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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.)

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.)

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.

DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9.

The phosphatase inhibitor okadaic acid exerted a biphasic effect on the shape of spontaneously polarized Walker carcinosarcoma cells. At lower concentrations, the drug suppressed cell polarity (IC50 = 0.14 microM) and the cells reverted to a spherical shape. At higher concentrations (> 0.25 microM), cells developed large blebs (IC50 = 0.4 microM). Furthermore, 0.2 microM okadaic acid completely suppressed spontaneous cell locomotion. Two specific inhibitors of protein kinase C did not prevent the actions of okadaic acid on cell shape, showing that this enzyme is very likely not involved. Another phosphatase inhibitor, calyculin A, also suppressed polarity (IC50 = 60 nM) and produced blebbing cells (IC50 = 70 nM). 1 microM okadaic acid induced a 40- to 70-fold increase in phosphorylation of the intermediate filament protein vimentin in intact cells. Increased phosphorylation of this major phosphoprotein correlated with the generation of blebbing cells, rather than with inhibition of polarity and may thus be involved in generating the marked shape changes. We conclude that constitutive phosphatase activity is required for motility and control of shape in Walker carcinosarcoma cells.

In 50-90% of cases, humoral hypercalcemia of malignancy (HHM) is due to tumor secretion of parathyroid hormone-related protein (PTHrP). Glucocorticoids are sometimes used as calcium lowering agents and there are in vitro results showing that glucocorticoids diminish PTHrP production. In this study we tested whether the serum-calcium-lowering effect of glucocorticoids is due to decreased PTHrP production by the tumor. As an animal and cell culture model we used the Walker carcinosarcoma (WCS) 256, a rat mammary carcinoma cell line producing PTHrP. In vitro, dexamethasone caused a dose-dependent inhibition of PTHrP production, whereby already 1-5 nmol/L revealed a significant decrease by WCS 256 cells. In contrast to these in vitro results, in WCS 256 tumor-bearing rats, dexamethasone (4 mg/kg body weight on day 4, and 1 mg/kg body weight from day 5 until day 7 after WCS transplantation; circulating dexamethasone levels > 20 nmol/L) did not decrease PTHrP production, PTHrP secretion, serum calcium, or tumor weight in vivo. We conclude that, in this PTHrP-mediated model of humoral hypercalcemia of malignancy, glucocorticoids do not decrease PTHrP production and secretion in vivo and do not show a calcium-lowering effect.

1. Rats treated with a single dose of benzpyrene when newborn and inoculated with Walker's ascitic tumor cells when 6 weeks old showed 6 and 9 days later an unequivocal increase, whereas 15 and 20 days later an unequivocal decrease, in dexamethasone binding capacity (receptor number) relative to the control, i.e. a reversion of receptor activity in the course of tumor genesis. 2. The reversion of receptor activity showed a parallelism with the increase of tumor mortality over the control. 3. The experimental observations support the conclusion that neonatal exposure to benzpyrene has a depressive effect on general resistance that is reflected (or probably caused?) among others by a decrease in the binding capacity of glucocorticoid receptors.

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.

In Wistar rats bearing a Walker-256 carcinoma flank tumor, a standard skin wound was inflicted on the backs of the animals. Nitrogen intake and nitrogen balance measures were obtained. The standard wound did not promote tumor growth. Wound contraction in tumor-bearing animals was not different from that in tumor-free animals. Tumor-bearing rats had anorexia, lower nitrogen intake and a tendency to lower their nitrogen balance. These findings suggest that both cancer and wound healing are privileged metabolic events, having as a consequence sacrifice of the host.

The utilization of acetate is fundamental to numerous cellular processes. At the level of chromatin, histone acetylation is thought to regulate transcription, and acetate is a major source of cellular energy as it is the substrate for the citric acid cycle. The present work investigates the incorporation of [1-14C]sodium acetate in alkylating agent sensitive (WS) and resistant (WR) Walker 256 carcinosarcoma cells. WR bound the labeled acetate four to six times faster than WS cells, as determined by incorporation of [1-14C]sodium acetate into trichloroacetic acid precipitable material. This difference was consistently observed in both nuclear and cytoplasmic fractions of the cells and in cells permeabilized prior to incubation with radioactive acetate. WS and WR cells did not differ from each other in content of either reduced or acetylated CoA. Since adding exogenous CoA-SH to cell lysates did not alter acetate binding or reduce the differences between WS and WR, increased acetylation in WR cells was independent of CoA levels. Using [1-14C]acetyl CoA to label lysolecithin-permeabilized WS and WR cells revealed no difference between the sensitive and resistant lines, in contrast to the 5-fold greater binding of [1-14C]sodium acetate in permeabilized WR cells. This suggests that WR cells formed acetyl CoA more rapidly than did the WS cells from acetate plus endogenous CoA. Chlorambucil treatment (24 hr) did not affect acetylation of nuclear proteins in log phase cells. Finally, 3-fold greater acetylation in a line of Yoshida sarcoma cells that is resistant to alkylating agents, compared to the sensitive line, supported the generality of the phenomenon.

Rats bearing the Walker-256 (W-256) tumor display an anorexic profile which resembles that of normal animals forced to drink 2% NaCl [2,24], a regimen which depletes neurohypophyseal dynorphin-A (DYN) [3,9]. As expected, the naloxone reversible feeding induced by 2-deoxy-D-glucose (2-DG) was attenuated (36%) in the W-256 tumor bearing rats (TBR). Interestingly, immunoreactive (ir) levels of dynorphin-A 1-17 (DYN-17) and its postulated breakdown product, dynorphin-A 1-18 (DYN-8), were also reduced in the neurohypophysis of W-256 TBR by 42 and 50%, respectively. However, ir-DYN levels were not reduced in TBR in those brain regions which are probably involved in the regulation of appetite (e.g., hypothalamus). 2-DG itself did not consistently affect ir-DYN levels in any tissue for either controls or TBR. The ratio of DYN-8 to DYN-17 did not change in response to any treatment, including the depletion of both peptides from the NIL of TBR. In summary, the present data do not support DYN depletion as being a factor which contributes to the anorexia of the W-256 TBR.

Dipyridamole is a non-specific inhibitor of nucleoside transport into mammalian cells. It is currently undergoing clinical evaluation in combination with various antimetabolites in an attempt to enhance the activity of these anticancer drugs by blocking the salvage of extracellular nucleosides, an important determinant of their cytotoxicity. In the present study, the effect of i.v. infusions of dipyridamole on [3H]thymidine incorporation into DNA has been examined in the anaesthetized rat. The tissues studied were bone marrow, gastrointestinal tract epithelium and the ascitic form of the Walker carcinosarcoma. Dipyridamole at 10 mg/kg, given over 3 hr, led to plasma levels of less than 5 microM and did not reduce [3H]thymidine incorporation into any of the tissues studied. At 40 mg/kg dipyridamole (plasma levels 10-15 microM) [3H]thymidine incorporation into the DNA of bone marrow and gastrointestinal tract epithelium was reduced to 20-30% of control values. Increasing the dose to 100 mg/kg did not lead to a further suppression of incorporation. Measurement of [3H]thymidine plasma pharmacokinetics and the intracellular distribution of tritium suggested that the inhibition of [3H]thymidine incorporation was due to reduced cellular uptake. In contrast to the effects on normal tissues, even at a lethal dose (200 mg/kg) dipyridamole did not significantly inhibit [3H]thymidine incorporation into Walker tumour cells. The levels of dipyridamole found in the ascitic fluid, at 100 mg/kg approximately half those in plasma, argue against a pharmacokinetic basis for this difference. Dipyridamole was found to bind extensively (97%) to rat plasma proteins, which may explain the discrepancy between the concentrations of dipyridamole required to inhibit nucleoside incorporation in vitro, in serum-free media, and those needed in vivo. From a comparison of the plasma levels of dipyridamole which cause an inhibition of [3H]thymidine incorporation in the rat with those which can be achieved safely in patients, it is concluded that dipyridamole is unlikely to markedly reduce nucleoside salvage in man.

The growth inhibitory effects of N6-monobutyryl adenosine 3',5'-monophosphate (mbcAMP) and N6,O2'-dibutyryl adenosine 3',5' monophosphate (dbcAMP) towards Walker carcinoma in vitro are significantly potentiated by the addition of L-homocysteine to the culture medium. This effect is not seen with L-cysteine or when exogenous cAMP or prostaglandin E2(PGE2) replace the butyrylated cyclic nucleotide. Combinations of mbcAMP or dbcAMP and L-homocysteine significantly inhibit nucleic acid methylations. Both the butyrylated cyclic nucleotides cause an elevation of the intracellular level of S-adenosyl-L-homocysteine (SAH), a potent inhibitor of S-adenosyl-L-methionine (SAM) dependent methyltransferases, and this is significantly enhanced in combination with L-homocysteine. The increase in SAH level produced by such combinations is proportional to the inhibition of methyl group incorporation into 5-methyl cytosine and 7-methyl guanine. These results suggest that L-homocysteine potentiates accumulation of SAH in the presence of mbcAMP and dbcAMP and that the resultant inhibition of methylation accounts for the enhanced growth inhibition.

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.