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Scope: Nutritional supplementation of the maternal diet can modify the cancer susceptibility in adult offspring. Therefore, the authors evaluate the effects of a fish-oil diet administered to a long-term, during pre-mating, gestation, and lactation, in reducing cancer-cachexia damages in adult Walker-256 tumor-bearing offspring., Methods and Results: Female rats receive control or fish oil diet during pre-mating, gestation, and lactation. After weaning, male offspring are fed the control diet until adulthood and distributed in (C) control adult-offspring; (W) adult tumor-bearing offspring; (OC) adult-offspring of maternal fish oil diet; (WOC) adult tumor-bearing offspring of maternal fish oil diet groups. Fat body mass is preserved, muscle expression of mechanistic target of rapamicin (mTOR) and eukariotic binding protein of eukariotic factor 4E (4E-BP1) is modified, being associated with lower 20S proteasome protein expression, and the liver alanine aminotransferase (ALT) enzyme content maintained in the WOC group. Also, the OC group shows reduced triglyceridemia., Conclusion: In this experimental model of cachexia, the long-term maternal supplementation is a positive strategy to improve liver function and lipid metabolism, as well as to modify muscle proteins expression in the mTOR pathway and also reduce the 20S muscle proteasome protein, without altering the tumor development and muscle wasting in adult tumor-bearing offspring., (© 2021 Wiley-VCH GmbH.)

Context: Cancer 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., Objectives: Evaluate hyperalgesia, edema, COX-1, COX-2, IL-10, and Bdkrb1 mRNA in low-level laser irradiated Walker-256 tumor-bearing rats., Methods: Rat 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/cm 2 , Ø = 0.028 cm 2 . Mechanical hyperalgesia was evaluated by Randall-Selitto test. Plethysmography measured edema; mRNA levels of COX-1, COX-2, IL-10, and Bdkrb1were analyzed., Results: It 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., Conclusion: Results 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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: To assess oxidative stress and structural changes of the serum albumin in rats with transplanted Walker-256 carcinosarcoma (W256) strains with varying sensitivity to doxorubicin (Dox)., Materials and Methods: The study was performed on female Wistar rats with transplanted W256. On the 9 th day after tumor cell transplantation an analysis of peripheral blood, oxidative stress parameters, and structural changes of serum albumin of experimental animals was performed., Results: On the 9th day after W256 transplantation a significant increase in the leukocyte counts was observed in the groups of animals with the Dox-resistant and parental (Dox-sensitive) W256 tumors compared with the group of the intact animals: up to 14.24 ± 1.92 • 10 3 /μl and 9.78 ± 1.03 • 10 3 /μl, vs 8.92 ± 1.04 • 10 3 /μl, respectively, due to the increase of granulocyte and monocyte counts. The number of lymphocytes was within the normal range. The level of hemoglobin and the erythrocyte counts were also within normal limits, but hematocrit in both groups of animals with tumors somewhat increased against the background of 1.2-fold elevation of the mean erythrocyte volume. In the group of rats with Dox-resistant W256, there was observed a decrease in the plateletcrit by almost 22% and thrombocyte counts - by 28%. Analysis of oxidative stress indices revealed a significant increase in the level of reactive oxygen species, 2-fold increase of malonic dialdehyde level and the degree of oxidative damage of blood plasma proteins, as well as a decrease in the activity of catalase in hemolysates (by 12-15%) in both groups of tumor-bearing rats. With the use of differential scanning calorimetry, UV and fluorescence spectroscopy we have revealed anomalous conformational changes of albumin caused by tumor development: structural rearrangements in the region of its first drug binding site located in the IIA domain, separation of globular parts of albumin molecule, and partial "opening" in a protein molecular three-domain structure resulting a loss of its thermal resistance., Conclusion: The development of transplanted Walker-256 carcinosarcoma, especially its Dox-resistant variant, results in severe metabolic intoxication reflected in alteration of hematological parameters, and indices of oxidative stress, as well as architectonic changes of serum albumin.

Purpose: The aim of this study was to investigate the effects of creatine supplementation on muscle wasting in Walker-256 tumor-bearing rats., Methods: 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 21 days. After 11 days 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 10 days 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., Results: 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., Conclusion: Creatine supplementation prevents skeletal muscle atrophy by attenuating tumor-induced pro-inflammatory environment, a condition that minimizes Atrogin-1 and MuRF-1-dependent proteolysis.

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 ( 18 F-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 ( 18 F-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.

Background: Patients with primary and metastatic brain cancer have an extremely poor prognosis, mostly due to the late diagnosis of disease. Urine, which lacks homeostatic mechanisms, is an ideal biomarker source that accumulates early and highly sensitive changes to provide information about the early stage of disease., Methods: A rat model mimicking the local tumor growth process in the brain was established with intracerebral Walker 256 (W256) cell injection. Urine samples were collected on days 3, 5, and 8 after injection, and then analyzed by liquid chromatography coupled with tandem mass spectrometry., Results: In the intracerebral W256 model, no obvious clinical manifestations or abnormal magnetic resonance imaging (MRI) signals were found on days 3 or 5; at these time points, 9 proteins were changed significantly in the urine of all eight tumor rats. On day 8, when tumors were detected by MRI, 25 differential proteins were identified, including 10 that have been reported to be closely related to brain metastasis or primary tumors. The differential urinary proteome was compared with those from the subcutaneous W256 model and the intracerebral C6 model. Few differential proteins overlapped, and specific differential protein patterns were observed among the three models., Conclusions: These findings demonstrate that early changes in the urine proteome can be detected in the intracerebral W256 model. The urinary proteome can reflect the difference when tumor cells with different growth characteristics are inoculated into the brain and when identical tumor cells are inoculated into different areas, specifically, the subcutis and the brain., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Aim: To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes - ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma (BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF)., Materials and Methods: The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256 carcinosarcoma treated with LF at the doses of 1 and 10 mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO - by unified biochemical method., Results: The introduction of LF at the doses of 1 and 10 mg/kg resulted in a decrease in the ratio of Cu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10 mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10 mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content., Conclusions: The administration of LF, especially at a dose of 10 mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

Background: The mechanisms of drug resistance of cancer have not been yet elucidated in details. Recently, the role of mast cells (MCs) in the development of drug resistance has been brought in the limelight. The aim of the study was to examine the morphological features of doxorubicin (DOX)-resistant Walker 256 carcinosarcoma and to assess the response of MCs and histamine content in these cells in relation to the development of resistance to DOX as well as in DOX-resistant tumors., Materials and Methods: The DOX resistance was induced by serial passages of Walker 256 carcinosarcoma in rats in the setting of DOX treatment in vivo. MCs in tumors were detected in the sections by staining with Toluidine Blue O. Histamine content in MCs stained with solution of Water Blue-Orcein was assessed by Astaldi semiquantitative method taking into account different staining intensity., Results: Formation of DOX resistance in the course of serial passages of Walker 256 carcinosarcoma was accompanied by the increase in the number of MCs in tumors and histamine content. Nevertheless, in tumors with phenotype of complete DOX resistance the number of histamine-containing MCs decreased to the same level as in tumors of the original strain that are DOX-sensitive., Conclusion: MCs are involved in formation of DOX resistance in Walker 256 carcinosarcoma.

Hedyotis diffusa, a traditional Chinese herbal medicine, is widely used for oncotherapy and shows a positive effect in the clinical treatment. But its mechanism of anticancer activities is complicated and unclear. This study was undertaken to assess the therapeutic effects and reveal detailed mechanisms of H. diffusa for oncotherapy. A Walker 256 tumor-bearing rat model was established, and metabolomic profiles of plasma and urine were obtained from 1 H NMR technique. Multivariate statistical analysis methods were used to characterize the discriminating metabolites between control (C), Walker 256 tumor-bearing rats model (M), and H. diffusa treatment (H) groups. Finally, 13 and 10 metabolomic biomarkers in urine and plasma samples were further identified as characteristic metabolites in M group, whereas H group showed a partial metabolic balance recovered, such as ornithine, N-acetyl-l-aspartate, l-aspartate, and creatinine in urine samples, and acetate, lactate, choline, l-glutamine, and 3-hydroxybutyrate in plasma samples. On the basis of the methods above, we hypothesized H. diffusa treatment reduced the injury caused by Walker 256 tumor and maintained a metabolic balance. Our study demonstrated that this method provided new insights into metabolic alterations in tumor-bearing biosystems and researching on the effects of H. diffusa on the endogenous metabolism in tumor-bearing rats., (Copyright © 2017 John Wiley & Sons, Ltd.)

This study investigated the effect of epinephrine (a vasoconstrictor) and hydralazine (a vasodilator) on the hepatic disposition of 5-fluorouracil (5-FU) after application to the surface of the liver in rats. Normal livers were compared with a Walker 256 carcinoma cell tumor model. A cylindrical diffusion cell was attached to the liver surface. 5-Fluorouracil was added into the diffusion cell in combination with vasomodulators or after pretreatment with epinephrine. After selected treatment times, the 5-FU concentrations were assayed at three sites in the excised livers. The 5-FU concentration in the region under the cell attachment site (site 1) was significantly higher after concomitant application of 5-FU and epinephrine, compared with 5-FU alone, and increased in an epinephrine dose-dependent manner. On the other hand, preferential distribution of 5-FU at site 1 was not seen when applied in combination with hydralazine. After 10 min of epinephrine pretreatment, the concentration of 5-FU at site 1 was approximately two times higher than that for the control. Furthermore, the 5-FU concentration at site 1 of the tumor model was greatly increased compared with the normal liver. These results suggest that application of epinephrine to the liver surface might enhance the accumulation of 5-FU at the desired target site., (Copyright © 2017 John Wiley & Sons, Ltd.)

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 × 10 7 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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cancer cachexia is characterised by involuntary weight loss associated with systemic inflammation and metabolic changes. Studies aimed at maintaining lean body mass in cachectic tumour-bearing hosts have made important contributions reducing the number of deaths and improving the quality of life. In recent years, leucine has demonstrated effective action in maintaining lean body mass by decreasing muscle protein degradation. Currently, there is a growing need to understand how leucine stimulates protein synthesis and acts protectively in a cachectic organism. Thus, this study aimed to assess the effects of a leucine-rich diet on protein degradation signalling in muscle over the course of tumour growth. Animals were distributed into four experimental groups, which did or did not receive 2×10 6 viable Walker-tumour cells. Some were fed a leucine-rich diet, and the groups were subsequently sacrificed at three different time points of tumour evolution (7th, 14th, and 21st days). Protein degradation signals, as indicated by ubiquitin-proteasome subunits (11S, 19S, and 20S) and pro- and anti-inflammatory cytokines, were analysed in all experimental groups. In tumour-bearing animals without nutritional supplementation (W7, W14, and W21 groups), we observed that the tumour growth promoted a concurrent decrease in muscle protein, a sharp increase in pro-inflammatory cytokines (TNFα, IL-6, and IFNγ), and a progressive increase in proteasome subunits (19S and 20S). Thus, the leucine-supplemented tumour-bearing groups showed improvements in muscle mass and protein content, and in this specific situation, the leucine-rich diet led to an increase on the day in cytokine profile and proteasome subunits mainly on the 14th day, which subsequently had a modulating effect on tumour growth on the 21st day. These results indicate that the presence of leucine in the diet may modulate important aspects of the proteasomal pathway in cancer cachexia and may prevent muscle wasting due to the decrease in the cachexia index., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bone cancer pain (BCP) is the most common complication in patients with bone cancer. Glial cell line-derived neurotrophic factor (GDNF) is believed to be involved in chronic pain conditions. In this article, the expression and roles of GDNF were studied in a rat model of BCP induced by tibia injection of Walker 256 rat mammary gland carcinoma cells. Significant mechanical and thermal hyperalgesia and ongoing pain were observed beginning as early as day 5 post injection. The expression level of GDNF protein examined on day 16 after tibia injection was decreased in the L3 dorsal root ganglion (DRG) and lumbar spinal cord, but not in other spinal levels or the anterior cingulate cortex. Phosphorylation of Ret, the receptor for GDNF family ligands, was also decreased. Furthermore, normalizing GDNF expression with lentiviral vector constructs in the spinal cord significantly reduced mechanical and thermal hyperalgesia, spinal glial activation, and pERK induction induced by tibia injection, but did not affect ongoing pain. Together these findings provide new evidence for the use of GDNF as a therapeutic treatment for bone cancer pain states., (© 2016 UICC.)

The purpose of this study was to establish a population pharmacokinetic/pharmacodynamic (PK/PD) model linking etoposide free tumor and total plasma concentrations to the inhibition of solid tumor growth in rats. Walker-256 tumor cells were inoculated subcutaneously in the right flank of Wistar rats, which were randomly divided in control and two treated groups that received etoposide 5 or 10mg/kg i.v. bolus every day for 8 and 4days, respectively, and tumor volume was monitored daily for 30days. The plasma and intratumoral concentrations-time profiles were obtained from a previous study and were modeled by a four-compartment population pharmacokinetic (popPK) model. PK/PD analysis was conducted using MONOLIX v.4.3.3 on average data and by mean of a nonlinear mixed-effect model. PK/PD data were analyzed using a modification of Simeoni Tumor Growth Inhibition (TGI) model by introduction of an E max function to take into account the concentration dependency of k 2variable parameter (variable potency). The Simeoni TGI-E max model was capable to fit schedule-dependent antitumor effects using the tumor growth curves from the control and two different administered schedules. The PK/PD model was capable of describing the tumor growth inhibition using total plasma or free tumor concentrations, resulting in higher k 2max (maximal potency) for free concentrations (25.8mL·μg -1 ·day -1 - intratumoral vs. 12.6mL·μg -1 ·day -1 total plasma). These findings indicate that the plasma concentration may not be a good surrogate for pharmacologically active free tumor concentrations, emphasizing the importance of knowing drug tumor penetration to choose the best antitumor therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to investigate changes in homocysteine (Hcy) metabolism and redox balance in response to exercise treatment in a tumor-bearing rat model., Methods: Male Wistar rats were exposed, or not, to a resistance exercise program 6 wk before inoculation with Walker-256 tumor cells or vehicle. After application, rats maintained their routine for 12 d and were then sacrificed for plasma and liver analyses., Results: Impaired Hcy metabolism was evident after 12 d of tumor cell inoculation as demonstrated by significantly increased (P < 0.05) plasma total homocysteine (tHcy) concentration (53%) and decreased plasma cysteine, methionine, and vitamin B12 concentrations. Decreased hepatic cystathionine β-synthase (CBS) and betaine-homocysteine S-methyltransferase mRNA levels were found in tumor-bearing rats but not in controls. Tumor inoculation also decreased levels of liver reduced glutathione (GSH) and increased hepatic oxidative stress compared with non-tumor controls. However, resistance exercise prevented the tumor-impaired transsulfuration pathway as demonstrated by the decreased plasma tHcy, hepatic CBS expression, and increased GSH in tumor-exercised versus tumor-sedentary rats. Remarkably, all measures of liver oxidative stress were suppressed by exercise training. Tumor weight was unchanged between groups., Conclusion: Resistance exercise prevented tHcy accumulation and liver oxidative damage caused by Walker-256 tumor cell inoculation; the modulatory effects of resistance exercise on Hcy metabolism appear to be at the level of transsulfuration pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tumor metastasis to bone can subsequently lead to bone cancer pain (BCP). Currently, BCP is difficult to conquer due to a poor understanding of the potential mechanisms. Several studies have indicated that astrocyte-specific connexin 43 (Cx43) was involved in the neuropathic pain, and Cx43 induced the release of chemokine CXCL12 in bone marrow stromal cells. However, whether spinal Cx43 mediates the production of CXCL12 to participate in the maintenance of BCP is still unknown. Here we showed that Walker 256 tumor cells inoculation into the tibia induced a significant mechanical allodynia, which was accompanied by upregulation of spinal p-Cx43 and CXCL12 expression levels from day 6 to day 18 after inoculation. Spinal Cx43 was mainly expressed in astrocytes, and intrathecal (43)Gap26 (a selective Cx43 blocker) markedly attenuated mechanical allodynia as well as reduced p-Cx43 and CXCL12 expression at day 18 after inoculation. Pre-intrathecal administration of CXCL12 almost abolished the attenuated mechanical allodynia by (43)Gap26. Furthermore, intrathecal injection of anti-CXCL12 neutralizing antibody could ameliorate mechanical allodynia with concomitant inhibition of upregulation of CXCL12 expression, but not influence on p-Cx43 expression. Our results indicate that Cx43 mediates CXCL12 production from spinal dorsal horn in astrocytes to maintain bone cancer pain in rats. These findings may improve our understanding of the underlying mechanisms of BCP and provide a novel target for the treatment of BCP.

Background: Morphine is widely used in patients with moderate and severe cancer pain, whereas the development of drug tolerance remains a major problem associated with opioid use. Previous studies have shown that cannabinoid type 2 (CB2) receptor agonists induce morphine analgesia, attenuate morphine tolerance in normal and neuropathic pain animals, induce transcription of the μ-opioid receptor (MOR) gene in Jurkat T cells, and increase morphine analgesia in cancer pain animals. However, no studies of the effects of CB2 receptor agonists on morphine tolerance in cancer pain have been performed. Therefore, we investigated the effect of repeated intrathecal (IT) injection of the low-dose CB2 receptor agonist AM1241 on the development of morphine tolerance in walker 256 tumor-bearing rats. We also tested the influence of the CB2 receptor agonist AM1241 on MOR protein and messenger ribonucleic acid (mRNA) expression in the rat spinal cord and dorsal root ganglia (DRG)., Methods: Walker 256 cells were implanted into the plantar region of each rat's right hindpaw. Tumor-bearing rats received IT injection of the CB2 receptor agonist AM1241 or antagonist AM630 with or without morphine subcutaneously twice daily for 8 days. Rats receiving drug vehicle only served as the control group. Mechanical paw withdrawal threshold and thermal paw withdrawal latency were assessed by a von Frey test and hot plate test 30 minutes after drug administration every day. MOR protein and mRNA expression in the spinal cord and DRG were detected after the last day (day 8) of drug administration via Western blot and real-time reverse transcription polymerase chain reaction. The data were analyzed via analysis of variance followed by Student t test with Bonferroni correction for multiple comparisons., Results: Repeated morphine treatments reduced the mechanical withdrawal threshold and thermal latency. Coadministration of a nonanalgetic dose of the CB2 receptor agonist AM1241 with morphine significantly inhibited the development of morphine tolerance and increased the MOR protein expression in the spinal cord and DRG and mRNA expression in the spinal cord in tumor-bearing rats., Conclusions: Our findings indicate that IT injection of a nonanalgetic dose of a CB2 receptor agonist increased the analgesia effect and alleviated tolerance to morphine in tumor-bearing rats, potentially by regulating MOR expression in the spinal cord and DRG. This receptor may be a new target for prevention of the development of opioid tolerance in cancer pain.

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement-specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1-3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.

The study was aimed at determining the changes of metal-containing proteins in blood serum and tumor tissue of animals with parental and doxorubicin-resistant strains of Walker-256 carcinosarcoma before and after the cytostatic administration. It has been shown that upon doxorubicin action the levels of total iron and transferrin in the tissues from the both groups of animals decreased while that of ferritine simultaneously increased with more pronounced pattern in the group of animals with resistant tumor strain. It has been shown that upon the action of doxorubicin in tumor tissue of animals with different sensitivity to the cytostatic there could be observed oppositely directed changes in the redox state of these cells that in turn determined the content of “ free iron” complexes, RO S generation and concentration of active forms of matrix metaloproteinase- 2 and matrix metaloproteinase-9, namely, the increase of these indexes in animals with parental strain and their decrease in animals with the resistant one. So, our study has demonstrated the remodulating effect of doxorubicin on the state of metal-containing proteins and redox characteristics of tumor dependent on its sensitivity to cytostatic, at the levels of the tumor and an organism. These data may serve as a criterion for the development of programs for the correction of malfunction of iron metabolism aimed at elevating tumor sensitivity to cytostatic agents.

The tumor microenvironment is characterized by hypoxia, acidosis as well as other metabolic and biochemical alterations. Its role in cancer progression is increasingly appreciated especially on invasive capacity and the formation of metastasis. The effect of acidosis on metastasis formation of two rat carcinoma cell lines was studied in the animal model. In order to analyze the pH dependency of different steps of metastasis formation, invasiveness, cell adhesion and migration of AT-1 prostate cancer cells as well as possible underlying cell signaling pathways were studied in vitro. Acidosis significantly increased the formation of lung metastases of both tumor cell lines in vivo. In vitro, extracellular acidosis neither enhanced invasiveness nor affected cell adhesion to a plastic or to an endothelial layer. However, cellular motility was markedly elevated at pH 6.6 and this effect was sustained even when extracellular pH was switched back to pH 7.4. When analyzing the underlying mechanism, a prominent role of ROS in the induction of migration was observed. Signaling through the MAP kinases ERK1/2 and p38 as well as Src family kinases was not involved. Thus, cancer cells in an acidic microenvironment can acquire enhanced motility, which is sustained even if the tumor cells leave their acidic microenvironment e.g. by entering the blood stream. This increase depended on elevated ROS production and may contribute to the augmented formation of metastases of acidosis-primed tumor cells in vivo.

Background: Cachexia is a significant problem in patients with cancer. The effect of cancer on interstitial cells of Cajal (ICC) and neurons of the gastrointestinal tract have not been studied previously. Although supplementation with L-glutamine 2% may have beneficial effects in cancer-related cachexia, and be protective of ICC in models of oxidative stress such as diabetes, its effects on ICC in cancer have also not been studied., Methods: Twenty-eight male Wistar rats were divided into four groups: control (C), control supplemented with L-glutamine (CG), Walker 256 tumor (WT), and Walker 256 tumor supplemented with L-glutamine (WTG). Rats were implanted with tumor cells or injected with saline in the right flank. After 14 days, the jejunal tissues were collected and processed for immunohistochemical techniques including whole mounts and cryosections and Western blot analysis., Key Results: Tumor-bearing rats demonstrate reduced numbers of Myenteric ICC and deep muscular plexus ICC and yet increased Ano1 protein expression and enhanced ICC networks. In addition, there is more nNOS protein expressed in tumor-bearing rats compared to controls. L-glutamine treatment had a variety of effects on ICC that may be related to the disease state and the interaction of ICC and nNOS neurons. Regardless, L-glutamine reduced the size of tumors and also tumor-induced cachexia that was not due to altered food intake., Conclusions & Inferences: There are significant effects on ICC in the Walker 256 tumor model. Although supplementation with L-glutamine has differential and complex effects of ICC, it reduces tumor size and tumor-associated cachexia, which supports its beneficial therapeutic role in cancer., (© 2015 John Wiley & Sons Ltd.)

Background: Several studies have been shown pro-apoptotic effects of fish oil (FO), rich in n-3 polyunsaturated fatty acids (n-3 PUFA) on cancer cells. Nevertheless, few in vivo experiments have provided data of its ability on apoptosis protein expression in tumor tissue. Thus, in this study we investigate the effect of FO supplementation on apoptosis protein expression in Walker 256 tumor bearing rats. Male Wistar rats were randomly assigned to three groups: fed with regular chow (W); fed regular chow supplemented with FO (WFO) or coconut fat (WCO) (1 g/kg body weight/daily). After thirty days, all animals were inoculated subcutaneously with Walker 256 tumor cells., Findings: Protein expression was done by western blotting in Walker 256 tumor tissue samples. FO decreased the Bcl-2/Bax ratio (p < 0.05) and increased the p53 (p < 0.05), cleaved caspase-7 (p < 0.05) and cleaved caspase-3 (p < 0.05) in Walker 256 tumor tissue., Conclusions: Our data suggest that the pro-apoptotic effect of FO in Walker 256 tumor is related with specifics cleaved caspases.

Background: The aim of the present study paper was to identify the role of reactive oxygen species (ROS) in apoptosis signaling mechanisms. We used for this purpose two ruthenium complex compounds based on that overproduce these reactive species by their metabolism thus manifesting their antitumor activity too., Materials and Methods: In vivo studies were performed in Walker 256 carcinoma-bearing Wistar rats treated with two ruthenium (III) (Ru(III)) complexes with -fluoroquinolones norfloxacin and ofloxacin. The treatment started 7 days after tumor grafting. We assayed the dynamics of apoptosis by flow-cytometry and the biochemical oxidative status parameters. The biological samples used were serum and whole-tumor tissues; the results were compared to the untreated control group., Results: The results showed an increase of apoptosis from 14.79% to 59.72% 14.79% to 59.72% in tumor cells treated with the most active combination, ruthenium complex with norfloxacin. We also noted an increase of the oxidative status and ROS production during treatment., Conclusion: The newly-synthesized complexes are less toxic and their activity is based on the induction of oxidative stress., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Walker 256 carcinosarcoma is a transplantable model of rat carcinoma that originally appeared spontaneously in mammary glands. The growth rate of Walker 256 carcinosarcoma in vasopressin-deficient Brattleboro rats is lower than in WAG rats and their congenic hybrids with normal vasopressin levels. Study of tumor proteins detected essential alterations. Tumor regression starting at the 14th day in Brattleboro rats was accompanied by changes in the laminin pattern. At the 21st day, the concentration of α-chains became twice as low, while β-chains of laminin showed a sixfold increase compared to the initial equimolar correlation of bands. Congenic hybrids having one active copy of the vasopressin gene to provide a physiological level of hormone against the genetic background of Brattleboro rats show the same laminin alterations as WAG rats. They demonstrated a similar moderate increase of γ-chains and threefold growth of α- and β-chains of laminin in tumor tissue. It is supposed that vasopressin may be involved in the regulation of relevant local stimuli to trigger renovation of the laminin composition in a course of growing Walker 256 carcinosarcoma.

The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development ofdoxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage) in Walker-256 carc'inosarcoma tissue. We observed decreased serumferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases withfully developed resistance phenotype. Transferrin content showed changes opposite to that offerritin. During the development of resistance phenotype the tumor tissue also exhibited increased 'free iron' concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.

Expression of VEGF-A in Walker 256 carcinosarcoma and mandibular salivary gland of rats during paraneoplastic process and various regimens of chemotherapy with melatonin and cyclophosphamide were studied by immunohistochemical methods. VEGF-A expression increased in the tumor node and salivary gland after monotherapy with melatonin and cyclophosphamide during progression of tumor growth and paraneoplastic syndrome. A decrease in VEGF-A expression in the tumor node and salivary gland was found after monotherapy with melatonin and cyclophosphamide and, especially, after combined treatment, which proves maximum therapeutic efficiency of combined administration of chemical agents with various mechanisms of action. Overexpression of VEGF-A in the mandibular salivary gland can have diagnostic and predictive value in early diagnostics of neoplasms.

The contribution of anti-inflammatory property of celecoxib in the improvement of metabolic disorders in cancer is unknown. The purpose of this study was to compare the effects of celecoxib and ibuprofen, non-steroidal anti-inflammatory drugs (NSAIDs), on several metabolic changes observed in Walker-256 tumor-bearing rats. The effects of these NSAIDs on the tumor growth were also assessed. Celecoxib or ibuprofen (both at 25 mg/Kg) was administered orally for 12 days, beginning on the day the rats were inoculated with Walker-256 tumor cells. Celecoxib treatment prevented the losses in body mass and mass of retroperitoneal adipose tissue, gastrocnemius, and extensor digitorum longus muscles in tumor-bearing rats. Celecoxib also prevented the rise in blood levels of triacylglycerol, urea, and lactate, the inhibition of peripheral response to insulin and hepatic glycolysis, and tended to attenuate the decrease in the food intake, but had no effect on the reduction of glycemia induced by the tumor. In addition, celecoxib treatment increased the number of Walker-256 cells with signs of apoptosis and the tumor necrosis area and prevented the tumor growth. In contrast, ibuprofen treatment had no effect on metabolic parameters affected by the Walker-256 tumor or tumor growth. It can be concluded that celecoxib, unlike ibuprofen, ameliorated several metabolic changes in rats with Walker-256 tumor due to its anti-tumor effect and not its anti-inflammatory property.

The endogenous electric field may provide an important signal for directional cell migration during cancer metastasis but the mechanism of cell electrotaxis is poorly understood. It was postulated that microtubules play a central role in the polarization and directional migration of several types of cells. In this paper we investigated the role of microtubules in electrotaxis of rat Walker carcinosarcoma WC256 cells. We found that colchicine-stimulated disassembly of microtubules caused the formation of blebs instead of lamellipodia at the front of about 45% of cells. Most of the remaining cells contracted and became rounded or transformed into non-polar cells. Depolymerization of microtubules in both subpopulations of cells reduced the directionality of cell migration to about 50% of the control, but bleb- forming cells migrated much more efficiently than lamellipodia-forming cells. The analysis of microtubules architecture in the presence of an endogenous electric field showed that there is no relationship between the direction of migration and the polarization of microtubules. These results suggest that microtubules are not indispensable for electrotaxis of WC256 cells, however they may improve the directionality of cell migration.

In cancer cachexia, the role of nitric oxide (NO) in the central nervous system remains unclear. Cerebellar degeneration has been reported in cancer patients, but the participation of NO has not been studied. Thus, this study investigated the mechanism of oxidative cerebellar injury in a time-course cancer cachexia experimental model. The cachexia index is progressive and evident during the evolution of the tumor. Nitric oxide and lipid hydroperoxidation quantification was performed using a very sensitive and precise chemiluminescence method, which showed that both analyzed parameters were increased after tumor implantation. In the day 5 group, NO was significantly increased, and this experimental time was chosen to treat the rats with the NO inhibitors N-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG). When treated with NO inhibitors, a significant decrease in both NO and lipid hydroperoxide levels occurred in the cerebellum. 3-nitrotyrosine was also analyzed in cerebellar tissue by immunohistochemistry; it was increased at the three experimental time points studied, and decreased when treated with L-NAME and AG. Besides demonstrating that lipid hydroperoxidation in the cerebellum of rats with cachexia increases in a time-dependent manner, this study is the first to describe the participation of NO and its oxidized product 3-NT in the cerebellum of cachectic rats bearing the Walker 256 solid tumor.

The dynamics of lipoprotein content during Walker 256 tumor growth in rats was studied. Moderate changes in HDL and LDL were paralleled by significant changes in VLDL level. A 2-fold increase of VLDL in comparison with the intact control was recorded on day 10 of tumor growth. Exposure to total hyperthermia additionally stimulated VLDL synthesis and this parameter increased by 4 times and more in rats with tumors in comparison with controls. This effect of hyperthermia correlated with significant subsequent decrease of rat mortality caused by the lethal effect of the tumor.

Treatment for bone cancer pain remains a clinical challenge due to a poor understanding of the underlying mechanisms. Protease-activated receptor 2 (PAR2), a receptor for inflammatory proteases, has been implicated in nociceptive signaling under both normal and pathologic pain states. However, little is known of the role of PAR2 in cancer-induced bone pain. Here we investigated the potential role of PAR2 in a rat model of bone cancer pain. The model of bone cancer pain was induced by inoculating Walker 256 into the tibia bone cavity of rats and verified by X-ray imaging, pathology, and behavior assessments. The rats with bone cancer exhibited marked mechanical allodynia, thermal hyperalgesia, and signs of spontaneous nocifensive behavior. Subcutaneous administration of the PAR2 antagonist FSLLRY-NH2 almost completely abolished mechanical allodynia and thermal hyperalgesia but had no effects on spontaneous pain behavior in the rats with bone cancer. Immunohistochemical study revealed that the expression of PAR2 was significantly increased in large- and medium-sized dorsal root ganglia (DRG) neurons but not in small-sized neurons after Walker 256 inoculation. These results suggest that the increased expression of PAR2 in the DRG may contribute to the development of mechanical allodynia and thermal hyperalgesia associated with bone cancer rats. PAR2 might become a novel target for the treatment of pain in patients with bone cancer.

Background: Cancer-cachexia induces a variety of metabolic disorders of protein turnover and is more pronounced when associated with pregnancy. Tumour-bearing pregnant rats have impaired protein balance, which decreases protein synthesis and increases muscle breakdown. Because branched-chain amino acids, especially leucine, stimulate protein synthesis, we investigated the effect of a leucine-rich diet on protein metabolism in the foetal gastrocnemius muscles of tumour-bearing pregnant rats., Methods: Foetuses of pregnant rats with or without Walker 256 tumours were divided into six groups. During the 20 days of the experiment, the pregnant groups were fed with either a control diet (C, control rats; W, tumour-bearing rats; Cp, rats pair-fed the same normoprotein-diet as the W group) or with a leucine-rich diet (L, leucine rats; LW, leucine tumour-bearing rats; and Lp, rats pair-fed the same leucine-rich diet as the LW group). After the mothers were sacrificed, the foetal gastrocnemius muscle samples were resected, and the protein synthesis and degradation and tissue chymotrypsin-like, cathepsin and calpain enzyme activities were assayed. The muscle oxidative enzymes (catalase, glutathione-S-transferase and superoxide dismutase), alkaline phosphatase enzyme activities and lipid peroxidation (malondialdehyde) were also measured., Results: Tumour growth led to a reduction in foetal weight associated with decreased serum protein, albumin and glucose levels and low haematocrit in the foetuses of the W group, whereas in the LW foetuses, these changes were less pronounced. Muscle protein synthesis (measured by L-[3H]-phenylalanine incorporation) was reduced in the W foetuses but was restored in the LW group. Protein breakdown (as assessed by tyrosine release) was enhanced in the L and W groups, but chymotrypsin-like activity increased only in group W and tended toward an increase in the LW foetuses. The activity of cathepsin H was significantly higher in the W group foetuses, but the proteolytic calcium-dependent pathway showed similar enzyme activity. In parallel, an intense oxidative stress process was observed only in the group W foetuses., Conclusions: These data suggested that the proteasomal and lysosomal proteolytic pathways and oxidative stress are likely to participate in the process of foetal muscle catabolism of Walker's tumour-bearing pregnant rats. The present work shows that foetal muscle can be protected by supplementation with a leucine-rich diet.

Background/aims: The objective of the current work was to test the effect of metformin on the tumor growth in rats with metabolic syndrome., Methods: We obtained pre-diabetic hyperinsulinemic rats by neonatal treatment with monosodium L-glutamate (MSG), which were chronically treated every day, from weaning to 100 day old, with dose of metformin (250 mg/kg body weight). After the end of metformin treatment, the control and MSG rats, treated or untreated with metformin, were grafted with Walker 256 carcinoma cells. Tumor weight was evaluated 14 days after cancer cell inoculation. The blood insulin, glucose levels and glucose-induced insulin secretion were evaluated., Results: Chronic metformin treatment improved the glycemic homeostasis in pre-diabetic MSG-rats, glucose intolerance, tissue insulin resistance, hyperinsulinemia and decreased the fat tissue accretion. Meanwhile, the metformin treatment did not interfere with the glucose insulinotropic effect on isolated pancreatic islets. Chronic treatment with metformin was able to decrease the Walker 256 tumor weight by 37% in control and MSG rats. The data demonstrated that the anticancer effect of metformin is not related to its role in correcting metabolism imbalances, such as hyperinsulinemia. However, in morphological assay to apoptosis, metformin treatment increased programmed cell death., Conclusion: Metformin may have a direct effect on cancer growth, and it may programs the rat organism to attenuate the growth of Walker 256 carcinoma.

Photodynamic therapy (PDT) has been clinically used for liver cancer. The pharmacokinetics of a photosensitizer needs to be monitored so that PDT can be performed at the most favorable time and with the proper dose to increase the cure rate. As mTHPC is a fluorescent compound, we investigate its pharmacokinetics, distribution, and elimination in the rat orthotropic liver cancer model in order to confirm an optimal treatment opportunity of liver cancer PDT. After intravenous administration at a single dose of 300 μg/kg, mTHPC was extracted from tissue homogenates or plasma. Then, mTHPC concentrations were assessed by fluorescence spectroscopy and the data were processed with PK-GRAPH pharmacokinetic procedure. The plasma concentration-time profile of mTHPC showed a short distribution half-life (T½α = 0.082 h) and a relatively longer elimination half-life (T½β = 28.23 h), which quite fitted with a two-compartment model. The results of mTHPC tissue distributions showed that the highest drug accumulation was in tumor tissue, and successively decreased in liver, heart, spleen, muscle, and skin tissues. The drug distribution ratio of tumor to normal tissue reached the peak at 24 h after mTHPC administration. mTHPC was eliminated at a suitable rate in rat orthotropic liver cancer model, and there was no long-term accumulation of mTHPC in rat tissues. For PDT of orthotropic liver cancer, 24 h after mTHPC intravenous injection may be the optimal treatment time point, which might provide higher clinical efficacy and reduce side effects.

Background: Shark liver oil (SLOil) and fish oil (FOil), which are respectively rich in alkylglycerols (AKGs) and n-3 polyunsaturated fatty acids (PUFAs), are able to reduce the growth of some tumors and the burden of cachexia. It is known that FOil is able to reduce proliferation rate and increase apoptotic cells and lipid peroxidation of tumor cells efficiently. However, there are few reports revealing the influence of SLOil on these parameters. In the current study, effects of FOil chronic supplementation on tumor growth and cachexia were taken as reference to compare the results obtained with SLOil supplementation. Also, we evaluated if the association of SLOil and FOil was able to promote additive effects., Methods: Weanling male Wistar rats were divided into 4 groups: fed regular chow (C), supplemented (1 g/kg body weight) with SLOil (CSLO), FOil (CFO) and both (CSLO + FO). After 8 weeks half of each group was inoculated with Walker 256 cells originating new groups (W, WSLO, WFO and WSLO + FO). Biochemical parameters of cachexia, tumor weight, hydroperoxide content, proliferation rate and percentage of apoptotic tumor cells were analysed. Fatty acids and AKG composition of tumor and oils were obtained by high performance liquid chromatography and gas chromatography - mass spectrometry, respectively. Statistical analysis was performed by unpaired t-test and one-way ANOVA followed by a post hoc Tukey test., Results: Fourteen days after inoculation, SLOil was able to restore cachexia parameters to control levels, similarly to FOil. WSLO rats presented significantly lower tumor weight (40%), greater tumor cell apoptosis (~3-fold), decreased tumor cell proliferation (35%), and higher tumor content of lipid hydroperoxides (40%) than observed in W rats, but FOil showed more potent effects. Supplementation with SLOil + FOil did not promote additive effects. Additionally, chromatographic results suggested a potential incorporation competition between the n-3 fatty acids and the AKGs in the tumor cells' membranes., Conclusions: SLOil is another marine source of lipids with similar FOil anti-cachectic capacity. Furthermore, despite being less potent than FOil, SLOil presented significant in vivo antitumor effects. These results suggest that the chronic supplementation with SLOil may be adjuvant of the anti-cancer therapy.

Cancer-cachexia causes severe weight loss, particularly from the wasting of skeletal muscle, which occurs due to increased protein catabolism and/or decreased protein synthesis. The muscle protein degradation observed in cancer patients is mediated by a specific cytokine, proteolysis-inducing factor (PIF), which is produced by the tumour. This protein increases the ubiquitin-proteasome pathway activity, and the synthesis of muscle protein in these patients can be affected by several factors, including nutrient-related signalling. Some nutrients, such as leucine, can decrease the ubiquitin-proteasome pathway activity and increase the skeletal muscle protein content in cachectic animals. In this study, we investigated the effects of leucine on cell viability, morphology, functional proteasome activity, enzymatic activity, and protein synthesis and degradation in C2C12 myotubes exposed to the proteolysis-inducing factor (PIF)-like protein purified from Walker tumour-bearing rats. Walker factor (WF) had no cytotoxic effects on myotube cells and morphological characteristics were not altered in the presence of WF and/or leucine. However, increased alkaline phosphatase activity was observed. At higher WF concentrations, chymotrypsin-like activity, cathepsin B activity and 20S proteasome gene expression increased. Treating myotubes with leucine before exposure to WF causes leads to a decrease in proteasome activity as well as the activity of chymotrypsin and cathepsin enzymes. Total protein synthesis decreased in WF-treated cells concomitantly as protein degradation increased. After leucine exposure, the observed effects of WF were minimal or even reverted in some cases. Taken together, these results suggest an important modulatory effect for leucine on the effects of WF in C2C12 myotube cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

It is essential to choose one preprocessing method for liquid chromatography-mass spectrometry (LC-MS)-based metabolomics studies of urine samples in order to overcome their variability. However, the commonly used normalization methods do not substantially reduce the high variabilities arising from differences in urine concentration, especially for signal saturation (abundant metabolites exceed the dynamic range of the instrumentation) or missing values. Herein, a simple preacquisition strategy based on differential injection volumes calibrated by creatinine (to reduce the concentration differences between the samples), combined with normalization to "total useful MS signals" or "all MS signals", is proposed to overcome urine variabilities. This strategy was first systematically compared with other popular normalization methods by application to serially diluted urine samples. Then, the method has been verified using rat urine samples of pre- and postinoculation of Walker 256 carcinoma cells. The results showed that the calibration of injection volumes based on creatinine values could effectively eliminate intragroup differences caused by variations in the concentrations of urinary metabolites, thus giving better parallelism and clustering effects. In addition, peak area normalization could further eliminate intraclass differences. Therefore, the strategy of combining peak area normalization with calibration of injection volumes of urine samples based on their creatinine values is effective for solving problems associated with urinary metabolomics.

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3 × 10(7) cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2(-ΔΔCT) method. FO significantly decreased tumor growth (W=13.18 ± 1.58 vs WFO=5.40 ± 0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1 ± 1.62 vs WFO=59.39 ± 5.53, P<0.001) and PPARγ (W=100.4 ± 1.04 vs WFO=88.22 ± 1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06 ± 0.022 vs WFO=0.31 ± 0.04 (P<0.001) for COX-2, W=1.08 ± 0.02 vs WFO=0.52 ± 0.08 (P<0.001) for PPARγ, and W=1.04 ± 0.02 vs WFO=0.82 ± 0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.

Few studies have investigated liver gluconeogenesis in cancer and there is no agreement as to whether the activity of this pathway is increased or decreased in this disease. The aim of this study was to evaluate gluconeogenesis from alanine, pyruvate and glycerol, and related metabolic parameters in perfused liver from Walker-256 tumour-bearing rats on days 5 (WK5 group), 8 (WK8 group) and 12 (WK12 group) of tumour development. There was reduction (P < 0.05) of liver glucose production from alanine and pyruvate in WK5, WK8 and WK12 groups, which was accompanied by a decrease (P < 0.05) in oxygen consumption. Moreover, there was higher (P < 0.05) pyruvate and lactate production from alanine in the WK5 group and a marked reduction (P < 0.05) of pyruvate and urea production from alanine in the WK12 group. In addition, liver glucose production and oxygen consumption from glycerol were not reduced in WK5, WK8 and WK12 groups. Thus the, the results show inhibition of hepatic gluconeogenesis from alanine and pyruvate, but not from glycerol, on days 5, 8 and 12 of Walker-256 tumour development, which can be attributed to the metabolic step in which the substrate enters the gluconeogenic pathway., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

This study aimed to compare the anti-neoplastic effects of an Uncaria tomentosa (UT) brute hydroethanolic (BHE) extract with those of two fractions derived from it. These fractions are choroformic (CHCl3) and n-butanolic (BuOH), rich in pentacyclic oxindole alkaloids (POA) and antioxidant substances, respectively. The cancer model was the subcutaneous inoculation of Walker-256 tumour cells in the pelvic limb of male Wistar rat. Subsequently to the inoculation, gavage with BHE extract (50 mg.kg(-1)) or its fractions (as per the yield of the fractioning process) or vehicle (Control) was performed during 14 days. Baseline values, corresponding to individuals without tumour or treatment with UT, were also included. After treatment, tumour volume and mass, plasma biochemistry, oxidative stress in liver and tumour, TNF-α level in liver and tumour homogenates, and survival rates were analysed. Both the BHE extract and its BuOH fraction successfully reduced tumour weight and volume, and modulated anti-oxidant systems. The hepatic TNF-α level indicated a greater effect from the BHE extract as compared to its BuOH fraction. Importantly, both the BHE extract and its BuOH fraction increased the survival time of the tumour-bearing animals. Inversely, the CHCl3 fraction was ineffective. These data represent an in vivo demonstration of the importance of the modulation of oxidative stress as part of the anti-neoplastic activity of UT, as well as constitute evidence of the lack of activity of isolated POAs in the primary tumour of this tumour lineage. These effects are possibly resulting from a synergic combination of substances, most of them with antioxidant properties.

The dynamics of expression of the RT1A antigen of the class I major histocompatibility complex (MHC) in a Walker 256 tumor after its transplantation into Brattleboro rats with a genetic defect of Arginine-Vasopressin synthesis in the hypothalamus was studied. Expression of the RT1A antigen was detected by means of Western-blotting and flow cytometry in the tumor cells on the 14th-17th days after transplantation. In addition, a simultaneous increase in the portion of cells that express the RT1A antigen and in the level of its expression per cell was observed. It is presupposed that at a deficiency of Arginine-Vasopressin, a renewal of expression of the class I MHC antigens, which results in an increase of immunogenicity of this tumor and regression, occurs in the Walker 256 tumor in the Brattleboro rats.

It is not yet known how tumour cells traverse the blood-brain barrier (BBB) to form brain metastases. Substance P (SP) release is a key component of neurogenic inflammation which has been recently shown to increase the permeability of the BBB following CNS insults, making it a possible candidate as a mediator of tumour cell extravasation into the brain. This study investigated the properties of the BBB in the early stages of tumour cell invasion into the brain, and the possible involvement of SP. Male Wistar rats were injected with Walker 256 breast carcinoma cells via the internal carotid artery and euthanised at 1, 3, 6 and 9 days post tumour inoculation. Culture medium-injected animals served as controls at 1 and 9 days. Evidence of tumour cell extravasation across the BBB was first observed at 3 days post-inoculation, which corresponded with significantly increased albumin (p < 0.05) and SP immunoreactivity (p < 0.01) and significantly reduced endothelial barrier antigen labelling of microvessels when compared to culture medium control animals (p < 0.001). By day 9 after tumour cell inoculation, 100 % of animals developed large intracranial neoplasms that had significantly increased albumin in the peri-tumoral area (p < 0.001). The increased SP immunoreactivity and altered BBB properties at 3 days post-inoculation that coincided with early tumour invasion may be indicative of a mechanism for tumour cell extravasation into the brain. Thus, extravasation of tumour cells into the brain to form cerebral metastases may be a SP-mediated process.

Objective: To study the main mechanisms of Aitongxiao Recipe (ATXR) for anti-tumor at the molecular level, and to clarify different efficient drugs' roles in anti-tumor, thus in-depth explaining the objectivity and substance of "cancer toxic" theory., Methods: Walker-256 tumor strain was used for Wistar rat transplanted liver cancer modeling. After successful modeling rats were randomly divided into 5 groups, i. e., the ATXP group, the qi regulating and blood circulating group (as the assembled I group), the heat clearing and detoxification group (as the assembled II group), the body resistance strengthening and cultivating group (as the assembled III group), and the model group, 10 in each group. Corresponding medication was given to rats in each group for 14 successive days. Finally rats were sacrificed and the tumor mass was taken out. The apoptosis rate and the cell cycle of tumor cells were detected by flow cytometry Annexin V/PI. The protein and mRNA expressions of Bcl-2 and survivin were detected using immunohistochemistry and real-time fluorescent quantitative PCR., Results: (1) The apoptosis of hepatoma carcinoma cells could be obviously promoted in the ATXP group. The cell cycle could also be affected, making major cells arrest at G0/G1 phase. The proliferation of hepatoma carcinoma cells was effectively prevented. The efficacy in the assembled II group was in line with that in the ATXP group with no statistical difference (P>0.05). It was also effective in the assembled III group, but its efficacy was not as good as that in the former two groups, showing statistical difference (P<0.01). (2) ATXP could obviously down-regulate the protein and mRNA expressions of Bcl-2 and survivin in hepatoma carcinoma cells. Drugs for heat clearing and detoxification showed significant effects on down-regulating the protein and mRNA expressions of Bcl-2 and survivin in hepatoma carcinoma cells. Their effects were similar to that of ATXP (P>0.05). The effects of drugs for body resistance strengthening and cultivating were not as good as the former two, showing statistical difference (P<0.01). Drugs for blood circulating and stasis removing could up-regulate the protein and mRNA expressions of Bcl-2 and survivin to some extent., Conclusions: (1) ATXP could increase the apoptosis ratio of hepatoma carcinoma cells obviously through down-regulating the protein and mRNA expressions of Bcl-2 and survivin, thus inhibiting their proliferation. (2) Drugs for heat clearing and detoxification played the most important roles in ATXP. The evil heat and dampness (damp-heat insidious pathogen) is the most fundamental carcinogenic factors. The insufficiency of vital qi is also one of the pathogenic factors. The mechanisms of phlegm, stasis, and other pathological products are not clear and await further studies.

The hepatic response to cyclic adenosine monophosphate (cAMP) and N6-monobutyryl-cAMP (N6-MB-cAMP) in the glucose and glycogen catabolism and hepatic glycogen levels were evaluated in Walker-256 tumor-bearing rats, on days 5 (WK5), 8 (WK8), and 11 (WK11) after the implantation of tumor. Rats without tumor fed ad libitum (fed control rats) or that received the same daily amount of food ingested by anorexics tumor-bearing rats (pair-fed control rats) or 24 h fasted (fasted control rats) were used as controls. Glucose and glycogen catabolism were measured in perfused liver. Hepatic glycogen levels were lower (p < 0.05) in WK5, WK8, and WK11 rats in comparison with fed control rats, but not in relation to the pair-fed control rats. However, the stimulatory effect of cAMP (3 and 9 μM) in the glycogen catabolism was lower (p < 0.05), respectively, in WK5 and WK8 rats compared to the pair-fed and fed control rats. Accordingly, the suppressive effect of cAMP (6 μM) in the glucose catabolism, under condition of depletion of hepatic glycogen (24 h fast), was lower (p < 0.05) in WK5 and WK11 rats than in fasted control rats. Similarly, the suppressive effect of N6-MB-cAMP (1 μM), a synthetic analogue of cAMP that it is not degraded by phosphodiesterase 3B (PDE3B), in the glucose catabolism was lower (p < 0.05) in WK5 rats compared to fasted control rats. In conclusion, livers of Walker-256 tumor-bearing rats showed lower response to cAMP in the glucose and glycogen catabolism in various stages of tumor development (days 5, 8 and 11), which was probably not due to the lower hepatic glycogen levels nor due to the increased activity of PDE3B.

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K(+)ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K(+)ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E(2)- (PGE(2), 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE(2)) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE(2)) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE(2); 8 µg/paw) and the ATP-sensitive K(+) channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE(2); 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

Blood metabolic parameters of Walker-256 tumour-bearing rats, on days 5, 8, 11 and 14 after implantation of tumour, were compared with those of rats without tumour fed ad libitum (free-fed control) or with reduced feeding (pair-fed control), similar to the anorexic tumour-bearing rats. Cachexia parameters and tumour mass also were investigated. In general, especially on day 14 after implantation of tumour, there was reduction of body mass, gastrocnemius muscle mass, food intake and glycemia and increase of blood triacylglycerol, free fatty acids, lactate and urea, compared with free-fed controls rats. These changes did not occur in pair-fed control, except a slight reduction of glycemia. Pair-fed control showed no significant changes in blood cholesterol and glycerol in comparison with free-fed control, although there was reduction of cholesterol and increase of blood glycerol on day 14 after tumour implantation compared with pair-fed control. The results demonstrate that, besides the characteristic signs of the cachexia syndrome such as anorexia, weight loss and muscle catabolism, Walker-256 tumour-bearing rats show several blood metabolic alterations, some of which begin as early as day 5 after implantation of tumour, and are accentuated during the development of cachexia. Evidence that the alterations of blood metabolic parameters of tumour-bearing rats were not found in pair-fed control indicate that they were not caused by decreased food intake. These changes were probably mediated by factors produced by tumour or host tissue in response to the presence of tumour., (Copyright © 2011 John Wiley & Sons, Ltd.)

It remains unclear as to whether P2Y1 purinergic receptor (P2Y1R) and the molecules that act downstream, such as extracellular signal-regulated protein kinase 1/2 (ERK1/2), are involved in the development of cancer-induced bone pain (CIBP) in vivo. Here, we investigated the role of the P2Y1R in the modulation of CIBP-associated nociception in spinal cord and dorsal root ganglia (DRG). A CIBP model was established by inoculating Walker 256 gland carcinoma cells into the tibia of female rats. Tactile allodynia and spontaneous pain were assessed using von Frey filaments and ambulatory scores. The results showed that both the paw withdrawal latency to tactile allodynia and the ambulatory score to spontaneous pain were significantly different between the CIBP group and the sham group on days 7-9 post-inoculation (P< 0.01). Furthermore, rats in the CIBP group also showed a progressive increase in ambulatory score, which is different from the sham group (P< 0.01). Furthermore, P2Y1R mRNA and phosphorylated ERK1/2 (p-ERK1/2) protein expression levels were increased in the spinal dorsal horn and DRG of the CIBP group relative to the sham group. However, intrathecal injection of the P2Y1R antagonist MRS2179 decreased P2Y1R mRNA and p-ERK1/2 protein expression in the spinal dorsal horn and DRG (P< 0.01). These results provide evidence that the inhibition of P2Y1R-mediated ERK1/2 phosphorylation in the spinal dorsal horn and DRG can attenuate nociception transmission.

The flavonoid naringin is a polyphenolic compound that naturally occurs in citrus. Patients with cancer generally present features of malnutrition and cachexia. Levels of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) are raised in patients with cancer. This study was designed to analyze the in vivo effect of naringin in the therapeutic treatment of rats bearing Walker 256 carcinosarcoma (W256). Rats were treated intraperitoneally with different doses of naringin (10, 25 and 35 mg/kg), for 50 days. At 25 mg/kg, naringin inhibited tumor growth by ~75%. With this treatment, TNF-α and IL-6 levels decreased (p<0.05) in comparison with the control. In addition, two rats presented complete tumor regression. Inhibition of tumor growth, survival increase and the reduction of TNF-α and IL-6 levels in rats bearing W256 treated with naringin strongly suggest that this compound has potential as an anticarcinogenic drug.

This study was undertaken to estimate antioxidative status of two malignant-mammary gland carcinoma (Walker 256/B) and malignant-prostate carcinoma cells (MatLyLu) disseminated in ascitic fluids. Malignant carcinoma cells (10(7) cells) were twice serially intraperitoneal injected in male Wistar rats to develop ascites. After 7 days, ascitic fluids were collected, and cells in suspension were usable for biological assays. Cellular lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS) levels. Some antioxidant parameters: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were also assessed. Comparisons with control baseline (cells maintained in normal culture medium) were analyzed. TBARS levels were found to be significantly decreased in both ascitic cancer cells compared to the baseline except for in the ascite I of MatLyLu cells. On the other hand, SOD and CAT activities were found to be statistically increased in the two malignant ascitic passages. GSH-Px levels were elevated in the first and in the second ascitic passages (p<0.05 and p<0.01, respectively). Our results suggest that malignant ascites are associated not only with reduced levels of TBARS but also with increased antioxidant parameters, indicating the increasing antioxidant potency of two cancer cells during malignancies process., (Copyright © 2010 Elsevier GmbH. All rights reserved.)