Pd2Spermine as an Alternative Therapeutics for Cisplatin-Resistant Triple-Negative Breast Cancer.

Cisplatin (cDDP) resistance is a matter of concern in triple-negative breast cancer therapeutics. We measured the metabolic response of cDDP-sensitive (S) and -resistant (R) MDA-MB-231 cells to Pd2Spermine(Spm) (a possible alternative to cDDP) compared to cDDP to investigate (i) intrinsic response/resistance mechanisms and (ii) the potential cytotoxic role of Pd2Spm. Cell extracts were analyzed by untargeted nuclear magnetic resonance metabolomics, and cell media were analyzed for particular metabolites. CDDP-exposed S cells experienced enhanced antioxidant protection and small deviations in the tricarboxylic acid cycle (TCA), pyrimidine metabolism, and lipid oxidation (proposed cytotoxicity signature). R cells responded more strongly to cDDP, suggesting a resistance signature of activated TCA cycle, altered AMP/ADP/ATP and adenine/uracil fingerprints, and phospholipid biosynthesis (without significant antioxidant protection). Pd2Spm impacted more markedly on R/S cell metabolisms, inducing similarities to cDDP/S cells (probably reflecting high cytotoxicity) and strong additional effects indicative of amino acid depletion, membrane degradation, energy/nucleotide adaptations, and a possible beneficial intracellular γ-aminobutyrate/glutathione-mediated antioxidant mechanism.

Disclosing a metabolic signature of cisplatin resistance in MDA-MB-231 triple-negative breast cancer cells by NMR metabolomics.

This work compared the metabolic profile of a parental MDA-MB-231 cisplatin-sensitive triple negative breast cancer (TNBC) cell line with that of a derived cisplatin-resistant line, to characterize inherent metabolic adaptations to resistance, as a means for marker and new TNBC therapies discovery. Supported by cytotoxic, microscopic and biochemical characterization of both lines, Nuclear Magnetic Resonance (NMR) metabolomics was employed to characterize cell polar extracts for the two cell lines, as a function of time (0, 24 and 48 h), and identify statistically relevant differences both between sensitive and resistant cells and their time course behavior. Biochemical results revealed a slight increase in activation of the NF-κB pathway and a marked decrease of the ERK signaling pathway in resistant cells. This was accompanied by lower glycolytic and glutaminolytic activities, possibly linked to glutamine being required to increase stemness capacity and, hence, higher survival to cisplatin. The TCA cycle dynamics seemed to be time-dependent, with an apparent activation at 48 h preferentially supported by anaplerotic aromatic amino acids, leucine and lysine. A distinct behavior of leucine, compared to the other branched-chain-amino-acids, suggested the importance of the recognized relationship between leucine and in mTOR-mediated autophagy to increase resistance. Suggested markers of MDA-MB-231 TNBC cisplatin-resistance included higher phosphocreatine/creatine ratios, hypotaurine/taurine-mediated antioxidant protective mechanisms, a generalized marked depletion in nucleotides/nucleosides, and a distinctive pattern of choline compounds. Although the putative hypotheses generated here require biological demonstration, they pave the way to the use of metabolites as markers of cisplatin-resistance in TNBC and as guidance to develop therapies.

Impact of Conventional and Potential New Metal-Based Drugs on Lipid Metabolism in Osteosarcoma MG-63 Cells.

This work investigated the mechanisms of action of conventional drugs, cisplatin and oxaliplatin, and the potentially less deleterious drug Pd2Spermine (Spm) and its Pt(II) analog, against osteosarcoma MG-63 cells, using nuclear-magnetic-resonance metabolomics of the cellular lipidome. The Pt(II) chelates induced different responses, namely regarding polyunsaturated-fatty-acids (increased upon cisplatin), suggesting that cisplatin-treated cells have higher membrane fluidity/permeability, thus facilitating cell entry and justifying higher cytotoxicity. Both conventional drugs significantly increased triglyceride levels, while Pt2Spm maintained control levels; this may reflect enhanced apoptotic behavior for conventional drugs, but not for Pt2Spm. Compared to Pt2Spm, the more cytotoxic Pd2Spm (IC50 comparable to cisplatin) induced a distinct phospholipids profile, possibly reflecting enhanced de novo biosynthesis to modulate membrane fluidity and drug-accessibility to cells, similarly to cisplatin. However, Pd2Spm differed from cisplatin in that cells had equivalent (low) levels of triglycerides as Pt2Spm, suggesting the absence/low extent of apoptosis. Our results suggest that Pd2Spm acts on MG-63 cells mainly through adaptation of cell membrane fluidity, whereas cisplatin seems to couple a similar effect with typical signs of apoptosis. These results were discussed in articulation with reported polar metabolome adaptations, building on the insight of these drugs' mechanisms, and particularly of Pd2Spm as a possible cisplatin substitute.

Effect of Pd2Spermine on Mice Brain-Liver Axis Metabolism Assessed by NMR Metabolomics.

Cisplatin (cDDP)-based chemotherapy is often limited by severe deleterious effects (nephrotoxicity, hepatotoxicity and neurotoxicity). The polynuclear palladium(II) compound Pd2Spermine (Pd2Spm) has emerged as a potential alternative drug, with favorable pharmacokinetic/pharmacodynamic properties. This paper reports on a Nuclear Magnetic Resonance metabolomics study to (i) characterize the response of mice brain and liver to Pd2Spm, compared to cDDP, and (ii) correlate brain-liver metabolic variations. Multivariate and correlation analysis of the spectra of polar and lipophilic brain and liver extracts from an MDA-MB-231 cell-derived mouse model revealed a stronger impact of Pd2Spm on brain metabolome, compared to cDDP. This was expressed by changes in amino acids, inosine, cholate, pantothenate, fatty acids, phospholipids, among other compounds. Liver was less affected than brain, with cDDP inducing more metabolite changes. Results suggest that neither drug induces neuronal damage or inflammation, and that Pd2Spm seems to lead to enhanced brain anti-inflammatory and antioxidant mechanisms, regulation of brain bioactive metabolite pools and adaptability of cell membrane characteristics. The cDDP appears to induce higher extension of liver damage and an enhanced need for liver regeneration processes. This work demonstrates the usefulness of untargeted metabolomics in evaluating drug impact on multiple organs, while confirming Pd2Spm as a promising replacement of cDDP.

Metabolic profiling of induced acute pancreatitis and pancreatic cancer progression in a mutant Kras mouse model.

Untargeted Nuclear Magnetic Resonance (NMR) metabolomics of polar extracts from the pancreata of a caerulin-induced mouse model of pancreatitis (Pt) and of a transgenic mouse model of pancreatic cancer (PCa) were used to find metabolic markers of Pt and to characterize the metabolic changes accompanying PCa progression. Using multivariate analysis a 10-metabolite metabolic signature specific to Pt tissue was found to distinguish the benign condition from both normal tissue and precancerous tissue (low grade pancreatic intraepithelial neoplasia, PanIN, lesions). The mice pancreata showed significant changes in the progression from normal tissue, through low-grade and high-grade PanIN lesions to pancreatic ductal adenocarcinoma (PDA). These included increased lactate production, amino acid changes consistent with enhanced anaplerosis, decreased concentrations of intermediates in membrane biosynthesis (phosphocholine and phosphoethanolamine) and decreased glycosylated uridine phosphates, reflecting activation of the hexosamine biosynthesis pathway and protein glycosylation.

Metabolic Impact of Anticancer Drugs Pd2Spermine and Cisplatin on the Brain of Healthy Mice.

The new palladium agent Pd2Spermine (Spm) has been reported to exhibit promising cytotoxic properties, while potentially circumventing the known disadvantages associated to cisplatin therapeutics, namely acquired resistance and high toxicity. This work presents a nuclear magnetic resonance (NMR) metabolomics study of brain extracts obtained from healthy mice, to assess the metabolic impacts of the new Pd2Spm complex in comparison to that of cisplatin. The proton NMR spectra of both polar and nonpolar brain extracts were analyzed by multivariate and univariate statistics, unveiling several metabolite variations during the time course of exposition to each drug (1-48 h). The distinct time-course dependence of such changes revealed useful information on the drug-induced dynamics of metabolic disturbances and recovery periods, namely regarding amino acids, nucleotides, fatty acids, and membrane precursors and phospholipids. Putative biochemical explanations were proposed, based on existing pharmacokinetics data and previously reported metabolic responses elicited by the same metal complexes in the liver of the same animals. Generally, results suggest a more effective response of brain metabolism towards the possible detrimental effects of Pd2Spm, with more rapid recovery back to metabolites' control levels and, thus, indicating that the palladium drug may exert a more beneficial role than cDDP in relation to brain toxicity.

Impact of the Pd2Spm (Spermine) Complex on the Metabolism of Triple-Negative Breast Cancer Tumors of a Xenograft Mouse Model.

The interest in palladium(II) compounds as potential new anticancer drugs has increased in recent years, due to their high toxicity and acquired resistance to platinum(II)-derived agents, namely cisplatin. In fact, palladium complexes with biogenic polyamines (e.g., spermine, Pd2Spm) have been known to display favorable antineoplastic properties against distinct human breast cancer cell lines. This study describes the in vivo response of triple-negative breast cancer (TNBC) tumors to the Pd2Spm complex or to cisplatin (reference drug), compared to tumors in vehicle-treated mice. Both polar and lipophilic extracts of tumors, excised from a MDA-MB-231 cell-derived xenograft mouse model, were characterized through nuclear magnetic resonance (NMR) metabolomics. Interestingly, the results show that polar and lipophilic metabolomes clearly exhibit distinct responses for each drug, with polar metabolites showing a stronger impact of the Pd(II)-complex compared to cisplatin, whereas neither drug was observed to significantly affect tumor lipophilic metabolism. Compared to cisplatin, exposure to Pd2Spm triggered a higher number of, and more marked, variations in some amino acids, nucleotides and derivatives, membrane precursors (choline and phosphoethanolamine), dimethylamine, fumarate and guanidine acetate, a signature that may be relatable to the cytotoxicity and/or mechanism of action of the palladium complex. Putative explanatory biochemical hypotheses are advanced on the role of the new Pd2Spm complex in TNBC metabolism.

Novel Insights into Mice Multi-Organ Metabolism upon Exposure to a Potential Anticancer Pd(II)-Agent.

Pd(II)-compounds are presently regarded as promising anticancer drugs, as an alternative to Pt(II)-based drugs (e.g., cisplatin), which typically trigger severe side-effects and acquired resistance. Dinuclear Pd(II) complexes with biogenic polyamines such as spermine (Pd2Spm) have exhibited particularly beneficial cytotoxic properties, hence unveiling the importance of understanding their impact on organism metabolism. The present study reports the first nuclear magnetic resonance (NMR)-based metabolomics study to assess the in vivo impact of Pd2Spm on the metabolism of healthy mice, to identify metabolic markers with possible relation to biotoxicity/side-effects and their dynamics. The changes in the metabolic profiles of both aqueous and lipophilic extracts of mice kidney, liver, and breast tissues were evaluated, as a function of drug-exposure time, using cisplatin as a reference drug. A putative interpretation was advanced for the metabolic deviations specifically triggered by Pd2Spm, this compound generally inducing faster metabolic response and recovery to control levels for all organs tested, compared to cisplatin (except for kidney lipid metabolism). These results constitute encouraging preliminary metabolic data suggestive of potential lower negative effects of Pd2Spm administration.

Metabolic Aspects of Palladium(II) Potential Anti-Cancer Drugs.

This mini-review reports on the existing knowledge of the metabolic effects of palladium [Pd(II)] complexes with potential anticancer activity, on cell lines and murine models. Most studies have addressed mononuclear Pd(II) complexes, although increasing interest has been noted in bidentate complexes, as polynuclear structures. In addition, the majority of records have reported in vitro studies on cancer cell lines, some including the impact on healthy cells, as potentially informative in relation to side effects. Generally, these studies address metabolic effects related to the mechanisms of induced cell death and antioxidant defense, often involving the measurement of gene and protein expression patterns, and evaluation of the levels of reactive oxygen species or specific metabolites, such as ATP and glutathione, in relation to mitochondrial respiration and antioxidant mechanisms. An important tendency is noted toward the use of more untargeted approaches, such as the use of omic sciences e.g., proteomics and metabolomics. In the discussion section of this mini-review, the developments carried out so far are summarized and suggestions of possible future developments are advanced, aiming at recognizing that metabolites and metabolic pathways make up an important part of cell response and adaptation to therapeutic agents, their further study potentially contributing valuably for a more complete understanding of processes such as biotoxicity or development of drug resistance.

Multi-Organ NMR Metabolomics to Assess In Vivo Overall Metabolic Impact of Cisplatin in Mice.

This work describes, to our knowledge, the first NMR metabolomics analysis of mice kidney, liver, and breast tissue in response to cisplatin exposure, in search of early metabolic signatures of cisplatin biotoxicity. Balb/c mice were exposed to a single 3.5 mg/kg dose of cisplatin and then euthanized; organs (kidney, liver, breast tissue) were collected at 1, 12, and 48 h. Polar tissue extracts were analyzed by NMR spectroscopy, and the resulting spectra were studied by multivariate and univariate analyses. The results enabled the identification of the most significant deviant metabolite levels at each time point, and for each tissue type, and showed that the largest metabolic impact occurs for kidney, as early as 1 h post-injection. Kidney tissue showed a marked depletion in several amino acids, comprised in an overall 13-metabolites signature. The highest number of changes in all tissues was noted at 12 h, although many of those recovered to control levels at 48 h, with the exception of some persistently deviant tissue-specific metabolites, thus enabling the identification of relatively longer-term effects of cDDP. This work reports, for the first time, early (1-48 h) concomitant effects of cDDP in kidney, liver, and breast tissue metabolism, thus contributing to the understanding of multi-organ cDDP biotoxicity.

NMR metabolomics to study the metabolic response of human osteoblasts to non-poled and poled poly (L-lactic) acid.

Untargeted nuclear magnetic resonance (NMR) metabolomics was employed, for the first time to our knowledge, to characterize the metabolome of human osteoblast (HOb) cells and extracts in the presence of non-poled or negatively poled poly-L-lactic acid (PLLA). The metabolic response of these cells to this polymer, extensively used in bone regeneration strategies, may potentially translate into useful markers indicative of in vivo biomaterial performance. We present preliminary results of multivariate and univariate analysis of NMR spectra, which have shown the complementarity of lysed cells and extracts in terms of information on cell metabolome, and unveil that, irrespective of poling state, PLLA-grown cells seem to experience enhanced oxidative stress and activated energy metabolism, at the cost of storage lipids and glucose. Possible changes in protein and nucleic acid metabolisms were also suggested, as well as enhanced membrane biosynthesis. Therefore, the presence of PLLA seems to trigger cell catabolism and anti-oxidative protective mechanisms in HOb cells, while directing them towards cellular growth. This was not sufficient, however, to lead to a visible cell proliferation enhancement in the presence of PLLA, although a qualitative tendency for negatively poled PLLA to be more effective in sustaining cell growth than non-poled PLLA was suggested. These preliminary results indicate the potential of NMR metabolomics in enlightening cell metabolism in response to biomaterials and their properties, justifying further studies of the fine effects of poled PLLA on these and other cells of significance in tissue regeneration strategies.

Saliva NMR metabolomics: Analytical issues in pediatric oral health research.

OBJECTIVES: Saliva metabolome is a promising diagnostic tool concerning oral and systemic diseases. We aimed at establishing a suitable protocol for saliva collection and gauging the relative impacts of gender, dentition stage, and caries on the saliva metabolome of a small children cohort. SUBJECTS AND METHODS: A nuclear magnetic resonance-based metabolomics cross-sectional study of children saliva (n = 38) compared the effects of: (a) stimulation and unstimulation conditions, and (b) collection through passive drool and using an absorbing device. Multivariate and univariate statistical analyses were applied to evaluate such effects and those related to gender, dentition stage and caries. RESULTS: No significant differences were found between unstimulated and stimulated saliva, and the former was used for subsequent studies. Swab collection induced significant changes in sample composition, indicating passive drool as preferential. The impacts of gender and dentition stage were not significant compared to that of caries, which induced variations in the levels of 21 metabolites. These comprised amino acids and monosaccharides observed for the first time to our knowledge regarding children caries, suggesting protein hydrolysis and deglycosylation. CONCLUSIONS: Unstimulated passive drool saliva metabolome may carry a caries signature.

Urine Nuclear Magnetic Resonance (NMR) Metabolomics in Age-Related Macular Degeneration.

Biofluid biomarkers of age-related macular degeneration (AMD) are still lacking, and their identification is challenging. Metabolomics is well-suited to address this need, and urine is a valuable accessible biofluid. This study aimed to characterize the urinary metabolomic signatures of patients with different stages of AMD and a control group (>50 years). It was a prospective, cross-sectional study, where subjects from two cohorts were included: 305 from Coimbra, Portugal (AMD patients n = 252; controls n = 53) and 194 from Boston, United States (AMD patients n = 147; controls n = 47). For all participants, we obtained color fundus photographs (for AMD staging) and fasting urine samples, which were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy. Our results revealed that in both cohorts, urinary metabolomic profiles differed mostly between controls and late AMD patients, but important differences were also found between controls and subjects with early AMD. Analysis of the metabolites responsible for these separations revealed that, even though distinct features were observed for each cohort, AMD was in general associated with depletion of excreted citrate and selected amino acids at some stage of the disease, suggesting enhanced energy requirements. In conclusion, NMR metabolomics enabled the identification of urinary signals of AMD and its severity stages, which might represent potential metabolomic biomarkers of the disease.