Study Of The Cardiotoxicity Of Anti-tumor Targeting Drug ENDOSTAR In Zebrafish

Objective ENDOSTAR(Recombinant Human Endostatin Injection,rh-ES)is the first antitumor targeting drug of the world.It belongs to the category of the anti-angiogenic agents.Scientist carried out protein engineering on endogenous endostatin to increase its protein activity,stability and medicinal properties.Rh-ES has become the first-line clinical treatment of non small cell lung cancer since 2006.Its mechanism is to inhibit tumor growth by restraining the formation of angiogenesis within the tumor.In clinical practice,the most common adverse effect of rh-ES is the reversible cardiotoxicity,such as abnormal cardiac electrical conduction and pathological abnormalities in the myocardial tissue.However irreversible cardiac toxicity can happens,there was a case of a patient diagnosed of advanced non-small cell lung cancer,died from acute left-heart failure on the seventh day of rh-ES treatment.Thus the incidence and severity of rh-ES induced cardiac toxicity might be more serious than what we have thought.The cardiactoxicity of rh-ES still needs further evaluation.In this study,we use zebrafish embryos and adult zebrafish as an experimental model to evaluate the cardiotoxicity of rh-ES and explore the pathological mechanisms of the cardiotoxicity.The contents and results are shown as follows:1.The results in 72 hpf zebrafish embryos experiment have shown:after 24 hours exposure,the embryonic mortality of different experimental groups increased in a dose relationship.LC50 is 2.15%.After 72 hours exposure,there are different degrees of yolk sac absorption delaying,body section curving,pericardial edema,heart cyclization failure in different experimental group.Where in,the incidence rate of body section carving and pericardial edema in 0.1%and 1%concentration groups had significant difference compared to the control group;After exposure,the pericardial area and SV-BA distance of 0.1%,1%concentration experimental group has significantly increased in a dose and time dependent manner compare with the control group;The heart rate of zebrafish embryos in the experimental group of 0.1%and 1%concentrations decreased significantly compared with the control group;Through the frozen section and HE staining,we found zebrafish embryonic heart of 1%concentration experimental group:atrial ventricular position change,The area of the lumen of the atrium is significantly reduced,and the thin and small tubular heart is formed.The wall of the heart becomes thinner and the number of cells is reduced.The whole embryo acridine orange staining indicated myocardial apoptosis in the experimental group of 0.1%and 1%concentration.2.The results in adult zebrafish embryos experiment have shown:there was a significant increase in the mortality of the experimental group compared with the control group in seven times and ten times injection regimen.Through the frozen section and HE staining,we found:the shape of the cardiac ventricle has subtle changes in the rh-ES injected fishes;myocardial fibers were disarranged in these fishes;the whole nucleus became fuzzy;intercellular space has enlarged;vacuoles within the myocardial tissues associated with less myocardial cell layers and thinner myocardial wall.The most obvious change is the vacuoles in the cardiac muscle tissue increased significantly;Experiments shows that:in the regimen of seven times and ten times rh-ES injection,the heart weight/body weight ratio in the experimental group has no significant difference compared to that of the control group.The results suggested that rh-ES may not lead to the occurrence of small heart in zebrafish;By calculating the cardiac muscle cell density in the pathological sections of zebrafish heart we found:there was a significant reduction in myocardial cell density in the experimental group compared with the control group.TUNEL/DAPI double fluorescence staining results have shown:There was obvious cardiomyocyte apoptosis of the experimental group in the 10 times injection regimen.3.The experimental results of fluorescence quantitative PCR in adult zebrafish have shown:Compared with the control group,the expression level of Bcl-2 gene in the experimental group was significantly lower than that in the control group.The Bcl-2 gene plays a critical role in the regulation of apoptosis,down-regulation of Bcl-2 gene may initiate apoptosis,increase the level of apoptosis,and further lead to Cardiac dysfunction and cardiac toxicity in zebrafish.The expression level of Beclinl gene in the experimental group was significantly higher than that in the control group;The expression level of Beclinl gene is response to the level of cell autophagy,zebrafish may protect cells by up regulation autophagy to antagonist apoptosis caused by rh-ES;The expression level of?-catenin gene in the experimental group was significantly decreased,?-catenin gene is the marker gene in the Wnt signaling pathway,rh-ES may affect Wnt signal pathway by down regulate the expression of ?-catenin gene.