Phytochemicals, antioxidant activity and hepatoprotective effect of ginger (Zingiber officinale) on diethylnitrosamine toxicity in rats.

Context: Chronic liver damage has serious medical consequences. Objective: To investigate the hepatoprotective effect of dry Zingiber officinale (ginger) and its essential (volatile) oil against diethylnitrosamine (DEN) toxicity in rats. Materials and methods: Phenols and flavonoids components were characterized in dry ginger using HPLC-UV instrument while ginger essential oil (E.O.) was investigated via GC-MS technique. Antioxidant activity was determined in vitro. In rat model, ginger was administrated for 2 months. Lipid profile, antioxidant biomarkers, liver functions and histopathology were assessed. Results: Chlorogenic acid (63.85 ppm) and hesperidin (156.91 ppm) are among the major phenolic and flavonoid constituents in dry ginger. Curcumene (15.21%) and linalool (13.47%) represent the main E.O. constituents. In rats treated with ginger E.O., a significant elevation in serum HDL (31.14%) was accompanied by a decrease in LDL (55.14%). A significant decrease in serum ALT and ALP was reported (56.85% and 53.84%, respectively). Serum GSH-Px activity has significantly increased 75.06%. Meanwhile, E.O. showed anticancer potential against HepG2 cell line (IC50 = 40 µg/mL). Liver histopathological examinations confirmed the protective effect against abnormalities. Conclusion: Ginger was able to reduce the severity of DEN-cytotoxicity in rats, which suggests a novel antioxidant role originating from this medicinal plant.

Therapeutic Efficacy of Vitamin D in Experimental c-MET-ß-Catenin-Driven Hepatocellular Cancer.

Aberrant activation of ß-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/ß-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant ß-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH)2D3] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH)2D3 is reported to inhibit ß-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with ß-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.

Octahydrocurcumin, a final hydrogenated metabolite of curcumin, possesses superior anti-tumor activity through induction of cellular apoptosis.

The biological activity of curcumin (CUR), a promising naturally occurring dietary compound for the treatment of hepatocellular carcinoma (HCC), was closely associated with its metabolite. Octahydrocurcumin (OHC) is the final hydrogenated metabolite of CUR and has been reported to have potential biological activities. However, difficulties in access have hampered its biological studies. In the current investigation, we designed an efficient synthesis method to produce OHC, and comparatively explored the anti-cancer effect and potential mechanism of OHC and CUR in an H22 ascites tumor-bearing mice model. The results indicated that OHC had a relatively wide margin of safety, and exhibited superior effects to CUR in suppressing the tumor growth, including ascending weight, abdominal circumference, ascites volume and cancer cell viability. OHC significantly induced H22 cell apoptosis by upregulating the p53 expression and downregulating the MDM2 expression. OHC also remarkably decreased the Bcl-2 and Bcl-xl protein expressions, and increased the Bax and Bad expressions in ascitic cells. Furthermore, THC substantially induced the release of cytochrome C, caspase-3, caspase-9 and the cleavage of PARP to induce H22 cell apoptosis. Taken together, OHC was more effective than CUR in suppressing H22-induced HCC through the activation of the mitochondrial apoptosis pathway. OHC may thus be a promising anti-HCC agent.

Caloric Restriction Prevents Carcinogen-Initiated Liver Tumorigenesis in Mice.

Caloric restriction (CR) and endurance exercise elicit wide-ranging health benefits including reduced risk of select cancers. In addition, diet composition influences oncogenesis, although its interactions with exercise and CR are not well understood. Therefore, to investigate the potential interactions between diet and lifestyle interventions on liver tumorigenesis, the carcinogen diethylnitrosamine was administered to 72 male C57Bl/6 mice that were subsequently fed diets enriched with lard (CTL) or olive oil and were further stratified to voluntary wheel running (Ex) or 30% CR for 49 weeks. Although Ex and diet composition did not influence liver oncogenesis, CR prevented hepatic tumor formation. In addition, CR reduced steatosis, hepatocyte ballooning, inflammation, and immune cell infiltration, all of which are hallmarks in the progression of nonalcoholic fatty liver disease to liver tumorigenesis. RNA sequencing of nontransformed liver tissues from CR mice revealed changes in metabolic pathways and reduced inflammation, cytokine production, stellate cell activation and migration, and genes associated with liver injury and oncogenesis. These data demonstrate that CR protects against steatosis, liver inflammation, and liver injury and is a robust deterrent of carcinogen-induced hepatic oncogenesis. Cancer Prev Res; 10(11); 660-70. ©2017 AACR.

Inhibition of Diethylnitrosamine-Induced Hepatocarcinogenesis in Mice by a High Dietary Protein Intake.

Epidemiological and experimental evidence supports the key role of diet in the development of many types of cancer. Recent studies have suggested that dietary modifications may be beneficial for individuals at high risk for hepatocellular carcinoma (HCC). In this study, we investigated the effect of a high-protein (HP; 20% casein) dietondiethylnitrosamine (DEN)-induced hepatocarcinogenesis. Mice were given free access to water with 30 µg/ml DEN and fed a normal or HP diet for 22 wk. The results showed mice consuming HP diets had reduced mortality rates and body weights and lower hepatic enzyme activity compared to DEN-treated mice on a normal diet. HP consumption also promoted collagen accumulation in the liver, and reduced numbers of proliferating hepatocytes and infiltrating inflammatory cells, as well as decreased expression of inflammatory factor interleukin-1ß, and nuclear factor κB activation. These data indicate that HP diets can inhibit DEN-induced hepatocarcinogenesis via suppression of the inflammatory response and provide a new evidence for the dietary management of clinical patients with hepatocellular carcinoma.

Effect of dietary soy protein on tumor necrosis factor productivity in macrophages from nephritic and hepatoma-bearing rats.

The present study investigated the effect of dietary soy protein isolate (SPI) on tumor necrosis factor-alpha (TNF) productivity in peritoneal macrophages from nephritic and hepatoma-bearing rats. Dietary SPI significantly inhibited the elevated production of TNF by lipopolysaccharide-stimulated macrophages in nephritic and hepatoma-bearing rats compared with dietary casein, while it exerted no influence on the TNF productivity in normal rats. Removal of the minor components contained in SPI by ethanol extraction could significantly or partially restore the reduced TNF production caused by SPI in nephritic and hepatoma-bearing rats, respectively. These results suggest that dietary SPI could suppress the enhanced productivity of TNF associated with the progression of nephritis and hepatoma, and some factors existing in the ethanol extract of SPI are suggested to be involved in suppressing TNF productivity by macrophages.

Dietary PUFA modulate the expression of proliferation and differentiation markers in Morris 3924A hepatoma cells.

The effect of dietary polyunsaturated fatty acids on the expression of differentiation and proliferation markers in Morris 3924A hepatoma cells was investigated. ACT/I rats were conditioned 10 days with diets enriched with linoleic acid or alpha-linolenic acid before subcutaneous hepatoma cell transplantation. After 19 days from the inoculum, the mRNA levels of liver-enriched transcription factors and of their target genes were quantified. Both linoleic acid- and linolenic acid-enriched diets induced a decrease of beta-actin, AFP, PCNA, c-myc and of hepatocyte nuclear factors HNF-1alpha and HNF-4alpha mRNA levels in tumor tissue whereas HNF-3beta expression was induced by both dietary treatments. Only the alpha-linolenic acid-enriched diet was effective in reducing c-jun and increasing albumin mRNA levels. Since albumin is a C/EBPalpha target gene, C/EBPalpha gene transcription was evaluated at both protein and mRNA levels. It was found that alpha-linolenic acid-enriched diet did not enhance the C/EBPalpha mRNA content in hepatoma tissue while inducing C/EBPalpha protein expression with an isoform pattern similar to the hepatic phenotype. This evidence implies that alpha-linolenic acid or one of its metabolic products induce albumin synthesis in hepatoma cells by modulating C/EBPalpha gene expression at post-transcriptional level.

Nontoxic potentiation of cancer chemotherapy by dietary oligofructose or inulin.

Our previously published results indicated that dietary treatment with oligofructose or inulin inhibited malignant tumor growth in experimental animals. Thus it appeared to be interesting to investigate whether the same treatment could have a positive influence on tumor chemotherapy. The chemotherapy-potentiating effect of 15% oligofructose or inulin incorporated into the basal diet for experimental animals was investigated on a transplantable mouse liver tumor. This dietary adjuvant therapy was started seven days before intraperitoneal transplantation of transplantable liver tumor and was continued until the end of experiments. A single, subtherapeutic dose of six different cytotoxic drugs commonly utilized in treatment of human cancer was intraperitoneally injected 48 hours after tumor transplantation. In all experiments, dietary oligofructose or inulin significantly potentiated the therapeutic effects of six different cytotoxic drugs. Such dietary treatment potentiating cancer chemotherapy could be introduced into classical protocols of human cancer treatment as a new, nontoxic, and easily applicable adjuvant cancer therapy without any supplementary risk for patients.

Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis.

The effect of individual administration of low doses of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1 g/kg body weight) on the growth of Morris hepatocarcinoma 3924A transplanted in ACI/T rats was investigated. Both EPA and DHA inhibited growth of the hepatocarcinoma (50% reduction of tumor weight or volume at the 19th day after transplantation for both of the n-3 PUFA groups). EPA treatment reduced the percentage of proliferating tumor cells labeled with BUdR (10-fold), whereas DHA did not. Conversely, DHA supplementation induced a doubling of the number of cells undergoing apoptosis (labeled by TUNEL), whereas EPA treatment was much less effective. Analysis of changes in phospholipid fatty acids in tumor-cell membranes after both treatments with EPA and DHA showed a significant reduction in arachidonic-acid levels. EPA and docosapentaenoic acid (DPA), its elongation product, were increased in the phospholipids from EPA-treated animals. DHA and EPA, but not DPA, were increased in the DHA-treated group. It is concluded from the results of the present study that the anti-tumoral effect of EPA is related mainly to its inhibition of cell proliferation, whereas that of DHA corresponds with its induction of apoptosis. The alterations in fatty-acid composition induced by EPA or DHA appear to be factors underlying their differential actions on cell proliferation and apoptosis.

Inhibition of aflatoxin Ml excretion in rat urine during dietary intervention with oltipraz.

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz) is an effective chemopreventive agent against several classes of carcinogens in many target organs. Induction of carcinogen detoxication enzymes, particularly glutathione S-transferases, appears to be an important component of the protective actions of oltipraz. It has recently been observed that addition of oltipraz to rat liver microsomes or to cultured human hepatocytes blocks the oxidative metabolism of aflatoxin B1 (AFB1) to its 8,9-oxide and the hydroxylated derivative aflatoxin Ml (AFM1). 0ltipraz is a competitive and perhaps irreversible inhibitor of cytochromes P450 1A2 and 3A4. To determine whether oltipraz can affect cytochrome P450-dependent metabolism of AFB1 in vivo we have assessed the effect of oltipraz on the urinary excretion of oxidative metabolites of AFB1 before, during and after a transient intervention. Male F344 rats, housed individually in glass metabolism cages, were gavaged daily with 25 microg [3H]AFB1 for 28 consecutive days. Starting on day 6 and extending to day 16 half of the rats were fed a diet supplemented with 0.075% oltipraz. Sequential 24 h urine samples were collected and a subset analyzed for AFB1 metabolites. AFM1 was the major metabolite detected in all urine samples, accounting for 2-6% of the administered dose. The excretion of AFM1 was greatly reduced (77%) during the active phase of the intervention, when oltipraz was added to the diet, but rapidly returned to control levels after cessation of oltipraz administration. This inhibition of AFM1 excretion was not seen in animals receiving oltipraz by gavage 24 h prior to dosing with AFB1. Collectively these data are consistent with the view that oltipraz or a short-lived metabolite inhibits cytochrome P450 1A2 in vivo.

Growth inhibition of subcutaneously transplanted hepatomas by alterations of the dietary arginine-methionine balance.

We hypothesized that alteration of the dietary arginine-methionine balance might inhibit tumor growth and suggest nutritional strategies for cancer therapy. The Morris hepatoma 3924A was subcutaneously transplanted in ACI rats. Control diets containing normal levels of arginine, methionine, and other amino acids in replacement of protein (24%), carbohydrates (59%), fat (10%), and fiber, vitamins, and minerals (7%) were fed for 28 days. Six experimental diets were adjusted to maintain amino acids at 23-25% and carbohydrates at 58-60%; these diets were 1%-2% deficient in arginine or supplemented with 1-2% arginine (expressed as percent amino acid content of diet) in combination with normal, deficient, and supplementary levels of methionine. Daily food intake was unaffected by the experimental diets. The control groups gained 26.4 +/- 2.8 g body weight, and small body weight decrements ranged from 3.5% to 8.4% in the groups fed the experimental diets. Tumor weight of controls was 8.5 +/- 1.5% of body weight. The experimental diets that produced significant tumor growth inhibition (TGI) were 1) the arginine-methionine-deficient diet, 2) the arginine-excess-methionine-deficient diet, 3) the arginine-deficient diet, and 4) the excess-arginine diet. Diets containing excess methionine failed to produce TGI. TGI resulted in tumor weights 41-46% of control values. TGI was associated with significantly lower blood urea nitrogen, plasma protein, and tumor spermidine-to-spermine ratio than in tumor-bearing controls. It is concluded that dietary alteration of a single amino acid, arginine, might be a potentially useful nutritional strategy for controlling tumor growth.

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.

Inhibition of AFB1-induced liver cancer and induction of increased microsomal enzyme activity by dietary constituents.

A long-term study, using male Wistar rats, was initiated to determine whether the effects of dietary constituents on AFB1-induced liver cancer could be associated with altered microsomal enzyme activity. They were maintained on mice pellets mixed with specific dietary constituents for 7 days and then given a single carcinogenic dose of AFB1 (500 micrograms/rat). After three months, the dietary constituents were discontinued and the animals were left on mice pellets and drinking water only for a period of about 20 months. At the end of the trial period, it was observed that dietary mixtures containing small quantities of either beta-carotene, ascorbic acid, GSH, vitamin E, selenium salt, or uric acid, effectively inhibited the development of AFB1-induced liver cancer and induced increased microsomal enzyme activity. Whereas beta-carotene and uric acid were the most effective inhibitors, vitamin E was the least, yet a significant inhibitor of liver cancer. Hepatic levels of cytochrome P-450, aniline hydroxylase and chlorpromazine demethylase were significantly induced in rats fed fortified food followed by AFB1 treatment than in control animals. The inhibition of liver cancer by dietary factors was probably due to their ability to induce the activity of hepatic microsomal enzymes. Increased enzyme activity could lead to rapid activation of AFB1 metabolism, resulting in loss of activated AFB1 metabolites that attack cell components. Inhibition of liver cancer is therefore associated with induction of increased microsomal enzyme activity.

Anti-cancer therapy with valine-depleted amino acid imbalance solution.

For the purpose of developing amino acid imbalance solution applicable to cancer treatment, we prepared seven kinds of amino acid imbalance solutions based on a 10% balanced amino acid solution and investigated the anti-tumor effects of each solution. The administration of valine-depleted amino acid solution for 8 days at a daily dose of 53 ml/rat (79.5 kcal/rat) resulted in the most significant inhibitory effects on the growth of hepatoma (AH109A) and mammary tumors (MRMT-1), the rate being 82.8% and 90.8%, respectively. Side effects observed were inhibition of increases in host body weight, weight loss of the spleen and thymus, loss of hair, and a decrease in the amounts of total plasma protein and albumin. When the daily dosage of valine-depleted amino acid solution was reduced to 40 ml/rat (58.3 kcal/rat), anti-tumor effects were still noticed, while side effects were abated. These findings indicate that side effects accompanying the use of this solution can be alleviated by controlling the ingredients of the solution as well as the amount administered. It is thus suggested that valine-depleted amino acid imbalance solution is a valuable tool in the treatment of cancer.