The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate.

Background/purpose: 5' Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methods: MC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. Results: The results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. Conclusion: AMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.

AICAr, a Widely Used AMPK Activator with Important AMPK-Independent Effects: A Systematic Review.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway.

Acadesine Circumvents Azacitidine Resistance in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Myelodysplastic syndrome (MDS) defines a group of heterogeneous hematologic malignancies that often progresses to acute myeloid leukemia (AML). The leading treatment for high-risk MDS patients is azacitidine (Aza, Vidaza®), but a significant proportion of patients are refractory and all patients eventually relapse after an undefined time period. Therefore, new therapies for MDS are urgently needed. We present here evidence that acadesine (Aca, Acadra®), a nucleoside analog exerts potent anti-leukemic effects in both Aza-sensitive (OCI-M2S) and resistant (OCI-M2R) MDS/AML cell lines in vitro. Aca also exerts potent anti-leukemic effect on bone marrow cells from MDS/AML patients ex-vivo. The effect of Aca on MDS/AML cell line proliferation does not rely on apoptosis induction. It is also noteworthy that Aca is efficient to kill MDS cells in a co-culture model with human medullary stromal cell lines, that mimics better the interaction occurring in the bone marrow. These initial findings led us to initiate a phase I/II clinical trial using Acadra® in 12 Aza refractory MDS/AML patients. Despite a very good response in one out 4 patients, we stopped this trial because the highest Aca dose (210 mg/kg) caused serious renal side effects in several patients. In conclusion, the side effects of high Aca doses preclude its use in patients with strong comorbidities.

AMPK activator acadesine fails to alleviate isoniazid-caused mitochondrial instability in HepG2 cells.

Isoniazid (INH) is a first-line antituberculosis drug that is adversely associated with hepatotoxicity. Recently, impairment of mitochondrial homeostasis involved in this side effect has been noticed. Mitochondrial homeostasis is achieved by the balance between the generation of functional mitochondria by biogenesis and elimination of dysfunctional mitochondria by autophagy. AMP-activated protein kinase (AMPK) can maintain mitochondrial stability through positive control of these two processes. In this study, we showed that AMPK activator acadesine (AICAR) alleviated INH-caused impairment of mitochondrial biogenesis by activation of silent information regulator two ortholog 1 (SIRT1)-peroxisome proliferator-activated receptor γ coactivator 1α (PGC1 α) pathway in HepG2 cells. However, mitochondrial instability and apoptosis were caused by AICAR along with an unexpected decrease in INH-induced cytoprotective autophagy. Therefore, AICAR failed to alleviate INH-caused mitochondrial instability in HepG2 cells due to its inhibitory effect on autophagy induced by INH. Copyright © 2017 John Wiley & Sons, Ltd.

Effects of Acadesine on Myocardial Energy Metabolism of Model Dogs with Myocardial Ischemia-reperfu-sion Injury after Cardiopulmonary Bypass / 中国药房

OBJECTIVE:To explore the effects of acadesine on myocardial energy metabolism of model dogs with myocardial ischemia-reperfusion injury (MIRI) after cardiopulmonary bypass (CPB). METHODS:Dogs were randomly divided into control group,model group,acadesine low-dose,high-dose groups(0.8,3.2 mg/kg),6 in each group. All dogs received CPB. Except for control group,dogs in other groups were reduced for MIRI model,and perfused St.Thomas cardiac cardioplegia lipid containing rel-evant drugs 60 min after main artery block. The uptake rates of myocardial glucose and free fatty acid(FFA),creatine kinase isoen-zyme(CK-MB)concent in venous sinus plasma and adenosine triphosphate(ATP)content in mitochondria were detected and calcu-lated before bypass and after 15,60,90 min of reperfusion. Left ventricular systolic pressure(LVSP)and left ventricular end dia-stolic pressure(LVEDP)were analyzed,and mRNA expression of adenylate-activated protein kinase(AMPK)and protein expres-sion of phosphorylated AMPK(p-AMPK)in myocardial tissue were detected. RESULTS:Before bypass,all indexes in each group had no statistic significances(P>0.05). After bypass,compared with control group,uptake rates of myocardial glucose and FAA, ATP content,mRNA expression of AMPK and protein expression of p-AMPK and LVSP in 3 time points in model group and each administration group were obviously decreased(P<0.05);LVEDP and CK-MB concent in plasma were obviously increased(P<0.05). Compared with model group,uptake rates of myocardial glucose and FAA,ATP content,mRNA expression of AMPK and protein expression of p-AMPK and LVSP in 3 time points in each administration group were obviously increased (P<0.05);LVEDP and CK-MB concent in plasma were obviously decreased (P<0.05);and high-dose group showed more obvious change than that of low-dose group (P<0.05). CONCLUSIONS:Acadesine can promote the AMPK phosphorylation,contribute to the myocardial glucose and FFA uptake to promote the increase of ATP in myocardial mitochondria and relieve MIRI after CPB.

Effects of Acadesine on Myocardial Energy Metabolism of Model Dogs with Myocardial Ischemia-reperfu-sion Injury after Cardiopulmonary Bypass / 中国药房

OBJECTIVE:To explore the effects of acadesine on myocardial energy metabolism of model dogs with myocardial ischemia-reperfusion injury (MIRI) after cardiopulmonary bypass (CPB). METHODS:Dogs were randomly divided into control group,model group,acadesine low-dose,high-dose groups(0.8,3.2 mg/kg),6 in each group. All dogs received CPB. Except for control group,dogs in other groups were reduced for MIRI model,and perfused St.Thomas cardiac cardioplegia lipid containing rel-evant drugs 60 min after main artery block. The uptake rates of myocardial glucose and free fatty acid(FFA),creatine kinase isoen-zyme(CK-MB)concent in venous sinus plasma and adenosine triphosphate(ATP)content in mitochondria were detected and calcu-lated before bypass and after 15,60,90 min of reperfusion. Left ventricular systolic pressure(LVSP)and left ventricular end dia-stolic pressure(LVEDP)were analyzed,and mRNA expression of adenylate-activated protein kinase(AMPK)and protein expres-sion of phosphorylated AMPK(p-AMPK)in myocardial tissue were detected. RESULTS:Before bypass,all indexes in each group had no statistic significances(P>0.05). After bypass,compared with control group,uptake rates of myocardial glucose and FAA, ATP content,mRNA expression of AMPK and protein expression of p-AMPK and LVSP in 3 time points in model group and each administration group were obviously decreased(P<0.05);LVEDP and CK-MB concent in plasma were obviously increased(P<0.05). Compared with model group,uptake rates of myocardial glucose and FAA,ATP content,mRNA expression of AMPK and protein expression of p-AMPK and LVSP in 3 time points in each administration group were obviously increased (P<0.05);LVEDP and CK-MB concent in plasma were obviously decreased (P<0.05);and high-dose group showed more obvious change than that of low-dose group (P<0.05). CONCLUSIONS:Acadesine can promote the AMPK phosphorylation,contribute to the myocardial glucose and FFA uptake to promote the increase of ATP in myocardial mitochondria and relieve MIRI after CPB.

Bcl-2high mantle cell lymphoma cells are sensitized to acadesine with ABT-199.

Acadesine is a nucleoside analogue with known activity against B-cell malignancies. Herein, we showed that in mantle cell lymphoma (MCL) cells acadesine induced caspase-dependent apoptosis through turning on the mitochondrial apoptotic machinery. At the molecular level, the compound triggered the activation of the AMPK pathway, consequently modulating known downstream targets, such as mTOR and the cell motility-related vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation by acadesine was concomitant with a blockade of CXCL12-induced migration. The inhibition of the mTOR cascade by acadesine, committed MCL cells to enter in apoptosis by a translational downregulation of the antiapoptotic Mcl-1 protein. In contrast, Bcl-2 protein levels were unaffected by acadesine and MCL samples expressing high levels of Bcl-2 tended to have a reduced response to the drug. Targeting Bcl-2 with the selective BH3-mimetic agent ABT-199 sensitized Bcl-2high MCL cells to acadesine. This effect was validated in vivo, where the combination of both agents displayed a more marked inhibition of tumor outgrowth than each drug alone. These findings support the notions that antiapoptotic proteins of the Bcl-2 family regulate MCL cell sensitivity to acadesine and that the combination of this agent with Bcl-2 inhibitors might be an interesting therapeutic option to treat MCL patients.

Acadesine Triggers Non-apoptotic Death in Tumor Cells.

We studied the cytotoxicity of acadesine (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside) for tumor and normal cells of various species and tissue origin. In tumor cells, acadesine triggered non-apoptotic death; the potency of the compound to normal cells was substantially lower. Acadesine was toxic for tumor cells with multidrug resistant phenotypes caused by the transmembrane transporter Р-glycoprotein or lack of proapoptotic p53. Activity of adenosine receptors was required for acadesine-induced cell death, whereas functioning of АМР-dependent protein kinase was not required. A more pronounced cytotoxicity for tumor cells, as well as the non-canonical death mechanism(s), makes acadesine a promising candidate for antitumor therapy.