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Placental dysfunction leads to foetal damage, which jeopardises the exchange between the maternal and foetal systems. We evaluated the effects of tumour growth on the activity of antioxidant enzymes and oxidative stress in placental tissue and cell culture from tumour-bearing pregnant rats compared to non-tumour-bearing pregnant rats that were ascitic fluid injected. Ascitic fluid is obtained from Walker tumour-bearing rats and contains a cytokine called Walker factor (WF), which is a molecule similar to proteolysis-inducing factor (PIF), and induces changes in protein metabolism and oxidative stress. Pregnant Wistar rats were distributed into control (C), tumour-bearing (W) and ascitic fluid injected (A) groups and were sacrificed on days 16, 19 and 21 of pregnancy to analyse the profile of enzyme activities (glutathione-S-transferase (GST), catalase (CAT), alkaline phosphatase (AP)) and malondialdehyde (MDA) content in placental tissue. Meanwhile, placenta samples from all groups were obtained on day 21, placed in primary culture and treated with WF for 72 h. The presence of tumour or ascitic fluid reduced the protein content of the placental tissue. On day 16 there was a significant reduction in AP activity in W rats, and on day 19, CAT activity and MDA content significantly increased. These results indicate that the presence of cancer decreased antioxidant enzyme capacity in the placenta, increasing the amount of oxidation in these cells, which may contribute to irreversible placental damage and compromisefoetal development. WF treatment induces similar changes in placental cells in primary culture, resulting in less cell viability and increased oxidative stress. These results indicate that WF, provided by the tumour or inoculation of ascitic fluid, has negative effects on placental homeostasis, which impairs foetal health., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin-proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S alpha proteasome subunit, 19S MSSI ATPase regulator subunit and 11S alpha subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin-proteasome system during tumour growth.

The placenta provides all energy and nutrient requirements for healthy fetal development. The placenta in rats is capable of storing glycogen, although the placenta cells must therefore mobilize stored glycogen to its own glucose supply. Moreover, maternal glucose and/or placental lactate furnished the fetal growth. Adult female Wistar rats were divided into three groups: Control-C, tumour bearing-W; injected ascitic fluid-A. The rats were sacrificed on the 16th, 19th or 21st day of gestation, analysing the placenta and fetus weights and placental tissue samples was aliquoted for biochemical assays of glycogen and protein content and alkaline phosphatase activity. Placental sections were morphometrically analysed and glycogen positive cells were counted. The placental and fetal weight were significantly reduced in both W and A rats from 16th up to 21st day of gestation, which showed high levels of fetal reabsorption sites. Significant reduction in labyrinth zone at day 21 in both tumour bearing and ascitic fluid injected groups was shown, suggesting less substrate exchange at the maternal/fetal surface. The alkaline phosphatase activity as well total protein content were found to be reduced in W and A group. The total placental glycogen and glycogen cells decreased during tumour bearing and ascitic fluid injection, suggesting reduction in its own stored energy. Ascitic fluid injected group, representing an indirect tumour effect, presented similar reduction changes in the placenta to the tumour-bearing group. In conclusion, the tumour growth and, especially, ascitic fluid injection promoted irreversible placental tissue damage altering homeostasis and compromising fetal development.

Cancer cachexia induces host protein wastage but the mechanisms are poorly understood. Branched-chain amino acids play a regulatory role in the modulation of both protein synthesis and degradation in host tissues. Leucine, an important amino acid in skeletal muscle, is higher oxidized in tumor-bearing animals. A leucine-supplemented diet was used to analyze the effects of Walker 256 tumor growth on body composition in young weanling Wistar rats divided into two main dietary groups: normal diet (N, 18% protein) and leucine-rich diet (L, 15% protein plus 3% leucine), which were further subdivided into control (N or L) or tumor-bearing (W or LW) subgroups. After 12 days, the animals were sacrificed and their carcass analyzed. The tumor-bearing groups showed a decrease in body weight and fat content. Lean carcass mass was lower in the W and LW groups (W = 19.9 0.6, LW = 23.1 1.0 g vs N = 29.4 1.3, L = 28.1 1.9 g, P < 0.05). Tumor weight was similar in both tumor-bearing groups fed either diet. Western blot analysis showed that myosin protein content in gastrocnemius muscle was reduced in tumor-bearing animals (W = 0.234 0.033 vs LW = 0.598 0.036, N = 0.623 0.062, L = 0.697 0.065 arbitrary intensity, P < 0.05). Despite accelerated tumor growth, LW animals exhibited a smaller reduction in lean carcass mass and muscle myosin maintenance, suggesting that excess leucine in the diet could counteract, at least in part, the high host protein wasting in weanling tumor-bearing rats.

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C(2)C(12) myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 microM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the alpha-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E2(14k)), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia., (comCopyright 2002 Cancer Research UK)