Evaluation of Glucose Uptake in Normal and Cancer Cell Lines by Positron Emission Tomography.

To date, there is no definitive demonstration of the utility of positron emission tomography (PET) in studying glucose metabolism in cultured cell lines. Thus, this study was designed to compare PET to more standardized methods for the quantitative assessment of glucose uptake in nontransformed and transformed living cells and to validate PET for metabolic studies in vitro. Human colon and breast carcinoma cell lines and mouse embryo fibroblasts were evaluated for [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) uptake by PET and autoradiography and 2-deoxyglucose (2-DG) incorporation by colorimetric assay and analyzed for the radiotoxic effects of [ 18 F]FDG and the expression levels of glucose transporters. Indeed, [ 18 F]FDG incorporation on PET was comparable to [ 18 F]FDG uptake by autoradiography and 2-DG incorporation by colorimetric assay, although radiotracer-based methods exhibited more pronounced differences between individual cell lines. As expected, these data correlated with glucose transporters 1 to 4 and hexokinase II expression in tumor cell lines and mouse fibroblasts. Notably, [ 18 F]FDG incorporation resulted in low apoptotic rates, with fibroblasts being slightly more sensitive to radiotracer-induced cell death. The quantitative analysis of [ 18 F]FDG uptake in living cells by PET represents a valuable and reproducible method to study tumor cell metabolism in vitro, being representative of the differences in the molecular profile of normal and tumor cell lines

Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells.

Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis.Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation.These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells

TRAP1 regulates stemness through Wnt/β-catenin pathway in human colorectal carcinoma

Colorectal carcinoma (CRC) is a common cause of cancer-related death worldwide. Indeed, treatment failures are triggered by cancer stem cells (CSCs) that give rise to tumor repopulation upon initial remission. Thus, the role of the heat shock protein TRAP1 in stemness was investigated in CRC cell lines and human specimens, based on its involvement in colorectal carcinogenesis, through regulation of apoptosis, protein homeostasis and bioenergetics. Strikingly, co-expression between TRAP1 and stem cell markers was observed in stem cells located at the bottom of intestinal crypts and in CSCs sorted from CRC cell lines. Noteworthy, TRAP1 knockdown reduced the expression of stem cell markers and impaired colony formation, being the CSC phenotype and the anchorage-independent growth conserved in TRAP1-rich cancer cells. Consistently, the gene expression profiling of HCT116 cells showed that TRAP1 silencing results in the loss of the stem-like signature with acquisition of a more-differentiated phenotype and the downregulation of genes encoding for activating ligands and target proteins of Wnt/β-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/β-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of β-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of β-catenin and several Wnt/β-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of β-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/β-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics

New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway

Structural studies of Helicobacter pylori proteins relevant for gastric colonization

Gastric cancer is the second leading cause of cancer death worldwide. This is in part a consequence of the increased human longevity, although the rate of gastric cancer has actually been declining from the early 20th Century. Since the relationship between H. pylori and chronic gastritis was established, investigators began to take interest in the role played by H. pylori in this type of cancer. From the first studies to examine this association, several epidemiological data have confirmed the relationship between the regional prevalence of H. pylori and the incidence of stomach cancer. H. pylori is a Gram-negative spiral-shaped bacterium that is characterized by two aspects: the use of several unipolar flagella that confer motility and its ureasic activity. H. pylori is a highly variable bacterial species, both genotypically and phenotypically, and is highly adapted for survival in the gastric niche. The development of effective treatment options to eradicate the H. pylori infection has resulted in an immense change in the clinical management of upper gastrointestinal diseases. The wide use of antibiotic therapies against H. pylori infection, however, is related to the number of therapeutic failures. Recent data show a decreasing efficacy of these therapies worldwide. The research for new therapeutic strategies and new drugs is necessary. The project presented here aims to determine not only the three dimensional structure of proteins playing a role in the virulence of the bacterium, but also their analysis and characterization using an approach of structural genomics. The three-dimensional structure of a protein, in addition to clarify its function, may be useful for the design of potential new drugs. This thesis presents several potential drug targets, each consisting of proteins involved in pathogenicity or essential to the survival of the bacterium. All genes were cloned as described by using different strategies; 7 out of 9 proteins were expressed, 3 out of 7 were soluble in an heterologous enviromen; these three proteins were structurally characterized, two with a crystallographic approach and one with SAXS. The results of the studies on the latter three proteins are presented in detail: CagL, a protein belonging to cagPAI; HP1286, a periplasmic protein belonging to the family YceI that is induced by osmotic stress created by high NaCl or high acidity; and finally HP0797, an adhesin. CagL is a protein localized on the surface of the pilus and acts as a specialized adhesin that connects the T4SS with target cells. The integrin complex α5β1 binds to a small protein of 26 kDa, CagL precisely, which is encoded by the gene hp0539 of the cagPAI. CagL seems to be a functional ortholog of VirB5, a structural component of the T4SS of Agrobacterium tumefaciens. In this thesis we propose a structural model of the oligomerization state of CagL in solution, using the technique of small angle x-ray scattering (SAXS). This model is supported by other experiments, such as crosslinking and gel filtration. Regarding the HP1286 enzyme, crystals were grown and diffraction data measured. Crystals belong to space group P212121 and contain a dimer per asymmetric unit. Each monomer has a hydrophobic pocket in which there is an electron density indicating the presence of a ligand. The latter was identified by mass spectrometry as erucamide, the amide of a fatty acid of 22 carbon atoms, erucic acid (Z-CH3 (CH2) 7CH = CH (CH2) 11CONH2). The recombinant protein HpaA (HP0797), an adhesin, was expressed in E. coli in soluble form without the signal peptide. We have obtained crystals that diffract only at a resolution of 4 Å and attempts to improve them are in progress. A molecular model was built by homology modelling and the putative domain of glycolipids of HpaA was predicted based on its amino acid sequence.Il tumore gastrico è la seconda causa di morte per cancro nel mondo. Questo fatto è in parte una conseguenza della maggiore longevità umana, anche se il tasso di cancro gastrico è effettivamente in calo rispetto ai primi anni del 20° secolo. Da quando è stata stabilita una relazione tra H. pylori e gastrite cronica, i ricercatori hanno cominciato ad interessarsi al ruolo svolto da H. pylori in questi tipi di tumore. Dai primi studi volti ad esaminare tale associazione, numerosi dati epidemiologici hanno confermato la relazione tra la prevalenza regionale di H. pylori e l'incidenza dei tumori allo stomaco. H. pylori è un batterio Gram-negativo a forma di spirale che si caratterizza per due aspetti: l’utilizzo di numerosi flagelli unipolari che gli conferiscono motilità e l’attività ureasica. H. pylori è una specie batterica altamente eterogenea, sia genotipicamente che fenotipicamente, che si è adattata alla sopravvivenza nella nicchia gastrica. Diversi aspetti della biologia delle adesine di H. pylori esemplificano l'importanza dell’eterogeneità in questo sistema: (i) nessuna adesina in particolare è essenziale per il fissaggio alla mucosa gastrica, il che indica la ridondanza del meccanismo adesivo; (ii) l’espressione delle adesine è diversificata tra ceppi ed è variabile nel tempo all'interno di un unico ceppo; (iii) le interazioni adesive contribuiscono all'infiammazione ed è probabile che siano coinvolte nella progressione della malattia. Lo sviluppo di diversi trattamenti per sradicare l'infezione da H. pylori si è tradotto in un enorme cambiamento nella gestione clinica delle patologie del tratto gastrointestinale superiore. L'ampio uso di terapie antibiotiche contro l'infezione da H. pylori è, però, correlato al numero di fallimenti terapeutici. Dati recenti mostrano una diminuzione dell'efficacia di queste terapie in tutto il mondo. Ciò ha reso necessaria la ricerca di nuove strategie terapeutiche e di nuovi farmaci. Il progetto qui presentato mira a determinare non solo la struttura tridimensionale delle proteine descritte, ma anche la loro analisi e caratterizzazione attraverso un approccio di genomica strutturale. La struttura tridimensionale di una proteina, infatti, oltre a chiarire le funzioni della proteina target, può essere utile per la progettazione di potenziali nuovi farmaci. In questa tesi vengono presentati diversi potenziali bersagli farmacologici, tutti consistenti in proteine coinvolte nella patogenicità o comunque essenziali per la sopravvivenza del batterio. Tutti i geni descritti sono stati clonati utilizzando strategie diverse; 7 su 9 proteine sono state espresse, 3 su 7 sono risultate solubili in ambiente eterologo; queste tre proteine sono state infine caratterizzate strutturalmente: due con approccio cristallografico ed una con SAXS. Sono presentati in dettaglio i risultati degli studi su queste ultime tre proteine: CAGL, una proteina appartenente alla cagPAI; HP1286, una proteina periplasmatica appartenente alla famiglia YceI che è indotta da stress osmotico creato da elevato NaCl o da elevata acidità; infine HP0797, un’altra adesina. CAGL è una proteina presente sulla superficie del pilus e funge da adesina specializzata che collega il T4SS con le cellule bersaglio. Il complesso di integrine α5β1 si lega ad una piccola proteina di 26 kDa, CAGL appunto, che è codificata dal gene hp0539 della cagPAI. CAGL sembra essere un ortologo funzionale di VirB5, un componente strutturale del T4SS di Agrobacterium tumefaciens. In questa tesi viene proposto un modello strutturale dello stato di oligomerizzazione di CAGL in soluzione, utilizzando la tecnica di “small angle x-ray scattering” (SAXS). Questo modello viene supportato da numerosi esperimenti, quali crosslinking e gel filtrazione. Per l’enzima HP1286 sono stati ottenuti cristalli ed i dati di diffrazione sono stati misurati. I cristalli appartengono al gruppo spaziale P212121; essi contengono un dimero per unità asimmetrica. Ogni monomero presenta una tasca idrofobica nella quale c’è una densità elettronica che indica la presenza di un ligando. Quest’ultimo è stato identificato dalla spettrometria di massa, come erucamide, l’ammide di un acido grasso di 22 atomi carbonio, acido erucico (Z-CH3 (CH2) 7CH = CH (CH2) 11CONH2). La proteina ricombinante HpaA (HP0797), un’adesina, è stata espressa in E. coli in forma solubile, senza il peptide segnale. Si sono ottenuti cristalli che per ora diffrangono solo alla risoluzione di 4 Å e si sta cercando di migliorarli. Il putativo dominio glicolipidico della HpaA è stato predetto sulla base dalla sequenza aminoacidica. La struttura atomica HpaA potrebbe essere utilizzata anche per caratterizzare i domini glicolipidici in proteine che sono racchiuse in un guscio di lipidi

The role of human chorionic gonadotropin as tumor marker: Biochemical and clinical aspects

Tumor markers are biological substances that are produced/released mainly by malignant tumor cells, enter the circulation in detectable amounts and are potential indicators of the presence of a tumor. The most useful biochemical markers are the tumor-specific molecules, i.e., receptors, enzymes, hormones, growth factors or biological response modifiers that are specifically produced by tumor cells and not, or minimally, by the normal counterpart (Richard et al. Principles and practice of gynecologic oncology. Wolters Kluwer Health, Philadelphia, 2009). Based on their specificity and sensitivity in each malignancy, biomarkers are used for screening, diagnosis, disease monitoring and therapeutic response assessment in clinical management of cancer patients.This chapter is focused on human chorionic gonadotropin (hCG), a hormone with a variety of functions and widely used as a tumor biomarker in selected tumors. Indeed, hCG is expressed by both trophoblastic and non-trophoblastic human malignancies and plays a role in cell transformation, angiogenesis, metastatization, and immune escape, all process central to cancer progression. Of note, hCG testing is crucial for the clinical management of placental trophoblastic malignancies and germ cell tumors of the testis and the ovary. Furthermore, the production of hCG by tumor cells is accompanied by varying degrees of release of the free subunits into the circulation, and this is relevant for the management of cancer patients (Triozzi PL, Stevens VC, Oncol Rep 6(1):7-17, 1999).The name chorionic gonadotropin was conceived: chorion derives from the latin chordate meaning afterbirth, gonadotropin indicates that the hormone is a gonadotropic molecule, acting on the ovaries and promoting steroid production (Cole LA, Int J Endocrinol Metab 9(2):335-352, 2011). The function, the mechanism of action and the interaction between hCG and its receptor continue to be the subject of intensive investigation, even though many issues about hCG have been well documented (Tegoni M et al., J Mol Biol 289(5):1375-1385, 1999)

Dual EGFR and BRAF blockade overcomes resistance to vemurafenib in BRAF mutated thyroid carcinoma cells

Abstract Background BRAF inhibitors are effective anticancer agents in BRAF-mutated melanomas. By contrast, evidences about sensitivity of thyroid carcinomas to BRAF inhibition are conflicting and it has been proposed that BRAF V600E thyroid carcinoma cells are less sensitive to BRAF inhibitors due to activation of parallel signaling pathways. This study evaluated the hypothesis that feedback activation of EGFR signaling counteracts the cytostatic activity of vemurafenib (PLX4032) in BRAF V600E thyroid carcinoma cells. Methods Cell proliferation, cell cycle distribution, induction of apoptosis and EGFR and AKT signaling were evaluated in thyroid carcinoma cell lines bearing the BRAF V600E mutation in response to PLX4032. Results A partial and transient cytostatic response to PLX4032 was observed in thyroid carcinoma cell lines bearing the BRAF V600E mutation, with lack of full inhibition of ERK pathway. Interestingly, the exposure of thyroid carcinoma cells to PLX4032 resulted in a rapid feedback activation of EGFR signaling with parallel activation of AKT phosphorylation. Consistently, the dual inhibition of EGFR and BRAF, through combination therapy with PLX4032 and gefitinib, resulted in prevention of EGFR phosphorylation and sustained inhibition of ERK and AKT signaling and cell proliferation. Of note, the combined treatment with gefitinib and vemurafenib or the exposure of EGFR-silenced thyroid carcinoma cells to vemurafenib induced synthetic lethality compared to single agents. Conclusions These data suggest that the dual EGFR and BRAF blockade represents a strategy to by-pass resistance to BRAF inhibitors in thyroid carcinoma cells

Helicobacter pylori acidic stress response factor HP1286 is a YceI homolog with new binding specificity

HP1286 from Helicobacter pylori is among the proteins that play a relevant role in bacterial colonization and persistence in the stomach. Indeed, it was demonstrated to be overexpressed under acidic stress conditions, together with other essential virulence factors. Here we describe its crystal structure, determined at 2.1 A \u2da resolution. The molecular model, a dimer characterized by two-fold symmetry, shows that HP1286 structurally belongs to the YceIlike protein family, which in turn is characterized by the lipocalin fold. The latter characterizes proteins possessing an internal cavity with the function of binding and \u2044 or transport of amphiphilic molecules. Surprisingly, a molecule of erucamide was found bound in the internal cavity of each monomer of recombinant HP1286, cloned and expressed in an Escherichia coli heterologous system. The shape and length of the cavity indicate that, at variance with other members of the family, HP-YceI has a binding specificity for amphiphilic compounds with a linear chain of about 22 carbon atoms. These features, along with the fact that the protein is secreted by the bacterium and is involved in adaptation to an acidic environment, suggest that its function could be that of sequestering specific fatty acids or amides from the environment, either to supply the bacterium with the fatty acids necessary for its metabolism, or to protect and detoxify it from the detergent-like antimicrobial activity of fatty acids that are eventually present in the external milieu

Heat shock proteins in cancer stem cell maintenance: a potential therapeutic target?

Cancer stem cells (CSCs) are a subpopulation of tumor cells with unlimited self-renewal capability, multilineage differentiation potential and long-term tumor repopulation capacity. CSCs reside in anatomically distinct regions within the tumor microenvironment, called niches, and this favors the maintenance of CSC properties and preserves their phenotypic plasticity. Indeed, CSCs are characterized by a flexible state based on their capacity to interconvert between a differentiated and a stem-like phenotype, and this depends on the activation of adaptive mechanisms in response to different environmental conditions. Heat Shock Proteins (HSPs) are molecular chaperones, upregulated upon cell exposure to several stress conditions and are responsible for normal maturation, localization and activity of intra and extracellular proteins. Noteworthy, HSPs play a central role in several cellular processes involved in tumor initiation and progression (i.e. cell viability, resistance to apoptosis, stress conditions and drug therapy, EMT, bioenergetics, invasiveness, metastasis formation) and, thus, are widely considered potential molecular targets. Furthermore, much evidence suggests a key regulatory function for HSPs in CSC maintenance and their upregulation has been proposed as a mechanism used by CSCs to adapt to unfavorable environmental conditions, such as nutrient deprivation, hypoxia, inflammation. This review discusses the relevance of HSPs in CSC biology, highlighting their role as novel potential molecular targets to develop anticancer strategies aimed at CSC targeting

IL6/STAT3 axis mediates resistance to BRAF inhibitors in thyroid carcinoma cells

Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 26-53% of human thyroid malignancies and, differently from melanomas, are poorly sensitive to BRAF inhibitors (BRAFi), and develop acquired resistance through activation of alternative signaling pathways. A whole-genome gene expression analysis of TC BRAF V600E cells exposed to PLX4032 identified JAK/STAT among the most significantly modulated signaling pathways. Interestingly, both transient exposure and chronic adaptation to PLX4032 resulted in upregulation of IL6/STAT3 axis and this impaired the cytostatic activity of PLX4032. Mechanistically, exposure to PLX4032 enhanced IL6 secretion and this, in turn, was responsible for STAT3 upregulation, activation of ERK signaling and poor sensitivity to BRAF inhibition. Consistently, the dual blockade of STAT3 (by siRNA or pharmacological inhibition) or IL6 signaling (by the humanized anti-human IL6 receptor antibody, tocilizumab) and BRAF (by PLX4032) improved the inhibition of cell cycle progression compared to PLX4032 single agent. These data support the role of IL6/STAT3 signaling pathway in modulating TC cell response to PLX4032 and candidate IL6 targeting as a strategy to improve the activity of PLX4032 in BRAF V600E TC cells