Activating AMP-activated Protein Kinase Signaling Research Articles

Silencing protein kinase C ζ by microRNA-25-5p activates AMPK signaling and inhibits colorectal cancer cell proliferation.

Developing novel strategies against human colorectal cancer (CRC) cells is needed. Activation of AMP-activated protein kinase (AMPK) could possibly inhibit CRC cells. Protein kinase C ζ (PKCζ) is an AMPK negative regulator. Here we found that PKCζ expression was significantly elevated in human colon cancer tissues and CRC cells. PKCζ upregulation was correlated with AMPK in-activation and mTOR complex 1 (mTORC1) over-activation. Reversely, PKCζ shRNA knockdown activated AMPK signaling and inhibited HT-29 cell proliferation. Significantly, downregulation of microRNA-25-5p (miR-25-5p), a PKCζ-targeting miRNA, could be the cause of PKCζ upregulation. Exogenous expression of miR-25-5p silenced PKCζ to activate AMPK signaling, which inhibited HT-29 cell proliferation. In vivo studies showed that HT-29 xenograft growth in mice was inhibited after expressing PKCζ shRNA or miR-25-5p. Collectively, PKCζ could be a novel oncogenic protein of human CRC. PKCζ silence, by targeted-shRNA or miR-25-5p expression, activates AMPK and inhibits HT-29 cell proliferation.

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC.

AntagomiR-451 inhibits oxygen glucose deprivation (OGD)-induced HUVEC necrosis via activating AMPK signaling.

Oxygen glucose deprivation (OGD) application in cultured human umbilical vein endothelial cells (HUVECs) mimics ischemic injuries. AntagomiR-451, the miroRNA-451 (“miR-451”) inhibitor, could activate pro-survival AMP-activated protein kinase (AMPK) signaling. In the current study, we showed that forced-expression of antagomiR-451 depleted miRNA-451 and significantly attenuated OGD-induced necrosis of HUVECs. Activation of AMPK was required for antagomiR-451-mediated pro-survival actions. AMPK inhibition, by AMPKα shRNA or dominant negative mutation, almost completely abolishedantagomiR-451-mediated HUVEC protection again OGD. Reversely, forced-activation of AMPK by exogenous expression of constructively-active AMPKα inhibited OGD-induced HUVEC necrosis. At the molecular level, antagomiR-451 expression in HUVECs inhibited OGD-induced programmed necrosis, the latter was evidenced by mitochondrial p53-cyclophilinD (Cyp-D) association, mitochondrial depolarization as well as reactive oxygen species (ROS) production and lactate dehydrogenase (LDH) breach. Together, we suggest that antagomiR-451 activates AMPK to inhibit OGD-induced programmed necrosis in HUVECs.

Liraglutide, a GLP-1 receptor agonist, inhibits vascular smooth muscle cell proliferation by enhancing AMP-activated protein kinase and cell cycle regulation, and delays atherosclerosis in ApoE deficient mice

Background and aimsSeveral studies have demonstrated that both native glucagon-like peptide-1 (GLP-1) and GLP-1 receptor agonists suppress the progression of atherosclerosis in animal models. MethodsWe investigated whether liraglutide, a GLP-1 analogue, could prevent the development of atherosclerosis in apolipoprotein E knockout mice (ApoE−/−) on a high-fat diet. We also examined the influence of liraglutide on angiotensin II-induced proliferation of rat vascular smooth muscle cells (VSMCs) via enhancement of AMP-activated protein kinase (AMPK) signaling and regulation of cell cycle progression. ResultsTreatment of ApoE−/− mice with liraglutide (400 μg/day for 4 weeks) suppressed atherosclerotic lesions and increased AMPK phosphorylation in the aortic wall. Liraglutide also improved the endothelial function of thoracic aortas harvested from ApoE−/− mice in an ex vivo study. Furthermore, liraglutide increased AMPK phosphorylation in rat VSMCs, while liraglutide-induced activation of AMPK was abolished by exendin 9-39, a GLP-1 antagonist. Moreover, angiotensin (Ang) II-induced proliferation of VSMCs was suppressed by liraglutide in a dose-dependent manner, and flow cytometry of Ang II-stimulated VSMCs showed that liraglutide reduced the percentage of cells in G2/M phase (by arrest in G0/G1 phase). ConclusionsThese findings suggest that liraglutide may inhibit Ang II-induced VSMC proliferation by activating AMPK signaling and inducing cell cycle arrest, thus delaying the progression of atherosclerosis independently of its glucose-lowering effect.

Cyclosporine A attenuates cardiac dysfunction induced by sepsis via inhibiting calcineurin and activating AMPK signaling.

The aim of the present study was to investigate whether cyclosporine A (CSA) improved cardiac dysfunction at an early stage of sepsis. Male Wistar rats were randomly divided into the following three groups: the sham‑operated control group, the cecal ligation puncture (CLP) procedure‑induced sepsis group and the CSA intervention group. Cecal ligation was performed to generate a sepsis model. At different time points (2, 6, 12, 24 and 72h) following sepsis induction, blood pressure, cardiac function, and non‑esterified free fatty acid (NEFA) levels in the plasma and myocardia were measured, and the expression levels of components associated with the AMP‑activated protein kinase (AMPK)‑acetyl CoA carboxylase (ACC)‑carnitine palmitoyl transferase 1 (CPT1) signaling pathway were compared among the three groups. Sepsis induced a decrease in blood pressure and cardiac function at 24h following sepsis induction in the CLP group, and CSA treatment ameliorated these pathophysiological alterations. In addition, rats in the CLP group exhibited significant increases in calcineurin activity and NEFA accumulation in the heart when compared with those in the sham group. These effects were attenuated by CSA treatment. Mechanistically, the activity of the AMPK‑ACC‑CPT1 pathway was enhanced by CSA treatment. The present study revealed that CSA treatment increases cardiac function at an early stage of sepsis in rats. This treatment partially suppresses calcineurin activity while activating the AMPK‑TCC‑CPT1 pathway.

Sestrin 2 induces autophagy and attenuates insulin resistance by regulating AMPK signaling in C2C12 myotubes

Impaired insulin-stimulated glucose uptake in skeletal muscle serves a critical role in the development of insulin resistance (IR), whereas the precise mechanism of the process remains unknown. Recently, the evolutionarily conserved, stress-inducible protein Sestrin2 (Sesn2) has been proposed to play a protective role against obesity-induced IR and diabetes. Activation of Sesn2 may activate AMP-activated protein kinase (AMPK) accompanied by suppression of mammalian target of rapamycin (mTOR), which may ultimately lead to autophagy induction. In view of the potential protective effects of autophagy on the physiological and the pathological regulatory processes via the regulation of energy homeostasis and metabolism, we investigated the effects of Sesn2 on the components of the insulin signaling pathway and insulin-stimulated glucose uptake in palmitate-induced insulin-resistant C2C12 myotubes. We showed that Sesn2 effectively restored the impaired insulin signaling. Moreover, autophagic activity decreased in response to palmitate, whereas Sesn2 significantly reversed the palmitate-suppressed autophagic signaling in this context. Our findings further revealed that Sesn2-induced autophagy contributed to restore the impaired insulin signaling through the activation of AMPK signal. Even in the presence of palmitate, Sesn2 up-regulation maintained insulin sensitivity and glucose metabolism via AMPK-dependent autophagic activation.

NPC-26 kills human colorectal cancer cells via activating AMPK signaling.

NPC-26 is novel mitochondrion-interfering compound. The current study tested its potential effect against colorectal cancer (CRC) cells. We demonstrated that NPC-26 induced potent anti-proliferative and cytotoxic activities against CRC cell lines (HCT-116, DLD-1 and HT-29). Activation of AMP-activated protein kinase (AMPK) signaling mediated NPC-26-induced CRC cell death. AMPKα1 shRNA knockdown or dominant negative mutation abolished NPC-26-induced AMPK activation and subsequent CRC cell death. NPC-26 disrupted mitochondrial function, causing mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) production. ROS scavengers (NAC or MnTBAP) and mPTP blockers (cyclosporin A or sanglifehrin A) blocked NPC-26-induced AMPK activation and attenuated CRC cell death. Significantly, intraperitoneal injection of NPC-26 potently inhibited HCT-116 tumor growth in severe combined immuno-deficient (SCID) mice. Yet, its anti-tumor activity was significantly weakened against AMPKα1-silenced HCT-116 tumors. Together, we conclude that NPC-26 kills CRC cells possibly via activating AMPK signaling.

White to beige conversion in PDE3B KO adipose tissue through activation of AMPK signaling and mitochondrial function

Understanding mechanisms by which a population of beige adipocytes is increased in white adipose tissue (WAT) reflects a potential strategy in the fight against obesity and diabetes. Cyclic adenosine monophosphate (cAMP) is very important in the development of the beige phenotype and activation of its thermogenic program. To study effects of cyclic nucleotides on energy homeostatic mechanisms, mice were generated by targeted inactivation of cyclic nucleotide phosphodiesterase 3b (Pde3b) gene, which encodes PDE3B, an enzyme that catalyzes hydrolysis of cAMP and cGMP and is highly expressed in tissues that regulate energy homeostasis, including adipose tissue, liver, and pancreas. In epididymal white adipose tissue (eWAT) of PDE3B KO mice on a SvJ129 background, cAMP/protein kinase A (PKA) and AMP-activated protein kinase (AMPK) signaling pathways are activated, resulting in “browning” phenotype, with a smaller increases in body weight under high-fat diet, smaller fat deposits, increased β-oxidation of fatty acids (FAO) and oxygen consumption. Results reported here suggest that PDE3B and/or its downstream signaling partners might be important regulators of energy metabolism in adipose tissue, and potential therapeutic targets for treating obesity, diabetes and their associated metabolic disorders.

Isoorientin induces apoptosis, decreases invasiveness, and downregulates VEGF secretion by activating AMPK signaling in pancreatic cancer cells.

Isoorientin (or homoorientin) is a flavone, which is a chemical flavonoid-like compound, and a 6-C-glucoside of luteolin. Isoorientin has been demonstrated to have anti-cancer activities against various tumors, but its effects on pancreatic cancer (PC) have not been studied in detail. In this study, we aim to investigate whether isoorientin has potential anti-PC effects and its underlying mechanism. In PC, isoorientin strongly inhibited the survival of the cells, induced cell apoptosis, and decreased its malignancy by reversing the expression of epithelial–mesenchymal transition and matrix metalloproteinase and decreased vascular endothelial growth factor expression. Meanwhile, we investigated the activity of the AMP-activated protein kinase (AMPK) signaling pathway after isoorientin treatment, which was forcefully activated by isoorientin, as expected. In addition, in the PC cells that were transfected with lentivirus to interfere with the expression of the gene PRKAA1, there were no differences in the apoptosis rate and the expression of malignancy biomarkers in the tumors of the isoorientin-treated and untreated groups. Thus, we demonstrated that isoorientin has potential antitumor effects via the AMPK signaling pathway, and isoorientin merits further investigation.

MicroRNA-25 targets PKCζ and protects osteoblastic cells from dexamethasone via activating AMPK signaling

AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζ's 3’ untranslated regions (UTRs). Forced-expression of miR-25 downregulated PKCζ and activated AMPK in human osteoblastic cells (OB-6 and hFOB1.19 lines), which thereafter protected cells from Dex. Reversely, expression of antagomiR-25, the miR-25 inhibitor, upregulated PKCζ and inhibited AMPK activation, exacerbating Dex damages. Notably, PKCζ shRNA knockdown similarly activated AMPK and protected osteoblastic cells from Dex. AMPK activation was required for miR-25-induced osteoblastic cell protection. AMPKα shRNA or dominant negative mutation almost completely blocked miR-25-induced cytoprotection against Dex. Further studies showed that miR-25 expression increased NADPH activity and suppressed Dex-induced oxidative stress in osteoblastic cells. Such effects by miR-25 were abolished with AMPKα knockdown or mutation. Significantly, miR-25-5p level was increased in patients’ necrotic femoral head tissues, which was correlated with PKCζ downregulation and AMPK hyper-activation. These results suggest that miR-25-5p targets PKCζ and protects osteoblastic cells from Dex possibly via activating AMPK signaling.

GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production

LPS stimulation in macrophages/monocytes induces TNFα production. We here tested the potential effect of GSK621, a novel AMP-activated protein kinase (AMPK) activator, against the process. In RAW264.7 macrophages, murine bone marrow-derived macrophages (BMDMs), and chronic obstructive pulmonary disease (COPD) patients' monocytes, GSK621 significantly inhibited LPS-induced TNFα protein secretion and mRNA synthesis. Inhibition of AMPK, through AMPKα shRNA knockdown or dominant negative mutation (T172A), almost abolished GSK621's suppression on TNFα in RAW264.7 cells. Reversely, forced-expression of a constitutively-active AMPKα (T172D) mimicked GSK621 actions and reduced LPS-induced TNFα production. Molecularly, GSK621 suppressed LPS-induced reactive oxygen species (ROS) production and nuclear factor kappa B (NFκB) activation. In vivo, GSK621 oral administration inhibited LPS-induced TNFα production and endotoxin shock in mice. In summary, GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production in macrophages/monocytes.

FGF21 activates AMPK signaling: impact on metabolic regulation and the aging process.

Fibroblast growth factor 21 (FGF21) has a significant role in the regulation of energy metabolism, e.g., in the control of systemic glucose and lipid metabolism. For instance, FGF21 enhances insulin sensitivity, increases glucose uptake, and thus can decrease serum hyperglycemia, while it also increases lipid oxidation and inhibits lipogenesis. AMP-activated protein kinase (AMPK) is a tissue energy sensor involved in maintaining the energy balance and tissue integrity. It is known that AMPK signaling generates an energy metabolic profile which displays a remarkable overlap with that of FGF21. There is convincing evidence that endocrine FGF21 signaling activates the AMPK pathway, either directly through FGFR1/β-klotho signaling or indirectly by stimulating the secretion of adiponectin and corticosteroids, which consequently can activate AMPK signaling in their target tissues. By activating AMPK, FGF21 can promote a healthy aging process and thus extend mammalian lifespan. We will examine the signaling mechanisms through which FGF21 can activate the AMPK pathway and then discuss the significance of the close connection between FGF21 and AMPK signaling in the control of metabolic disorders and the aging process.

MiR-135b expression downregulates Ppm1e to activate AMPK signaling and protect osteoblastic cells from dexamethasone.

Activation of AMP-activated protein kinase (AMPK) could potently protect osteoblasts/osteoblastic cells from dexamethasone (Dex). We aim to induce AMPK activation via microRNA (“miRNA”) downregulation of its phosphatase Ppm1e. We discovered that microRNA-135b (“miR-135b”) targets the 3' untranslated regions (UTRs) of Ppm1e. In human osteoblasticOB-6 cells and hFOB1.19 cells, forced-expression of miR-135b downregulated Ppm1e and activated AMPK signaling. miR-135b also protected osteoblastic cells from Dex. shRNA-induced knockdown of Ppm1e similarly activated AMPK and inhibited Dex-induced damages. Intriguingly, in the Ppm1e-silenced osteoblastic cells, miR-135b expression failed to offer further cytoprotection against Dex. Notably, AMPK knockdown (via shRNA) or dominant negative mutation abolished miR-135b-induced AMPK activation and cytoprotection against Dex. Molecularly, miR-135b, via activating AMPK, increased nicotinamide adenine dinucleotide phosphate (NADPH) activity and inhibited Dex-induced oxidative stress. At last, we found that miR-135b level was increased in human necrotic femoral head tissues, which was correlated with Ppm1e downregulation and AMPK activation. There results suggest that miR-135b expression downregulates Ppm1e to activate AMPK signaling, which protects osteoblastic cells from Dex.

OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling.

Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling.

Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation

This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0–2) and late (days 4–8), but not middle (days 2–4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process.

GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings.

Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent.

Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling.

Adiponectin has been demonstrated to protect the cardiovascular system and bone marrow mesenchymal stem cells (BMSCs). However, it is unclear whether adiponectin can protect BMSCs against flow shear stress (FSS). In this study, our aim was to explore the effects of adiponectin on BMSCs and to explore the role of AMP-activated protein kinase (AMPK) signaling in this process. Shear stress significantly inhibits the survival and increases the apoptosis of BMSCs in an intensity-dependent manner. The expression levels of TGF-β, bFGF, VEGF, PDGF, and Bcl2 are simultaneously reduced, and the phosphorylation levels of AMPK and ACC, as well as the expression level of Bax, are increased. Supplementation with adiponectin promotes the survival of BMSCs; reverses the changes in the expression levels of TGF-β, bFGF, VEGF, PDGF, Bcl2, and Bax; and further amplifies the phosphorylation of AMPK and ACC. Furthermore, the protective effects of adiponectin can be partially neutralized by AMPK siRNA. In summary, we have demonstrated for the first time that adiponectin can effectively protect BMSCs from FSS and that this effect depends, at least in part, on the activation of AMPK signaling.

Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings.

Here we evaluated the anti-cancer activity of aqueous Oldenlandia diffusa (OD) extracts (ODE) in colorectal cancer (CRC) cells. We showed that ODE exerted potent anti-proliferative, cytotoxic and pro-apoptotic activities against a panel of established CRC lines (HCT-116, DLD-1, HT-29 and Lovo) and primary (patient-derived) human CRC cells. ODE activated AMP-activated protein kinase (AMPK) signaling, which led to subsequent mTORC1 inhibition and Bcl-2/HIF-1α downregulation in CRC cells. In ODE-treated CRC cells, AMPKα1 formed a complex with p53. This might be important for p53 activation and subsequent cancer cell apoptosis. Inhibition of AMPK signaling, though dominant negative (dn) mutation or shRNA/siRNA knockdown of AMPKα1 attenuated ODE-exerted CRC cytotoxicity. In vivo, i.p. administration of ODE inhibited HCT-116 xenograft tumor growth in SCID mice. In addition, AMPK activation, mTORC1 inhibition and p53 activation were observed in ODE-treated HCT-116 xenograft tumors. These results suggest that ODE inhibits CRC cells in vitro and in vivo, possibly via activation of AMPK-dependent signalings.

YiQiFuMai Powder Injection Attenuates Ischemia/Reperfusion-Induced Myocardial Apoptosis Through AMPK Activation.

The YiQiFuMai powder injection (YQFM), a traditional Chinese medicine (TCM) prescription re-developed based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in cardiovascular diseases in clinics. However, its role in protection against myocardial ischemia/reperfusion (MI/R) injury has not been elucidated. The present study not only evaluated the cardioprotective effect of YQFM from MI/R injury but also investigated the potential molecular mechanisms both in vivo and in vitro. The myocardium infarct size, production of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac function, TUNEL staining, and caspase-3 activity were measured. Cell viability was determined, and cell apoptosis was measured by Hoechst 33342 staining and flow cytometry. Mitochondrial membrane potential (ΔΨm) was measured, and ATP content was quantified by bioluminescent assay. Expression of apoptosis-related proteins, including Caspase-3, Bcl-2, Bax, AMPKα, and phospho-AMPKα, was analyzed by western blotting. AMPKα siRNA transfection was also applied to the mechanism elucidation. YQFM at a concentration of 1.06 g/kg significantly reduced myocardium infarct size and the production of LDH, CK in serum, improved the cardiac function, and also produced a significant decrease of apoptotic index. Further, combined treatment with compound C partly attenuated the anti-apoptotic effect of YQFM. In addition, pretreatment with YQFM ranging from 25 to 400 μg/mL markedly improved cell viability and decreased LDH release. Moreover, YQFM inhibited H9c2 apoptosis, blocked the expression of caspase-3, and modulated Bcl-2 and Bax proteins, leading to an increased mitochondrial membrane potential and cellular ATP content. Mechanistically, YQFM activated AMP-activated protein kinase (AMPK) signaling pathways whereas pretreatment with AMPK inhibitor Compound C and application of transfection with AMPKα siRNA attenuated the anti-apoptotic effect of YQFM. Our results indicated that YQFM could provide significant cardioprotection against MI/R injury, and potential mechanisms might suppress cardiomyocytes apoptosis, at least in part, through activating the AMPK signaling pathways.

AMPK promotes tolerance to Ras pathway inhibition by activating autophagy.

Targeted inhibitors of oncogenic Ras (rat sarcoma viral oncogene)-Raf signaling have shown great promise in the clinic, but resistance remains a major challenge: 30% of tumors with pathway mutations do not respond to targeted inhibitors, and of the 70% that do respond, all eventually develop resistance. Before cancer cells acquire resistance, they respond to initial drug treatment either by undergoing apoptosis ('addiction') or by surviving treatment albeit with reduced growth ('tolerance'). As these drug-tolerant cells serve as a reservoir from which resistant cells eventually emerge, inhibiting the pathways that confer tolerance could potentially delay or even prevent recurrence. Here, we show that melanomas and other cancers acquire tolerance to Ras-Raf pathway inhibitors by activating autophagy, which is mediated by the cellular energy sensor AMP-activated protein kinase (AMPK). Blocking this AMPK-mediated autophagy sensitizes drug-tolerant melanomas to Ras-Raf pathway inhibitors. Conversely, activating AMPK signaling and autophagy enables melanomas that would otherwise be addicted to the Ras-Raf pathway to instead tolerate pathway inhibition. These findings identify a key mechanism of tolerance to Ras-Raf pathway inhibitors and suggest that blocking either AMPK or autophagy in combination with these targeted inhibitors could increase tumor regression and decrease the likelihood of eventual recurrence.