Murine interleukin-12 prevents the development of cancer cachexia in a murine model

Murine colon 26 carcinoma causes cachexia even when the tumor burden is small. In this tumor model, murine IL-12 suppressed the induction of cancer cachexia and also inhibited tumor growth. IL-12 reduced the serum levels of IL-6, a cachexia mediator in this model, and alleviated the body weight loss and other abnormalities associated with cachexia, such as adipose tissue wasting and hypoglycemia. The anticachectic activity was observed even at low doses of IL-12, insufficient to inhibit tumor growth. IL-12 greatly increased levels of IFN-γ in the tumor tissue and, to a lesser extent, in the circulation. IFN-γ given intraperitoneally also prevented cancer cachexia, although it did not reduce IL-6 levels either in the tumor or in the circulation. In athymic mice bearing the same colon 26 tumor, IL-12 was no longer anticachectic and did not induce IFN-γ. These results indicate that the anticachectic activity of IL-12 is T-cell-dependent and results from at least 2 mechanisms, the down-regulation of IL-6 and the up-regulation of IFN-γ. © 1996 Wiley-Liss, Inc.     

Tumour-associated hypoglycaemia in a murine cachexia model.

Animals bearing a cachexia-inducing tumour, the MAC16 adenocarcinoma, showed a progressive decrease in blood glucose levels with increasing weight loss, while animals bearing a histologically similar tumour, the MAC13 adenocarcinoma, showed no change in either body weight or blood glucose levels with growth of the tumour. The effect of the MAC16 tumour on blood glucose levels appeared to be unrelated to food intake, glucose consumption by the tumour, or to the production of increased levels of IGF-I and IGF-II mRNA by the tumour cells. The relationship between the induction of cachexia and alteration in blood glucose levels remains unknown.     

Effect of insulin on weight loss and tumour growth in a cachexia model

A comparison has been made between the effects of daily insulin injection and a ketogenic diet on weight loss and tumour weight in an experimental model of cancer cachexia (MAC16). Weight loss associated with the MAC16 tumour was significantly reduced both by a ketogenic diet (80% MCT) and by daily insulin injections without an increase in either food or water consumption. Animals fed the 80% MCT diet had a significantly reduced tumour weight compared with controls fed a normal laboratory diet, while in animals administered 20 U insulin kg-1 day-1 the tumour weight was 50% greater than in saline infused controls. The stimulation of tumour growth by insulin was counteracted by the inclusion of 3-hydroxybutyrate in the drinking water without any alteration in the extent of weight loss. Depletion of both carcass fat and muscle dry weight in animals bearing the MAC16 tumour was reversed in animals administered either insulin or an 80% MCT diet. Animals bearing the MAC16 tumour had a reduced nitrogen balance compared with non-tumour-bearing controls, mainly due to excess urea excretion, and this was reversed towards control values in animals fed an 80% MCT diet, but not in animals administered insulin. These results suggest that a ketogenic diet is more effective than insulin administration in reversing the cachectic process and has the advantage of a concomitant reduction in tumour weight.     

Doxifluridine decreases serum levels of interleukin-6 in a cancer cachexia model]

Cancer cachexia is a common paraneoplastic syndrome in patients with advanced malignancies. However, the mechanisms of the development of cancer cachexia remain to be elucidated. Interleukin (IL)-6 is known to be involved in the development of cancer cachexia. The KPL-4 human breast cancer cell line, which was recently established in our laboratory, secretes IL-6 into the culture medium. Oral administration of doxifluridine (5'-DFUR, 60 mg/kg or 120 mg/kg) significantly inhibited the growth of KPL-4 tumors, reduced the tissue levels of IL-6, and alleviated body weight loss. Serum IL-6 levels were also lowered by 5'-DFUR in nude mice bearing KPL-4 tumors. Additionally, it is suggested that tumor necrosis factor (TNF)-alpha is involved in the cachexia induced by KPL-4 tumors. We suggest that 5'-DFUR suppresses cancer cachexia by lowering IL-6 levels in the tumor tissues and serum.     

Reduction of weight loss and tumour size in a cachexia model by a high fat diet

An attempt has been made to reverse cachexia and to selectively deprive the tumour of metabolic substrates for energy production by feeding a ketogenic regime, since ketone bodies are considered important in maintaining homeostasis during starvation. As a model we have used a transplantable mouse adenocarcinoma of the colon (MAC 16) which produces extensive weight loss without a reduction in food intake. When mice bearing the MAC16 tumour were fed on diets in which up to 80% of the energy was supplied as medium chain triglycerides (MCT) with or without arginine 3-hydroxybutyrate host weight loss was reduced in proportion to the fat content of the diet, and there was also a reduction in the percentage contribution of the tumour to the final body weight. The increase in carcass weight in tumour-bearing mice fed high levels of MCT was attributable to an increase in both the fat and the non-fat carcass mass. Blood levels of free fatty acids (FFA) were significantly reduced by MCT addition. The levels of both acetoacetate and 3-hydroxybutyrate were elevated in mice fed the high fat diets, and tumour-bearing mice fed the normal diet did not show increased plasma levels of ketone bodies over the non-tumour-bearing group despite the loss of carcass lipids. Both blood glucose and plasma insulin levels were reduced in mice bearing the MAC16 tumour and this was not significantly altered by feeding the high fat diets. The elevation in ketone bodies may account for the retention of both the fat and the non-fat carcass mass. This is the first example of an attempt to reverse cachexia by a diet based on metabolic differences between tumour and host tissues, which aims to selectively feed the host at the expense of the tumour.     

Acceleration of a murine T-cell leukemia associated with loss of interleukin-3 producing activity

L-8313 is a murine T-cell leukemia cell line the cells of which constitutively produce interleukin-3 (IL-3). S2-8313 is a unique subline of L-8313 which has lost IL-3 producing activity. Although the growth of S2-8313 cells in the bone marrow is comparable to that of L-8313 cells, the survival time of C3H mice grafted with S2-8313 cells is significantly shorter than that of C3H mice grafted with L-8313 cells. The accelerated death observed in C3H mice bearing S2-8313 cells is attributable to early development of granulocytopenia and thrombocytopenia, which resulted from the loss of the IL-3 producing activity.     

Effect of a fluorinated pyrimidine on cachexia and tumour growth in murine cachexia models: relationship with a proteolysis inducing factor

The fluorinated pyrimidine nucleoside, 5'-deoxy-5-fluorouridine (5'-dFUrd) has been shown to effectively attenuate the progress of cachexia in the murine adenocarcinomas MAC16 and colon 26 as well as in the human uterine cervical carcinoma xenograft, Yumoto. Although concomitant inhibition of tumour growth was observed in all three models this was not sufficient to account for the preservation of body weight. An attempt has been made to correlate the anti-cachectic activity of 5'-dFUrd with the presence of a tumour produced proteolysis-inducing factor (PIF), thought to be responsible for the development of cachexia in the MAC16 model. Two variants of colon 26 adenocarcinoma were employed, clone 20 which produces profound cachexia, and clone 5 which produces no change in body weight in recipient animals. Mice bearing the colon 26, clone 20 variant showed evidence for the presence of PIF in tumour, serum and urine, while there was no evidence for the presence of PIF in tumour or body fluids of mice bearing the clone 5 tumours. Treatment of animals bearing the clone 20 variant with 5'-dF Urd led to the disappearance of PIF from the tumour, serum and urine concomitant with the attenuation of the development of cachexia. The human cervical carcinoma, Yumoto, which also induced cachexia in recipiant animals, showed expression of PIF in tumour, serum and urine in control and vehicle-treated mice, but was absent in mice treated with 5'-dFUrd. Thus in these experimental models cachexia appears to be correlated with the presence of PIF.     

Flavopiridol-related proinflammatory syndrome is associated with induction of interleukin-6.

BACKGROUND: Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins. PURPOSE: The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines. METHODS: Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h). RESULTS: There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells. CONCLUSION: Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.     

Serum soluble tumour necrosis factor receptor 55 is increased in patients with haematological neoplasias and is associated with immune activation and weight loss

Enhanced concentrations of soluble forms of the receptor for tumour necrosis factor (TNF)-α have been detected in the serum of cancer patients. We determined serum concentrations of soluble TNF receptor p55 (sTNF-R55) in patients with haematological neoplasias, 50 patients suffering from non-Hodgkin's lymphoma (n = 35), Hodgkin's disease (n = 10) and multiple myeloma (n = 5). Compared with healthy controls and with patients with potential thyroid disease, significantly elevated concentrations of sTNF-R55 were found (mean ± standard error: 2.68 ± 0.22 vs. 1.23 ± 0.21 ng/ml, P < 0.0001 and 2.18 ± 0.32 ng/ml, P = 0.03). Likewise, neopterin concentrations were raised (19.6 ± 3.66 vs. 5.3 ± 0.25 nmol/l in controls, P < 0.0001). We found a significant correlation between sTNF-R55 and neopterin concentrations (Rs = 0.544, P < 0.001). Patients with weight loss showed higher sTNF-R55 concentrations than patients with stable weight. Our results confirm the relevance of sTNF-R55 concentrations in serum of patients with cancer.     

Enhancement of in vivo antitumor activity of a novel antimitotic 1-phenylpropenone derivative, AM-132, by tumor necrosis factor-alpha or interleukin-6.

TK5048 and its derivatives, AM-132, AM-138, and AM-97, are recently developed antimitotic (AM) compounds. These 1-phenylpropenone derivatives induce cell cycle arrest at the G2 / M phase of the cell cycle. TK5048 inhibited tubulin polymerization in human lung cancer PC-14 cells in a concentration-dependent manner. In a polymerization assay using bovine brain tubulin, AM-132 and AM-138 were quite strong, AM-97 was moderately strong, and TK5048 was a relatively weak inhibitor of tubulin polymerization. A murine leukemia cell line resistant to a sulfonamide antimitotic agent, E7010, which binds to colchicine-binding sites on tubulin, was cross-resistant to the in vitro growth-inhibitory effect of AM compounds. Inhibition of tubulin polymerization is therefore one of the mechanisms of action of these AM compounds against tumor cells. To profile the antitumor effect of AM compounds, the in vivo antitumor effect of AM-132 was evaluated against cytokine-secreting Lewis lung carcinoma (LLC). Tumor-bearing mice were treated with intravenous AM-132 using three different treatment schedules. LLC tumors expressing tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-6 were very sensitive to AM-132. In particular, LLC tumors expressing IL-6 were markedly reduced by AM-132 treatment, and showed coloring of the tumor surface and unusual hemorrhagic necrosis. These results suggest a combined effect of AM-132 and cytokines on the blood supply to tumors.     

A comparison of long-chain triglycerides and medium-chain triglycerides on weight loss and tumour size in a cachexia model

A comparison has been made between the ability of long-chain triglycerides (LCT) and medium-chain triglycerides (MCT) to prevent weight loss induced by the cachexia-inducing colon adenocarcinoma (MAC16) and to reduce tumour size. There was no difference in calorie consumption or nitrogen intake between the various groups. When compared with a normal control high carbohydrate, low fat diet, animals fed MCT showed a reduced weight loss and a marked reduction in tumour size. In contrast neither weight loss nor tumour size differed significantly from the controls in animals fed the LCT diet. An elevated plasma level of 3-hydroxybuturate was found only in the animals fed the MCT diets. Administration of LCT caused an increase in the plasma level of FFA, which was not observed in the MCT group. These results suggest that diets containing MCT would provide the best ketogenic regime to reverse the weight loss in cancer cachexia with a concomitant reduction in tumour size.     

Serum levels of tumour necrosis factor-alpha and interleukin-6 and their correlation with body mass index, weight loss, appetite and survival rate--preliminary data of Viennese outpatients with metastatic cancer during palliative chemotherapy

The serum cytokine levels (in particular interleukine-6 (IL-6) and tumour necrosis factor-α (TNF-α)) of 61 advanced stage cancer patients receiving palliative chemotherapy as outpatients were determined with quantikine immunoassays. The values were correlated with body mass index (BMI), weight loss and appetite. Furthermore cytokine levels of patients who have died within one year were compared with those of patients who have survived more than a year. Serum levels of IL-6 (median: 1.93 pg/ml, range: 0.32–42.87) and of TNF-α (median: 2.55 pg/ml, range: 1.03–34.06) did not correlate with BMI, weight loss and appetite. Serum IL-6 levels of patients with survival time less than one year were significantly higher than the levels of patients who survived more than one year, no significant differences in TNF-α serum levels were evident. The data of this observation are consistent with current literature. Due to changes in serum levels of proinflammatory cytokines in response to chemotherapy and additional therapy, it is unlikely that IL-6 and TNF-α can be used as independent indicators for weight loss and appetite. Nevertheless, high serum levels of IL-6 correlate with short-time mortality.     

Inhibition of pro-inflammatory cytokines in tumour associated macrophages is a potential anti-cancer mechanism of carboxyamidotriazole

Carboxyamidotriazole (CAI) has not only direct anti-cancer activities, but also anti-inflammation effects in a variety of inflammatory animal models. In the present study, we investigated whether macrophages, which are important both in cancer and inflammation, could be regulated by CAI. The results showed that CAI could inhibit tumour necrosis factor-α (TNF-α) production in macrophages in various environments, including those isolated from peritoneal cavity of adjuvant-induced arthritis (AA) rats, from Lewis lung carcinoma (LLC) transplanted tumours and those induced by LLC cells in vitro. Dexamethasone (DEX), one of the pro-inflammatory cytokines inhibitors, could enhance CAI’s inhibition of LLC cells proliferation and invasion in macrophages and LLC cells co-culture systems, as well as the tumour growth in vivo. However, DEX failed to enhance CAI’s inhibition of LLC cells proliferation when LLC cells were cultured alone, suggesting that the combination of CAI and DEX exerted great anti-tumour effects probably by acting on macrophages in the tumour environment. Over all, we found CAI could act on macrophages and regulate the production of TNF-α not only in inflammatory diseases but also in tumour microenvironment, which might be another anti-tumour mechanism of CAI.     

Interleukin-6, tumour necrosis factor alpha and interleukin-1beta in patients with renal cell carcinoma

As regulators of malignant cell behaviour and communication with stroma, cytokines have proved useful in understanding cancer biology and developing novel therapies. In renal cell carcinoma, patients with inflammatory reactions are known to have poor prognosis. In order to elucidate the relation between renal cell carcinoma and the host, serum levels of inflammatory cytokines, interleukin-6, tumour necrosis factor alpha, interleukin-1beta, were measured. One hundred and twenty-two patients with renal cell carcinoma and 21 healthy control subjects were studied, and serum cytokine levels were measured using a highly sensitive ELISA kit. As a result, in the control group, interleukin-6, tumour necrosis factor alpha and interleukin-1beta levels were 1.79+/-2.03, 2.74+/-0.94 and 0.16+/-0.17 pg ml(-1), respectively. In the renal cell carcinoma patients, they were 8.91+/-13.12, 8.44+/-4.15 and 0.53+/-0.57 pg ml(-1), respectively, and significantly higher. In the comparison of stage, interleukin-6 level was significantly higher in the stage IV group compared to the other stage groups including the control group, while tumour necrosis factor alpha level was significantly higher in each stage group compared to the control group. As for grade, interleukin-6 level was significantly higher in the grade 3 group compared to the control, grade 1 and grade 2 groups, while tumour necrosis factor alpha level was significantly higher in each grade group compared to the control group. All cytokines had a positive correlation with tumour size. In regard to the correlation with CRP, all cytokines had a positive correlation with CRP, while interleukin-6 had a particularly strong correlation. In conclusion, interleukin-6 may be one of the factors for the poor prognosis of patients with renal cell carcinoma. In addition, tumour necrosis factor alpha may be useful in the early diagnosis of renal cell carcinoma and post-operative follow-up.     

NM23-H1 immunostaining is inversely associated with tumour staging but not overall survival or disease recurrence in colorectal carcinomas.

The NM23-H1 gene product has been recently identified as a potential metastasis suppressor. Studies on breast carcinomas have shown an inverse correlation between NM23-H1 status and stage of carcinogenesis and overall survival. However, in colorectal cancer, conflicting data have been reported. This study aimed to investigate whether NM23-H1 immunostaining is correlated with tumour stage, overall survival, disease recurrence, tumour differentiation, age and sex in colorectal carcinomas for the Singapore population using chi-square analysis. The staining was performed on 141 paraffin-embedded surgical specimens collected between 1991 and 1992 using a monoclonal anti-NM23-H1 antibody. Follow-up of patients was until time of death or for 5 years. There was a very significant inverse association between tumour staging and NM23-H1 status (P = 0.0004). However, NM23-H1 expression was not significantly correlated to overall 5-year survival, disease recurrence, tumour differentiation, age or sex. Thus, although NM23-H1 may be involved in suppressing metastasis, NM23-H1 immunohistochemistry has no prognostic value in colorectal cancer. This is the first report of a significant inverse association of NM23-H1 status with tumour staging in colorectal cancer which showed no correlation with overall survival or disease recurrence. Our result thus cautions against the practice of equating an inverse relation of genetic markers with tumour staging to survival or disease recurrence.     

Rosiglitazone and imidapril alone or in combination alleviate muscle and adipose depletion in a murine cancer cachexia model

Rosiglitazone (RGZ) and imidapril improve cancer cachexia via different mechanisms. Therefore, we hypothesized that combination therapy of RGZ+imidapril would further attenuate cancer cachexia in vivo. After injection with colon-26 adenocarcinoma for 9 days, BALB/c mice were randomly divided into the following four treatment groups for 7 days (n?=?8 per group): (1) placebo, (2) RGZ, (3) imidapril, and (4) RGZ+imidapril. Eight healthy control animals were also assessed. Body weight, tumor volume, gastrocnemius muscle and epididymal adipose mass, serum metabolic markers and cytokines, and the expression of nuclear factor-κB and two E3 ubiquitin ligases, atrogin-1 and MuRF-1, were measured. From days 14 to 16, all treatments significantly reduced tumor volume (P?<?0.05). From days 10 to 16, improvements in the tumor-free body weight were observed in the RGZ and RGZ+imidapril groups. In addition, significant improvements in both gastrocnemius muscle and epididymal adipose mass were observed in all treatment groups (all, P?<?0.05). Furthermore, all treatments significantly increased tumor necrosis factor alpha levels as compared to those observed in the healthy control animals (P?<?0.001). Insulin levels significantly increased in the placebo group as compared to those in the healthy control group (P?<?0.05), which were reduced in all the treatment groups (P?<?0.05). Finally, whereas all treatments significantly reduced atrogin-1 levels as compared to the placebo group (all, P?<?0.05), significant reductions in MuRF-1 levels were only observed in the RGZ and RGZ+imidapril groups (both, P?<?0.05). Thus, all three treatments reduce tumor growth and alleviate cancer cachexia; however, synergistic effects of RGZ+imidapril combination therapy were not observed.     

Isolated limb perfusion with tumour necrosis factor-alpha and melphalan for unresectable bone sarcomas of the lower extremity.

AIMS: Isolated limb perfusion (ILP) with recombinant tumour necrosis factor-alpha (rTNF-alpha) and melphalan has recently been reported to induce major tumour responses and permit limb salvage in over 80% of patients with unresectable soft-tissue sarcomas of the extremities. We investigated whether TNF-based ILP could allow limb-sparing surgery in patients with primary, recurrent or metastatic bone sarcoma to the lower extremity who met the criteria for an amputation and had failed or refused chemotherapy. METHODS: From August 1992 to December 1997, we employed ILP with rTNF-alpha and melphalan in 13 patients with unresectable bone sarcoma of the lower extremity, all of whom were candidates for amputation. The aim was to reduce tumour size and allow the performance of a limb-sparing surgery (LSS). RESULTS: Following ILP, none of the patients had severe local toxicity and only one patient experienced significant systemic side-effects. LSS was subsequently performed in nine of the 13 patients. LSS was feasible in an additional three patients but was not performed because of the emergence of diffused metastatic disease. CONCLUSIONS: ILP with rTNF-alpha and melphalan can allow limb salvage in patients wih locally advanced bone sarcomas who had failed standard treatment options. Its potential role in the treatment of unresectable bone sarcomas of the extremities merits further evaluation.