Walker-256 Tumor-bearing Rats Research Articles

Pharmacokinetic-Pharmacodynamic Analysis of pH-Responsive Doxorubicin-Releasing Micelles with Anticancer Activity.

This study aimed to investigate the effect of in vivo pH-responsive doxorubicin (DOX) release and the targetability of pilot molecules in folic acid (FA)-modified micelles using a pharmacokinetic-pharmacodynamic (PK-PD) model. The time profiles of intratumoral DOX concentrations in Walker256 tumor-bearing rats were monitored using a microdialysis probe, followed by compartmental analysis, to evaluate intratumoral tissue pharmacokinetics. Maximal DOX was released from micelles 350 min after the administration of pH-responsive DOX-releasing micelles. However, FA modification of the micelles shortened the time to peak drug concentration to 150 min. Additionally, FA modification resulted in a 27-fold increase in the tumor inflow rate constant. Walker256 tumor-bearing rats were subsequently treated with DOX, pH-responsive DOX-releasing micelles, and pH-responsive DOX-releasing FA-modified micelles to monitor the tumor growth-time profiles. An intratumoral threshold concentration of DOX (55-64 ng/g tumor) was introduced into the drug efficacy compartment to construct a PD model, followed by PK-PD analysis of the tumor growth-time profiles. Similar results of threshold concentration and drug potency of DOX were obtained across all three formulations. Cell proliferation was delayed as the drug delivery ability of DOX was improved. The PK model, which was developed using the microdialysis method, revealed the intratumoral pH-responsive DOX distribution profiles. This facilitated the estimation of intratumoral PK parameters. The PD model with threshold concentrations contributed to the estimation of PD parameters in the three formulations, with consistent mechanisms observed. We believe that our PK-PD model can objectively assess the contributions of pH-responsive release ability and pilot molecule targetability to pharmacological effects.

Effects of cancer-induced cachexia and administration of l-glutathione on the intestinal mucosa in rat

Walker-256 tumor is an experimental model known to promote cachexia syndrome, oxidative stress, and systemic inflammation. This study evaluated the duodenal mucosa of rats with Walker-256 tumor administered with 1% l-glutathione, intending to evaluate the damage caused by cancer-associated cachexia in the gastrointestinal tract and the effects of antioxidant administration on mucosal protection. Twenty-four 55-day-old male Wistar rats were distributed into four groups: control (C); control administered with 1% l-glutathione (C-GSH); Walker-256 tumor (W) and Walker-256 tumor administered with 1% l-glutathione (W-GSH). After 14 days of treatment, the duodenum was harvested for morphometric analysis of the mucosa, proliferation, apoptosis, immunostaining of varicosities immunoreactive (IR) to vasoactive intestinal peptide (VIP) and 5-HT-IR cells, and quantification of mast cells and goblet cells. Walker-256 tumor-bearing rats showed cachexia syndrome, mucosal atrophy, reduced cell proliferation, reduced 5-HT-IR cells, and increased goblet cells and VIPergic varicosities, which were not reversed by l-glutathione. On the other hand, l-glutathione caused a reduction of cells in apoptosis and mast cell recruitment, demonstrating a partial recovery of the damage detected in the intestinal mucosa.

L-glutathione 1% promotes neuroprotection of nitrergic neurons and reduces the oxidative stress in the jejunum of rats with Walker-256-bearing tumor.

Our main goals were to investigate the effects of L-glutathione (1%) treatment in Walker-256 tumor-bearing rats by analyzing immunoreactive neurons (IR), responsive to the nNOS enzyme and 3-Nitrotyrosine, in their jejunum myenteric plexus. Moreover, the oxidative state and inflammatory process in these animals were investigated. Four experimental groups were utilized: control (C), control treated with L-glutathione (CGT), Walker-256 tumor-bearing rats (TW), and Walker-256 tumor-bearing rats treated with L-glutathione (TWGT). After 14 days of tumor inoculation, the jejunum was collected for immunohistochemical techniques and assessment of oxidative status. Plasma was collected to evaluate oxidative status and measure cytokines. The TW group exhibited a decrease of reduced glutathione in their jejunum, which was prevented in the L-glutathione treated TWGT group. TW animals presented pronounced oxidative stress by increasing levels of lipoperoxidation in their jejunum and malondialdehyde in their plasma; however, the L-glutathione treatment in TWGT group was not able to avoid it. The total antioxidant capacity was altered in groups TW and TWGT, yet the last one had a better index in their plasma. The IL-10, and TNF-α levels increased in TWGT animals. The nNOS-IR neuron density decreased in the jejunum myenteric plexus of the TW group, which was avoided in the TWGT group. The nNOS +3-Nitrotyrosine neurons quantification did not show significative alterations. The treatment with L-glutathione (1%) imposed an important defense to some parameters of oxidative stress induced by TW-256, leading to neuroprotection to the loss in the nNOS-IR neuron density.

Insulin in combination with pioglitazone prevents advanced cachexia in 256-Walker tumor-bearing rats: effect is greater than treatment alone and is associated with improved insulin sensitivity.

Insulin (INS) resistance and hypoinsulinemia commonly observed in cancer-carrying, can contribute to cachexia. However, the effects of INS and INS sensitizers, such as pioglitazone (PIO), particularly when used in combination therapy, on cancer cachexia have not been evaluated sufficiently. We investigated the effects of INS and PIO, at various doses, either isolated or combined, on cachexia in Walker-256 tumor-bearing rats (TB rats). INS or INS + PIO were administered in TB rats, for 6 or 12days, starting from the day of tumor cells inoculation. INS at 18 or 27 U/kg (12-days treatment), but not 9 U/kg, reduced fat loss and slightly prevented weight loss. However, INS 18 U/kg + PIO 5, 10, 20, or 40mg/kg (6 or 12-day treatment) reduced fat loss and markedly prevented weight loss but did not affect muscle wasting. While TB rats lost weight (37.9% in 12days), TB rats treated with INS 18 U/kg + PIO 5mg/kg showed pronounced weight gain (73.7%), which was greater than the sum (synergism) of the weight gains promoted by isolated treatments with INS 18 U/kg (14.7%) or PIO 5mg/kg (13.1%). The beneficial effect of the INS 18 U/kg + PIO 5mg/kg on weight loss was associated with improved INS sensitivity, as indicated by the higher blood glucose clearance constant (kITT), decreased levels of free fatty acids and triacylglycerols (INS resistance-inducing factors) in the blood, and increased expression of p-Akt (INS signaling pathway protein) in adipose tissue. The combined treatment with INS 18 U/kg + PIO 5mg/kg was more effective in preventing advanced cachexia in TB rats than each treatment alone, emerging as the best approach, considering the lower dosage and higher efficacy. This combination completely preserved adipose mass and markedly reduced weight loss through a synergistic mechanism linked to improved insulin sensitivity. These findings provide new insights into the importance of drug combinations in effectively combating fat loss in advanced cachexia.

Peripheral insulin resistance is early, progressive, and correlated with cachexia in Walker-256 tumor-bearing rats.

Insulin (INS) resistance is often found in cancer-bearing, but its correlation with cachexia development is not completely established. This study investigated the temporal sequence of the development of INS resistance and cachexia to establish the relationship between these factors in Walker-256 tumor-bearing rats (TBrats). INS hepatic sensitivity and INS resistance-inducing factors, such as free fatty acids (FFA) and tumor necrosis factor-α (TNF-α), were also evaluated. Studies were carried out on Days 2, 5, 8, and/or 12 after inoculation of tumor cells in rats. The peripheral INS sensitivity was assessed by theINS tolerance testand the INS hepatic sensitivity in in situ liver perfusion. TBrats with 5, 8, and 12 days of tumor, but not 2 days, showed decreased peripheral INS sensitivity (INS resistance), retroperitoneal fat, and body weight, compared to healthy rats, which were more pronounced on Day 12. Gastrocnemius muscle wasting was observed only on Day 12 of tumor. The peripheral INS resistance was significantly correlated (r = -.81) with weight loss. Liver INS sensitivity of TBrats with 2 and 5 days of tumor was unchanged, compared to healthy rats. TBrats with 12 days of tumor showed increased plasma FFA and increased TNF-α in retroperitoneal fat and liver, but not in the gastrocnemius, compared to healthy rats. In conclusion, peripheral INS resistance is early, starts along with fat and weight loss and before muscle wasting, progressive, and correlated with cachexia, suggesting that it may play an important role in the pathogenesis of the cachectic process in TBrats. Therefore, early correction of INS resistance may be a therapeutic approach to prevent and treat cancer cachexia.

Influence of red wine polysaccharides on cytochrome P450 enzymes and inflammatory parameters in tumor models

The soluble fraction of polysaccharides from cabernet franc red wine (SFP) previously showed antitumoral effects by modulating the immune system. The present study tested the hypothesis that the SFP can regulate CYPs in vitro in HepG2 cells and in vivo in Walker-256 tumor-bearing rats. The SFP was used in the following protocols: (i) solid tumor, (ii) liquid tumor, and (iii) chemopreventive solid tumor. The SFP reduced solid tumor growth in both solid tumor protocols but did not inhibit liquid tumor development. The SFP reduced total CYP levels in the solid and liquid tumor protocols and reduced the gene expression of Cyp1a1 and Cyp2e1 in rats and CYP1A2 in HepG2 cells. An increase of N-acetylglucosaminidase activity was observed in all SFP-treated rats, and TNF-α levels increased in the solid tumor protocol in the vehicle, SFP, and vincristine (positive control) groups. The chemopreventive solid tumor protocol did not modify CYP levels in the liver or intestine or N-acetylglucosaminidase and myeloperoxidase activity in the liver. The in vitro digestion and nuclear magnetic resonance analyses suggested that SFP was minimally modified in the gastrointestinal system. In conclusion, SFP inhibited CYPs both in vivo and in vitro, likely as a result of its immunoinflammatory actions.

Insulin secretion decline in Walker-256 tumor-bearing rats is early, follows the course of cachexia, and is not improved by lixisenatide.

Lixisenatide, a glucagon-like peptide-1 receptor agonist, is used to stimulate insulin secretion in patients with type 2 diabetes mellitus. However, its effect on insulin secretion in cancer patients, particularly during the cachexia course, has not yet been evaluated. The purpose of this study was to investigate the lixisenatide effect on INS secretion decline during the cachexia course (2, 6, and 12 days of tumor) in pancreatic islets isolated from Walker-256 tumor-bearing rats. Pancreatic islets of healthy and tumor-bearing rats were incubated in the presence or absence of lixisenatide (10 nM). Tumor-bearing rats showed reduction of body weight and fat and muscle mass, characterizing the development of cachexia, as well as reduction of insulinemia and INS secretion stimulated by glucose (5.6, 8.3, 11.1, 16.7, and 20 mM) on days 2, 6, and/or 12 of tumor. Lixisenatide increased the 16.7 mM glucose-stimulated insulin secretion, but not by 5.6 mM glucose, in the islets of healthy rats, without changing the insulin intracellular content. However, lixisenatide did not prevent the decreased 16.7 mM glucose-stimulated insulin secretion in the pancreatic islets of rats with 2, 6, and 12 days of tumor and neither the decreased insulin intracellular content of rats with 12 days of tumor. In consistency, in vivo treatment with lixisenatide (50 μg kg-1, SC, once daily, for 6 days) visually increased insulinemia of healthy fasted rats, but did not prevent hypoinsulinemia of tumor-bearing rats. In conclusion, Walker-256 tumor-bearing rats showed early decline (2 days of tumor) of insulin secretion, which followed the cachexia course (6 and 12 days of tumor) and was not improved by lixisenatide, evidencing that this insulin secretagogue, used to treat type 2 diabetes, does not have beneficial effect in cancer bearing-rats.

Laser-photobiomodulation on experimental cancer pain model in Walker Tumor-256

ContextCancer Pain is considered a common and significant clinical problem in malignant neoplasms, comprising 20% to 50% of all patients with tumor progression. Laser photobiomodulation (L-PBM) has been used in a multitude of pain events, ranging from acute trauma to chronic articular. However, L-PBM has never been tested in cancer pain. ObjectivesEvaluate hyperalgesia, edema, COX-1, COX-2, IL-10, and Bdkrb1 mRNA in low-level laser irradiated Walker-256 tumor-bearing rats. MethodsRat hind paw injected with Walker Tumor-256 (W-256) and divided into six groups of 6 rats: G1 (control) - W-256 injected, G2- W-256 + Nimesulide, G3- W-256 + 1 J, G4- W-256 + 3 Jand G5- W256 + 6 J. Laser parameters: λ = 660 nm, 3.57 W/cm2, Ø = 0.028 cm2. Mechanical hyperalgesia was evaluated by Randall–Selitto test. Plethysmography measured edema; mRNA levels of COX-1, COX-2, IL-10, and Bdkrb1were analyzed. ResultsIt was found that the W-256 + 1 J group showed a decrease in paw edema, a significant reduction in pain threshold. Higher levels of IL-10 and lower levels of COX-2 and Bdkrb1 were observed. ConclusionResults suggest that 1 J L-PBM reduced the expression of COX-2 and Bdkrb1 and increasing IL-10 gene expression, promoting analgesia to close levels to nimesulide.

Creatine supplementation does not promote tumor growth or enhance tumor aggressiveness in Walker-256 tumor-bearing rats.

This study aimed to analyze the effect of creatine (Cr) supplementation on tumor microenvironment, evaluating the parameters of tumor aggressiveness. Sixteen male Wistar rats were randomly assigned to 2 groups (n=8/group): Tumor-bearing (T) and tumor-bearing supplemented with Cr (TCr). Cr supplementation was provided in drinking water for a total of 21 d. After 11 d of Cr supplementation (TCr group) or water (T group), Walker-256 tumor cells were inoculated subcutaneously in the right flank of all rats, which kept receiving Cr supplementation (TCr group) or water (T group) for 10 more days. The total period of the experiment was 21 d. Tumor weight corresponded with approximately 3.5% ± 0.9% of animal body weight in the T group. Cr supplementation did not accelerate tumor growth or increase tumor size. The histopathological analysis demonstrated the presence of nuclear pleomorphisms and atypical nuclei, with the presence of low-differentiated tumor cells, in both groups. Cr supplementation did not alter apoptosis and cell proliferation markers, nor tumor capsule thickness and viable tumor area. Cr supplementation in Walker-256 tumor-bearing rats did not induce significant changes in tumor development, and did not interfere with the parameters of tumor aggressiveness, such as the level of cell differentiation and proliferation.

Tumor development in rats and cancer cachexia are reduced by treatment with botryosphaeran by increasing apoptosis and improving the metabolic profile

AimsCancer is a multifactorial disease characterized by an uncontrolled growth of cells that can lead to cachexia-anorexia syndrome. Botryosphaeran, a fungal (1 → 3)(1 → 6)-β-D-glucan produced by Botryosphaeria rhodina MAMB-05, has presented antimutagenic, antiproliferative, pro-apoptotic, hypoglycemic and hypocholesterolemic effects. This study evaluated the effects of botryosphaeran (30 mg/kg b.w./day) on tumor development and cachexia syndrome in Walker-256 tumor-bearing rats, and also the metabolic and hematological profiles of these animals. Materials and methodsMale Wistar rats were divided into 3 groups: control (C), control tumor (CT) and control tumor botryosphaeran (CTB). On the first day, 1 × 107 Walker-256 tumor cells were inoculated subcutaneously into the right flank of the CT and CTB rats, and concomitantly treatment with botryosphaeran (30 mg/kg b.w./day) started. After the 15th day of treatment, biological parameters, tumor development, cachexia, glucose and lipid profiles, hemogram and protein expression were analyzed. Key findingsBotryosphaeran significantly reduced tumor development (p = 0.0024) and cancer cachexia, modulated the levels of glucose, triglycerides and HDL-cholesterol, and corrected macrocytic anemia. Botryosphaeran also increased significantly the bax expression in the tumor tissue (p = 0.038) demonstrating that this (1 → 3)(1 → 6)-β-D-glucan is increasing the apoptosis of tumor cells. p53, p27, bcl-2, caspase-3 and Forkhead transcription factor 3a (FOXO3a) protein expression were similar among the groups. SignificanceThis study demonstrated that botryosphaeran was effective in decreasing tumor development and cachexia by direct and indirect mechanisms increasing apoptosis and improving the metabolic and hematological profiles.

Akt activation by insulin treatment attenuates cachexia in Walker-256 tumor-bearing rats.

Cancer-bearing often exhibits hypoinsulinemia, insulin (INS) resistance and glutamine depletion associated with cachexia. However, INS and glutamine effects on cachexia metabolic abnormalities, particularly on tumor-affected proteins related to INS resistance, are poorly known. The main purpose of this study was to investigate the effects of INS and glutamine dipeptide (GDP) treatments on phospho-protein kinase B (p-Akt), and phospho-hormone sensitive lipase (p-HSL) in Walker-256 tumor-bearing rats. INS (NPH, 40 UI/kg, subcutaneous), GDP (1.5 g/kg, oral), INS+GDP or vehicle (control rats) were administered for 13 days, once a day, starting at the day of inoculation of tumor cells. The experiments were performed 4 hours after the last treatment to evaluate acute effects of INS and GDP, besides the chronic effects. INS and/or INS+GDP treatments, which markedly increased the insulinemia, increased the p-Akt: total Akt ratio and prevented the increased p-HSLSer552 : total HSL ratio in the retroperitoneal fat of tumor-bearing rats, without changing the INS resistance and increased expression of factor tumor necrosis-α (TNF-α) in this tissue. INS and INS+GDP also increased the p-Akt: total Akt ratio, whereas GDP and INS+GDP increased the GLUT4 glucose transporter gene expression, in the gastrocnemius muscle of the tumor-bearing rats. Accordingly, treatments with INS and INS+GDP markedly reduced glycemia, increased retroperitoneal fat and attenuated the body mass loss of tumor-bearing rats. In conclusion, hyperinsulinemia induced by high-dose INS treatments increased Akt phosphorylation and prevented increased p-HSLSer552 : total HSL ratio, overlapping INS resistance. These effects are consistent with increased fat mass gain and weight loss (cachexia) attenuation of tumor-bearing rats, evidencing that Akt activation is a potential strategy to prevent loss of fat mass in cancer cachexia.

Resistance Exercise Counteracts Tumor Growth in Two Carcinoma Rodent Models.

Although resistance exercise (RE) is now recognized as an adjuvant in cancer treatment because of its capacity to prevent muscle wasting, weakness, and cachexia, it is unknown whether RE can mitigate tumor development. Two solid adenocarcinoma models (Walker-256 and Ehrlich) were used to investigate the effects of RE on tumor cell proliferation, growth, and aggressiveness parameters in tumor-bearing animals' life span. Walker-256 tumor-bearing rats and Ehrlich tumor-bearing mice were subjected to RE, which consisted of climbing a ladder apparatus with loads tied to their tails. After 4 wk, animals were euthanized, and tumors were excised and assessed for tumor microenvironment evaluation such as cell proliferation and apoptosis determination, collagen deposit, and presence of malignant tumor morphology. Our data demonstrate that RE mitigated tumor growth and favored tumor end points such as lower Scarff-Bloom-Richardson histological grade tumor, denoting slow cell aberrant form and division, decreased tumor cell proliferation (evaluated by nucleus marked with antigen ki-67), and lower viable tumor area in both types of tumors studied. In addition, RE stimulated tumor microvessel density in Walker-256 tumor-bearing rats, but there was no change in their life span. RE may mitigate tumor growth and tumor malignancy parameters such as lower histopathological grade, assuming less nuclear pleomorphism and mitotic cells, smaller viable tumor area, and decreased tumor cell proliferation in both adenocarcinomas. In addition, RE induced tumor vascularization.

Creatine supplementation in Walker-256 tumor-bearing rats prevents skeletal muscle atrophy by attenuating systemic inflammation and protein degradation signaling.

The aim of this study was to investigate the effects of creatine supplementation on muscle wasting in Walker-256 tumor-bearing rats. Wistar rats were randomly assigned into three groups (n = 10/group): control (C), tumor bearing (T), and tumor bearing supplemented with creatine (TCr). Creatine was provided in drinking water for a total of 21days. After 11days of supplementation, tumor cells were implanted subcutaneously into T and TCr groups. The animals' weight, food and water intake were evaluated along the experimental protocol. After 10days of tumor implantation (21 total), animals were euthanized for inflammatory state and skeletal muscle cross-sectional area measurements. Skeletal muscle components of ubiquitin-proteasome pathways were also evaluated using real-time PCR and immunoblotting. The results showed that creatine supplementation protected tumor-bearing rats against body weight loss and skeletal muscle atrophy. Creatine intake promoted lower levels of plasma TNF-α and IL-6 and smaller spleen morphology changes such as reduced size of white pulp and lymphoid follicle compared to tumor-bearing rats. In addition, creatine prevented increased levels of skeletal muscle Atrogin-1 and MuRF-1, key regulators of muscle atrophy. Creatine supplementation prevents skeletal muscle atrophy by attenuating tumor-induced pro-inflammatory environment, a condition that minimizes Atrogin-1 and MuRF-1-dependent proteolysis.

A Population Dynamic Energy Budget-Based Tumor Growth Inhibition Model for Etoposide Effects on Wistar Rats.

This work aimed to develop a population PK/PD tumor-in-host model able to describe etoposide effects on both tumor cells and host in Walker-256 tumor-bearing rats. Etoposide was investigated on thirty-eight Wistar rats randomized in five arms: two groups of tumor-free animals receiving either placebo or etoposide (10mg/kg bolus for 4days) and three groups of tumor-bearing animals receiving either placebo or etoposide (5 or 10mg/kg bolus for 8 or 4days, respectively). To analyze experimental data, a tumor-in-host growth inhibition (TGI) model, based on the Dynamic Energy Budget (DEB) theory, was developed. Total plasma and free-interstitial tumor etoposide concentrations were assessed as driver of tumor kinetics. The model simultaneously describes tumor and host growths, etoposide antitumor effect as well as cachexia phenomena related to both the tumor and the drug treatment. The schedule-dependent inhibitory effect of etoposide is also well captured when the intratumoral drug concentration is considered as the driver of the tumor kinetics. The DEB-based TGI model capabilities, up to now assessed only in mice, are fully confirmed in this study involving rats. Results suggest that well designed experiments combined with a mechanistic modeling approach could be extremely useful to understand drug effects and to describe all the dynamics characterizing in vivo tumor growth studies.

ANTITUMOR AND GENOTOXIC EFFECTS OF LACTOFERRIN IN WALKER-256 TUMOR-BEARING RATS

To investigate the influence of exogenous lactoferrin (LF) on tumor growth, energy and lipid metabolism of Walker-256carcinosarcoma and to assess genotoxic effects of LF. The study was performed on Walker-256 tumor-bearing rats. Total lipids and phospholipids were determined by thin-layer chromatography. Comet assay was used to investigate the genotoxic effects of LF. Daily i.p. administrations of exogenous LF at concentrations of 1mg/kg and 10mg/kg starting from the 4th day after tumor transplantation suppressed growth of Walker-256carcinosarcoma by almost 44%. After treatment with recombinant LF in both doses, the phospholipid composition of Walker-256carcinosarcoma cells was changed (3-fold increase of phosphatidylethanolamine, 3.4-fold increase of phosphatidylcholine, and 1.8-fold increase of sphingomyelin, while the cardiolipin content decreased by 67%. Exogenous LF was not genotoxic for bone marrow cells (as assessed by the ratio of PCE/NCE, number of micronuclei) and peripheral blood lymphocytes (percentage of DNA in the tail of a comet) in Walker-256carcinosarcoma-bearing rats. Exogenous LF caused the inhibition of Walker-256carcinosarcoma growth and a decrease in the microviscosity of plasma cell membranes, and exerted no genotoxicity toward bone marrow cells and peripheral blood of experimental animals.

Decreased hepatic response to glucagon, adrenergic agonists, and cAMP in glycogenolysis, gluconeogenesis, and glycolysis in tumor-bearing rats.

The response to glucagon and adrenaline in cancer cachexia is poorly known. The aim of this study was to investigate the response to glucagon, adrenergic agonists (α and β) and cyclic adenosine monophosphate (cAMP) on glycogenolysis, gluconeogenesis, and glycolysis in liver perfusion of Walker-256 tumor-bearing rats with advanced cachexia. Liver ATP content was also investigated. Rats without tumor (healthy) were used as controls. Agonists α (phenylephrine) and β (isoproterenol) adrenergic, instead of adrenaline, and cAMP, the second messenger of glucagon and isoproterenol, were used in an attempt to identify mechanisms involved in the responses. Glucagon (1 nM) stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis in the liver of healthy and tumor-bearing rats, but their effects were lower in tumor-bearing rats. Isoproterenol (20 µM) stimulated glycogenolysis, gluconeogenesis, and glycolysis in healthy rats and had virtually no effect in tumor-bearing rats. cAMP (9 µM) also stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis in healthy rats but had practically no effect in tumor-bearing rats. Phenylephrine (2 µM) stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis and these effects were also lower in tumor-bearing rats than in healthy. Liver ATP content was lower in tumor-bearing rats. In conclusion, tumor-bearing rats with advanced cachexia showed a decreased hepatic response to glucagon, adrenergic agonists (α and β), and cAMP in glycogenolysis, gluconeogenesis, and glycolysis, which may be due to a reduced rate of regulatory enzyme phosphorylation caused by the low ATP levels in the liver.

Maternal nutritional supplementation with fish oil and/or leucine improves hepatic function and antioxidant defenses, and minimizes cachexia indexes in Walker-256 tumor-bearing rats offspring

In this study, we hypothesized that throughout the pregnancy/weaning period, nutritional supplementation with leucine (which improves protein synthesis) and/or fish oil (rich in omega-3, which modulates oxidative stress) can minimize/improve cachexia-induced damage in rat offspring. Thus, we investigated the maternal supplementation with these nutrients over the modulation of cachexia index and liver function in tumor-bearing rats offspring. Pregnant rats were fed control, leucine, omega-3, and leucine/omega-3 diets, which were given throughout the gestational and weaning periods. The male offspring were subjected to a control diet until adulthood (120 days) and then distributed into 5 groups (n=4-6 per group): C, Control; W, tumor-bearing; WL, tumor-bearing group with a maternal leucine-rich diet; WO, tumor-bearing group with a maternal omega-3 diet; and WLO, tumor-bearing group with a maternal leucine-rich and omega-3 diet. The W group had a higher cachexia index (31.83 ± 2.9%), but this parameter decreased in the WO (P=0.0380) and WLO groups (P=0.0187). In addition, the W group had a lower survival rate, and the WLO group exhibited a trend toward increased survival (P=0.0505). The hepatic function in maternal supplemented groups was preserved, while the W group exhibited an increased aspartate-aminotransferase/alanine-aminotransferase ratios (P=0.0152) and also enhanced liver oxidative stress, with higher alkaline phosphatase (P=0.0190) and superoxide dismutase (P=0.0190) activities, and trended toward to higher malondialdehyde content (P=0.0556). In contrast, the maternal-supplemented groups had similar liver enzymes and malondialdehyde contents. Thus, we concluded that supplementing the maternal diet modulated/improved liver antioxidant responses and ameliorated the cachexia state in tumor-bearing rat offspring.

Ruthenium complex exerts antineoplastic effects that are mediated by oxidative stress without inducing toxicity in Walker-256 tumor-bearing rats

The present study evaluated the in vivo antitumor effects and toxicity of a new Ru(II) compound, cis-(Ru[phen]2[ImH]2)2+ (also called RuphenImH [RuC]), against Walker-256 carcinosarcoma in rats. After subcutaneous inoculation of Walker-256 cells in the right pelvic limb, male Wistar rats received 5 or 10mgkg−1 RuC orally or intraperitoneally (i.p.) every 3 days for 13 days. A positive control group (2mgkg−1 cisplatin) and negative control group (vehicle) were also used. Tumor progression was checked daily. After treatment, tumor weight, plasma biochemistry, hematology, oxidative stress, histology, and tumor cell respiration were evaluated. RuC was effective against tumors when administered i.p. but not orally. The highest i.p. dose of RuC (10mgkg−1) significantly reduced tumor volume and weight, induced oxidative stress in tumor tissue, reduced the respiration of tumor cells, and induced necrosis but did not induce apoptosis in the tumor. No clinical signs of toxicity or death were observed in tumor-bearing or healthy rats that were treated with RuC. These results suggest that RuC has antitumor activity through the modulation of oxidative stress and impairment of oxidative phosphorylation, thus promoting Walker-256 cell death without causing systemic toxicity. These effects make RuC a promising anticancer drug for clinical evaluation.

Resistance exercise attenuates skeletal muscle oxidative stress, systemic pro-inflammatory state, and cachexia in Walker-256 tumor-bearing rats

The aim of this study was to investigate the effects of resistance exercise training (RET) on oxidative stress, systemic inflammatory markers, and muscle wasting in Walker-256 tumor-bearing rats. Male (Wistar) rats were divided into 4 groups: sedentary controls (n = 9), tumor-bearing (n = 9), exercised (n = 9), and tumor-bearing exercised (n = 10). Exercised and tumor-bearing exercised rats were exposed to resistance exercise of climbing a ladder apparatus with weights tied to their tails for 6 weeks. The physical activity of control and tumor-bearing rats was confined to the space of the cage. After this period, tumor-bearing and tumor-bearing exercised animals were inoculated subcutaneously with Walker-256 tumor cells (11.0 × 107 cells in 0.5 mL of phosphate-buffered saline) while control and exercised rats were injected with vehicle. Following inoculation, rats maintained resistance exercise training (exercised and tumor-bearing exercised) or sedentary behavior (control and tumor-bearing) for 12 more days, after which they were euthanized. Results showed muscle wasting in the tumor-bearing group, with body weight loss, increased systemic leukocytes, and inflammatory interleukins as well as muscular oxidative stress and reduced mTOR signaling. In contrast, RET in the tumor-bearing exercised group was able to mitigate the reduced body weight and muscle wasting with the attenuation of muscle oxidative stress and systemic inflammatory markers. RET also prevented loss of muscle strength associated with tumor development. RET, however, did not prevent the muscle proteolysis signaling via FBXO32 gene messenger RNA expression in the tumor-bearing group. In conclusion, RET performed prior tumor implantation prevents cachexia development by attenuating tumor-induced systemic pro-inflammatory condition with muscle oxidative stress and muscle damage.

L-Glutamine supplementation promotes an improved energetic balance in Walker-256 tumor-bearing rats.

We evaluated the effects of supplementation with oral l-glutamine in Walker-256 tumor-bearing rats. A total of 32 male Wistar rats aged 54 days were randomly divided into four groups: rats without Walker-256 tumor, that is, control rats (C group); control rats supplemented with l-glutamine (CG group); Walker-256 tumor rats without l-glutamine supplementation (WT group); and WT rats supplemented with l-glutamine (WTG group). l-Glutamine was incorporated into standard food at a proportion of 2 g/100 g (2%). After 10 days of the experimental period, the jejunum and duodenum were removed and processed. Protein expression levels of key enzymes of gluconeogenesis, that is, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, were analyzed by western blot and immunohistochemical techniques. In addition, plasma corticosterone, glucose, insulin, and urea levels were evaluated. The WTG group showed significantly increased plasma glucose and insulin levels ( p < 0.05); however, plasma corticosterone and urea remained unchanged. Moreover, the WTG group showed increased immunoreactive staining for jejunal phosphoenolpyruvate carboxykinase and increased expression of duodenal glucose-6-phosphatase. Furthermore, the WTG group presented with less intense cancer cachexia and slower tumor growth. These results could be attributed, at least partly, to increased intestinal gluconeogenesis and insulinemia, and better glycemia maintenance during fasting in Walker-256 tumor rats on a diet supplemented with l-glutamine.