in standard medium, melanocytes were able to grow in soft agar colonies. Treated melanocytes had increased DNA content (+8%, P < 0.05), chromosomal aberrations, and modified oncoprotein profile: p-Akt expression increased (+32%, P < 0.01), Akt decreased, and c-Myc increased (+40%, P < 0.05). PP2A protein expression increased (+42, P < 0.05), while PP2A methylation decreased (-42%, P < 0.05), and PP2A activity was reduced (-27%, P < 0.05). PP2A transcription level was increased (ppp2r1a, PP2A subunit A, +44%, P < 0.05). Also, transcriptomic data revealed modifications in insr (insulin receptors, +10%, P < 0.05) and Il8 (inflammation protein, +99%, P < 0.01). Glycolysis was modified with increased transcription of Pgk1 and Hif1a (P < 0.05), decreased transcription of Pfkfb3 (P < 0.05), decreased activity of pyruvate kinase (P < 0.01), and decreased pyruvate cell content as assessed by (1)H-NMR spectroscopy. In addition, methyl group metabolism was altered with decreased global DNA methylation (-51%, P < 0.01), increased cytosolic protein methylation (+18%, P < 0.05), and consistent changes in methylated species on (1)H-NMR spectra. In conclusion, exposure to chronic insulin and glucose supplementation induces oncogenic changes and methyl group metabolism redistribution, which may be a biomarker of transformation.

Cell cycle-dependent conjugation of endogenous BRCA1 protein with SUMO-2/3.

BACKGROUND: BRCA1, the main breast and ovarian cancer susceptibility gene, has a key role in maintenance of genome stability, cell cycle and transcription regulation. Interestingly, some of the numerous proteins which interact with BRCA1 protein undergo conjugation with small ubiquitin-like modifiers (SUMO). This post-translational modification is related to transcription, DNA repair, nuclear transport, signal transduction, and to cell cycle stress response. METHODS AND RESULTS: Protein sequence analysis suggests that sumoylation target sites belong to the RING finger and BRCT domains (BRCA1 C-terminus), two crucial regions for BRCA1 function. Moreover putative SUMO interacting motifs are present in the sequence of many proteins of BRCA1 network. Using immunoprecipitations and western blotting, we show the conjugation of endogenous nuclear BRCA1 protein with SUMO-2/3. BRCA1 conjugation with SUMO-2/3 is linked to the cell cycle in a cell line dependent manner since no cell cycle dependence of sumoylation is observed in MCF7 breast cancer cells. In contrast, BRCA1 conjugation with SUMO-2/3 is linked to the oxidative stress independently to the cell line, in DU145, MCF7 and 293 T cells. CONCLUSION AND GENERAL SIGNIFICANCE: Our data reveal a new BRCA1 regulation pathway implying sumoylation in response to cell cycle progression and oxidative stress, providing a possible mechanism for the involvement of BRCA1 gene in tumorigenesis.

Quantitative two-dimensional HRMAS 1H-NMR spectroscopy-based metabolite profiling of human cancer cell lines and response to chemotherapy.

NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping.

A model of phospholipid biosynthesis in tumor in response to an anticancer agent in vivo.

We study, in this paper, a model for the core of the system of the Glycerophospholipid metabolism in the murine cells. It comprises the simple and enzymatic reactions of PhosphatidylEthanolamine and the PhosphatidylCholine. The model's general structure is taken from a number of books and articles. We translate this model into a set of ordinary differential equations (ODEs), to propose a quantitative explanation of the experimental experiences and the observed results. In order to make it usable as a basis for simulations and mathematical analysis we need to make precise the various constants present in the equations but which are usually not directly accessible in the literature. In a first step we considered experimental data of rat's liver cells obtained by NMR spectroscopy: given the values of metabolite concentrations we find appropriate parameter values which allow us to describe the system with ODEs. We have then performed several analyses using the developed model such as stability analysis. A first interesting result is the global stability of the system which was observed by simulation and then proved by mathematical arguments. A second important result is that we observe on the diagrams that the steady state for normal cells is precisely a singular point of order two, whereas tumoral cells present different characteristics; this fact has been proved for PhosphatidylEthanolamine N-Methyl transferase (PEMT), an enzyme which seems to be identified for the first time as a crucial element in the tumoral process. In a second step we applied our model to experimental data of proton HRMAS NMR spectroscopy for solid B16 melanoma and Lewis lung (3LL) 3LL carcinoma cells treated by Chloroethyl Nitrosourea (CENU). We performed a complete comparative analysis of parameters in order to learn the predictive statements to explain increases and decreases which one can observe in concentrations.

Combined methionine deprivation and chloroethylnitrosourea have time-dependent therapeutic synergy on melanoma tumors that NMR spectroscopy-based metabolomics explains by methionine and phospholipid metabolism reprogramming.

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met-sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Mitochondrial bioenergetic background confers a survival advantage to HepG2 cells in response to chemotherapy.

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (DeltaPsim). It thus may be predicted that DeltaPsim regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O(2) consumption/ATP production coupling, DeltaPsim maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered DeltaPsim, leading to an increased energy request to regenerate DeltaPsim in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.

Chemotherapy-induced bystander effect in response to several chloroethylnitrosoureas: an origin independent of DNA damage?

Chloroethylnitrosourea (CENU) chemotherapy is used for the treatment of melanoma tumors. The main mechanism of action of this anticancer agent is via DNA damage. We recently showed in murine experiments using a parental double B16 melanoma tumor model that, after treatment of primary tumors with cystemustine (CENU agent), untreated secondary tumors exhibited growth inhibition and metabolism disorders. The response of secondary untreated tumor was called the chemotherapy-induced bystander effect. To see whether chemotherapy-induced bystander effects were induced with other members of the CENU family, we compared three CENU(s) used in melanoma treatment: cystemustine, carmustine and fotemustine. Our results demonstrate that fotemustine, like cystemustine, but not carmustine induced a protective effect against secondary untreated tumors including alterations in phospholipid derivative and glutathione which are the metabolic signature of the bystander effect. From these data we may conclude that DNA damage to the primary tumor is not sufficient to explain chemotherapy-induced bystander effects.

Optimal methionine-free diet duration for nitrourea treatment: a Phase I clinical trial.

In animal models, methionine (MET) restriction in association with chloroethylnitrosoureas led to a substantial improvement. On this basis, we initiated a Phase I clinical trial of dietary MET restriction in association with chloroethylnitrosourea (cystemustine) treatment for patients with recurrent glioma or metastatic melanoma. Our purpose was 1) to determine the optimal MET-free diet duration for a maximum depletion of plasma MET and 2) to evaluate the feasibility of this association. A total of 10 patients received 4 cycles of 2 wk of an association of a MET-free diet of 1, 2, 3, or 4 consecutive days and cystemustine (60 mg/m(2)). For each cycle, plasma MET concentrations, nutritional status (weight, albumin, prealbumin) and toxicity were measured. Conversely, fed-state concentrations of plasma MET (12 AM) were reduced by dietary MET restriction, with an optimal depletion of 41% at the 1st day of MET-free diet without effect of the extending MET-free diet period. Indeed, we demonstrated the feasibility, that is, good diet acceptability and good tolerance (nutritional status and toxicity), of the association of a MET-free diet and cystemustine treatment. Based on these results, a Phase II clinical trial has been initiated to test the activity of the association of a 1-day MET-free diet with cystemustine treatment.

PP2A activity is controlled by methylation and regulates oncoprotein expression in melanoma cells: a mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment.

Protein phosphatase 2A (PP2A), an Akt pathway inhibitor, is considered to be activated by methylation of its catalytic subunit. Also PP2A downregulation was proposed to take part in carcinogenesis. Recently, PP2A activation was shown to be activated in response to DNA damage. To obtain further information on the role of PP2A in tumors and response to DNA damage, we investigated the relationship between PP2A methylation and activity, cell proliferation, Akt activation, c-Myc expression and PTEN activity in B16 melanoma cells untreated and after chloroethylnitrosourea (CENU) treatment. In untreated cells, okadaic acid, an antagonist of PP2A methylation, inhibited PP2A activity, stimulated cell proliferation, increased Akt activation and c-Myc expression. Xylulose-5-phosphate, an agonist of PP2A methylation, increased PP2A activity, decreased cell proliferation, Akt activation and c-Myc expression. However, both PP2A methylation modulators increased PTEN activity. During the response to CENU treatment, PP2A methylation and activity were strongly increased, Akt activation and c-Myc expression were decreased. However PTEN activity was increased. After tumor cell growth recovery, these modifications were moderately decreased. PP2A methylation was quantified and correlated positively with PP2A activity, and negatively with criteria for cell aggressiveness (cell proliferation, Akt activation, c-Myc expression). Based on these data, PP2A methylation status controls PP2A activity and oncoproteins expression and PP2A is strongly activated after CENU treatment thus partly explaining the growth inhibition in response to this agent. It follows that PP2A promethylating agents are potential candidates for anticancer drugs.

A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

BACKGROUND: Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). RESULTS: MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.