L-leucine dietary supplementation modulates muscle protein degradation and increases pro-inflammatory cytokines in tumour-bearing rats.

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 2×106 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.

Study of antitumor activity of antibodies to TNF-α on Walker carcinosarcoma model.

The effect of release-active antibodies to TNF-α (Artrofoon) on the development of Walker carcinosarcoma 256 was studied in Wistar rats. Intragastric dose of this drug significantly inhibited the growth of tumor node (55% inhibition of tumor growth), but less effectively than the reference drug cyclophosphamide (80%). The studied drug significantly prolonged animal' lifespan (+97%) and its efficiency in this respect was comparable to that of cyclophosphamide (+96%).

[Expression of the RT1A molecule of the class I major histocompatibility complex in a Walker 256 tumor after transplantation into Brattleboro rats with a genetic defect of arginine-vasopressin synthesis].

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.

Immunohistochemistry of the uterine cervix of rats bearing the Walker 256 tumor treated with copaiba balsam.

PURPOSE: To investigate the immunohistochemistry of the uterine cervix of 20 Wistar rats (Rattus norvegicus) bearing the Walker 256 tumor, treated with copaiba oil (Copaifera officinalis). METHODS: The animals were grouped into four subgroups, with five rats each: the GCT and GCopT received distilled water and topically copaiba, respectively, while the GCG and GCopG received distilled water and copaiba by gavage, respectively. The substances were administered for nine days. On the 12th day, after euthanasia, the tumor pieces were sent to the identification of T CD4+, T CD8+ and Natural Killer cells. RESULTS: It was found that the pattern of expression for specific markers of phenotypes of cells involved in tumor immune response was similar in all groups, regardless the administration way of copaiba oil (topical or gavage). CONCLUSION: Copaiba balsam, administered either topically or by gavage, did not alter the pattern of tumor immune response in rats bearing Walker 256 Tumor.

Energy controllable steep pulse (ECSP) treatment suppresses tumor growth in rats implanted with Walker 256 carcinosarcoma cells through apoptosis and an antitumor immune response.

Electrochemotherapy has been widely used for the treatment of solid tumors, although the underlying mechanism remains unclear. We aimed to investigate the effects of energy controllable steep pulse (ECSP) on the regulation of tumor growth and apoptosis in rats implanted with Walker 256 carcinosarcoma cells. A rat tumor model was established by injection of Walker 256 carcinosarcoma cells into the inguinal area. H&E staining, transmission electron microscopy, and the TUNEL assay were used to detect apoptosis. Concanavalin A-induced lymphocyte transformation and MTT assays were used to assess lymphocyte proliferation. ELISA was used to determine serum cytokine levels. After 2 weeks of ECSP treatment, tumor growth in rats was effectively suppressed, while tumor cell apoptosis was significantly induced compared to the control tumor group. Moreover, ECSP treatment enhanced proliferation and activation of lymphocytes and natural killer (NK) cells. Serum IL-2 and IFN-gamma levels were significantly decreased, and IL-4 and 1-10 levels dramatically increased in rats with control tumors compared to rats without tumors and lacking treatment (p < 0.05). In contrast, ECSP treatment increased IL-2 and IFN-gamma levels, but reduced IL-4 and IL-10 levels to normal values. Moreover, ECSP also increased TNF-alpha production, possibly from peritoneal microphages. Our current study demonstrates that ECSP treatment is able to effectively reduce tumors in rats via induction of apoptosis and activation of the rat antitumor immune response. These data provide insightful information for the future application of ECSP-based electrochemotherapy in clinical trials against solid tumors.

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis.

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.

Granulocytes as effective anticancer agent in experimental solid tumor models.

The aim of the study was to elucidate the effects of murine granulocytes on the growth of solid murine tumors when administrated in the vicinity of W256 carcinoma growing in Sprague Dawley rats, and in the vicinity of Ehrlich ascites tumor (EAT) growing in BALBc mice. The administration of granulocytes significantly improved the survival of W256-bearing rats, and increased the tumor regression incidence from 17% up to 75%. Rats with regressing tumors had 2.5 times increased levels of granulocytes in peripheral blood, which were also cytotoxic in vitro for W256 carcinoma cells. However, blood levels of cytokine-induced neutrophil chemoattractant-2, tumor necrosis factor α and interleukin 6 were similar between rats with regressing tumors and control healthy rats, suggesting that the observed regression of W256 carcinoma was caused by specific anticancer effects of the applied granulocytes. Anticancer effects of granulocytes were also found in BALBc mice bearing solid form of EAT, resulting in a 20% increase of survival in EAT-bearing mice. Therefore, the administration of granulocytes, isolated from healthy animals and applied at the site of solid tumors in rats and in mice, reduced experimental tumor growth, and extended the survival of tumor-bearing animals, while in some rats it even caused a W256 regression.

Effect of fish oil supplementation for two generations on changes of lymphocyte function induced by Walker 256 cancer cachexia in rats.

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-alpha and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-gamma plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

[Effects of electroacupuncture on tumor growth and immune function in the Walker-256 model rat].

OBJECTIVE: To study the effect of acupuncture on tumor and its mechanism. METHODS: Liver cancer, gastric cancer and hypodermic tumor rat models were made by implantation of replicated Walker-256 cell strain. The 3 model rats were respectively divided into two groups at random, a model control group and an electroacupuncture group. The electroacupuncture groups were treated with electroacupuncture (EA) at "Zusanli" (ST 36), "Hegu" (LI 4) and "San-yinjiao" (SP 6), once each day, 15 min one session, for 15 days. The gross tumor volume and the tumor inhibitory rate, and the levels of humoral immunity index, including serum 1gG, IgM, IgA and C3, C4, and the levels of cellular immunity index, including CD4+, CD8+ and CD4+/CD8+ in the peripheral blood in each group were detected. RESULTS: The gross tumor volumes in the EA groups were significantly smaller than those in the model control group (P<0.01). The contents of IgG, IgM and C3 in the EA groups increased significantly compared with those in the model control group (P<0.05 or P<0.01). The contents of IgA and C4 in the EA groups did not significantly change (P>0.05). The content of CD4+ and CD4+/CD8+ in the EA groups are significantly higher than those in the model control group (P<0.05 or P<0.01). CONCLUSION: Acupuncture at "Zusanli" (ST 36), "Hegu" (LI 4) and "Sanyinjiao" (SP 6) can increase immune function and inhibit tumor growth in Walker-256 liver cancer, gastric cancer and hypodermic tumor rats.

The involvement of granulocytes in spontaneous regression of Walker 256 carcinoma.

We described before that oxidative burst of granulocytes is cytotoxic for melanoma B16F10 and for Walker 256 carcinoma (W256). Therefore, we assumed that granulocytes could also be important mechanism of the host defence against tumour. In current study we report massive granulocyte infiltration at the site of W256 transplanted in the hind limb of Sprague-Dawley associated with spontaneous tumour regression observed for 22/25 rats (87%). Peripheral blood granulocytes of these animals were highly cytotoxic for W256 cells cultured in vitro. After the tumour disappearance the inflammatory oxidative burst of the granulocytes ended. Distraction of granulocytes from the tumour by s.c. Sephadex injection decreased the incidence of the W256 regression to only 7/25 animals (30%). These results suggest that innate immunity based on immune competent granulocytes may be the cause of well known phenomenon of spontaneous regression of W256 carcinoma.

[Effect of steep pulsed electric fields on the immune response of tumor-bearing Wistar mice].

This study sought to evaluate the effect of steep pulsed electric fields (SPEFs) on the immune response of Wistar mice inoculated with Walker256 sarcoma. Thirty mice were randomly divided into three groups: control group (group A, inoculated with Walker256 sarcoma, not treated), treatment group (group B, inoculated with Walker256 sarcoma, treated by SPEFs), and normal control group (group C, inoculated with normal saline, not treated). Tumor size was measured before and every 3 days after treatment by vernier caliper. MTT methods were used to assess the lymphocytes proliferation and the natural killer (NK) cells activity. TNF-a activity was measured by ELISA. Statistical analysis was performed utilizing the SPSS10.0 software package. The experiment results revealed that tumor growth was significantly inhibited in group B as compared with group A (P < 0.01), and that lymphocytes proliferation, NK cells activity and TNF-a activity in group B were not significantly different from those in group C (P = 0.953, P = 0.130, P = 0.080, respectively) but markedly higher than those in group A (P < 0.05). The results also showed that SPEFs could not only kill tumor cells but also induce antitumor immune response and improve the immune function of the host efficiently.

A comparison of the effects of pneumoperitoneum and laparotomy on natural killer cell mediated cytotoxicity and Walker tumor growth in Wistar rats.

BACKGROUND: Surgical stress promotes impaired immunological function, which contributes to tumor growth. Natural killer activity (NKA) has a protective role in immunity to tumors. So, the aim of this experimental study was to assess tumor growth and (NKA) after pneumoperitoneum and laparotomy. METHODS; Sixty male Wistar rats were divided into three groups (anesthesia, CO2 pneumoperitoneum and laparotomy) plus ten controls. All experimental animals were inoculated subcutaneously with 8 x 105 Walker carcinosarcoma 256 cells. Animals were sacrificed on 1st(POD1) and 8th (POD8) postoperative day. Tumors were excised and weighed. RESULTS: On POD1 all animals had diminished NKA when compared to controls; NKA after pneumoperitoneum was significantly greater than after laparotomy. On POD8 all animals, except after laparotomy, reached NKA at controls levels. Tumor weight was significantly greater after laparotomy when compared to pneumoperitoneum. CONCLUSIONS: Pneumoperitoneum causes a less depressed NKA and less tumor growth when compared to laparotomy.

Effect of immunisation with cancer procoagulant on the growth of Walker 256 carcinosarcoma cells in rats.

The effect of preimmunisation with cancer procoagulant (CP) on the growth of Walker 256 carcinosarcoma cells in Wistar Lew/Han/IMP rats in vivo has been analysed. One group of rats was immunised with CP purified from human amnion-chorion membranes. The rest of the animals (control groups) were injected with 0.9% NaCl in Freund's adjuvant or were not immunised at all. When the presence of polyclonal anti-CP antibody was detected in CP-immunised rats' serum, all (CP-immunised and control) animals were injected i.p. with 4.8 x 10(5) Walker 256 cells per rat. After 5 days ascitic fluid was collected and viable cells were counted. The complete lack of viable Walker 256 carcinosarcoma cells in 24% of the CP-immunised rats was observed. However, the viable neoplastic cells were present in all control (NaCl-injected and nonimmunised) animals. It seems possible that CP plays an important role in tumour progression, so immunisation with CP can reduce the risk of development of malignant disease.

Role of mitochondria in the immune response to cancer: a central role for Ca2+.

This study demonstrates that Ca2+ stimulates mitochondrial energy metabolism during spleen lymphocyte activation in response to the ascitic Walker 256 tumor in rats. Intracellular Ca2+ concentrations, phosphorylated protein kinase C (pPKC) levels, Bcl-2 protein contents, interleukin-2 (IL-2) levels, mitochondrial uncoupling protein-2 (UCP-2) contents and reactive oxygen species (ROS) were significantly elevated in these activated lymphocytes. Mitochondria of activated lymphocytes exhibited high free Ca2+ concentrations in the matrix and enhanced oligomycin-sensitive oxygen consumption, indicating an increased rate of oxidative phosphorylation. The production of ROS was largely decreased by diphenylene iodinium in the activated lymphocytes, suggesting that NADPH oxidase is the prevalent source of these species. Accumulation of UCP-2 and the anti-apoptotic protein Bcl-2 is probably important to prevent mitochondrial dysfunction and cell death elicited by the sustained high levels of intracellular Ca2+ and ROS and may explain the observed higher resistance from activated lymphocytes against the opening of the mitochondrial membrane permeability pore (MPT). All these changes were blocked by pretreatment of the rats with verapamil, an L-type Ca2+ channel antagonist. These data demonstrate a central role of Ca2+ in the control of mitochondrial bioenergetics in spleen lymphocytes during the immune response to cancer.

Fish oil alters T-lymphocyte proliferation and macrophage responses in Walker 256 tumor-bearing rats.

OBJECTIVE: We investigated the effect of the dietary ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) from postweaning until adulthood on T-lymphocyte proliferation, T-lymphocyte subpopulations (helper and cytotoxic), and production of cytotoxic mediators by macrophages in tumor-bearing rodents. METHODS: Weanling male Wistar rats received a normal low-fat (40 g/kg of diet) chow diet or a high-fat (300 g /kg) diet that included fish or sunflower oil or blends of fish and sunflower oils to yield omega-6:omega-3 PUFA ratios of approximately 6:1, 30:1, and 60:1 ad libitum. After 8 wk, 50% of rats in each group were inoculated with 1 mL of 2 x 10(7) Walker 256 cells. Fourteen days after tumor inoculation, animals were killed and lymphocytes and macrophages were obtained for study. RESULTS: The diets richest in omega-6 PUFA resulted in higher proliferation of thymus, spleen, and gut-associated lymphocytes compared with the chow diet irrespective of tumor burden. In contrast, the fish oil diet resulted in lower proliferation of thymus and spleen lymphocytes compared with the chow diet. Diets rich in omega-6 PUFA decreased the proportion of CD8+ lymphocytes. In non-tumor-bearing and tumor-bearing rats, hydrogen peroxide production by macrophages was highest in rats that consumed diets high in omega-3 PUFAs. Superoxide and nitric oxide production were little affected by the dietary ratio of omega-6 to omega-3 PUFAs. CONCLUSION: Dietary omega-6 and omega-3 PUFA contents alter immune function in non-tumor-bearing and tumor-bearing rats. The omega-3 PUFAs decreased T-cell proliferation but increased hydrogen peroxide production compared with omega-6 PUFAs. Decreased tumor growth and cachexia and increased survival previously reported for fish oil in Walker 256 tumor-bearing rats may be related to improved macrophage function rather than to improved T-cell function.

Oxidative burst and anticancer activities of rat neutrophils.

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256. Survival and tumor growth dynamics were monitored in vivo, while tumor cell proliferation when mixed with neutrophils was studied in vitro together with the generation/release of neutrophil respiratory burst products, primarily 1O2. Neutrophils were collected upon Sephadex injection. The survival of Sephadex injected animals was slightly improved, while their tumors grew less than in controls. The presence of tumor cells in vitro activated neutrophils to produce singlet oxygen similar to phorbol ester. Neutrophils from Sephadex-bearing animals diminished tumor cell proliferation in vitro (measured by 3H-TdR incorporation), while neutrophils from Sephadex and the tumor-bearing animals did not show such activity in vitro. Our results confirm that in the case of rapidly growing tumors such as murine W256 carcinoma neutrophils have antitumorous effects in the early phase of tumor development.

Cachexia induced by Walker 256 tumor growth causes rat lymphocyte death.

Death induction by Walker 256 tumor cachexia in non-tumor-infiltrating lymphocytes was investigated. Lymphocytes from cachectic tumor-bearing rats presented a higher proportion of cells with ruptured membranes, indicating necrotic cell death. The cachexia induced by Walker 256 tumor also increased by 3.6-fold the percentage of cells with fragmented DNA, suggestive of apoptotic cell death. The mitochondria involvement was examined by analysis of mitochondria transmembrane potential using rhodamine 123. Lymphocytes from cachectic tumor-bearing rats presented a more pronounced depolarization of mitochondrial transmembrane potential in comparison with cells from the control group. The expression of important proapoptotic (Bcl-xs, Bax, p53, caspase-3) and antiapoptotic genes (Bcl-2 and Bcl-xL) was also altered by tumor cachexia. These results suggest that the immunosuppression induced by Walker 256 tumor cachexia is at least in part a result of lymphocyte death. Evidence was found for the involvement of mitochondria and important proapoptotic genes in the process of lymphocyte death by Walker 256 tumor cachexia.

Adipose tissue in Walker 256 tumour-induced cachexia: possible association between decreased leptin concentration and mononuclear cell infiltration.

The adipose tissue (AT) is severely affected by cachexia, a paraneoplastic syndrome, which increases the morbidity and mortality of cancer. There is, however, a heterogeneous response to the condition, according to the AT depot. As plasma leptin concentration has been often reported to vary in cachexia, we have measured (species specific radioimmunoassay) the local concentration of leptin in three AT depots: retroperitoneal (RPAT), epididymal (EAT) and mesenteric (MES) of Walker 256 tumour-bearing rats. A reduced concentration of leptin ( P<0.0001) was found in all the depots and in the plasma of the cachectic rats, compared with controls already from day 4 after tumour cell injection. The presence of a cell infiltrate was observed in all AT obtained from the tumour-bearing animals. Ultrastructural analysis, along with immunocytochemistry for RT1B (indicating the presence of MHCII) and using antibody against mononuclear phagocytes, showed the cells to be macrophages. The profile of TNFalpha and PGE2 secretion by the infiltrate was investigated (commercial kits). There was increased production of both factors by the cells of all AT ( P<0.05) compared with peritoneal macrophages obtained from the cachectic rats, while the cells isolated from MES showed the highest synthesis of TNFalpha. The results suggest a possible modulation of the chronic locally produced TNFalpha and PGE2 upon leptin synthesis by the AT of the cachectic rats.

Walker 256 tumor MHC class I expression during the shift from A variant to the immunogenic AR variant.

Novel tumor cell variants can be obtained by serially passaging tumor cells in different media and/or environments. Serial intraperitoneal (ip) passages of the Walker 256 tumor A variant was followed for studying the generation of its regressive AR variant. MHC class I molecule expression was assessed since variations in this molecule would explain changes in tumor cell immunogenicity and therefore, the shift from progressive A variant to the regressive AR variant. Within 25 ip passages all serial repetitions shifted from A to AR variant, which was characterized by a significant increase in red blood cell (RBC) osmotic fragility with marked spleen hypertrophy in the host. In one serial repetition AR tumor cells were rejected (ip passage number 36) and immunity against the AR and A variants was conferred. Flow cytometry analysis showed a significant increase in the number MHC class I positive cells in AR variant (n = 15, 14.21 +/- 1.32) compared with A variant (n = 10, 9.10 +/- 1.22). These data provide evidence that the generation of the AR variant could result from factors present in the ip environment leading to an increase in the number of Walker 256 MHC class I positive tumor cells, probably due to immune selection of MHC class I negative tumor cells.