Triticum vulgare Extract Modulates Protein-Kinase B and Matrix Metalloproteinases 9 Protein Expression in BV-2 Cells: Bioactivity on Inflammatory Pathway Associated with Molecular Mechanism Wound Healing.

Matrix metalloproteinases (MMPs) are a large family of ubiquitously expressed zinc-dependent enzymes with proteolitic activities. They are expressed in physiological situations and pathological conditions involving inflammatory processes including epithelial to mesenchymal transition (EMT), neuronal injury, and cancer. There is also evidence that MMPs regulate inflammation in tumor microenvironment, which plays an important role in healing tissue processes. Looking at both inflammatory and neuronal damages, MMP9 is involved in both processes and their modulation seems to be regulated by two proteins: tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6). However other important genes are involved in molecular regulation of transcription factors, protein-kinase B (AKT), and p65. In addition, Triticum vulgare extract (TVE) modulated the biological markers associated with inflammatory processes, including p65 protein. While there are no evidence that TVE might be involved in the biological modulation of other inflammatory marker as AKT, we would like to assess whether TVE is able to (1) modulate phosphorylation of AKT (pAKT) as an early marker of inflammatory process in vitro and (2) affect MMP9 protein expression in an in vitro model. The BV-2 cells (microglial of mouse) have been used as an in vitro model to simulate both inflammatory and neuronal injury pathologies. Here, MMP9 seems to be involved in cellular migration through inflammatory marker activation. We simulate an inflammatory preclinical model treating BV-2 cells with lipopolysaccharide (LPS) to induce proinflammatory activation affecting pAKT and p65 proteins. TVE is revealed to restore the native expression of AKT and p65. Additionally, TVE extract modulates also the protein concentration of MMP9. Nevertheless, immunofluorescence confocal analyses revealed that both AKT and MMP9 are regulated together, synchronously. This work seems to demonstrate that two important genes can be used to monitor the beginning of an inflammatory process, AKT and MMP9, in which TVE seems able to modulate their expression of inflammation-associated molecules.

Triticum vulgare extract exerts an anti-inflammatory action in two in vitro models of inflammation in microglial cells.

Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing.

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

Synergistic activity of the c-Met and tubulin inhibitor tivantinib (ARQ197) with pemetrexed in mesothelioma cells.

Malignant pleural mesothelioma (MPM) is a lethal disease with scarce therapeutic options, and preclinical studies on new targeted-agents are warranted. Because previous studies reported high c-Met expression and alterations in the microtubules network in most MPM samples, we evaluated the activity of tivantinib, which has been recently suggested to affect microtubule polymerization in addition to inhibiting c-Met. In four MPM cell lines tivantinib inhibited both c-Met activity and microtubule polymerization, resulting in inhibition of cell-growth with IC50s ranging between 0.3 µM (MSTO-211H) and 2.4 µM (H2052). Furthermore tivantinib synergistically enhanced the antiproliferative and proapoptotic activity of pemetrexed, as detected by sulforhodamine-B-assay and flow cytometry. The synergistic interaction was associated with reduction of thymidylate synthase expression and inhibition of migratory activity. In aggregate, these data show the ability of tivantinib to specifically target key pathways in MPM cells and synergistically interact with pemetrexed, supporting further studies on this therapeutic approach.

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.

Loss of heterozygosity status of D9S105 marker is associated with downregulation of Krüppel-like factor 4 expression in pancreatic ductal adenocarcinoma and pancreatic intraepithelial lesions.

The transcription factor Krüppel-like factor 4 (KLF4) may act both as an oncogene and a tumor suppressor in a tissue-dependent manner, and further studies on its role in pancreatic ductal adenocarcinoma (PDAC) progression and clinical outcome are warranted. Therefore, we investigated the loss of heterozygosity (LOH) in the 9q22.3-32 region and loss of KFL4 gene expression in epithelial cells from 35 PDAC, 6 pancreatic intraductal neoplasias (PanINs) and 6 normal ducts, isolated by laser microdissection, as well as their correlation with overall survival (OS) in patients treated with gemcitabine in the adjuvant setting. LOH was evaluated with 4 microsatellite markers and in situ hybridization, while KLF4 expression was studied by reverse transcription-PCR and immunohistochemistry. LOH in at least 1 locus was observed in 25 of 35 PDAC cases and in 5 of 6 PanINs, respectively. In particular, the loss of the D9S105 marker was present in 46.9% of PDAC and 83.3% of PanINs, becoming the most deleted marker, while no LOH in D9S105 was observed in normal Wirsung pancreatic duct. Lack of KLF4 mRNA expression was significantly associated with: (1) genomic deletion flanking KLF4 in PDAC and in PanINs (with LOH of D9S105), (2) low-grade PDAC-associated PanIN, (3) lack of KLF4 protein expression, and (4) shorter OS. These results strongly suggest a relationship between D9S105 deletion and downregulation of KLF4 gene expression as an early event in PDAC progression, as well as a possible role of KLF4 as a prognostic biomarker in gemcitabine-treated patients. and IAP.

Identification of microRNA-21 as a biomarker for chemoresistance and clinical outcome following adjuvant therapy in resectable pancreatic cancer.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS SIGNIFICANCE: Low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased anticancer drug activity in vitro. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC.