Cristina Iuga

Soy Isoflavones and Breast Cancer Cell Lines: Molecular Mechanisms and Future Perspectives

The potential benefit of soy isoflavones in breast cancer chemoprevention, as suggested by epidemiological studies, has aroused the interest of numerous scientists for over twenty years. Although intensive work has been done in this field, the preclinical results continue to be controversial and the molecular mechanisms are far from being fully understood. The antiproliferative effect of soy isoflavones has been commonly linked to the estrogen receptor interaction, but there is growing evidence that other pathways are influenced as well. Among these, the regulation of apoptosis, cell proliferation and survival, inhibition of angiogenesis and metastasis or antioxidant properties have been recently explored using various isoflavone doses and various breast cancer cells. In this review, we offer a comprehensive perspective on the molecular mechanisms of isoflavones observed in in vitro studies, emphasizing each time the dose-effect relationship and estrogen receptor status of the cells. Furthermore, we present future research directions in this field which could provide a better understanding of the inner molecular mechanisms of soy isoflavones in breast cancer.

Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ3 and focal adhesion signaling pathways

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics.

Proteomic identification of potential prognostic biomarkers in resectable pancreatic ductal adenocarcinoma

Pancreatic cancer is a devastating disease with a mortality rate almost identical with its incidence. In this context, the investigation of the pancreatic cancer proteome has gained considerable attention because profiles of proteins may be able to identify disease states and progression more accurately. Therefore, our objective was to investigate the changes in the proteome of patients suffering from pancreatic ductal adenocarcinoma (PDAC) by a comprehensive quantitative approach. Comparative proteomic profiling by label‐free LC‐MS/MS analysis of nine matched pairs of tumor and nontumor pancreas samples was used to identify differences in protein levels characteristic for PDAC. In this analysis, 488 proteins were quantified by at least two peptides of which 99 proteins displayed altered levels in PDAC (p < 0.01, fold change >1.3). Screening of data revealed a number of molecules that had already been related to PDAC such as galectin‐1 (LEG1), major vault protein, adenylyl cyclase‐associated protein 1 (CAP1), but also a potential new prognostic biomarker prolargin (PRELP). The Kaplan–Meier survival analysis revealed a significant correlation of protein abundance of PRELP with postoperative survival of patients with PDAC. For selected proteins the findings were verified by targeted proteomics (SRM), validated by immunohistochemistry and Western blotting and their value as candidate biomarkers is discussed.

The Impact of Soy Isoflavones on MCF-7 and MDA-MB-231 Breast Cancer Cells Using a Global Metabolomic Approach

Despite substantial research, the understanding of the chemopreventive mechanisms of soy isoflavones remains challenging. Promising tools, such as metabolomics, can provide now a deeper insight into their biochemical mechanisms. The purpose of this study was to offer a comprehensive assessment of the metabolic alterations induced by genistein, daidzein and a soy seed extract on estrogen responsive (MCF-7) and estrogen non-responsive breast cancer cells (MDA-MB-231), using a global metabolomic approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that all test compounds induced a biphasic effect on MCF-7 cells and only a dose-dependent inhibitory effect on MDA-MB-231 cells. Proton nuclear magnetic resonance (1H-NMR) profiling of extracellular metabolites and gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites confirmed that all test compounds shared similar metabolic mechanisms. Exposing MCF-7 cells to stimulatory concentrations of isoflavones led to increased intracellular levels of 6-phosphogluconate and ribose 5-phosphate, suggesting a possible upregulation of the pentose phosphate pathway. After exposure to inhibitory doses of isoflavones, a significant decrease in glucose uptake was observed, especially for MCF-7 cells. In MDA-MB-231 cells, the glutamine uptake was significantly restricted, leading to alterations in protein biosynthesis. Understanding the metabolomic alterations of isoflavones represents a step forward in considering soy and soy derivates as functional foods in breast cancer chemoprevention.

Paclitaxel-loaded poly(lactic-co-glycolic acid) microspheres: preparation and in vitro evaluation

Poly(lactic-co-glycolic acid) (PLGA) microspheres containing paclitaxel were prepared by an oil-in-water (o/w) emulsification solvent evaporation method. Box Benhken experimental design was used for studying the parameters that influence the microsphere characteristics and for calculating the optimal formulation. The resulting microspheres were characterized regarding their physicochemical properties, drug content and in vitro drug release. The stirring rate had the greatest influence on particle size and particle size distribution. PLGA and polyvinyl alcohol concentrations had an important influence on drug loading: drug loading increased linearly with PLGA concentration and decreased linearly with PVA concentration. The differential scanning calorimetry proved that there are interactions between paclitaxel and polymer. The release behavior of paclitaxel from the polymer matrix exhibited a biphasic pattern characterized by a slow initial release during the first 26 days (less than 10% paclitaxel), followed by a faster and uniform release (the accumulative amount of paclitaxel released in 92 days was between 17 and 44%).

Impact of blood sample collection methods on blood protein profiling studies

Pre-analytical factors have a significant impact on the integrity of blood samples used for qualitative and quantitative protein profiling. Important factors are the type of the blood collection tube and the anticoagulant used. Only a few studies have been performed to assess these variables by comparing only serum and EDTA plasma collection tubes with some target proteins or peptides. In this study, we investigated the protein profile of blood samples collected in serum, EDTA-, heparin-, and citrate plasma tubes. Furthermore, we compared the depletion efficiency of 6 high abundant proteins, the detectable blood protein profile, the variance, and the coverage of the detectable protein sets. The largest differences were found between serum and plasma samples with respect to the peptide number and the occurrence of classical blood proteins. The heparin plasma evidenced a high number of detectable proteins, low global variance and a high similarity to EDTA- and citrate plasma and may therefore be also a useful test tube for blood protein profiling. In addition, a core set of blood proteins were described and the portions and compositions of sampling specific proteins were disclosed. Therefore, pre-analytical issues such as the sample collection method should be considered for protein profiling studies.

Assessment of isoflavone aglycones variability in soy food supplements using a validated HPLC-UV method

Background and aims Soy supplements are often recommended in the management of menopause symptoms. The declared content of soy supplements is commonly expressed as total isoflavones per dosage form. Given that soy isoflavones have different estrogenic potencies, pharmacokinetics and metabolism, the aim of this study was to evaluate the total isoflavone content and the aglycone profile of seven soy supplements and one soy seed extract. Label accuracy was assessed, in relation to the precise content and the recommended posology for estimating whether the optimal dose is achieved for alleviating menopause symptoms. Methods A high performance liquid chromatography method was developed for evaluating the aglycone content (genistein, daidzein, glycitein). After extraction and acidic hydrolysis, the aglycones were separated on a C18 column, using 0.1% acetic acid and acetonitrile as mobile phases. The flow rate was 1.5mL min−1 and the UV detector wavelength was set at 260nm. A linear relationship was found in the range 5–80μg mL−1. The method was validated using the accuracy profile methodology. Results The total isoflavone content ranged from 6.07 to 41.68mg dosage form−1. Various aglycone profiles were obtained for each supplement which can result in a different estrogenic activity, bioavailability and finally, in a different efficiency in alleviating menopause symptoms. In most clinical trials where soy isoflavones were evaluated, little attention was paid to determining the exact aglycone profile of the employed soy extracts. Conclusions As clinical outcomes continue to be controversial, this study highlights the need of standardization in genistein, rather than total isoflavones and labeling accuracy for soy supplements.

Plasma protein profiling of patients with intraductal papillary mucinous neoplasm of the pancreas as potential precursor lesions of pancreatic cancer

Efforts for the early diagnosis of the pancreatic ductal adenocarcinoma (PDAC) have recently been driven to one of the precursor lesions, namely intraductal papillary mucinous neoplasm of the pancreas (IPMN). Only a few studies have focused on IPMN molecular biology and its overall progression to cancer. Therefore, IPMN lacks comprehensive characterization which makes its clinical management controversial. In this study, we characterized plasma proteins in the presence of IPMNs in comparison to healthy controls, chronic pancreatitis, and PDAC by a proteomics approach using data-independent acquisition based mass spectrometry. We describe several protein sets that could aid IPMN diagnosis, but also differentiation of IPMN from healthy controls, as well as from benign and malignant diseases. Among all, high levels of carbonic anhydrases and hemoglobins were characteristic for the IPMN group. By employing ELISA based quantification we validated our results for human tissue inhibitor of metalloproteinase inhibitor 1 (TIMP-1). We consider IPMN management directed towards an early potential cancer development a crucial opportunity before PDAC initiation and thus its early detection and cure.

Data on the impact of the blood sample collection methods on blood protein profiling studies

Complete blood protein profiles of 4 different blood sample collection methods (EDTA-, heparin- and citrate plasma and serum) were investigated and the data presented herein is an extension of the research article in Ilies et al. [1]. Specimens were depleted of 6 highly abundant proteins and protein profiling was assessed by nano-LC UDMSE. Exhaustive protein sets and protein abundances before and after depletion are presented in tables and figures. Also, the core protein set and the unique proteins for each sample collection method previously described [1] are disclosed.

Plasma protein absolute quantification by nano-LC Q-TOF UDMSE for clinical biomarker verification

Background and aims Proteome-based biomarker studies are targeting proteins that could serve as diagnostic, prognosis, and prediction molecules. In the clinical routine, immunoassays are currently used for the absolute quantification of such biomarkers, with the major limitation that only one molecule can be targeted per assay. The aim of our study was to test a mass spectrometry based absolute quantification method for the verification of plasma protein sets which might serve as reliable biomarker panels for the clinical practice. Methods Six EDTA plasma samples were analyzed after tryptic digestion using a high throughput data independent acquisition nano-LC Q-TOF UDMSE proteomics approach. Synthetic Escherichia coli standard peptides were spiked in each sample for the absolute quantification. Data analysis was performed using ProgenesisQI v2.0 software (Waters Corporation). Results Our method ensured absolute quantification of 242 non redundant plasma proteins in a single run analysis. The dynamic range covered was 105. 86% were represented by classical plasma proteins. The overall median coefficient of variation was 0.36, while a set of 63 proteins was found to be highly stable. Absolute protein concentrations strongly correlated with values reviewed in the literature. Conclusions Nano-LC Q-TOF UDMSE proteomic analysis can be used for a simple and rapid determination of absolute amounts of plasma proteins. A large number of plasma proteins could be analyzed, while a wide dynamic range was covered with low coefficient of variation at protein level. The method proved to be a reliable tool for the quantification of protein panel for biomarker verification in the clinical practice.