Ultrasound- and Microbubble-Assisted Gemcitabine Delivery to Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death worldwide. Poor drug delivery to tumours is thought to limit chemotherapeutic treatment efficacy. Sonoporation combines ultrasound (US) and microbubbles to increase the permeability of cell membranes. We assessed gemcitabine uptake combined with sonoporation in vitro in three PDAC cell lines (BxPC-3, MIA PaCa-2 and PANC-1). Cells were cultured in hypoxic bioreactors, while gemcitabine incubation ± sonoporation was conducted in cells with operational or inhibited nucleoside membrane transporters. Intracellular active metabolite (dFdCTP), extracellular gemcitabine, and inactive metabolite (dFdU) concentrations were measured with liquid chromatography tandem mass spectrometry. Sonoporation with increasing US intensities resulted in decreasing extracellular gemcitabine concentrations in all three cell lines with inhibited membrane transporters. In cells with inhibited membrane transporters, without sonoporation, dFdCTP concentrations were reduced down to 10% of baseline. Sonoporation partially restored gemcitabine uptake in these cells, as indicated by a moderate increase in dFdCTP concentrations (up to 37% of baseline) in MIA PaCa-2 and PANC-1. In BxPC-3, gemcitabine was effectively inactivated to dFdU, which might represent a protective mechanism against dFdCTP accumulation in these cells. Intracellular dFdCTP concentrations did not change significantly following sonoporation in any of the cell lines with operational membrane transporters, indicating that the gemcitabine activation pathway may have been saturated with the drug. Sonoporation allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.

Preanalytical Stability of Gemcitabine and its Metabolite 2', 2'-Difluoro-2'-Deoxyuridine in Whole Blood-Assessed by Liquid Chromatography Tandem Mass Spectrometry.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Liquid chromatography/tandem mass spectrometry method for simultaneous quantification of eight endogenous nucleotides and the intracellular gemcitabine metabolite dFdCTP in human peripheral blood mononuclear cells.

Quantification of endogenous nucleotides is of interest for investigation of numerous cellular biochemical processes, such as energy metabolism and signal transduction, and may also be applied in cancer and antiretroviral therapies in which nucleoside analogues are used. For these purposes we developed and validated a sensitive and high accuracy ion-pair liquid chromatography tandem mass spectrometry (IP LC-MS/MS) method for simultaneous quantification of eight endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP) and 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP), an intracellular metabolite of the nucleoside analogue gemcitabine. The assay was validated using 200µL aliquots of peripheral blood mononuclear cell (20×10(6)cells/ml, 4×10(6)cells) extracts, pretreated with activated charcoal and spiked with unlabeled nucleotides, deoxynucleotides and dFdCTP. Analytes were extracted by simple precipitation with cold 60% methanol containing isotope labeled internal standards and separated on a porous graphitic carbon column. For method validation, the concentration ranges were: 0.125-20.8pmol injected for deoxynucleotides, 0.25-312.5pmol injected for dFdCTP and 5-3200pmol injected for nucleotides. The highest coefficients of variation (CV) were 12.1% for within run assay and 11.4% for between run assay, both representing the precision at the lowest analyte concentrations. The method was applied to monitor dFdCTP and changes in endogenous nucleotides in patients who were receiving gemcitabine infusions.

Sensitivity and accuracy of organic matrix-assisted laser desorption and ionisation mass spectrometry of FeCl3 is higher than in in matrix-free approach.

We compare the quality and reliability of laser desorption and ionisation mass spectra of FeCl3 acquired without the assistance of the matrix with the spectra acquired with different organic matrix molecules. Generally, inorganic salts tend to form clusters upon laser irradiation, the signals of which can be easily distinguished from ions arising from the matrix. In the presence of a matrix, cluster ions are, however, mostly suppressed. We have compared the number of analyte signals, accuracy of determination of isotope composition of the analyte and the sensitivity of FeCl3 detection between different approaches. The results obtained imply that the sensitivity of mass spectrometric analysis of FeCl3 is somewhat higher when matrices are applied than in the matrix-free approach. Among all matrices tested in this work, F20TPP seems to be the most promising for further applications as a matrix for mass spectrometry of inorganic salts.

Interactions of platinum and ruthenium coordination complexes with pancreatic phospholipase A(2) and phospholipids investigated by MALDI TOF mass spectrometry.

Phospholipase A2 is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA2 activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA2 inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA2 and phospholipids, as well as the application of MALDI-TOF mass spectrometry for screening PLA2 inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.

Inhibitory effect of cisplatin and [Pt(dach)Cl2] on the activity of phospholipase A2.

This work has been focused on testing the influence of two selected Pt(II) complexes cisplatin, Pt(NH3)2Cl2, and [Pt(dach)Cl2] on the activity of porcine pancreatic phospholipase A2 (PLA2). It has been assumed that this enzyme plays a role in carcinogenesis and that it could be a target in the tumour therapy. The results of this study show that both Pt(II) complexes inhibit the activity of the enzyme, though they bind to it in a different manner. While cisplatin interacts with the enzyme in an acompetitive manner, the stable interaction of [Pt(dach)Cl2] with PLA2 could not be detected under our experimental conditions.

Estradiol enhances effects of fructose rich diet on cardiac fatty acid transporter CD36 and triglycerides accumulation.

Fructose rich diet increases hepatic triglycerides production and has deleterious cardiac effects. Estrogens are involved in regulation of lipid metabolism as well, but their effects are cardio beneficial. In order to study effects of fructose rich diet on the main heart fatty acid transporter CD36 and the role of estrogens, we subjected ovariectomized female rats to the standard diet or fructose rich diet, with or without estradiol (E2) replacement. The following parameters were analyzed: feeding behavior, visceral adipose tissue mass, plasma lipids, cardiac CD36 expression, localization and insulin regulation, as well as the profile of cardiac lipids. Results show that fructose rich diet significantly increased plasma triglycerides and decreased plasma free fatty acid (FFA) concentration, while E2 additionally emphasized FFA decrease. The fructose diet increased cardiac plasma membrane content of CD36 in the basal and insulin-stimulated states, and decreased its low density microsomes content. The E2 in fructose-fed rats raised the total cardiac protein content of CD36, its presence in plasma membranes and low density microsomes, and cardiac deposition of triglycerides, as well. Although E2 counteracts fructose in some aspects of lipid metabolism, and separately they have opposite cardiac effects, in combination with fructose rich diet, E2 additionally enhances CD36 presence in plasma membranes of cardiac cells and triglycerides accumulation, which paradoxically might promote deleterious effects of fructose diet on cardiac lipid metabolism. Taken together, the results presented in this work are of high importance for clinical administration of estrogens in females with a history of type 2 diabetes.

Colloidal TiO2 nanoparticles as substrates for M(S)ALDI mass spectrometry of transition metal complexes.

RATIONALE: Nanoparticles as substrates for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have advantages over organic matrices, since they enable acquisition of spectra in the low-mass range. It has been previously shown that TiO(2) nanoparticles can be used as substrate for MALDI-TOF MS analysis of phospholipids and for other types of molecules, but none of them was applied to the analysis of transition metal complexes. METHODS: The MALDI-TOF mass spectra of potential anti-tumor drugs [AuCl(2)(bipy)]Cl, [PtCl(4)(bipy)], and [RuCl(2)(bipy)(2) ]Cl acquired with organic matrices have been compared with spectra acquired with colloidal titanium dioxide nanoparticles. Colloidal TiO(2) nanoparticles (NPs) with average diameter of 5 nm were synthesized and characterized by microscopy. For some experiments, the TiO(2) NPs were treated at 60 °C. Suspensions of matrix and the analyte were premixed, applied to the MALDI target and left at room temperature. Mass spectra were acquired with a 50-Hz pulsed nitrogen laser emitting at a wavelength of 337 nm. RESULTS: The MALDI spectra of transition metal complexes acquired with TiO(2) NPs exhibited somewhat lower sensitivity than those with organic matrices; on the other hand, they are characterized by significantly lower number of signals arising from the tested complexes than the organic matrices. Whereas adducts between organic matrices and the analytes were detectable in the spectra, this was not the case for the TiO(2)-assisted mass spectra. CONCLUSIONS: We have shown that colloidal TiO(2) NPs can be used as substrates for MALDI-TOF MS of transition metal complexes. Although the sensitivity of this approach in comparison with the use of organic matrices might still be a problem, the potential of the applications of NPs for the mass spectrometric characterization of transition metal complexes is clearly demonstrated.

Inhibitory effect of platinum and ruthenium bipyridyl complexes on porcine pancreatic phospholipase A2.

Pancreatic phospholipase A(2) (PLA(2)) plays an important role in cellular homeostasis as well as in the process of carcinogenesis. Effects of metallo-drugs used as chemotherapeutics on the activity of this enzyme are unknown. In this work, the interaction between porcine pancreatic PLA(2) and two selected transition metal complexes--tetrachloro(bipyridine) platinum(IV) ([PtCl(4)(bipy)]) and dichloro (bipyridine) ruthenium(III)chloride ([RuCl(2)(bipy)(2)]Cl)--was studied. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence spectroscopy have been used to analyse the enzyme activity in the absence and presence of metal complexes and to verify potential binding of these drugs to the enzyme. The tested metal complexes decreased the activity of phospholipase A(2) in an uncompetitive inhibition mode. A binding of the ruthenium complex near the active site of the enzyme could be evidenced and possible modes of interaction are discussed.

FAB, ESI and MALDI Mass Spectrometric methods in the study of metallo-drugs and their biomolecular interactions.

This review surveys the soft ionisation mass spectrometric methods that are most commonly used for the investigation of the mechanism of interaction between metallo-drugs and biomolecules. In the first part of the review, an overview of the applications of transition metal complexes in the therapy of various diseases (arthritis, cancer, diabetes) is given, whereas the second part focuses on the obtained results dealing with various aspects of the interaction between metal complexes and different types of biomolecules. Possibilities and limitations of each mass spectrometric method-namely, fast atom bombardment (FAB), electrospray ionisation (ESI) and matrix-assisted laser desorption and ionisation (MALDI)--are discussed in the third part of this review along with the examples of their application for the analysis of metal complexes, as well as of the products of their interaction with biomolecules.

Laser desorption and ionization time-of-flightversus matrix-assisted laser desorption and ionization time-of-flight mass spectrometry of and metal complexes.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful, "soft" ionization technique for the analysis of both transition metal complexes, which are used as metallo-drugs in the therapy of various types of tumors, and biomolecules. Since some metal complexes absorb light in the UV range, it should be possible to analyse them without additional matrices, i.e. using LDI-TOF MS. In this study, the matrix-free approach was tested for the analysis of [PtCl2(dach)] (dichloride(1,2-diamincyclohexane) platinum(ii)), [RuCl2(en)2]Cl (dichloridobis(ethylenediamine) ruthenium(iii) chloride) and [RuCl2(bipy)2]Cl (bis(bipyridine)dichloridoruthenium(iii) chloride) and the detection limit for these compounds was determined. In summary, the LDI-TOF mass spectra of [PtCl2(dach)] and [RuCl2(en)2]Cl are rather simple, whereas in the presence of 2,5-DHB as a matrix, additional peaks are generated. On the other hand, the standard MALDI-TOF mass spectrum of [RuCl2(bipy)2]Cl exhibits only one peak arising from the complex, in contrast to six peaks detectable in the LDI-TOF mass spectrum. The detection limit in the MALDI-TOF MS analysis of [PtCl2(dach)] and [RuCl2(bipy)2]Cl complexes was lower than that determined in LDI-TOF MS. Taking all into account, in this paper, we have demonstrated some advantages and drawbacks of the matrix-free LDI-TOF mass spectrometric analysis of transition metal complexes.