Publications by authors named "Marta Palmieri"

47 Publications

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine.

Int J Mol Sci 2020 Dec 22;22(1). Epub 2020 Dec 22.

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, 70126 Bari, Italy.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance.

Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs.

Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions.

Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.
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http://dx.doi.org/10.3390/ijms22010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792955PMC
December 2020

Progressively De-Differentiated Pancreatic Cancer Cells Shift from Glycolysis to Oxidative Metabolism and Gain a Quiescent Stem State.

Cells 2020 06 28;9(7). Epub 2020 Jun 28.

Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, 37134 Verona, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.
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http://dx.doi.org/10.3390/cells9071572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408749PMC
June 2020

Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells.

Arch Biochem Biophys 2020 01 5;679:108219. Epub 2019 Dec 5.

Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biochemistry, University of Verona, Verona, Italy. Electronic address:

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 isoforms can acquire oncogenic properties referred to as gain-of-function (GOF). In this study, we used wild-type (A375) and mutant p53 (MeWo) melanoma cell lines to assess the regulation of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) by mutant p53. The effects of mutant p53 were evaluated by qPCR, immunoblotting, enzyme activity assay, cell proliferation assay, reactive oxygen species (ROS) assay after cellular transfection. We demonstrate that mutant p53 induces MnSOD expression, which is recovered by the ROS scavenger N-acetyl-l-cysteine. This suggests MnSOD induction as a defense mechanism of melanoma cells to counterbalance the pro-oxidant conditions induced by mutant p53. We also demonstrate that mutant p53 induces the expression of Sirtuin3 (SIRT3), a major mitochondrial NAD-dependent deacetylase, stimulating MnSOD deacetylation and enzymatic activity. Indeed, the restoration of SIRT3 reverses MnSOD activity decrease by mutant p53 knock-down. Finally, MnSOD knock-down further enhances mutant p53-mediated ROS increase, counteracting mutp53-dependent cell hyperproliferation. This indicates that SIRT3 and MnSOD act to maintain ROS levels controlled to promote cell proliferation and survival, providing new therapeutic opportunities to be further considered for clinical studies in cancer patients bearing mutant TP53 gene.
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http://dx.doi.org/10.1016/j.abb.2019.108219DOI Listing
January 2020

Regulation of succinate dehydrogenase and role of succinate in cancer.

Semin Cell Dev Biol 2020 02 1;98:4-14. Epub 2019 May 1.

Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biochemistry, University of Verona, Verona, Italy.

Succinate dehydrogenase (SDH) has been classically considered a mitochondrial enzyme with the unique property to participate in both the citric acid cycle and the electron transport chain. However, in recent years, several studies have highlighted the role of the SDH substrate, i.e. succinate, in biological processes other than metabolism, tumorigenesis being the most remarkable. For this reason, SDH has now been defined a tumor suppressor and succinate an oncometabolite. In this review, we discuss recent findings regarding alterations in SDH activity leading to succinate accumulation, which include SDH mutations, regulation of mRNA expression, post-translational modifications and endogenous SDH inhibitors. Further, we report an extensive examination of the role of succinate in cancer development through the induction of epigenetic and metabolic alterations and the effects on epithelial to mesenchymal transition, cell migration and invasion, and angiogenesis. Finally, we have focused on succinate and SDH as diagnostic markers for cancers having altered SDH expression/activity.
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http://dx.doi.org/10.1016/j.semcdb.2019.04.013DOI Listing
February 2020

Regulation of Autophagy by Nuclear GAPDH and Its Aggregates in Cancer and Neurodegenerative Disorders.

Int J Mol Sci 2019 Apr 26;20(9). Epub 2019 Apr 26.

Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biochemistry, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein-protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.
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http://dx.doi.org/10.3390/ijms20092062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539768PMC
April 2019

Oncometabolites in cancer aggressiveness and tumour repopulation.

Biol Rev Camb Philos Soc 2019 08 10;94(4):1530-1546. Epub 2019 Apr 10.

Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, 37134, Verona, Italy.

Tumour repopulation is recognized as a crucial event in tumour relapse where therapy-sensitive dying cancer cells influence the tumour microenvironment to sustain therapy-resistant cancer cell growth. Recent studies highlight the role of the oncometabolites succinate, fumarate, and 2-hydroxyglutarate in the aggressiveness of cancer cells and in the worsening of the patient's clinical outcome. These oncometabolites can be produced and secreted by cancer and/or surrounding cells, modifying the tumour microenvironment and sustaining an invasive neoplastic phenotype. In this review, we report recent findings concerning the role in cancer development of succinate, fumarate, and 2-hydroxyglutarate and the regulation of their related enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase. We propose that oncometabolites are crucially involved in tumour repopulation. The study of the mechanisms underlying the relationship between oncometabolites and tumour repopulation is fundamental for identifying efficient anti-cancer therapeutic strategies and novel serum biomarkers in order to overcome cancer relapse.
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http://dx.doi.org/10.1111/brv.12513DOI Listing
August 2019

Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes.

Biochim Biophys Acta Gen Subj 2019 01 26;1863(1):61-72. Epub 2018 Sep 26.

Department of Drug Science and Technology, University of Torino, Italy. Electronic address:

Background: Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches.

Methods: Hyaluronic acid (HA) decorated liposomes, containing diethyldithiocarbamate‑copper (Cu(DDC)), able to target the specific CSC marker CD44 receptor were prepared by ion gradient technique and fully characterized. Their antiproliferative effect was evaluated on pancreatic CSCs derived from PDAC cell lines or patients. To clarify the mechanism of action of Cu(DDC) liposomes, ROS level neutralization assay in the presence of N-acetyl-L-cysteine was performed.

Results: Liposomes showed high encapsulation efficiency and Cryo-TEM analysis revealed the presence of Cu(DDC) crystals in the aqueous core of liposomes. In vitro test on pancreatic CSCs derived from PDAC cell lines or patients showed high ROS mediated anticancer activity of HA decorated liposomes. The sphere formation capability of CSCs obtained from patients was drastically reduced by liposomal formulations containing Cu(DDC).

Conclusions: The obtained results show that the encapsulation of Cu(DDC) complex in HA decorated liposomes strongly increases its anti-proliferative activity on pancreatic CSCs.

General Significance: This paper describes for the first time the use of HA decorated liposomes containing Cu(DDC) against pancreatic CSCs and opens the way to the development of nanomedicine based CSC-targeted therapeutic approaches.
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http://dx.doi.org/10.1016/j.bbagen.2018.09.018DOI Listing
January 2019

Extracellular matrix composition modulates PDAC parenchymal and stem cell plasticity and behavior through the secretome.

FEBS J 2018 06 27;285(11):2104-2124. Epub 2018 Apr 27.

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Its aggressiveness is driven by an intense fibrotic desmoplastic reaction in which the increasingly collagen I-rich extracellular matrix (ECM) and several cell types, including cancer stem cells (CSCs), create a tumor-supportive environment. However, how ECM composition regulates CSC dynamics and their relationship with the principle parenchymal tumor population to promote early invasive growth is not yet characterized. For this, we utilized a platform of 3D organotypic cultures composed of laminin-rich Matrigel, representative of an early tumor, plus increasing concentrations of collagen I to simulate malignant stroma progression. As ECM collagen I increases, CSCs progress from a rapidly growing, vascular phenotype to a slower growing, avascular phase, while maintaining their endothelial-like gene signatures. This transition is supported autocrinically by the CSCs and paracrinically by the parenchymal cells via their ECM-dependent secretomes. Indeed, when growing on an early tumor ECM, the CSCs are dedicated toward the preparation of a vascular niche by (a) activating their growth program, (b) secreting high levels of proangiogenic factors which stimulate both angiogenesis and vasculogenic mimicry, and (c) overexpressing VEGFR-2, which is activated by VEGF secreted by both the CSC and parenchymal cells. On Matrigel, the more differentiated parenchymal tumor cell population had reduced growth but a high invasive capacity. This concerted high local invasion of parenchymal cells into the CSC-derived vascular network suggests that a symbiotic relationship between the parenchymal cells and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis.
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http://dx.doi.org/10.1111/febs.14471DOI Listing
June 2018

UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism.

Free Radic Biol Med 2017 12 27;113:176-189. Epub 2017 Sep 27.

Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona, Verona, Italy. Electronic address:

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.09.022DOI Listing
December 2017

Secreted molecules inducing epithelial-to-mesenchymal transition in cancer development.

Semin Cell Dev Biol 2018 06 30;78:62-72. Epub 2017 Jun 30.

Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona, Verona, Italy.

The epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. EMT is involved in embryo development, wound healing, tissue regeneration, organ fibrosis and has also been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells. These cells have been shown to acquire a mesenchymal phenotype when localized at the invasive front of primary tumours increasing aggressiveness, invasiveness, metastatic potential and resistance to chemotherapy. There is now increasing evidence demonstrating that a crucial role in the development of this process is played by factors secreted by cells of the tumour microenvironment or by the tumour cells themselves. This review summarises the current knowledge of EMT induction in cancer by paracrine or autocrine mechanisms, by exosomes or free proteins and miRNAs.
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http://dx.doi.org/10.1016/j.semcdb.2017.06.027DOI Listing
June 2018

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways.

J Proteomics 2017 01 13;150:310-322. Epub 2016 Oct 13.

University of Verona, Department of Neuroscience, Biomedicine and Movement, Verona 37134, Italy.

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability.

Biological Significance: To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies.
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http://dx.doi.org/10.1016/j.jprot.2016.10.002DOI Listing
January 2017

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

Mol Oncol 2016 08 12;10(7):1008-29. Epub 2016 Apr 12.

Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona, Verona, Italy. Electronic address:

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.
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http://dx.doi.org/10.1016/j.molonc.2016.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423176PMC
August 2016

Secretome protein signature of human pancreatic cancer stem-like cells.

J Proteomics 2016 Mar 2;136:1-12. Epub 2016 Feb 2.

University of Verona, Department of Biotechnology, Proteomics and Mass Spectrometry Laboratory, Verona 37134, Italy.

Unlabelled: Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified.

Biological Significance: The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered.
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http://dx.doi.org/10.1016/j.jprot.2016.01.017DOI Listing
March 2016

The metabolic landscape of cancer stem cells.

IUBMB Life 2015 Sep 4;67(9):687-93. Epub 2015 Sep 4.

Department of Life and Reproduction Sciences, Section of Biochemistry, University of Verona, Verona, Italy.

Cancer stem cells (CSCs) are a sub-population of quiescent cells endowed with self-renewal properties that can sustain the malignant behavior of the tumor mass giving rise to more differentiated cancer cells. For this reason, the specific killing of CSCs represents one of the most important challenges of the modern molecular oncology. However, their particular resistance to traditional chemotherapy and radiotherapy imposes a thorough understanding of their biological and biochemical features. The metabolic peculiarities of CSCs may be a therapeutic and diagnostic opportunity in cancer research. In this review, we summarize the most significant discoveries on the metabolism of CSCs describing and critically analyzing the studies supporting either glycolysis or mitochondrial oxidative phosphorylation as a primary source of energy for CSCs.
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http://dx.doi.org/10.1002/iub.1426DOI Listing
September 2015

Antioxidant Mechanisms and ROS-Related MicroRNAs in Cancer Stem Cells.

Oxid Med Cell Longev 2015 29;2015:425708. Epub 2015 Apr 29.

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.

Increasing evidence indicates that most of the tumors are sustained by a distinct population of cancer stem cells (CSCs), which are responsible for growth, metastasis, invasion, and recurrence. CSCs are typically characterized by self-renewal, the key biological process allowing continuous tumor proliferation, as well as by differentiation potential, which leads to the formation of the bulk of the tumor mass. CSCs have several advantages over the differentiated cancer cell populations, including the resistance to radio- and chemotherapy, and their gene-expression programs have been shown to correlate with poor clinical outcome, further supporting the relevance of stemness properties in cancer. The observation that CSCs possess enhanced mechanisms of protection from reactive oxygen species (ROS) induced stress and a different metabolism from the differentiated part of the tumor has paved the way to develop drugs targeting CSC specific signaling. In this review, we describe the role of ROS and of ROS-related microRNAs in the establishment and maintenance of self-renewal and differentiation capacities of CSCs.
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http://dx.doi.org/10.1155/2015/425708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429193PMC
April 2016

Mitochondrial uncoupling protein 2 and pancreatic cancer: a new potential target therapy.

World J Gastroenterol 2015 Mar;21(11):3232-8

Massimo Donadelli, Ilaria Dando, Elisa Dalla Pozza, Marta Palmieri, Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, 37134 Verona, Italy.

Overall 5-years survival of pancreatic cancer patients is nearly 5%, making this cancer type one of the most lethal neoplasia. Furthermore, the incidence rate of pancreatic cancer has a growing trend that determines a constant increase in the number of deceases caused by this pathology. The poor prognosis of pancreatic cancer is mainly caused by delayed diagnosis, early metastasis of tumor, and resistance to almost all tested cytotoxic drugs. In this respect, the identification of novel potential targets for new and efficient therapies should be strongly encouraged in order to improve the clinical management of pancreatic cancer. Some studies have shown that the mitochondrial uncoupling protein 2 (UCP2) is over-expressed in pancreatic cancer as compared to adjacent normal tissues. In addition, recent discoveries established a key role of UCP2 in protecting cancer cells from an excessive production of mitochondrial superoxide ions and in the promotion of cancer cell metabolic reprogramming, including aerobic glycolysis stimulation, promotion of cancer progression. These observations together with the demonstration that UCP2 repression can synergize with standard chemotherapy to inhibit pancreatic cancer cell growth provide the molecular rationale to consider UCP2 as a potential therapeutic target for pancreatic cancer. In this editorial, recent advances describing the relationship between cancer development and mitochondrial UCP2 activity are critically provided.
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http://dx.doi.org/10.3748/wjg.v21.i11.3232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363752PMC
March 2015

Pancreatic ductal adenocarcinoma cell lines display a plastic ability to bi‑directionally convert into cancer stem cells.

Int J Oncol 2015 Mar 12;46(3):1099-108. Epub 2014 Dec 12.

Department of Life and Reproduction Sciences, Section of Biochemistry, University of Verona, Verona, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.
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http://dx.doi.org/10.3892/ijo.2014.2796DOI Listing
March 2015

Regulation of miR-23b expression and its dual role on ROS production and tumour development.

Cancer Lett 2014 Jul 19;349(2):107-13. Epub 2014 Apr 19.

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Verona, Italy. Electronic address:

Among the wide family of microRNAs, microRNA 23b (miR-23b) intriguingly assumes opposite roles on regulation of reactive oxygen species (ROS) and on the development of human cancers. In this review we describe novel findings concerning the molecular events involved in miR-23b gene activation or repression and in both ROS regulation and tumour development. In particular, we define the molecular targets of miR-23b that determine its function as either a tumour suppressor or oncomir in different cell types. Finally, we analyze the involvement of miR-23b in cancer cell metabolism, including autophagy, and in biomarker signatures of microRNAs allowing a prognostic and therapeutic evaluation in various human cancers.
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http://dx.doi.org/10.1016/j.canlet.2014.04.012DOI Listing
July 2014

Roles for microRNA 23b in regulating autophagy and development of pancreatic adenocarcinoma.

Gastroenterology 2013 Nov 23;145(5):936-8. Epub 2013 Sep 23.

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Verona, Italy. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2013.09.037DOI Listing
November 2013

UCP2, a mitochondrial protein regulated at multiple levels.

Cell Mol Life Sci 2014 Apr 27;71(7):1171-90. Epub 2013 Jun 27.

Section of Biochemistry, Deparment of Life and Reproduction Sciences, University of Verona, Strada Le Grazie 8, 37134, Verona, Italy,

An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.
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http://dx.doi.org/10.1007/s00018-013-1407-0DOI Listing
April 2014

Hyaluronic acid-coated liposomes for active targeting of gemcitabine.

Eur J Pharm Biopharm 2013 Nov 18;85(3 Pt A):373-80. Epub 2013 Jun 18.

Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Italy. Electronic address:

The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine lipophilic prodrug [4-(N)-lauroyl-gemcitabine, C12GEM]. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors. For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800 Da (12 disaccharidic units) and 12,000 Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation. Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44(+)) and that the uptake increases with increase in the polymer molecular weight. Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes. Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand.
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http://dx.doi.org/10.1016/j.ejpb.2013.06.003DOI Listing
November 2013

Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists.

Electrophoresis 2013 May 11;34(9-10):1359-68. Epub 2013 Apr 11.

Proteomics and Mass Spectrometry Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.

The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up- and down-regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.
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http://dx.doi.org/10.1002/elps.201200402DOI Listing
May 2013

Targeting gemcitabine containing liposomes to CD44 expressing pancreatic adenocarcinoma cells causes an increase in the antitumoral activity.

Biochim Biophys Acta 2013 May 4;1828(5):1396-404. Epub 2013 Feb 4.

Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.

Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.
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http://dx.doi.org/10.1016/j.bbamem.2013.01.020DOI Listing
May 2013

Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis.

Apoptosis 2013 Mar;18(3):337-46

Department of Life and Reproduction Sciences, Section of Biochemistry, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.

TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.
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http://dx.doi.org/10.1007/s10495-012-0790-6DOI Listing
March 2013

UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells.

Biochim Biophys Acta 2013 Mar 2;1833(3):672-9. Epub 2012 Nov 2.

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Verona, Italy.

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.
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http://dx.doi.org/10.1016/j.bbamcr.2012.10.028DOI Listing
March 2013

Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival.

Am J Pathol 2012 Nov 31;181(5):1524-9. Epub 2012 Aug 31.

Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.
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http://dx.doi.org/10.1016/j.ajpath.2012.07.016DOI Listing
November 2012

Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine.

Biochim Biophys Acta 2012 Oct 15;1823(10):1856-63. Epub 2012 Jun 15.

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Verona, Italy.

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.
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http://dx.doi.org/10.1016/j.bbamcr.2012.06.007DOI Listing
October 2012

Gemcitabine response in pancreatic adenocarcinoma cells is synergistically enhanced by dithiocarbamate derivatives.

Free Radic Biol Med 2011 Apr 12;50(8):926-33. Epub 2011 Jan 12.

Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.

Pancreatic adenocarcinoma is a common malignancy that remains refractory to all available therapies, including the gold standard drug gemcitabine (GEM). We investigated the effect of the combination of GEM and each of the ionophore compounds pyrrolidine dithiocarbamate (PDTC) and disulfiram [DSF; 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethylmethanethioamide] on p53(-/-) pancreatic adenocarcinoma cell growth. PDTC or DSF synergistically inhibited cell proliferation when used in combination with GEM by inducing apoptotic cell death. This effect was associated with an increased mitochondrial O(2)(•-) production and was further enhanced by zinc ions. Basal levels of mitochondrial O(2)(•-) or manganese superoxide dismutase (MnSOD) strictly correlated with the IC(50) for GEM or the percentage of synergism. Thus, the most relevant values of the antiproliferative synergism were obtained in GEM-resistant pancreatic adenocarcinoma cell lines. Interestingly, the GEM-sensitive T3M4 cells transfected with MnSOD expression vector showed mitochondrial O(2)(•-) and IC(50) for GEM similar to those of resistant cell lines. In vivo experiments performed on nude mice xenotransplanted with the GEM-resistant PaCa44 cell line showed that only the combined treatment with GEM and DSF/Zn completely inhibited the growth of the tumoral masses. These results and the consideration that DSF is already used in clinics strongly support the GEM and DSF/Zn combination as a new approach to overcoming pancreatic cancer resistance to standard chemotherapy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2011.01.001DOI Listing
April 2011