The Role And Mechanism Of CDC20 In DOX-induced Cardiotoxicity

Background:Doxorubicin(DOX)is an anthracycline antibiotic that can be used to treat breast cancer,ovarian cancer,gastrointestinal malignant tumors,lymphoma and leukemia in adults and children,but it can cause time-and dose-dependent cardiotoxicity,leading to severe cardiomyopathy and congestive heart failure,which limits its clinical application.Cell division cycle 20 homologue(CDC20)is an activator of the anaphase-promoting complex(APC),which belongs to E3 ubiquitin ligase complex,which plays a key role in apoptosis,tumorigenesis and cardiac hypertrophy.However,the role of CDC20 in DOX-induced cardiotoxicity(DIC)is unclear.Objective:To establish a cardiotoxicity model induced by DOX in vivo to study the potential role of CDC20 in the pathogenesis of DIC and to determine whether CDC20interferes with the anti-tumor effect of DOX.To provide potential new strategies and targets for the prevention and treatment of DOX-induced cardiotoxicity in clinic.Methods:1.Caspase 3 activity analysisCaspase 3 activity in H9C2 cells,heart tissues and tumor tissues was detected by caspase 3 activity detection kit.2.Transcriptional analysisH9C2 cells were treated with DOX,and the downstream genes regulated by DOX were analyzed by transcriptomics.3.Gene and protein expression analysisRNA was extracted from heart tissue with Trizol reagent,and c DNA was synthesized after reverse transcription.The expression level of CDC20 gene was detected by real-time PCR.Protein from heart and tumor tissues was extracted by protein lysate,and the expression levels of CDC20,Bax,PCNA andβ-actin were analyzed by western blot.4.Animal model and treatment(1)C57BL/6J mice were injected with AAV9-c TNT-CDC20 via tail vein to overexpression CDC20 specifically in the myocardium.After 4 weeks,DOX(5mg/kg)was injected via tail vein every week for 4 weeks.Cardiac function and pathological changes were evaluated by echocardiography and pathological staining.(2)C57BL/6J mice were injected with AAV9-c TNT-si CDC20 via tail vein to knockdown CDC20 specifically in the myocardium.After 4 weeks,DOX(5mg/kg)was injected via tail vein every week for 4 weeks.Cardiac function and pathological changes were evaluated by echocardiography and pathological staining.(3)C57BL/6J mice were intraperitoneally injected with CDC20 inhibitor Apcin(2.5mg/kg)every day until the mice were sampled,and DOX(5mg/kg)was injected via tail vein every week for 4 weeks.Cardiac function and pathological changes were evaluated by echocardiography and pathological staining.(4)C57BL/6J mice were injected with AAV9-c TNT-CDC20 through the tail vein.After 4 weeks,8×10~5 B16-F10 melanoma cells were subcutaneously inoculated into the right thigh of mice.When the tumor volume was about 300mm~3,DOX was injected into the tail vein once a week with a dose of 5mg/kg each time.Two weeks later,the mice were sampled.5.Cardiac function detectionAfter depilating the chest with depilatory cream,anesthetize the mice,coat a layer of ultrasonic coupling agent on the chest,and use Vevo2100 high-resolution imaging system to perform echocardiogram examination on the mice to obtain echocardiogram spectrum,and measure parameters such as ejection fraction EF%,short axis shortening rate FS%,left ventricular anterior wall(LVAW)and left ventricular posterior wall thickness(LVPW).6.Histopathological analysisAfter the heart was removed,the heart was fixed with 4%paraformaldehyde for 24hours,and the tissue was embedded and cut into 4μm tissue sections for histopathological staining,including hematoxylin-eosin(H&E)staining,wheat germ agglutinin(WGA)staining,Masson staining,TUNEL staining,immunofluorescence staining and immunohistochemical staining.Results:1.DOX can cause myocardial damageThrough in vitro and in vivo experiments,we found that DOX induced a decrease in LVEF%and a decrease in cardio-weight/tibia ratio in mice.DOX induced the increase of vacuoles in H9C2 cells and the enhancement of caspase 3 activity.2.Downstream genes regulated by DOX were analyzed by transcriptomicsIn order to clarify the mechanism of myocardial injury caused by DOX,the downstream genes regulated by DOX were analyzed using transcriptomic techniques using DOX-treated H9C2 cells in this study.It was found that the expression of CDC20decreased significantly after DOX treatment.3.Myocardial specific overexpression of CDC20 alleviates DOX-induced cardiotoxicityHistopathology showed that cardiomyocyte specific overexpression of CDC20could effectively alleviates DOX-induced cardiomyocyte apoptosis,inflammation,fibrosis,and cell atrophy after DOX administration for four weeks.4.Myocardial specific knockdown of CDC20 aggravated DOX-induced cardiotoxicityHistopathology showed that inhibition of CDC20 could aggravate DOX-induced cardiomyocyte apoptosis,inflammation,fibrosis and cell atrophy four weeks after DOX administration.5.Apcin aggravates DOX-induced cardiotoxicityHistopathology showed that Apcin,a CDC20-specific inhibitor,aggravated DOX-induced myocardial apoptosis,inflammation,fibrosis,and cell atrophy after DOX administration for 4 weeks.6.Myocardial specific overexpression of CDC20 can alleviate the cardiotoxicity induced by DOX,and does not affect the tumor growth inhibition effect of DOXHistopathology showed that cardiomyocyte specific overexpression of CDC20could effectively relieve cardiomyocyte apoptosis,inflammation,fibrosis,and cell atrophy in DOX-induced tumor bearing mice after four weeks of DOX administration,and did not affect the inhibitory effect of DOX on tumor.Conclusion:the current results show that overexpression of CDC20 can alleviate myocardial injury induced by DOX,and overexpression of CDC20 does not affect the effect of DOX on inhibiting tumor proliferation and promoting tumor apoptosis.