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Introduction. Sphingosine 1-phosphate (S1P) is involved in many processes such as cell survival, growth, migration, and cancer. S1P is formed by the phosphorylation of sphingosine by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). While the role of SK1 in cancer is well established, the role of SK2 in regulating apoptosis is still a matter of debate, even though emerging evidence has highlighted the importance of SK2 in cancer. Therefore, SKs represent a promising target for cancer therapy. Recently, the importance of S1P in hematological malignancies has been described. Here, we analyze the therapeutic effects of 2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), an SK1/2 inhibitor, and (R)-FTY720 methyl ether (ROMe), a SK2-selective inhibitor, in T-ALL cell lines. Methods. T- ALL cell lines (Molt-4, Jurkat, CEM-R) were treated with SKi and ROMe for 40 h, then MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining. Protein expression was studied by western blot (WB). Results. SKi and ROMe induced a decrease in cell viability, as demonstrated by MTT assays. Annexin-V/PI staining and flow cytometric analysis of cells treated with a SKi concentration equivalent to the IC50 documented the occurrence of apoptotic cell death. This was confirmed by WB analysis for caspase and PARP cleavage in all the cell lines. However, we also observed a SKi-induced autophagy by means of WB and transmission electron microscopy analysis in Jurkat and CEM-R cells. We then analyzed the expression of ER stress/UPR hallmarks. SKi activated the ER stress/UPR pathway and this occurred following apoptosis and correlated with autophagy. The combination of SKi and chloroquine (an autophagy inhibitor) induced a decrease in cell viability, indicating that the UPR/autophagic response is likely to be a protective mechanism. In Molt-4 cells, we detected an increase in SK1 expression after 40 h of treatment with SKi, that could represent an attempt of cells to escape SKi-induced apoptosis, as high SK1 expression is known to enhance cell growth and survival. Notably, SKi synergized with vincristine at concentrations of SKi that were much below its respective IC50, suggesting that vincristine sensitized T-ALL cells to SKi and that combining chemotherapeutic agents with SK inhibitors could be feasible in the treatment of T-ALL. Finally, ROMe treatment induced an autophagic type of cell death. Conclusions. We report herein that SKi and ROMe affect T-ALL cell viability, but they exert their effects through different mechanisms. While ROMe induces an autophagic cell death, SKi induces apoptosis. Moreover, for the first time, we demonstrated that SKi activates an ER stress/UPR pathway in T-ALL cells and this is linked with a protective autophagic response. Thus, our findings indicate that SK1 or SK2 may represent potential targets for treating T-ALL, thereby enabling better management of this cancer.

Inositide-specific phospholipase Cbeta1 (PLCbeta1) signaling in cell proliferation has been investigated thoroughly in the G(1) cell cycle phase. However, little is known about its involvement in G(2)/M progression. We used murine erythroleukemia cells to investigate the role of PLCbeta1 in G(2)/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G(2)/M progression. We report that PLCbeta1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si-RNA mediated silencing showed a role of JNK, PKCalpha, PKCbetaI, and the beta1 isoform of PI-PLC in cell accumulation in G(2)/M [as observed by fluorescence-activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCalpha, or PLCbeta1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCbeta1, and, foremost, the finding that the PLCbeta1-mediated pathway represents a functional nuclear inositide signaling in the G(2)/M transition.

Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene—PNA oligonucleotide (BGA002)—as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC.

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated β-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.

Hydrogels based on short peptide molecules are interesting biomaterials with wide present and prospective use in biotechnologies. A well-known possible drawback of these materials can be their limited mechanical performance. In order to overcome this problem, we prepared Fmoc-Phe3self-assembling peptides by a biocatalytic approach, and we reinforced the hydrogel with graphene oxide nanosheets. The formulation here proposed confers to the hydrogel additional physicochemical properties without hampering peptide self-assembly. We investigated in depth the effect of nanocarbon morphology on hydrogel properties (i.e. morphology, viscoelastic properties, stiffness, resistance to an applied stress). In view of further developments towards possible clinical applications, we have preliminarily tested the biocompatibility of the composites. Our results showed that the innovative hydrogel composite formulation based on FmocPhe3 and GO is a biomaterial with improved mechanical properties that appears suitable for the development of biotechnological applications.

Osteoarthritis is a degenerative disease that strongly correlates with age and promotes the breakdown of joint cartilage and subchondral bone. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stromal cells (MSCs) isolated from adult tissues. It seems that MSCs derived from synovial joint tissues exhibit superior chondrogenic ability, but their unclear distribution and low frequency actually limit their clinical application. To date, the influence of aging on synovial joint derived MSCs&rsquo, biological characteristics and differentiation abilities remains unknown, and a full understanding of the mechanisms involved in cellular aging is lacking. The aim of this study was therefore to investigate the presence of age-related alterations in synovial fluid MSCs and their influence on the potential ability of MSCs to differentiate toward chondrogenic phenotypes. Synovial fluid MSCs, isolated from healthy equine donors from 3 to 40 years old, were cultured in vitro and stimulated towards chondrogenic differentiation for up to 21 days. An equine model was chosen due to the high degree of similarity of the anatomy of the knee joint to the human knee joint and as spontaneous disorders develop that are clinically relevant to similar human disorders. The results showed a reduction in cell proliferation correlated with age and the presence of age-related tetraploid cells. Ultrastructural analysis demonstrated the presence of morphological features correlated with aging such as endoplasmic reticulum stress, autophagy, and mitophagy. Alcian blue assay and real-time PCR data showed a reduction of efficiency in the chondrogenic differentiation of aged synovial fluid MSCs compared to young MSCs. All these data highlighted the influence of aging on MSCs&rsquo, characteristics and ability to differentiate towards chondrogenic differentiation and emphasize the importance of considering age-related alterations of MSCs in clinical applications.

// Sandra Durante 1, * , Silvia Vecchiarelli 2, * , Annalisa Astolfi 1 , Elisa Grassi 2 , Riccardo Casadei 3 , Donatella Santini 4 , Riccardo Panzacchi 4 , Claudio Ricci 3 , Salvatore Serravalle 5 , Giuseppe Tarantino 1 , Mirella Falconi 6 , Gabriella Teti 6 , Valentina Indio 1 , Andrea Pession 5 , Francesco Minni 3 , Guido Biasco 1, 2 , Mariacristina Di Marco 2 1 Giorgio Prodi Cancer Research Centre, University of Bologna, Bologna, Italy 2 Department of Experimental, Diagnostic and Specialty Medicine University of Bologna, Sant’Orsola-Malpighi Hospital, Bologna, Italy 3 Department of Medical and Surgical Sciences, University of Bologna, Sant’Orsola-Malpighi Hospital, Bologna, Italy 4 Pathology Unit, Sant'Orsola-Malpighi Hospital, Bologna, Italy 5 Department of Medical and Surgical Sciences, “Lalla Seragnoli” Hematology-Oncology Unit, University of Bologna, Bologna, Italy 6 DIBINEM—Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy * These authors contributed equally to this work Correspondence to: Annalisa Astolfi, email: annalisa.astolfi@unibo.it Keywords: PDA, IPMN, chromosome 3, NGS, PIK3CA Received: April 11, 2016 Accepted: August 10, 2016 Published: August 22, 2016 ABSTRACT Background: Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic preneoplastic lesion of pancreatic cancer. We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with clinically-oriented bioinformatic interpretation of data to understand the most relevant alterations of precursor lesions at different stages to identify new diagnostic markers. Results: We identified multiple copy number alterations, particularly in lesions with severe dysplasia, with 7 IPMN with low-intermediate dysplasia carrying a nearly normal karyotype and 13 IPMN with complex Karyotype (> 4 alterations), showing high grade dysplasia. A specific gain of chromosome arm 3q was found in IPMN with complex Karyotype (92%). This gain of 3q is particularly interesting for the presence of oncogenes such as PIK3CA, GATA2 and TERC that are part of pathways that deregulate cell growth and promote disease progression. Quantitative PCR and FISH analysis confirmed the data . Further demonstration of the overexpression of the PIK3CA gene supports the identification of this alteration as a possible biomarker in the early identification of patients with IPMN at higher risk for disease progression. Materials and methods: High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMN by Oncoscan FFPE assay. Results were validated by qPCR and FISH analysis. Conclusions: The identification of these markers at an early stage of disease onset could help to identify patients at risk for cancer progression and new candidates for a more specific targeted therapy.

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes thein vitrobehavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

Estimating the post mortem interval (PMI) is still a crucial step in Forensic Pathology. Although several methods are available for assessing the PMI, a precise estimation is still quite unreliable and can be inaccurate. The present study aimed to investigate the immunohistochemical distribution and mRNA expression of hypoxia inducible factor (HIF‐1α) in post mortem gingival tissues to establish a correlation between the presence of HIF‐1α and the time since death, with the final goal of achieving a more accurate PMI estimation. Samples of gingival tissues were obtained from 10 cadavers at different PMIs (1–3 days, 4–5 days and 8–9 days), and were processed for immunohistochemistry and quantitative reverse transcription‐polymerase chain reaction. The results showed a time‐dependent correlation of HIF‐1α protein and its mRNA with different times since death, which suggests that HIF‐1α is a potential marker for PMI estimation. The results showed a high HIF‐1α protein signal that was mainly localized in the stratum basale of the oral mucosa in samples collected at a short PMI (1–3 days). It gradually decreased in samples collected at a medium PMI (4–5 days), but it was not detected in samples collected at a long PMI (8–9 days). These results are in agreement with the mRNA data. These data indicate an interesting potential utility of Forensic Anatomy‐based techniques, such as immunohistochemistry, as important complementary tools to be used in forensic investigations.