New insights into metabolic alterations and mitochondria re-arrangements in pancreatic adenocarcinoma

Among the most aggressive cancer types, pancreatic ductal adenocarcinoma (PDAC) represents one with the highest lethality due to its resistance to therapies and to the frequent metastatic spread [...]

Meccanismi molecolari dell'attivit\ue0 antitumorale associata alla modulazione degli ioni zinco in cellule di adenocarcinoma pancreatico

Lo zinco \ue8 il secondo metallo maggiormente abbondante nel corpo umano. Questo ione \ue8 essenziale in un\u2019ampia variet\ue0 di processi cellulari, in quanto ha un ruolo sia funzionale agendo da cofattore per pi\uf9 di 300 enzimi, sia strutturale per la stabilizzazione della struttura terziaria di molte proteine. L\u2019omeostasi dello zinco \ue8 un processo cellulare ben regolato dall\u2019espressione di trasportatori di membrana e di proteine sequestranti zinco. A livello intracellulare esiste un \u201cpool\u201d labile di zinco, molto dinamico soggetto al flusso ionico ed influenzato dalla deprivazione e dalla \u201csupplementazione\u201d di questo ione; questo \u201cpool\u201d labile sembra essere importante per la citoprotezione e la regolazione dell\u2019apoptosi. Le specie reattive dell\u2019ossigeno (ROS), prodotte costantemente da una variet\ue0 di processi cellulari, causano, a bassi livelli, un aumento della progressione del ciclo cellulare, mentre a livelli pi\uf9 elevati, un arresto del ciclo cellulare e successivamente apoptosi o necrosi. L\u2019utilizzo di molecole capaci di indurre stress ossidativo potrebbe essere un\u2019efficace strategia terapeutica contro il cancro, in quanto, nelle cellule tumorali, aventi un livello basale di ROS pi\uf9 elevato, un ulteriore aumento di stress ossidativo determinerebbe lo scatenarsi di fenomeni di morte cellulare che non avverrebbero nelle cellule normali. In questa tesi sono esposti gli effetti ottenuti in linee cellulari di adenocarcinoma pancreatico umano trattate con due molecole: la pirrolidina ditiocarbammato (PDTC) [1] e l\u2019N,N,N\u2019,N\u2019- tetrakis(2-piridilmetil)etilenediammina (TPEN) [2]. Il PDTC, grazie alla sua attivit\ue0 ionofora, \ue8 in grado di aumentare la quantit\ue0 di zinco intracellulare, a bassi livelli se utilizzato da solo o a livelli pi\uf9 elevati se utilizzato in associazione con zinco esogeno. L\u2019aumento di zinco intracellulare \ue8 direttamente proporzionale all\u2019aumento di ROS nella cellula e causa fenomeni diversi a seconda dell\u2019entit\ue0 dell\u2019incremento. Il PDTC causa l\u2019attivazione di ERK1/2 e l\u2019induzione di P21, con conseguente blocco della progressione del ciclo cellulare in fase S. Il trattamento associato PDTC e zinco determina il danneggiamento della membrana mitocondriale con il rilascio di AIF, che trasloca nel nucleo e causa una forte apoptosi caspasi indipendente. Il TPEN, grazie alla sua attivit\ue0 chelate, \ue8 in grado di diminuire i livelli di zinco intracellulare, determinando lo scatenarsi di fenomeni indipendenti dallo stress ossidativo. La sua forte citotossicit\ue0 \ue8 dovuta sia al blocco della progressione del ciclo cellulare in fase G1 sia a morte apoptotica. Il TPEN \ue8 in grado di regolare l\u2019espressione di vari geni, in particolare, per quanto riguarda il ciclo cellulare, aumenta il rapporto tra i livelli di espressione dei geni inibitori dei complessi ciclina-cdk e i geni delle cicline, mentre per l\u2019apoptosi, incrementa il rapporto tra i livelli di espressione dei geni mitocondriali apoptotici e quelli anti-apoptotici. Inoltre, il TPEN determina danno mitocondriale e attivazione delle caspasi. Sia il PDTC che il TPEN sono risultati essere pi\uf9 efficienti nell\u2019inibizione della vitalit\ue0 cellulare delle linee di adenocarcinoma pancreatico rispetto al chemioterapico standard, la gemcitabina, e inoltre risultano essere poco citotossiche su cellule normali di fibroblasti primari.not availabl

Divergent roles of mitochondria dynamics in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors; it is often diagnosed at an advanced stage and is hardly treatable. These issues are strictly linked to the absence of early diagnostic markers and the low efficacy of treatment approaches. Recently, the study of the metabolic alterations in cancer cells has opened the way to important findings that can be exploited to generate new potential therapies. Within this scenario, mitochondria represent important organelles within which many essential functions are necessary for cell survival, including some key reactions involved in energy metabolism. These organelles remodel their shape by dividing or fusing themselves in response to cellular needs or stimuli. Interestingly, many authors have shown that mitochondrial dynamic equilibrium is altered in many different tumor types. However, up to now, it is not clear whether PDAC cells preferentially take advantage of fusion or fission processes since some studies reported a wide range of different results. This review described the role of both mitochondria arrangement processes, i.e., fusion and fission events, in PDAC, showing that a preference for mitochondria fragmentation could sustain tumor needs. In addition, we also highlight the importance of considering the metabolic arrangement and mitochondria assessment of cancer stem cells, which represent the most aggressive tumor cell type that has been shown to have distinctive metabolic features to that of differentiated tumor cells

Mitochondrial Dynamics as Potential Modulators of Hormonal Therapy Effectiveness in Males

Worldwide the incidence of andrological diseases is rising every year and, together with it, also the interest in them is increasing due to their strict association with disorders of the reproductive system, including impairment of male fertility, alterations of male hormones production, and/or sexual function. Prevention and early diagnosis of andrological dysfunctions have long been neglected, with the consequent increase in the incidence and prevalence of diseases otherwise easy to prevent and treat if diagnosed early. In this review, we report the latest evidence of the effect of andrological alterations on fertility potential in both young and adult patients, with a focus on the link between gonadotropins' mechanism of action and mitochondria. Indeed, mitochondria are highly dynamic cellular organelles that undergo rapid morphological adaptations, conditioning a multitude of aspects, including their size, shape, number, transport, cellular distribution, and, consequently, their function. Since the first step of steroidogenesis takes place in these organelles, we consider that mitochondria dynamics might have a possible role in a plethora of signaling cascades, including testosterone production. In addition, we also hypothesize a central role of mitochondria fission boost on the decreased response to the commonly administrated hormonal therapy used to treat urological disease in pediatric and adolescent patients as well as infertile adults

Secretome protein signature of human pancreatic cancer stem-like cells

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-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 72 kDa 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

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro

The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 μg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 μg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs

Promising 3D in vitro models for studying tumour heterogeneity and testing novel therapeutic approaches in pancreatic cancer

In this study we produced 3D organotypic cultures and spheroids to mimic the complex microenvironment of pancreatic cancer and to test alternative therapeutic strategies

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

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

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

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-\u3baB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-\u3baB 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