Filomena Botelho

2-Bromo-5-hydroxyphenylporphyrins for photodynamic therapy: Photosensitization efficiency, subcellular localization and in vivo studies

BACKGROUND Photodynamic therapy (PDT) is a therapeutic modality capable of inducing cell death by oxidative stress through activation of a sensitizer by light. Aryl-porphyrin with hydroxyl groups are good photosensitizers and presence of bromine atoms can enhance the photodynamic activity through heavy atom effect. These facts and our previous work made pertinent to compare the photodynamic capacity of tetraaryl brominated porphyrin (TBr4) with the corresponding diaryl (BBr2) derivative. METHODS Cell cultures were incubated with the sensitizers, ranging from 50nM to 10μM and irradiated until 10J. Cell proliferation was analysed by MTT assay. Flow cytometry studies evaluated cell death pathways, mitochondrial membrane potential and ROS. For in vivo studies Balb/c nu/nu mice were injected with 4×10(6)cells. After PDT, monitoring was carried out for 12 days to establish Kaplan-Meier survival curves. Tumours were excised and histological analysis was performed. RESULTS Both sensitizers seem to accumulate in the mitochondria. The molecules have no intrinsic cytotoxicity or in non-tumour cells at therapeutic concentrations. Both sensitizers induced a significant decrease of cell proliferation and growth of xenografts of melanoma and colorectal adenocarcinoma. Diaryl BBr2 is more efficient than tetraaryl TBr4, concerning intracellular ROS production, mitochondrial disruption and induction of cell death. The main cell death pathway is necrosis. CONCLUSIONS TBr2 and BBr4 are promising sensitizers with good photodynamic properties and have the ability to induce cell death in human melanoma and colorectal adenocarcinoma in vitro and in vivo. We consider that BBr2 is a molecule that should be the subject of extensive studies towards clinical use.

Synthetic porphyrins bearing β-propionate chains as photosensitizers for photodynamic therapy

Porphyrins with different numbers of β-propionate chains mimicking natural porphyrins were prepared via the 2+2 MacDonald type approach. Photodynamic activity against WiDr colon adenocarcinoma cells showed that activity is related to the number of β-propionate chains, with the derivatives with two carboxylic groups showing higher activity.

Estrogen Receptor Ligands for Targeting Breast Tumours: A Brief Outlook on Radioiodination Strategies

The design and development of radiolabelled estradiol derivatives has been an important area of research due to their recognized value in breast cancer management. The estrogen receptor (ER) is a relevant biomarker in the diagnosis, prognosis and prediction of the therapeutic response in estrogen receptor positive breast tumours. Hence, many radioligands based on estradiol derivatives have been proposed for targeted functional ER imaging. The main focus of this review is to survey the current knowledge on estradiol-based radioiodinated receptor ligands synthesis for breast tumour functional imaging. The main preclinical and clinical achievements in the field will also be briefly presented to make the manuscript more comprehensive.

Evaluation of Novel Radioiodinated C7-substituted Δ6,7 – estradiol Derivatives for Molecular Recognition of ER-Positive Breast Tumours

The estrogen receptor is a striking target for molecular imaging and therapy of certain malignancies. In the case of cancer, the ability of imaging the expression of the ER would offer a noninvasive means of classifying tumours guiding the optimal choice of therapy. The importance of estradiol based radioligands as potential imaging agents for estrogen receptor rich tumours and the growing interest in Auger electron emitters for potential radiotherapy of estrogen receptorpositive breast cancers lead to the synthesis and evaluation of a series of radioiodinated derivatives of estradiol. Seeking for new probes for targeting the estrogen receptor two novel radioiodinated Δ6,7 – estradiol derivatives containing different C7-alkyl chains were prepared and labelled with 125I. To evaluate the potential use of their 123I-analogues for imaging breast tumours the biological behaviour of the new radioligands was studied in immature female rats to determine their uptake in and selectivity for tissues containing estrogen receptors. The different alkyl chains induced major modifications in the uptake efficiency, selectivity and in the metabolization degree of the compounds. It is not very clear, however, whether the alterations in uptake efficiency and selectivity are the result of differences in lipophilicity or altered patterns of metabolism. Even so, these radioligands exhibit different biological patterns that may be helpful in optimizing the imaging of ER positive tumours.

Dynamics of CD86 Expression on Allergic Inflammation - New Insights

CD86 is a well-known costimulatory molecule in its interaction with CD28 and/or CTLA present on T cells, and is essential for full activation of naïve T-cell and subsequent differentiation. Usually the B7 molecules are expressed mainly on APCs and B cells and in specific conditions on other activated cells. These costimulatory molecules are involved in the development of allergic inflammation and airways hyperreactivity (AHR) in allergen-challenged mice. Activated T cells, CD4(+)CD25(+), express CD86 in the first 60 minutes after the specific inhalatory exposure. These T cells can be relevant in IgE mediated allergic reaction possibly by an autocrine costimulation via CD28/CTLA activation pathway. The blockage of the expression of CD86 could be a potential therapeutical target to reduce the magnitude or the progression of the allergic reaction. The review article also discussed relevant patents.

Radioiodinated ligands for the estrogen receptor: Effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols

Three novel 17 alpha-ethynyl-Delta(6,7)-estra-3,17beta-diols and their 17 alpha-[(125)I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17 alpha-[(125)I]-iodovinyl-Delta(6,7)-estra-3,17beta-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated.

Glycolysis inhibition as a strategy for hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is the most frequently detected primary malignant liver tumor, representing a worldwide public health problem due to its high morbidity and mortality rates. The HCC is commonly detected in advanced stage, precluding the use of treatments with curative intent. For this reason, it is crucial to find effective therapies for HCC. Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Such dependence provides a reliable possible strategy to specifically target cancer cells based on the inhibition of glycolysis. HCC, such as other cancer types, presents a clinically well-known upregulation of several glycolytic key enzymes and proteins, including glucose transporters particularly glucose transporter 1 (GLUT1). Such enzymes and proteins constitute potential targets for therapy. Indeed, for some of these targets, several inhibitors were already reported, such as 2-Deoxyglucose, Imatinib or Flavonoids. Although the inhibition of glycolysis presents a great potential for an anticancer therapy, the development of glycolytic inhibitors as a new class of anticancer agents needs to be more explored. Herein, we propose to summarize, discuss and present an overview on the different approaches to inhibit the glycolytic metabolism in cancer cells, which may be very effective in the treatment of HCC.

Specific Immunotherapy and Central Immune System

Despite the current knowledge, the mechanisms by which the specific immunotherapy (SIT) achieves clinical improvement remains unclear. However, it is now clear that the immune tolerance is one of the major targets of this kind of treatment. Immune tolerance depends on different mechanisms, including T-cell anergy, T-cell depletion by apoptosis, and active immune suppression (Akdis & Akdis, 2011). One of the goals of SIT is the induction of tolerance to allergens to which the patient is sensitized. IL-10 is probably a relevant cytokine induced by this treatment and is associated to regulatory T cells (T-regs) that actively control or suppress the function of other cells, generally in an inhibitory pattern (Frew, 2010). The changes in microenvironment due to the decrease in histamine and PGE2 release by mast cell, and the IL-10 and TGF- release by dendritic cells (DC) could switch the T-cell population into T-regs. These alterations will then lead to tolerance (Schmidt-Weber & Blaser 2005). Much of the knowledge about SIT has been based on studies using subcutaneous route of administration (SCIT), but increasing data is now available based on studies using sublingual immunotherapy (SLIT). The sublingual mucosa, where the deposition of the extract occurs, has very particular characteristics, quite different from the cellular subcutaneous tissue. The dendritic cells present in the buccal region are distinct from the Langerhans cells present in the skin. These cells present, constitutively, receptors with high affinity for IgE, FcRI+, MHC class I and II molecules, as well as co-stimulation molecules, namely CD40, CD80/B7.1 and CD86/B7.2 (Allam et al., 2006). There is also expression of CD14, a lipopolysaccharide (LPS) receptor, which is relevant for the modulation of Th2 and Th1. In SLIT, the allergen is captured by the DC cell by C-lectin endocytosis receptors and/or by ligation to the IgE on the surface. After the internalization, the migration to the regional lymphoid nodules occurs, and it is then presented to the T cells (Geijtenbeek, 2006). A study that compares the DC population in the oral and nasal mucosa was able to demonstrate that only the DC myeloid type is profusely present in the oral region, in contrast with the nasal mucosa where both populations are present (Allam et al., 2006). Another very important difference is the high expression levels of FcRI in the oral mucosa, which is almost absent in the skin’s Langerhans cells (Allam et al., 2003). Furthermore, the expression levels of MHC class I and II molecules, CD40, CD80 and CD86 is significantly higher in oral DC than in the skin.

Dental caries and bacterial load in saliva and dental biofilm of type 1 diabetics on continuous subcutaneous insulin infusion

Abstract Objectives Since most of the studies evaluates diabetics on multiple daily injections therapy and continuous subcutaneous insulin infusion may help gain better metabolic control and prevent complications, the objective of this study was to evaluate the prevalence of dental caries, the unstimulated salivary flow rate and the total bacteria load, Streptococcus spp. levels and Lactobacillus spp. levels in saliva and supragingival dental biofilm of type 1 diabetics on insulin pump. Material and Methods Sixty patients with type 1 diabetes on insulin pump and 60 nondiabetic individuals were included. The dental caries evaluation was performed using ICDAS and the oral hygiene was assessed according to Greene and Vermillion Simplified Oral Hygiene Index. Unstimulated saliva and supragingival dental biofilm were collected. Total bacteria, Streptococcus spp. and Lactobacillus spp. was quantified by qPCR. Results Patients with type 1 diabetes had a higher prevalence of dental caries and filled and missing teeth when compared with the control group. These patients were associated with more risk factors for the development of dental caries, namely a lower unstimulated salivary flow rate and a higher bacterial load in saliva and dental biofilm. Conclusion Some risk factors related to dental caries were associated with type 1 diabetics. An early diagnosis combined with the evaluation of the risk profile of the diabetic patient is imperative, allowing the dental caries to be analyzed through a perspective of prevention and the patient to be integrated into an individualized oral health program.

Abstract 2603: Cancer stem cells and dedifferentiation: the stromal match-point.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Bypassing all the research advances in the last decades, cancer remains as a major public health problem affecting more than 1.5 million (M) new individuals each year just in the USA, and killing more than 0.5 M. Recent research emphasized the major role of cancer stem cells (CSCs) in the metastatic disease, the main cause of cancer patients mortality. CSCs drive tumorigenesis and differentiation, contributing to tumors’ heterogeneity and to their relative chemo- and radiotherapy resistance and eventually relapse. Following CSCs identification, targeted therapeutic approaches have been developed to abolish them. However, CSCs can reemerge through dedifferentiation of tumor-committed stromal cells condemning this therapeutics. The mechanisms behind dedifferentiation are still unclear and are the main focus of our investigation. Lung cancer is one of the main causes of cancer-related deaths worldwide. Its prevalence is increasing due to the widespread smoking habits and increasing accumulation of atmosphere pollutants. In this work hexavalent chromium [Cr(VI)] was selected as a model of cancer carcinogenesis mainly due to is increasing occupational relevance. The non-malignant human bronchial epithelial cell line BEAS-2B was malignantly transformed into the RenG2 system using low density culture in the presence of Cr(VI). A parallel control cell line (Cont1) was produced under the same conditions, though, in the absence of Cr(VI). Two additional cell lines (DRenG2 and DDRenG2) were attained following serial rounds of injection in nude mice. Metabolic studies using [18F]fluoro-2-deoxyglucose) and nuclear magnetic resonance spectroscopy performed in all the cell lines revealed a more glycolytic phenotype for the derivatives (DRenG2 and DDRenG2), compatible with a quiescent phenotype. Subsequent karyotype and real time PCR-based cellular characterization identified different cellular sub-populations within each cell line, strengthening the hypothesis on the CSCs presence. The sphere-formation assay, used to search for CSCs presence, revealed the presence of CSCs only DRenG2 and in DDRenG2 cell lines. This suggested that a dedifferentiation process featured the formation of CSCs during RenG2 derivation in nude mice. The involvement of mice stroma in this process was uncovered by surgical isolation of mouse stromal cells of the subcutaneous compartment and subsequent co-culture with RenG2 cells for 30-60 days (time needed to induce tumors in mice with RenG2), which resulted in the emergence of a CSCs sub-population. A cytokine multiplex array analysis performed in the conditioned medium of the co-cultured cells in parallel with comparative genome hybridization array (aCGH) analysis performed in all the cell lines under study revealed a panel of potential paracrine orchestrators of this stromal-induced dedifferentiation process. Citation Format: Carlos F. Rodrigues, Mariana Val, Ines P. Rodrigues, Susana Ferreira, Filomena Botelho, Jose Ramalho, Anatoly Zhitkovich, Isabel M. Carreira, Carmen Alpoim. Cancer stem cells and dedifferentiation: the stromal match-point. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2603. doi:10.1158/1538-7445.AM2013-2603