João Casalta-Lopes

Cardiac troponin I: Prothrombotic risk marker in non-valvular atrial fibrillation

BACKGROUND Evidence of a link between small rises in cardiac troponin I (cTnI) and an increased risk of thromboembolic events (TE) in atrial fibrillation (AF) is currently scarce. OBJECTIVES We aimed to assess the relation between cTnI and findings of an increased thromboembolic risk in patients with non-valvular AF using transesophageal echocardiography. METHODS We have included 245 patients performing transthoracic and transesophageal echocardiogram, alongside with laboratory assessment (including cTnI) in a cross-sectional survey. Changes associated to TE were sought on transesophageal echocardiogram: left atrial or left atrial appendage thrombus, dense spontaneous echocardiographic contrast, low flow velocities in the left atrial appendage and protuberant aortic plaques. Comparisons were performed according to the baseline concentration of cTnI, regarding the prevalence of these changes. We have added cTnI to CHADS2 and CHA2DS2-VASc scores in order to assess its capability to refine risk stratification using transesophageal markers as surrogate endpoints and assessed it by means of ROC-curve analysis and Net Reclassification Improvement (NRI). RESULTS A direct relation between rising concentrations of cTnI and a higher prevalence of transesophageal echocardiogram changes was found. Furthermore, the addition of cTnI to CHADS2 and CHA2DS2-VASc scores improved their ability to predict changes associated to TE on transesophageal echocardiography both through ROC-curve analysis and NRI. CONCLUSION cTnI seems to be associated to thromboembolic risk in patients with AF. The possible role of cTnI in the refinement of risk stratification schemes needs to be tested in further prospective studies using clinical endpoints.

Hepatocellular Carcinoma and Chemotherapy: The Role of p53

Background: Hepatocellular carcinoma (HCC) is the most common primary neoplasm of the liver. A major proportion of HCCs also present mutation of the gene that encodes p53, which confers chemoresistance. The main goal of this work is to investigate the effect of cisplatin, doxorubicin and 5-fluoruracil (5-FU) in three human HCC cell lines which differ in p53 expression. Methods: HepG2 (expressing normal p53), HuH7 (expressing mutated p53) and Hep3B2.1-7 (not expressing p53) cell lines were cultivated in the presence of cisplatin, doxorubicin and 5-FU. Cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The type of cell death and Bax and Bcl2 activation were assessed by flow cytometry. Results: It was found that for all of the cell lines studied, the agent that gave the most satisfactory results was doxorubicin. 5-FU demonstrated no activity in these cell lines. Conclusions: For all the cell lines studied, doxorubicin was the most satisfactory agent. In HepG2 and HuH7 cell lines, it can activate Bax with statistical significance.

Cytotoxicity of Ascorbic Acid in a Human Colorectal Adenocarcinoma Cell Line (WiDr): In Vitro and In Vivo Studies

Vitamin C, available in its reduced form (ascorbic acid; AA) and in its oxidized form (dehydroascorbic acid; DHA), may act in physiological conditions as an antioxidant or pro-oxidant. The aim of this study is to evaluate the cytotoxic effects of pharmacological doses of AA in a human colorectal adenocarcinoma cell line (WiDr) in vitro, through spectrophotometry, clonogenic assays and flow cytometry, and in vivo with xenotransplanted Balb/c nu/nu mice. The results show that the reduced form of vitamin C induces an anti-proliferative and cytotoxic effect in adenocarcinoma colorectal cells under study. The results obtained in vivo after treatment with AA showed a large reduction in the rate of tumor growth. Such understanding can guide decisions about which colorectal cancer patients might potentially benefit from vitamin C pharmacologic therapy.

New Approach for Treatment of Primary Liver Tumors: The Role of Quercetin

abstract Hepatocellular carcinoma (HCC) is the most common primary liver tumor (PLT), with cholangiocarcinoma (CC) being the second most frequent. Glucose transporter 1 (GLUT-1) expression is increased in PLTs and therefore it is suggested as a therapeutic target. Flavonoids, like quercetin, are GLUT-1 competitive inhibitors and may be considered as potential therapeutic agents for PLTs. The objective of this study was evaluation of quercetin anticancer activity in three human HCC cell lines (HepG2, HuH7, and Hep3B2.1–7) and in a human CC cell line (TFK-1). The possible synergistic effect between quercetin and sorafenib, a nonspecific multikinase inhibitor used in clinical practice in patients with advanced HCC, was also evaluated. It was found that in all the cell lines, quercetin induced inhibition of the metabolic activity and cell death by apoptosis, followed by increase in BAX/BCL-2 ratio. Treatment with quercetin caused DNA damage in HepG2, Hep3B2.1–7, and TFK-1 cell lines. The effect of quercetin appears to be independent of P53. Incubation with quercetin induced an increase in GLUT-1 membrane expression and a consequent reduction in the cytoplasmic fraction, observed as a decrease in 18F-FDG uptake, indicating a GLUT-1 competitive inhibition. The occurrence of synergy when sorafenib and quercetin were added simultaneously to HCC cell lines was noticed. Thus, the use of quercetin seems to be a promising approach for PLTs through GLUT-1 competitive inhibition.

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.

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine fused chlorins as very active photodynamic agents for melanoma cells.

The development of new stable 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins with high absorption properties at 650 nm, and their impressive photosensitizer ability against melanotic and amelanotic cancer cells is described. Comparison between a diester-substituted chlorin with the corresponding dihydroxymethyl derivative demonstrated that the increased hydrophilicity of the latter is crucial to ensure nanomolar activity against melanoma cells. The new photosensitizer leads to death of human melanoma cells being both apoptosis and necrosis in equal parts involved in the treatment response. The dihydroxymethyl-chlorin was particularly active against human melanocytic melanoma A375 cells, which can be viewed as a solution to overcome the resistance of melanotic melanoma to photodynamic therapy.

Influence of P53 on the radiotherapy response of hepatocellular carcinoma

Background/Aims Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. Methods Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. Results The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. Conclusions These results suggest that P53 plays a key role in the radiotherapy response of HCC.

Platinum(II) Ring-Fused Chlorins as Near-Infrared Emitting Oxygen Sensors and Photodynamic Agents

Novel near-infrared luminescent compounds based on platinum(II) 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins are described. These compounds have high photostability and display light emission, in particular simultaneous fluorescence and phosphorescence emission in solution at room temperature, in the biologically relevant 700-850 nm red and near-infrared (NIR) spectral region, making them excellent materials for biological imaging. The simultaneous presence of fluorescence and phosphorescence emission at room temperature, with the phosphorescence strongly quenched by oxygen whereas fluorescence remains unaffected, allows these compounds to be used as ratiometric oxygen sensors in chemical and biological media. Both steady-state (fluorescence vs phosphorescence intensities) and dynamic (dependence of phosphorescence lifetimes upon oxygen concentration) luminescence approaches can be used. Photocytotoxicity studies against human melanocytic melanoma cells (A375) indicate that these compounds display potential as photosensitizers in photodynamic therapy.

Beyond the limits of oxygen: effects of hypoxia in a hormone-independent prostate cancer cell line.

Prostate cancer (PCa) has a high incidence worldwide. One of the major causes of PCa resistance is intratumoral hypoxia. In solid tumors, hypoxia is strongly associated with malignant progression and resistance to therapy, which is an indicator of poor prognosis. The antiproliferative effect and induced death caused by doxorubicin, epirubicin, cisplatin, and flutamide in a hormone-independent PCa cell line will be evaluated. The hypoxia effect on drug resistance to these drugs, as well as cell proliferation and migration, will be also analyzed. All drugs induced an antiproliferative effect and also cell death in the cell line under study. Hypoxia made the cells more resistant to all drugs. Moreover, our results reveal that long time cell exposure to hypoxia decreases cellular proliferation and migration. Hypoxia can influence cellular resistance, proliferation, and migration. This study shows that hypoxia may be a key factor in the regulation of PCa.

Evaluation of a 99mTc‐labelled meso‐bisphenylporphyrin as a tumour image agent

Porphyrins are excellent agents for photodynamic treatment of various types of cancer and also good metal chelators that form highly stable metallo-complexes with different radionuclides. Therefore, radiolabelled porphyrins could also be potentially used as tumour imaging agents. In this context, the aim of this work was the radiolabelling of meso-bis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, 2CPP, with Technetium-99 m ((99m) Tc) and the evaluation of its radiochemical and biological properties in vitro and in vivo. The labelling procedure was optimized resulting in an efficiency of 92.52 ± 0.48%. The complex (99m) TC-2CPP remained stable for more than 4 h. The biodistribution showed that (99m) Tc-2CPP is eliminated by gastrointestinal and urinary pathways. The tumour/muscle ratio increases over time, being 3.33 ± 1.22 and 3.55 ± 1.29 in WiDr-bearing tumours mice and in H1299-bearing tumours mice, respectively, 6 h post-injection, showing the tumour specificity of the (99m) Tc-2CPP complex. The favourable tumour/muscle ratio of (99m) Tc-2CPP shows that this complex could potentially be used as tumour imaging agent. Moreover, it could be used to follow the progression or regression of tumours before, during and after the radiotherapy, chemotherapy and photodynamic therapy.