Bax/Bcl-2 protein expression ratio and leukocyte function are related to reduction of Walker-256 tumor growth after ß-hydroxy-ß-methylbutyrate (HMB) administration in Wistar rats.

This study investigated the mechanisms by which ß-hydroxy-ß-methylbutyrate (HMB) administration in rats reduces Walker-256 tumor growth. Male Wistar rats were supplemented with HMB (76 mg/kg/day) (HW), or a placebo (W), during 8 wk by gavage. At the 6th wk, rats were inoculated with a suspension of Walker 256 tumor cells (3 × 10(7)/mL). Fifteen days after inoculation, the HW group showed higher glycemia (109.4 ± 5.53 vs. 89.87 ± 7.02 mg/dL, P < 0.05) and lower spleen (1.35 ± 0.05 vs. 1.65 ± 0.12 g, P < 0.05) and tumor weights (9.64 ± 1.07 vs. 13.55 ± 1.19 g, P < 0.05) compared to the W group. Tumor cells extracted from the HMB-treated rats displayed a 36.9% decrement in rates of proliferation ex vivo and a significant increase in the Bax/Bcl-2 protein expression ratio in comparison to those extracted from the placebo-treated rats (P < 0.05). Both phagocytic capacity and H(2)O(2) production rates were higher in polymorphnuclear cells that were obtained from the blood of the HW rats in comparison to those from the W rats (P < 0.05). Reduction of necrotic regions and an intense infiltration of leukocytes and activated granulocytes in HW were evident by transmission electron microscopy. Our findings suggest that HMB supplementation decreases tumor burden by modifying the inner environment of tumor cells and by interfering with blood leukocyte function.

Beta-hydroxy-beta-methylbutyrate supplementation reduces tumor growth and tumor cell proliferation ex vivo and prevents cachexia in Walker 256 tumor-bearing rats by modifying nuclear factor-kappaB expression.

Cancer cachexia syndrome contributes to wasting and weight loss leading to inefficacy of anticancer therapy. In this study, the anticatabolic agent beta-hydroxy-beta-methylbutyrate (HMB) was supplemented to adult Walker 256 tumor-bearing rats during 8 weeks aiming to determine if tumor burden could be reduced. Male Wistar rats were randomly assigned to nontumor and tumor-bearing groups and fed regular chow or regular chow plus HMB supplemented (76 mg/kg body weight). Beta-hydroxy-beta-methylbutyrate supplementation induced a lower tumor weight and tumor cell proliferation ex vivo, totally prevented glycemia reduction, as well as blunted the increase in the serum lactate concentrations and also preserved glycogen stores in tumor-bearing rats. Reduction in tumor cell proliferation ex vivo was accompanied by increased nuclear factor-kappaB inhibitor-alpha content by more than 100%. In contrast, nuclear factor-kappaB p65 subunit content was suppressed by 17% with HMB supplementation. In conclusion, HMB supplementation, at a similar dose used in humans to increase muscle mass, caused antitumor and anticachectic effects, with tumor-cell nuclear factor-kappaB pathway participation, which might be a potential nutritional strategy in cancer therapy.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes.

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

[A novel model for isolation of Walker's tumoral cells using the Ficoll-Hypaque gradient]. / Um novo modelo de isolamento do tumor de Walker utilizando o gradiente de Ficoll-Hypaque.

PURPOSE: The purpose was to evaluate a novel technique for isolation of Walker's tumoral cells using a Ficoll-Hypaque gradient and its further influence on tumor development. METHODS: Twenty male Wistar rats have been divided in 2 groups: G1= without ficoll, G2= with ficoll. Tumor was excised, homogenized and suspended in lactate ringer. A sample of the cell suspension was adjusted at a concentration of 1x10(6) cells/ml (G1). A second sample was centrifuged on a Ficoll-Hypaque gradient and the cell concentration was then adjusted (G2). Tumor was implanted by subcutaneous injection of 1.0 ml in the right armpit of rats. Tumor volume (TV) and tumor weight (TW) were compared in two groups. RESULTS: There were no differences between the two groups in TV (G1=17.9+/-3.8 cm3 vs. G2=17.2+/-4.4 cm3; p=0.190) and TW (G1=7.0+/-1.8 g vs. G2=7.3+/-2.8 g; p=0.569). The histological analysis showed similar patterns of infiltration by small-undifferentiated cells and necrosis in both groups. However, a mild to moderate granulocytic exudate was more frequent in the animals whose tumors derived from Ficoll-isolated cells. Hemorrhage from slight to moderate was only observed in this group. CONCLUSION: A Ficoll-Hypaque gradient can provide more adequate isolation of Walker's tumor and the cell suspension obtained by this technique has lower contamination by other cell types.

Decorin is one of the proteoglycans expressed in Walker 256 rat mammary carcinoma.

Proteoglycan and glycosaminoglycan content was analyzed in a model of rat mammary carcinoma to study the roles of these compounds in tumorigenesis. Hyaluronic acid and proteoglycans bearing chondroitin and/or dermatan sulfate chains were detected in solid tumors obtained after subcutaneous inoculation of Walker 256 rat carcinoma cells. About 10% of sulfated glycosaminoglycan chains corresponded to heparan sulfate. The small leucine-rich proteoglycan, decorin, was identified as one of the proteoglycans, in addition to others of higher molecular weight, by cross-reaction with an antiserum raised against pig laryngeal decorin and by N-terminal amino acid sequencing. Decorin was separated from other proteoglycans by hydrophobic chromatography and its complete structure was determined. It has a molecular weight of about 85 kDa and a dermatan chain of 45 kDa with 4-sulfated disaccharides. After degradation of the glycosaminoglycan chain, three core proteins of different molecular weight (36, 46 and 56 kDa) were identified. The presence of hyaluronic acid and decorin has been reported in a variety of tumors and tumor cells. In the Walker 256 mammary carcinoma model, hyaluronic acid may play an important role in tumor progression, since it provides a more hydrated extracellular matrix. On the other hand, decorin, which is expressed by stromal cells, represents a host defense response to tumor growth.

H1 histone sub-type distribution and DNA topoisomerase activity in skeletal muscle of tumour-bearing rats.

In an investigation of possible causes of the transcriptional defect which has been observed in muscles atrophying in response to tumour growth, the amounts of histone H1 subtypes and the activities of DNA topoisomerase I and II, factors which can affect the structural organisation of chromatin, were studied in nuclei of skeletal muscle of rats bearing a Walker 256 carcinoma. The H1 histones were separated by SDS/PAGE into three fractions, H1.1, H1.2 and H1(0). The level of H1.2 but not that of H1.1 or H1(0) was increased in the tumour-bearing animal. Tumour growth did not affect the activity of either DNA topoisomerase I or II.

Effects of passive immunization against parathyroid hormone-related protein: PTHrP is the responsible factor in mediating hypercalcemia in the Walker carcinosarcoma 256 rat model.

The Walker carcinosarcoma (WCS) 256 is a well-characterized rat model of humoral hypercalcemia of malignancy (HHM). We addressed the question of whether parathyroid hormone-related protein (PTHrP) is the factor responsible for mediating HHM in this model. WCS 256 cells were subcutaneously implanted in female rats. We examined the plasma at days 0, 2, 4, 6, and 8. The midregional PTHrP measured by radioimmunoassay (RIA) and the plasma calcium increased significantly. Measuring PTHrP by a two-site immunoradiometric assay (IRMA) showed comparable results. There was a strong positive correlation between plasma calcium and midregional PTHrP (r = 0.85, p < 0.0001). A strong positive correlation between tumor weight and both midregional PTHrP (r = 0.83, p < 0.0001) and plasma calcium (r = 0.87, p < 0.0001) was also found. After surgical removal of the tumor at day 5, both plasma calcium and plasma PTHrP levels fell to within the normal range. Ip administration of native polyclonal antiserum against PTHrP(53-84) led to a significant decrease of plasma calcium. Extracted WCS 256 tumor showed 5-fold increased levels of midregional PTHrP compared with liver. Immunohistochemistry and Western blot were positive for PTHrP. RNA from the WCS 256 tumor was positive for PTHrP whereas liver tissue RNA was negative. WCS 256 cells grown in vitro also secreted PTHrP into the medium. We conclude that PTHrP is synthesized and secreted by WCS 256 and that PTHrP is the factor responsible for mediating hypercalcemia in the WCS 256 rat model.

Actin and actin-binding proteins in plasma membranes derived from Walker 256 ascites or solid tumour cells.

Plasma membranes from Walker 256 carcinoma cells grown ascitically or as a solid tumour were examined with respect to actin content, [3H]cytochalasin B-binding and the binding of 125I-labelled G-actin to membrane proteins separated by SDS-PAGE. Differences were observed both in cytochalasin B-binding to membrane actin and affinity of 125I-labelled G-actin for specific membrane proteins.

Isolation of a 18,000-Da molecular weight form of parathyroid hormone-related protein from the rat Walker carcinosarcoma 256.

The hypercalcaemic Walker carcinosarcoma 256 is a rat model for humoral hypercalcaemia of malignancy (HHM). Tumour products such as parathyroid hormone-related proteins (PTHRP) and various growth factors appear to be responsible for this syndrome. Recently, PTHRP immunoreactivity has been detected in the conditioned medium of Walker tumour cells. The present report describes the isolation of a 18,000-Da molecular weight form of PTHRP from Walker tumour homogenates, by using a relatively simple immunoaffinity purification method. Our results suggest that this PTHRP form is similar to that purified from other HHM-related tumours.

Effects of glucocorticoid and chemotherapy on the peritumoral edema and astrocytic reaction in experimental brain tumor.

To study the effects of glucocorticoids and chemotherapeutic agents on the pathophysiology of the tumor-induced brain edema, the site of Evans blue-albumin extravasation, the distribution of extravasated serum albumin, and the extent of local astrocytic reaction were examined in a rat model of implanted brain tumor. Experimental brain tumors were produced by implanting small pellets of Walker 256 carcinosarcoma into the cerebral cortex of Wistar rats. In the steroid group, rats were administered with intraperitoneal methylprednisolone succinate (15 mg/kg) daily on and after the 6th day postimplantation, and sacrificed on the 14th day. In the chemotherapy group, rats were given an intravenous injection of cyclophosphamide (30 mg/kg) on the 14th day, and sacrificed on the 21st day. Rats in the untreated group were sacrificed on the 14th day without any therapy. Each animal was sacrificed by the transcardiac perfusion with paraformaldehyde 30 min after intravenous injection of Evans blue. Firstly, coronal blocks of the brain were examined for Evans blue staining macroscopically. Paraffin embedded sections were studied for the Evans blue fluorescence and for the immunohistochemical reaction to serum albumin and GFAP. The examination of Evans blue demonstrated that the origin of extravasation of serum albumin was the tumor and the adjacent brain with dense tumor cell infiltration in any group of rats. The extravasated serum albumin distributed widely and the astrocytic reaction was prominent in the brain of the untreated group. A positive correlation was observed between the intensity of albumin immunoreaction and the degree of astrocytic proliferation. Chemotherapy effectively decreased the size of tumor and reduced the extravasation of serum albumin. The astrocytic reaction was however, not reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Co-purification of calcium transport-stimulating and DNA synthesis-stimulating agents with parathormone-like activity isolated from the hypercalcaemic strain of the Walker 256 tumour.

One of the strains of the Walker 256 carcinosarcoma induces in the rat a humoral hypercalcaemia of malignancy (HHM) syndrome which is similar to that reported in human patients. We have isolated from this tumour a chromatographic fraction which displays an adenylate cyclase stimulating activity in dog kidney cortical membranes, similar to that of a parathormone (PTH) related protein isolated from various HHM related tumours. In addition, this fraction stimulated initial calcium (Ca) uptake in confluent Madin-Darby canine kidney (MDCK) cell monolayers in a dose-dependent manner. Maximal stimulation of Ca uptake was associated with an enhanced Ca efflux from MDCK cells preloaded with the cation, and with an increased DNA synthesis in these cells. These activities might be involved in development of increased tubular calcium reabsorption in Walker 256 tumour-bearing rats.

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine.

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.