Vitamin D Deficiency Promotes Liver Tumor Growth in Transforming Growth Factor-ß/Smad3-Deficient Mice Through Wnt and Toll-like Receptor 7 Pathway Modulation.

Disruption of the TGF-ß pathway is associated with liver fibrosis and suppression of liver tumorigenesis, conditions associated with low Vitamin D (VD) levels. However, potential contributions of VD to liver tumor progression in the context of TGF-ß signaling remain unexplored. Our analyses of VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3(+/-) mice, with a three-fold increase in TLR7 expression compared to controls. ChIP and transcriptional assays confirm Smad3 binding at two TLR7 promoter SBE sites. Molecular interactions between TGF-ß pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-ß pathway member expression levels and ß-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-ß member expression with lower ß-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-ß pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and ß-catenin activation. VD could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling.

Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.

UNLABELLED: Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. CONCLUSION: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

Dietary supplementation with conjugated linoleic acid does not improve nutritional status of tumor-bearing rats.

Tumor necrosis factor-alpha (TNF) is an immunoregulatory cytokine that plays a major role in tumor-induced anorexia and weight loss. Conjugated linoleic acids (CLA) are naturally occurring isomers of linoleic acid that, when added to the diet, improve food intake and body weight in mice injected with TNF. The purpose of the present study was to examine the effects of a diet supplemented with 0.5% CLA on the nutritional status of rats implanted with the Morris 7777 hepatoma. Body weight, food intake, serum levels of insulin-like growth factor, and splenocyte synthesis of TNF were not different in tumor-bearing animals fed CLA versus the control diet. However, insulin levels were increased in both tumor-bearing and control animals given CLA. The 0.5% CLA did not improve the nutritional status nor alter TNF synthesis in hypophagic tumor-bearing rats. The biological significance of increased insulin levels in animals given CLA remains to be determined.

Suppression of hypercholesterolemia in hepatoma-bearing rats by cabbage extract and its component, S-methyl-L-cysteine sulfoxide.

The effect of cabbage extract on cholesterol metabolism was studied in Donryu rats subcutaneously implanted with an ascites hepatoma cell line (AH109A). The hepatoma-bearing rats exhibited hypercholesterolemia induced by increasing cholesterogenesis in the host liver and decreasing steroid excretion into feces. The cabbage extract intake or administration reduced serum cholesterol level and enhanced fecal bile acid excretion and cholesterol 7alpha-hydroxylase activity, the rate-limiting enzyme of bile acid biosynthesis, in the microsomal fraction of the liver. Furthermore, S-methyl-L-cysteine sulfoxide, a component of cabbage, could mimic the effect of cabbage extract when orally administered. These results suggest that cabbage suppresses hypercholesterolemia responding to hepatoma growth by upregulating cholesterol catabolism and that S-methyl-L-cysteine sulfoxide in cabbage is one of the factors suppressing hypercholesterolemia in the hepatoma-bearing rats.

Supplemental nutrition with ornithine alpha-ketoglutarate in rats with cancer-associated cachexia: surgical treatment of the tumor improves efficacy of nutritional support.

We investigated the use of ornithine alpha-ketoglutarate in treatment of rats bearing Morris hepatoma 7777. Rats received diets containing either ornithine alpha-ketoglutarate, which has been used in other catabolic states (i.e. injury, sepsis), or an isonitrogenous, isocaloric diet containing glycine. Untreated tumors grew to a mass of 11 g/100 g body weight over the 3-wk period after implantation and induced progressive anorexia, negative nitrogen balance, and body and tissue wasting. Compared with glycine, ornithine alpha-ketoglutarate had no effect on tumor growth, but also did not alter the catabolic effects of the tumor on its host. We hypothesized that capture of amino acids by the tumor limited the efficacy of supplemental nutrition here and in published reports in which tumor burden comprised 4-30% of body weight. This is supported by our observation that a 3-wk of implantation the rate of protein deposition plus amino acid oxidation by the tumor was equivalent to approximately 70% of the host's daily protein intake. To parallel the clinical situation in which tumor burden is small at diagnosis and initiation of treatment, the same diets were tested in rats treated by excision of the tumor at a limited stage of the disease. Rats received 3 d preoperative nutrition with ornithine alpha-ketoglutarate or glycine, and continued on the same diets for 3 or 6 d postoperatively. Compared with glycine-fed rats, ornithine alpha-ketoglutarate-fed rats showed a more positive nitrogen balance, higher concentrations of glutamine and branched-chain amino acids in muscle, and accelerated protein deposition in small intestine (P < 0.05). Our results explain the lack of success of nutritional support in untreated cancer and underline the need for clinically relevant animal models for further studies.

Reduction of hyperlipidemia in hepatoma-bearing rats by dietary fish oil.

The effect of dietary fish oil on serum lipid levels was studied by comparing it with dietary corn oil in Donryu rats subcutaneously implanted with an ascites hepatoma cell line (AH109A). The hepatoma-bearing rats exhibited hyperlipidemia characterized by a rise in both serum cholesterol and triglyceride levels. Increased cholesterogenesis in the host liver and decreased steroid excretion into feces are suggested to be responsible for the hepatoma-induced hypercholesterolemia, and increased fatty acid mobilization from peripheral adipose tissues and decreased triglyceride clearance from the blood circulation are considered causes for the hepatoma-induced hypertriglyceridemia. Dietary fish oil reduced the hyperlipidemia in these animals, suppressed the hepatoma-induced increase in hepatic cholesterogenesis and fatty acid mobilization from adipose tissue. Dietary fish oil also tended to increase fatty acid oxidation in the liver. Such diverse effects of fish oil may lead to the reduction of the hepatoma-induced hyperlipidemia. These results suggest that studies on dietary fish oil may be warranted in patients with cancer-related hyperlipidemia.

Effects of dietary supplemented amino acids on endogenous hypercholesterolemia in rats.

Effects of additions of amino acids to a 20% casein diet on serum cholesterol (Ch) were studied in hypothyroid and hepatoma-bearing rats with endogenous hypercholesterolemia as well as in normal rats. In normal Wistar rats, methionine (Met) was hypercholesterolemic at the "nutritional" level (0.2-0.4%), but hypocholesterolemic at the "excess" level (1.2-2.4%). In Wistar rats with hypothyroidism induced by thiouracil, the addition of excess (1.2%) Met to the 20% casein diet reduced an endogenous hypercholesterolemia due to hypothyroidism by suppressing an elevation in (VLDL + LDL)-Ch with no significant influence on HDL-Ch. In Donryu rats received a subcutaneous implantation of AH109A cells (an ascites hepatoma line), either 1.2% Met, 1.2% cystine (Cys), or 1.2% Met and 2.5% glycine (Gly) in combination improved a hepatoma-induced hypercholesterolemia and abnormal serum lipoprotein profiles by suppressing a hepatoma-induced increase in (VLDL + LDL)-Ch. From Ch turnover studies in hepatoma-bearing rats, an impaired catabolism of Ch in the liver was suggested to be one cause for the hepatoma-induced elevation in (VLDL + LDL)-Ch. One of the dietary manipulations. met and Gly in combination (Met + Gly), was found to improve the impaired Ch catabolism, this leading to a reduction of the (VLDL + LDL)-Ch level by Met + Gly in hepatoma-bearing rats.

Effect of cancer cachexia and amiloride treatment on the intracellular sodium content in tissue cells.

This study was designed to investigate the effects of a growing H6 hepatoma on the intracellular element content in three distinctly different tissue cell populations of the mouse host (hepatocytes, fibroblasts, and crystal enterocytes). X-ray microanalysis measurements of the intranuclear concentrations of several elements (sodium, magnesium, phosphorus, sulfur, chlorine, and potassium) were made. Briefly, the tumor presence significantly increased intranuclear sodium concentration but not the concentration of magnesium, phosphorus, sulfur, chlorine, or potassium in three tissue cell types of mice that were anorectic and cachectic. A second aim of the study was to see if injections of the diuretic amiloride, a drug reported to block passive influx of sodium into mammalian cells, would counteract the effect of the tumor presence and lower the intranuclear concentration of sodium towards that of a non-tumor-bearing host. Amiloride did significantly lower the intranuclear level of sodium in the host tissues to that of non-tumor-bearing mice. The amiloride-caused decrease on intracellular sodium was correlated to a decreased cell proliferation activity in the tumor cells and duodenal enterocytes. A possible relationship between the intracellular concentration of sodium in tissue cells and cancer cachexia is discussed.

Antirheumatic drugs and macrophage function: effects on tumour cell growth in vitro.

The ability of the antirheumatic drugs D-penicillamine, chloroquine and levamisole to modify macrophage-mediated inhibition of tumour cell growth in vitro was investigated. Increasing numbers of rat peritoneal macrophages were cocultured with a fixed number of ascites hepatoma AH-13 rat tumour cells. Tumour cell growth was assessed as the uptake of 3H-thymidine (3H-TdR) by AH-13 cells at the end of a 24 h period of coculture with macrophages treated in vitro or in vivo with the various drugs. In vitro, preincubation of macrophages with D-penicillamine or chloroquine (50 - 250 micrograms/ml) increased tumour cell 3H-TdR incorporation, compared to cultures with untreated macrophages. Macrophages from rats treated with D-penicillamine or chloroquine (50 mg/kg/day orally) for 4 days similarly increased tumour cell 3H-TdR incorporation, compared to cultures with macrophages from untreated rats. These effects persisted for at least 3 to 4 weeks of treatment. Preincubation with levamisole (10 - 100 micrograms/ml) in vitro had no effect on macrophage-mediated inhibition of tumour cells, whereas increased tumour cell 3H-TdR incorporation was observed in cultures with macrophages from rats treated with levamisole (5 mg/kg/day orally) in vivo. Macrophages from rats with experimentally induced chronic inflammation, i.e. adjuvant arthritis, were found to increase tumour cell 3H-TdR incorporation, compared to macrophages from nonarthritic rats. This trend was further enhanced by treatment with D-penicillamine, chloroquine or levamisole.