Publications by authors named "Guido Gambara"

23 Publications

  • Page 1 of 1

Reactive Jumps Preserve Skeletal Muscle Structure, Phenotype, and Myofiber Oxidative Capacity in Bed Rest.

Front Physiol 2019 15;10:1527. Epub 2020 Jan 15.

Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Institute of Integrative Neuroanatomy, Berlin, Germany.

Identification of countermeasures able to prevent disuse-induced muscle wasting is crucial to increase performance of crew members during space flight as well as ameliorate patient's clinical outcome after long immobilization periods. We report on the outcome of short but high-impact reactive jumps (JUMP) as countermeasure during 60 days of 6° head-down tilt (HDT) bed rest on myofiber size, type composition, capillarization, and oxidative capacity in tissue biopsies (pre/post/recovery) from the knee extensor (VL) and deep calf (SOL) muscle of 22 healthy male participants (Reactive jumps in a sledge, RSL-study 2015-2016, DLR:envihab, Cologne). Bed rest induced a slow-to-fast myofiber shift (type I ->II) with an increased prevalence of hybrid fibers in SOL after bed rest without jumps (control, CTRL, = 0.016). In SOL, JUMP countermeasure in bed rest prevented both fast and slow myofiber cross-sectional area (CSA) decrements ( = 0.005) in CTRL group. In VL, bed rest only induced capillary rarefaction, as reflected by the decrease in local capillary-to-fiber ratio (LCFR) for both type II (pre vs. post/R + 10, = 0.028/0.028) and type I myofibers (pre vs. R + 10, = 0.012), which was not seen in the JUMP group. VO (pL × mm × min) calculated from succinate dehydrogenase (SDH)-stained cryosections (OD) showed no significant differences between groups. High-impact jump training in bed rest did not prevent disuse-induced myofiber atrophy in VL, mitigated phenotype transition (type I - >II) in SOL, and attenuated capillary rarefaction in the prime knee extensor VL however with little impact on oxidative capacity changes.
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http://dx.doi.org/10.3389/fphys.2019.01527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974579PMC
January 2020

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model.

Int J Cancer 2020 02 1;146(4):1031-1041. Epub 2019 Aug 1.

Department of Pediatric Hematology and Oncology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.
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http://dx.doi.org/10.1002/ijc.32572DOI Listing
February 2020

From Chemotherapy to Combined Targeted Therapeutics: and Models to Decipher Intra-tumor Heterogeneity.

Front Pharmacol 2018 14;9:77. Epub 2018 Feb 14.

cpo - Cellular Phenomics & Oncology Berlin-Buch GmbH, Berlin, Germany.

Recent advances in next-generation sequencing and other omics technologies capable to map cell fate provide increasing evidence on the crucial role of intra-tumor heterogeneity (ITH) for cancer progression. The different facets of ITH, from genomic to microenvironmental heterogeneity and the hierarchical cellular architecture originating from the cancer stem cell compartment, contribute to the range of tumor phenotypes. Decoding these complex data resulting from the analysis of tumor tissue complexity poses a challenge for developing novel therapeutic strategies that can counteract tumor evolution and cellular plasticity. To achieve this aim, the development of and cancer models that resemble the complexity of ITH is crucial in understanding the interplay of cells and their (micro)environment and, consequently, in testing the efficacy of new targeted treatments and novel strategies of tailoring combinations of treatments to the individual composition of the tumor. This challenging approach may be an important cornerstone in overcoming the development of pharmaco-resistances during multiple lines of treatment. In this paper, we report the latest advances in patient-derived 3D (PD3D) cell cultures and patient-derived tumor xenografts (PDX) as and models that can retain the genetic and phenotypic heterogeneity of the tumor tissue.
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http://dx.doi.org/10.3389/fphar.2018.00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817069PMC
February 2018

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

Malar J 2017 09 12;16(1):366. Epub 2017 Sep 12.

Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs.

Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca levels.

Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca oscillations suggests a potential role of NAADP in regulating Ca signalling of P. falciparum.
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http://dx.doi.org/10.1186/s12936-017-2013-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596470PMC
September 2017

Kinase-driven metabolic signalling as a predictor of response to carboplatin-paclitaxel adjuvant treatment in advanced ovarian cancers.

Br J Cancer 2017 Aug 29;117(4):494-502. Epub 2017 Jun 29.

Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle, Manassas, VA 20110, USA.

Background: The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin-paclitaxel treatment.

Methods: Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1-AMPK and AKT-mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin-paclitaxel-sensitive and -resistant tumours.

Results: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK-AKT-mTOR axis compared to early EOC (P<0.05 for AMPKα T172, AMPKα1 S485, AMPKβ1 S108, AKT S473 and T308, mTOR S2448, p70S6 S371, 4EBP1 S65, GSK-3 α/β S21/9, FOXO1 T24/FOXO3 T32, and FOXO1 S256). Advanced tumours with low relative activation of the metabolic signature and increased FOXO1 T24/FOXO3 T32 phosphorylation (P=0.041) were associated with carboplatin-paclitaxel resistance.

Conclusions: If validated in a larger cohort of patients, the decreased AMPK-AKT-mTOR activation and phosphorylation of FOXO1 T24/FOXO3 T32 may help identify carboplatin-paclitaxel-resistant EOC patients.
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http://dx.doi.org/10.1038/bjc.2017.195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558684PMC
August 2017

Microgravity-Induced Transcriptome Adaptation in Mouse Paraspinal Muscle Highlights Insulin Resistance-Linked Genes.

Front Physiol 2017 5;8:279. Epub 2017 May 5.

Center of Space Medicine Berlin, Charité Universitätsmedizin BerlinBerlin, Germany.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (Mafb), protein kinase C delta (Prkcd), Muscle Atrophy F-box (MAFbx/Atrogin-1/Fbxo32), and Muscle RING Finger 1 (MuRF-1) were further validated by real time qPCR analysis. In conclusion, our study highlighted the regulation of transcripts mainly linked to insulin sensitivity and metabolism in following 30 days of microgravity exposure. The apparent absence of robust signs of back muscle atrophy in space-flown mice, despite the overexpression of Atrogin-1 and MuRF-1, opens new questions on the possible role of microgravity-sensitive genes in the regulation of peripheral insulin resistance following unloading and its consequences on paraspinal skeletal muscle physiology.
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http://dx.doi.org/10.3389/fphys.2017.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418220PMC
May 2017

Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer.

Clin Cancer Res 2017 Aug 26;23(16):4919-4928. Epub 2017 Apr 26.

George Mason University, Manassas, Virginia.

Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis. Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions ( < 0.01, = 0.056, and = 0.053, respectively). However, mutations alone were insufficient in predicting protein activation ( = 0.32 and = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); < 0.01, = 0.02, and = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) < 0.01, mTOR (S2448) < 0.01, 4EBP1 (S65) = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of mutations and activation of the PI3K-AKT-mTOR signaling network. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564311PMC
August 2017

Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown Mice.

PLoS One 2017 11;12(1):e0169314. Epub 2017 Jan 11.

Center for Space Medicine Berlin, Neuromuscular Group, Charité Universitätsmedizin Berlin, Berlin, Germany.

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169314PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226721PMC
August 2017

The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response.

Infect Immun 2016 Nov 17;84(11):3105-3113. Epub 2016 Oct 17.

Department of Public Health and Infectious Diseases, Sapienza University, Rome, Italy.

Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.
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http://dx.doi.org/10.1128/IAI.00438-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067744PMC
November 2016

Vibration mechanosignals superimposed to resistive exercise result in baseline skeletal muscle transcriptome profiles following chronic disuse in bed rest.

Sci Rep 2015 Nov 24;5:17027. Epub 2015 Nov 24.

Center of Space Medicine Berlin, Neuromuscular System, Institute of Anatomy, Charité Universitätsmedizin Berlin, Germany.

Disuse-induced muscle atrophy is a major concern in aging, in neuromuscular diseases, post-traumatic injury and in microgravity life sciences affecting health and fitness also of crew members in spaceflight. By using a laboratory analogue to body unloading we perform for the first time global gene expression profiling joined to specific proteomic analysis to map molecular adaptations in disused (60 days of bed rest) human soleus muscle (CTR) and in response to a resistive exercise (RE) countermeasure protocol without and with superimposed vibration mechanosignals (RVE). Adopting Affymetrix GeneChip technology we identified 235 differently transcribed genes in the CTR group (end- vs. pre-bed rest). RE comprised 206 differentially expressed genes, whereas only 51 changed gene transcripts were found in RVE. Most gene transcription and proteomic changes were linked to various key metabolic pathways (glycolysis, oxidative phosphorylation, tricarboxylic acid (TCA) cycle, lipid metabolism) and to functional contractile structures. Gene expression profiling in bed rest identified a novel set of genes explicitly responsive to vibration mechanosignals in human soleus. This new finding highlights the efficacy of RVE protocol in reducing key signs of disuse maladaptation and atrophy, and to maintain a close-to-normal skeletal muscle quality outcome following chronic disuse in bed rest.
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http://dx.doi.org/10.1038/srep17027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657004PMC
November 2015

Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways.

Biomed Res Int 2015 4;2015:965271. Epub 2015 Jun 4.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy.

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca(2+) signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by "scratch assay," was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca(2+) release specifically regulates distinct Angs-mediated angiogenic steps.
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http://dx.doi.org/10.1155/2015/965271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471310PMC
March 2016

TLR3 engagement induces IRF-3-dependent apoptosis in androgen-sensitive prostate cancer cells and inhibits tumour growth in vivo.

J Cell Mol Med 2015 Feb 2;19(2):327-39. Epub 2014 Dec 2.

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.
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http://dx.doi.org/10.1111/jcmm.12379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407608PMC
February 2015

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.

Proc Natl Acad Sci U S A 2014 Nov 20;111(44):E4706-15. Epub 2014 Oct 20.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.
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http://dx.doi.org/10.1073/pnas.1406029111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226099PMC
November 2014

Functional characterization of epithelial ovarian cancer histotypes by drug target based protein signaling activation mapping: implications for personalized cancer therapy.

Proteomics 2015 Jan;15(2-3):365-73

Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

Epithelial ovarian carcinoma (EOC) is a deadly disease, with a 5-year survival of 30%. The aim of the study was to perform broad-scale protein signaling activation mapping to evaluate if EOC can be redefined based on activated protein signaling network architecture rather than histology. Tumor cells were isolated using laser capture microdissection (LCM) from 72 EOCs. Tumors were classified as serous (n = 38), endometrioid (n = 13), mixed (n = 8), clear cell (CCC; n = 7), and others (n = 6). LCM tumor cells were lysed and subjected to reverse-phase protein microarray to measure the expression/activation level of 117 protein drug targets. Unsupervised hierarchical clustering analysis was utilized to explore the overall signaling network. ANOVA was used to detect significant differences among the groups (p < 0.05). Regardless of histology, unsupervised analysis revealed five pathway-driven clusters. When the EOC histotypes were compared by ANOVA, only CCC showed a distinct signaling network, with activation of EGFR, Syk, HER2/ErbB2, and SHP2 (p = 0.0007, p = 0.0021, p < 0.0001, and p = 0.0410, respectively). The histological classification of EOC fails to adequately describe the underpinning protein signaling network. Nevertheless, CCC presents unique signaling characteristics compared to the other histotypes. EOC may need to be characterized by functional signaling activation mapping rather than pure histology.
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http://dx.doi.org/10.1002/pmic.201400214DOI Listing
January 2015

Toll-like receptors in prostate infection and cancer between bench and bedside.

J Cell Mol Med 2013 Jun 4;17(6):713-22. Epub 2013 Apr 4.

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

Toll-Like receptors (TLRs) are a family of evolutionary conserved transmembrane proteins that recognize highly conserved molecules in pathogens. TLR-expressing cells represent the first line of defence sensing pathogen invasion, triggering innate immune responses and subsequently priming antigen-specific adaptive immunity. In vitro and in vivo studies on experimental cancer models have shown both anti- and pro-tumoural activity of different TLRs in prostate cancer, indicating these receptors as potential targets for cancer therapy. In this review, we highlight the intriguing duplicity of TLR stimulation by pathogens: their protective role in cases of acute infections, and conversely their negative role in favouring hyperplasia and/or cancer onset, in cases of chronic infections. This review focuses on the role of TLRs in the pathophysiology of prostate infection and cancer by exploring the biological bases of the strict relation between TLRs and prostate cancer. In particular, we highlight the debated question of how reliable mutations or deregulated expression of TLRs are as novel diagnostic or prognostic tools for prostate cancer. So far, the anticancer activity of numerous TLR ligands has been evaluated in clinical trials only in organs other than the prostate. Here we review recent clinical trials based on the most promising TLR agonists in oncology, envisaging a potential application also in prostate cancer therapy.
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http://dx.doi.org/10.1111/jcmm.12055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823175PMC
June 2013

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells.

Blood 2011 May 1;117(18):4968-77. Epub 2011 Mar 1.

Department of Anatomy, Sapienza University of Rome, Rome, Italy.

A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.
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http://dx.doi.org/10.1182/blood-2010-02-266338DOI Listing
May 2011

A novel biomarker harvesting nanotechnology identifies Bak as a candidate melanoma biomarker in serum.

Exp Dermatol 2011 Jan;20(1):29-34

Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA.

Background: Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. This manuscript applies several new measurement technologies with the purpose of elucidating molecular signatures of melanoma aggressiveness.

Purpose: We sought to determine whether low-abundant serum proteins related to apoptotic pathways could be measured and correlated with defined melanoma subtypes. Hydrogel core shell nanoparticles, a new technology capable of selectively entrapping low molecular weight proteins and protecting them from enzymatic degradation, were used to capture candidate serum biomarkers. Biomarker levels were correlated with confocal microscopy, thereby representing a combination of new technologies for in vivo histologic documentation.

Results: Among a panel of analyzed serum proteins, Bak was differentially expressed between nevi and melanomas. Melanomas with higher Bak serum levels exhibited more pronounced junctional activity on confocal imaging, whereas lesions with 'sparse' dermal nests had weak Bak expression.

Conclusions: Our study links serum proteome analysis with confocal microscopic clinical in vivo histologic classification of melanomas. Bak has not been previously measured in serum. Bak differential expression among melanoma subtypes confirms the importance of the apoptotic pathway as a contributor to melanoma aggressiveness.
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http://dx.doi.org/10.1111/j.1600-0625.2010.01187.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777620PMC
January 2011

Tumorigenic and metastatic activity of human thyroid cancer stem cells.

Cancer Res 2010 Nov 19;70(21):8874-85. Epub 2010 Oct 19.

Department of Surgical and Oncological Sciences, Cellular and Molecular Pathophysiology Laboratory, University of Palermo, Palermo, Italy.

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres, which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor, including the aggressive metastatic features of undifferentiated thyroid carcinomas, which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-1994DOI Listing
November 2010

Investigation of the ovarian and prostate cancer peptidome for candidate early detection markers using a novel nanoparticle biomarker capture technology.

AAPS J 2010 Dec 12;12(4):504-18. Epub 2010 Jun 12.

Department of Urology, S. Giovanni Bosco Hospital, Turin, Italy.

Current efforts to identify protein biomarkers of disease use mainly mass spectrometry (MS) to analyze tissue and blood specimens. The low-molecular-weight "peptidome" is an attractive information archive because of the facile nature by which the low-molecular-weight information freely crosses the endothelial cell barrier of the vasculature, which provides opportunity to measure disease microenvironment-associated protein analytes secreted or shed into the extracellular interstitium and from there into the circulation. However, identifying useful protein biomarkers (peptidomic or not) which could be useful to detect early detection/monitoring of disease, toxicity, doping, or drug abuse has been severely hampered because even the most sophisticated, high-resolution MS technologies have lower sensitivities than those of the immunoassays technologies now routinely used in clinical practice. Identification of novel low abundance biomarkers that are indicative of early-stage events that likely exist in the sub-nanogram per milliliter concentration range of known markers, such as prostate-specific antigen, cannot be readily detected by current MS technologies. We have developed a new nanoparticle technology that can, in one step, capture, concentrate, and separate the peptidome from high-abundance blood proteins. Herein, we describe an initial pilot study whereby the peptidome content of ovarian and prostate cancer patients is investigated with this method. Differentially abundant candidate peptidome biomarkers that appear to be specific for early-stage ovarian and prostate cancer have been identified and reveal the potential utility for this new methodology.
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http://dx.doi.org/10.1208/s12248-010-9211-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977006PMC
December 2010

Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH.

Drug Test Anal 2009 Sep;1(9-10):447-54

Department of Urology, S. Giovanni Bosco Hospital, Turin, Italy.

Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy.
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http://dx.doi.org/10.1002/dta.96DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869624PMC
September 2009

NAADP-induced Ca(2+ signaling in response to endothelin is via the receptor subtype B and requires the integrity of lipid rafts/caveolae.

J Cell Physiol 2008 Aug;216(2):396-404

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, University of Rome La Sapienza, Rome, Italy.

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.
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http://dx.doi.org/10.1002/jcp.21407DOI Listing
August 2008

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins.

J Mol Biol 2007 Aug 14;371(5):1174-87. Epub 2007 Jun 14.

Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.
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http://dx.doi.org/10.1016/j.jmb.2007.06.019DOI Listing
August 2007

Endothelin induces functional hypertrophy of peritubular smooth muscle cells.

J Cell Physiol 2007 Jul;212(1):264-73

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, "La Sapienza" University of Rome, Rome, Italy.

When chronically stimulated with agonists of contraction, smooth muscle cells (SMCs) undergo cell hypertrophy, a process defined as increase in size and potentiation of the contractile phenotype in the absence of proliferation. Hypertrophic response has long been associated to a number of pathologies of the cardiovascular and respiratory systems. We have investigated the phenotypic and functional response of SMCs to long-term treatment with endothelin. Our model was primary cultures of peritubular smooth muscle cells (PSMC) a testicular cell type target of locally produced endothelin and characterized by an unusual phenotypic stability when cultured in simple medium in complete absence of serum. We report the following responses of PSMC to 4-day exposure to ET-1: (i) increased protein synthesis without induction of cell proliferation; (ii) increase in cell size (evaluated by means of flow cytometry) and increased expression of SM-alpha-actin, desmin, caldesmon and calponin, markers of the contractile phenotype. In experiments of selective stimulation of either ETA or ETB receptor subtypes, both proved to be involved in inducing the observed hypertrophic responses. The hypertrophic cells exhibit the ultrastructural features of differentiated SMCs and are capable of calcium mediated contractile response when acutely stimulated with ET-1 specifically through ETA and/or ETB receptors, as evaluated by calcium imaging and scanning electron microscopy. These observations demonstrate that engagement of ET receptors is capable of inducing potentiation of the contractile phenotype and functional hypertrophy of PSMC.
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http://dx.doi.org/10.1002/jcp.21028DOI Listing
July 2007