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Multiple myeloma (MM) is the second most prevalent hematological malignancy, characterized by infiltration of the bone marrow by malignant plasma cells. Extramedullary disease (EMD) represents a more aggressive condition involving the migration of a subclone of plasma cells to paraskeletal or extraskeletal sites. Liquid biopsies could improve and speed diagnosis, as they can better capture the disease heterogeneity while lowering patients' discomfort due to minimal invasiveness. Recent studies have confirmed alterations in the proteome across various malignancies, suggesting specific changes in protein classes. In this study, we show that MALDI-TOF mass spectrometry fingerprinting of peripheral blood can differentiate between MM and primary EMD patients. We constructed a predictive model using a supervised learning method, partial least squares-discriminant analysis (PLS-DA) and evaluated its generalization performance on a test dataset. The outcome of this analysis is a method that predicts specifically primary EMD with high sensitivity (86.4%), accuracy (78.4%), and specificity (72.4%). Given the simplicity of this approach and its minimally invasive character, this method provides rapid identification of primary EMD and could prove helpful in clinical practice.
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Mieloma Múltiplo , Humanos , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Biópsia Líquida/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biomarcadores Tumorais/sangueRESUMO
ABSTRACT: Pancreas is a vital gland of gastro-intestinal system with exocrine and endocrine secretory functions, interweaved into essential metabolic circuitries of the human body. Pancreatic ductal adenocarcinoma (PDAC) represents one of the most lethal malignancies, with a five-year survival rate of 11%. This poor prognosis is primarily attributed to the absence of early symptoms, rapid metastatic dissemination, and the limited efficacy of current therapeutic interventions. Despite recent advancements in understanding the etiopathogenesis and treatment of PDAC, there remains a pressing need for improved individualized models, the identification of novel molecular targets, and the development of unbiased predictors of disease progression. Here we aim to explore the concept of precision medicine utilizing three-dimensional, patient-specific cellular models of pancreatic tumors and discuss their potential applications in uncovering novel druggable molecular targets and predicting clinical parameters for individual patients.
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Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.
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This work aims to demonstrate the potential of pulsed laser ablation synthesis (PLA) of tellurium nanoparticles (Te NPs) for use in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) applications. An experimental laboratory setup for PLA synthesis of fresh Te NPs was designed to prevent unwanted aggregation of uncoated Te NPs and avoid the need to use additional modifiers. Performing pulsed laser ablation synthesis in liquid (PLAL) using acetone was found to be the optimal way of preparing Te NPs. Another possibility is to use commercially available laser ablation devices for laser ablation - inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform PLA in a helium atmosphere, but this approach is less efficient and results in the formation of unwanted larger particles. The prepared Te NPs were studied using the transmission electron microscopy (TEM) and dynamic light scattering (DLS) methods. TEM images showed the formation of Te NP nanochains composed of many crystallized Te NPs with sizes ranging from 8 to 15 nm. The various size distributions of the synthesized Te NPs identified using the DLS method correspond to the size distributions of aggregations rather than individual Te NPs. The synthesized Te NPs were used for a pilot study of their possible use with the MALDI-MS technique. An important effect was observed when Te NPs were used to perform a MALDI-MS analysis of the α-cyclodextrin (α-CD) and cucurbit[7]uril (CB7) macrocycles, which consisted in a decline in the formation of matrix adducts. Furthermore, several changes in MALDI-MS mass spectra of intact cells and a positive effect of Te NPs on the crystallization of the MALDI-MS matrix were observed.
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Monoclonal gammopathies are a group of blood diseases characterized by presence of abnormal immunoglobulins in peripheral blood and/or urine of patients. Multiple myeloma and plasma cell leukemia are monoclonal gammopathies with unclear etiology, caused by malignant transformation of bone marrow plasma cells. Mass spectrometry with matrix-assisted laser desorption/ionization and time-of-flight detection is commonly used for investigation of the peptidome and small proteome of blood plasma with high accuracy, robustness, and cost-effectivity. In addition, mass spectrometry coupled with advanced statistics can be used for molecular profiling, classification, and diagnosis of liquid biopsies and tissue specimens in various malignancies. Despite the fact there have been fully optimized protocols for mass spectrometry of normal blood plasma available for decades, in monoclonal gammopathy patients, the massive alterations of biophysical and biochemical parameters of peripheral blood plasma often limit the mass spectrometry measurements. In this paper, we present a new two-step extraction protocol and demonstrated the enhanced resolution and intensity (>50×) of mass spectra obtained from extracts of peripheral blood plasma from monoclonal gammopathy patients. When coupled with advanced statistics and machine learning, the mass spectra profiles enabled the direct identification, classification, and discrimination of multiple myeloma and plasma cell leukemia patients with high accuracy and precision. A model based on PLS-DA achieved the best performance with 71.5% accuracy (95% confidence interval, CI = 57.1-83.3%) when the 10× repeated 5-fold CV was performed. In summary, the two-step extraction protocol improved the analysis of monoclonal gammopathy peripheral blood plasma samples by mass spectrometry and provided a tool for addressing the complex molecular etiology of monoclonal gammopathies.
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Leucemia Plasmocitária , Mieloma Múltiplo , Paraproteinemias , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Mieloma Múltiplo/diagnóstico , Paraproteinemias/diagnóstico , PlasmaRESUMO
A new heteroleptic copper(II) compound named C0-UDCA was prepared by reaction of [Cu(phen)2(OH2)](ClO4)2 (C0) with the bile ursodeoxycholic acid (UDCA). The resulting compound is able to inhibit the lipoxygenase enzyme showing more efficacy than the precursors C0 and UDCA. Molecular docking simulations clarified the interactions with the enzyme as due to allosteric modulation. The new complex shows antitumoral effect on ovarian (SKOV-3) and pancreatic (PANC-1) cancer cells at the Endoplasmic Reticulum (ER) level by activating the Unfolded Protein Response. In particular, the chaperone BiP, the pro-apoptotic protein CHOP and the transcription factor ATF6 are upregulated in the presence of C0-UDCA. The combination of Intact Cell MALDI-MS and statistical analysis have allowed us to discriminate between untreated and treated cells based on their mass spectrometry fingerprints.
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Inibidores de Lipoxigenase , Neoplasias , Inibidores de Lipoxigenase/farmacologia , Ácido Ursodesoxicólico/farmacologia , Fenantrolinas/química , Cobre/farmacologia , Cobre/química , Simulação de Acoplamento Molecular , Estresse do Retículo Endoplasmático , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Apoptose , Neoplasias PancreáticasRESUMO
RATIONALE: Carbides, including tellurium carbides (TeC), play crucial roles in diverse applications, but TeC synthesis has not been described in the literature. Laser ablation synthesis (LAS) coupled with mass spectrometry was used here for in situ TeC clusters synthesis and identification of the reaction products to better understand TeC formation. METHODS: Laser desorption ionization time-of-flight mass spectrometry (LDI-TOFMS) was used to generate the TeC clusters and determine their stoichiometry via computer modeling of isotopic patterns. RESULTS: A simple one-pot procedure was developed for Te-nanodiamond nanocomposite preparation. A suspension of fine-powdered Te was mixed with a suspension of nanodiamonds (both in acetonitrile), and the resulting precipitated nanocomposite was suitable for the synthesis of TemCn clusters using LDI. Various unary and binary clusters were formed. The stoichiometry of the novel TemCn clusters, determined via computer modeling of isotopic patterns, is reported here for the first time. CONCLUSIONS: The Te-nanodiamond composite was found to be the most suitable precursor for the generation of TemCn clusters. In total, 35 binary TemCn clusters were identified, when several of them were not obtained using commercial TeC material.
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Pathological pain subtypes can be classified as either neuropathic pain, caused by a somatosensory nervous system lesion or disease, or nociplastic pain, which develops without evidence of somatosensory system damage. Since there is no gold standard for the diagnosis of pathological pain subtypes, the proper classification of individual patients is currently an unmet challenge for clinicians. While the determination of specific biomarkers for each condition by current biochemical techniques is a complex task, the use of multimolecular techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), combined with artificial intelligence allows specific fingerprints for pathological pain-subtypes to be obtained, which may be useful for diagnosis. We analyzed whether the information provided by the mass spectra of serum samples of four experimental models of neuropathic and nociplastic pain combined with their functional pain outcomes could enable pathological pain subtype classification by artificial neural networks. As a result, a simple and innovative clinical decision support method has been developed that combines MALDI-TOF MS serum spectra and pain evaluation with its subsequent data analysis by artificial neural networks and allows the identification and classification of pathological pain subtypes in experimental models with a high level of specificity.
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Inteligência Artificial , Redes Neurais de Computação , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Dor/diagnósticoRESUMO
BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
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Células-Tronco Embrionárias Humanas , Diferenciação Celular , Epitélio , Humanos , Pulmão/metabolismo , Tensoativos/metabolismoRESUMO
BACKGROUND: Recent discoveries in cancer therapeutics have proven combination therapies more effective than individual drugs. This study describes the efficacy of the combination of Cinnamomum zeylanicum and doxorubicin against benzene-induced leukemia. METHODS AND RESULTS: Brine shrimp assay was used to assess the cytotoxicity of C. zeylanicum, doxorubicin and their combination. After AML induction in Sprague Dawley rats, the same drugs were given to rat groups. Changes in organ weight, haematological profile, and hepatic enzymes were determined. Real-time PCR was used to elucidate the effect on the expression of STMN1, GAPDH, P53 and various TRAIL and NF-kappaB components. C. zeylanicum reduced the cytotoxicity of doxorubicin. The combination treatment showed better anti-leukemic results than any of the individual drugs as evident from STMN1 expression (p < 0.001). It was particularly effective in reducing total white blood cell counts and recovering lymphocytes, monocytes and eosinophils along with hepatic enzymes ALT and AST (p < 0.001). All doses recovered relative organ weights and improved blood parameters. The combination therapy was particularly effective in inducing apoptosis, inhibition of proliferation marker GAPDH (p < 0.001) and NF-kappaB pathway components Rel-A (p < 0.001) and Rel-B (p < 0.01). Expressions of TRAIL components c-FLIP (p < 0.001), TRAIL ligand (p < 0.001) and caspase 8 (p < 0.01) were also altered. CONCLUSION: Cinnamomum zeylanicum in combination with doxorubicin helps to counter benzene-induced cellular and hepatic toxicity and improves haematological profile. The anti-leukemic effects are potentially due to inhibition of GAPDH and NF-kappa B pathway, and through regulation of TRAIL pathway. Our data suggests the use of C. zeylanicum with doxorubicin to improve anti-leukemic therapeutic regimes.
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Leucemia , Óleos Voláteis , Animais , Apoptose , Benzeno/farmacologia , Cinnamomum zeylanicum/metabolismo , Doxorrubicina/farmacologia , Leucemia/tratamento farmacológico , NF-kappa B/metabolismo , Óleos Voláteis/farmacologia , Ratos , Ratos Sprague-Dawley , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.
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Fósforo/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aminoácidos/análise , Aminoácidos/química , Técnicas de Cultura de Células/métodos , Linhagem Celular , Células-Tronco Embrionárias Humanas , Humanos , Peptídeos/análise , Peptídeos/química , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
Copper is an endogenous metal ion that has been studied to prepare a new antitumoral agent with less side-effects. Copper is involved as a cofactor in several enzymes, in ROS production, in the promotion of tumor progression, metastasis, and angiogenesis, and has been found at high levels in serum and tissues of several types of human cancers. Under these circumstances, two strategies are commonly followed in the development of novel anticancer Copper-based drugs: the sequestration of free Copper ions and the synthesis of Copper complexes that trigger cell death. The latter strategy has been followed in the last 40 years and many reviews have covered the anticancer properties of a broad spectrum of Copper complexes, showing that the activity of these compounds is often multi factored. In this work, we would like to focus on the anticancer properties of mixed Cu(II) complexes bearing substituted or unsubstituted 1,10-phenanthroline based ligands and different classes of inorganic and organic auxiliary ligands. For each metal complex, information regarding the tested cell lines and the mechanistic studies will be reported and discussed. The exerted action mechanisms were presented according to the auxiliary ligand/s, the metallic centers, and the increasing complexity of the compound structures.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Complexos de Coordenação/química , Cobre/química , Fenantrolinas/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Química Sintética , Complexos de Coordenação/síntese química , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Ligantes , Estrutura MolecularRESUMO
The novel copper complex [Cu(phen)2(salubrinal)](ClO4)2 (C0SAL) has been synthesised and characterised. Copper(ii) is coordinated by salubrinal through the thionic group, as shown by the UV-Vis, IR, ESI-MS and tandem mass results, together with the theoretical calculations. The formed complex showed a DPPH radical scavenging ability higher than that of salubrinal alone. Studies on lipid oxidation inhibition showed that the C0SAL concentration, required to inhibit the enzyme, was lower than that of salubrinal. The inhibition of the enzyme could take place via allosteric modulation, as suggested by docking calculations. C0SAL showed a good cytotoxic activity on A2780 cells, 82 fold higher than that of the precursor salubrinal and 1.4 fold higher than that of [Cu(phen)2(H2O)](ClO4)2. Treatment with C0SAL in SKOV3 ovarian cancer cells induced expression of GRP-78 and DDIT3 regulators of ER-stress response. The cytotoxic effect of C0SAL was reverted in the presence of TUDCA, suggesting that C0SAL induces cell death through ER-stress. In A2780 cells treated with C0SAL γ-H2AX was accumulated, suggesting that DNA damage was also involved.
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Cinamatos/farmacologia , Cobre/farmacologia , Fenantrolinas/farmacologia , Tioureia/análogos & derivados , Antivirais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Ácido Tauroquenodesoxicólico/farmacologia , Tioureia/farmacologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
There is an ongoing need for the development of new cancer therapeutics that combine high cytotoxic efficiency with low side effects, and also override resistance to the first-line chemotherapeutics. Copper(ii)-phenanthroline complexes are promising compounds that were shown previously to induce an immediate cytotoxic response over a panel of tumor cell lines in vitro. The molecular mechanism, however, remained unresolved. In this work we performed a thorough study of the copper(ii)-phenanthroline complexes containing different imidazolidine-2-thione ligands in ovarian cancer cells, and revealed that these complexes induce endoplasmic reticulum (ER) stress and subsequently cell death mediated by the unfolded protein response. Alleviation of the ER-stress by tauroursodeoxycholic acid (TUDCA) attenuated the cytotoxic effects. In summary, we have identified a novel, ER-dependent, molecular mechanism mediating cytotoxic effects of copper(ii)-phenanthroline complexes.
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Antineoplásicos/farmacologia , Cobre/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fenantrolinas/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Antineoplásicos/química , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Fenantrolinas/químicaRESUMO
Multiple myeloma (MM) is a highly heterogeneous disease of malignant plasma cells. Diagnosis and monitoring of MM patients is based on bone marrow biopsies and detection of abnormal immunoglobulin in serum and/or urine. However, biopsies have a single-site bias; thus, new diagnostic tests and early detection strategies are needed. Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry (MALDI-TOF MS) is a powerful method that found its applications in clinical diagnostics. Artificial intelligence approaches, such as Artificial Neural Networks (ANNs), can handle non-linear data and provide prediction and classification of variables in multidimensional datasets. In this study, we used MALDI-TOF MS to acquire low mass profiles of peripheral blood plasma obtained from MM patients and healthy donors. Informative patterns in mass spectra served as inputs for ANN that specifically predicted MM samples with high sensitivity (100%), specificity (95%) and accuracy (98%). Thus, mass spectrometry coupled with ANN can provide a minimally invasive approach for MM diagnostics.
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Inteligência Artificial , Metaboloma , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Redes Neurais de Computação , Idoso , Idoso de 80 Anos ou mais , Inteligência Artificial/estatística & dados numéricos , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Conjuntos de Dados como Assunto , Feminino , Humanos , Imunoglobulinas/sangue , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Análise de Componente Principal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Resistencia a Medicamentos Antineoplásicos , Mitocôndrias/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Esfingomielinas/biossíntese , Idoso , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Peróxido de Hidrogênio/química , Lipídeos/química , Metabolômica , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismoRESUMO
RATIONALE: Carbon-phosphides are new and promising strategic materials with applications e.g. in optoelectronics. However, their chemistry and methods of synthesis are not completely understood, and only a limited number of C-P clusters have been detected up to now. Laser ablation synthesis (LAS) or laser desorption ionisation (LDI) has great potential to generate Cm Pn clusters in the gas phase and to act as the basis for the development of new technology. METHODS: The LAS of carbon phosphides using mixtures of nano-carbon sources (graphene, nanodiamonds) with phosphorus allotropes (red, black, and phosphorene) was examined. Since phosphorene is not commercially available, it was synthesised. A reflectron time-of-flight mass spectrometer was used to produce and identify the C-P clusters. A transmission electron microscope was used to characterise the prepared composites. RESULTS: LDI of various carbon-phosphorus composites generated a range of carbon-phosphides. From graphene-red phosphorus, Cm P+ (m = 3-47), Cm P2 + (m = 2-44), Cm P3 + (m = 1-42), Cm P4 + (m = 1-39), Cm P5 + (m = 1-37), Cm P6 + (m = 1-34), Cm P7 + (m = 1-31), Cm P8 + (m = 1-29), Cm P9 + (m = 1-26), Cm P10 + (m = 1-24), Cm P11 + (m = 1-21), and Cm P12 + (m = 1-19) clusters were detected, while nanodiamond composites with red/black phosphorus and with phosphorene yielded C24 P5 + 2n + (n = 0-28), C24 P5 + 2n + (n = 0-16), and C24 P5 + 2n + (n = 0-14) clusters, respectively. In total, over 300 new carbon-phosphide clusters were generated. CONCLUSIONS: The novel series of carbon-phosphide clusters generated from graphene or nanodiamond composites with red/black phosphorus or with phosphorene demonstrated rich carbon-phosphide chemistry that might inspire the development of novel nano-materials with specific properties.
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RATIONALE: Development of therapy-resistant cancer is a major problem in clinical oncology, and there is an urgent need for novel markers identifying development of the resistant phenotype. Lipidomics represents a promising approach to discriminate lipid profiles of malignant phenotype cells. Alterations in phospholipid distribution or chemical composition have been reported in various pathologies including cancer. Here we were curious whether quantitative differences in phospholipid composition between cisplatin-resistant and -sensitive model cancer cell lines could be revealed by mass spectrometric means. METHODS: The phospholipid contents of cell membranes of the cancer cell lines CCRF-CEM and A2780, both responsive and resistant to cisplatin, were analyzed by solid-phase extraction (SPE) and electrospray ionization mass spectrometry (ESI-MS and tandem mass spectrometry (MS/MS)). Extracts were obtained by disruption of cells with a dounce tissue grinder set followed by centrifugation. To minimize the enzymatic activity, phospholipids were extracted from cell extracts by SPE immediately after the cell lysis and analyzed by MS. Both supernatant and pellet fractions of cell extracts were analyzed. RESULTS: A phospholipid profile specific for cell lines and their phenotypes was revealed. We have documented by quantitative analysis that phosphocholines PC P-34:0, PC 34:1, PC 20:2_16:0, LPC 18:1 and LPC 16:0 PLs were present in the 200-400 µM concentration range in CCRF-CEM cisplatin-responsive cells, but absent in their cisplatin-resistant cells. Similarly, PC 34:1, LPC 18:1 and LPC 16:0 were increased in cisplatin-responsive A2780 cells, and PC 20:2_16:0 was downregulated in cisplatin-resistant A2780 cells. CONCLUSIONS: In this work we showed that the ESI-MS analysis of the lipid content of the therapy-resistant and -sensitive cells can clearly distinguish the phenotypic pattern and determine the potential tumor response to cytotoxic therapy. Lipid entities revealed by mass spectrometry and associated with development of therapy resistance can thus support molecular diagnosis and provide a potential complementary cancer biomarker.
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Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fosfolipídeos/análise , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Fosfolipídeos/química , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Ovarian surface epithelium (OSE) forms a single layer of mostly cuboidal cells on surface of mammalian ovaries that is inherently exposed to cell stress evoked by tissue damage every ovulation and declines morphologically after menopause. Endoplasmic reticulum (ER) is a principal cell organelle involved in proteosynthesis, but also integrating various stress signals. ER stress evokes a conserved signaling pathway, the unfolded protein response (UPR), leading to cell death or adaptation to stress conditions. In this work, we document that mouse OSE suffers from ER stress during replicative senescence in vitro, develops abnormalities in ER and initiates UPR. Attenuation of ER stress in senescent OSE by tauroursodeoxycholic acid (TUDCA) reconditions ER architecture and leads to delayed onset of senescence. In summary, we show for the first time a mutual molecular link between ER stress response and replicative senescence leading to phenotypic changes of non-malignant ovarian surface epithelium.
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Senescência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Epitélio/patologia , Ovário/patologia , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Regulação para Baixo/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/ultraestrutura , Feminino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Encurtamento do Telômero/efeitos dos fármacos , Tunicamicina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.