Delta-tocotrienol enhances the anti-tumor effects of interferon alpha through reactive oxygen species and Erk/MAPK signaling pathways in hepatocellular carcinoma cells.

The complexity of hepatocellular carcinoma (HCC) signaling and the failure of pharmacological therapeutics reveal the significance of establishing new anti-cancer strategies. Interferon alpha (IFN-α) has been used as adjuvant therapy for reducing HCC recurrence and improving survival. Delta-tocotrienol (δ-tocotrienol), a natural unsaturated isoform of vitamin E, is a promising candidate for cancer treatment. In this study, we evaluated whether the combination of δ-tocotrienol with IFN-α displays significant advantages in the treatment of HCC cells. Results showed that the combination significantly decreased cell viability, migration and invasion of HCC cells compared with single therapies. Combining δ-tocotrienol and IFN-α enhanced the decrease in proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase (MMP) 7 and MMP-9. The combination also produced an enhancement of apoptosis together with increased Bax/Bcl-xL ratio and reactive oxygen species (ROS) generation. δ-tocotrienol induced Notch1 activation and changes in Erk and p38 MAPK signaling status. Blocking experiments confirmed that ROS and Erk are involved, at least in part, in the anti-cancer effects of the combined treatment. In conclusion, the combination of δ-tocotrienol with IFN-α therapy showed promising results for HCC cell treatment, which makes the combination of cytokine-based immunotherapy with natural products a potential strategy against liver cancer.

IFN-α-2b treatment protects against diet-induced obesity and alleviates non-alcoholic fatty liver disease in mice.

IFN-α is used for inflammatory purposes, and obesity and NAFLD are strongly correlated with inflammatory processes. We wondered whether IFN-α-2b can attenuate obesity development and its associated NAFLD in mice fed high fat diet (HFD) for 10 weeks. IFN-α-2b had a robust effect on body weight loss associated with NAFLD amelioration by decreasing hepatic inflammation. IFN-α-2b-treated mice showed increased plasma cholesterol levels together with decreased hepatic cholesterol, both on chow and HF diets. Interestingly, mice on IFN-α-2b treatment secreted smaller VLDL particles highly enriched in cholesterol. Mechanistically, we found that IFN-α-2b antiobesity effects were related to increased fatty acid oxidation; and its effects on cholesterol metabolism were due to both a decrease in the master cholesterogenic transcription factor SREBP-2 and in the rate limiting enzyme in cholesterol synthesis, HMGCR. To our knowledge, this is the first report showing the effects of IFN-α-2b on the prevention of the development of HFD-induced body weight gain and dyslipidemia through a mechanism that involves fatty acid oxidation and cholesterol decrease. These studies comprise necessary steps for understanding the amelioration of obesity and NAFLD. Results shed some light into the mechanism of action of natural cytokines, and their effects on ameliorating obesity and its related diseases.

Attenuation of liver cancer development by oral glycerol supplementation in the rat.

PURPOSE: Glycerol usage is increasing in food industry for human and animal nutrition. This study analyzed the impact of glycerol metabolism when orally supplemented during the early stage of rat liver carcinogenesis. METHODS: Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group). IP animals also received glycerol by gavage (200 mg/kg body weight, IPGly group). RESULTS: Glycerol treatment reduced the volume of preneoplastic lesions by decreasing the proliferative status of liver foci, increasing the expression of p53 and p21 proteins and reducing the expression of cyclin D1 and cyclin-dependent kinase 1. Besides, apoptosis was enhanced in IPGly animals, given by an increment of Bax/Bcl-2 ratio, Bad and PUMA mitochondrial expression, a concomitant increase in cytochrome c release and caspase-3 activation. Furthermore, hepatic levels of glycerol phosphate and markers of oxidative stress were increased in IPGly rats. Oxidative stress intermediates act as intracellular messengers, inducing p53 activation and changes in JNK and Erk signaling pathways, with JNK activation and Erk inhibition. CONCLUSION: The present work provides novel data concerning the preventive actions of glycerol during the development of liver cancer and represents an economically feasible intervention to treat high-risk individuals.

Hepatic carboxylesterase 3 (Ces3/Tgh) is downregulated in the early stages of liver cancer development in the rat.

It is accepted that cancer development is associated with metabolic changes. Previously, we established a model of hepatic preneoplasia in which adult rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP) for 6weeks until they develop altered hepatic foci (AHF). Here, we found that a whole metabolic shift occurs in order to favor cancer development. IP animals presented with increased plasma lipids due to increased VLDL secretion as well as increased liver lipid accretion due to stimulated transacetylase activity rather than lipogenesis, compared to control rats. We found that carboxylesterase 3/triacylglycerol hydrolase (Ces3/Tgh) presented with a perilobular distribution surrounding lipid droplets in normal livers. However, it is downregulated both at the protein and mRNA level in liver homogenates and is almost undetectable inside the AHF with no changes in the surrounding tissue. Ces3/Tgh expression is regulated by ω-3 fatty acids, thus, supplementation of diet with fish oil, allowed the restoration of Ces3/Tgh expression inside the foci and, more interestingly, led to the decrease in number and volume of the AHF. These studies show a preventive role of Ces3/Tgh in liver cancer development.

Role of eicosanoids in liver repair, regeneration and cancer.

Eicosanoids are lipid signaling molecules derived from the oxidation of ω-6 fatty acids, usually arachidonic acid. There are three major pathways, including the cyclooxygenase (COX), lipoxygenase (LOX), and P450 cytochrome epoxygenase (CYP) pathway. Prostanoids, which include prostaglandins (PG) and thromboxanes (Tx), are formed via the COX pathway, leukotrienes (LT) and lipoxins (LX) by the action of 5-LOX, and hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) by CYP. Although eicosanoids are usually associated with pro-inflammatory responses, non-classic eicosanoids, as LX, have anti-inflammatory and pro-resolving properties. Eicosanoids like PGE2, LTB4 and EETs have been involved in promoting liver regeneration after partial hepatectomy. PGE2 and LTB4 have also been reported to participate in the regenerative phase after ischemia and reperfusion (I/R), while cysteinyl leukotrienes (Cys-LT) contribute to the inflammatory process associated with I/R and are also involved in liver fibrosis and cirrhosis. However, LX, another product of 5-LOX, have the opposite effect, acting as pro-resolving mediators in these pathologies. In liver cancer, most studies show that eicosanoids, with the exception of LX, promote the proliferation of hepatocellular carcinoma cells and favor metastasis. This review summarizes the synthesis of different eicosanoids in the liver and discusses key findings from basic research linking eicosanoids to liver repair, regeneration and cancer and the impact of targeting eicosanoid cascade. In addition, studies in patients are presented that explore the potential use of eicosanoids as biomarkers and show correlations between eicosanoid production and the course and prognosis of liver disease.

The chemoprotective effects of IFN-α-2b on rat hepatocarcinogenesis are blocked by vitamin E supplementation.

Many cancer patients receive their classical therapies together with vitamin supplements. However, the effectiveness of these strategies is on debate. Here we aimed to evaluate how vitamin E supplementation affects the anticancer effects of interferon (IFN-α) using an early-model of liver cancer development (initiation-promotion, IP). Male Wistar rats subjected to this model were divided as follows: untreated (IP), IP treated with recombinant IFN-α-2b (6.5  ×  105 U/kg), IP treated with vitamin E (50 mg/kg), and IP treated with combination of vitamin E and IFN-α-2b. After treatments rats were fasted and euthanized and plasma and livers were collected. Combined administration of vitamin E and IFN-α-2b induced body weight drop, increased liver apoptosis, and low levels of hepatic lipids. Interestingly, vitamin E and IFN-α-2b combination also induced an increase in altered hepatic foci number, but not in size. It seems that vitamin E acts on its antioxidant capability in order to block the oxidative stress induced by IFN-α-2b, blocking in turn its beneficial effects on preneoplastic livers, leading to harmful final effects. In conclusion, this study shows that vitamin E supplementation in IFN-α-2b-treated rats exerts unwanted effects; and highlights that in spite of being natural, nutritional supplements may not always exert beneficial outcomes when used as complementary therapy for the treatment of cancer.

Hepatic glycerol metabolism is early reprogrammed in rat liver cancer development.

Evidence shows that oral glycerol supplementation during the early stages of rat liver cancer reduces the growth of preneoplastic lesions. Besides, human hepatocellular carcinoma (HCC) cells display decreased expression of glycerol channel aquaporin 9 (AQP9) and also diminished glycerol-3-phosphate (G3P) content. According to this, we analyzed glycerol metabolism during the initial stages of rat liver carcinogenesis. Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group) or left untreated (control, C group). Different features of glycerol metabolism were compared between both groups. IP animals showed increased plasma free glycerol levels and liver AQP9 protein expression. Also, IP rats showed increased glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) hepatic activities. Gluconeogenesis from glycerol both in vivo and in isolated perfused liver was higher in rats having liver preneoplasia. Nevertheless, preneoplastic foci notably reduced AQP9 and GK protein expressions, displaying a reduced ability to import glycerol and to convert it into G3P, as a way to preserve preneoplastic hepatocytes from the deleterious effect of G3P. In conclusion, the metabolic shift that takes place in the initial stages of liver cancer development comprises an increased hepatic utilization of glycerol for gluconeogenesis. Enhanced glucose production from glycerol is mostly carried out by the surrounding non-preneoplastic tissue and can be used as an energy source for the early transformed liver cells.

Vitamin K2 supplementation blocks the beneficial effects of IFN-α-2b administered on the early stages of liver cancer development in rats.

OBJECTIVE: Vitamin K2, which is present in dairy products and has been recommended as a micronutrient supplement in humans, contains anticancer properties. Interferon (IFN)-α-2b administered during development of hepatic preneoplasia decreased both number and volume percentage of altered hepatic foci (AHF) by increasing apoptosis in the foci. The aim of this study was to evaluate the effects of IFN-α-2b treatment supplemented with vitamin K2 in the early stages of liver cancer development in rats. METHODS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted [IP] group). Animals were divided into four groups: untreated (IP), IP treated with IFN-α-2b (6.5 × 105 U/kg), IP treated with vitamin K2 (10 mg/kg), and IP treated with both compounds. RESULTS: The study results demonstrated that vitamin K2 blocked IFN-α-2b-induced reduction in size and volume of the altered hepatic foci and inhibited IFN-α-2b-induced apoptosis. Its inhibition of IFN-α-2b-induced apoptosis was mediated by increased levels of total hepatic Bcl-2 in rat preneoplastic livers. CONCLUSION: These findings demonstrate that supportive vitamin supplements or therapies are not always safe because they could put the life of patients treated with IFN-α-2b at risk.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy.

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

Inhibition of sirtuins 1 and 2 impairs cell survival and migration and modulates the expression of P-glycoprotein and MRP3 in hepatocellular carcinoma cell lines.

Sirtuins (SIRTs) 1 and 2 deacetylases are overexpressed in hepatocellular carcinoma (HCC) and are associated with tumoral progression and multidrug resistance (MDR). In this study we analyzed whether SIRTs 1 and 2 activities blockage was able to affect cellular survival and migration and to modulate p53 and FoxO1 acetylation in HepG2 and Huh7 cells. Moreover, we analyzed ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 3 (MRP3) expression. We used cambinol and EX-527 as SIRTs inhibitors. Both drugs reduced cellular viability, number of colonies and cellular migration and augmented apoptosis. In 3D cultures, SIRTs inhibitors diminished spheroid growth and viability. 3D culture was less sensitive to drugs than 2D culture. The levels of acetylated p53 and FoxO1 increased after treatments. Drugs induced a decrease in ABC transporters mRNA and protein levels in HepG2 cells; however, only EX-527 was able to reduce MRP3 mRNA and protein levels in Huh7 cells. This is the first work demonstrating the regulation of MRP3 by SIRTs. In conclusion, both drugs decreased HCC cells survival and migration, suggesting SIRTs 1 and 2 activities blockage could be beneficial during HCC therapy. Downregulation of the expression of P-gp and MRP3 supports the potential application of SIRTs 1 and 2 inhibitions in combination with conventional chemotherapy.