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.

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.

IFN-α-2b induces apoptosis by decreasing cellular cholesterol levels in rat preneoplastic hepatocytes.

IFN-α administration to patients has been long discouraged and pushed back by new and apparently better drugs; however the adverse secondary effect, the high costs and the lack of specific action, make these new drugs hard to be used and put IFN-α again in the eye of the researchers. IFN-α-2b was demonstrated to induce apoptosis and modulation of lipid metabolism and the mechanisms are still unknown. Here, we sought to find the link between these features using a model of early stage cancer development. Using in vitro and in vivo approaches, we evaluated apoptosis and lipid metabolism. IFN-α-2b induced changes in hepatic cholesterol mass due to decreased synthesis and increased secretion. Interestingly, the drop in cellular cholesterol levels was necessary for IFN-α-2b to induce apoptosis. Results presented in this paper show the complexity of the action of IFN-α-2b on the early stages of liver cancer development. We show for the first time an interrelationship between cholesterol, apoptosis and IFN-α-2b. This makes clear the need for a reevaluation of IFN-α-2b action in order to develop softer, safer and more bearable derivatives. In this regard, knowing the molecular mechanisms by which IFN-α exerts its cellular actions is of crucial importance, and it is the main condition for therapy success for classical and new malignancies.

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.

Metformin and glucose starvation decrease the migratory ability of hepatocellular carcinoma cells: targeting AMPK activation to control migration.

Hepatocellular carcinoma (HCC) is a highly metastatic cancer with very poor prognosis. AMP activated kinase (AMPK) constitutes a candidate to inhibit HCC progression. First, AMPK is downregulated in HCC. Second, glucose starvation induces apoptosis in HCC cells via AMPK. Correspondingly, metformin activates AMPK and inhibits HCC cell proliferation. Nevertheless, the effect of AMPK activation on HCC cell invasiveness remains elusive. Here, migration/invasion was studied in HCC cells exposed to metformin and glucose starvation. Cell viability, proliferation and differentiation, as well as AMPK and PKA activation were analyzed. In addition, invasiveness in mutants of the AMPKα activation loop was assessed. Metformin decreased cell migration, invasion and epithelial-mesenchymal transition, and interference with AMPKα expression avoided metformin actions. Those antitumor effects were potentiated by glucose deprivation. Metformin activated AMPK at the same time that inhibited PKA, and both effects were enhanced by glucose starvation. Given that AMPKα(S173) phosphorylation by PKA decreases AMPK activation, we hypothesized that the reduction of PKA inhibitory effect by metformin could explain the increased antitumor effects observed. Supporting this, in AMPK activating conditions, cell migration/invasion was further impaired in AMPKα(S173C) mutant cells. Metformin emerges as a strong inhibitor of migration/invasion in HCC cells, and glucose restriction potentiates this effect.

AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation.

The signaling pathways that govern survival response in hepatic cancer cells subjected to nutritional restriction have not been clarified yet. In this study we showed that liver cancer cells undergoing glucose deprivation both arrested in G0/G1 and died mainly by apoptosis. Treatment with the AMPK activator AICAR phenocopied the effect of glucose deprivation on cell survival, whereas AMPK silencing in HepG2/C3A, HuH-7 or SK-Hep-1 cells blocked the cell cycle arrest and the increase in apoptotic death induced by glucose starvation. Both AMPK and PKA were promptly activated after glucose withdrawal. PKA signaling had a dual role during glucose starvation: whereas it elicited an early decreased in cell viability, it later improved this parameter. We detected AMPK phosphorylation (AMPKα(Ser173)) by PKA, which was increased in glucose starved cells and was associated with diminution of AMPK activation. To better explore this inhibitory effect, we constructed a hepatocarcinoma derived cell line which stably expressed an AMPK mutant lacking that PKA phosphorylation site: AMPKα1(S173C). Expression of this mutant significantly decreased viability in cells undergoing glucose starvation. Furthermore, after 36 h of glucose deprivation, the index of AMPKα1(S173C) apoptotic cells doubled the apoptotic index observed in control cells. Two main remarks arise: 1. AMPK is the central signaling kinase in the scenario of cell cycle arrest and death induced by glucose starvation in hepatic cancer cells; 2. PKA phosphorylation of Ser173 comes out as a strong control point that limits the antitumor effects of AMPK in this situation.

Centrosomal AKAP350 modulates the G1/S transition.

AKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participate in the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1/S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.

Protein kinase A signals apoptotic activation in glucose-deprived hepatocytes: participation of reactive oxygen species.

Glucose deprivation entails oxidative stress and apoptosis in diverse cell types. Liver tissue shows high tolerance to nutritional stress, however regulation of survival in normal hepatocytes subjected to glucose restriction is unclear. We assessed the survival response of cultured hepatocytes subjected to glucose deprivation and analyzed the putative participation of protein kinase A (PKA) in this response. Six hours glucose deprivation induced a PKA dependent activation of apoptosis in cultured hepatocytes, without having an impact on non apoptotic death. Apoptotic activation associated to glucose restriction was secondary to an imbalance in cellular reactive oxygen species (ROS). In this condition, PKA inhibition led to an early prevention in mitochondrial ROS production and a late increase in scavenging enzymes transcript levels. These results supported the hypothesis that PKA could modulate glucose deprivation induced apoptotic activation by conditioning mitochondrial ROS production during glucose fasting. We presented additional evidence sustaining this model: First, glucose withdrawal led to a 95% increase in mitochondrial cAMP levels in cultured hepatocytes; second, activation of PKA significantly augmented hepatic mitochondrial ROS generation, whereas PKA inhibition elicited the opposite effect. Mitochondrial PKA signaling, previously proposed as an autonomic pathway adjusting respiration rate, emerges as a mechanism of controlling cell survival during glucose restriction.

Nutritional stress in eukaryotic cells: oxidative species and regulation of survival in time of scarceness.

The survival response to glucose limitation in eukaryotic cells involves different signaling pathways highly conserved from yeasts to mammals. Upon nutritional restriction, a network driven by kinases such as the AMP dependent protein kinase (AMPK/Snf1), the Target of Rapamycin kinase (TOR), the Protein kinases A (PKA) or B (PKB/Akt) control stress defenses, cell cycle regulators, pro and anti apoptotic proteins, respiratory complexes, etc. In this work we review the state of the art in this scenario of kinase pathways, i.e. their principal effectors and links, both in yeasts and mammals. We also focus in downstream actors such as sirtuins and the Forkhead box class O transcription factors. Besides, we particularly analyze the participation of these kinases on the balance of Reactive Oxygen Species and their role in the regulation of survival during glucose deprivation. Key results on yeast stationary phase survival and the contribution of such genetics studies are discussed.