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Cancer-induced cachexia is a complex and poorly understood life-threatening syndrome that is characterized by progressive weight loss due to metabolic alterations, depletion of lipid stores, and severe loss of skeletal muscle protein. Gaining the ability to noninvasively image the presence or onset of cachexia is important to better treat this condition, to improve the design and optimization of therapeutic strategies, and to detect the responses to such treatments. In this study, we employed noninvasive magnetic resonance spectroscopic imaging (MRSI) and [(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) positron emission tomography (PET) to identify metabolic signatures typical of cachectic tumors, using this information to analyze the types and extents of metabolic changes induced by the onset of cachexia in normal tissues. Cachexia was confirmed by weight loss as well as analyses of muscle tissue and serum. In vivo, cachexia-inducing murine adenocarcinoma (MAC)16 tumors were characterized by higher total choline (tCho) and higher (18)FDG uptake than histologically similar noncachectic MAC13 tumors. A profound depletion of the lipid signal was observed in normal tissue of MAC16 tumor-bearing mice but not within the tumor tissue itself. High-resolution (1)H magnetic resonance spectroscopy (MRS) confirmed the high tCho level observed in cachectic tumors that occurred because of an increase of free choline and phosphocholine. Higher succinate and lower creatine levels were also detected in cachectic tumors. Taken together, these findings enhance our understanding of the effect of cancer on host organs and tissues as well as promote the development of noninvasive biomarkers for the presence of cachexia and identification of new therapeutic targets., (©2011 AACR)

Approximately one-third of cancer deaths are caused by cachexia, a severe form of skeletal muscle and adipose tissue wasting that affects men more than women. The heart also undergoes atrophy in cancer patients, but the mechanisms and the basis for apparent sex differences are unclear. In a mouse colon-adenocarcinoma model, cancer causes a loss of cardiac mass due to a decrease in cardiac myocyte size that is associated with reduced levels of all sarcomeric proteins. Unlike skeletal muscle cachexia, atrophic hearts do not upregulate the ubiquitin-proteasome system or its activity but increase autophagy. Thus, cancer causes cardiac atrophy by a mechanism distinct from that in skeletal muscle. Male tumor-bearing mice have a more severe phenotype than females, including greater cardiac mass loss and mortality, a more robust pro-inflammatory response to the tumor, and greater cardiac autophagy. In females, estrogen protects against cancer-induced cardiac atrophy and body weight loss by signaling through its receptor. Sex differences in cardiac atrophy need to be considered during the treatment of patients suffering from chemotherapy-induced cardiomyopathy to prevent exacerbation of cardiac dysfunction., (©2010 AACR.)

The potential interaction between cyclooxygenase (Cox) and NO metabolic pathways in the control of local tumor growth was evaluated. Mice bearing either a sarcoma-derived tumor (C57B1; MCG 101) or a malignant melanoma (C3H/HeN; K1735-M2) were used. These models were principally different because they demonstrate, in tumor hosts, conditions with and without cancer cachexia, seemingly related to high and low production of prostanoids, respectively. Cox inhibitors (Cox-1 and Cox-2) decreased tumor growth by 35-40% in MCG 101-bearing mice but had no such effect on melanoma-bearing mice, despite the expression of the Cox-2 protein in melanoma cells. Indomethacin reduced prostanoid production in both tumor (MCG 101) and host tissues and reduced tumor cell proliferation, mainly in vivo. Nitric oxide synthase (NOS) inhibitors (N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine) reduced tumor growth in vivo by approximately 50% in both tumor models. Tumor growth reduction, related to NOS inhibition, was unrelated to prostanoid production and was an in vivo phenomenon in both tumor models. Specific inhibitors of inducible NOS activity, unexpectedly, had no effect in any tumor model, although inducible NOS protein was present in tumor tissues in large amounts. A combination of Cox and NOS inhibitors had no additive effect on tumor growth (MCG 101). Cox inhibition increased tumor tissue (MCG 101) expression of cNOS mRNA but had no significant effect on tumor tissue expression of the transferrin receptor, vascular endothelial growth factor, or basic fibroblast growth factor. NOS inhibition increased tumor tissue content of cNOS mRNA but showed as well a trend to increase mRNA content of the transferrin receptor and vascular endothelial growth factor. Our results suggest that NOS inhibitors can decrease the local growth of tumors that are either responsive or unresponsive to Cox inhibition. This effect may reflect cross-talk between Cox and NOS pathways within or among tumor cells, or it may represent unrelated effects on tumor and host cells. Whether NO inhibition may be used therapeutically in clinical tumors that are unresponsive to eicosanoid intervention remains to be evaluated.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Parathyroid hormone related peptide (PTHRP) is the major causal agent in the syndrome of malignancy-associated hypercalcemia (MAH). Several studies have shown that PTHRP production is increased in response to growth factors and oncogenes, such as Tpr-Met, that are associated with the tyrosine kinase signaling pathway. Using site-directed mutagenesis of Tpr-Met and chemical inhibitors of phosphotidylinositol-3 kinase and Ras isoprenylation, we demonstrated previously that induction of PTHRP is mediated via the Ras signaling pathway. In the present study, we have directly investigated the role of the Ras oncogene in MAH. As a model system, we used Fisher rat 3T3 fibroblasts stably transfected with a Ras oncogene (Ras-3T3). Ras transfection enhanced PTHRP production 5-10-fold in these cells, and inoculation of this cell line into nude mice led to the development of hypercalcemia within 2 weeks. We used this system to evaluate the effect of a potent inhibitor of Ras processing, B-1086, on cell growth, PTHRP production, plasma calcium, and tumor growth. Treatment of Ras-3T3 cells in vitro with B-1086 at 0.1-10 microg/ml produced a significant reduction in PTHRP mRNA expression and PTHRP secretion and a significant decrease in cell proliferation. Treatment in vivo of BALB/c/nu/nu mice bearing Ras-3T3 tumors with B-1086 resulted in a significant inhibition in tumor growth. In addition, this treatment produced near normalization of serum Ca2+, a significant decrease in plasma PTHRP, and a reduction in tumoral PTHRP mRNA levels. These results show that the Ras pathway is involved in PTHRP production by tumors, identifies Ras as a potential target for treatment of MAH, and demonstrates Ras processing inhibitors as candidate therapeutic agents against this syndrome.

A s.c. injection of a mouse colon adenocarcinoma cell line, colon 26 clone 20, induced cachexia, as evidenced by progressive weight loss and severe hypoglycemia. Several lines of evidence indicate that a pro-inflammatory cytokine, interleukin 6 (IL-6), plays a major role, albeit partially, in the establishment of cachexia in this model. Because IL-10 can potentially inhibit the production of pro-inflammatory cytokines including IL-6, we evaluated the effects of IL-10 gene transfer on the establishment of cachexia. IL-6 transcript was detected at tumor sites of mice inoculated with parental or control vector transfectant cells, and serum IL-6 levels were markedly increased in these mice. The injection of parental cells into IL-6-deficient mice induced cachexia with elevated serum IL-6 levels comparable to wild-type mice, indicating that tumor cells are a major source of IL-6. The inoculation of IL-10-transfectant cells kept IL-10 mRNA expression at tumor sites and induced the elevation in serum IL-10 levels without affecting the growth rates of colon 26 cells both in vitro and in vivo. However, the implantation with IL-10-transfectant cells reduced the expression of IL-6 mRNA at the tumor sites and the elevation in serum IL-6 levels. Concomitantly, mice inoculated with IL-10-transfectant cells did not exhibit progressive weight loss, a reduction in food intake, or severe hypoglycemia, which was observed in mice inoculated with parental or control vector-transfectant cells. Collectively, these results suggest that IL-10 gene transfer prevented the occurrence of cachexia with a concomitant inhibition of IL-6 production at the tumor sites.

Growth, blood flow, oxygenation, and bioenergetic status of experimental tumors were investigated in normal (control) and anemic animals after administration of recombinant human erythropoietin (rhEPO). DS sarcomas were implanted s.c. onto the hind foot dorsum of Sprague-Dawley rats. Tumor-associated anemia was induced by the development of an i.p. hemorrhagic ascites. rhEPO (1000 IU/kg) was administered s.c. three times per week over 14 days, after which it was found to have significantly increased hematocrit values in both normal and anemic animals. Tumor growth in anemic animals was slower than in normal animals, and rhEPO administration did not influence tumor growth in either group. Tumor blood flow in anemic animals was lower than in control animals and was only increased in larger tumors in animals in which anemia was prevented by prophylactic rhEPO application. Tumor oxygenation, determined using polarographic needle electrodes and oxygen partial pressure histography, was poorer in anemic animals than in normal animals. This reduction could be reversed partially, but not compensated fully by rhEPO treatment in smaller tumors (< or = 1.4 ml). These changes suggest that rhEPO, by improving tumor oxygenation, may increase the efficacy of standard radiotherapy in anemic animals and may be of use in anemic tumor patients in whom the success of radiotherapy or O2-dependent chemotherapy might be limited by tumor hypoxia.

Humoral hypercalcemia of malignancy (HHM) is caused by the secretion of parathyroid hormone-related protein (PTHrP) by tumor cells, and tumors of squamous histology are the ones most commonly complicated by HHM. To determine why some squamous tumors cause HHM and others do not, we quantitated the levels of PTHrP mRNA expression and PTHrP secretion in a series of eight squamous tumor lines. As anticipated, we found that the level of PTHrP mRNA expression in individual lines correlated with their PTHrP secretion rates. However, PTHrP mRNA levels varied widely in individual lines, and only those tumor lines with the highest levels of PTHrP gene expression were able to cause hypercalcemia in athymic mice. We found that a specific segment of the PTHrP promoter could reproduce the relative pattern of PTHrP gene expression when cloned in front of a chloramphenicol acetyltransferase reporter gene and transiently transfected into these squamous lines. Deletional analysis confirmed that specific sequences within the PTHrP gene promoter appeared to be involved in the transactivation of the gene in tumor lines expressing high levels of PTHrP mRNA. These data suggest that the ability of a given squamous tumor to cause HHM is ultimately a function of its level of PTHrP gene expression, which in turn appears to be a function of the ability of specific transcription factors to transactivate PTHrP gene expression.

Murine gamma-interferon (MuIFN-gamma) is a potent inhibitor of bone resorption induced by interleukin 1 and parathyroid hormone-related protein in vitro. To investigate whether MuIFN-gamma is also effective in vivo, the cytokine was injected s.c. into hypercalcemic, tumor (EC-GI)-bearing nude mice, in which parathyroid hormone-related protein and interleukin 1 alpha are synergistically responsible for causing humoral hypercalcemia. When MuIFN-gamma was injected s.c. at a dose of 1 to 20 x 10(4) units for 5 days consecutively, serum calcium concentrations in the tumor-bearing mice decreased in a dose-dependent manner. The minimal effective dose was 5 x 10(4) units/mouse. Unlike calcitonin, which decreased the serum calcium concentration for only 1 to 2 days despite continuous daily injections, MuIFN-gamma decreased it for more than 7 days even after the injections had been stopped. Human gamma-interferon was completely ineffective. The decrease in serum calcium concentration was accompanied by a decrease in urinary calcium excretion. Histological examination of the femur revealed a decreased number of osteoclasts in the MuIFN-gamma-treated mice. Furthermore, MuIFN-gamma, when injected into nude mice or normal mice at a dose of 15 x 10(4) units for 3 days, almost completely abolished the formation of multinucleated osteoclast-like cells in vitro. These findings suggest that MuIFN-gamma suppresses the formation and maturation of osteoclasts and inhibits osteoclastic bone resorption, resulting in the prolonged decrease of serum calcium concentration seen in hypercalcemic, tumor-bearing nude mice. Therefore, bone resorption inhibitors like MuIFN-gamma, which ameliorate humoral hypercalcemia without an escape phenomenon, are potentially useful for the treatment of malignancy-associated hypercalcemia.

Administration of repetitive sublethal doses of either recombinant human tumor necrosis factor (TNF) or recombinant murine gamma-interferon (IFN) to non-tumor-bearing (NTB) rats caused a significant decline in food intake and body weight. After 3 days rats became resistant to the anorectic and weight loss effects of TNF but maintained persistent diminished food intake and diminished body weight gain while receiving recombinant murine IFN. Passive immunization against recombinant rat gamma-interferon allowed cachectic tumor-bearing (TB) rats to eat more food, have a lesser decline in body weight, live longer, and tolerate larger tumors than similar TB rats given nonspecific control antibody. TB rats treated with an antisera to recombinant murine TNF, which was 100% protective when given to NTB rats 6 h before a lethal endotoxin challenge, did not differ significantly from TB rats treated with control antibody with respect to food intake, body weight, survival, or tumor size. Serum levels of TNF or IFN were not detectable in cachectic tumor-bearing rats. The data indicate that the administration of exogenous IFN can simulate cachexia in NTB rats and that passive immunization against it can partially reverse the cachectic changes associated with cancer and prolong survival. These findings suggest that gamma-interferon may be an important mediator of cachexia in this rat tumor model.

A glomerulopathy in hamsters bearing human epithelial tumors is described. The tumors resulted from the s.c. implantation of human heteroploid cells (MA160) derived originally from a benign prostatic adenoma. The renal immunomorphological and ultrastructural changes include expansion of mesangial matrix and cellularity, electron-dense mesangial deposits, and mesangial deposits of immunoglobulin G and C'3. Renal eluates obtained from the tumor-bearing animals contained substantial amounts of immunoglobulins. Our immunopathological studies demonstrated that virtually all of the eluate immunoglobulin G was tumor-specific antibody directed exclusively against surface determinants of the MA160 cells. There was no demonstrable activity against cytoplasmic or nuclear constituents of the MA160 cells or against certain hamster antigenic determinants. The antibody in the eluate could be removed by repetitive absorptions with viable MA160 cells but not with similar absorptions with either normal human diploid fibroblasts, human heteroploid HeLa cells, or BHK21 hamster cells. Our data support the concept of a specific nephritogenic immunopathological process occurring as a result of immunity to tumor-specific surface determinants.

Cachexia is rare in nude mice bearing human malignant tumors even when the transplanted tumors become as large as the body size of the host. In our series on heterotransplantation of a variety of human malignant tumors into nude mice, a malignant melanoma (SEKI) was found to induce severe body weight loss in the host at the early stage of transplantation. There was no electrolyte disturbance, hyper- or hypoadrenocorticism, hyperthyroidism, or destruction of cells of vital organs to account for the weight loss. Moreover, no evidence was obtained for concomitant infection with bacteria, Mycoplasma or fungi. These cachectic mice revealed remarkably increased levels of serum sialyltransferase and decreased liver catalase activity. The removal of tumor tissues from these mice resulted in prompt recovery of body weight, serum sialyltransferase, and liver catalase activity within 1 to 2 weeks. On the basis of the results obtained, the SEKI melanoma was thought to have produced a pathophysiological state in host nude mice which was very similar to that of cachexia in cancer patients. Nude mice bearing transplants of SEKI melanoma may provide a useful system for the study of cancer cachexia in humans.

The effect of vitamin A deficiency on the sensitivity of the colon to the carcinogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting carcinogen, given intrarectally was studied in female Fischer rats. Animals maintained on Purina laboratory chow, semipurified vitamin A-free diet, or semipurified vitamine A-supplemented diet were given intrarectally 1.25, 0.63, or 0.31 mg MNNG 3 times weekly for 30 weeks and autopsied at the 45th week. The number of large bowel tumors per tumor-bearing rat was higher in animals receiving 1.25 mg MNNG compared to those given 0.63 or 0.31 mg. Vitamin A deficiency in rats given 1.25 mg MNNG significantly suppressed the large bowel tumor induction compared to rats fed adequate vitamin A. A high incidence of squamous cell papillomatosis of the urinary bladder was observed in rats fed vitamin A-free diet and given 1.25 mg MNNG. The present experiment suggests that the large intestine has a susceptibility that is different from that of the respiratory and urinary tracts to tumorigenic stimulation in vitamin A-deficient status.

Daily food intake and corresponding feeding activity (measured as duration) and feeding efficiency (amount of food ingested per unit of feeding activity) were measured both in normal Sprague-Dawley and Buffalo rats and during growth of Walker 256 and 4M mammary carcinomas in Sprague-Dawley rats and of Morris 5123 hepatoma in Buffalo rats. Estimates of meal size and frequency were also obtained. Growth of the carcinomas produced a decline in feeding activity accompanied, early in tumor growth, by a compensatory increase in feeding efficiency with no resultant effect on food intake. This compensated decline in feeding activity was due to reduction in average meal duration. Later, meal frequency was also reduced, with further reduction in feeding activity and reduction in food intake. There was little change in average meal size. The hepatoma produced a different detailed pattern of effect on feeding behavior. These effects are not nonspecific reactions to foreign tissue. The effects imply behavioral compensation for the breakdown of a rapidly responding physiological control of food intake and can be interpreted in terms of successive impairment of feeding control mechanisms that have different response rates and different behavioral modes.

To demonstrate that the anorexia and depletion of cachexia reverses on tumor removal, F344 rats underwent sarcoma resection when their food intake fell to 0 g/day. In survivors of surgery, reversal in food intake was apparent within 3 days postoperatively, followed after 2 days by gain in host weight. To detect whether the transmission of anorexia/cachexia in these tumor-bearing (TB) rats was via the circulation, four groups were studied: single non-tumor bearing (NTB); single TB; parabiotic NTB; and parabiotic TB. The measured blood exchange rate between parabiotic halves was 1.2-1.5%/min. No cachectic effect was detected in either half of the NTB parabionts. There was no evidence of sarcoma metastases in the tumor-free half of the parabiotic TB pair. All the rats associated with the presence of tumor showed cachectic effects but the degree and timing of effect varied among the three conditions, single TB, parabiotic TB half, and parabiotic tumor-free half. In all variables examined (fall in food intake, time of first fall in food intake, host weight loss, elevation of blood urea nitrogen) the severities were always in the same sequence: single TB greater than parabiotic TB half greater than parabiotic tumor-free half greater than NTB. In addition, the TB parabiotic pair had a significantly longer survival time and grew a significantly larger tumor than did the single TB animal. The parabiotic tumor had a slower initial growth rate and a slower deceleration rate than the singlet tumor. These results provide evidence for the humoral mediation of cancer-associated cachexia.

The i.p. inoculation of C3H mice with 5 times 10-6 cells of a transplantable ovarian carcinoma invariably evokes accumulation of large amounts of ascitic fluid. Histological and pharmacotherapeutic studies indicate that obstruction to peritoneal lymphatic drainage is a key factor in the formation of carcinomatous ascites in this model. In the early stages of ascites formation, an intense inflammatory reaction appears to occlude the condusts that connect the peritoneal cavity to the subdiaphragmatic lymphatic plexus. This inflammatory reaction, elicited by the presence of tumor cells within the peritoneal cavity, can be inhibited with high-dose systemic corticosteroid therapy. Ascitic fluid accumulation in animals so treated is markedly retarded. Tumor cells do not gain access to lymphatic capillaries draining the peritoneal cavity until ascitic fluid accumulation is massive. Systemic anticoagulation with heparin or sodium warfarin does not prevent lodgment of tumor cells within these lymphatic capillaries, nor does it alter the pattern of ascitic fluid accumulation. Various considerations suggest that excess production of ascitic fluid is not a likely pathogenetic factor in murine carcinomatous ascites.

The anabolic effects of exogenous neutral protamine hagedorn insulin on tumor-bearing (TB) and non-tumor-bearing (NTB) rats were examined. Exogenous insulin (2 units/100 g/day) produced similar hypoglycemia in TB and NTB rats. Food intake and body weight gain were significantly increased by insulin in NTB rats. In TB rats in an early stage of cachexia, insulin increased food intake and host weight (total body weight minus tumor weight). In TB rats with severe cachexia, insulin increased food intake and stabilized host weight when untreated TB controls were not eating and were losing weight. When daily insulin administration was started at an early stage of tumor growth and continued until death, there was again significant enhancement of host weight and food intake. Heart and adrenal weights were significantly reduced in insulin-treated TB animals. Tumor growth was not stimulated by insulin treatment. Survival time was slightly reduced in TB rats treated with long-term insulin. Survival time in TB rats randomized to insulin during late cachectic decline was not different from untreated TB controls. Insulin did not have any measurable effect on energy expenditure or the motor activity compartment of energy expenditure in either TB or NTB rats. Insulin treatment can reverse experimental cancer cachexia. It is a nutritional therapy which preferentially feeds the host over the tumor. As yet, its beneficial effects have not prolonged survival of tumor-bearing animals.

These studies examined the fate of Factor XIII (fibrin-stabilizing factor) in mice with plasmacytoma (MOPC-300, MOPC-384, MOPC-467, and J-558). Plasma Factor XIII levels in these mice decreased progressively with tumor expansion. No plasma inhibitors of Factor XIII activity could be detected. Factor XIII was found on plasmacytoma cell membranes and in the cytoplasm of the malignant cells by immunofluorescence. The inverse relationship between tumor load and plasma Factor XIII levels suggested that the fibrin-stabilizing factor was absorbed by the malignant cells.

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.