Effect And Mechanism Of Calpain Inhibitors On Skeletal Muscle Wasting In Cancer Cachectic Mice

Background: Cancer cachexia,which occurs in patients with advanced malignant tumor,is always associated with metabolic disorders of nutrients,especially progressive loss of skeletal muscle proteins.Although mounting evidence indicates that the ubiquitin-proteasome pathway plays a prominent role in the process of muscle wasting,the proteasome cannot disassemble myofibrillar proteins until they have been released from the myofibrils as myofilaments by the calpain system.However,it has been clarified that calpain inhibitors can prevent calpain-mediated protein degradation and cell apoptosis.With the help of an animal model of cancer-induced cachexia and in vitro cell models,we intended to explore:(1)Effect of different doses of calpain inhibitors on skeletal muscle wasting in cancer cachectic mice;(2)Combined effect of different kinds of calpain inhibitors on skeletal muscle wasting in cancer cachectic mice;(3)Mechanism of calpain inhibitors ameliorating skeletal muscle wasting;(4)Effect and mechanism of calpain inhibitors on cultured C2C12 myotubes in tumor microenvironment.Part1.Effect of different doses of calpain inhibitors on skeletal muscle wasting in cancer cachectic miceObjective: This study was to explore the effect of different doses of calpain inhibitors on skeletal muscle wasting in cancer cachectic mice.Methods: Animal experiment: sixty-six male BALB/c mice(body weight 20-24 g)were randomly divided into eleven groups: Group A(healthy controls),Group B(cachexia controls),Group C-E(low,medium,high doses of calpastatin),Group F-H(low,medium,high doses of calpain inhibitor IV),Group I-K(low,medium,high doses of calpeptin).Physical activity,fur condition,body weight,food intake,and tumor growth were recorded daily.After administration of certain dose of inhibitors,gastrocnemius muscles from each group were collected for further analysis,including the total weight,the amount of easily releasable myofilaments and the calpain activity of skeletal muscle.Results:(1)All the tumor-bearing groups had a significant decrease in gastrocnemius muscle mass as compared to the healthy controls,and the gastrocnemius muscle mass in middle and high dose groups were higher than the cachexia controls,while low dose groups remained unchanged;the ratio of gastrocnemius muscle/tumor-free bodyweight of the cachexia controls and the low dose groups is lower than the healthy controls,while the middle and high dose groups remained unchanged.(2)All the tumor-bearing groups had a significant increase in the amount of easily releasable myofilaments and the calpain activity as compared to the healthy controls,and all treatment groups were lower than the cachexia controls,especially in the middle and high dose groups.Part2.Combined effect of different kinds of calpain inhibitors on skeletal muscle wasting in cancer cachectic miceObjective: This study was to explore the effect of different kinds of calpain inhibitors on skeletal muscle wasting and myocyte apoptosis in cancer cachectic mice,and to observe the effect of these inhibitors on the growth of xenografted tumor,the nutrition index and overall survival time of these mice.Methods: Animal experiment: one hundred and sixty-two male BALB/c mice(body weight 20-24 g)were randomly divided into nine groups: Group A(positive controls),Group B(cachexia controls),Group C(calpastatin),Group D(calpain inhibitor IV),Group E(calpeptin),Group F(calpastatin+calpain inhibitor IV),Group G(calpastatin+calpeptin),Group H(calpain inhibitor IV+calpeptin),Group I(calpastatin+calpain inhibitor IV+calpeptin).The body weight,food intake,and tumor growth of all the mice were recorded daily.After administration of certain kinds of inhibitors,ten mice in each group were sacrificed,and their blood,tumors,and gastrocnemius muscles were collected for further analysis,including: Gastrocnemius muscle mass,tumor weight and tumor-free bodyweight;Morphological changes of skeletal muscle observed under light microscope;Serum biochemical markers of nutritional status;Amount of easily releasable myofilaments,calpain activity and 26 S proteasome activity of skeletal muscle;Protein abundance of actin,myosin,Mu RF-1,atrogin-1,μ-calpain,m-calpain and calpastatin in different groups determined using western blot analysis;m RNA levels of Mu RF-1 and atrogin-1 determined using q RT-PCR;Apoptotic index in gastrocnemius muscle detected using TUNEL assay;Expression of apoptosis-related protein,such as cleaved caspase-3,BAX,BCL-2,PARP,Bid and cytochrome C,determined using western blot analysis;Expression of Ki67 antigen in different tumor tissue detected using immunohistochemisty.In addition,to evaluate the effect of the different treatments,we also recorded the survival timeof eight mice remained in each group.Results:(1)There were significant differences in final bodyweight,gastrocnemius muscle mass and cross-section area of gastrocnemius muscle between the cachexia controls and the healthy controls,while all the treatment groups can improve these indicators,especially the combined treatment groups.(2)There were significant differences in the amount of easily releasable myofilaments,the protein expression of actin,myosin,atrogin-1 and Mu RF-1,the m RNA levels of atrogin-1 and Mu RF-1 and the 26 S proteasome activity between the cachexia controls and the healthy controls,and all the treatment groups can improve these indicators,while the combined treatment better than the monotherapy.(3)Expression of both kinds of calpains in gastrocnemius muscle seemed unchanged between the cachexia controls and the healthy controls,while the IV and the Calpeptin group downregulated their expression,and the CAST group upregulated;Expression of calpastatin in the cachexia controls was lower than the healthy controls,and the level of protein in each treatment group depending on whether administrated with calpastatin peptide or not;Compared with the healthy controls,the calpain activity of the cachexia controls was increased,while it could be downgraded by all kinds of inhibitors,especially combination of two or three kinds of the inhibitors;Calpain activity was positively correlated with the ratio of calpain/calpastatin to some extent.(4)Protein expressions of cleaved caspase-3,BAX and apoptotic index in gastrocnemius muscle of the cachexia controls was higher than the healthy controls,while the IV,the Calpeptin and the IV+Calpeptin groups had lower protein expressions and apoptotic index than the cachexia controls;but the groups involves calpastatin increased both of them;Protein expression trend of nuclear cleaved PARP,cytosolic cytochrome C and mitochondrial bid were in accord with the apoptosis-related protein above.(5)Mice from the cachexia group had lower levels of total protein,albumin,and glucose,and higher levels of triglycerides compared with the healthy controls;the administration of different calpain inhibitors could reverse these metabolic changes,especially the combination treatment.(6)Calpain inhibitors could reduce the expression of Ki67 antigen.(7)The tumor-bearing mice could benefit from calpain inhibition according to the data from Kaplan-Meier survival curves.Part 3.Mechanism of calpain inhibitors ameliorating skeletal muscle wastingObjective: This study was to explore the effect of different kinds of calpain inhibitors on serum inflammatory cytokines,oxidative stress indicators and metabolism-related and apoptosis-related signaling pathways of skeletal muscle.Methods: Animal experiment: To explore the mechanism of calpain inhibitors ameliorating skeletal muscle wasting,we did further analysis with the serum and muscle samples above,including: Serum TNF-α,IL-6,IL-1β,CRP and IGF-1 levels determined using ELISA;Content of MDA,activities of SOD and GSH-PX in skeletal muscle;Total and phosphorylation of Akt,m TOR,GSK-3β,Fox O3 a,ERK,p38 and JNK in the skeletal muscle of different groups detected by Western blot analysis;Changes in nuclear and cytosolic distribution of NFκB p65 in different groups.Results:(1)There were significant differences in serum TNF-α,IL-6,IL-1β,CRP levels between the cachexia controls and the healthy controls,while all the treatment groups could improve these indicators,especially the combined treatment groups;The tumor-bearing mice had a decreased level of serum IGF-1 compared with the healthy controls,but the inhibitor treatment had little effect on this cytokine.(2)MDA content of skeletal muscle in tumor-bearing mice was significant higher than the healthy controls,but the activities of SOD and GSH-PX were lower;There was significantdifference in the protein level of i NOS between the cachexia controls and the healthy controls,while the treatment groups could downregulate it,especially the combined treatment groups.(3)Protein level of p-IRS-1 in tumor-bearing mice was significant lower than the healthy controls,and protein levels of p-Akt,p-FOXO3 a,p-GSK3β and p-m TOR were much higher,however,the inhibitor treatment had little effect on these phosphorylated proteins.(4).Higher levels of skeletal muscle cell p-IκBα and nuclear p65 were observed in the cachexia controls than the healthy controls,while the treatment groups could downregulate it,especially the combined treatment groups.(5)Higher level of skeletal muscle p-p38 was found in the cachexia controls than the healthy controls,while all the treatment groups could downregulate it,especially the combined treatment groups;There was a higher level of skeletal muscle p-ERK in the cachexia controls compared to the healthy controls,but the p-JNK level seemed unchanged,but to our surprise,the groups involves calpastatin increased both of them.Part4.Effect and mechanism of calpain inhibitors on cultured C2C12 myotubes in tumor microenvironment.Objective: This study was to observe the changes of atrophy-related and apoptosis-related proteins and signaling pathway components of the C2C12 myotubes after incubated with different calpain inhibitors or different signal pathway inhibitors,and to explore their possible mechanisms.Methods: Cell culture experiment: We induced cultured C2C12 myoblasts to differentiate into mature myotubes.To ensure an effective myogenic differentiation,we also detected the m RNA levels of muscle-associated gene by q RT-PCR.Furthermore,C2C12 myotubes were cultured in tumor cell-derived conditioned media,and their changes of morphology,protein expression of m-calpain,Mu RF-1,atrogin-1 and cleaved caspase-3 and calpain activity were monitored at different time points.Finally,together with the detection above,inhibitors of different signal pathways,such as PD98059,SB203580,PDTC and SP600125,were added to the culture medium,in order to clarify the mechanism of calpain inhibitors alleviating cultured C2C12 myotubes atrophy.Results:(1)After induction of differentiation,m RNA expressions of desmin,Myo D and myogenin in C2C12 myotubes were higher than myoblast,but Pax7,MRF4 unchanged;As brighter green fluorescence,myotubes in negetive controls expressed more myosin Ⅱb protein than positive controls,while the different intervention groups could alleviate the effect.(2)After incubated in TCCM for 24 hours,expressions of m-calpain,calpastatin and calpain activity in the intervention groups showed significant differences compare to the positive controls,and last until 48h;After incubated in TCCM for 48 hours,expressions of Mu RF-1,atrogin-1 in the intervention groups showed significant differences compare to the positive controls,and last until 72h;After incubated in TCCM for 48 hours,expressions of cleaved caspase-3 in the IV and the calpeptin groups began to decrease compare to the positive controls,and after 72 hour,expressions of cleaved caspase-3 in the CAST group significantly increased,while the IV and the calpeptin groups were still at a low level.(3)After incubated in TCCM for 48 hours,expressions of nuclear p65,p-ERK,p-p38,p-C/EBPβ,Mu RF-1 and atrogin-1 in Group C were higher than Group A,while Calpain inhibitor IV could downregulate all the above,PDTC could reduce nuclear p65,Mu RF-1,PD98059 could alleviate p-ERK,atrogin-1,and SB203580 could downregulate p-p38、p-C/EBPβ and atrogin-1.(4)After incubated in TCCM for 72 hours,both TCCM and calpastatin could upregulate p-JNK,c-Fos,c-Jun levels,especially when administered together,however,pretreatment with SP600125 reduced all of them in Group E and G;Both TCCM and calpastatin could upregulate expressions of cleaved caspase-3 and BAX,especially when administered together,however,pretreatment with SP600125 downregulated their expressions in Group E,and pretreatment with both SP600125 and PD98059 had a better effect.Conclusions:(1)The mechanism of muscle wasting in a mouse model of cancer cachexia might involve activated calpain,excessive release of myofilaments.The treatment with different calpain inhibitors can alleviate cachexia-associated symptomsin a dose-dependent manner.(2)Calpain inhibitors reduce calpain activity and regulate degradation of myofibrils by changing the ratio of calpain to calpastatinactivated calpains enhance the expressions of Mu RF-1,atrogin-1 in skeletal muscle,upregulate 26 S proteasome activity,and further increase the degradation of muscle protein,while calpain inhibitors can alleviate them,especially when administrated together;Skeletal muscle apoptosis induced by activated calpains might be through mitochondrial apoptotic pathway;irreversible inhibitors derived from chemical synthesis can alleviate skeletal muscle apoptosis,while excessive calpastatin(an endogenous calpain inhibitor)promotes apoptosis;Calpain inhibitor can improve concentrations of serum nutritional markers in tumor-bearing mice and prolong theirsurvival time.(3)Calpain inhibitors have little effect on oxidative stress status or IGF-1/PI3K/Akt pathway in skeletal muscle of tumor-bearing mice,but they can reduce serum proinflammatory cytokines to a certain extent;NFκB pathway,MAPK/ERK pathway and p38 MAPK pathway activated in skeletal muscle of tumor-bearing mice,which seem positive correlated with the expression of Mu RF-1,atrogin-1,might contribute to muscle wasting in cancer cachexia,however,calpain inhibitors can downregulate their activity.(4)NFκB pathway in C2C12 myotubes is activated in tumor microenvironment,enhancing expression of Mu RF-1,while calpain inhibitor IV can prevent IκBα from being dissociated from NFκB p65/p50 dimer,further reducing expression of Mu RF-1 and ameliorating muscle wasting;p38MAPK pathway in C2C12 myotubes is also activated in tumor microenvironment,upregulating transcription factor C/EBPβ and enhancing expression of atrogin-1,while calpain inhibitor IV can block this pathway,further reducing expression of atrogin-1 and ameliorating muscle wasting;Apoptosis promoted by excessive calpastatin in tumor microenvironment may be mediated through JNK signaling pathway,while MAPK/ERK pathway playing a synergistic role in the process.