Ernährung 2006 International Cachexia Workshop Berlin, June 2006

Activation Peptides ATP E1 ATP Ubiquitin Proteolysis E2 Proteín Proteasome 26S E2 E3 Conjugation Ernährung 2006 International Cachexia Workshop Berlin, June 2006 Antiproteolytic strategies Prof. Dr. Josep M. Argilés Departamento de Bioquímica y Biología Molecular Universidad de Barcelona, Spain

Hippocrates of Kos: (460-370 BC) The flesh is consumed and becomes water,, the shoulders, clavicles, chest and thights melt away. The illness is fatal

Multiorgan syndrome Systemic disorder

T h e C a c h e x i a P y r a m i d T r e a t m e n t T u m o u r F o o d i n t a k e M e t a b o l i c a b n o r m a l i t i e s T u m o u r f a c t o r s C y t o k i n e s H o r m o n e s B r a i n I m m u n e s y s t e m S k e l e t a l A d i p o s e L i v e r m u s c l e t i s s u e B l o o d G u t I m m o b i l i t y Q u a l i t y W e i g h t o f l i f e l o s s A n e m i a O e d e m a W e a k n e s s

Skeletal muscle

MUSCLES AA LIVER Acute-phase proteins Gluconeogenesis

Skeletal muscle - Decreased AA uptake - Increased AA oxidation - Decreased glucose transport - Alterations in mitochondrial function (uncoupling) - Apoptosis - Alterations in satellite cell proliferation/differentiation - Increased proteolysis

ESTIMATION OF PROTEIN TURNOVER IN VIVO 14 C-PROTEIN LOADING 400mC i /kg body weight NaH 14 CO 3

SKELETAL MUSCLE (GASTROCNEMIUS) Ks Kd Ka Control 11.6 5.63 5.14 AH-130 12.0 13.9-1.97 Costelli et al., J Clin Invest. 1993;92(6):2783-9.

PROTEINS synthesis degradation c y c l ohe x im i de ( - ) TYROSINE TYROSINE TOTAL PRQTEOLYSIS = DEGRADATION NET PROTEOLYSIS = DEGRADATION - SYNTHESIS

Tyrosine released by isolated soleus rat muscles (nmoles/g.hr) PAIR-FED TUMOUR 194 251 *** (Martínez et al, 1994)

CANCER CACHEXIA PROTEIN Kd Ks AMINO ACIDS

Proteolytic systems in muscle Lysosomal Cathepsins Non-lysosomal Ca ++ -dependent Calpains ATP-dependent Ubiquitin

In vitro proteolysis (nmol Tyr released/g/h) 0 100 200 CONTROL TUMOR lisosomal Ca 2+ -dependent ubiquitin-dependent

Activation Peptides ATP E1 ATP Ubiquitin Proteolysis E2 Proteín Proteosome 26S E2 E3 Conjugation

Muscle-specific ubiquitin ligases MAFbx (atrogin-5) MURF-1

Ubiquitin mrna levels in skeletal muscle of gastric cancer patients Total Ub mrna levels (arbitrary units) 4000 3500 3000 2500 2000 1500 1000 500 0 Control subjects Stage I-II Stage III Stage IV Patients lost 5.57% of body weight (n= 20) Bossola et al. American Journal of Physiology 280: R1518-R1523 (2001)

Cytokine network Wasting TUMOUR Neuroendocrine response Muscle proteolysis Tumour-specific products Treatment

Therapeutic approaches

Pharmacological strategies MUSCLE WASTING Nutritional approaches

Anticatabolic + Anabolic

Using nutrition Leu β-hydroxy-β-methylbutyrate

Busquets et al., J Cell Physiol. 2002 191(3):283-9.

Chymotrypsin-like activity in proteasome Smith et al., Cancer Res. 2005;65(1):277-83

Smith et al., Cancer Res. 2005;65(1):277-83

Using nutraceuticals EPA Resveratrol

Whitehouse et al., Cancer Res. 2001;61(9):3604-9.

ω-3 FATTY ACIDS Wigmore et al., 1996 Treated a group of pancreatic cancer patients with fish oil capsules (containing 18% EPA and 12% DHA) for three months and observed an increase in body weight, a reduction of acute-phase proteins (C-reactive protein) and a stabilization of the resting energy expenditure. Fearon et al., 2003 A randomized double blind trial in pancreatic cancer patients using a protein and energy dense supplement enriched with ω-3 fatty acids and antioxidants. The trial demonstrates weight and LBM gain and improved quality of life. Gut. 52(10):1479-86 2003.

Resveratrol Diphenol found in grapes, peanuts nf-κb inhibitor COX-2 inhibitor Br J Cancer. 2004 91(9):1742-50. Wyke SM, Russell ST, Tisdale MJ. Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NFkappaB activation.

Using drugs β2 agonists Anticachectic cytokines

BETA-2 ADRENERGIC AGONISTS OH HO CH - CH 2 - NH - CH 3 ADRENALINE (EPINEPHRINE) HO Cl OH CH 3 H 2 N CH - CH 2 - NH - C - CH 3 CLENBUTEROL Cl CH 3 HOCH 2 OH CH 3 HO CH - CH 2 - NH - C - CH 3 SALBUTAMOL CH 3 HOCH 2 OH HO CH - CH 2 - NH - (CH 2 ) 6 - O - (CH 2 ) 4 SALM ETEROL HO OH CH 3 CH - CH 2 - NH - CH - CH 2 OH FENOTEROL HO O CH HN OH CH 3 HO CH - CH 2 - NH - CH - CH 2 OCH 3 FORMOTEROL

EFFECT OF FORMOTEROL TREATMENT ON SKELETAL MUSCLE WEIGHTS OF TUMOUR-BEARING RATS 1000 GASTROCNEMIUS ** 400 TIBIALIS 750 ** 300 ** 500 200 ** mg / 100 g IBW 250 0 75 SOLEUS 100 0 100 EDL control formoterol 50 75 50 ** *** 25 25 0 0 control tumour control tumour Busquets et al., 2005

EFFECT OF FORMOTEROL TREATMENT ON GENE EXPRESSION OF PROTEOLYTIC SYSTEMS IN RAT GASTROCNEMIUS ubiquitin 2.4 kb 300 400 ubiquitin 1.2 kb 200 proteasome C8 subunit arbitrary units 200 100 0 400 * control tumour m-calpain 300 200 100 0 150 * control tumour cathepsin B 150 100 50 0 * control *** tumour 300 100 control 200 *** formoterol 100 50 0 control tumour 0 control tumour Busquets et al., 2005

EDL or Soleus Krebs-Henseleit buffer 35ºC

EFFECTS OF BETA-AGONISTS ON IN VITRO PROTEOLYTIC RATE IN ISOLATED EDL MUSCLES nmol tyrosine released / g. 2 hours 300 200 100 0 *** *** control formoterol clenbuterol Busquets et al., 2005

Anticachectic cytokines: IL-15

70 Protein degradation in C2 myotubes % of total protein degradation 60 50 40 30 20 10 * Control IL-15 (20ng/ml) 0 Busquets et al., 2005

Proteolytic rate in isolated EDL rat muscles 140 120 Control IL-15 nmoles tyr/g x h 100 80 60 40 * 20 0 Busquets et al., 2005

EFFECTS OF IL-15 TREATMENT ON SKELETAL MUSCLE FROM TUMOUR-BEARING RATS MUSCLE WEIGHTS (% of control) PROT. DEGRADATION RATE (% / day) mrna EXPRESSION (% of control) 100 75 * ** ** 6 4 * 400 300 ** * ** 50 200 25 2 100 0 GSN SOL TIB 0 CONTROL TUMOUR 0 UB 2.4 UB 1.2 C8 Non-treated Treated Carbó et al., 2000

Using proteolytic inhibitors Proteasome inhibitors Lactacystin Bortezomib (PS-341) MG-132 Calpain inhibitors Calpeptin BN82270

Chymotrypsin-like activity in proteasome 10mg /kg body weight MG-132 Lolezek et al., Biochem. Biophys. Res. Commun. 345:38-42 2006.

Fareed et al., Am. J. Physiol. 290: R1589-97, 2006

Fareed et al., Am. J. Physiol. 290: R1589-97, 2006

Molecular mechanisms potentially involved in muscle wasting Preproteasomal Paraproteasomal Proteasomal Postproteasomal

Cancer Myofibrils Ca ++ /Calpain dependent release of myofilaments Reincorporation Myofilaments Substrate modification Ub/proteasome dependent proteolysis COP9 signalosome CHIP VCP Peptides TPP, aminopeptidases other exopeptidases Amino acids

CACHEXIA TREATMENT ANTIPROTEOLYTIC STRATEGIES Nutrition + Nutraceuticals + Drugs Anabolic +Anticatabolic

Thank you. Danke.