Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 µM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 µM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis.

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na(3)VO(4)) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na(3)VO(4) was cytotoxic against T24 cells (EC(50)=5.8 µM at 24 h), but in the presence of ascorbate (100 µM) the EC(50) fell to 3.3 µM. Na(3)VO(4) plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na(3)VO(4) did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na(3)VO(4) and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na(3)VO(4) alone, or combined with ascorbate, increased catalase activity, but only Na(3)VO(4) plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na(3)VO(4) plus ascorbate (2-3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na(3)VO(4). The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na(3)VO(4) in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.

Aminopyrimidoisoquinolinequinone (APIQ) redox cycling is potentiated by ascorbate and induces oxidative stress leading to necrotic-like cancer cell death.

Several phenylaminopyrimidoisoquinolinequinones (APIQs) were tested for their cytotoxicity against different cancer cell lines (K562, T24, HepG2) in the presence or absence of ascorbate. Ascorbate enhanced the toxic effects of quinones with first half-wave potential E(I) (1/2) values in the range of -480 to -660 mV. Phenylaminoquinones that were unsubstituted at position 6 exhibited greater cytotoxic activity than did their 6-methyl-substituted analogues. Two groups of compounds were further selected, namely 8-10 and 20-22, to study the cellular mechanisms involved in quinone cytotoxicity. Indeed, these compounds have the same range of redox potentials but differed considerably in their capacity to induce cell death. In the presence of ascorbate, the cell demise induced by compounds 8-10 was not caspase-3 dependent, as shown by the lack of activation of caspase-3 and the absence of cleaved PARP fragments. In addition, an index of ER stress (eIF2α phosphorylation) was activated by these compounds. Quinones 8-10 decreased the cellular capacity to reduce MTT dye and caused marked ATP depletion. Taken together, our results show that ascorbate enhances quinone redox-cycling and leads to ROS formation that inhibits cell proliferation and provokes caspase-independent cell death. Interestingly, we also observed that quinone 8 had a rather selective effect given that freshly isolated peripheral blood leukocytes from human healthy donors were more resistant than human leukemia K562 cells.

Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice.

Tumors contain oxygenated and hypoxic regions, so the tumor cell population is heterogeneous. Hypoxic tumor cells primarily use glucose for glycolytic energy production and release lactic acid, creating a lactate gradient that mirrors the oxygen gradient in the tumor. By contrast, oxygenated tumor cells have been thought to primarily use glucose for oxidative energy production. Although lactate is generally considered a waste product, we now show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. There is therefore a symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We identified monocarboxylate transporter 1 (MCT1) as the prominent path for lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism. Inhibiting MCT1 with alpha-cyano-4-hydroxycinnamate (CHC) or siRNA in these cells induced a switch from lactate-fueled respiration to glycolysis. A similar switch from lactate-fueled respiration to glycolysis by oxygenated tumor cells in both a mouse model of lung carcinoma and xenotransplanted human colorectal adenocarcinoma cells was observed after administration of CHC. This retarded tumor growth, as the hypoxic/glycolytic tumor cells died from glucose starvation, and rendered the remaining cells sensitive to irradiation. As MCT1 was found to be expressed by an array of primary human tumors, we suggest that MCT1 inhibition has clinical antitumor potential.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Delivery of soluble VEGF receptor 1 (sFlt1) by gene electrotransfer as a new antiangiogenic cancer therapy.

Since tumor growth is highly dependent on the formation of new blood vessels, angiogenesis inhibitors have become important players in anticancer treatments. Although less cytotoxic than conventional chemotherapy, most of the available antiangiogenic agents may provoke severe adverse effects which can limit their use. The design of new antiangiogenic strategies therefore requires integrating an early evaluation of possible interference with quiescent endothelial cells and nontumor angiogenesis. Here, we describe such a novel antiangiogenic approach based on the in vivo delivery by gene electrotransfer of a negative regulator of angiogenesis, namely, sFlt1. We found that this soluble variant of the vascular endothelial growth factor receptor 1 (Flt1, also known as VEGFR1), which acts as a VEGF trap, differentially influences tumor and postischemic hind limb angiogenesis in mice. sFlt1 gene electrotransfer in tibial cranial muscle leads to high sFlt1 protein expression and secretion, leading to a significant delay in the growth of syngeneic tumors but not altering the revascularization of ischemic peripheral tissue. The higher sensitivity of tumor-bearing animals toward sFlt1 trapping effects (vs ischemia-recovering animals) might be explained by a distinct pattern of VEGF release, as shown by VEGF measurements in plasma and tissue. In conclusion, our data support sFlt1 gene electrotransfer as a novel and safe modality to target VEGF-driven tumor angiogenesis and to maintain unaltered the recovery potential of ischemic tissues.

Comparison of methods for measuring oxygen consumption in tumor cells in vitro.

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.

Synthesis and antitumor evaluation of 8-phenylaminopyrimido[4,5-c]isoquinolinequinones.

A series of 8-phenylaminopyrimido[4,5-c]isoquinoline-7,10-quinone derivatives were prepared by regioselective amination reaction of pyrimido[4,5-c]isoquinoline-7,10-quinones with arylamines in the presence of a Lewis acid catalyst. Preliminary evaluation of the members of the series against cancer cell lines and assays of activation of their cytotoxic activity on K562 cells with ascorbic acid are reported.

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

Botulinum toxin potentiates cancer radiotherapy and chemotherapy.

PURPOSE: Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. EXPERIMENTAL DESIGN: To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells. RESULTS: Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature. CONCLUSIONS: The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.

Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems.

It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a "hypothesis-driven" approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a "data-driven" approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) and genomic gain of the NQO1 locus modulates breast cancer cell sensitivity to quinones.

AIMS: Alterations in the expression of antioxidant enzymes are associated with changes in cancer cell sensitivity to chemotherapeutic drugs (menadione and ß-lapachone). Mechanisms of acquisition of resistance to pro-oxidant drugs were investigated using a model of oxidative stress-resistant MCF-7 breast cancer cells (Resox cells). MAIN METHODS: FISH experiments were performed in tumor biopsy and breast cancer cells to characterize the pattern of the NQO1 gene. SNP-arrays were conducted to detect chromosomal imbalances. Finally, the importance of NQO1 overexpression in the putative acquisition of either drug resistance or an increased sensitivity to quinones by cancer cells was investigated by immunoblotting and cytotoxicity assays. KEY FINDINGS: Genomic gain of the chromosomal band 16q22 was detected in Resox cells compared to parental breast cancer MCF-7 cells and normal human mammary epithelial 250MK cells. This genomic gain was associated with amplification of the NQO1 gene in one tumor biopsy as well as in breast cancer cell lines. Using different breast cell models, we found that NQO1 overexpression was a main determinant for a potential chemotherapy resistance or an increased sensitivity to quinone-bearing compounds. SIGNIFICANCE: Because NQO1 is frequently modified in tumors at genomic and transcriptomic levels, the impact of NQO1 modulation on breast cancer cell sensitivity places NQO1 as a potential link between cancer redox alterations and resistance to chemotherapy. Thus, the NQO1 gene copy number and NQO1 activity should be considered when quinone-bearing molecules are being utilized as potential drugs against breast tumors.

Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study.

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.

Regulation of catalase expression in healthy and cancerous cells.

Catalase is an important antioxidant enzyme that dismutates hydrogen peroxide into water and molecular oxygen. The catalase gene has all the characteristics of a housekeeping gene (no TATA box, no initiator element sequence, high GC content in promoter) and a core promoter that is highly conserved among species. We demonstrate in this review that within this core promoter, the presence of DNA binding sites for transcription factors, such as NF-Y and Sp1, plays an essential role in the positive regulation of catalase expression. Additional transcription factors, such as FoxO3a, are also involved in this regulatory process. There is strong evidence that the protein Akt/PKB in the PI3K signaling pathway plays a major role in the expression of catalase by modulating the activity of FoxO3a. Over the past decade, other transcription factors (PPARγ, Oct-1, etc.), as well as genetic, epigenetic, and posttranscriptional processes, have emerged as crucial contributors to the regulation of catalase expression. Altered expression levels of catalase have been reported in cancer tissues compared to their normal counterparts. Deciphering the molecular mechanisms that regulate catalase expression could, therefore, be of crucial importance for the future development of pro-oxidant cancer chemotherapy.

The association of vitamins C and K3 kills cancer cells mainly by autoschizis, a novel form of cell death. Basis for their potential use as coadjuvants in anticancer therapy.

Deficiency of alkaline and acid DNase is a hallmark in all non-necrotic cancer cells in animals and humans. These enzymes are reactivated at early stages of cancer cell death by vitamin C (acid DNase) and vitamin K(3) (alkaline DNase). Moreover, the coadministration of these vitamins (in a ratio of 100:1, for C and K(3), respectively) produced selective cancer cell death. Detailed morphological studies indicated that cell death is produced mainly by autoschizis, a new type of cancer cell death. Several mechanisms are involved in such a cell death induced by CK(3), they included: formation of H(2)O(2) during vitamins redox cycling, oxidative stress, DNA fragmentation, no caspase-3 activation, and cell membrane injury with progressive loss of organelle-free cytoplasm. Changes in the phosphorylation level of some critical proteins leading to inactivation of NF-kappaB appear as main intracellular signal transduction pathways. The increase knowledge in the mechanisms underlying cancer cells death by CK(3) may ameliorate the techniques of their in vivo administration. The aim is to prepare the introduction of the association of vitamins C and K(3) into human clinics as a new, non-toxic adjuvant cancer therapy.

p38(MAPK)-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2-antioxidant signaling.

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38(MAPK). In line with this, genetic or pharmacological p38(MAPK) inhibition reduced p62 and NBR1 levels and aggregate formation and impaired Nrf2 activation, thus increasing photo-oxidative stress and cell death. p62-deficient cells, or cells lacking p62 and with reduced levels of NBR1 (through siRNA knockdown), also displayed reduced aggregate formation but exhibited attenuated ROS levels, reduced caspase activation, and improved survival after Hyp-PDT. The increased resistance to photo-oxidative stress exhibited by cells lacking p62 and/or NBR1 was overruled by the inhibition of p38(MAPK), which restored cytotoxic ROS levels, thus indicating the relevance of this signal in the control of cell viability. Taken together these findings provide evidence that in photodynamically treated cells a p38(MAPK)-regulated pathway coordinates the p62/NBR1-mediated clearance of cytosolic aggregates and mitigates PDT-induced proteotoxicity. They also reveal that a functional p38(MAPK)-Nrf2 signal is required to keep ROS levels in check and protect against PDT-induced proteotoxicity, independent of aggregate formation.

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.