Bárbara Gomes

De novo expression of CD44 variants in sporadic and hereditary gastric cancer

CD44 is the major ubiquitously expressed cell surface receptor for hyaluronate. The CD44 gene encodes several protein isoforms due to extensive alternative splicing and post-translational modifications. Some of these CD44 variable isoforms have been foreseen as key players in malignant transformation and their expression is highly restricted and highly specific, unlike the canonical CD44 standard isoform. In this study, we aimed at dissecting the mRNA splicing pattern of CD44 in normal stomach and gastric cancer (GC) cell lines (n=9) using cloning and quantitative mRNA amplification assays. Moreover, we assessed the RNA levels and protein expression pattern of relevant splicing forms in distinct premalignant and malignant gastric lesions (sporadic (n=43) and hereditary (n=3) forms) using real-time RT-PCR and immunohistochemistry. We also explored the association of CD44 and E-cadherin expression by immunohistochemistry, as E-cadherin has a pivotal functional role in GC. We established the pattern of CD44 variant forms in normal stomach and gastric malignancy. We observed that although exon v6-containing isoforms were rarely expressed in normal gastric mucosa, they became increasingly expressed both in gastric premalignant (hyperplastic polyps, complete and incomplete intestinal metaplasia, low- and high-grade dysplasia) and malignant lesions (cell lines derived from GCs, primary sporadic GCs and hereditary diffuse GCs (HDGCs)). Moreover, we verified that whenever E-cadherin expression was absent, exon v6-containing CD44 isoforms were overexpressed. The lack of expression of CD44 isoforms containing exon v6 in the surface and foveolar epithelia of normal stomach and, its de novo expression in premalignant, as well as in sporadic and hereditary malignant lesions of the stomach, pinpoint CD44 v6-containg isoforms as potential biomarkers for early transformation of the gastric mucosa. Further, our results raise the hypothesis of using CD44v6 as a marker of early invasive intramucosal carcinoma in HDGC CDH1 mutation carriers that lack CDH1 expression in their tumors.

Targeting nanoparticles across the blood-brain barrier with monoclonal antibodies

Development of therapeutics for brain disorders is one of the more difficult challenges to be overcome by the scientific community due to the inability of most molecules to cross the blood-brain barrier (BBB). Antibody-conjugated nanoparticles are drug carriers that can be used to target encapsulated drugs to the brain endothelial cells and have proven to be very promising. They significantly improve the accumulation of the drug in pathological sites and decrease the undesirable side effect of drugs in healthy tissues. We review the systems that have demonstrated promising results in crossing the BBB through receptor-mediated endocytic mechanisms for the treatment of neurodegenerative disorders such as Alzheimers and Parkinsons disease.

Helicobacter pylori colonization of the adenotonsillar tissue: fact or fiction?

OBJECTIVE The transmission of the gastric pathogen Helicobacter pylori involves the oral route. Molecular techniques have allowed the detection of H. pylori DNA in samples of the oral cavity, although culture of H. pylori from these type of samples has been sporadic. Studies have tried to demonstrate the presence of H. pylori in adenotonsillar tissue, with contradictory results. Our aim was to clarify whether the adenotonsillar tissue may constitute an extra gastric reservoir for H. pylori. METHODS Sixty-two children proposed for adenoidectomy or tonsillectomy were enrolled. A total of 101 surgical specimens, 55 adenoid and 46 tonsils, were obtained. Patients were characterized for the presence of anti-H. pylori antibodies by serology. On each surgical sample rapid urease test, immunohistochemistry, fluorescence in situ hybridization (FISH) with a peptide nucleic acid probe for H. pylori, and polymerase chain reaction-DNA hybridization assay (PCR-DEIA) directed to the vacA gene of H. pylori were performed. RESULTS Thirty-nine percent of the individuals had anti-H. pylori antibodies. Rapid urease test was positive in samples of three patients, all with positive serology. Immunohistochemistry was positive in samples of two patients, all with negative serology. All rapid urease test or immunohistochemistry positive cases were negative by FISH. All samples tested were negative when PCR-DEIA for H. pylori detection was used directly in adenotonsillar specimens. CONCLUSIONS The adenotonsillar tissue does not constitute an extra gastric reservoir for H. pylori infection, at least a permanent one, in this population of children. Moreover, techniques currently used for detecting gastric H. pylori colonization are not adequate to evaluate infection of the adenotonsillar tissues.

Cellular uptake of PLGA nanoparticles targeted with anti-amyloid and anti-transferrin receptor antibodies for Alzheimer’s disease treatment

During the last few decades, relevant efforts have been reported to design nanocarriers for drug transport through the blood brain barrier (BBB). New drugs, such as peptide iAβ5, capable to inhibit the aggregates associated with Alzheimeŕs disease (AD) are being tested but the most frequent drawback is to reach the brain in the desired concentrations due to the low BBB permeability-surface area. Our approach, as a proof of concept to improve drug transport through the BBB, is based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles with surface functionalized with anti-transferrin receptor monoclonal antibody (OX26) and anti-Aβ (DE2B4) to deliver encapsulated iAβ5 into the brain. Porcine brain capillary endothelial cells (PBCECs) were used as a BBB model to evaluate the system efficacy and toxicity. The uptake of immune nanoparticles with a controlled delivery of the peptide iAβ5 was substantially increased compared to the nanoparticles (NPs) without monoclonal antibody functionalization.

E-cadherin impairment increases cell survival through Notch-dependent upregulation of Bcl-2

The role of E-cadherin in tumorigenesis has been attributed to its ability to suppress invasion and metastization. However, E-cadherin impairment may have a wider impact on tumour development. We have previously shown that overexpression of mutant human E-cadherin in Drosophila produces a phenotype characteristic of downregulated Notch. Hence, we hypothesized that Notch signalling may be influenced by E-cadherin and may mediate tumour development associated with E-cadherin deficiency. De novo expression of wild-type E-cadherin in two cellular models led to a significant decrease in the activity of the Notch pathway. In contrast, the ability to inhibit Notch-1 signalling was lost in cells transfected with mutant forms of E-cadherin. Increased Notch-1 activity in E-cadherin-deficient cells correlated with increased expression of Bcl-2, and increased resistance to apoptotic stimuli. After Notch-1 inhibition, E-cadherin-deficient cells were re-sensitized to apoptosis in a similar degree to wild-type E-cadherin cells. We also show that Notch-inhibiting drugs are able to significantly inhibit the growth of E-cadherin-deficient cells xenografted into nude mice. This effect was comparable with the one observed in animals treated with the chemotherapeutic agent taxol, a chemical inducer of cell death. In conclusion, our results show that aberrant Notch-1 activation, Bcl-2 overexpression and increased cell survival are likely to play a crucial role in neoplastic transformation associated with E-cadherin impairment. These findings highlight the possibility of new targeted therapeutical strategies for the treatment of tumours associated with E-cadherin inactivation.

C/EBPα expression is associated with homeostasis of the gastric epithelium and with gastric carcinogenesis

Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family are fundamental for the control of differentiation and proliferation of many adult tissues. C/EBPα has a crucial role in inducing terminal differentiation and is an established tumor suppressor gene in several cancer models. The objective of this study was to analyze the putative role of C/EBPα in gastric carcinoma (GC). We analyzed the expression of C/EBPα in normal and neoplastic gastric tissues, and assessed the role of C/EBPα on proliferation and differentiation of GC cells. In normal gastric mucosa, C/EBPα is expressed in the foveolar epithelium and co-localizes with the gastric differentiation marker trefoil factor 1 (TFF1). The expression of C/EBPα was found to be lost in 30% of GC cases. To evaluate the role of C/EBPα in cell proliferation and differentiation, we transfected GC cells with a full-length C/EBPα protein. We observed a significant decrease in proliferation in C/EBPα-transfected cells. This was accompanied by a decrease in Cyclin D1, an increase in P27 expression, and an increased expression of TFF1. Finally, we showed that inhibition of the Ras/MAPK pathway leads to increased C/EBPα and TFF1 expression, and decreased cell proliferation and cyclin D1 expression in GC cells. Our results suggest that C/EBPα (together with other members of the C/EBP family) has an active role in the control of differentiation and proliferation in normal gastric mucosa. In GC, loss of C/EBPα may be associated with the switch from a cellular differentiation to a cellular proliferation program, presumably as a consequence of Ras/MAPK pathway activation.

Dual ligand immunoliposomes for drug delivery to the brain

Drug delivery systems that can reach brain areas affected by amyloid deposits are still underdeveloped. We propose pegylated liposomes functionalized with two antibodies, the anti-transferrin receptor monoclonal antibody (OX26MAb) and the anti-amyloid beta peptide antibody (19B8MAb), as nanocarriers of drugs for Alzheimers disease therapy. Two distinct conjugation methods are investigated. In one formulation, the OX26MAb is conjugated to the tip of polyethylene glycol molecules through the maleimide group and the 19B8MAb is bound through the streptavidin-biotin complex. In the second system the conjugation reagents are swapped between the antibodies. Fluorescence spectroscopy experiments on porcine brain capillary endothelial cells show that the cellular uptake of the immunoliposomes is substantially more efficient if OX26MAb antibody is conjugated through the streptavidin-biotin complex instead of the maleimide group. The ability of the immunoliposomes to cross the blood brain barrier was established by in vivo studies in wild type rats. Our results demonstrate the importance of the conjugation method used to bind the antibody that targets the blood brain barrier to immunoliposomes for efficient drug delivery to the brain.

PLGA nanoparticles as a platform for vitamin D-based cancer therapy.

Summary Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were studied as drug delivery vehicles for calcitriol, the active form of vitamin D3. In vitro effects of calcitriol encapsulated in PLGA nanoparticles were evaluated with respect to free calcitriol on human pancreatic cell lines, S2-013 and hTERT-HPNE, and the lung cancer cell line A549. Encapsulated calcitriol retained its biological activity, reducing the cell growth. Cytotoxicity assays demonstrated that encapsulation of calcitriol enhanced its inhibitory effect on cell growth at a concentration of 2.4 μM for the S2-013 cells (91%) and for A549 cells (70%) comparared to the free calcitriol results. At this concentration the inhibitory effect on nontumor cells (hTERT-HPNE) decreased to 65%. This study highlights the ability of PLGA nanoparticles to deliver vitamin D3 into cancer cells, with major effects regarding cancer cell cycle arrest and major changes in the cell morphological features.

Pyranoflavylium derivatives extracted from wine grape as photosensitizers in solar cells

Pyranoflavylium derivatives, namely cyanidin-3-glucoside, malvidin 3-O-glucoside, malvidin 3-O-coumaroylglucoside, cyanidin 3-glucoside-pyruvic acid adduct and malvidin 3-glucoside-pyruvic acid adduct, extracted from red grapes were used as sensitizers in dye-sensitized solar cells (DSSCs). With these natural dyes, cells efficiencies ranged up to 0.08%. The cyanidin based dyes were more efficient as sensitizers than the malvidin molecules and the presence of the pyruvic acid adduct decreased efficiency. Furthermore, the use of drops of Port wine in the production of DSSCs resulted in an efficiency of 0.025%. Although the obtained efficiencies still need improvements for an industrial application, it is given further evidence that wine dyes can harvest and convert sunlight into electricity through a process that mimics natural photosynthesis.

PLGA nanoparticles for calcitriol delivery

Calcitriol, the active metabolite of Vitamin D3, is a potential anticancer agent but exhibits several drawbacks. Therefore, new therapeutic strategies, as specific drug delivery systems, must be developed in order to overcome those limitations. The aim of this work was to prepare poly(lactide-coglycolide) nanoparticles as drug delivery vehicles for calcitriol. Two formulation parameters, vitamin/polymer ratio and sonication time, were firstly studied and discussed using cholecalciferol as a drug model. Then, calcitriol-loaded poly(lactide-coglycolide) nanoparticles with controlled sizes and properties were prepared. The nanoparticles remained stable at storage conditions for several weeks and they were successfully lyophilized to increase their shelf-life using a crioprotectant. In vitro studies using two human cancer cell lines, S2-013 and A549, demonstrated that bare PLGA NPs are biocompatible.