Flaubert Mbeunkui

Cancer and the tumor microenvironment: a review of an essential relationship

PurposeThe role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of relatively new knowledge for improved molecular diagnostics and therapeutics.MethodsThis review focuses on: (1) the approaches of preparing and analyzing secreted proteins, (2) the contribution of tumor microenvironment elements in cancer, and (3) the potential molecular targets for cancer therapy.ResultsThe microenvironment of a tumor is an integral part of its physiology, structure, and function. It is an essential aspect of the tumor proper, since it supplies a nurturing environment for the malignant process. A fundamental deranged relationship between tumor and stromal cells is essential for tumor cell growth, progression, and development of life threatening metastasis. Improved understanding of this interaction may provide new and valuable clinical targets for cancer management, as well as risk assessment and prevention. Non-malignant cells and secreted proteins from tumor and stromal cells are active participants in cancer progression.ConclusionsMonitoring the change in the tumor microenvironment via molecular and cellular profiles as tumor progresses would be vital for identifying cell or protein targets for cancer prevention and therapy.

SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis

Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta) using tomato plants (Solanum lycopersicum cv. Rutgers) as a method that allows soil-grown plants to be efficiently labeled using a 14N/15N isotope coding strategy. After 2 months of growth on 14N- and 15N-enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data-dependent acquisition, DDA) and alternating low- and elevated-energy MS scans (data-independent acquisition, MS(E)). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M-1 isotopologues of 70 identified 15N-labeled peptides from 16 different proteins indicated that 15N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15N-enriched nitrate used in the soil-based medium. Values for the various tissues ranged from 98.2 +/- 0.3% 15N incorporation in leaves to 98.8 2 +/- 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root-knot nematode (Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant-microorganism systems.

Efficient Quantification of the Health-Relevant Anthocyanin and Phenolic Acid Profiles in Commercial Cultivars and Breeding Selections of Blueberries (Vaccinium spp.)

Anthocyanins and phenolic acids are major secondary metabolites in blueberry with important implications for human health maintenance. An improved protocol was developed for the accurate, efficient, and rapid comparative screening for large blueberry sample sets. Triplicates of six commercial cultivars and four breeding selections were analyzed using the new method. The compound recoveries ranged from 94.2 to 97.5 ± 5.3% when samples were spiked with commercial standards prior to extraction. Eighteen anthocyanins and 4 phenolic acids were quantified in frozen and freeze-dried fruits. Large variations for individual and total anthocyanins, ranging from 201.4 to 402.8 mg/100 g, were assayed in frozen fruits. The total phenolic acid content ranged from 23.6 to 61.7 mg/100 g in frozen fruits. Across all genotypes, freeze-drying resulted in minor reductions in anthocyanin concentration (3.9%) compared to anthocyanins in frozen fruits. However, phenolic acids increased by an average of 1.9-fold (±0.3) in the freeze-dried fruit. Different genotypes frequently had comparable overall levels of total anthocyanins and phenolic acids, but differed dramatically in individual profiles of compounds. Three of the genotypes contained markedly higher concentrations of delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and malvidin 3-O-glucoside, which have previously been implicated as bioactive principles in this fruit. The implications of these findings for human health benefits are discussed.

Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE†

To evaluate the implementation of various denaturants and their efficacy in bottom‐up membrane proteomic methods using LC‐MS analysis, microsomes isolated from tomato roots were treated with MS‐compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data‐independent LC‐MSE analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC‐MSE. These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom‐up analysis of membrane proteomes than can be achieved using only a single denaturant.

In vitro antiplasmodial activity of indole alkaloids from the stem bark of Geissospermum vellosii.

ETHNOPHARMACOLOGICAL RELEVANCE The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the antiplasmodial constituents have not yet been described. As part of our ongoing investigations of new bioactive compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of isolated fractions and purified alkaloids from Geissospermum vellosii. MATERIALS AND METHODS Indole alkaloids were isolated and identified from a methanolic crude extract of Geissospermum vellosii bark using a combination of high performance counter current chromatography, mass spectrometry and nuclear magnetic resonance technologies. The methanolic extract, the crude alkaloid fractions and the purified compounds were tested for in vitro antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10). RESULTS An indole alkaloid (4) along with four known indole alkaloids, geissolosimine (1), geissospermine (2), geissoschizoline (3), and vellosiminol (5) were isolated and structure elucidated. The antiplasmodial activity (IC(50)) of the methanolic crude extract was 2.22 μg/mL, while for the isolated compounds it ranged from 0.96 μM to 13.96 μM except for (5) which showed a low activity (157 μM). Geissolosimine (1) showed the highest antiplasmodial activity (0.96 μM). CONCLUSIONS This study provides evidence to support the use of Geissospermum vellosii as an antimalarial agent, as used by the native populations. Geissolosimine (1) is a lead molecular structure for possible antimalarial drug development.

Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS.

The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes-ethyl acetate-methanol-water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), ¹H nuclear magnetic resonance (¹H NMR) and ¹³C nuclear magnetic resonance (¹³C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity.

Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry.

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.

Isolation and structural elucidation of indole alkaloids from Geissospermum vellosii by mass spectrometry.

Alkaloids from the stem bark of Geissospermum vellosii possess a variety of therapeutic properties including antimalarial activities, activity as a sexual stimulant and inhibition of the proliferation of HIV and herpes viruses. Methods currently used to isolate the active components from G. vellosii are time-consuming, labor intensive, and result in low recovery. In addition, there is a lack of sensitive and accurate analytical methods for the structural characterization and identification of alkaloid components in minor quantities. A combination of high performance counter-current chromatography and ESI tandem mass spectrometry (MS(n)) was established to isolate alkaloids from the stem bark of G. vellosii, and study their electrospray ionization mass spectrometry fragmentation behavior. Five indole alkaloids were successfully isolated and identified by nuclear magnetic resonance and mass spectrometry. The multi-stage tandem mass spectrometric data were used to study their fragmentation pattern and set a model for detailed structure characterization of related indole alkaloids. The presence of the even mass fragment ion suggestive of an odd number of nitrogen at m/z 144 corresponding to C(10)H(9)N was characteristic to indole alkaloids. The results of the experiments demonstrated that the combination of high performance counter current chromatography and ESI-MS(n) is a sensitive, selective and effective approach for rapid isolation and characterization of alkaloids from G. vellosii.

Leishmanicidal activity of a daucane sesquiterpene isolated from Eryngium foetidum

Abstract Context: Eryngium foetidum L. (Apiaceae) is a traditional herb that has been used for numerous medicinal applications, including as a treatment for parasitic infections, especially in the Neotropics from where it originates. Objective: This study evaluates the in vitro leishmanicidal and cytotoxicity activities of isolated compounds based on a bioassay-guided fractionation approach. Materials and methods: Defatted aerial parts of E. foetidum were subjected to extraction with methanol followed by partitioning with n-hexane, ethyl acetate and 50% methanol. Then, the first two fractions were subsequently fractionated by column chromatography and HPLC. Compound identity was confirmed by mass spectrometry and NMR spectroscopy. Leishmania tarentolae (promastigotes) and L. donovani (amastigotes) were used as testing parasites. L6 rat myoblasts were used for cytotoxicity. All extracts and fractions were tested at 20 μg/mL. Results: The initial methanol extract showed 20% growth inhibition of L. tarentolae. Then, the n-hexane and ethyl acetate fractions were also active showing approximately 40% growth inhibition. From these two fractions, the following compounds were isolated: lasidiol p-methoxybenzoate (1), a daucane sesquiterpene; and 4-hydroxy-1,1,5-trimethyl-2-formyl-cyclohexadien-(2,5)-[α-acetoxymethyl-cis-crotonate] (2), a terpene aldehyde ester derivative. Compound 1 inhibited the growth of both L. tarentolae and L. donovani with IC50 values of 14.33 and 7.84 μM, respectively; and showed no cytotoxicity (IC50 > 50 μM). Compound 2 was inactive in the L. tarentolae assay (IC50 > 50 μM). Discussion and conclusion: This study presented the bioassay-guided fractionation with the leishmanicidal and cytotoxicity activities of two compounds isolated for the first time from an Eryngium species.