Effect And Mechanism Of Ghrelin On Skeletal Muscle Wasting Of Cancer Cachexia

Objective : Cancer cachexia is a multifactorial metabolic syndrome mainly characterized by progressive muscle wasting.It severely reduces the quality of life and worsens the prognosis of cancer patients.Muscle atrophy of cancer cachexia is induced by multiple mechanisms,including an imbalance between muscle protein synthesis and degradation,myocyte damage,and muscle regeneration dysfunction,and no effective therapies currently exist.Ghrelin is a multifunctional small peptide,both acylated ghrelin(AG)and unacylated ghrelin(UnAG)can inhibit muscle wasting caused by multiple diseases.But few studies showed that AG and UnAG could inhibit cancer cachectic muscle wasting,and the mechanism remains unknow.The purpose of this study was to verify whether ghrelin could protect cocultured myotubes against atrophy,protect cocultured myoblasts against apoptosis,and inhibit skeletal muscle wasting in cancer cachectic mice,and the possible mechanisms involved.Our study extends the understanding of therapeutic effect and mechanisms of ghrelin on cancer cachectic muscle atrophy.At the same time,this study provides more evidences to suggest its therapeutic potential against cancer cachexia.Methods:Part 1: C2C12 myoblasts were induced to differentiate to myotubes.Cocultured myotubes with CT26 colon carcinoma cells for 6,12,24 and 48 hours to observe the metabolic changes of muscle protein in myotubes.Treated myotubes and CT26 cells separately with different concentrations of AG or UnAG to observe whether AG or UnAG affected the activity and proliferation of these cells.Treated cocultured myotubes with different concentrations of AG or UnAG for 24 hours to observe the metabolic changes of muscle protein in myotubes.Treated myotubes with AG or UnAG to observe whether AG or UnAG affected the expression and activity of factors regulating muscle protein metabolism in myotubes,and whether AG or UnAG affected the concentrations of inflammatory factors and myostatin in culture medium.Part 2: Cocultured myoblasts with CT26 cells for 6,12,24 and 48 hours to observe whether coculture induced myoblasts apoptosis.Treated cocultured myoblasts with different concentrations of AG or UnAG for 24 hours to observe whether AG or UnAG affected the apoptosis of cocultured myoblasts.Treated myoblasts with AG or UnAG to observe whether AG or UnAG protected the mitochondrial function,and whether AG or UnAG affected the expression and activity of factors that mediated or regulated cell apoptosis in myoblasts.Moreover,we treated myoblasts together with JNK signaling pathway inhibitor SP600125 and Akt signaling pathway inhibitor LY294002 to ensure that these signaling pathways were actually involved.Part 3: CT26 cells were injected subcutaneously into the dorsal side of male BALB/c mice to develop cancer cachetic mice model.Treated these mice with AG or UnAG to observe whether AG or UnAG protected muscle against atrophy and affected the concentrations of serum nutritional markers and serum inflammatory factors,also we detected whether AG or UnAG affected the expression and activity of factors that regulated muscle protein metabolism in skeletal muscle.Results:Part 1:(1)Coculture induced myotubes atrophy and decreased the MHC level in myotubes,these changes were partly reversed by AG/UnAG administration.(2)AG/UnAG decreased the activities of calpain,NF-?B and FoxO3 a,increased the activity of Akt,reduced the expressions of atrogin-1 and MuRF1,and down-regulated the autophagy level in cocultured myotubes.(3)AG/UnAG decreased the expressions and secretion of TNF-? and myostatin in cocultured myotubes,which led to the decreased concentrations of TNF-? and myostatin in culture medium.Part 2:(1)Coculture induced the loss of mitochondrial membrane potential,which led to the release of toxic proteins from the mitochondria into cytosol,activated caspase-3 and PARP,and caused myoblasts apoptosis.AG/UnAG administration could partly reverse these changes.(2)Coculture increased JNK activity,decreased Akt activity,and raised tBid protein level in myoblast cytoplasm,it also increased the Bax protein level and decreased the Bcl-2 protein level in mitochondrial,and AG/UnAG could partly inhibited these changes.Part 3:(1)AG/UnAG administration increased skeletal muscle mass,epididymal fat mass,and muscle fiber cross-sectional area in tumour-bearing mice.(2)AG/UnAG decreased the calpain activity,the myofilament level and the expression of atrogin-1 in skeletal muscle of tumour-bearing mice.(3)AG/UnAG increased Akt activity and decreased the expression and avtivity of FoxO3 a in skeletal muscle of tumour-bearing mice.(4)Compared with normal mice,tumour-bearing mice has lower serum levels of total protein,albumin and glucose,and higher serum level of triglycerides,TNF-? and IL-1?,and these changes could partly be reversed by AG/UnAG administration.Conclusions:(1)The cell coculture model used in part 1 could induce the myotubes atrophy and abnormal muscle protein metabolism,this co-culture model can,at least in part,simulate the myocyte environment in cancer cachexia in vitro.(2)The myotubes and CT26 cells in this coculture model could interact with each other via secreted factors.These factors could affect muscle protein metabolism via multiple mechanisms and induced myotubes atrophy.AG/UnAG administration could act on these mechanisms via inhibiting the activity of calpain,thus prevented cocultured myotubes atrophy by increasing muscle protein synthesis and reducing muscle protein degradation,indicated that both AG and UnAG have therapeutic potential against cancer cachectic muscle protein metabolism disregulation.(3)The cell coculture model used in part 2 could induce the myoblasts apoptosis,this co-culture model can,at least in part,simulate the myoblast environment in cancer cachexia in vitro.(4)Coculture could increased JNK activity and decreased Akt activity to act on Bcl 2 protein family members,which led to the activation of mitochondrial apoptosis pathway and induced cocultured myoblasts apoptosis.AG/UnAG administration could prevent these changes and protected cocultured myoblasts against apoptosis,indicated that both AG and UnAG have therapeutic potential against cancer cachectic muscle regeneration dysfunction.(5)AG/UnAG administration could decrease the muscle calpain activity to reduce myofibril resolvent in tumour-bearing mice.It also could increase Akt activity to reduce FoxO3 a activity via decreasing the muscle calpain activity,thus down-regulate the expression of atrogin-1,which inhibited muscle protein degradation.AG/UnAG administration could also improve nutrient metabolism and decrease serum level of proinflammatory factors in tumour-bearing mice.Both AG and UnAG could treat cancer cachexia through multiple mechanisms,which might be a new therapy for cancer cachexia.