Sara Crotti

Some thoughts on electrospray ionization mechanisms.

Electrospray ionization (ESI) mechanisms are highly complex, due to a series of physical and chemical phenomena taking place on a complex system, as a solution is. In fact, even if the solution of an analyte in a protic medium can be considered at first sight to be a two-component system, the presence of solvent dissociation equilibria and the possible interactions solvent–solvent dissociation products, solvent dissociation products–analyte make this system highly complex, also for the presence of possible ionic compounds (for example, Na+, K+) which strongly affect the above equilibria. A high number of research articles have been published, mainly devoted to charged droplet production and to gas-phase ion generation. They all show the high complexity of the processes affecting electrospray measurements related to either the chemical equilibria present in the condensed phase and to electrolysis processes at the emitter tip or to the processes occurring in the sprayed droplets. As a result, the chemical composition inside the small droplets from which the analyte ions are generated can be significantly different from those in sprayed solution. In this review, after a short survey of the proposed ESI mechanisms, some experiments are described. They were performed to examine if ion mobility in solution, before the formation of the sprayed charged droplets, can affect the ESI results. The data, obtained by studying both inorganic and organic analytes, indicate that the ESI spectra are dependent on the analyte dimension and charge state which, as a consequence, affect their ion mobility in solution.

Extracellular Matrix and Colorectal Cancer: How Surrounding Microenvironment Affects Cancer Cell Behavior?

Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells, and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis. Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. J. Cell. Physiol. 232: 967–975, 2017.

Predictive response biomarkers in rectal cancer neoadjuvant treatment

Locally advanced rectal cancer (RC) treatment is a challenge, because RC has a high rate of local recurrence. To date preoperative chemoradiotherapy (pCRT) is widely accepted as standard protocol of care for middle-low RC, but complete tumour response rate ranges from 4 to 44% and 5-year local recurrence rate is 6%. Better understanding of molecular biology and carcinogenesis pathways could be used both for pre-neoplastic lesions and locally recurrence diagnosis, and for tumour response prediction to therapy. Circulating molecules, gene expression and protein signature are promising sources to biomarker discovery. Several studies have evaluated potential predictors of response and recently, cell-free Nucleic Acid levels have been associated to tumour response to neoadjuvant therapies. Alternative method is the serum or plasma proteome and peptidome analysis. It may be ideally suited for its minimal invasiveness and it can be repeated at multiple time points throughout the treatment in contrast to tissue-based methods which still remain the most reliable and specific approach. Many studies have analyzed preoperative rectal tissue prognostic factor, but data are controversial or not confirmed.

Aspects of the Role of Surfaces in Ionization Processes

The operating principles and some applications of atmospheric pressure desorption electrospray ionization (DESI) and surface activated chemical ionization (SACI) methods are described in detail. The former technique allows one to obtain information on the chemical composition (in terms of organic compounds) present on a surface of interest. The latter, SACI, provides chemical information as a result of the interaction of a vaporised solution of the analyte with a metallic surface. Both techniques typically lead to the production of abundant protonated molecules. The data available in the literature indicate that both DESI and SACI are highly promising techniques with the former giving to mass spectrometry new application fields, and the latter an increasing sensitivity and a lowering of chemical noise that, in the case of biological samples, represent a weak point in many analytical measurements.

Compartmentalized activities of the pyruvate dehydrogenase complex sustain lipogenesis in prostate cancer

The mechanisms by which mitochondrial metabolism supports cancer anabolism remain unclear. Here, we found that genetic and pharmacological inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of the pyruvate dehydrogenase complex (PDC), inhibits prostate cancer development in mouse and human xenograft tumor models by affecting lipid biosynthesis. Mechanistically, we show that in prostate cancer, PDC localizes in both the mitochondria and the nucleus. Whereas nuclear PDC controls the expression of sterol regulatory element-binding transcription factor (SREBF)-target genes by mediating histone acetylation, mitochondrial PDC provides cytosolic citrate for lipid synthesis in a coordinated manner, thereby sustaining anabolism. Additionally, we found that PDHA1 and the PDC activator pyruvate dehydrogenase phosphatase 1 (PDP1) are frequently amplified and overexpressed at both the gene and protein levels in prostate tumors. Together, these findings demonstrate that both mitochondrial and nuclear PDC sustain prostate tumorigenesis by controlling lipid biosynthesis, thus suggesting this complex as a potential target for cancer therapy.Inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of the pyruvate dehydrogenase complex (PDC) regulating mitochondrial metabolism, inhibits lipid biosynthesis and prostate cancer development in mouse and human xenograft tumor models.

Mass spectrometry in the pharmacokinetic studies of anticancer natural products

In the history of medicine, nature has represented the main source of medical products. Indeed, the therapeutic use of plants certainly goes back to the Sumerian and Hippocrates and nowadays nature still represents the major source for new drugs discovery. Moreover, in the cancer treatment, drugs are either natural compounds or have been developed from naturally occurring parent compounds firstly isolated from plants and microbes from terrestrial and marine environment. A critical element of an anticancer drug is represented by its severe toxicities and, after administration, the drug concentrations have to remain in an appropriate range to be effective. Anyway, the drug dosage defined during the clinical studies could be inappropriate for an individual patient due to differences in drug absorption, metabolism and excretion. For this reason, personalized medicine, based on therapeutic drug monitoring (TDM), represents one of most important challenges in cancer therapy. Mass spectrometry sensitivity, specificity and fastness lead to elect this technique as the Golden Standard for pharmacokinetics and drug metabolism studies therefore for TDM. This review focuses on the mass spectrometry-based methods developed for pharmacokinetic quantification in human plasma of anticancer drugs derived from natural sources and already used in clinical practice. Particular emphasis was placed both on the pre-analytical and analytical steps, such as: sample preparation procedures, sample size required by the analysis and the limit of quantification of drugs and metabolites to give some insights on the clinical practice applicability.

Matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization and carbon nanohorns in the detection of antineoplastic drugs. 1. The cases of irinotecan, sunitinib and 6-alpha-hydroxy paclitaxel

The development of surface-assisted laser desorption/ionization (SALDI) methodologies in mass spectrometry allows, in principle, the development of new analytical approaches to qualitative and quantitative measurements on small molecules. Some of these methods have been applied to characterize two antineoplastic drugs: irinotecan (1) and sunitinib (2), and also 6-α-hydroxy-paclitaxel (3), the main metabolite of paclitaxel. Three different SALDI approaches have been tested employing nanostructure-assisted laser desorption/ionization (NALDI), carbon nanohorns (NHs) and carbon NHs covered by liquid additives. The results so obtained have been compared to those observed under matrix-assisted laser desorption/ionization (MALDI) conditions. Compounds 1 and 2 show the easy formation of protonated molecular species under all the experimental conditions, but the highest absolute intensity was achieved by NALDI. On the contrary, ionic species of low intensity are present for 3, among which are those that exhibit the highest intensity caused by [M + K]+ ions. After a critical evaluation of the obtained data, the linear response of the [M + H]+ ion intensity of 1 versus different deposited sample amounts was investigated, and the best results (R2 = 0.9889) were obtained under MALDI conditions. The analysis of plasma samples spiked with 1 showed, again, that the MALDI approach was the best one (R2 = 0.9766). The failure of NALDI measurements could be rationalized by the presence of ion-suppression effects.

Alterations of the Plasma Peptidome Profiling in Colorectal Cancer Progression

Early detection of colorectal cancer (CRC) remains a challenge. It has been highlighted that the pathological alterations within an organ and tissues might be reflected in serum or plasma proteomic/peptidic patterns. The aim of the study was to follow the changes in the plasma peptides associated to colorectal cancer progression by mass spectrometry. This study included 27 adenoma, 67 CRC (n = 33 I–II stage and n = 34 III–IV stage), 23 liver metastasis from CRC patients and 34 subjects disease‐free as controls. For plasma peptides analysis, samples purification was performed on the Nanoporous Silica Chips technology followed by matrix‐assisted laser desorption/ionisation‐time of flight analysis. Since the high complexity of the obtained dataset, multivariate statistical analysis, and discriminant pattern recognition were performed for study groups classification. Forty‐four of 88 ionic species were successfully identified as fragments of peptides and proteins physiologically circulating in the blood and belonging to immune and coagulation systems and inflammatory mediators. Many peptides clustered into sets of overlapping sequences with ladder‐like truncation clearly associated to proteolytic processes of both endo‐ and exoproteases activity. Comparing to controls, a different median ion intensity of the group‐type fragments distribution was observed. Moreover, the degradation pattern obtained by proteolytic cleavage was different into study groups. This pattern was specific and characteristic of each group: controls, colon tumour disease (including adenoma and CRC), and liver metastasis, revealing a role as biomarker in early diagnosis and prognosis. Our findings highlighted peculiar changes in protease activity characteristic of CRC progression from pre‐cancer lesion to metastatic disease. J. Cell. Physiol. 231: 915–925, 2016.

Liposomal delivery of a Pin1 inhibitor complexed with cyclodextrins as new therapy for high-grade serous ovarian cancer

ABSTRACT Pin1, a prolyl isomerase that sustains tumor progression, is overexpressed in different types of malignancies. Functional inactivation of Pin1 restrains tumor growth and leaves normal cells unaffected making it an ideal pharmaceutical target. Although many studies on Pin1 have focused on malignancies that are influenced by sex hormones, studies in ovarian cancer have lagged behind. Here, we show that Pin1 is an important therapeutic target in high‐grade serous epithelial ovarian cancer. Knock down of Pin1 in ovarian cancer cell lines induces apoptosis and restrains tumor growth in a syngeneic mouse model. Since specific and non‐covalent Pin1 inhibitors are still limited, the first liposomal formulation of a Pin1 inhibitor was designed. The drug was efficiently encapsulated in modified cyclodextrins and remotely loaded into pegylated liposomes. This liposomal formulation accumulates preferentially in the tumor and has a desirable pharmacokinetic profile. The liposomal inhibitor was able to alter Pin1 cancer driving‐pathways trough the induction of proteasome‐dependent degradation of Pin1 and was found to be effective in curbing ovarian tumor growth in vivo.

Peptide Patterns as Discriminating Biomarkers in Plasma of Patients With Familial Adenomatous Polyposis

BACKGROUND Familial adenomatous polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer. So far, no accepted prognostic markers are present to monitor patients with FAP. Consequently, the major problem in managing patients with FAP is the difficulty to predict when the switch between adenoma and malignant carcinoma occurs, leading to the necessity of preventive surgery. Proteomics is one of the most suitable approaches to identify biomarkers, and it is widely used in cancer research. In this investigation, we studied the circulating plasma peptides in samples collected from patients with FAP and compared the obtained results with adenoma, colorectal cancer, and control samples to discover peptides able to distinguish different phenotypes. MATERIALS AND METHODS The peptide fingerprint was obtained by matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry. After statistical analysis, a subset of 45 ionic species was found differently expressed in the 4 groups considered, 12 of them peculiar to patients with FAP. Moreover, 4 ionic species were found significantly changed in the switch between adenoma and malignant carcinoma. RESULTS Potentially prognostic peptides identified by this study derive mainly from circulating proteins, some of which are involved in the inflammatory response, such as complement C3 and C4 subjected to an exoprotease activity that seemed pathology related. CONCLUSIONS In this study, we defined for the first time a specific panel of peptides for monitoring patients with FAP that could be profitably used to monitor and predict the pathologic evolution in adenocarcinoma malignancy.