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Con il termine cancro (o tumori) si indica un insieme di patologie nelle quali le cellule hanno perso i normali meccanismi di controllo della crescita. Queste cellule, che proliferano in modo incontrollato, generano masse che alterano la struttura e il funzionamento del tessuto o dell’organo in cui si trovano, invadono i tessuti circostanti e, entrando nel flusso sanguigno e linfatico, si diffondono generando nuovi tumori. E’ ormai assodato che il cancro è il risultato di alterazioni genetiche ed epigenetiche che portano alla modifica dell’espressione e della funzione di geni che regolano la crescita e la sopravvivenza cellulare e che possono esser trasmesse per via ereditaria. L’esposizione ad agenti di diversa natura come contaminanti ambientali, quali policloro bifenili o metalli pesanti; cancerogeni chimici che alterano il DNA; il fumo di sigaretta; la luce ultravioletta; la presenza di agenti virali e gli effetti a lungo termine di uno stile alimentare non ben bilanciato; possono indurre lo sviluppo di alterazioni del profilo genico e favorire, pertanto, l’insorgenza della patologia cancerosa. Diverse evidenze sperimentali indicano che un tumore può impiegare anche decenni per svilupparsi e raggiungere gli stadi più aggressivi. La progressione tumorale, dunque, è un processo graduale che evolve grazie all’acquisizione successiva di mutazioni geniche che conferiscono alle cellule la capacità di sopravvivere nel microambiente tumorale e di progredire verso stadi di maggiore malignità. La progressione sembra seguire le stesse leggi della evoluzione delle specie Darwiniana (Figura 1). In una popolazione tumorale le cellule possono acquisire mutazioni differenti che determinano la presenza di sotto-popolazioni di cellule con un patrimonio genetico leggermente diverso tra loro e dalle cellule normali da cui si sono originate. Certi genotipi mutanti possono conferire un vantaggio di crescita selettivo ad alcuni subcloni cellulari favorendo la loro proliferazione e l’eventuale dominanza nel microambiente. Ad esempio, alcune mutazioni possono conferire alle cellule la capacità di dividersi più velocemente rispetto alle cellule normali oppure di sopravvivere anche in condizioni critiche, come la scarsità di nutrienti. La progressione tumorale è pertanto il risultato di un processo adattativo, dove è avvantaggiato il clone di cellule che si adegua meglio alle condizioni ambientali. Fintanto che le cellule tumorali rimangono raggruppate in un’unica massa nella loro posizione originale, i tumori sono considerati benigni; quando, invece, le cellule acquisiscono la capacità di invadere il tessuto circostante acquisiscono carattere maligno. L’invasività implica di solito la possibilità di evadere, di entrare nel flusso sanguigno o nei vasi linfatici e di formare tumori secondari o metastasi in altre parti del corpo distanti rispetto alla sede di origine. Il cancro si classifica sulla base del tipo di tessuto o di cellula da cui deriva. Col termine di carcinomi si indicano quei tumori che derivano da cellule epiteliali che coprono la superficie della nostra pelle e gli organi interni. Essi sono le forme tumorali più frequenti. I sarcomi, invece, derivano da cellule del tessuto muscolare o connettivo, mentre leucemie e linfomi derivano da cellule ematopoietiche o del sistema immunitario. Col termine di gliomi, neuroblastomi e retinoblastomi, infine, si intendono tumori derivati da cellule del sistema nervoso.

Sono stati sintetizzati composti a struttura policiclica complessa. Tutti i composti sintetizzati sono stati saggiati dall'NCI (USA) su un panel di 60 linee cellulari tumorali umane. Sula base dei risultati ottenuti è stato possibile effettuare uno studio SAR che ha evidenziato come la presenza di gruppi idrofili è necessaria ai fini dell'attività antitumorale. E' stato inoltre studiato il meccanismo d'azione di queste molecole. E' stato notato un arresto delle cellule MDA-MB231 in fase G0/G1 correlato con la defosforilazione ed attivazione, indotta dai composti, della proteina pRb. Il meccanismo comunque varia arrestando il ciclo cellulare in fase G2/M quando per effetto della glicosilazione del derivato.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

Introduction: Breast cancers (BC) are the major cause of death in women. More than 70% of BCs express high levels of estrogen receptor-α (ERα) and are sustained for their growth by the hormone. Estrogens seem to protect BC cells from apoptosis mediated by immunosurveillance associated with cytotoxic T lymphocytes and NK cells granzyme B release. However, the production of granzyme B inhibitor PI-9 by tumor cells causes a short-circuit in immunosurveillance’s signalling. Although it has been shown the role of PI-9 in BC cells, its presence has not been investigated in tumor stem cells so far. Methods: Cell viability was evaluated by MTT, cell cycle by propidium iodide staining; mRNA and protein levels by qPCR and western blotting. Tumorspheres from ERα+BC MCF7 cells were isolated in ultra-low attachment conditions. The higher expression of stemness markers (Nanog, Oct3/4 and Sox2) was found in tertiary tumorspheres which were used in our study. Results: Low doses (10 nM-10 μM) of 17-β estradiol consistently increased the number of MCF7 cells more than tumorspheres, while higher doses (50-100 μM) reduced cell number as a consequence of G2/M cell cycle arrest. The analysis of ERα disclosed the presence of three different isoforms (66, 46 and 36 kDa) in MCF7 cells. In contrast, tumorspheres exhibited an increase in ERα36, which lacks transcriptional activity, while the level of ERα66 was undetectable. Then, we analyzed the level of PI-9, which is transcriptionally regulated by ERα66. Surprisingly, we found that tertiary tumorspheres, express higher levels of both PI-9 protein and mRNA than MCF7 cells. Conclusions: Our data provided evidence that the high level of PI-9 in ER+ tertiary tumorspheres could supply a selective advantage to BC stem cells by interfering with immune-surveillance systems. Ongoing studies aim to elucidate the relationship between the levels of different ERα isoforms and PI-9 high expression in BC-stem cells.

A new polycylic compound was synthesized and evaluated for its antiproliferative activity against the NCI panel of human tumoral cell lines showing IC50 values in the range 0.47-5.43 µM. The compound induced a decrease of the level of cyclin B1, whereas increased the level of the inhibitor p21 WAF1

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in traditional medicine for its anti-inflammatory activity. In addition, parthenolide has been considered as a novel and effective anti-tumor agent because it induces cytotoxic effects in several tumor cell lines. Our studies demonstrated that parthenolide exerted strong cytotoxic effects in osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 33342 revealed in most cells after brief periods of treatments (3-5h) chromatin condensation and fragmentation, while only few cells were PI-positive. Prolonging the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective, thus suggesting a caspase-independent cell death. The study of the mechanism of action provided evidence that treatment with parthenolide rapidly stimulated (1-2 h) ROS generation, in particular by inducing activation of extracellular signal-regulated kinase1/2 and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, JNK activation and cell detachment from the matrix. ROS generation together with mitochondrial accumulation of Ca2+ favoured dissipation of Δψm, which appeared primarily determined by the opening of the permeability transition pore (PTP), since Δψm loss was partially prevented by cyclosporin A, an inhibitor of PTP opening. Recently, we focused our attention on MDA-MB231 cells, a very aggressive and poorly differentiated breast cancer cell line, which is negative for estrogen receptor alpha. Preliminary results suggested that parthenolide induced cell death in these cells with a mechanism similar to that demonstrated in osteosarcoma and melanoma cells. Interestingly, we demonstrated that in MDA-MB231 cells the effect of parthenolide was potentiated by the addition of z-VAD-fmk, a general inhibitor of caspases. Studies are in progress to elucidate the mechanism of this interaction which could suggest new strategies for the treatment of ER-α negative breast cancer.

Parthenolide, the major bioactive sesquiterpene lactone present in Feverfew (Tanacetum parthenium), has recently attracted considerable attention because of its complex pharmacological action involving anti-microbial, anti-inflammatory and anti-cancer effects. However, the mechanism of its cytotoxic effect on tumor cells still remains scarcely defined today. The aim of this study was to analyse the mechanism of parthenolide action on two lines of cancer cells, the human osteosarcoma MG63 and the melanoma SK-MEL-28 cells, on which parthenolide exerted its action inducing similar effects. Staining with Hoechst 33342 showed that parthenolide induced in the first phase of treatment (0-5 h) in most of cells of both the lines condensation of chromatin while only few cells were PI-positive. Moreover, cells assumed a rounded shape, detached from substrate and showed a reduction of their volume. In the second phase of treatment (5-15 h) a progressive increase in the percentages of PI-positive cells was observed, suggesting that extensive damage of cellular plasma membranes occurred only after long periods of treatment. All these events were not counteracted by z-VAD-fmk and other caspase inhibitors, but were dependent on oxidative stress. In fact the study of mechanism of action of partenolide revealed that all cytotoxic effects were prevented by NAC and after a short period of time (1-2 h) ROS production occurred by inducing activation of extracellular signal-regulated kinase1/2 (ERK1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-kB inhibition, JNK activation, cell detachment from the matrix and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca2+ also favoured dissipation of ∆ψm, which seemed primarily determined by PTP opening, since ∆ψm loss was partially prevented by the inhibitor cyclosporin A. In addition, immunofluorescence analysis revealed that at this stage AIF translocated from mitochondria to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.

Partenolide, a natural compound used in traditional medicine for its anti-inflammatory activity, has recently shown anti-tumor and apoptotic effects. Our studies demonstrated that HepG2; Hep3B and SK-Hep1 hepatocarcinoma cells, which are resistant to human recombinant TRAIL, are potently sensitized to TRAIL-induced apoptosis by low doses of parthenolide resulting in a marked synergist effect. To clarify the mechanism that accounts for this interaction, we demonstrated that parthenolide/TRAIL combination markedly increased DR4 and DR5. These effects might be correlated with STAT proteins modifications. In fact parthenolide and parthenolide/TRAIL combination decreased STAT3 and STAT5 and their phosphorylated forms. These lowering effects could be responsible for increased expression of death receptors, as suggested by the observation that down-regulation of STAT proteins by siRNA, stimulated the expression of both DR4 and DR5. Although the anti-tumor activity of parthenolide was identified recently, this research area appears promising as it is likely that parthenolide/TRAIL combination may contribute to the rational design of novel targeted therapies.

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Several studies highlighted the beneficial value of natural compounds in the prevention and treatment of obesity. Here, we investigated the anti-obesity effects of extracts of peel and seed of mango (Mangifera indica L.) cultivated in Sicily (Italy) in 3T3-L1 cells. Mango Peel (MPE) and Mango Seed (MSE) extracts at a 100 µg/mL concentration significantly reduced lipid accumulation and triacylglycerol contents during 3T3-L1 adipocyte differentiation without toxicity. HPLC-ESI-MS analysis showed that both the extracts contain some polyphenolic compounds that can account for the observed biological effects. The anti-adipogenic effect of MPE and MSE was the result of down-regulation of the key adipogenic transcription factor PPARγ and its downstream targets FABP4/aP2, GLUT4 and Adipsin, as well SREBP-1c, a transcription factor which promotes lipogenesis. In addition, both MPE and MSE significantly activated AMPK with the consequent inhibition of Acetyl-CoA-carboxylase (ACC) and up-regulated PPARα. The addition of compound C, a specific AMPK inhibitor, reduced the effects of MPE and MSE on AMPK and ACC phosphorylation, suggesting a role of AMPK in mediating MPE and MSE anti-lipogenic effects. Notably, MPE and MSE possess an elevated radical scavenging activity, as demonstrated by DPPH radical scavenging assay, and reduced ROS content produced during adipocyte differentiation. This last effect could be a consequence of the increase in the antioxidant factors Nrf2, MnSOD and HO-1. In conclusion, MPE and MSE possesses both anti-adipogenic and antioxidant potential, thus suggesting that the bio-waste products of mango are promising anti-obesity natural compounds.

A lifelong adherence to a gluten-free (GF) diet is currently the only treatment for Celiac disease (CD), an autoimmune disorder that arises after gluten ingestion in individuals who are genetically predisposed. The gluten intake exerts toxic effects through several pathways involving gut barrier integrity, intestinal microbiota composition and immune system stimulation. However, despite the great benefit of GF diet for CD patients, its use has been debated. Indeed, individuals who adopt this diet regime may be at risk of nutrient deficiencies. Emerging evidence supports a beneficial effect of a GF diet also for other pathological conditions, including gluten-related disorders (GRD) often associated to CD, such as Non celiac gluten sensitivity (NCGS) and Dermatitis Herpetiforme (DH) as well as Irritable bowel syndrome (IBS) and Diabetes. This suggests a pathogenic role of gluten in these conditions. Despite the growing popularity of GF diet among consumers, to date, there are limited evidences supporting its use for the management of non-celiac diseases. Therefore, in this review, we discuss whether the GF diet could really improve the general quality of life of patients with GRD and non-GRD conditions, keeping in mind its sensorial limitations and nutritional inadequacies. In addition, we discuss the current motivations, leading to the use of a GF diet, despite the inferior quality of GF products respect to those containing gluten.

Fatty acid–binding proteins (FABPs) are lipid chaperones assisting in the trafficking of long-chain fatty acids with functions in various cell compartments, including oxidation, signaling, gene-transcription regulation, and storage. The various known FABP isoforms display distinctive tissue distribution, but some are active in more than one tissue. Quantitative and/or qualitative changes of FABPs are associated with pathological conditions. Increased circulating levels of FABPs are biomarkers of disorders such as obesity, insulin resistance, cardiovascular disease, and cancer. Deregulated expression and malfunction of FABPs can result from genetic alterations or posttranslational modifications and can be pathogenic. We have assembled the disorders with abnormal FABPs as chaperonopathies in a distinct nosological entity. This entity is similar but separate from that encompassing the chaperonopathies pertaining to protein chaperones. In this review, we discuss the role of FABPs in the pathogenesis of metabolic syndrome, cancer, and neurological diseases. We highlight the opportunities for improving diagnosis and treatment that open by encompassing all these pathological conditions within of a coherent nosological group, focusing on abnormal lipid chaperones as biomarkers of disease and etiological-pathogenic factors.

Programmed cell death is fundamental for multicellular organisms either in animal or plant kingdom. Classic apoptosis, which represents the best studied form of cell death, is dependent on caspase protease activity in animals. These proteases are not present in plants, where caspase-like activities, including metacaspases, are involved in the execution of plant cell death. Beyond apoptosis, various non-apoptotic forms of cell death also exist, including autophagy, necroptosis, pyroptosis, and ferroptosis. These types of cell death can be activated independently of apoptosis and sometimes occur when apoptosis is inhibited. Non-apoptotic forms of cell death are best characterized in animals, whereas, in plants, the literature is less extensive, but the molecular executioners of plant cell death are becoming clearer. The aim of this review is to describe different types of cell death and the mechanisms involved in animals and plants, highlighting similarities and differences in the two kingdoms. Moreover, implications of cell death pathways in cancer are discussed.

Ethanolic extracts from Mangifera indica L. have been proved to possess anti-tumor properties in many cancer systems. However, although most effects have been demonstrated with fruit pulp extract, the underlying molecular mechanisms of mango peel are still unclear. This study was designed to explore the effects of mango peel extract (MPE) on colon cancer cell lines. MPE affected cell viability and inhibited the colony formation trend of tumor cells, while no effects were observed in human dermal fibroblasts used as a non-cancerous cell line model. These events were a consequence of the induction of apoptosis associated to reactive oxygen species (ROS) production, activation of players of the oxidative response such as JNK and ERK1/2, and the increase in Nrf2 and manganese superoxide dismutase (MnSOD). Significantly, mango peel-activated stress triggered a DNA damage response evidenced by the precocious phosphorylation of histone 2AX (&gamma, H2AX), as well as phosphorylated Ataxia telangiectasia-mutated (ATM) kinase and p53 upregulation. Mango peel extract was also characterized, and HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis unveiled the presence of some phenolic compounds that could be responsible for the anti-cancer effects. Collectively, these findings point out the importance of the genotoxic stress signaling pathway mediated by &gamma, H2AX in targeting colon tumor cells to apoptosis.

Il Partenolide (PN) è un sesquiterpene lattone, estratto dalla pianta medicinale Tanacetum Parthenium, noto per le sue proprietà antiinfiammatorie, antimicrobiche e antitumorali. A tal proposito, recentemente è stato dimostrato che il PN è in grado di esercitare un potente effetto citotossico in cellule di “carcinoma mammario umano triplo negativo” (TNBC) MDA-MB231. Tuttavia quando il composto è impiegato a basse dosi determina effetti citotossici molto limitati, anche dopo lunghi periodi di trattamento. Questi risultati suggeriscono che il PN nelle cellule MDA-MB231 è in grado di indurre effetti citoprotettivi che si evidenziano quando impiegato a basse dosi. Poiché tale caratteristica potrebbe rappresentare un ostacolo al potenziale impiego in terapia del PN il mio studio è stato rivolto alla individuazione di composti in grado di potenziare l’effetto di basse dosi di PN in cellule MDA-MB231. L’attenzione è stata rivolta al SAHA, un inibitore delle HDAC. I risultati ottenuti hanno dimostrato che il pre-trattamento con il SAHA incrementa in modo sinergico l’effetto citotossico indotto da basse dosi di PN. In particolare è interessante osservare che il PN da solo stimola la via di sopravvivenza Akt/mTOR e la traslocazione al nucleo di Nrf2, il SAHA quando impiegato da solo induce produzione di ROS e attivazione del meccanismo autofagico. Quando le cellule sono sottoposte al trattamento con l’associazione dei due composti, il SAHA contrasta l’attivazione del pathway Akt/mTOR indotta dal PN e quest’ultimo riduce la produzione di ROS e il meccanismo autofagico indotto dal SAHA. I due composti inducono, inoltre, modifiche epigenetiche: il SAHA determina iperacetilazione degli istoni H3 e H4 e il PN riduce i livelli della DNMT1. E’ interessante osservare che tali modifiche si mantengono anche quando i due composti agiscono in associazione e potrebbero essere responsabili degli incrementati livelli osservati di prodotti di geni oncosoppressori come p21 e p27 e della caduta della proteina anti-apoptotica Bcl-2 e di p65. A questi eventi si accompagna la deplezione del GSH che determina caduta del Δψm, con conseguente fuoriuscita del citocromo c, attivazione della caspasi 3 e morte per apoptosi degli elementi cellulari.