Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

BACKGROUND: Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. METHODS: The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 µmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. RESULTS: Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells.

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells. When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism.

Catalase expression in MCF-7 breast cancer cells is mainly controlled by PI3K/Akt/mTor signaling pathway.

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3ß and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

Downregulation of Sox9 expression associates with hepatogenic differentiation of human liver mesenchymal stem/progenitor cells.

Understanding the mechanisms triggering hepatogenic differentiation of stem/progenitor cells would be useful for studying postnatal liver regeneration and development of liver cell therapies. Many evidences support the involvement of Sox9 transcription factor in liver development. Here, we investigate the possibility of liver mesenchymal stem/progenitor cells to constitutively express Sox9 by using reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and western blotting. The involvement of Sox9 in hepatogenic differentiation was assessed by following its expression at different steps of the process, evaluating the impact of its altered expression, and analyzing its expression in human liver disease specimen. Liver mesenchymal stem/progenitor cells constitutively express Sox9 at both the mRNA and protein levels. Upon hepatogenic differentiation, Sox9 expression is downregulated mainly in the maturation step after oncostatin M treatment. Induction of Sox9 expression using transforming growth factor beta is accompanied with a decrease of the quality of hepatogenic differentiation. Blunting Sox9 expression using specific ShRNA clearly alters the levels of several hepatic markers, an effect confirmed in HepG2 cells. In human liver disease specimen, Sox9 expression is enhanced at both the mRNA and protein levels compared with healthy donors. The current data demonstrate that Sox9 may play a pivotal role in hepatocyte lineage development, including adult liver mesenchymal stem/progenitor cells. Further studies on the identification of pathways regulated by or regulating Sox9 will certainly gain insight into the molecular networks controlling hepatogenic differentiation.

Role of AMPK activation in oxidative cell damage: Implications for alcohol-induced liver disease.

Chronic alcohol consumption is a well-known risk factor for liver disease. Progression of alcohol-induced liver disease (ALD) is a multifactorial process that involves a number of genetic, nutritional and environmental factors. Experimental and clinical studies increasingly show that oxidative damage induced by ethanol contributes in many ways to the pathogenesis of alcohol hepatoxicity. Oxidative stress appears to activate AMP-activated protein kinase (AMPK) signaling system, which has emerged in recent years as a kinase that controls the redox-state and mitochondrial function. This review focuses on the most recent insights concerning the activation of AMPK by reactive oxygen species (ROS), and describes recent evidences supporting the hypothesis that AMPK signaling pathways play an important role in promoting cell viability under conditions of oxidative stress, such as during alcohol exposure. We suggest that AMPK activation by ROS can promote cell survival by inducing autophagy, mitochondrial biogenesis and expression of genes involved in antioxidant defense. Hence, increased intracellular concentrations of ROS may represent a general mechanism for enhancement of AMPK-mediated cellular adaptation, including maintenance of redox homeostasis. On the other hand, AMPK inhibition in the liver by ethanol appears to play a key role in the development of steatosis induced by chronic alcohol consumption. Although more studies are needed to assess the functions of AMPK during oxidative stress, AMPK may be a possible therapeutic target in the particular case of alcohol-induced liver disease.

Overexpression of GRP94 in breast cancer cells resistant to oxidative stress promotes high levels of cancer cell proliferation and migration: implications for tumor recurrence.

Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H(2)O(2)-generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94-silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similar to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers.

Intracellular ATP levels determine cell death fate of cancer cells exposed to both standard and redox chemotherapeutic agents.

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined.

Catalase overexpression in mammary cancer cells leads to a less aggressive phenotype and an altered response to chemotherapy.

Because reactive oxygen species (ROS) are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated set of antioxidant molecules to prevent the toxic accumulation of these species. However, compared with normal cells, malignant cells often exhibit increased levels of intracellular ROS and altered levels of antioxidant molecules. The resulting endogenous oxidative stress favors tumor growth by promoting genetic instability, cell proliferation and angiogenesis. In this context, we assessed the influence of catalase overexpression on the sensitivity of breast cancer cells towards various anticancer treatments. Our data show that catalase overexpression in MCF-7 cells leads to a 7-fold increase in catalase activity but provokes a 40% decrease in the expression of both glutathione peroxidase and peroxiredoxin II. Interestingly, proliferation and migration capacities of MCF-7 cells were impaired by the overexpression of catalase, as compared to parental cells. Regarding their sensitivity to anticancer treatments, we observed that cells overexpressing catalase were more sensitive to paclitaxel, etoposide and arsenic trioxide. However, no effect was observed on the cytotoxic response to ionizing radiations, 5-fluorouracil, cisplatin or doxorubicin. Finally, we observed that catalase overexpression protects cancer cells against the pro-oxidant combination of ascorbate and menadione, suggesting that changes in the expression of antioxidant enzymes could be a mechanism of resistance of cancer cells towards redox-based chemotherapeutic drugs.

Stimulation of human and mouse erythrocyte Na(+)-K(+)-2Cl(-) cotransport by osmotic shrinkage does not involve AMP-activated protein kinase, but is associated with STE20/SPS1-related proline/alanine-rich kinase activation.

This study was undertaken to investigate whether the mechanism of increased Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter activity by osmotic shrinkage involved AMP-activated protein kinase (AMPK) activation. AMPK was found to phosphorylate a recombinant GST-dogfish (1-260) NKCC1 fragment at Ser38 and Ser214, corresponding to Ser77 and Ser242 in human NKCC1, respectively. Incubation of human erythrocytes with 20 microM A769662 AMPK activator increased Ser242 NKCC1 phosphorylation but did not stimulate (86)Rb(+) uptake. Under hypertonic conditions in human red blood cells (RBCs) incubated with 0.3 M sucrose, NKCC1 activity increased as measured by bumetanide-sensitive (86)Rb(+) uptake and AMPK was activated. However, there was no effect of AMPKalpha1 deletion in mouse RBCs on the increased rate of (86)Rb(+) uptake induced by hyperosmolarity. AMPK activation by osmotic shrinkage of mouse RBCs was abrogated by 10 microM STO-609 CaMKKbeta inhibitor, but incubation with STO-609 did not affect the increase in (86)Rb(+) uptake induced by hyperosmolarity. Osmotic shrinkage of human and mouse RBCs led to activation loop phosphorylation of the STE20/SPS1-related proline/alanine-rich kinase (SPAK) at Thr233, which was accompanied by phosphorylation of NKCC1 at Thr203/207/212, one of which (Thr207) is responsible for cotransporter activation. Therefore, phosphorylation-induced activation of NKCC1 by osmotic shrinkage does not involve AMPK and is likely to be due to SPAK activation.

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells.

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca(2+)-dependent AMPK activation via calmodulin-dependent protein kinase kinase-beta(CaMKKbeta), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKbeta inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

LRP-1 silencing prevents malignant cell invasion despite increased pericellular proteolytic activities.

The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

Thrombospondin-1 enhances human thyroid carcinoma cell invasion through urokinase activity.

Previous studies reported that modification in the expression of the matricellular multidomain glycoprotein thrombospondin-1 (TSP-1) could play a critical role in the control of tumor progression and metastasis development. The function of this multimodular protein in cancers appears highly dependent on the cellular context and thus remains to date very difficult to accurately characterize. Controversial results indeed exist reporting either pro- or anti-invasive properties of TSP-1. Since it appeared that TSP-1 could be of prognostic value for certain specific types of cancers, we examined in this study the prospective function of TSP-1 in the control of human follicular thyroid carcinoma (FTC) cell invasiveness. First, we established that the aggressive behavior of human thyroid malignant cells is closely correlated to the TSP-1 amount. We demonstrated that exogenously added TSP-1 stimulates by two-fold the capacity of FTC cells to invade Matrigel-coated wells. The use of specific anti-TSP-1 blocking antibodies led to a drastic inhibition of the basal FTC cell invasion. Zymography experiments revealed that the uPA-dependent proteolytic activity is directly controlled by TSP-1, MMPs activity is not. The TSP-1-mediated stimulation of uPA appears to occur at post-transcriptional level. Finally, we established that the TSP-1-stimulated FTC cell invasion is wholly abolished under anti-uPA blocking antibodies or aprotinin treatments whereas MMP inhibitors have no effect. All together, we evidenced in the present study that TSP-1 promotes human follicular thyroid carcinoma cell invasion mainly through up-regulation of the urokinase-dependent activity.

Human thyroid carcinoma cell invasion is controlled by the low density lipoprotein receptor-related protein-mediated clearance of urokinase plasminogen activator.

The low density lipoprotein receptor-related protein (LRP), a large scavenger receptor reported to mediate the uptake and degradation of various ligands, emerges as a promising receptor for targeting the invasive behaviour of human cancer cells. However, the accurate function of LRP during tumor invasion seems to be highly dependent on cellular context and remains controversial. The expression patterns of both this receptor and the main proteolytic systems involved in cell invasion were examined in two follicular thyroid carcinoma cell lines exhibiting different invasive phenotypes. We established that a low expression of LRP at the cell surface was associated to elevated extracellular MMP2 and urokinase plasminogen activator (uPA) activities as well as to high invasiveness properties. Surprisingly, neither exogenously added receptor-associated protein, an antagonist of LRP, nor LRP blocking antibodies significantly modified the amount of extracellular MMP2. Furthermore, the invasive phenotype of thyroid carcinoma cells was not related to their matrix metalloproteinases amount since different specific inhibitors of these proteases failed to affect the invasive properties of both cell lines. Additionally, blocking LRP-mediated clearance led to a further increase of the uPA amount and activities and to increased invasiveness in both cell lines. Finally thyroid carcinoma cells aggressiveness was widely increased by exogenous uPA; and anti-uPA antibodies treatments abolished both basal and receptor-associated protein-induced thyroid cell invasion. Overall our results identified the LRP-mediated clearance of uPA as one of the mechanisms involved during the control of human thyroid carcinoma cell invasion.