Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields

We investigated the interaction between magnetic carbon nanotubes (CNTs) and mesenchymal stem cells (MSCs), and their ability to guide these intravenously injected cells in living rats by using an external magnetic field. MATERIALS & METHODS: Multiwalled CNTs were used to treat MSCs derived from rat bone marrow. Cytotoxicity induced by nanotubes was studied using the WST-1 proliferation and Hoechest 33258 apoptosis assays. The effects of nanotubes on MSCs were evaluated by monitoring the effects on cellular growth rates, immunophenotyping and differentiation, and on the arrangement of cytoskeletal actin. MSCs loaded with nanotubes were injected in vivo in the portal vein of rats driving their localization in the liver by magnetic field. An histological analysis was performed on the liver, lungs and kidneys of all animals. RESULTS: CNTs did not affect cell viability and their ability to differentiate in osteocytes and adipocytes. Both the CNTs and the magnetic field did not alter the cell growth rate, phenotype and cytoskeletal conformation. CNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the biodistribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). CONCLUSION: Multiwalled CNTs hold the potential for use as nanodevices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for the manipulation/guidance of MSCs in regenerative medicine and cell transplantation

The Titanium Surface Modulates the Expression of Beta-Catenin and DLX5 Genes in Pancreatic Ductal Carcinoma in Vitro. Can the Metallic Stent Increase PDAC Aggressiveness?

Context Adenoductal pancreas (PDAC) is a fatal cancer. Its aggressiveness is associated in part with the EMT process of metastasis. Two genes specifically involved in these phenomena are b-catenin and DLX5. While the first gene has been widely studied also in pancreatic cancer, few data are associated with DLX5. However, its over-expression has been recently associated with the formation of metastases in breast cancer in vivo. An exogenous factor involved in the modulation of the expression of these genes seems to be titanium. This compound is usually employed for the pallia action of patients with PDAC, to reduce choledochal stenosis due to compression. Objective The purpose of this study was to assess whether titanium is able to modulate the expression of these two genes in vitro. Methods We used a primary cell culture of PADC (PP78). The cells were seeded and cultivated in contact with two different titanium surfaces for 10 days. After this period the total mRNA was extracted and the quantification of β-catenin and DLX5 genes was performed by RT-PCR according to the ΔΔCt analysis. Then cells were stained using the immunofluorescence technique (IF) to quantify the b-catenin protein expression .using a computerized high-resolution acquisition system (D-Sight, Menarini, Florence, Italy) The cells were scored evaluating the cytoplasmic positivity as follows (0 absent, 1 low, 2 middle, 3 strong). The experiment was carried out in triplicate and untreated cells (without titanium contact) were used as control. Results Quantitative analyses showed that both titanium surfaces positively affected beta-catenin (mean 2.8 fold) and DLX5 (2.0 fold) mRNA expressions with respect to the controls (P<0.0007). Both titanium surfaces also increased the protein score 3 values of β-catenin in treated cells with respect to their controls (P=0.0158). Conclusion Our data showed that several titanium surfaces positively modulated the expression of two genes associated with the increase of the aggressiveness of PDAC in vitro. Clinical studies are needed to find out which type of stent can be used in the surgical operation with palliative intent

Vascular Dysfunction in a Mouse Model of Rett Syndrome and Effects of Curcumin Treatment

Mutations in the coding sequence of the X-linked gene MeCP2 (Methyl CpG–binding protein) are present in around 80% of patients with Rett Syndrome, a common cause of intellectual disability in female and to date without any effective pharmacological treatment. A relevant, and so far unexplored feature of RTT patients, is a marked reduction in peripheral circulation. To investigate the relationship between loss of MeCP2 and this clinical aspect, we used the MeCP2 null mouse model B6.129SF1-MeCP2tm1Jae for functional and pharmacological studies. Functional experiments were performed on isolated resistance mesenteric vessels, mounted on a pressurized myograph. Vessels from female MeCP2(+/−) mice show a reduced endothelium-dependent relaxation, due to a reduced Nitric Oxide (NO) availability secondary to an increased Reactive Oxygen Species (ROS) generation. Such functional aspects are associated with an intravascular increase in superoxide anion production, and a decreased vascular eNOS expression. These alterations are reversed by curcumin administration (5% (w/w) dietary curcumin for 21 days), which restores endothelial NO availability, decreases intravascular ROS production and normalizes vascular eNOS gene expression. In conclusion our findings highlight alterations in the vascular/endothelial system in the absence of a correct function of MeCP2, and uncover related cellular/molecular mechanisms that are rescued by an anti-oxidant treatment

Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary and gemcitabine-resistant pancreatic cancer cells

A new series of eighteen imidazo [2,1-b] [1,3,4]thiadiazole derivatives was efficiently synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably cytotoxic activity with the half maximal inhibitory concentration values (IC50) ranging from 0.23 to 11.4 mM, and 0.29e12.2 mM, respectively. However, two additional compounds, 12b and 13g, displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma (PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and gemcitabine-resistant (Panc-1R), eliciting IC50 values ranging from micromolar to sub-micromolar level, associated with significant reduction of cell-migration and spheroid shrinkage. These remarkable results might be explained by modulation of key regulators of epithelial-to-mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one of the possible mechanisms of action, using a specific ELISA. In conclusion, novel imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and PTK2/FAK

Pancreatoduodenectomy without vascular resection in patients with primary resectable adenocarcinoma and unilateral venous contact:A matched case study

Purpose. To investigate the oncological outcome and survival of patients following a conservative approach on the portal- mesenteric axis, in an intraoperative ultrasound-selected group of pancreatoduodenectomy (PD), performed on patients with primary resectable with vascular contact (prVC) pancreatic ductal adenocarcinoma (PDAC). Methods. A consecutive series of patients who underwent PD for PDAC at our tertiary care center, between 2008 and 2017, were reviewed. A total of 156 PDs and 88 total pancreatectomies were performed during the study period, including 35 vascular resections. We identified a group of 40 (25.6%) patients with prVC-PDAC in whom after checking the feasibility with intraoperative ultrasound, we were able to perform PD by separation of the tumor from the portomesenteric axis avoiding vascular resection, without residual macroscopic disease (no vascular resection, nvrPD), and compared this group, using case-matched methodology, with the standard PD (sPD) group of primary resectable without vascular contact- (prwVC-) PDAC. Results. The median follow-up was 28.5 ± 23.2 months in the sPD group and 23.8 ± 20.8 months in the nvrPD group (p = 0 35). Isolated local recurrence rate was 2/40 (5%) in both groups. Additionally, there were no statistical differences in the systemic progression of the disease (42.5% sPD vs. 45% nvrPD, p = 0 82) or local plus synchronous systemic disease rates (2.5% sPD vs. 7.5% nvrPD, p = 0 30). The median survival was 22 months for the sPD group and 23 months for the nvrPD group, p = 0 86. The overall survival was similar in the two groups (1 y: 76.3% sPD vs. 70.0% nvrPD; 3 y: 35.6% vs. 31.6%; and 5 y: 28.5% vs. 25.3%; p = 0 80). Conclusions. PD without vascular resection can be considered safe and oncologically acceptable in selected patients with preoperative diagnosis of prVC-PDAC. The poor prognosis of PDAC is related to the aggressive biology and systemic spread of the tumor, rather than the local control of the disease

A polymorphism in the promoter is associated with EZH2 expression but not with outcome in advanced pancreatic cancer patients

Aim: EZH2 expression is a prognostic marker in radically resected pancreatic ductal adenocarcinoma (PDAC) patients. Here we investigated its role in locally advanced/metastatic patients, as well as candidate polymorphisms. Materials & methods: EZH2 expression and polymorphisms were evaluated by quantitative reverse transcription PCR in 32 laser microdissected tumors, while polymorphisms were also studied in blood samples from two additional cohorts treated with gemcitabine monotherapy (n = 93) or polychemotherapeutic regimens (n = 247). Results: EZH2 expression correlated with survival and with the rs6958683 polymorphism in the first cohort of patients, but this polymorphism was not associated with survival in our larger cohorts. Conclusion: EZH2 is a prognostic factor for locally advanced/metastatic PDACs, while candidate polymorphisms cannot predict clinical outcome. Other factors involved in EZH2 regulation, such as miR-101, should be investigated in accessible samples in order to improve the clinical management of advanced PDAC

SLC22A3 polymorphisms do not modify pancreatic cancer risk, but may influence overall patient survival

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted

Common germline variants within the CDKN2A/2B region affect risk of pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PNETs) are heterogeneous neoplasms which represent only 2% of all pancreatic neoplasms by incidence, but 10% by prevalence. Genetic risk factors could have an important role in the disease aetiology, however only a small number of case control studies have been performed yet. To further our knowledge, we genotyped 13 SNPs belonging to the pleiotropic CDKN2A/B gene region in 320 PNET cases and 4436 controls, the largest study on the disease so far. We observed a statistically significant association between the homozygotes for the minor allele of the rs2518719 SNP and an increased risk of developing PNET (ORhom = 2.08, 95% CI 1.05-4.11, p = 0.035). This SNP is in linkage disequilibrium with another polymorphic variant associated with increased risk of several cancer types. In silico analysis suggested that the SNP could alter the sequence recognized by the Neuron-Restrictive Silencer Factor (NRSF), whose deregulation has been associated with the development of several tumors. The mechanistic link between the allele and the disease has not been completely clarified yet but the epidemiologic evidences that link the DNA region to increased cancer risk are convincing. In conclusion, our results suggest rs2518719 as a pleiotropic CDKN2A variant associated with the risk of developing PNETs

Identification of MicroRNA-21 as a Biomarker for Chemoresistance and Clinical Outcome Following Adjuvant Therapy in Resectable Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy.Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells.. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC