Application of Perinatal Derivatives on Oncological Preclinical Models: A Review of Animal Studies.

The increasing cancer incidence has certified oncological management as one of the most critical challenges for the coming decades. New anticancer strategies are still needed, despite the significant advances brought to the forefront in the last decades. The most recent, promising therapeutic approaches have benefitted from the application of human perinatal derivatives (PnD), biological mediators with proven benefits in several fields beyond oncology. To elucidate preclinical results and clinic outcomes achieved in the oncological field, we present a narrative review of the studies resorting to animal models to assess specific outcomes of PnD products. Recent preclinical evidence points to promising anticancer effects offered by PnD mediators isolated from the placenta, amniotic membrane, amniotic fluid, and umbilical cord. Described effects include tumorigenesis prevention, uncontrolled growth or regrowth inhibition, tumor homing ability, and adequate cell-based delivery capacity. Furthermore, PnD treatments have been described as supportive of chemotherapy and radiological therapies, particularly when resistance has been reported. However, opposite effects of PnD products have also been observed, offering support and trophic effect to malignant cells. Such paradoxical and dichotomous roles need to be intensively investigated. Current hypotheses identify as explanatory some critical factors, such as the type of the PnD biological products used or the manufacturing procedure to prepare the tissue/cellular treatment, the experimental design (including human-relevant animal models), and intrinsic pathophysiological characteristics. The effective and safe translation of PnD treatments to clinical practice relies on the collaborative efforts of all researchers working with human-relevant oncological preclinical models. However, it requires proper guidelines and consensus compiled by experts and health workers who accurately describe the methodology of tissue collection, PnD isolation, manufacturing, preservation, and delivery to the final user

Localização subcelular do novo fotossensibilizador em células do melanoma ocular através de microscopia confocal

Introdução: O melanoma ocular é o objetivo de várias investigações em medicina e medicina veterinária devido à baixa taxa de resposta aos tratamentos conservadores convencionais1. A terapia fotodinâmica (PDT) é uma terapia mais conservadora para os tecidos não neoplásicos, que induz e ativa as vias de morte celular nos tecidos neoplásicos alvo2,3. O fotossensibilizador, luz e oxigenio são os três componentes vitais não tóxicos da reação fotodinâmica. Quando o fotossensibilizador é ativado por uma fonte de luz (600-800 nm), ocorre a produção de espécies reativas de oxigenio (ROS) que exerce efeito terapêutico sobre a neoplasia2. A oxidação irreversível leva à morte da célula tumoral por apoptose, necrose e autofagia2. A morte celular pode ser ativada pela via extrínseca, que envolve recetores da membrana plasmática, ou pela via intrínseca, em que a mitocôndria tem um papel central2,3. A localização subcelular dos fotossensibilizadores constitui o local de dano primário da terapia fotodinâmica e é determinante nas vias de atuação ativadas pela reação fotodinâmica3. Recentemente, desenvolvemos um grupo de novas clorinas, que são fotossensibilizadores muito eficazes em células de melanoma2,3,4. Dessa forma o objetivo do trabalho foi avaliar a localização subcelular (núcleo e mitocôndria) da nova clorina em células do melanoma ocular. Métodos: Células da linha celular de melanoma ocular (MP-41) cultivadas em meio RPMI com 25% de FBS, foram semeadas em placas de 24 poços com lamelas esterilizadas. As células foram incubadas com 500 nM da nova clorina (derivado dihidroximetilo de 4,5,6,7-tetra-hidropirazolo[1,5-a]piridina fundido com tetrafenilclorina) por 24 h. Após este período, as culturas foram submetidas a duas lavagens com PBS, de modo a remover todo o fotossensibilizador no exterior das células, e procedeu-se à marcação dos organelos. As células que foram marcadas com o corante nuclear Hoechst 33252 (blue) e incubadas com uma solução de 5 μg/ml da sonda em PBS durante 15 minutos no escuro. As culturas celulares que foram marcadas com a sonda mitocondrial MitoTracker® Green FM foram incubadas com uma solução de 200 nM da sonda em PBS durante 30 minutos, a 37ºC, no escuro. Após as incubações com as respetivas sondas as células foram lavadas com PBS as lamelas foram montadas sobre lâminas e observadas ao microscópio confocal (Carl Zeiss MicroImaging LSM710, objetiva 40X) e fotografadas. As imagens foram analisadas no software ImageJ e a colocalização avaliada pelo coeficiente de correlação de Pearson. Resultados: Os estudos de localização subcelular confirmaram a internalização celular do novo fotossensibilizador. A média para coeficiente de Pearson foi -0,114 para colocalização núclear, certificando a existência 43% de correlação negativa, para as imagens analisadas, demostrando que não ocorre internalização nuclear do novo fotossensibilizador. No que respeita a mitocôndria, verificou-se a existência de 66%, correlação de Pearson positiva, que foi igual a média 0,366 para as imagens analisadas, demostrando que ocorre internalização mitocondrial do novo fotossensibilizador. Conclusão: Verificou-se que o novo fotossensibilizador é captado pelas células de melanoma ocular e que se acumula nas mitocôndrias, mas não penetra no núcleo celular

Open-Air Cold Plasma Device Leads to Selective Tumor Cell Cytotoxicity

The need for effective and safe therapies for cancer is growing as aging is modifying its epidemiology. Cold atmospheric plasma (CAP) has gained attention as a potential anti-tumor therapy. CAP is a gas with enough energy to ionize a significant fraction of its constituent particles, forming equal numbers of positive ions and electrons. Timely-resolved output voltage measurement, emission spectroscopy, and quantification of reactive species (RS) in plasma-activated media (PAM) were performed to characterize the physical and chemical properties of plasma. To assess the cytotoxicity of cold atmospheric plasma in human tumors, different cell lines were cultured, plated, and exposed to CAP, followed by MTT and SRB colorimetric assays 24 h later. Human fibroblasts, phenotypically normal cells, were processed similarly. Plasma cytotoxicity was higher in cells of breast cancer, urinary bladder cancer, osteosarcoma, lung cancer, melanoma, and endometrial cancer. Cytotoxicity was time-dependent and possibly related to the increased production of hydrogen peroxide in the exposed medium. Sixty seconds of CAP exposure renders selective effects, preserving the viability of fibroblast cells. These results point to the importance of conducting further studies of the therapy with plasma

Diabetes mellitus and prostate cancer metabolism: Is there a relationship?

Objective: Our aim was to evaluate the effects of glucose levels and diabetes mellitus in prostate cancer (PCa) biology. Materials and methods: Two PCa cell lines (LNCap and PC3) were cultured in RPMI medium with different glucose concentrations [5mM (LG) and 25mM (HG)]. Expressions of androgen receptor, Her2/neu and glucose transporters (GLUT1, 3, 5 and 12) were evaluated by flow cytometry. Proliferation rate was assessed by colorimetric assay MTT and cellular characterization was performed by haematoxylin and eosin staining. Additionally, we performed a cross sectional analysis of 704 patients undergoing radical prostatectomy who were divided into two groups (diabetic and non-diabetic). An analysis of clinical and histological data seeking to identify the differences on tumor aggressiveness between the two groups was performed. Results: In LNCaP cell line, when the glucose concentration in the medium increased, there was an increased in AR expression. Regarding expression of Her2/neu receptor, medium’s glucose concentration significantly changed the expression of this receptor in both PC3 and LNCaP cell lines. Growth rate was higher on the HG medium for both cell lines. The clinical study of patients undergoing radical prostatectomy revealed no relationship between the presence of diabetes and the development of more aggressive tumours. Diabetic patients had significantly higher prostatic volumes, however, no significant difference was found between the relapse risk classification or the ISUP classification between the two groups. Conclusions: Our results showed that medium glucose concentration could influence prostate cancer cells growing but not the aggressiveness

Terapia Fotodinâmica: Uma Abordagem no Retinoblastoma

Dissertação de mestrado em Bioquímica, apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A terapia fotodinâmica apresenta-se como uma abordagem terapêutica emergente no tratamento de cancro, podendo induzir morte celular através de um mecanismo que envolva stresse oxidativo por ativação luminosa de moléculas fotossensibilizadoras. Esta dissertação permitiu avaliar a ação fotodinâmica dos fotossensibilizadores BBr2HPP, BBr2HPC, BBr2HPP-O(CH2)2OH e BBr2HPPGluc-OAC em retinoblastoma humano in vitro. Os estudos de captação não demonstraram diferenças de captação entre os diferentes fotossensibilizadores. Os estudos de proliferação celular demonstraram que os quatro fotossensibilizadores têm efeitos semelhantes, apresentando valores de IC50 dentro da mesma ordem de grandeza. Foi também verificado que o tratamento fotodinâmico induz um decréscimo do conteúdo proteico na linha celular de retinoblastoma, compatível com diminuição da viabilidade celular. A morte celular ocorre preferencialmente por apoptose, corroborada pelo surgimento de um pico pré-G1, na análise do ciclo celular e paragem na fase G2/M. Neste trabalho foi também possível verificar que o tratamento fotodinâmico com os quatro fotossensibilizadores estudados não induz danos significativos no DNA. Verificou-se que induz um aumento dos níveis intracelulares de peróxidos, sem alteração dos níveis do radical superóxido. Verificou-se existir uma tendência para que os quatro compostos estudados neste trabalho promovam a disrupção do potencial de membrana mitocondrial, após ativação luminosa. O tratamento fotodinâmico com os quatro fotossensibilizadores referidos não leva à ativação das defesas antioxidantes, nomeadamente da enzima superóxido dismutase e do glutatião reduzido. Este trabalho revela o potencial do tratamento fotodinâmico aplicado ao retinoblastoma. Os compostos fotossensibilizadores estudados são de grande interesse e a avaliação dos mesmos em modelos in vivo hetero e ortotópicos constitui perspetiva futura.Photodynamic therapy is presented as an emerging therapeutic modality for the treatment of cancer by inducing cell death through a mechanism involving oxidative stress after activation of photosensitizing molecules by light. This work allowed us to evaluate the photodynamic action of photosensitizers BBr2HPP, BBr2HPC, BBr2HPP-O(CH2)2OH) and BBr2HPPGluc-OAC in human retinoblastoma in vitro. The uptake studies showed no differences in uptake between different photosensitizers. Cell proliferation studies demonstrated that all four photosensitizers have a very similar effect in the retinoblastoma cells, showing IC50 values in the same order of magnitude. It was also found that the photodynamic treatment induces a decrease in protein content in retinoblastoma cell line compatible with decreased cell viability. Cell death occurs preferentially by apoptosis, supported by the appearance of a pre-G1 peak and cell cycle arrest at G2/M phase. In this work it was also possible to verify that photodynamic treatment with the four photosensitizers studied did not induce significant damage to DNA. It was found that treatment with the four photosensitizers induces an increase in intracellular levels of peroxides, exhibiting no ability to alter the levels of superoxide radical. It has been found that there is a tendency for the four compounds studied in this work to promote the disruption of the mitochondrial membrane potential after light activation. The photodynamic treatment with four referred photosensitizers does not lead to the activation of antioxidant defenses, including superoxide dismutase and reduced glutathione. This work presents great potential in photodynamic treatment of retinoblastoma based on the presented porphyrin and chlorin compounds being of great interest to evaluate the therapeutic potential of these in hetero and orthotopic models in vivo

Oncological Applications of Photodynamic Therapy in Dogs and Cats

Photodynamic therapy (PDT) in small animals’ oncology has been under research focus, pointing to new treatment possibilities. Moreover, several animal studies constitute experimental human disease models due to the similarity of tumor biology between animals and man. PDT uses photosensitizing compounds without toxicity per se. When subjected to a specific wavelength, the photosensitizers are activated, triggering the production of reactive oxygen species (ROS) that lead to cell death. Additionally, antiangiogenic effects and immune stimulation may also be elicited. PDT is minimally invasive, non-toxic, and does not induce carcinogenic or mutagenic side effects. Thus, it is safe for non-neoplastic tissues compared with other neoplasms treatment modalities. This review describes the applications of PDT in the cancer treatment of small animals, particularly dogs and cats, focusing on the respective photosensitizers and treatment protocols used in trials in this therapeutic modality

Microleakage Evaluation of Temporary Restorations Used in Endodontic Treatment-An Ex Vivo Study

(1) Background: Coronal microleakage can lead to endodontic treatment failure. This study aimed to compare the sealing ability of different temporary restorative materials used during endodontic treatment. (2) Methods: Eighty sheep incisors were collected, uniformized in length, and access cavities were performed, except for in the negative control group, where the teeth were left intact. The teeth were divided into six different groups. In the positive control group, the access cavity was made and left empty. In the experimental groups, access cavities were restored with three different temporary materials (IRM®, Ketac™ Silver, and Cavit™) and with a definitive restorative material (Filtek Supreme™). The teeth were submitted to thermocycling, and two and four weeks later, they were infiltrated with 99mTcNaO4, and nuclear medicine imaging was performed. (3) Results: Filtek Supreme™ obtained the lowest infiltration values. Regarding the temporary materials, at two weeks, Ketac™ Silver presented the lowest infiltration, followed by IRM®, whereas Cavit™ presented the highest infiltration. At four weeks, Ketac™ Silver remained with the lowest values, whereas Cavit™ decreased the infiltration, comparable to IRM®. (4) Conclusion: Regarding temporary materials, Ketac™ Silver had the lowest infiltration at 2 and 4 weeks, whereas the highest infiltration was found in the Cavit™ group at two weeks and in the IRM® group at 4 weeks

Hypoglycaemic and Antioxidant Properties of Acrocomia aculeata (Jacq.) Lodd Ex Mart. Extract Are Associated with Better Vascular Function of Type 2 Diabetic Rats

Oxidative stress is involved in the metabolic dysregulation of type 2 diabetes (DM2). Acrocomia aculeata (Aa) fruit pulp has been described for the treatment of several diseases, and recently we have proved that its leaves have phenolic compounds with a marked antioxidant effect. We aimed to assess whether they can improve metabolic, redox and vascular functions in DM2. Control Wistar (W-Ctrl) and non-obese type 2 diabetic Goto-Kakizaki (GK-Ctrl) rats were treated for 30 days with 200 mg.kg-1 aqueous extract of Aa (EA-Aa) (Wistar, W-EA-Aa/GK, GK-EA-Aa). EA-Aa was able to reduce fasting glycaemia and triglycerides of GK-EA-Aa by improving proteins related to glucose and lipid metabolism, such as GLUT-4, PPARγ, AMPK, and IR, when compared to GK-Ctrl. It also improved viability of 3T3-L1 pre-adipocytes exposed by H2O2. EA-Aa also increased the levels of catalase in the aorta and kidney, reduced oxidative stress and increased relaxation of the aorta in GK-treated rats in relation to GK-Ctrl, in addition to the protective effect against oxidative stress in HMVec-D cells. We proved the direct antioxidant potential of the chemical compounds of EA-Aa, the increase in antioxidant defences in a tissue-specific manner and hypoglycaemic properties, improving vascular function in type 2 diabetes. EA-Aa and its constituents may have a therapeutic potential for the treatment of DM2 complications.This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved