Background
Cancer is the second leading cause of disease-mediated death. The most common types of cancer in women are breast and cervical cancers [
1,
2]. Neoplasias are difficult to control and treat because they are associated with several biochemical and metabolic changes. These changes increase overall metabolic consumption in the host, especially in the skeletal muscle, and decrease lean body mass. This culminates in reduced quality of life and decreased survival in affected patients.
Moreover, the prevalence of cancer during pregnancy is not rare, and the coexistence of these two complex metabolic and hormonal conditions can negatively impact the mother and foetus [
3,
4]. To date, few studies have reported on the influence of pregnancy on cancer and vice versa [
3,
4]. Recent publications have indicated that pregnancy offers a protective effect against cancer, but other reports suggest that pregnancy can accelerate the growth and development of neoplasias. Furthermore, there are difficulties associated with diagnosing early-stage tumours in pregnant women [
5,
6]. Zemlickis and colleagues [
7] found that patients diagnosed with breast cancer during pregnancy exhibited more advanced tumours and a higher incidence of metastasis compared to non-pregnant controls. King and colleagues [
8] found a greater incidence of advanced stage tumours in patients who were pregnant. Pregnant patients with cancer can exhibit a more advanced evolution of the disease due to delays in diagnosis and because the hormonal and physiological changes that accompany pregnancy can result in a more aggressive disease response.
The endocrine changes associated with cancer evolution during pregnancy have not been well studied because of the many physiological adaptations that concomitantly occur in the maternal organism. These adaptations are primarily driven by the maternal/foetal unit known as the placenta [
9,
10]. Thus, additional studies examining how neoplastic progression is affected by the complex alteration of hormonal homeostasis imposed by pregnancy are needed. The aim of the current study was to evaluate how tumour development affects cellular activity in the placenta and how these changes interfere with placental protein metabolism. Previous studies have also shown that humoural factors produced by tumours and/or host cells can indirectly interfere with placental and foetal integrity. The consumption of a leucine-rich diet can enhance cellular activity; as such, this study also aimed to analyse the modulatory effect of nutritional supplementation of leucine on tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and by assessing anti-oxidant enzyme activity in placental tissues collected from pregnant tumour-bearing rats.
Discussion
Tumour development, especially development of Walker 256 tumours, which serve as an experimental model of cachexia [
21,
22], promotes the breakdown of structural tissue, including skeletal muscle mass [
23], and increases the production of protein waste in the host. Tumour growth also affects other tissues and metabolic processes [
24,
25]. In our previous experimental studies [
20,
26‐
28] and in the present study, we found that tumour growth causes severe damage during the course of pregnancy. This damage is particularly harmful to placental tissue and results in significant foetal weight loss and increased foetal resorption [
17,
20,
28]. Our previous results indicated that supplementation with a leucine-rich diet could prevent some of the effects induced by tumour evolution. Consumption of a leucine-rich diet also minimised the indirect effects produced by injections with ascitic fluid. Animals consuming this diet exhibited improvements in placental tissue health, leading to increased foetal numbers and enhanced expression of genes involved in placental cell signalling.
Foetal growth is primarily determined by nutrient availability, which is related to the capacity of the placenta to transport nutrients [
29,
30]. Foetal macronutrient requirements for oxidative metabolism and growth are met by placental transport, a process that is greatly influenced by the maternal bloodstream and by placental metabolism. The foetal nutrient supply is also considerably affected by the conversion of glucose to lactate (or fructose in some species) in the placenta and by the extensive transamination of amino acids. Placental capacity for nutrient transport increases with foetal demand. As such, maternal-foetal transport kinetics are tightly associated with the expression and distribution of specific transporters among placental cell types and with placental antioxidant response capacity against damage [
17] or reduced nutritional supply [
29]. Abnormal placental functioning affects foetal growth, especially during late gestation [
29‐
34]. Here, in pregnant, tumour-bearing rats, the consequences of tumour evolution were demonstrated. These included decreased body weight and deleterious effects on foetal growth. These changes suggest that the nutritional supply to the placenta and foetus was diverted to benefit tumour growth. Indirect tumour effects were assessed using an ascitic fluid injection model. This enabled the exclusion of nutritional factors from the observed placental and foetal damage and reinforced that substances produced by tumour and/or host cells can jeopardise the placenta and foetal development.
Several reports have suggested that placental oxidative stress is involved in the etiopathogenesis of pregnancy-related diseases, such as preeclampsia, as well as in foetal growth restriction [
17,
29,
35‐
37]. Reduced perfusion as a result of abnormal placentation leads to ischemic injury and to increased oxidative stress in preeclampsia [
35,
36]. The activity of GST, an important detoxification enzyme related to reactive oxygen species processing, can greatly contribute to both foetal and placental antioxidative responses. Consistent with this fact, the activity of this enzyme increased in the groups inoculated with ascites fluid and in the groups supplemented with a leucine-rich diet (L, WL and LA). This result suggests that placental tissue and foetal development were improved. In a previous study, MDA and xanthine oxidase (XO) levels were higher in maternal plasma, umbilical cord plasma, and placental tissues of patients with intrauterine growth restriction (IUGR) than in a control group [
17,
35,
36]. Conversely, patients with IUGR exhibited lower antioxidant levels in maternal plasma, umbilical cord plasma, and placental tissues [
36]. This reduction suggests that oxidative stress increases in patients with IUGR [
35,
38]. As cancer also increases oxidative stress in host tissues [
17,
23], in the current study, we inferred that oxidative stress-related damage in placental tissue resulted from tumour growth effects. The observed increases in foetal resorption and reductions in foetal weight likely resulted from decreased placental antioxidative responses. In the W group, increased lipid peroxidation was also observed. Additionally, AP activity, which is generally related to cellular activity and cellular transport, was significantly reduced in the W group. This reduction suggests that tumour growth has deleterious effects on pregnancy. Conversely, placental dysfunction associated with increased lipid peroxidation (MDA) was significantly reduced in the groups fed a leucine-rich diet (L, LW and LA). This reduction was likely associated with the observed positive responses and improvements in foetal and placenta parameters in these groups [
17,
20,
28].
Placental function is regulated by numerous factors of foetal, maternal, and placental origin [
34,
39‐
43]. These factors may impact the placenta itself in an autocrine/paracrine manner by integrating numerous and sometimes divergent intracellular regulatory signalling pathways [
34,
41‐
43]. Moreover, changes in placental nutrient transport capacity or supply and/or placental cell activities may not be the primary factors responsible for altered foetal growth, as we verified here in the W group. Other factors could be involved in this harmful process, as evidenced by the similar alterations observed in the A group and the W group. Thus, the most important factor influenced by tumour evolution may be the regulation of placental transport function by maternal and foetal signalling molecules and placental cell signalling. It is important to note that the placenta produces many cytokines and hormones that can act in a paracrine or autocrine manner. These compounds can be affected by humoural factors in ascitic fluid and likely jeopardise placental activity, inducing foetal damage. However, in the A group, several tumour effects were either minimised or abolished, suggesting that placental cell activity can be specifically regulated and that regulatory factors may have opposing effects on different placental process, such as placental transport and nutrient provision to the foetus. Thus, the integration of multiple stimuli is critical for adjusting placental function to accommodate maternal and foetal welfare.
Placental trophoblast cells must integrate numerous and possibly divergent maternal and foetal stimuli and modify cellular functioning according to the host-environment interaction, as we have previously reported [
17,
28]. Previous studies have demonstrated that placental signalling requires the integration of multiple pathways, including the regulation of nutrient transport and the regulation of the mTOR signalling pathway, which is the main pathway regulating placental amino acid transport [
39‐
43]. Additionally, alterations in the activity and/or expression of placental mTOR and PPARγ have been observed in human pregnancies complicated by altered foetal growth [
31,
42]. It is likely that the reduced foetal growth and increased foetal resorption observed in tumour-bearing rats had multifactorial causes and were potentially associated with decreases in the activities of cell signalling proteins. The above changes are likely affected by placental protein synthesis and degradation, two processes that can be differentially affected by tumour factors (such as proteolysis-inducing factor (PIF) or Walker factor (WF)) to cause foetal impairment in different manners [
31,
34]. The mTOR/eIF2 signalling pathway controls protein synthesis in response to nutrient availability. Moreover, mTOR is a positive regulator of placental nutrient transport and is involved in the regulation of foetal growth. These functions are consistent with the results reported here: the levels of mTOR, p70S6K1 and other related proteins decreased in the W group, suggesting failure in placental delivery of nutrition. Conversely, other regulatory processes related may have been responsible for the foetal impairment observed in group A independent of nutrient supply.
The inoculation of ascitic fluid and the effects of humoural factors mimicked the results of PIF and WF in tumour-bearing rats [
44]. We previously reported that WF significantly alters protein metabolism and induces foetal damage [
2,
11,
44]. It is reasonable to infer that the more severe effects of tumour growth, whether direct or indirect, result from the activation of the proteolytic pathway in placental tissue (increased MuRF-1 and ubiquitin gene and protein expression), especially in the W group and, to a lesser extent, in the A group. This process is responsible for placental failure and consequent foetal damage.
Our study highlights the important role of a leucine-rich diet in modulating placental cell activity and maintaining antioxidative response during tumour growth. As many other studies have proposed, leucine, one of the three branched-chain amino acids, can act as a cellular signal to increase protein synthesis downstream of mTOR, p70S6K1 and eIF4G in skeletal muscle [
45‐
47]. In a previous report, we also demonstrated that consumption of a leucine-rich diet enhances protein synthesis in muscle. Moreover, leucine can improve cellular responses to antioxidative stress and reduce proteolysis by inhibiting the ubiquitin-proteasome pathway [
27,
48‐
51]. Despite foetal weight loss, the tumour-bearing group that received leucine nutritional supplementation exhibited improvements in foetal number and resorption as well as in placental tissue, the most crucial factor in maintaining a normal pregnancy course.
As in previous studies in our laboratory, we found that tumour growth promotes intense mobilisation of muscle proteins associated with diminished protein synthesis [
25‐
27]. Tumour-induced placenta damage was related to changes in key protein-synthesis proteins and increased proteolysis. Similar to other studies, nutritional leucine supplementation did not prevent all harmful effects on foetal development, but it did minimise some of the harmful effects on placental cell activity, enhance protein synthesis and reduce placental proteolysis caused by the direct and indirect effects of tumour growth. However, consistent with increased foetal numbers and reduced foetal absorption rates, alterations in protein synthesis and degradation were observed, as reflected by placental protein balance. This demonstrates that tumour growth (both directly, as in groups W and WL, and indirectly, as in groups A and LA) promotes important changes in the maternal-foetal unit, and these changes can be ameliorated with nutritional leucine supplementation.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
BLGC acquired, analysed and interpreted data. PCS acquired and analysed data. RT performed PCRs molecular assay and analysed and interpreted data. AGO performed Western Blot assay and analysed and interpreted data. LRV acquired, analysed and interpreted data. EMS performed protein and degradation assay and analysed and interpreted data. MCCGM conceived and coordinated the study, aided in the interpretation and discussion of the results and prepared and revised the final manuscript. All authors have read and approved the final text of the manuscript.