Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury

Abstract: Chronic organ‐donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia–reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL‐1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia–reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL‐1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia–reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well‐known cytoprotective factors as hemeoxygenase 1 (HO‐1). Fatty livers preserved in IGL‐1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL‐1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL‐1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e‐NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL‐1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia–reperfusion injury.     

Trimetazidine： Is it a promising drug for use in steatotic grafts？

AIM:Chronic organ-donor shortage has led to theacceptance of steatotic livers for transplantation,despitethe higher risk of graft dysfunction or nonfunctionassociated with the ischemic preservation period ofthese organs.The present study evaluates the effects oftrimetazidine(TMZ)on an isolated perfused liver model.METHODS:Steatotic and non-steatotic livers werepreserved for 24 h in the University of Wisconsin(UW)solution with or without TMZ.Hepatic injury and function(transaminases,bile production and sulfobromophthalein(BSP)clearance)and factors potentially involved in thesusceptibility of steatotic livers to ischemia-reperfusion(I/R)injury,including oxidative stress,mitochondrialdamage,microcirculatory diseases,and ATP depletionwere evaluated.RESULTS:Steatotic livers preserved in UW solutionshowed higher transaminase levels,lower bile productionand BSP clearance compared with non-steatotic livers.Alterations in perfusion flow rate and vascular resistance,mitochondrial damage,and reduced ATP content weremore evident in steatotic livers.TMZ addition to UWsolution reduced hepatic injury and ameliorated hepaticfunctionality in both types of the liver and protectedagainst the mechanisms potentially responsible for thepoor tolerance of steatotic livers to I/R.CONCLUSION:TMZ may constitute a useful approachin fatty liver surgery,limiting the inherent risk of steatoticliver failure following transplantation.     

Sirtuin 1 in rat orthotopic liver transplantation:An I GL-1 preservation solution approach

AIM:To investigate the possible involvement of Sirtuin1(SIRT1)in rat orthotopic liver transplantation(OLT),when Institute Georges Lopez 1(IGL-1)preservation solution is enriched with trimetazidine(TMZ).METHODS:Male Sprague-Dawley rats were used as donors and recipients.Livers were stored in IGL-1 preservation solution for 8h at 4℃,and then underwent OLT according to Kamada’s cuff technique without arterialization.In another group,livers were stored in IGL-1 preservation solution supplemented with TMZ,at10-6 mol/L,for 8 h at 4℃and then underwent OLT.Rats were sacrificed 24 h after reperfusion,and liver and plasma samples were collected.Liver injury(transaminase levels),mitochondrial damage(glutamate dehydrogenase activity)oxidative stress(malondialdehyde levels),and nicotinamide adenine dinucleotide(NAD+),the cofactor necessary for SIRT1 activity,were determined by biochemical methods.SIRT1 and its substrates(acFox O1,ac-p53),the precursor of NAD+,nicotinamide phosphoribosyltransferase(NAMPT),as well as the phosphorylation of adenosine monophosphate activated protein kinase(AMPK),p-m TOR,p-p70S6K(direct substrate of m TOR),autophagy parameters(beclin-1,LC3B)and MAP kinases(p-p38 and p-ERK)were determined by Western blot.RESULTS:Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preservedin IGL-1 solution alone.In addition,livers preserved in IGL-1+TMZ presented reduced levels of oxidative stress.This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity,as indicated by decreased acetylation of p53 and Fox O1.The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD+/NADH levels.Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of m TOR and its direct substrate(p-p70S6K).As a consequence,autophagy mediators(beclin-1 and LC3B)were overexpressed.Furthermore,MAP kinases were regulated in livers preserved with IGL-1+TMZ,as they were characterized by enhanced p-ERK and decreased p-p38protein expression.CONCLUSION:Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT,through SIRT1 up-regulation.     

Impact of ischaemic preconditioning on experimental steatotic livers following hepatic ischaemia–reperfusion injury: a systematic review

Background

Steatotic livers are vulnerable to the deleterious effects of ischaemia–reperfusion injury (IRI) that occur after hepatic surgery. Ischaemic preconditioning (IPC) has been shown to abrogate the effects of IRI in patients undergoing hepatic surgery. Experimental studies have suggested that IPC may be beneficial in steatotic livers subjected to IRI.

Objective

The aim of this systematic review was to evaluate the effects of IPC on steatotic livers following hepatic IRI in experimental models.

Methods

An electronic search of the OVID Medline and EMBASE databases was performed to identify studies that reported clinically relevant outcomes in animal models of hepatic steatosis subjected to IPC and IRI.

Results

A total of 1093 articles were identified, of which 18 met the inclusion criteria. There was considerable heterogeneity in the type of animal model, and duration and type of IRI. Increased macrovesicular steatosis (> 30%) was associated with a poor outcome following IRI. Ischaemic preconditioning was found to be beneficial in > 30% steatotic livers and provided for decreased histological damage, improved liver function findings and increased survival.

Conclusions

Experimental evidence supports the use of IPC in steatotic livers undergoing IRI. These findings may be applicable to patients undergoing liver surgery. However, clinical studies are required to validate the efficacy of IPC in this setting.     

Insulin like growth factor-1 increases fatty liver preservation in IGL-1 solution

AIM: To investigate the benefits of insulin like growth factor-1 (IGF-1) supplementation to serum-free institut georges lopez-1 (IGL-1) solution to protect fatty liver against cold ischemia reperfusion injury. METHODS: Steatotic livers were preserved for 24 h in IGL-1  solution supplemented with or without IGF-1 and then perfused "ex vivo " for 2 h at 37℃. We examined the effects of IGF-1 on hepatic damage and function (transaminases, percentage of sulfobromophthalein clearance in bile and vascular resistance). We also studied other factors associated with the poor tolerance of fatty livers to cold ischemia reperfusion injury such as mitochondrial damage, oxidative stress, nitric oxide, tumor necrosis factor-α (TNF-α) and mitogen-activated protein kinases.RESULTS: Steatotic livers preserved in IGL-1 solutionsupplemented with IGF-1 showed lower transaminase levels, increased bile clearance and a reduction in vascular resistance when compared to those preserved in IGL-1solution alone. These benefits are mediated by activation of AKT and constitutive endothelial nitric oxide synthase (eNOS), as well as the inhibition of inflammatory cytokines such as TNF-α. Mitochondrial damage and oxidative stress were also prevented.CONCLUSION: IGL-1  enrichment with IGF-1 increasedfatty liver graft preservation through AKT and eNOS activation, and prevented TNF-α release during normothermic reperfusion.     

Intermittent ischaemia maintains function after ischaemia reperfusion in steatotic livers

Background:

Ischaemic preconditioning (IPC) and intermittent ischaemia (INT) reduce liver injury after ischaemia reperfusion (IR). Steatotic livers are at a higher risk of IR injury, but the protection offered by IPC and INT is not well understood. The aim of the present study was to determine the effectiveness of IPC and INT in maintaining liver function in steatotic livers.

Material and methods:

A model of segmental hepatic ischaemia (45 min) and reperfusion (60 min) was employed using lean and obese Zucker rats. Bile flow recovery was measured to assess dynamic liver function, hepatocyte fat content quantified and blood electrolytes, metabolites and bile calcium measured to assess liver and whole body physiology. Liver marker enzymes and light and electron microscopy were employed to assess hepatocyte injury.

Results:

IPC was not effective in promoting bile flow recovery after IR in either lean or steatotic livers, whereas INT promoted good bile flow recovery in steatotic as well as lean livers. However, the bile flow recovery in steatotic livers was less than that in lean livers. In steatotic livers, ischaemia led to a rapid and substantial decrease in fat content. Steatotic livers were more susceptible to IR injury than lean livers, as indicated by increased blood ALT concentrations and major histological injury.

Conclusion:

INT is more effective than IPC in restoring liver function in the acute phase of IR in steatotic livers. In obese patients, INT may be useful in promoting better liver function after IR after liver resection.     

Steatotic livers are susceptible to normothermic ischemia-reperfusion injury from mitochondrial Complex-I dysfunction

AIM: To assess the effects of ischemic preconditioning (IPC, 10-min ischemia/10-min reperfusion) on steatotic liver mitochondrial function after normothermic ischemia-reperfusion injury (IRI). METHODS: Sixty male Sprague-Dawley rats were fed 8-wk with either control chow or high-fat/high-sucrose diet inducing > 60% mixed steatosis. Three groups (n = 10/group) for each dietary state were tested: (1) the IRI group underwent 60 min partial hepatic ischemia and 4 h reperfusion; (2) the IPC group underwent IPC prior to same standard IRI; and (3) sham underwent the same surgery without IRI or IPC. Hepatic mitochondrial function was analyzed by oxygraphs. Mitochondrial Complex-I, Complex-II enzyme activity, serum alanine aminotransferase (ALT), and histological injury were measured. RESULTS: Steatotic-IRI livers had a greater increase in ALT (2476 ± 166 vs 1457 ± 103 IU/L, P < 0.01) and histological injury following IRI compared to the lean liver group. Steatotic-IRI demonstrated lower Complex-I activity at baseline [78.4 ± 2.5 vs 116.4 ± 6.0 nmol/(min.mg protein), P < 0.001] and following IRI [28.0 ± 6.2 vs 104.3 ± 12.6 nmol/(min.mg protein), P < 0.001]. Steatotic-IRI also demonstrated impaired Complex-I function post-IRI compared to the lean liver IRI group. Complex-II activity was unaffected by hepatic steatosis or IRI. Lean liver mitochondrial function was unchanged following IRI. IPC normalized ALT and histological injury in steatotic livers but had no effect on overall steatotic liver mitochondrial function or individual mitochondrial complex enzyme activities. CONCLUSION: Warm IRI impairs steatotic liver Complex-I activity and function. The protective effects of IPC in steatotic livers may not be mediated through mitochondria.     

Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation

BACKGROUND/AIMS: Hepatic steatosis is a risk factor for transplantation. We examined the role of AMP-activated protein kinase (AMPK) and nitric oxide (NO) in the benefits of preconditioning in steatotic liver transplantation. METHODS: Steatotic liver transplantation with or without preconditioning was induced in Zucker rats. The activities of AMPK and NO synthase (NOS) were measured and altered pharmacologically. RESULTS: Preconditioning or AMPK activation with aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased AMPK and constitutive NOS activities and protected against lipid peroxidation, nitrotyrosine formation and hepatic injury in both grafts. Inhibition of AMPK activity removed the benefits of preconditioning. NO synthesis inhibition abolished the benefits of preconditioning or AICAR. Therefore, preconditioning or AICAR, through AMPK activation, may induce NO synthesis, thus protecting against hepatic injury in both steatotic and non-steatotic liver transplantation. In non-steatotic grafts, NO donors simulated the benefits of preconditioning. However, in steatotic grafts, NO supplementation was ineffective. CONCLUSIONS: These results indicate (a) a potential relationship between AMPK and NO in the benefits of preconditioning in steatotic liver transplantation, (b) AICAR as a new phamacological strategy in steatotic liver transplantation and (c) a differential effect of NO supplementation in both grafts.     

Current studies on therapeutic approaches for ischemia/reperfusion injury in steatotic livers

Steatotic livers are particularly vulnerable to ischemia/reperfusion (I/R) injury, resulting in poor outcomes following liver surgery and transplantation. Therapeutic approaches for I/R injury in steatotic livers are currently under intensive investigation. This review summarizes and discusses the approaches developed during the last few years to prevent hepatic I/R injury in steatotic livers. Among the proposed approaches, ischemic preconditioning and intermittent clamping are the two most promising approaches that have been applied in some clinical centers for liver surgery and transplantation, but most of others have not reached clinical application yet.     

Polyethylene glycol rinse solution:An effective way to prevent ischemia-reperfusion injury

AIM:To test whether a new rinse solution containing polyethylene glycol 35(PEG-35)could prevent ischemia-reperfusion injury(IRI)in liver grafts.METHODS:Sprague-Dawley rat livers were stored in University of Wisconsin preservation solution and then washed with different rinse solutions(Ringer’s lactate solution and a new rinse solution enriched with PEG-35 at either 1 or 5 g/L)before ex vivo perfusion with Krebs-Heinseleit buffer solution.We assessed the following:liver injury(transaminase levels),mitochondrial damage(glutamate dehydrogenase activity),liver function(bile output and vascular resistance),oxidative stress(malondialdehyde),nitric oxide,liver autophagy(Beclin-1 and LCB3)and cytoskeleton integrity(filament and globular actin fraction);as well as levels of metalloproteinases(MMP2 and MMP9),adenosine monophosphate-activated protein kinase(AMPK),heat shock protein 70(HSP70)and heme oxygenase 1(HO-1).RESULTS:When we used the PEG-35 rinse solution,reduced hepatic injury and improved liver function were noted after reperfusion.The PEG-35 rinse solution prevented oxidative stress,mitochondrial damage,and liver autophagy.Further,it increased the expression of cytoprotective heat shock proteins such as HO-1 and HSP70,activated AMPK,and contributed to the restoration of cytoskeleton integrity after IRI.CONCLUSION:Using the rinse solution containing PEG-35 was effective for decreasing liver graft vulnerability to IRI.     

Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy

Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-beta-d-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1(+/-) mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.     

Hypoxia inducible factor-1�� accumulation in steatotic liver preservation: Role of nitric oxide

AIM:To examine the relevance of hypoxia inducible factor(HIF-1)and nitric oxide(NO)on the preservation of fatty liver against cold ischemia-reperfusion injury(IRI). METHODS:We used an isolated perfused rat liver model and we evaluated HIF-1αin steatotic and non-steatotic livers preserved for 24 h at 4℃in University of Wisconsin and IGL-1 solutions,and then subjected to 2 h of normothermic reperfusion.After normoxic reperfusion,liver enzymes,bile production,bromosulfophthalein clearance,as well as HIF-1αand ...     

Pre‐ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress‐dependent autophagy via PERK and IRE1 signalings

Melatonin has demonstrated a potential protective effect in central nervous system. Thus, it is interesting to determine whether pre‐ischemia melatonin administration could protect against cerebral ischemia/reperfusion (IR)‐related injury and the underlying molecular mechanisms. In this study, we revealed that IR injury significantly activated endoplasmic reticulum (ER) stress and autophagy in a middle cerebral artery occlusion mouse model. Pre‐ischemia melatonin treatment was able to attenuate IR‐induced ER stress and autophagy. In addition, with tandem RFP‐GFP‐LC3 adeno‐associated virus, we demonstrated pre‐ischemic melatonin significantly alleviated IR‐induced autophagic flux. Furthermore, we showed that IR induced neuronal apoptosis through ER stress related signalings. Moreover, IR‐induced autophagy was significantly blocked by ER stress inhibitor (4‐PBA), as well as ER‐related signaling inhibitors (PERK inhibitor, GSK; IRE1 inhibitor, 3,5‐dibromosalicylaldehyde). Finally, we revealed that melatonin significantly alleviated cerebral infarction, brain edema, neuronal apoptosis, and neurological deficiency, which were remarkably abolished by tunicamycin (ER stress activator) and rapamycin (autophagy activator), respectively. In summary, our study provides strong evidence that pre‐ischemia melatonin administration significantly protects against cerebral IR injury through inhibiting ER stress‐dependent autophagy. Our findings shed light on the novel preventive and therapeutic strategy of daily administration of melatonin, especially among the population with high risk of cerebral ischemic stroke.     

Effects of Institut Georges Lopez-1 and Celsior preservation solutions on liver graft injury

AIM: To compare Institut Georges Lopez(IGL-1) and Celsior preservation solutions for hepatic endothelium relaxation and liver cold ischemia reperfusion injury(IRI).METHODS: Two experimental models were used.In the first one, acetylcholine-induced endotheliumdependent relaxation(EDR) was measured in isolated ring preparations of rat hepatic arteries preserved or not in IGL-1 or Celsior solutions(24 h at 4 ℃).To determine nitric oxide(NO) and cyclooxygenase EDR, hepatic arteries were incubated with L-NG-nitroarginine methyl ester(L-NAME), an inhibitor of endothelium nitric oxide synthase(e NOS), or with L-NAME plus indomethacin, an inhibitor of cyclooxygenase.In the second experiment, rat livers were cold-stored in IGL-1 or Celsior solutions for 24 h at 4 ℃ and then perfused "ex vivo " for 2 h at 37 ℃.Liver injury was assessed by transaminase measurements, liver function by bile production and bromosulfophthalein clearance, oxidative stress by malondialdehyde levels and catalase activity and alterations in cell signaling pathways by pA kt, pA MPK, eN OS and MAPKs proteins level.RESULTS: After cold storage for 24 h with either Celsior or IGL-1, EDR was only slightly altered.Infreshly isolated arteries, EDR was exclusively mediated by NO.However, cold-stored arteries showed NOand COX-dependent relaxation.The decrease in NO-dependent relaxation after cold storage was significantly more marked with Celsior.The second study indicated that IGL-1 solution obtained better liver preservation and protection against IRI than Celsior.Liver injury was reduced, function was improved and there was less oxidative stress.IGL-1 solution activated Akt and AMPK, which was concomitant with increased eN OS expression and nitrite/nitrate levels.Furthermore, MAPKs kinases were regulated in livers preserved with IGL-1 solution since reductions in p-p38, p-ERK and p-JNK protein levels were observed.CONCLUSION: IGL-1 solution preserved NO-dependent relaxation better than Celsior storage solution and enhanced liver graft preservation.     

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin‐1, ubiquitin‐like autophagy‐related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3‐I to autophagosome‐associated LC3‐II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome‐associated membrane protein (LAMP)‐1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase‐3 activity, and immunoglobulin‐heavy‐chain‐binding protein (BiP) and CCAAT/enhancer‐binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.     

Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega-3 fatty acids

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega-6 (n-6): omega-3 (n-3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline-deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega-3 and omega-6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega-3 PUFAs and compared with the control diet-fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega-6: omega-3 PUFA ratio. Dietary omega-3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. CONCLUSION: Macrosteatotic livers disclosed an abnormal omega-6: omega-3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega-3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool.     

Chlorinated adenosine analogue induces AMPK and autophagy in chronic lymphocytic leukaemia cells during therapy

8‐chloro‐adenosine (8‐Cl‐Ado) is currently in phase‐I clinical trials for acute myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Previously, we demonstrated that treatment with 8‐Cl‐Ado leads to diminished ATP levels. We hypothesized that AMP‐activated protein kinase (AMPK) signalling would be initiated in these cells, leading to induction of autophagy. AMPK activation and induction of autophagy were demonstrated during preclinical incubations in CLL cells with the analogues. Importantly, we extended similar observations in CLL lymphocytes during an 8‐Cl‐Ado phase‐I trial. In conclusion, 8‐Cl‐Ado treatment induces autophagy in CLL lymphocytes in vitro as well as in vivo during clinical trial.