Cardiotoxicity Of The Cancer Therapeutic Agent Recombinant Human Endostatin

Background and objectives:In 1971 Folkman, professor of Harvard Medical School, first proposed the hypothesis that all tumor growth and metastasis are angiogenesis-dependent, and blocking of angiogenesis is one of an effective strategy to prevent the growth of the tumor, which founded the field of angiogenesis research. In recent years, angiogenesis inhibitors, such as bevacizumab, sorafenib, sunitinib, which targeted tumor angiogenesis, achieved good effects. Endostatin (ES), a 20 kDa C-terminal globular domain of the collagenⅩⅧ, as a broad-spectrum angiogenesis inhibitor, which was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line for its ability to inhibit tumor angiogenesis, has been shown to regress tumors 93-97% in mice, then it attracted the attention of the world. Although endostatin has evident activity as anti-angiogenic agents, neither showed substantial benefit in early-phase clinical trials. Regrettably, low activity in the human body was founded, and scientists could not overcome technical problems of the protein renaturation meanwhile the drugs proved expensive to produce and their clinical development was shelved largely for economic considerations. Interestingly, however, a formulation of endostatin has been approved for treating lung cancer in China, suggesting its potential. Chinese scholars Dr. Luo changed the amino acid sequence of Endostatin and resolved the protein renaturation so that efficacy had been significantly enhanced. A new type recombinant human endostatin (Rh-endostatin, endostar), whicn was reaserched and developed independently by our country and was the first endostatin in the world, had gained formal approvalof State Food and Drug Administration (SFDA) as the first category of anticancer drug. Rh-endostatin had gained good effets on advanced lung cancer ever since the clinical application in July 2006.However, inⅠ—Ⅲclinical trials of Rh-endostatin, 30 patients (6.38%) of all 470 advanced NSCLC patients who were subjects in clinical trial had mild/moderate cardiologic adverse reactions of mainly myocardial ischemia within Day2-7 after the administration and posing no dangers to the patient's life. 6.4%o of these cases had more evident but reversible symptoms, which did not influence the administration continuation but could alleviate without any symptomatic treatment. In the patients with previous coronary heart disease and hypertension, rh-endostatin caused the following frequent cardiologic adverse reactions: sinus tachycardia, mild ST-T change, AV conduction blocking, atrial premature beats and rare ventricular premature beats. In our department of oncology, an advanced lung cancer patient, who was treated with rh-endostatin combined with gemcitabine. developed acute left failure on the day 6 of the first 2-week cycle. An electrocardiogram showed sinus tachycardia and premature ventricular contraction, and heart rates were 110-120 times per min, the concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP) in serum (15859ng/L) was beyond normal limits (349ng/L), at last she died. Although the patient experienced hypertension (stage I) for 8 years, her blood pressure was stable and heart function was nomal. The case reminded that caution on cardiotoxicity is therefore warranted on the cancer patients treated with rh-endostatin combined with chemotherapy, especially the patients with previous heart disease. Although past studies of cardiotoxicity have typically focused on anthracyclines or radiotherapy, more recently interest has turned to drugs that target agents. Cardiovascular side effect of molecular targeted drugs was found gradually. Cardiotoxicity of targeted agent was first reported for trastuzumab (Herceptin), the monoclonal antibody that targets the ERBB2 receptor (also known as HER2). In addition, cases of heart failure have been reported after treatment of patients with imatinib, and adverse cardiac effects are mentioned in the prescribing information for dasatinib (Sprycel), sunitinib (Sutent), sorafenib (Nexavar) and bevacizumab (Avastin). In the initial clinical trials it was evident that trastuzumab had significant cardiotoxicity, which manifests as a decrease in left ventricular ejection fraction (LVEF) or as symptomatic CHF. The incidence of cardiac dysfunction ranged from 4% to 7% with trastuzumab alone, to 27% when chemotherapy that included anthracyclines was dministered concurrently. The mechanism of tratumumab-induced cardiac dysfunction is not fully understood, but some results suggest that inhibition of cardiomyocyte ERBB2 signalling by trastuzumab might be a central mechanism of the cardiotoxicity. ErbB2 normally appears to protect against the development of cardiac myocyte apoptosis and dilated cardiomyopathy. Some researcher found that cardiac myocytes transduced with a tyrosine kinase mutant form of c-Abl that was resistant to imatinib were protected from imatinib-induced cell death suggesting that loss of signaling through c-Abl is critical for the toxicity of imatinib in myocytes. Although the mechanism is less clear, left ventricular dysfunction also develops in 15% of individuals receiving the receptor tyrosine kinase inhibitor sunitinib, used to treat people with metastatic renal carcinoma and stomach cancer. Recently, progress has been made in determining basic mechanisms underlying the cardiotoxicity of these drugs. There are two key features to clarify for each agent: first, the target responsible for cardiotoxicity and, second, the signalling pathway or pathways mediating the toxicity. It is clear, however, that cardiotoxicity is not a target agent 'class effect' because it seems to be uncommon with certain other target agents. Therefore, toxicity needs to be determined for each agent on a case-by-case basis.Although in most cases the overall cardiac risk of rh-endostatin does not seem to be excessive, the precise clinical magnitude of the problem is not clear and the potential reversibility of the dysfunction is unknown. It is necessary to monitor the myocardial damage in treatment of rh-endostatin combined with chemotherapy, and it is necessary to reseach the cardiaotoxicity mechanisms of rh-endostatin for further study. All these would be profit for clinical application of rh-endostatin in the future.Methods:1. Clinical data of 11 cancer patients with rh-endostatin combined with chemotherapy were analyzed retrospectively. The symptoms including cardiopalmus, chest distress, dyspnea and changes of electrocardiogram (ECG), myocardium enzymogram and LVEF were observed during treatment. Then, the cardiotoxicity of rh-endostatin was evaluated and markers were discovered to predict the heart damage in early.2. Prospective clinical trial was designed in order to screening comprehensively the early clinical diagnosis markers of cardotoxicity. Thirty cancer patients were divided into two groups, Group A (n=18) received rh-endostatin combined with chemotherapy and control group (Group B, n=12) received chemotherapy only, that all these chemotherapeutic agents were less cardiotoxicity. ECG,LVEF and CK-MB, cTnT levels in serum and NT-proBNP levels in plasma, were detected in all the patients before and after therapy in each cycle (do and dis) . The observation lasted two treatment cycles.3. Twenty four healthy rats were randomly devided into four groups, with six each group. Low dose, moderate dose and high dose group received rh-endostatin at a dose of 3mg/kg/d, 6mg/kg/d and 12mg/kg/d by ntraperitoneal injection respectly while control group received the same volume normal sodium alone. Half of rats in each group were killed 24 hour later after four weeks' administration, and the rest were killed 24 hour later after the whole eight weeks' administration. Pathomorphism changes in rat myocardial tissue stained with Hematoxylin and eosin were observed by microscope, and ultrastrucural changes in rat myocardial tissue were observed bytransmission electron microscope;cariomyocytes apoptosis were detected with TdT-mediated dUTP nick end labeling (TUNEL) assay; microvessel density (MVD)in myocardial tissue of was measured by immunohistochemistry marking endothelial cell with CD34.4. We cultured cardiac cell line H9c2 to study the cardiotoxicity in vitro. 100μg/ml, 200μg/ml, 400μg/ml rh-endostatin treated H9c2 cells respectly while control group untreated with rh-endostatin. The ratio of early apoptosis H9c2 cells were quantified with flow cytometry and DNA fragmentation of apoptosis cells detected by DNA ladderinging. Ultrastrucural changes in H9c2 cells were observed bytransmission electron microscope.5. We cultured cardiac cell line H9c2 to study the molecular mechanism of cardiotoxicity in vitro. 100μg/ml, 200μg/ml, 400μg/ml rh-endostatin treated H9c2 cells respectly while control group untreated with rh-endostatin. Mitochondrial membrane potential was analysised using laser scanning confocal microscope; Cytochrome C release was detected using immunocytochemistry; Changes on the ratio of ADP to ATP were measured using chemiluminescence; The protein level of phosphor-JNK and phosphor-eIf2αwas detected with western boltting; The expression and localization of nucleolin were detected by immunofluorescence. Results:1. We found the case number of abnormal electrocardiogram increased significantly at the end of treatment compared with on pretherapy (x~2 = 6.471, P=0.035) and LVEF had not significant change on posttherapy (t=2.083, P=0.075). Among myocardium enzymogram. CK-MB was higher on protherapy than on pretherapy and there was a significant difference (t=2.530, P=0.030) while the others myocardium enzyme had no significant change (P > 0.05).2. We found that in prospective trial, the rate of abnormal ECG cases was higher in Group A than in Group B on post-therapy (x~2 = 6.087, P=0.024)and LVEF by colored B Ultrasound had not significant difference between Group A and Group B during and after therapy (P>0.05). The value of CK-MB and cTnT were higher in Group A than in Group B during and after therapy and there was a significant difference (P<0.05). NT-proBNP in group A increased significantly during therapy and decreased sharply in intermission of therapy (P<0.01).3. Upon staining with hematoxylin and eosin stain, the rat cardiomyocyte of vehicle-treated group showed neat arrangement of myocardial fibers, rich adqulis cytoplasm, normal intercellular space, without cellular edema, and phanerous transverse striation, while he rat cardiomyocyte of both four-week and eight-week high dose rh-endostatin-treated group showed disorded arrangement of myocardial fibers, dilatation of intercellular space , damage of cardiomyocyte cytoarchitectonics, even degeneration and necrosis, karyolysis in the local myocardium, and infiltration of neutrophil, lymphomonocyte, and leukomonocyte below epicardium. Myocardial pathomorphism changes of eight-week high dose rh-endostatin-treated group were more serious than that of four-week high dose rh-endostatin-treated group. Myocardial pathomorphism changes in moderate and low dose rh-endostatin-treated groups were not observed. Seen from transmission electron micrographs, the rat cardiomyocyte of vehicle-treated group showed that myocardial myofilament arrange was in order and organelles had no hydropsia and denaturation, while the rat cardiomyocyte of high dose rh-endostatin-treated group showed that sarcoplasmic reticulum was dilated evidently, other abnormalities included slightly swollen cardiomyocyte mitochondria and partially effaced cristae. Damage of ultrastructure in moderate and low dose rh-endostatin-treated groups was not observed. TUNEL assay showed there were more positive cells in cardiac muscle from the hearts of rh-endostatin-treated rats than vehicle-treated rats, and there was statistical difference between two groups(P<0.05), while there were few positive cells in cardiac muscle from the hearts of moderate and low dose rh-endostatin-treated groups. It demonstrates that inducing cadiocyte apoptosis is one of likely ways for rh-endostatin to result in cardiotoxicity. In addition, compared with control group, microvascular density (MVD), detected by immunohistochemistry of CD34, decreased in high dose rh-endostatin-treated groups (P<0.05). It indicates myocardial ischemia followed by the decrease of MVD is another likely reason to explain cardiotoxicity of rh-endorstatin.4. We found that rh-endostatin induced dose-dependent apoptosis of cardiomyocytes cell line H9c2, as determined by staining with the potential-sensitive fluorochromes PI and Annexin V. Transmission electron micrographs of drug-treated H9c2 cells showed results similar to those seen in studies of rats on endostatin, including swollen cardiomyocyte mitochondria, partially effaced cristae and dilated endocytoplasmic reticulum. Aprat from these, karyopycnosis and karyorrhexis were seen clearly, and what is more important is that apoptotic body, small pieces that apoptotic cells shrink in size and break into, was found. There was discernible increase in DNA laddering in the H9c2 cells treated wih 400μg/ml rh-endostatin for 30h.5. We found that rh-endostatin produced dose-dependent collapse of the mitochondrial membrane potential, as determined by staining with the potential-sensitive fluorochromes JC-1. Loss of membrane potential was followed by pronounced release of cytochrome c into the cytosol. Compared with control group, there was also a marked rise on the ratio of ADP to ATP (P<0.05). The expression level of protein of p-JNK and p-eIf2αin the H9c2 cells treated wih 400μg/ml rh-endostatin for both 14h and 28h increased whilel low expression level in control group (P<0.01). Nucleolin expressed on the membrane of normal H9c2 cells using indirect immunofluorescence, and after treating with 400μg/ml rh-endostatin 2h, the expression of nucleolin increased on the membrane of H9c2 cells and nucleolin entered into kytoplasm.Conclusion:1. Recombinant human endostatin combined with chemotherapy had mild cardiac adverse reactions and heart damage, but patients" cardiac function had not changed significantly. It is considered CK-MB and ECG can be used as the markers of early detection on cardotoxicity of rh-endostatin and the cardotoxicity of rh-endostatin should be vigilant against in the course of recombinant human endostatin combined with chemotherapy.2. Rh-endostatin has mild cardiotoxicity. ECG,CK-MB, cTnT and NT-proBNP can predict the cardiotoxicity of rh-endostatin as markers. We suggest the three myocardial biochemical markers had better to be detected together, in order to improve the sensitivity and accuracy of prediction on the rh-endostatin-induced heart damage.3. High dose rh-endostatin would resulte in obviously rat myocardium pathological changes and organelles damage of ultrastructure. TUNEL positive cells in rat cardiomyocytes of high dose group increased obviously. These finding demonstrated that cardiomyocytes apoptosis mechanism involved in the pathological process of cardiac toxicity of rh-endostatin.4. In vitro high dose rh-endostatin treatment of H9c2 cells caused apoptosis as demonstrated by three methods, annexinv/PI isothiocyanate staining, DNA ladderinging, and appearance of apoptotic bodies and organelles damage of ultrastructure.5. Molecular mechanisms of endostrar-induced cardiotoxicity result from activation of the mitochondrial and the ER stress apoptosis pathway, causing cardiomyocytes apoptosis. The finding demonstrates that nucleolin express on the membrane of H9c2 cells, and may be a function membrane receptor for rh-endostatin cardiotoxicity.