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1.
FEBS J ; 281(19): 4421-38, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25065674

ABSTRACT

Numerous physiological functions are controlled by redox-responsive signaling pathways. Disruption of redox balance by oxidative stress is recognized as a major cause of many pathological conditions, including aging, highlighting the importance of investigating how antioxidants maintain redox homeostasis. AMP-activated protein kinase (AMPK) is activated in response to cellular conditions that accompany energy depletion and plays a central role in the regulation of energy homeostasis, tumorigenesis and longevity. Recently, several antioxidants have been reported to activate AMPK, although the mechanisms by which AMPK acts to adjust the levels of cellular reactive oxygen species are not fully characterized. In the present study, we investigated the role of AMPK in mediating resveratrol-induced antioxidant effects and the molecular mechanisms underlying its actions. We demonstrate that AMPK activity plays an indispensable role in the operation of the ROS defense system by inducing the expression of the antioxidant enzymes, manganese superoxide dismutase and catalase, in response to resveratrol or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-d-ribonucleotide. In addition, we identified the mechanism involved in the antioxidant function of AMPK, demonstrating that AMPK directly phosphorylates human FoxO1 (forkhead box O1) at Thr(649) in vitro and increases FoxO1-dependent transcription of manganese superoxide dismutase and catalase. Mutagenesis studies showed that this AMPK-mediated phosphorylation of FoxO1 is critical for FoxO1 stability and nuclear localization, establishing the molecular basis for the induction of FoxO1 transcriptional activity. Our results reveal a novel FoxO1-dependent mechanism by which AMPK controls the expression of antioxidant enzymes and suggest that AMPK has an important role in maintaining redox homeostasis.


Subject(s)
AMP-Activated Protein Kinases/physiology , Antioxidants/pharmacology , Forkhead Transcription Factors/metabolism , Stilbenes/pharmacology , Active Transport, Cell Nucleus , Animals , Forkhead Box Protein O1 , HEK293 Cells , Hep G2 Cells , Humans , Mice , Phosphorylation , Protein Processing, Post-Translational , Protein Stability , Reactive Oxygen Species/metabolism , Resveratrol , Transcription, Genetic , Transcriptional Activation
2.
Apoptosis ; 19(4): 615-28, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24173372

ABSTRACT

AMP-activated protein kinase (AMPK) performs a pivotal function in energy homeostasis via the monitoring of intracellular energy status. Once activated under the various metabolic stress conditions, AMPK regulates a multitude of metabolic pathways to balance cellular energy. In addition, AMPK also induces cell cycle arrest or apoptosis through several tumor suppressors including LKB1, TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are direct substrates of AMPK. Therefore, it is expected that activators of AMPK signal pathway might be useful for treatment or prevention of cancer. In the present study, we report that cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1, p53, TSC2, thereby leading to suppression of mTORC1 in a number of LKB1-expressing cancer cells including HepG2 human hepatoma, but not in LKB1-deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner, and a portion of cells underwent apoptosis as a result of long-term treatment. It also induced autophagic HepG2 cell death in an AMPK-dependent manner. Cryptotanshinone significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a substantial activation of AMPK signal pathways. Collectively, we demonstrate for the first time that cryptotanshinone harbors the therapeutic potential for the treatment of cancer through AMPK activation.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Antineoplastic Agents/pharmacology , Autophagy/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , Phenanthrenes/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Enzyme Activation , Hep G2 Cells , Heterografts , Humans , Male , Metformin/pharmacology , Mice , Mice, Nude , Neoplasm Transplantation , Phenanthrenes/therapeutic use , Signal Transduction
3.
Biochem Pharmacol ; 85(7): 965-76, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23337568

ABSTRACT

Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3ß. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and ß-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of ß-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment.


Subject(s)
Anti-Obesity Agents/pharmacology , Chromones/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Thiazoles/pharmacology , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipogenesis , Adiposity/drug effects , Aminophenols/pharmacology , Animals , Anti-Obesity Agents/chemistry , Body Weight/drug effects , Cell Differentiation , Chromones/chemistry , HEK293 Cells , Hep G2 Cells , Humans , Lipid Metabolism/drug effects , Lithium Chloride/pharmacology , Male , Maleimides/pharmacology , Mice , Mice, Inbred C57BL , Obesity/drug therapy , Obesity/pathology , Obesity/physiopathology , Protein Stability , Thiazoles/chemistry , beta Catenin/metabolism
4.
Apoptosis ; 17(9): 938-49, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22684842

ABSTRACT

Tumor hypoxia is considered the best validated target in clinical oncology because of its significant contribution to chemotherapy failure and drug resistance. As an approach to target hypoxia, we assessed the potential of quercetin, a flavonoid widely distributed in plants, as a anticancer agent under hypoxic conditions and examined its pharmacological mechanisms by primarily focusing on the role of AMP-activated protein kinase (AMPK). Quercetin significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model with a substantial reduction of AMPK activity. In a cell culture system, quercetin more dramatically induced apoptosis of HCT116 cancer cells under hypoxic conditions than normoxic conditions, and this was tightly associated with inhibition of hypoxia-induced AMPK activity. An in vitro kinase assay demonstrated that quercetin directly inhibits AMPK activity. Inhibition of AMPK by expressing a dominant-negative form resulted in an increase of apoptosis under hypoxia, and a constitutively active form of AMPK effectively blocked quercetin-induced apoptosis under hypoxia. Collectively, our data suggest that quercetin directly inhibits hypoxia-induced AMPK, which plays a protective role against hypoxia. Quercetin also reduced the activity of hypoxia-inducible factor-1 (HIF-1), a major transcription factor for adaptive cellular response to hypoxia. Moreover, quercetin sensitized HCT116 cancer cells to the anticancer drugs cisplatin and etoposide under hypoxic conditions. Our findings suggest that AMPK may serve as a novel target for overcoming tumor hypoxia-associated negative aspects.


Subject(s)
AMP-Activated Protein Kinases/antagonists & inhibitors , Apoptosis/drug effects , Cell Hypoxia/drug effects , Colonic Neoplasms/drug therapy , Quercetin/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Cell Line, Tumor , Cisplatin/pharmacology , Etoposide/pharmacology , Genes, Reporter , Humans , Hypoxia-Inducible Factor 1/drug effects , Hypoxia-Inducible Factor 1/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
5.
Arterioscler Thromb Vasc Biol ; 28(10): 1796-802, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18658046

ABSTRACT

OBJECTIVE: Hypoxia-inducible factor 1alpha (HIF-1alpha) is primarily involved in the adapting of cells to changes in oxygen levels, which is essential for normal vascular function. Recently, physiological roles for retinoic acid-related orphan receptor alpha (RORalpha) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of RORalpha in the hypoxia signaling pathway in connection with activation of HIF-1alpha. METHODS AND RESULTS: Under hypoxic conditions, expression of RORalpha was induced. When RORalpha was introduced exogenously, protein level as well as transcriptional activity of HIF-1alpha was enhanced. Putative ligands of RORalpha, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1alpha, which was abolished by RNA interference against RORalpha. RORalpha was physically associated with HIF-1alpha through DNA binding domain, which was required to the RORalpha-induced stabilization and transcriptional activation of HIF-1alpha. Finally, either infection with adenovirus encoding RORalpha or treatment with ROR ligands enhanced the formation of capillary tubes by human umbilical vascular endothelial cells. CONCLUSIONS: Our results provide a new insight for the function of RORalpha in amplification of hypoxia signaling and suggest a potential application of RORalpha ligands for the therapy of hypoxia-associated vascular diseases.


Subject(s)
Cell Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Trans-Activators/metabolism , Transcriptional Activation , Animals , Binding Sites , Cell Hypoxia/genetics , Cholesterol Esters/metabolism , Endothelial Cells/metabolism , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ligands , Melatonin/metabolism , Mice , NIH 3T3 Cells , Neovascularization, Physiologic , Nuclear Receptor Subfamily 1, Group F, Member 1 , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Signal Transduction/genetics , Trans-Activators/genetics , Transfection
6.
Carcinogenesis ; 29(4): 713-21, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18258605

ABSTRACT

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H(2)O(2) and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1alpha protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1alpha ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.


Subject(s)
Adenylate Kinase/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Prostatic Neoplasms/genetics , Reactive Oxygen Species/metabolism , Transcription, Genetic , Carcinoma, Hepatocellular , Cell Line, Tumor , Colonic Neoplasms , Enzyme Activation , Gene Transfer Techniques , Genes, Reporter , Genes, myc , HeLa Cells , Humans , Liver Neoplasms , Lung Neoplasms , Male , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin/metabolism
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