Protective Effects Of PKC Inhibitor Chelerythrine On Cardiotoxicity Induced By Sunitinib

Sunitinib(SUN)is a small molecule multi-targeted tyrosine kinase inhibitor(TKI).TKIs are mainly used for anti-cancer therapy since overactivation of tyrosine kinases is involved in the initiation and progression of many cancers.These agents have led to remarkable advances in the treatment of many cancers which previously had few therapeutic options.Development of TKI agents has become the hot spot in the field of pharmaceutical research and new drug development.Despite these advances,it is increasingly recognized that many TKIs have unintended cardiotoxicity.The clinical manifestations of cardiotoxicity reported with TKIs range from asymptomatic ECG changes through decrease of left ventricular ejection fraction to severe cardiac failure.Therefore,TKIs-induced cardiotoxicity is a key concern in determining the risk/benefit of this novel class of antineoplastic agents.At present,the molecular mechanism underlying the cardiotoxicity produced by TKIs is not well understood.Increasing evidence shows that cardiotoxicity may result from the inhibition of multiple tyrosine kinases in cardiomyocytes and then inhibiting signaling pathway mediated by these kinases.Cardiotoxicity seems continue to be unavoidable instances because of the similar prosurvival roles the kinases play in both cancer cells and cardiomyocytes.Based on the fact that overactivation of distinct subtypes of protein kinase C is involved in heart failure,our previous study has tried to test the effect of PKC isoenzyme inhibition on cardiotoxicity induced by sunitinib and imatinib in nonatal rat ventrcular myocytes.We have found that selective inhibition of novel PKC including PKCδ and ε isoenzymes,but not conventional PKC isoforms could antagonize cardiotoxicity.It suggests that over activation of nPKC signaling pathway may mediate cardiotoxicity induced by Sunitinib and Imatinib.Inhibition of nPKC may play the protective effects against cardiotoxicity.To test our hypothesis,this study was further designed to determine the effects of n PKC inhibitor chelerythrine(CHE)on cardiotoxicity induced by sunitinib(SUN)in vivo.We will expect to provide experimental evidence for exploring the specific mechanisms underlying the cardiotoxicity and finding the specific target for toxicity prevention and treatment.Objective: To observe the effects of different dose of PKC inhibitor CHE on cardiotoxicity induced by SUN in mice.By detecting the expression of PKCs in myocardium and try to understand the molecular mechanisms of myocardial protection by CHE.Methods: A total of 50 healthy adult male mice were randomly divided into five groups of five animals each.Control group: orally water(0.1 ml/10g)and intraperitoneal injection of saline(0.1 ml/10 g);SUN group: orally SUN(200 mg/kg/d)and intraperitoneal injection of saline(0.1 ml/10 g);SUN+CHE(L)group: orally SUN(200 mg/kg/d)and intraperitoneal injection of CHE(0.375 mg/kg/d);SUN+CHE(H)group: orally SUN(200 mg/kg/d)and intraperitoneal injection of CHE(0.75 mg/kg/d);SUN+OLM group: orally SUN(200 mg/kg/d)and orally OLM(conventional drug for heart failure treating)(3 mg/kg/d).All mice were treated for 3 weeks.On day 21,animals blood samples was collected and serum were obtained by centrifuging plasma at 3000 g for10 min.Brain natriuretic peptide(BNP)and cardiac troponin I(cTnI)levels in serum were determined by using commercial kits.The heart was excised and mitochondria from ventricular myocardium were extracted to measure the activity of mitochondrial complexes IV and V.Some heart tissues were taken to make the transmission electron microscopy slices and observe the ultrastructural changes.Part of left ventricular tissue samples were fixed for in10% formalin-saline and followed by paraffin embedding and were made H&E staining for microscopic observations.Myocardial tissue proteins were extracted to detect the expression of p-PKC ε,p-PKC δ and nPKC α.All the data were analyzed using OriginPro7.5 and were presented as means ± SEM.The statistical significance of the differences between groups was evaluated using one-way ANOVA.And differences with P < 0.05 were considered statistically significantResults:1 Effects of CHE on cardiotoxicity induced by Sunitinib in mice in vivo.(1)Weight indicators:None of the drugs caused significant changes in heart: body weight ratio or lung: body weight ratio.(2)Serum biomarkers: Compared with control,SUN significantly increased the concentration of BNP(P < 0.05),and meanwhile decreased cTnI level(P < 0.01)in serum.Co-administration of different dose of CHE or OLM significantly decreased the elevation of BNP caused by SUN(P < 0.05)and also abolished the reduction of cTnI(P < 0.01).(3)Mitochondrial function indicators: The activity of mitochondrial complexes IV and V was significantly reduced in SUN group compared with control group(P < 0.01).It was significantly increased(P < 0.01)after administered simultaneously different dose of CHE and OLM,even more than the control level in SUN treated animals.(4)Ultra-structure changes: Transmission electron microscopy showed that SUN induced the fusion of mitochondrial ridges and membrane,cytoplasm mild swelling.After administered simultaneously different dose of CHE and OLM,structure of mitochondrial was improved in different degree.High dose of CHE almost reverted the mitochondrial structure to control level.H&E stainings suggested that SUN caused interstitial congestion and nucleus slightly enlarging.After administered simultaneously different dose of CHE and OLM,interstitial congestion significantly alleviated.These results suggested that CHE could prevent myocardial from toxicity induced by SUN in mice and high dose of CHE is more powerful in improvement of structure of mitochondrial.2 The changes of expressions in p-PKC ε,p-PKC δ and p-PKC α.Posphorylation of PKCs representatives the activation of PKC.By using Western blot the expression of phosphorylated PKCs(p-PKCs)was detected to.The results showed that expression of p-PKC ε was significantly increased in SUN group compared with control group(P < 0.05)and the other two p-PKC isoforms remained no significant change.After administered simultaneously low dose of CHE and OLM,the over-expressed p-PKC ε has not been significantly inhibited,but the high dose of CHE has significantly inhibited the over-expressed p-PKC ε(P < 0.05).Neither CHE nor OLM has changed the expression of p-PKC δ or p-PKC α significantly.These results suggest that activation of PKC ε but not PKC δ or PKC α in heart could be a mechanism by which SUN produces cardiotoxicity.Selectively inhibition of PKC ε could antagonize cardiotoxicity induced by SUN.Conclusions:1 CHE can effectively prevent cardiotoxicity induced by SUN and this cardiopretection showed a dose-dependent trend.High dose of CHE is more effective.2 Cardiotoxicity induced by SUN is related to activation of PKC ε.Protective effects of CHE are achieved by inhibiting activation of PKC ε.