Juan Pablo Parody

Hyperglycemia induces apoptosis in rat liver through the increase of hydroxyl radical: new insights into the insulin effect

In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.

Quercetin prevents liver carcinogenesis by inducing cell cycle arrest, decreasing cell proliferation and enhancing apoptosis

SCOPE Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G₁ and S phases, accumulation in G₂, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.

Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.

Cross-talk between IFN-α and TGF-β1 signaling pathways in preneoplastic rat liver

Interferon-γ/transforming growth factor-β (IFN-γ/TGF-β) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-α/TGF-β. In previous studies, we showed that IFN-α2b increases TGF-β1 production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-α/TGF-β1 pathways was explored. We observed a positive cross-talk between IFN-α and TGF-β1 signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-β1 and IFN-α signals. Interestingly, we reported the novel finding that the autocrine TGF-β1 up-regulates TGF-βRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-α2b and by autocrine TGF-β1 are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.

Attenuation of the Wnt/b-catenin/TCF pathway by in vivo interferon-a2b (IFN-a2b) treatment in preneoplastic rat livers

Wnt/β-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for β-catenin binding, particularly under cellular oxidative stress conditions. Contrary to β-catenin/TCF, β-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-α2b (IFN-α2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-β1 (TGF-β1). This study was aimed to assess the status of the Wnt/β-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-α2b treatment on it. We demonstrated that the Wnt/β-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-α2b treatment inhibits Wnt/β-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.

Interferon-α2b (IFN-α2b)-induced apoptosis is mediated by p38 MAPK in hepatocytes from rat preneoplastic liver via activation of NADPH oxidase

It is still unclear how Interferon-alfa (IFN-α) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-α2b induces hepatocytic transforming growth factor-beta1 (TGF-β1) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-β1, alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-α induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-α2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-α2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-α2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-α2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-β1-dependent.

Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity

Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats

Abstract Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.

FoxO3a modulation and promotion of apoptosis by interferon-α2b in rat preneoplastic liver.

FoxO3a, a member of the FOXO family of transcription factors, is expressed in adult liver and modulates the expression of genes involved in apoptosis. FoxO3a is post‐translationally regulated, negatively by PI3K/Akt and MAPK/Erk and positively by oxidative stress/JNK pathways. In previous works, we have demonstrated that interferon‐α2b (IFN‐α2b) induces apoptosis of hepatic preneoplastic foci through the production of reactive oxygen species (ROS).

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.