Placental, Foetal, and Maternal Serum Metabolomic Profiles in Pregnancy-Associated Cancer: Walker-256 Tumour Model in a Time-Course Analysis.

Cancer during pregnancy presents a delicate coexistence, imposing ethical and professional challenges on both the patient and medical team. In this study, we aimed to explore in a pre-clinical model the impact of tumour evolution in serum, placental and foetal metabolomics profiles during pregnancy in a time-course manner. Pregnant Wistar rats were distributed into two experimental groups: Control (C) and Walker-256 tumour-bearing (W). The rats were euthanised on three different gestational periods: at 12 days post-conception (dpc), at 16 dpc, and at 19 dpc. Serum, placenta and foetal metabolomic profiles were performed by 1H-NMR spectra following the analyses using Chenomx NMR Analysis Software V8.3. The tumour evolution was exponential, affecting the placental metabolomic profile during all the pregnancy stages. The placental tissue in tumour-bearing dams developed at a lower speed, decreasing the foetus's weight. Associated with the serum metabolomic changes related to tumour growth, the placental metabolomic alterations impacted many metabolic pathways related to energy provision, protein synthesis and signalling, which directly harmed the foetus's development. The development of the foetus is clearly affected by the damage induced by the tumour evolution, which alters the metabolic profile of both the serum and the placenta, impairing early embryonic development.

Aerobic training prevents cardiometabolic changes triggered by myocardial infarction in ovariectomized rats.

This study aimed to evaluate the impact of aerobic training (AT) on autonomic, cardiometabolic, ubiquitin-proteasome activity, and inflammatory changes evoked by myocardial infarction (MI) in ovariectomized rats. Female Wistar rats were ovariectomized and divided into four groups: sedentary + sham (SS), sedentary + MI (SI), AT + sham surgery (TS), AT + MI (TI). AT was performed on a treadmill for 8 weeks before MI. Infarcted rats previously subjected to AT presented improved physical capacity, increased interleukin-10, and decreased pro-inflammatory cytokines. Metabolomic analysis identified and quantified 62 metabolites, 9 were considered significant by the Vip Score. SS, SI, and TS groups presented distinct metabolic profiles; however, TI could not be distinguished from the SS group. MI dramatically increased levels of dimethylamine, and AT prevented this response. Our findings suggest that AT may be useful in preventing the negative changes in functional, inflammatory, and metabolic parameters related to MI in ovariectomized rats.

A Metabolomic Approach to Predict Breast Cancer Behavior and Chemotherapy Response.

Although the classification of breast carcinomas into molecular or immunohistochemical subtypes has contributed to a better categorization of women into different therapeutic regimens, breast cancer nevertheless still progresses or recurs in a remarkable number of patients. Identifying women who would benefit from chemotherapy could potentially increase treatment effectiveness, which has important implications for long-term survival. Metabolomic analyses of fluids and tissues from cancer patients improve our knowledge of the reprogramming of metabolic pathways involved in resistance to chemotherapy. This review evaluates how recent metabolomic approaches have contributed to understanding the relationship between breast cancer and the acquisition of resistance. We focus on the advantages and challenges of cancer treatment and the use of new strategies in clinical care, which helps us comprehend drug resistance and predict responses to treatment.

Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats.

BACKGROUND: Cachexia is one of the most important causes of cancer-related death. Supplementation with branched-chain amino acids, particularly leucine, has been used to minimise loss of muscle tissue, although few studies have examined the effect of this type of nutritional supplementation on the metabolism of the tumour-bearing host. Therefore, the present study evaluated whether a leucine-rich diet affects metabolomic derangements in serum and tumour tissues in tumour-bearing Walker-256 rats (providing an experimental model of cachexia). METHODS: After 21 days feeding Wistar female rats a leucine-rich diet, distributed in L-leucine and LW-leucine Walker-256 tumour-bearing groups, we examined the metabolomic profile of serum and tumour tissue samples and compared them with samples from tumour-bearing rats fed a normal protein diet (C - control; W - tumour-bearing groups). We utilised 1H-NMR as a means to study the serum and tumour metabolomic profile, tumour proliferation and tumour protein synthesis pathway. RESULTS: Among the 58 serum metabolites examined, we found that 12 were altered in the tumour-bearing group, reflecting an increase in activity of some metabolic pathways related to energy production, which diverted many nutrients toward tumour growth. Despite displaying increased tumour cell activity (i.e., higher Ki-67 and mTOR expression), there were no differences in tumour mass associated with changes in 23 metabolites (resulting from valine, leucine and isoleucine synthesis and degradation, and from the synthesis and degradation of ketone bodies) in the leucine-tumour group. This result suggests that the majority of nutrients were used for host maintenance. CONCLUSION: A leucine rich-diet, largely used to prevent skeletal muscle loss, did not affect Walker 256 tumour growth and led to metabolomic alterations that may partially explain the positive effects of leucine for the whole tumour-bearing host.

Dietary leucine supplementation minimises tumour-induced damage in placental tissues of pregnant, tumour-bearing rats.

BACKGROUND: The occurrence of cancer during pregnancy merges two complex, poorly understood metabolic and hormonal conditions. This association can exacerbate the conditions of both the mother and the foetus. The branched-chain amino acid leucine enhances cellular activity, particularly by increasing protein synthesis. This study aimed to analyse the modulatory effect of a leucine-rich diet on direct and indirect tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and assessing anti-oxidant enzyme activity in placental tissues collected from pregnant, tumour-bearing rats. RESULTS: Pregnant rats were either implanted with Walker 256 tumour cells or injected with ascitic fluid (to study the indirect effects of tumour growth) and then fed a leucine-rich diet. Animals in a control group underwent the same procedures but were fed a normal diet. On the 20(th) day of pregnancy, tumour growth was observed. Dams fed a normoprotein diet showed the greatest tumour growth. Injection with ascitic fluid mimicked the effects of tumour growth. Decreased placental protein synthesis and increased protein degradation were observed in both the tumour-bearing and the ascitic fluid-injected groups that were fed a normoprotein diet. These effects resulted in low placental DNA and protein content and high lipid peroxidation (measured by malondialdehyde content). Decreased placental protein synthesis-related gene expression was observed in the tumour group concomitant with increased expression of genes encoding protein degradation-associated proteins and proteolytic subunits. CONCLUSIONS: Consumption of a leucine-rich diet counteracted the effects produced by tumour growth and injection with ascitic fluid. The diet enhanced cell signalling, ameliorated deficiencies in DNA and protein content, and balanced protein synthesis and degradation processes in the placenta. The improvements in cell signalling included changes in the mTOR/eIF pathway. In conclusion, consumption of a leucine-rich diet improved placental metabolism and cell signalling in tumour-bearing rats, and these changes reduced the deleterious effects caused by tumour growth.

A leucine-rich diet modulates the tumor-induced down-regulation of the MAPK/ERK and PI3K/Akt/mTOR signaling pathways and maintains the expression of the ubiquitin-proteasome pathway in the placental tissue of NMRI mice.

Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process.

Leucine-rich diet improves the serum amino acid profile and body composition of fetuses from tumor-bearing pregnant mice.

Pregnancy is a complex process that can be jeopardized when associated with cancer, because of the coexistence of two complex metabolic conditions: a fetus and cancer. The aim of this study was to evaluate fetal growth in association with cancer development as well as the indirect effects produced by tumors in pregnant mice subjected to a leucine-rich diet, knowing that leucine supplementation can minimize the tumor effects by acting as a cell signaling agent to improve the protein synthesis process. We evaluated fetuses (n = 6) from NMRI pregnant mice fed either a control or a leucine-rich diet in either the presence or absence of an MAC16 colon adenocarcinoma or ascitic fluid inoculation. The fetal serum amino acids were separated using high-performance liquid chromatography, and fetal cytokine levels were analyzed using a microsphere-based multiplex immunoassay (Luminex xMAP). Fetal body composition was measured as the water, fat, and protein total content and total serum protein, albumin, and glucose content. Tumor growth resulted in a severe reduction in fetal body weight and protein content and increased fetal resorption, associated with placental weight decrease; these effects were minimized by a leucine-rich diet. Serum total protein and glucose content were reduced in fetuses from tumor-bearing dams but were reverted by nutritional supplementation. The serum amino acid profiles differed significantly between the tumor-bearing mice fed with a leucine-rich diet and controls. Certain tumor effects were reproduced in fetuses from ascitic fluid-injected dams, suggesting indirect effects of tumor growth. We conclude that certain effects of tumor growth can be mimicked by ascitic fluid injection and can be modulated by a leucine-rich diet.

Serum miR-181d-5p levels in response to controlled ovarian stimulation: predictive value and biological function.

The response to controlled ovarian stimulation (COS) for in vitro fertilization (IVF) varies dramatically from one patient to another, affecting success rates. A previous large-scale study identified increased serum miR-181d-5p levels in patients with high response to COS prior to stimulation. We aim to evaluate whether the expression of miR-181d-5p differs according to the ovarian response to COS in women undergoing intracytoplasmic sperm injection (ICSI) cycles. Samples collected prior to COS for ICSI were split into three groups depending on the ovarian response to COS: poor response (PR), <4 oocytes retrieved (n=25); normal response (NR), ≥8 and ≤12 oocytes retrieved (n=21); and high response (HR), >25 oocytes retrieved (n=20). miR-181d-5p serum levels were compared among experimental groups. miR-181d-5p levels were increased in the HR group when compared to the PR (p=0.0001) and NR groups (p=0.0079). miR-181d-5p levels correlated with the number of aspirated follicles (p<0.0001), retrieved oocytes (p<0.0001), and mature oocytes (p=0.0002). Increased miR-181d-5p levels independently predict a high response (p=0.006), with Positive and Negative Predictive Values of 66.7% and 69.4%, respectively. miR-181d-5p was also detected in the ovarian tissue in a mouse model. Moreover, computational analysis of miR-181d-5p predicted targets and promoter region suggested that this miRNA might be involved in the regulation of key signaling pathways and biological processes for female reproductive biology. In conclusion, miR-181d-5p is a promising circulating predictor of high stimulation and potential mediator of the hypothalamus-pituitary-gonad axis, providing opportunities for the individualization of COS protocols.

Pregnancy and Cancer: Cellular Biology and Mechanisms Affecting the Placenta.

Cancer during pregnancy is rarely studied due to its low incidence (1:1000). However, as a result of different sociocultural and economic changes, women are postponing pregnancy, so the number of pregnant women with cancer has been increasing in recent years. The importance of studying cancer during pregnancy is not only based on maternal and foetal prognosis, but also on the evolutionary mechanisms of the cell biology of trophoblasts and neoplastic cells, which point out similarities between and suggest new fields for the study of cancer. Moreover, the magnitude of how cancer factors can affect trophoblastic cells, and vice versa, in altering the foetus's nutrition and health is still a subject to be understood. In this context, the objective of this narrative review was to show that some researchers point out the importance of supplementing branched-chain amino acids, especially leucine, in experimental models of pregnancy associated with women with cancer. A leucine-rich diet may be an interesting strategy to preserve physiological placenta metabolism for protecting the mother and foetus from the harmful effects of cancer during pregnancy.

A Time-Course Comparison of Skeletal Muscle Metabolomic Alterations in Walker-256 Tumour-Bearing Rats at Different Stages of Life.

Cancer cachexia is a severe wasting condition that needs further study to find ways to minimise the effects of damage and poor prognosis. Skeletal muscle is the most impacted tissue in cancer cachexia; thus, elucidation of its metabolic alterations could provide a direct clue for biomarker research and be applied to detect this syndrome earlier. In addition, concerning the significant changes in the host metabolism across life, this study aimed to compare the metabolic muscle changes in cachectic tumour-bearing hosts at different ages. We performed 1H-NMR metabolomics in the gastrocnemius muscle in weanling and young adult Walker-256 tumour-bearing rats at different stages of tumour evolution (initial, intermediate, and advanced). Among the 49 metabolites identified, 24 were significantly affected throughout tumour evolution and 21 were significantly affected regarding animal age. The altered metabolites were mainly related to increased amino acid levels and changed energetic metabolism in the skeletal muscle, suggesting an expressive catabolic process and diverted energy production, especially in advanced tumour stages in both groups. Moreover, these changes were more severe in weanling hosts throughout tumour evolution, suggesting the distinct impact of cancer cachexia regarding the host's age, highlighting the need to adopting the right animal age when studying cancer cachexia.

Walker-256 Tumour-Induced Cachexia Altered Liver Metabolomic Profile and Function in Weanling and Adult Rats.

Cancer cachexia occurs in up to 85% of advanced cancer patients, affecting different tissues and organs, mainly the liver, which plays a central role in body metabolism control. However, liver responses to cancer cachexia progression are still poorly understood. Considering the possible different challenges provided by the rodent's phase of life and the cachexia progression, we evaluated the liver metabolic alterations affected by Walker-256 tumour growth in weanling and young-adult rats. For this, we applied a metabolomics approach associated with protein and gene expression analyses. Higher amino acid levels and impaired glucose metabolism were important features in tumour-bearing animals' liver tissue. The weanling hosts had more pronounced cachexia, with higher carcass spoliation, liver lipid metabolism and impaired CII and CIV mitochondrial complexes. The liver alterations in young adult tumour-bearing rats were related to energy status and nucleotide metabolites, such as uridine, NAD+, xanthosine, hypoxanthine and inosine. In conclusion, the Walker-256 tumour-induced cachexia impaired liver metabolism, being more severe in the weanling hosts. Further studies are needed to correlate these changes in the preclinical model, which can be correlated to the clinical features of cancer cachexia, allowing for a translational potential involving the liver function and its responses to potential treatments.

Leucine-Rich Diet Improved Muscle Function in Cachectic Walker 256 Tumour-Bearing Wistar Rats.

Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia.

1H-NMR Based Serum Metabolomics Identifies Different Profile between Sarcopenia and Cancer Cachexia in Ageing Walker 256 Tumour-Bearing Rats.

Sarcopenia among the older population has been growing over the last few years. In addition, the incidence of cancers increases with age and, consequently, the development of cachexia related cancer. Therefore, the elucidation of the metabolic derangements of sarcopenia and cachexia are important to improve the survival and life quality of cancer patients. We performed the 1H-NMR based serum metabolomics in adult (A) and ageing (S) Walker 256 tumour-bearing rats in different stages of tumour evolution, namely intermediated (Wi) and advanced (Wa). Among 52 serum metabolites that were identified, 21 were significantly increased in S and 14 and 19 decreased in the Wi and Wa groups, respectively. The most impacted pathways by this metabolic alteration were related by amino acid biosynthesis and metabolism, with an upregulation in S group and downregulation in Wi and Wa groups. Taken together, our results suggest that the increase in metabolic profile in ageing rats is associated with the higher muscle protein degradation that releases several metabolites, especially amino acids into the serum. On the other hand, we hypothesise that the majority of metabolites released by muscle catabolism are used by tumours to sustain rapid cell proliferation and tumorigenesis.

Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia.

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia.

Leucine-rich diet induces a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, reducing glucose consumption and metastasis in Walker-256 tumour-bearing rats.

Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both in vivo and in vitro models of a Walker-256 tumour. Tumour-bearing Wistar rats were randomly distributed into a control group (W; normoprotein diet) and leucine group (LW; leucine-rich diet [normoprotein + 3% leucine]). After 20 days of tumour evolution, the animals underwent 18-fludeoxyglucose positron emission computed tomography (18F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (18F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.