Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model.

BACKGROUND: Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. RESULTS: Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. CONCLUSIONS: NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex.

Liver membrane proteome glycosylation changes in mice bearing an extra-hepatic tumor.

Cancer is well known to be associated with alterations in membrane protein glycosylation (Bird, N. C., Mangnall, D., and Majeed, A. W. (2006) Biology of colorectal liver metastases: A review. J. Surg. Oncol. 94, 68-80; Dimitroff, C. J., Pera, P., Dall'Olio, F., Matta, K. L., Chandrasekaran, E. V., Lau, J. T., and Bernacki, R. J. (1999) Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 256, 631-636; and Arcinas, A., Yen, T. Y., Kebebew, E., and Macher, B. A. (2009) Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns. J. Proteome Res. 8, 3958-3968). Equally, it has been well established that tumor-associated inflammation through the release of pro-inflammatory cytokines is a common cause of reduced hepatic drug metabolism and increased toxicity in advanced cancer patients being treated with cytotoxic chemotherapies. However, little is known about the impact of bearing a tumor (and downstream effects like inflammation) on liver membrane protein glycosylation. In this study, proteomic and glycomic analyses were used in combination to determine whether liver membrane protein glycosylation was affected in mice bearing the Engelbreth-Holm Swarm sarcoma. Peptide IPG-IEF and label-free quantitation determined that many enzymes involved in the protein glycosylation pathway specifically; mannosidases (Man1a-I, Man1b-I and Man2a-I), mannoside N-acetylglucosaminyltransferases (Mgat-I and Mgat-II), galactosyltransferases (B3GalT-VII, B4GalT-I, B4GalT-III, C1GalT-I, C1GalT-II, and GalNT-I), and sialyltransferases (ST3Gal-I, ST6Gal-I, and ST6GalNAc-VI) were up-regulated in all livers of tumor-bearing mice (n = 3) compared with nontumor bearing controls (n = 3). In addition, many cell surface lectins: Sialoadhesin-1 (Siglec-1), C-type lectin family 4f (Kupffer cell receptor), and Galactose-binding lectin 9 (Galectin-9) were determined to be up-regulated in the liver of tumor-bearing compared with control mice. Global glycan analysis identified seven N-glycans and two O-glycans that had changed on the liver membrane proteins derived from tumor-bearing mice. Interestingly, α (2,3) sialic acid was found to be up-regulated on the liver membrane of tumor-bearing mice, which reflected the increased expression of its associated sialyltransferase and lectin receptor (siglec-1). The overall increased sialylation on the liver membrane of Engelbreth-Holm Swarm bearing mice correlates with the increased expression of their associated glycosyltransferases and suggests that glycosylation of proteins in the liver plays a role in tumor-induced liver inflammation.

Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord.

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.

Melanocortin-4 receptor antagonist TCMCB07 ameliorates cancer- and chronic kidney disease-associated cachexia.

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.

Tumor anorexia: a learned food aversion?

Anorexia can occur when a specific diet is associated with a developing illness. The studies reported here show that the decline in food intake which accompanies tumor growth is accompanied by the development of aversions to the specific diet consumed during tumor growth. An immediate elevation in food consumption occurred when a novel diet was introduced. Therefore, the development of learned aversions to the specific diet eaten during tumor growth may be a causal factor in the development of tumor anorexia.

SB366791, a TRPV1 antagonist, potentiates analgesic effects of systemic morphine in a murine model of bone cancer pain.

BACKGROUND: Bone cancer pain has a major impact on the quality of life of cancer patients but is difficult to treat. Therefore, development of a novel strategy for bone cancer pain is needed for improvement of the patient quality of life. In this study, we examined the analgesic effects of the combination of a transient receptor potential vanilloid subfamily 1 (TRPV1) antagonist and morphine on pain-related behaviours in a murine model of bone cancer pain. METHODS: C3H/HeJ mice underwent injection of osteolytic sarcoma cells into the intramedullary space of the femur. The analgesic effects of intraperitoneal morphine and the analgesic effect of a TRPV1 antagonist, SB366791 [N-(3-methoxyphenyl)-4-chlorocinnamide], on bone cancer pain-related behaviours were examined. The analgesic effects of the combination of SB366791 and morphine on bone cancer pain were also examined. RESULTS: Intraperitoneal morphine significantly reduced the number of spontaneous flinches and improved ambulation only at the highest dose of 10 mg kg(-1) whereas weight-bearing was not improved. Intraperitoneal SB366791 at doses of 0.3 and 1.0 mg kg(-1), but not at a dose of 0.1 mg kg(-1), reduced the number of spontaneous flinches, whereas neither weight-bearing nor ambulation was improved. Addition of a sub-analgesic dose of SB366791 (0.1 mg kg(-1)) to morphine significantly reduced the number of flinches and improved weight-bearing compared with the effects of morphine alone. CONCLUSIONS: Our findings showed that the combination of morphine and SB366791 has potent analgesic effects on bone cancer pain. The findings of this study may lead to novel strategies for the treatment of bone cancer pain.

Effects of ghrelin on anorexia in tumor-bearing mice with eicosanoid-related cachexia.

Ghrelin is a novel brain-gut peptide that stimulates food intake and may secondarily increase body weight via a growth hormone secretagogue receptor (GHS-R). Tumor-bearing mice (MCG101), characterized by anorexia, fat loss and muscle wasting due to increased concentration of PGE2 and proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha), were provided ghrelin i.p. at a low (20 microg/day) and high dose (40 microg/day) to examine the ability of ghrelin to counteract tumor-induced anorexia. Immunohistochemical staining and Western blot analyses were used to identify GHS-R expression in the brain as well as its relationship to NPY expression in hypothalamic neurons. GHS-R mRNA in hypothalamus and ghrelin mRNA in gastric fundus were quantified by RT-PCR. Body composition was determined by carcass extractions. GHS-R expression in hypothalamus and plasma ghrelin levels were significantly increased in freely-fed tumor-bearing mice, while gastric fundus expression of ghrelin was unaltered compared to non-tumor-bearing mice (controls). Ghrelin treatment increased food intake, body weight and whole body fat at both low and high doses of ghrelin in normal controls, while tumor-bearing mice showed improved intake and body composition at the high dose of ghrelin only. Exogenous ghrelin normalized the GHS-R expression in hypothalamus from tumor-bearing mice without alterations in the gastric fundus expression of ghrelin. Tumor growth was not altered by exogenous ghrelin. Our results indicate that MCG 101-bearing mice became ghrelin resistant despite upregulation of hypothalamic GHS-R expression, which confirms similar indirect observations in cancer patients. Thus, other factors downstream of the ghrelin-GHS-R system appear to be more important than ghrelin to explain cancer-induced anorexia.

In vivo antigenic modification of tumor cells. I. Introduction of murine leukemia virus antigens on non-virus-producing murine sarcomas.

Murine oncovirus antigens represent excellent targets for immune recognition, and virus-associated tumors are generally susceptible to various immunotherapy protocols. Virus-negative tumors, however, are nonimmunogenic and refractory to immunologic control. Therefore, the feasibility of the introduction of antigens onto non-virus-expressing tumors in situ in inbred C57BL/6J mice by systemic administration of nononcogenic murine retroviruses was investigated. Two classes of murine fibrosarcomas were studied: a 3-methylcholanthrene-induced fibrosarcoma syngeneic to C57BL/6 mice (MCA-FS) and a Harvey murine sarcoma virus-transformed, nonproducer fibrosarcoma syngeneic to C57BL/6 mice (H-NP). Both were found to be devoid of infectious ecotropic murine leukemia virus (MuLV) or MuLV antigens. A single dose of Friend murine leukemia virus (F-MuLV) was used to superinfect MCA-FS- and H-NP-induced tumors in vivo and converted these tumors to a highly productive, virus-positive state. In vivo superinfected tumors were indistinguishable from their preinfected counterparts by competition radioimmunoassays for the virion's major envelope glycoprotein, gp71, and its group-specific antigen, p30, and by assays for infectious virus. Analysis of virus from tumor extracts proved that the antigenic specificity of the superinfected tumor was provided by F-MuLV administered systemically to the animals. Finally, an immunoperoxidase technique, applied to tumor cross sections, demonstrated the uniform appearance of viral antigens in the superinfected tumors.

Antipyretic and antiviral action of vitamin A in Moloney sarcoma virus- and poxvirus-inoculated mice.

Six week-old male CBA/J mice fed a commercial powdered laboratory chow or the same chow supplemented with vitamin A palmitate (150,000 U/kg) were inoculated with either the Moloney strain of murine sarcoma virus (M-MuSV) or poxvirus. Central body temperature was measured daily. Both viruses elicited fevers, but the fevers were less pronounced and of shorter duration in the mice ingesting the vitamin A-supplemented diet. Palpable M-MuSV-induced tumors appeared later, were less frequent, grew more slowly, and were resorbed sooner in the mice fed the vitamin A supplement. Similarly, in these mice the appearance of pox lesions was delayed, their numbers reduced, and their disappearance hastened.

Thyroid hormones and experimental cancer cachexia.

The relationship between circulating thyroid hormones and nutritional status was studied in sarcoma-bearing inbred C57BL/6J mice and control mice. Supplementation with exogenous thyroxine (T4) was also evaluated. Tumor-bearing animals had depressed levels of circulating thyroid hormones. This was also found in food-restricted (pair-fed and pair-weighed) controls. Plasma levels of thyroid hormones decreased with increased tumor burden. Thyrotropin-releasing hormone caused an increased response of thyroid-stimulating hormone in tumor-bearing animals. Low levels of thyroid hormones in sarcoma-bearing mice were due to depressed hormone production by the thyroid gland rather than to increased clearance rate of hormones. Plasma levels of triiodothyronine (T3) correlated to the amount of whole-body nitrogen among sarcoma-bearing mice and food-restricted controls. Exogenous T4 increased food intake by 20% in sarcoma-bearing mice. The benefit of this was probably counteracted by an increased metabolic rate, since reversal of plasma levels of T3 and free T4 had no net effect on body composition of freely eating sarcoma-bearing mice, although it had a negative effect on body and muscle composition in food-restricted controls. Exogenous T4 did not stimulate tumor growth. The results indicate that low circulating levels of thyroid hormones in experimental cancer cachexia are probably caused by the reduced food intake (anorexia), which is in agreement with findings in clinical cancer. Depression of thyroid hormones is probably a physiological means to reduce energy expenditure and to preserve substrates in progressive cancer disease.

Host genetic factors influencing the occurrence of leukemoid reaction in BALB/cMk mice bearing transplanted tumors.

Host genetic factors influencing the occurrence of leukemoid reaction in BALB/cMk strain mice bearing transplanted tumors were studied. A Mendelian hybridization experiment was performed with BALB/cMk and C57BL/6 strains, and their strain hybrids; the first filial (F1) generation hybrids; the second filial (F2) generation hybrids; and the backcrosses to the two parental strains. The results of these studies suggested a genetic regulation of leukemoid reaction occurrence in BALB/cMk mice bearing transplanted tumors. Genes permissive to the occurrence of the reaction seemed to be dominant to their nonpermissive alleles.

Role of whole-body lipids and nitrogen as limiting factors for survival in tumor-bearing mice with anorexia and cachexia.

This study was designed to ascertain whether the overall availability of whole-body lipids and nitrogen is a limiting factor for survival in tumor-bearing mice suffering from anorexia and cachexia. Three-month-old nongrowing mice (C57BL/6J) were given s.c. transplants of a methylcholanthrene-induced sarcoma. Freely fed, starved, and pair-fed animals were used. Body and lipid composition, tumor growth, and survival time were measured. Freely fed sarcoma-bearing mice died with profoundly altered body composition. This was not explained by the anorexia assessed in pair-feeding experiments. Starvation had caused a more severe depletion in body composition in both tumor-bearing and nontumor-bearing animals than the tumor alone did in freely fed tumor-bearing mice. Freely fed tumor-bearing animals had normal proportions of whole-body triglycerides, cholesterol, and polar lipids, but they lost palmitic acid quantitatively more than any other fatty acid. It is unlikely that any single fatty acid became limiting during tumor growth. The results show that the overall availability of lipids, nitrogen, and glucose precursors is not a limiting factor for survival in experimental tumor cachexia. Other factors considered to be more likely as determining factors for the death of tumor-bearing animals are discussed.

Effect of hyperglycemia on the thermal response of murine normal and tumor tissues.

The effect of an i.p. injection of glucose on the thermal response of murine tissues was studied. Animal tumors were early generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Tumors were transplanted into the foot pad, and hyperthermia was given by immersing the foot into a constant-temperature water bath. The tumor and normal tissue responses were studied by assays of the time required for half the tumors to reach 1000 cu mm from treatment day and of the treatment time required for one-half of the animals to develop a loss of one toe or greater reaction. The glucose administration enhanced tumor response more substantially than normal tissue response. The enhancement was greater for a large tumor than for a small tumor and also greater at 42.0 degrees than at 45.5 degrees. Presumably, the hyperglycemia induced acidosis which eventually enhanced thermal response. Present results suggested that the hyperglycemia is a potential method to specifically enhance tumor response at elevated temperatures.

Evaluation of anorexia as the cause of altered protein synthesis in skeletal muscles from nongrowing mice with sarcoma.

The importance of decreased food intake as the mechanism behind altered protein metabolism in skeletal muscle in cancer was evaluated. A methylcholanthrene-induced sarcoma (MCG 101) transplanted in weight-stable and nongrowing mice (C57BL/6J) was used as the tumor-animal model. Three study groups with appropriate control groups were used: sarcoma-bearing mice; pair-fed mice; and starved mice. The synthesis of myofibrillar and sarcoplasmic proteins was decreased in sarcoma-bearing mice. This was correlated to decreased content of RNA in the muscles and caused a net loss of muscle tissue was measured by dry weight of skeletal muscles. The incorporation rate of amino acids into myofibrillar and sarcoplasmic proteins was decreased to the same extent in the pair-fed mice as that in the sarcoma-bearing mice. This probably reflected decreased protein synthesis, since the radioactivity (dpm/mg) did not differ significantly in the crude transfer RNA fraction between the groups. Separation of soluble proteins from muscle tissue by means of ion-exchange chromatography showed that the pattern of decreased protein synthesis was not tumor specific when compared to muscle affected by starvation. The decrease in protein synthesis was more or less selective, since the synthesis of basic proteins was considerably decreased and was influenced more than were neutral and acidic proteins in both cancer and starvation. Anorexia of a tumor-bearing host is a sufficient trigger to induce decreased protein synthesis in skeletal muscles, but other factors may also be of quantitative importance.

Role of prostaglandins in the production of hypercalcemia by tumors.

This report summarizes the data from two animal and cell culture systems that serve as models that show how certain malignant tumors produce hypercalcemia by means of a humoral mechanism. Studies with the HSDM1 murine fibrosarcoma and the VX2 carcinoma in the rabbit have led to the conclusion that these two tumors produce hypercalcemia in the host by means of a mechanism that utilizes prostaglandin E2 as the mediator between the neoplasm and bone. Analogous or identical mechanisms may operate in a small number of human tumors.

Tumorilysin collected in diffusion chambers and restraint of foreign body tumorigenesis.

Tumorilysin was found in diffusion chambers implanted s.c. or i.p. in A/BiF/F50+, DBA/2J and C57BL/6J mice. Chambers with 0.45-micrometer pores implanted s.c. were most densely covered by a syncytium of macrophages and had the most consistently active tumorilysin, compared with smaller-pored chambers or those implanted i.p. The lysin acted on cultures of murine sarcomas induced by foreign bodies, murine and human mammary carcinoma, and human lymphoma. Lysis was demonstrable within 15 min. There was a dose-response relationship between residual cell counts in cultures and concentrations of diffusion chamber fluid between 1.5 and 12.5%. Murine fibroblasts in culture were not lysed by tumorilytic fluid. The incidence of sarcomas induced by 15-mm vinyl squares implanted s.c. in A/BiF/F50+ mice was significantly reduced at 64 weeks in each of 4 experiments by 2 or 3 injections of 0.05 ml diffusion chamber fluid within the capsule on each side at 2 to 42 weeks after implantation. Analyses of the fluid, compared with serum, for the following substances showed no correlation with tumorilysis: cathepsin D; neutral protease; complement C3 fraction; and arginase. Tumorilysin was preserved by lyophilization and was destroyed by heating to 56 degrees; it did not pass filters cutting off at m.w. 300,000.

Stimulation of tumor growth in adult rats in vivo during acute streptozotocin-induced diabetes.

The effects of acute diabetes mellitus on the growth of Morris hepatoma 7288CTC and Jensen sarcoma were studied in fed, young (less than 200 g), and adult (greater than 250 g) rats. Animals were matched for tumor size and growth; the rates of tumor growth were the same in fed, young and adult nondiabetic rats. Diabetes was induced by the i.v. injection of streptozotocin (65 mg/kg total body weight) into tumor-bearing rats and changes in arterial blood nutrient concentrations were compared to changes in the rates of tumor growth and DNA synthesis. In young rats acute diabetes did not increase the blood concentrations of the fat store-derived nutrients and did not increase the rate of tumor growth. In adult rats, however, acute diabetes raised the arterial blood free fatty acid, glycerol, triglyceride, and ketone body concentrations to high levels and increased the rate of tumor growth about three times over that observed in untreated rats. Progress curves for the mobilization of host fat stores and for incorporation of [methyl-3H]thymidine into tumor DNA during the onset of diabetes showed that these activities were closely correlated in adult rats. Both processes began to increase 2 to 4 h after streptozotocin treatment, reached an initial peak at 12 to 16 h, decreased to a low point at 18 to 20 h, and then increased again to the new steady state after 23 to 24 h. The results indicate that the rate of tumor growth in rats in vivo is limited by the availability of a substance(s) present in the hyperlipemic blood of adult diabetic rats. The tight relationship between host lipolysis and tumor growth suggests that the substance(s) is derived from host fat stores.

Prolonged survival of tumor-bearing rats with repetitive low-dose recombinant tumor necrosis factor.

Tumor necrosis factor may be a mediator of the syndrome of cancer cachexia. Tachyphylaxis or tolerance to the cachectic effects of recombinant tumor necrosis factor (rTNF) has been previously described. In this study, we investigate whether repetitive exposure to rTNF can induce similar tolerance in tumor-bearing (TB) rats and ameliorate cachexia induced by the tumor. In experiment 1, non-tumor-bearing (NTB) and TB rats were randomized to either escalating low doses of rTNF or saline i.p. twice daily for 9 consecutive days. NTB rats treated with rTNF demonstrated a significant decline in food intake and weight change (P less than 0.00001) but soon developed tolerance to the cachectic effects of rTNF; they consumed significantly more food than on the first day of treatment and had weight change similar to NTB rats treated with saline. TB rats treated with rTNF showed a similar significant decline in food intake and weight change (P less than 0.0001) and also demonstrated similar tolerance to the cachectic effects of rTNF with continued treatment. Following treatment, TB rats that had been treated with rTNF ate significantly more and lost less weight than TB rats that had been treated with saline (P less than 0.00001). rTNF treatment of TB rats also demonstrated antineoplastic activity, as estimated tumor weight of tumors from rats treated with rTNF were significantly less than controls (P = 0.003). The anticachexia and antineoplastic effects of rTNF resulted in prolonged survival of TB rats treated with rTNF compared to control TB rats (P = 0.015). Experiment 2 utilized two different rTNF treatment regimens in TB rats: one group received 12 days of escalating doses of rTNF, and another group received 15 days of rTNF treatment. TB rats treated with rTNF again had a significantly greater food intake (P less than 0.00001) and delayed weight loss (P = 0.0001) posttreatment that was further augmented by additional doses of rTNF. Antineoplastic activity of rTNF was less clear, and overall tumor growth curves were not affected by rTNF treatment. Survival of TB rats treated with rTNF was again significantly increased in a dose-dependent manner (P = 0.006). Repeated administration of low doses of rTNF to TB rats induces mild reduction in tumor growth, tolerance to the cachectic effects of rTNF that results in tolerance to the cachectic effects of tumor, and prolongation of survival.

Experimental cancer cachexia: the role of host-derived cytokines interleukin (IL)-6, IL-12, interferon-gamma, and tumor necrosis factor alpha evaluated in gene knockout, tumor-bearing mice on C57 Bl background and eicosanoid-dependent cachexia.

MCG 101 tumors were implanted sc. on wild-type C57 Bl and gene knockout mice to evaluate the role of host-produced cytokines [interleukin (IL)-6, IL-12, IFNgamma, tumor necrosis factor (TNF) receptor 1, and TNF receptor 2] to explain local tumor growth, anorexia, and carcass weight loss in a well-defined model with experimental cachexia. Indomethacin was provided in the drinking water to explore interactions between host and tumor-derived prostaglandins and proinflammatory cytokines for tumor growth. Wild-type tumor-bearing mice developed cachexia because of rapid tumor growth, which were both attenuated in IL-6 gene knockouts. Similar findings were observed after provision of anti-IL-6 to wild-type tumor-bearing mice. Alterations in food intake were not directly related to systemic IL-6 but rather secondarily to IL-6-dependent tumor growth. The absence of host-derived IL-12, IFN-gamma, or the TNF receptor 1 or receptor 2 gene did not attenuate tumor growth or improve subsequent cachexia. Thus, carcass weight loss was not improved by the omission of host cytokine (TNF-alpha, IL-12, or IFN-gamma) except for IL-6. Systemic indomethacin provision decreased plasma prostaglandin E2 in five of six groups of gene knockout tumor-bearing mice, which was associated with improved carcass weight in these groups. Indomethacin seemed to improve food intake to a similar extent in both wild-type and gene knockouts, which agree with the speculation that eicosanoids are more important to explain anorexia than host cytokines. Our results demonstrate that host- and tumor-derived cytokines and prostaglandins interact with tumor growth and promote cachexia in a more complex fashion than usually presented based on previous information in studies on either anti-cytokine experiments in vivo or on gene knockouts with respect to a "single cytokine model." Overall, host cytokines were quantitatively less important than tumor-derived cytokines to explain net tumor growth, which indirectly explains subsequent cachexia and anorexia.

In vivo oxygen consumption rate of DS sarcoma cells on inhibition of DNA synthesis.

The effect of inhibiting DNA synthesis on the cellular O2 consumption rate of tumor cells (DS sarcoma) in vivo was analyzed using a photometric technique. Five days after DS-sarcoma ascites was induced in SD rats, animals were treated either with fludarabine (400 mg/kg i.p., 6 h prior to measurements) or lovastatin (3 x 20 mg/kg i.p., 24, 15, and 3 h prior to measurements), drugs that can inhibit tumor cell proliferation. In addition to cellular O2 consumption, the cell cycle distribution and the fraction of DNA-synthesizing cells in the tumor ascites were measured. Both drugs lowered DNA synthesis significantly, an effect that was more pronounced with fludarabine. The cellular O2 consumption rate following lovastatin application was significantly impaired (approximately 33%), whereas fludarabine had practically no effect on the respiration rate of tumor cells. From these data, it is concluded that a reduction in DNA synthesis does not necessarily result in a decrease in the O2 consumption rate of tumor cells in vivo.