Alterações da sinalização purinérgica gastrointestinal na Diabetes

A dismotilidade entérica é uma complicação a longo prazo da Diabetes mellitus (DM) que causa desconforto significativo em 76% dos pacientes diabéticos. Sabendo que as purinas estão envolvidas na neuromodulação colinérgica e que no SNC de animais diabéticos foram encontradas alterações na expressão de recetores purinérgicos, decidimos investigar se na dismotilidade diabética a neuromodulação purinérgica se encontra preservada. O modelo animal escolhido de diabetes tipo I resultou da administração de estreptozotocina (STZ, 55 mg/kg, IP) a ratazanas (Rattus norvegicus, Wistar). Este modelo STZ provou ser adequado para o estudo, apresentando 2 semanas após a indução polidipsia, poliúria, polifagia, hiperglicemia e um atraso da motilidade gastrointestinal. A caraterização morfológica macroscópica dos animais STZ revelou um aumento significativo do cego e do intestino. Funcionalmente, estudos preliminares indicam que as contrações espontâneas do íleo dos animais STZ perdem ritmicidade e apresentam maior amplitude que as dos animais controlo de uma forma insensível à TTX, sugerindo o comprometimento das ICC. Paralelamente, estudos imagiológicos revelaram uma perda neuronal mioentérica, principalmente de neurónios nitrérgicos, sendo os colinérgicos preservados. Contudo, a resposta muscular do íleo de animais diabéticos à acetilcolina (ACh) foi inferior à dos controlos, estando a libertação de ACh modulada pela adenosina modificada. Verificou-se que a inibição promovida pelos recetores A1 se mantinha, mas que se perdia a facilitação mediada pela ativação de recetores A2A, cuja imunorreatividade também se encontrava diminuída. Curiosamente, apesar do catabolismo do ATP e dos seus metabolitos estar aumentado nos animais STZ, não se verificou um aumento dos níveis extracelulares de adenosina. Nos animais diabéticos a adenosina é rapidamente desaminada e recaptada por transportadores de nucleósidos, com principal relevância para os concentrativos, sendo os equilibrativos responsáveis pelo transporte da adenosina em animais controlo. Os resultados apresentados nesta tese sugerem que a dismotilidade diabética pode dever-se à perda da atividade nitrérgica, das ICC e da neuromodulação purinérgica mediada por recetores A2A, comprometendo assim a libertação de ACh e consequentemente a motilidade GI

Multi-organ NMR metabolomics to assess in vivo overall metabolic impact of cisplatin in mice

This work describes, to our knowledge, the first NMR metabolomics analysis of mice kidney, liver, and breast tissue in response to cisplatin exposure, in search of early metabolic signatures of cisplatin biotoxicity. Balb/c mice were exposed to a single 3.5 mg/kg dose of cisplatin and then euthanized; organs (kidney, liver, breast tissue) were collected at 1, 12, and 48 h. Polar tissue extracts were analyzed by NMR spectroscopy, and the resulting spectra were studied by multivariate and univariate analyses. The results enabled the identification of the most significant deviant metabolite levels at each time point, and for each tissue type, and showed that the largest metabolic impact occurs for kidney, as early as 1 h post-injection. Kidney tissue showed a marked depletion in several amino acids, comprised in an overall 13-metabolites signature. The highest number of changes in all tissues was noted at 12 h, although many of those recovered to control levels at 48 h, with the exception of some persistently deviant tissue-specific metabolites, thus enabling the identification of relatively longer-term effects of cDDP. This work reports, for the first time, early (1-48 h) concomitant effects of cDDP in kidney, liver, and breast tissue metabolism, thus contributing to the understanding of multi-organ cDDP biotoxicity.publishe

Novel insights into mice multi-organ metabolism upon exposure to a potential anticancer Pd(II)-agent

Pd(II)-compounds are presently regarded as promising anticancer drugs, as an alternative to Pt(II)-based drugs (e.g., cisplatin), which typically trigger severe side-effects and acquired resistance. Dinuclear Pd(II) complexes with biogenic polyamines such as spermine (Pd2Spm) have exhibited particularly beneficial cytotoxic properties, hence unveiling the importance of understanding their impact on organism metabolism. The present study reports the first nuclear magnetic resonance (NMR)-based metabolomics study to assess the in vivo impact of Pd2Spm on the metabolism of healthy mice, to identify metabolic markers with possible relation to biotoxicity/side-effects and their dynamics. The changes in the metabolic profiles of both aqueous and lipophilic extracts of mice kidney, liver, and breast tissues were evaluated, as a function of drug-exposure time, using cisplatin as a reference drug. A putative interpretation was advanced for the metabolic deviations specifically triggered by Pd2Spm, this compound generally inducing faster metabolic response and recovery to control levels for all organs tested, compared to cisplatin (except for kidney lipid metabolism). These results constitute encouraging preliminary metabolic data suggestive of potential lower negative effects of Pd2Spm administration.This research was developed within the scope of the CICECO—Aveiro Institute of Materials, with references UIDB/50011/2020 and UIDP/50011/2020, financed by national funds through the Portuguese Foundation for Science and Technology (FCT/MEC) and when appropriate co-financed by European Regional Development Fund (FEDER) under the PT2020 Partnership Agreement. This work was also funded by the FCT through UIDB/00070/2020 (ALMBC and MPMM), PO-CI-01-0145-FEDER-0016786, and Centro-01-0145-FEDER-029956 (co-financed by COMPETE 2020, Portugal 2020 and European Community through FEDER). It also received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project UIDB/50006/2020. We also acknowledge the Portuguese National NMR Network (PTNMR), supported by FCT funds as the NMR spectrometer used is part of PTNMR and partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL, and the FCT through PIDDAC). M.V. thanks the FCT and the PhD Program in the department of Medicines and Pharmaceutical Innovation (i3DU) for their PhD grant PD/BD/135460/2017 and T.J.C. thanks FCT for their PhD grant SFRH/BD/145920/2019, both grants were funded by the European Social Fund of the European Union and national funds FCT/MCTES.publishe

Patient-reported outcomes and experiences assessment in women with breast cancer: Portuguese case study

In 2020, female breast cancer was the most commonly diagnosed cancer worldwide, representing the type of cancer with the highest incidence among women and the second most common cause of cancer death among women in all OECD countries. The conventional measures addressing the burden of breast cancer by measuring mortality, incidence, and survival do not entirely reflect the quality of life and patients' experience when receiving breast cancer care. The main objective of this study is to capture patient-reported outcomes and experiences in women with breast cancer in Portugal using methods developed for international benchmarking purposes, such as the OECD Patient-reported Indicators Surveys. The study included 378 women with breast cancer, with the age distribution being 19.8% aged 15 to 49 years and 80.2% aged 50 years and over. The data collection procedure and analysis followed the "OECD Breast Cancer Patient Reported Outcomes Working Group" protocol, allowing subsequent comparability with data from other OECD member countries. Most women were satisfied with the treatment outcome regarding the shape of their lumpectomy breasts when wearing a bra (96.1%) and with the equal size of both breasts (78.3%). Findings on the WHO QOL-BREF showed that women manifest a lower score in well-being when compared with the general population or populations living with chronic diseases. This study shows the feasibility of implementing and using patient-reported metrics (PROM and PREM) in breast cancer services in Portugal. Measuring PROMs and PREMs from Portuguese women receiving breast cancer care provides insightful evidence of the quality and value of cancer care.info:eu-repo/semantics/publishedVersio

Preclinical pharmacokinetics and biodistribution of anticancer dinuclear Palladium(II)-Spermine Complex (Pd2Spm) in mice

Palladium-based compounds are regarded as potential analogs to platinum anticancer drugs with improved properties. The present study assessed the pharmacokinetics and biodistribution of a dinuclear palladium(II)-spermine chelate (Pd2Spm), which has previously been shown to possess promising in vitro activity against several therapy-resistant cancers. Using inductively coupled plasma-mass spectrometry, the kinetic profiles of palladium/platinum in serum, serum ultrafiltrate and tissues (kidney, liver, brain, heart, lungs, ovaries, adipose tissue and mammary glands) were studied in healthy female Balb/c mice after a single intraperitoneal bolus injection of Pd2Spm (3 mg/kg bw) or cisplatin (3.5 mg/kg bw) between 0.5 and 48 h post-injection. Palladium in serum exhibited biphasic kinetics with a terminal half-life of 20.7 h, while the free palladium in serum ultrafiltrate showed a higher terminal half-life than platinum (35.5 versus 31.5 h). Palladium was distributed throughout most of the tissues except for the brain, with the highest values in the kidney, followed by the liver, lungs, ovaries, adipose tissue and mammary glands. The in vitro cellular accumulation was also evaluated in breast cancer cells, evidencing a passive diffusion as a mechanism of Pd2Spm’s cellular entry. This study reports, for the first time, the favorable pharmacokinetics and biodistribution of Pd2Spm, which may become a promising pharmacological agent for cancer treatmentinfo:eu-repo/semantics/publishedVersio

Pd2Spermine Complex Shows Cancer Selectivity and Efficacy to Inhibit Growth of Triple-Negative Breast Tumors in Mice

Pd2Spm is a dinuclear palladium(II)-spermine chelate with promising anticancer properties against triple-negative breast cancer (TNBC), a breast carcinoma subset with poor prognosis and limited treatment options. The present study evaluated the in vitro and in vivo anticancer effects of Pd2Spm compared to the reference metal-based drug cisplatin. Triple-negative breast cancer MDA-MB-231 cells, non-cancerous MCF-12A breast cells and chorioallantoic membrane (CAM) assay were used for antiproliferative, antimigratory and antiangiogenic studies. For an in vivo efficacy study, female CBA nude mice with subcutaneously implanted MDA-MB-231 breast tumors were treated with Pd2Spm (5 mg/kg/day) or cisplatin (2 mg/kg/day) administered intraperitoneally during 5 consecutive days. Promising selective antiproliferative activity of Pd2Spm was observed in MDA-MB-231 cells (IC50 values of 7.3-8.3 µM), with at least 10-fold lower activity in MCF-12A cells (IC50 values of 89.5-228.9 µM). Pd2Spm inhibited the migration of MDA-MB-231 cells, suppressed angiogenesis in CAM and decreased VEGF secretion from MDA-MB-231 cells with similar potency as cisplatin. Pd2Spm-treated mice showed a significant reduction in tumor growth progression, and tumors evidenced a reduction in the Ki-67 proliferation index and number of mitotic figures, as well as increased DNA damage, similar to cisplatin-treated animals. Encouragingly, systemic toxicity (hematotoxicity and weight loss) observed in cisplatin-treated animals was not observed in Pd2Spm-treated mice. The present study reports, for the first time, promising cancer selectivity, in vivo antitumor activity towards TNBC and a low systemic toxicity of Pd2Spm. Thus, this agent may be viewed as a promising Pd(II) drug candidate for the treatment of this type of low-prognosis neoplasia

Impact of the Pd2Spm (Spermine) Complex on the Metabolism of Triple-Negative Breast Cancer Tumors of a Xenograft Mouse Model

The interest in palladium(II) compounds as potential new anticancer drugs has increased in recent years, due to their high toxicity and acquired resistance to platinum(II)-derived agents, namely cisplatin. In fact, palladium complexes with biogenic polyamines (e.g., spermine, Pd2Spm) have been known to display favorable antineoplastic properties against distinct human breast cancer cell lines. This study describes the in vivo response of triple-negative breast cancer (TNBC) tumors to the Pd2Spm complex or to cisplatin (reference drug), compared to tumors in vehicle-treated mice. Both polar and lipophilic extracts of tumors, excised from a MDA-MB-231 cell-derived xenograft mouse model, were characterized through nuclear magnetic resonance (NMR) metabolomics. Interestingly, the results show that polar and lipophilic metabolomes clearly exhibit distinct responses for each drug, with polar metabolites showing a stronger impact of the Pd(II)-complex compared to cisplatin, whereas neither drug was observed to significantly affect tumor lipophilic metabolism. Compared to cisplatin, exposure to Pd2Spm triggered a higher number of, and more marked, variations in some amino acids, nucleotides and derivatives, membrane precursors (choline and phosphoethanolamine), dimethylamine, fumarate and guanidine acetate, a signature that may be relatable to the cytotoxicity and/or mechanism of action of the palladium complex. Putative explanatory biochemical hypotheses are advanced on the role of the new Pd2Spm complex in TNBC metabolism

Metabolic Impact of Anticancer Drugs Pd2Spermine and Cisplatin on the Brain of Healthy Mice

The new palladium agent Pd2Spermine (Spm) has been reported to exhibit promising cytotoxic properties, while potentially circumventing the known disadvantages associated to cisplatin therapeutics, namely acquired resistance and high toxicity. This work presents a nuclear magnetic resonance (NMR) metabolomics study of brain extracts obtained from healthy mice, to assess the metabolic impacts of the new Pd2Spm complex in comparison to that of cisplatin. The proton NMR spectra of both polar and nonpolar brain extracts were analyzed by multivariate and univariate statistics, unveiling several metabolite variations during the time course of exposition to each drug (1-48 h). The distinct time-course dependence of such changes revealed useful information on the drug-induced dynamics of metabolic disturbances and recovery periods, namely regarding amino acids, nucleotides, fatty acids, and membrane precursors and phospholipids. Putative biochemical explanations were proposed, based on existing pharmacokinetics data and previously reported metabolic responses elicited by the same metal complexes in the liver of the same animals. Generally, results suggest a more effective response of brain metabolism towards the possible detrimental effects of Pd2Spm, with more rapid recovery back to metabolites' control levels and, thus, indicating that the palladium drug may exert a more beneficial role than cDDP in relation to brain toxicity

Role of Inflammation and Redox Status on Doxorubicin-Induced Cardiotoxicity in Infant and Adult CD-1 Male Mice

Doxorubicin (DOX) is a topoisomerase II inhibitor commonly used in the treatment of several types of cancer. Despite its efficacy, DOX can potentially cause fatal adverse effects, like cardiotoxicity. This work aimed to assess the role of inflammation in DOX-treated infant and adult mice and its possible link to underlying cardiotoxicity. Two groups of CD-1 male mice of different ages (infants or adults) were subjected to biweekly DOX administrations, to reach a cumulative dose of 18.0 mg/kg, which corresponds approximately in humans to 100.6 mg/m2 for infants and 108.9 mg/m2 for adults a clinically relevant dose in humans. The classic plasmatic markers of cardiotoxicity increased, and that damage was confirmed by histopathological findings in both groups, although it was higher in adults. Moreover, in DOX-treated adults, an increase of cardiac fibrosis was observed, which was accompanied by an increase in specific inflammatory parameters, namely, macrophage M1 and nuclear factor kappa B (NF-κB) p65 subunit, with a trend toward increased levels of the tumor necrosis factor receptor 2 (TNFR2). On the other hand, the levels of myeloperoxidase (MPO) and interleukin (IL)-6 significantly decreased in DOX-treated adult animals. In infants, a significant increase in cardiac protein carbonylation and in the levels of nuclear factor erythroid-2 related factor 2 (Nrf2) was observed. In both groups, no differences were found in the levels of tumor necrosis factor (TNF-α), IL-1β, p38 mitogen-activated protein kinase (p38 MAPK) or NF-κB p52 subunit. In conclusion, using a clinically relevant dose of DOX, our study demonstrated that cardiac effects are associated not only with the intensity of the inflammatory response but also with redox response. Adult mice seemed to be more prone to DOX-induced cardiotoxicity by mechanisms related to inflammation, while infant mice seem to be protected from the damage caused by DOX, possibly by activating such antioxidant defenses as Nrf2