重组人血管内皮抑素的心脏毒性及其机制研究
|
摘要
背景与目的:
1971年哈佛大学医学院Folkman教授首先提出“肿瘤生长和转移依赖于新生血管生成,阻断血管生成是遏制肿瘤生长的有效策略”的理论,由此开创了肿瘤抗血管生成治疗的研究。近年来,针对肿瘤新生血管这个靶点新的抗癌药物(如贝伐单抗、索拉非尼、舒尼替尼)已经从实验室走向临床应用,并取得了良好的疗效。除此之外,做为广谱内源性血管生长抑制因子—内皮抑素(Endostatin)在小鼠体内可使93～97%肿瘤停止生长,成为全球最引人注目的抗血管生成药物。然而在以后临床应用中却发现Endostatin在人体内活性很低,科学家无法攻克蛋白质复性的技术难关,生产成本过高无法实现Endostatin的大规模生产。我国学者罗永章等经过长期不懈的努力,解决该蛋白复性的技术难关,创造的改变了Endostatin的氨基酸序列,使该蛋白的药用性能和疗效得到了显著提高。就是这个我国自主研发的新型重组人血管内皮抑素(Recombinant human endostatin,rh-Endostatin,Endostar,恩度),于2005年9月获得了国家食品药品监督管理局颁发的生物制品一类新药证书,它是世界上首例血管内皮抑制素抗癌新药。2006年7月恩度在我国上市开始临床使用,对晚期肺癌的治疗显示出较好的疗效。
据恩度治疗说明书描述,Ⅰ～Ⅲ期临床试验安全性评价指出,470例受试晚期非小细胞肺癌患者中有30例(6.38%)出现了心脏不良反应,主要表现为用药初期少数患者出现轻度疲乏、胸闷、心慌等症状,用药后第2～7天内发生心肌缺血,以及心电图出现窦性心动过速、轻度ST-T改变、房室传导阻滞、房性早搏、偶发室性早搏等,研究者认为以上心脏不良反应均为轻.中度,未危及患者生命,其中6.4‰的患者症状较为明显,但均为可逆性,多数不影响治疗进行,不需要对症治疗即可缓解。然而,笔者在恩度临床使用过程中,却发现其心脏毒性发生率和严重程度要高于药物说明书的描述,部分患者因心脏毒性暂停治疗,部分患者需要经对症治疗心脏毒性才能缓解,甚至有一例晚期非小细胞肺癌患者在恩度联合健择治疗的第7天,出现急性左心衰,心电图提示窦性心动过速,心率110～120次/分,偶发室早,N端前脑利尿肽(NT-proBNP)升高到15859pg/ml,最终因心力衰竭医治无效死亡,患者既往有高血压病史8年,用药前心脏功能正常。综合以上,根据我肿瘤治疗中心的用药体会,认为恩度的心脏毒性发生比较广泛,严重性尚待进一步评价。目前临床医生对其心脏毒性的发生警惕性尚不够,关于心脏毒性的早期诊断和预测也应开展研究。
既往研究多集中于蒽环类药物或放疗所致的心脏毒性,然而,随着分子靶向药物临床逐渐广泛,越来越多的分子靶向药物心血管不良反应逐渐被发现并受到研究者重视,有关心脏毒性的研究热点也转向了分子靶向药物,其发生机制也有了初步的研究。研究表明单独使用曲妥珠单抗,有4-7%发生心功能不全,尤其是充血性心衰(GHF)和左室射血分数(LVEF)的减少,发生机制可能与心脏组织上的HER-2受体的分化和存活有关。Nature Medicine一篇文章指出,伊马替尼可能增加化疗患者发生心力衰竭的风险,研究者推测,伊马替尼可能是通过抑制心肌细胞上的ABL激酶,并影响细胞内蛋白质合成,从而导致了对心肌细胞的损伤。舒尼替尼也有心脏毒性的报道。
尽管临床前试验认为恩度心脏毒性不是十分严重,然而使用过程中出现了左心衰严重不良心血管事件。心脏的副作用是恩度最常见并且是最有可能危及生命的毒副反应,因此,有必要对恩度心脏毒性的严重程度再次评价,筛选临床早期诊断和预测的指标。从长远上考虑,对于心脏毒性的了解越深入越利于新药的国际推广应用。研究表明分子靶向药物的心脏毒性机制不同于蒽环类药物,前者心脏毒性多与药物作用的靶点密切相关,并且信号转导通路参与心脏毒性的机制。研究者也认为每种分子靶向药物的心脏毒性机制都不完全相同,故关于恩度发生心脏毒性的机制需要深入研究。
基于上述理论,本研究试图从临床观察和实验研究两方面评价rh-Endostatin的心脏毒性,筛选早期诊断和预测临床指标,并初步探讨其心脏损伤的可能机理。
方法:
1.将既往使用恩度联合化疗的11例晚期肿瘤患者的资料进行回顾性分析,包括治疗过程中有无心慌、胸闷、呼吸困难等临床症状及症状发生时间,治疗前后心电图(ECG),心肌酶谱及LVEF的改变,评价恩度心脏毒性,并初步筛选心脏毒性早期诊断临床指标。
2.为客观评价心脏毒性以及全面筛选心脏毒性早期临床诊断指标,设计了前瞻性临床实验,选择恩度联合化疗18例,单纯化疗12例为对照研究,每周期用药的d_0、d_15检测ECG、LVEF、CK-MB、cTnT和NT-proBNP等多项指标。
3.24只实验大鼠随机分成4组,每组6只。对照组以等体积生理盐水腹腔注射,rh-Endostatin低、中、高三个剂量组,分别给与3mg/kg、6mg/kg、12mg/kg腹腔注射,每日一次,每组各半大鼠(3只)分别给药4周和8周时间,均在末次给药后24h杀死。HE染色各组大鼠心肌组织,光镜下观察病理形态学改变;透射电镜观察大鼠心肌组织超微结构的病理改变;通过TUNEL方法检测大鼠心肌细胞凋亡;通过CD34来标记内皮细胞进行免疫组织化学检测,检测大鼠心肌组织的微血管密度改变。
4.分别以100μg/ml、200μg/ml、400μg/ml三个不同剂量的rh-Endostatin处理H9c2心肌细胞24h、48h,不加药物作为对照组,通过Annexin V/PI双染流式细胞仪检测心肌细胞凋亡率。400μg/ml rh-Endostatin处理H9c2心肌细胞24h,不加药物作为对照组,透射电镜观察心肌细胞超微结构的改变。400μg/mlrh-Endostatin处理H9c2心肌细胞,不加药物作为对照组,琼脂糖凝胶电泳DNA Laddering方法检测细胞凋亡。
5.体外培养H9c2心肌细胞进行细胞凋亡的发生机制研究。以100μg/ml、200μg/ml、400μg/ml rh-Endostatin三个不同剂量和不加药物对照组进行实验研究,通过共聚焦显微镜检测心肌线粒体膜电位变化,细胞免疫化学方法进行细胞色素C释放实验、化学发光法测心肌细胞胞内ADP与ATP比值的改变。400μg/ml rh-Endostatin处理H9c2心肌细胞14h、28h,不加药物为对照组,Western Blotting方法检测p-JNK和p-eIf2α的表达水平,研究rh-Endostatin产生细胞凋亡的信号转导机制。通过免疫荧光方法细胞定位检测H9c2心肌细胞核仁素的表达,初步探讨rh-Endostatin的心肌毒性作用受体。
结果:
1.回顾性研究结果提示,rh-Endostatin联合化疗治疗后与治疗前比较心电图异常率有统计学意义(χ~2=6.471,P=0.035)。治疗后与治疗前比较,心肌酶谱中CK-MB升高具有统计学性意义(t=2.530,P=0.030),其他几种心肌酶变化无统计学意义(P＞0.05)。治疗前与治疗后比较LVEF值的改变无统计学意义(t=2.083,P=0.075)。
2.前瞻性对照研究结果提示,rh-Endostatin联合化疗组患者治疗后ECG异常改变率以及CK-MB、cTnT和NT-proBNP均轻度升高,第一周期治疗后与单纯化疗组比较,ECG异常改变两组有统计学差异(χ~2=6.087,P=0.024),而LVEF值的改变两组比较无统计学差异(P＞0.05),两个周期治疗后两组比较CK-MB、cTnT均有统计学差异(P均＜0.05),两个周期治疗后两组比较NT-proBNP差异均有统计学意义(P均＜0.01)。
3.体内实验结果提示,HE染色光镜下观察显示正常对照组心肌纤维排列整齐,无心肌纤维的破坏,细胞质丰富均匀,细胞间隙正常,无水肿;Rh-Endostatin高剂量组4周和8周均可观察到心肌纤维排列紊乱,肌间隙扩大,心肌细胞结构模糊,变性坏死,局部心肌细胞核溶解或消失;心外膜下中性粒细胞及单核、淋巴细胞浸润,8周病理形态改变较4周损害程度加重。Rh-Endostatin中、低剂量组心肌组织无明显病理改变。透射电镜下观察对照组大鼠心肌肌丝排列整齐规则,细胞器无水肿和变性。Rh-Endostatin高剂量4周组和8周组均可见心肌线粒体肿胀、变性,嵴疏松、断裂,肌丝溶解,肌浆网扩张明显,8周超微结构改变较4周更为明显。Rh-Endostatin中剂量组和低剂量组无明显病理形态学改变。TUNEL法测心肌细胞凋亡,镜下观察对照组、rh-Endostatin低剂量组未见心肌凋亡细胞,中剂量组少见凋亡细胞,高剂量组可见较多凋亡细胞。将各组凋亡指数进行统计学分析,提示rh-Endostatin 4周高剂量组凋亡指数显著高于正常对照组,有统计学差异(P=0.004);8周中剂量和高剂量与对照组相比较有统计学差异(P=0.033、P=0.000)。8周高剂量组凋亡指数最高。用CD34免疫组织化学标记内皮细胞,对心肌组织微血管计数,结果提示高剂量rh-Endostatin组4周和8周可观察到血管密度增加,显著高于正常对照组,有统计学意义(P＜0.05)。4周高剂量组微血管密度最高。
4.Annexin V/PI双染法检测心肌细胞早期凋亡率,结果提示四组相比有统计学意义(F=126.492,P=0.000),其中200、400μg/ml组与对照组相比有显著统计学意义(P=0.000),400μg/ml组凋亡率最高,表明rh-Endostatin能诱导心肌细胞凋亡,并且具有剂量依赖性。结果也提示药物作用时间对凋亡的影响有统计学差异(F=2.927,P=0.092),24h凋亡率高于48h。用400μg/mlrh-Endostatin处理H9c2心肌细胞30h后,DNA被切成不同大小的条带,形成DNA Ladder,而未处理的细胞则是完整的一条带,没有Ladder产生。用400μg/ml rh-Endostatin处理24h后,电镜下可见心肌细胞内空泡增多,核固缩碎裂,凋亡小体产生,内质网扩张,线粒体肿胀,形态变圆,嵴疏松、断裂、消失,染色质块聚。而未处理的心肌细胞核大小形态正常,细胞器无损伤。
5.100μg/ml、200μg/ml、400μg/ml的rh-Endostatin处理H9c2细胞18h后,发现随着剂量的增加线粒体跨膜电位下降明显。细胞色素C释放实验提示未药物处理细胞的细胞色素C主要以胞内点状形式分布,表明它主要分布于线粒体中,400μg/ml处理24h后,细胞色素C移位,均匀分布于整个细胞中,表明它已从线粒体向胞浆释放。100μg/ml、200μg/ml、400μg/ml的rh-Endostatin处理H9c2细胞24h后,用无药物作为正常对照,四组之间ADP/ATP值比较有统计学意义(P=0.000),200μg/ml组和400μg/ml组与对照组相比有统计学意义(P均＜0.01)。分别于14h和28h收集400μg/ml rh-Endostatin处理细胞蛋白样本,发现药物处理后的p-JNK和p-eIF2a蛋白水平明显高于正常对照组,p-JNK和p-eIF2a的表达随着时间的延长而升高,药物处理14h和18h后p-JNK和p-eIF2a水平与正常对照组相比较均具有统计学差异(P均＜0.01)。免疫荧光定位提示H9c2细胞膜上表达核仁素,药物干预后表达量增多,胞浆表达明显增多。
结论:
1.初步显示重组人血管内皮抑素联合化疗存在轻度的心脏不良反应,对心肌有一定的损伤,但是短期内心功能未受到明显改变。CK-MB和心电图可作为早期监测心脏毒性的检查指标。在重组人血管内皮抑素联合化疗的过程中应该警惕心脏不良反应的发生。
2.心电图以及CK-MB、cTnT和NT-proBNP可作为早期监测心脏毒性的临床指标。CK-MB、cTnT和NT-proBNP三种心肌生化标志物联合检测,有助于提高预测rh-Endostatin心肌损伤的敏感性和准确性。
3.高剂量的rh-Endostatin对大鼠心肌有明显病理改变,并观察到超微结构的损伤,TUNEL方法也观察到高剂量组大鼠心肌组织凋亡细胞增多,提示心肌细胞凋亡机制参与了rh-Endostatin心脏毒性的病理过程。
4.体外实验从细胞的形态学和生化特征证实了rh-Endostatin对H9c2心肌细胞有凋亡作用,推测心肌细胞凋亡可能是导致心脏毒性的重要原因。
5.Rh-Endostatin对心肌细胞的毒性机制是因于rh-Endostatin激活了线粒体和内质网应激两条凋亡途径,造成了心肌细胞的凋亡。通过对核仁素的定位表达检测证实核仁素在心肌细胞膜表达,药物作用后核仁素转位,初步推测此膜表面核仁素可能是rh-Endostatin造成心脏毒性的作用受体。
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.
1971年哈佛大学医学院Folkman教授首先提出“肿瘤生长和转移依赖于新生血管生成,阻断血管生成是遏制肿瘤生长的有效策略”的理论,由此开创了肿瘤抗血管生成治疗的研究。近年来,针对肿瘤新生血管这个靶点新的抗癌药物(如贝伐单抗、索拉非尼、舒尼替尼)已经从实验室走向临床应用,并取得了良好的疗效。除此之外,做为广谱内源性血管生长抑制因子—内皮抑素(Endostatin)在小鼠体内可使93～97%肿瘤停止生长,成为全球最引人注目的抗血管生成药物。然而在以后临床应用中却发现Endostatin在人体内活性很低,科学家无法攻克蛋白质复性的技术难关,生产成本过高无法实现Endostatin的大规模生产。我国学者罗永章等经过长期不懈的努力,解决该蛋白复性的技术难关,创造的改变了Endostatin的氨基酸序列,使该蛋白的药用性能和疗效得到了显著提高。就是这个我国自主研发的新型重组人血管内皮抑素(Recombinant human endostatin,rh-Endostatin,Endostar,恩度),于2005年9月获得了国家食品药品监督管理局颁发的生物制品一类新药证书,它是世界上首例血管内皮抑制素抗癌新药。2006年7月恩度在我国上市开始临床使用,对晚期肺癌的治疗显示出较好的疗效。
据恩度治疗说明书描述,Ⅰ～Ⅲ期临床试验安全性评价指出,470例受试晚期非小细胞肺癌患者中有30例(6.38%)出现了心脏不良反应,主要表现为用药初期少数患者出现轻度疲乏、胸闷、心慌等症状,用药后第2～7天内发生心肌缺血,以及心电图出现窦性心动过速、轻度ST-T改变、房室传导阻滞、房性早搏、偶发室性早搏等,研究者认为以上心脏不良反应均为轻.中度,未危及患者生命,其中6.4‰的患者症状较为明显,但均为可逆性,多数不影响治疗进行,不需要对症治疗即可缓解。然而,笔者在恩度临床使用过程中,却发现其心脏毒性发生率和严重程度要高于药物说明书的描述,部分患者因心脏毒性暂停治疗,部分患者需要经对症治疗心脏毒性才能缓解,甚至有一例晚期非小细胞肺癌患者在恩度联合健择治疗的第7天,出现急性左心衰,心电图提示窦性心动过速,心率110～120次/分,偶发室早,N端前脑利尿肽(NT-proBNP)升高到15859pg/ml,最终因心力衰竭医治无效死亡,患者既往有高血压病史8年,用药前心脏功能正常。综合以上,根据我肿瘤治疗中心的用药体会,认为恩度的心脏毒性发生比较广泛,严重性尚待进一步评价。目前临床医生对其心脏毒性的发生警惕性尚不够,关于心脏毒性的早期诊断和预测也应开展研究。
既往研究多集中于蒽环类药物或放疗所致的心脏毒性,然而,随着分子靶向药物临床逐渐广泛,越来越多的分子靶向药物心血管不良反应逐渐被发现并受到研究者重视,有关心脏毒性的研究热点也转向了分子靶向药物,其发生机制也有了初步的研究。研究表明单独使用曲妥珠单抗,有4-7%发生心功能不全,尤其是充血性心衰(GHF)和左室射血分数(LVEF)的减少,发生机制可能与心脏组织上的HER-2受体的分化和存活有关。Nature Medicine一篇文章指出,伊马替尼可能增加化疗患者发生心力衰竭的风险,研究者推测,伊马替尼可能是通过抑制心肌细胞上的ABL激酶,并影响细胞内蛋白质合成,从而导致了对心肌细胞的损伤。舒尼替尼也有心脏毒性的报道。
尽管临床前试验认为恩度心脏毒性不是十分严重,然而使用过程中出现了左心衰严重不良心血管事件。心脏的副作用是恩度最常见并且是最有可能危及生命的毒副反应,因此,有必要对恩度心脏毒性的严重程度再次评价,筛选临床早期诊断和预测的指标。从长远上考虑,对于心脏毒性的了解越深入越利于新药的国际推广应用。研究表明分子靶向药物的心脏毒性机制不同于蒽环类药物,前者心脏毒性多与药物作用的靶点密切相关,并且信号转导通路参与心脏毒性的机制。研究者也认为每种分子靶向药物的心脏毒性机制都不完全相同,故关于恩度发生心脏毒性的机制需要深入研究。
基于上述理论,本研究试图从临床观察和实验研究两方面评价rh-Endostatin的心脏毒性,筛选早期诊断和预测临床指标,并初步探讨其心脏损伤的可能机理。
方法:
1.将既往使用恩度联合化疗的11例晚期肿瘤患者的资料进行回顾性分析,包括治疗过程中有无心慌、胸闷、呼吸困难等临床症状及症状发生时间,治疗前后心电图(ECG),心肌酶谱及LVEF的改变,评价恩度心脏毒性,并初步筛选心脏毒性早期诊断临床指标。
2.为客观评价心脏毒性以及全面筛选心脏毒性早期临床诊断指标,设计了前瞻性临床实验,选择恩度联合化疗18例,单纯化疗12例为对照研究,每周期用药的d_0、d_15检测ECG、LVEF、CK-MB、cTnT和NT-proBNP等多项指标。
3.24只实验大鼠随机分成4组,每组6只。对照组以等体积生理盐水腹腔注射,rh-Endostatin低、中、高三个剂量组,分别给与3mg/kg、6mg/kg、12mg/kg腹腔注射,每日一次,每组各半大鼠(3只)分别给药4周和8周时间,均在末次给药后24h杀死。HE染色各组大鼠心肌组织,光镜下观察病理形态学改变;透射电镜观察大鼠心肌组织超微结构的病理改变;通过TUNEL方法检测大鼠心肌细胞凋亡;通过CD34来标记内皮细胞进行免疫组织化学检测,检测大鼠心肌组织的微血管密度改变。
4.分别以100μg/ml、200μg/ml、400μg/ml三个不同剂量的rh-Endostatin处理H9c2心肌细胞24h、48h,不加药物作为对照组,通过Annexin V/PI双染流式细胞仪检测心肌细胞凋亡率。400μg/ml rh-Endostatin处理H9c2心肌细胞24h,不加药物作为对照组,透射电镜观察心肌细胞超微结构的改变。400μg/mlrh-Endostatin处理H9c2心肌细胞,不加药物作为对照组,琼脂糖凝胶电泳DNA Laddering方法检测细胞凋亡。
5.体外培养H9c2心肌细胞进行细胞凋亡的发生机制研究。以100μg/ml、200μg/ml、400μg/ml rh-Endostatin三个不同剂量和不加药物对照组进行实验研究,通过共聚焦显微镜检测心肌线粒体膜电位变化,细胞免疫化学方法进行细胞色素C释放实验、化学发光法测心肌细胞胞内ADP与ATP比值的改变。400μg/ml rh-Endostatin处理H9c2心肌细胞14h、28h,不加药物为对照组,Western Blotting方法检测p-JNK和p-eIf2α的表达水平,研究rh-Endostatin产生细胞凋亡的信号转导机制。通过免疫荧光方法细胞定位检测H9c2心肌细胞核仁素的表达,初步探讨rh-Endostatin的心肌毒性作用受体。
结果:
1.回顾性研究结果提示,rh-Endostatin联合化疗治疗后与治疗前比较心电图异常率有统计学意义(χ~2=6.471,P=0.035)。治疗后与治疗前比较,心肌酶谱中CK-MB升高具有统计学性意义(t=2.530,P=0.030),其他几种心肌酶变化无统计学意义(P＞0.05)。治疗前与治疗后比较LVEF值的改变无统计学意义(t=2.083,P=0.075)。
2.前瞻性对照研究结果提示,rh-Endostatin联合化疗组患者治疗后ECG异常改变率以及CK-MB、cTnT和NT-proBNP均轻度升高,第一周期治疗后与单纯化疗组比较,ECG异常改变两组有统计学差异(χ~2=6.087,P=0.024),而LVEF值的改变两组比较无统计学差异(P＞0.05),两个周期治疗后两组比较CK-MB、cTnT均有统计学差异(P均＜0.05),两个周期治疗后两组比较NT-proBNP差异均有统计学意义(P均＜0.01)。
3.体内实验结果提示,HE染色光镜下观察显示正常对照组心肌纤维排列整齐,无心肌纤维的破坏,细胞质丰富均匀,细胞间隙正常,无水肿;Rh-Endostatin高剂量组4周和8周均可观察到心肌纤维排列紊乱,肌间隙扩大,心肌细胞结构模糊,变性坏死,局部心肌细胞核溶解或消失;心外膜下中性粒细胞及单核、淋巴细胞浸润,8周病理形态改变较4周损害程度加重。Rh-Endostatin中、低剂量组心肌组织无明显病理改变。透射电镜下观察对照组大鼠心肌肌丝排列整齐规则,细胞器无水肿和变性。Rh-Endostatin高剂量4周组和8周组均可见心肌线粒体肿胀、变性,嵴疏松、断裂,肌丝溶解,肌浆网扩张明显,8周超微结构改变较4周更为明显。Rh-Endostatin中剂量组和低剂量组无明显病理形态学改变。TUNEL法测心肌细胞凋亡,镜下观察对照组、rh-Endostatin低剂量组未见心肌凋亡细胞,中剂量组少见凋亡细胞,高剂量组可见较多凋亡细胞。将各组凋亡指数进行统计学分析,提示rh-Endostatin 4周高剂量组凋亡指数显著高于正常对照组,有统计学差异(P=0.004);8周中剂量和高剂量与对照组相比较有统计学差异(P=0.033、P=0.000)。8周高剂量组凋亡指数最高。用CD34免疫组织化学标记内皮细胞,对心肌组织微血管计数,结果提示高剂量rh-Endostatin组4周和8周可观察到血管密度增加,显著高于正常对照组,有统计学意义(P＜0.05)。4周高剂量组微血管密度最高。
4.Annexin V/PI双染法检测心肌细胞早期凋亡率,结果提示四组相比有统计学意义(F=126.492,P=0.000),其中200、400μg/ml组与对照组相比有显著统计学意义(P=0.000),400μg/ml组凋亡率最高,表明rh-Endostatin能诱导心肌细胞凋亡,并且具有剂量依赖性。结果也提示药物作用时间对凋亡的影响有统计学差异(F=2.927,P=0.092),24h凋亡率高于48h。用400μg/mlrh-Endostatin处理H9c2心肌细胞30h后,DNA被切成不同大小的条带,形成DNA Ladder,而未处理的细胞则是完整的一条带,没有Ladder产生。用400μg/ml rh-Endostatin处理24h后,电镜下可见心肌细胞内空泡增多,核固缩碎裂,凋亡小体产生,内质网扩张,线粒体肿胀,形态变圆,嵴疏松、断裂、消失,染色质块聚。而未处理的心肌细胞核大小形态正常,细胞器无损伤。
5.100μg/ml、200μg/ml、400μg/ml的rh-Endostatin处理H9c2细胞18h后,发现随着剂量的增加线粒体跨膜电位下降明显。细胞色素C释放实验提示未药物处理细胞的细胞色素C主要以胞内点状形式分布,表明它主要分布于线粒体中,400μg/ml处理24h后,细胞色素C移位,均匀分布于整个细胞中,表明它已从线粒体向胞浆释放。100μg/ml、200μg/ml、400μg/ml的rh-Endostatin处理H9c2细胞24h后,用无药物作为正常对照,四组之间ADP/ATP值比较有统计学意义(P=0.000),200μg/ml组和400μg/ml组与对照组相比有统计学意义(P均＜0.01)。分别于14h和28h收集400μg/ml rh-Endostatin处理细胞蛋白样本,发现药物处理后的p-JNK和p-eIF2a蛋白水平明显高于正常对照组,p-JNK和p-eIF2a的表达随着时间的延长而升高,药物处理14h和18h后p-JNK和p-eIF2a水平与正常对照组相比较均具有统计学差异(P均＜0.01)。免疫荧光定位提示H9c2细胞膜上表达核仁素,药物干预后表达量增多,胞浆表达明显增多。
结论:
1.初步显示重组人血管内皮抑素联合化疗存在轻度的心脏不良反应,对心肌有一定的损伤,但是短期内心功能未受到明显改变。CK-MB和心电图可作为早期监测心脏毒性的检查指标。在重组人血管内皮抑素联合化疗的过程中应该警惕心脏不良反应的发生。
2.心电图以及CK-MB、cTnT和NT-proBNP可作为早期监测心脏毒性的临床指标。CK-MB、cTnT和NT-proBNP三种心肌生化标志物联合检测,有助于提高预测rh-Endostatin心肌损伤的敏感性和准确性。
3.高剂量的rh-Endostatin对大鼠心肌有明显病理改变,并观察到超微结构的损伤,TUNEL方法也观察到高剂量组大鼠心肌组织凋亡细胞增多,提示心肌细胞凋亡机制参与了rh-Endostatin心脏毒性的病理过程。
4.体外实验从细胞的形态学和生化特征证实了rh-Endostatin对H9c2心肌细胞有凋亡作用,推测心肌细胞凋亡可能是导致心脏毒性的重要原因。
5.Rh-Endostatin对心肌细胞的毒性机制是因于rh-Endostatin激活了线粒体和内质网应激两条凋亡途径,造成了心肌细胞的凋亡。通过对核仁素的定位表达检测证实核仁素在心肌细胞膜表达,药物作用后核仁素转位,初步推测此膜表面核仁素可能是rh-Endostatin造成心脏毒性的作用受体。
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.
引文
1.Folkman J.Tumor angiogenesis:therapeutic implications[J].N Engl J Med,1971,285(21):1182-1186.
2.Folkman J.Antiangiogenesis in cancer therapyendostatin and its mechanisms of action [J].Exp Cell Res,2006,312(5):594-607.
3.Kim Y M,Hwang S,Kim Y M,et al.Endostatin blocks vascular endot belial growth factor-mediated signaling via direct interaction wit h KDR/FIK-1[J].J Bio Chem,2002,277(31):27872-27879.
4.Urbich C,Reissner A.Chavakis E,et al.Dephosphorylation of endothelial nit ric oxide synt base contributes to t he anti-angiogenic effects of endostatin[J].FA S EB J,2002,16(7):706-708.
5.Hajitou A,Grignet C,Devy L,et al.The antitumoral effect of endostatin and angiostatin is associated wit h a down-reguration of vascular endothelial growth factor expression in tumor cells[J].FA S EB J,2002,16(13):1802-1804.
6.Rehn M,Veikkola T,Valdre K E,et al.Interaction of endostatin wit h integrins implicated in angiogenesis[J].Proc N at 1Acad Sci US A,2001,98(3):1024-1029.
7.Dixelius J,Larsson H,Sasaki T,et al.Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endot belial cell apoptosis[J].Blood,2000,95(11):3403-3411.
8. Dhanabal M ,Ramchandran R ,Waterman M J ,et al . Endostatin induces endothelial cell apoptosis [J ]. J B iol Chem, 1999,274 (17):11721-11726.
9. Kang H Y,Shim D ,Kang S S ,et al . Protein kinase B inhibits endostatin-induced apoptosis in HUVECs[J ]. J Biochem MolBiol, 2006,39 (1) :97-104.
10. MacDonald N J ,Shivers W Y,Narum D L , et al. Endostatin binds tropomyosin. A potential modulator of t he antitumor activity of endostatin [J]. J Biol Chem, 2001, 276 (27):25190-25196.
11. Hanai J, Dhanabal M, Karumanchi S A, et al. Endostatin causes G1 arrest of endothelial cells through inhibition of cyclinDl [J]. J B iol Chem, 2002,277 (19):16464-16469.
12. Y. Sun, J. Wang, Y. Liu, et al. Results of phase III trial of rh-endostatin (YH-16) in advanced non-small cell lung cancer (NSCLC) patients, J. Clin.Oncol, 2005,23
13. Jing Q, Xinyu Q, Rongcheng L. Recombinant Human Endostatin-associated Acute Left Heart Failure [J]. Clin Oncol, 2008, 20 (3): 268-268.
14. Force T, Krause DS, Van Etten RA. Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition [J].Nat Rev Cancer, 2007, 7(5):332-344.
15. Kerkela R, Grazette L, Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[J]. Nat Med,2006,12(8):908-916.
16. Seidman A, Hudis C, Pierri MK, et al. Cardiac dysfunction in the trastuzumab clinical trials experience [J].J Clin Oncol, 2002, 20(5): 1215-1221.
17. Ewer MS, Vooletich MT, Durand JB. Reversibility of trastuzumab-related cardiotoxicity: new insights based on clinical course and response to medical treatment [J]. J Clin Oncol. 2005, 23(31):7820-7826.
18. Grazette LP, Boecker W, Matsui T, et al. Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy [J]. J Am Coll Cardiol, 2004 Dec 7; 44(11):2231-2238.
19. Yeh ET, Tong AT, Lenihan DJ, et al. Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management [J].Circulation, 2004, 109(25):3122-3131.
1 O'Reilly MS,Boehm T,Shing Y,et al.Endostatin:an endogenous inhibitor of angiogenesis and tumor growth[J].Ceil,1997,88(2):277-285.
2 王金万,孙燕,刘永煜,等.重组人血管内皮抑素联合NP方案治疗晚期NSCLC随机、双盲、对照、多中Ⅲ期临床研究[J].中国肺癌杂志,2005,8(4):283-290.
3 杨林,王金万,孙燕,等.重组人血管内皮抑制素YH-16治疗晚期非小细胞肺癌的临床研究[J].中华肿瘤杂志,2006,28(2):138-141.
4 Force T,Krause DS,Van Etten RA.Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition[J].Nat Rev Cancer,2007,7(5):332-344.
5 Ewer MS,Vooletich MT,Durand JB,et al.Cardiac toxicity of trastuzumab in metastatic breast cancer patients previously treated with high-dose chemotherapy:a retrospective study[J].Br J Cancer,2006,94(7):1016-1020.
6 Perez EA,Rodeheffer R.Clinical cardiac tolerability of trastuzumab[J].J Clin Oncol,2004,22(2):322-329.
7 Michael S.Ewer,Mary Y.Vooletich,Jean-Bernard Durand,et al.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol,2005,23(31):7820-7826.
8 Slamon DJ,Leyland-Jones B,Shak S,et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[J].N Engl J Med,2001,344(11):783-792.
9 Seidman A,Hudis C,Pierri MK,et al.Cardiac dysfunction in the trastuzumab clinical trials experience[J].J Clin Oncol,2002,20(5):1215-1221.
10 Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[J].Nat Meal,2006,12(8):908-916.
1.Taniguchi R,Sato Y,Yamada T,et al.Combined measurements of cardiac troponin T and N-terminal pro-brain natriuretic peptide in patients with heart failure[J].Circ J,2004,68(12):1160-1164.
2.Tsutamoto T,Sakai H,Ishikawa C,et al.Direct comparison of transcardiac difference between brain natriuretic peptide(BNP) and N-terminal NT-proBNP in patients with chronic heart failure[J].Eur J Heart Fail,2007,9(6-7):667-673.
3.Koc M,Bozkurt A,Acart(u|¨)rk E,et al.Usefulness of N-Terminal Pro-B-Type Natriuretic Peptide Increase With Exercise for Predicting Cardiovascular Mortality in Patients With Heart Failure[J].Am J Cardiol,2008,101(8):1157-1162.
4.Yeh ET,Tong AT,Lenihan D J,et al.Cardiovascular complications of cancer therapy:diagnosis,pathogenesis,and management[J].Circulation,2004,109(25):3122-3131.
5.Slamon DJ,Leyland-Jones B,Shak S,et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overe0xpresses HER2[J].N Engl J Med,2001,344(11):783-792.
6.Park YH,Park H J,Kim BS,et al.BNP as a marker of the heart failure in the treatment of imatinib mesyiate [J]. Cancer Lett, 2006, 243(1):16-22.
7. Perik PJ, RikhofB, de Jong FA, et al. Results of plasma N-terminal pro B-type natriuretic peptide and cardiac troponin monitoring in GIST patients do not support the existence of imatinib -induced cardiotoxicity [J]. Ann. Oncol, 2008, 19(2):359-361.
8. Knobloch K, Tepe J, Lichtinghagen R, et al. Monitoring of cardiotoxicity during immunotherapy with Herceptin using simultaneous continuous wave Doppler depending on N-terminal pro-brain natriuretic peptide[J]. Clin Med, 2007, 7(1):88-89.
1.丁彦青.实用病理学七年制研究生试用教材[M](第一版).广州:第一军医大学出版.1999:29-30.
2.Weidner N,Semple JR Welch WR,et al.Tumor angiogenesis andmetastasis-correlation in invasive breast carcinoma[J].New Eng J Med,1991,324(1):1-8
3.Tanaka M,Ito H,Adachi S,et al.Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes[J].Circ Res,1994,75(3):426-433.
4.Abbate A,Biondi-Zoccai GG,Bussani R,et al.Increased myocardial apoptosis in patients with unfavorable left ventricular remodeling and early symptomatic post-infarction heart failure[J].J Am Coil Cardiol,2003,41(5):753-760.
5.Stoneman VE.Bennett MR.Role of apoptosis in atherosclerosis and its therapeutic implications[J].Clin Sci(Lond),2004,107(4):343354.
6.Gomez L,Chavanis N,Arqaud L,et al.Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia reperfusion[J].Am J Physiol Heart Circ Physiol,2005,289(5):H2153-H2158
7.Nakamura T,Ueda Y,Juan Y,et al.Fas-mediated apoptos is in adriamycin-induced cardiomyopathy in rats:in vivo s tudy[J].Circulation.2000,102(5):572-578.
8.Ewer MS,Vooletich MT,Durand JB.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol.2005,23(31):7820-7826.
9.Leri A,Glaudio PP,Li Q.Stretch myocyte apoptos is by activating P53 that enhances the local rennin-angiotens in system and decrease the Bcl-2-to-Bax Protein ratio in the cel[J]I.J Clin Invest,1998,101(7):1326-1342.
1.司徒镇强,吴军正.细胞培养[M].第二版.西安:世界图书出版公司 2007:217-219.
2.Williams GT.Programmed cell death:apoptosis and oncogenesis[J].Cell,1991,65(7):1097-1098.
3.Kerr JF,Wyllie AH.Currie AR.Apoptosis:a basic biological phenomenon with deranging implications in tissue kinetics[J].Br J Cancer,1972,26(4):239-257.
4.Gottlieb RA,Burleson KO,Kloner RA,et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes[J].J Clin Invest,1994,94(4):1621-1628.
5.Kawano H,Okada R,Kawano Y,et al.Apoptosis in acute and chronic myocarditis[J].Jpn Heart J,1994,35(6):745-750.
6.Mallat Z,Tedgui A,Fontaliran F,et al.Evidence of apoptosis in arrhythmogenic right ventricular dysplasia[J].N Engl J Med.1996,335(16):1190-1196.
7.Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[.l].Nat Med,2006,12(8):908-916.
8.Grazette LP,Boecker W,Matsui T,et al.Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes:implications for herceptin-induced cardiomyopathy[J].J Am Coll Cardiol,2004 Dec 7;44(11):2231-2238.
1.A shkenazi A,Dixit VM,Death receptors:signaling and modulation[J].Science,1998,281:1305-1308.
2.Crow MT,Mani K,Nam Y J,et al.The mitochondrial death pathway and cardiac myocyte apoptosis[J].Circ Res.2004,95(10):957-970.
3.Dumont EA,Hofstra L,van Heerde WL,et al.Cardiomyocyte death induced by myocardial ischemia and reperfusion:measurement with recombinant human Annexin-Ⅴ in a mouse model [J]. Circulation. 2000. 102: 1564 -1568.
4. Susin S A, Lorenzo H K, Zamzami N, et al. Mitochondrial release of caspase-2 and-9 during the apoptotic process [J]. J Exp Med, 1999, 189 (2):381-394.
5. Richter C, Schweizer M, Cossarizza A, et al. Control of apoptosis by the cellular ATP level [J]. FEBS Letters, 1996,378 (1): 107-110.
6. Kerkela R, Grazette L, Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate [J]. Nat Med, 2006, 12(8):908-916.
7. Grazette LP, Boecker W, Matsui T, et al. Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy [J]. J Am Coll Cardiol, 2004, 44(11 ):2231-2238.
1.Ferri KF,Kroemer G.Organelle-specific initiation of cell death pathways[J].Nat Cell Biol,2001,3(11):E255-E263.
2.Mehmet H.Caspases find a new place to hide[J].Nature,2000,403(6765):29-30.
3.Nakagawa T,Zhu H,Morishima N,et al.Caspase-12 mediates endoplasmic-reticulumspecific apoptosis and cytotoxicity by amyloidbeta[J].Nature,2000,403(6765):98-103.
4.Nakamura K,Bossy-Wetzel E,Burns K,et al.Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis[J].J Cell Biol,2000,150(4):731-740.
5.Kaufman R.J.Orchestrating the unfolded protein response in health and disease[J].J Clin Invest,2002,110(10):1389-1398.
6.Fon NC,Man BY,Ma X,et al.Celluler vacuolization and apoptosis induced by HBV large surface protein[J].Hepatolo,2002,36(6):1400-1407.
7.Tardif KD,Moil K,Siddiqui A.Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intercellular signaling pathway[J].J Virol.2002.76(15): 745-7459.
8. Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice [J]. Gastroenterology,2003, 124(5):1488-1499.
9. Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle[J]. Cell Calcium, 2002, 32: 235-249.
10. Szegezdi E, Duffy A, O'Mahoney ME, et al. ER stress contributes to ischemia-induced cardiomyocyte apoptosis [J]. Biochem Biophys Res Commun, 2006, 349(4):1406-1411.
11. Toth A, Nickson P, Mandl A, et al.Endoplasmic reticulum stress as a novel therapeutic target in heart diseases[J].Cardiovasc Hematol Disord Drug Targets,2007, 7(3):205-218.
12. Qi X, Vallentin A, Churchill E,et al.deltaPKC participates in the endoplasmic reticulum stress-induced response in cultured cardiac myocytes and ischemic heart[J].J Mol Cell Cardiol, 2007, 43(4):420-428.
13. Thuerauf DJ, Marcinko M, Gude N,et al. Activation of the unfolded protein response in infarcted mouse heart and hypoxic cultured cardiac myocytes[J].Circ Res. 2006,99(3): 275-282.
14. Martindale JJ, Fernandez R, Thuerauf D, et al. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6[J].Circ Res,2006,98(9):1186-1193.
15. Terai K, Hiramoto Y, Masaki M, et al.AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress[J].Mol Cell Biol,2005,25(21):9554-9575.
16. Nickson P, Toth A, Erhardt P. PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress[J].Cardiovasc Res, 2007,73(1):48-56.
17. Patil C,Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus the unfolded protein response in yeast and mammals. Curt Opin Cell Biol, 2001,13(3):349-355.
18. DeGracia D,Kumar R.Owen CR.et al. Molecular pathways of protein synthesis inhibition during brain reperfusion: implications for neuronal survival or death. J Cereb Blood Flow Metab,2002.22(2): 127-141.
19. lwawaki T, Hosoda A, Okuda T, etal. Translational control by the ER transmembrane kinase/ribonuclease IREI under ER stress. Nat Cell Biol, 2001, 3(2): 158-164.
20. Oyadomari S, Moil M. Roles of CHOP/GADDl 53 in endoplasmic reticulum stress. Cell Death Difer, 2004,11 (4):381-389.
21. Ruwhof C, vander Laarse A. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways [J],Cardiovasc Res ,2000,47 (1) :23-37.
22. Feuerstein GZ, Young PR. Apoptosis in cardiac diseases: stress and mitogen activated signaling pathways [J]. Cardiovasc Res ,2000,45 (3) :560
23. Sugden PH, Clerk A."Stress-responsive"mitogen-activated protein kinases(c- Jun N-termimal kinases and p38 mitogen-activated protein kinases) in the myocardium [J]. Circ Res, 1998; 83:345-352.
24. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation [J]. Sciense, 1998, 281:1305-1308.
25. Takemoto Y, Yoshiyama M,Takeuchi K. Increased JNK, AP-1 and NF-kB DNA binding activities in isoproterenol-induced cardiac remodeling[J]. J Mol Cell Cardiol. 1999, 31:2017-2030.
26. Kerkela R, Grazette L. Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate [J]. Nat Med, 2006, 12(8):908-916.
27. Rizzuto R, Pinton P, Carrington W, et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca~(2+) responses[J].Science,1998.280(5370):1763-1766.
1.Folkman J.Antiangiogenesis in cancer therapyendostatin and its mechanisms of action [J].Exp Cell Res,2006,312(5):594-607.
2.Shi H,Huang Y,Zhou H,et al.Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin[J].Blood,2007,110(8):2899-2906.
3.Ginisty H,Sicard H,Roger B,et al.Structure and functions of nucleolin[J].J Cell Sci,1999,112(Pt 6):761-772.
4.Andersen JS,Lyon CE,Fox AH,et al.Directed proteomic analysis of the human nucleolus [J].CUlT Biol,2002,12(1):1-11.
5.Mi Y,Thomas SD,Xu X,et al.Apoptosis in leukemia cells is accompanied by alterations in the levels and localization ofnucleolin[J].J Biol Chem,2003,278(10):8572-8579.
6.Kito S,Shimizu K,Okamura H,et al.Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells[J].Biochem Biophys Res Commun , 2003 , 300(4): 950-956.
7. Soundararajan S, Chen W, Spicer EK, et al. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells [J].Cancer Res. 2008, 68(7):2358-2365.
8. Zhang J, Tsaprailis G, Bowden GT.Nucleolin stabilizes Bcl-X L messenger RNA in response to UVA irradiation [J].Cancer Res. 2008,68(4): 1046-1054.
9. Tsou JH, Chang KY, Wang WC, et al. Nucleolin regulates c-Jun/Sp1-dependent transcriptional activation of cPLA-alpha in phorbol ester-treated non-small cell lung cancer A549 cells [J].Nucleic Acids Res. 2008,36(1):217-227.
10. Hirano K, Miki Y, Hirai Y,et al.A multifunctional shuttling protein nucleolin is a macrophage receptor for apoptotic cells[J].J Biol Chem. 2005,280(47):39284-39293.
11. Schulz M, Schneider S, Lottspeich F, et al. Identification of nucleolin as a glucocorticoid receptor interacting protein [J]. Biochem Biophys Res Commun. 2001,280(2):476-480.
12. Hovanessian AG, Puvion-Dutilleul F, Nisole S, et al. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton [J ]. Exp Cell Res, 2000, 261 (2):312-328.
13. Joo EJ, ten Dam GB, van Kuppevelt TH, et al.Nucleolin: acharan sulfate-binding protein on the surface of cancer cells [J].Glycobiology. 2005, 15(1): 1-9.
14. Chen X, Kube DM, Cooper MJ, et al. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA [J].Mol Ther. 2008, 16(2):333-342
15. Alete DE, Weeks ME, Hovanession AG, et al.Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-sigma [J]. FEBS J, 2006, 273(20):4668-4681.
16. Hovanessian AG. Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin [J].Cell Res. 2006, 16(2): 174-181.
17. Wang KK, Jiang L, Yi YX, et al. Effect of heat shock response on the cleavage of nucleolin induced by oxidative stress Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2004, 29(5):504-508.
18.王慷慨,方立,蒋碧梅,等.核仁素在不同细胞膜表面的表达[J].中国动脉硬化杂志,2007,15(1):63.
[1]Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[.1].Nat Med,2006,12(8):908-916.
[2]Jens Voortman,Giuseppe Giaccone.Severe reversible cardiac failure after bortezomib treatment combined with chemotherapy in a non-small cell lung cancer patient:a case report [J].BMC Cancer,2006,6:129.
[3]Seidman A,Hudis C,Pierri MK,et al.Cardiac dysfunction in the trastuzumab clinical trials experience[J].J Clin Oncol,2002,20(5):1215-1221.
[4]Ewer MS,Vooletich MT,Durand JB.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol.2005,23(31):7820-7826.
[5]Grazette LP,Boecker W,Matsui T,et al.Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes:implications for herceptin-induced cardiomyopathy[J].J Am Coll Cardiol,2004 Dec 7;44(11):2231-2238.
[6]徐扬,何斌.王雅杰,等.Herceptin与阿霉素联合应用对大鼠心脏毒性的影响[J].癌症,2004,23(4):367-371.
[7]Gordon M.S,Matei D,Aghajanianet C,et al.Clinical Activity of Pertuzumab(rhuMAb 2C4),a HER Dimerization Inhibitor,in Advanced Ovarian Cancer:potential predictive relationship with tumor HER2 activation status[J].J Clin Oncol.2006,24(26):4324-4332.
[8]Kaur A,Yu SS,Lee A J,et al.Thalidomide-induced sinus bradycardia[J].Ann Pharmacother,2003,37(7-8):1040-1043.
[9]Zhu X,Wu S,Dahut WL,et al.Risks of proteinuria and hypertension with bevacizumab,an antibody against vascular endothelial growth factor:systematic review and meta-analysis[J].Am J Kidney Dis,2007,49(2):186-193.
[10]Dincer M,A ltundag K.Angiotensin-converting enzyme inhibitors for bevacizumab induced hypertension[J].Ann Pharmacother,2006,40(12):2278-2279.
[11]Escudier B,Eisen T,Stadler WM,et al.Sorafenib in advanced clear-cell renal-cell carcinoma[J]._N Engl J Med,2007,356(2):125-134.
[12]Hahn O,Stadler W.Sorafenib[J].Curr Opin Oncol,2006,18(6):615-621.
[13]Maria Luisa Veronese,Ari Mosenkis,Keith T.Flaherty,et al.Mechanisms of Hypertension Associated With BAY 43-9006[J].J Clin Oncol,2006,24(9):1363-1369
[14]Park YH,Park HJ,Kim BS,et al.BNP as a marker of the heart failure in the treatment of imatinib mesylate[J].Cancer Lett.2006 Nov 8;243(1):16-22.
[15]Strumberg D,Awada A,Hirte H,el al.Pooled safety analysis of BAY 43-9006(sorafenib) monotherapy in patients with advanced solid tumours: Is rash associated with treatment outcome [J]. Eur J Cancer, 2006,42(4):548-556.
2.Folkman J.Antiangiogenesis in cancer therapyendostatin and its mechanisms of action [J].Exp Cell Res,2006,312(5):594-607.
3.Kim Y M,Hwang S,Kim Y M,et al.Endostatin blocks vascular endot belial growth factor-mediated signaling via direct interaction wit h KDR/FIK-1[J].J Bio Chem,2002,277(31):27872-27879.
4.Urbich C,Reissner A.Chavakis E,et al.Dephosphorylation of endothelial nit ric oxide synt base contributes to t he anti-angiogenic effects of endostatin[J].FA S EB J,2002,16(7):706-708.
5.Hajitou A,Grignet C,Devy L,et al.The antitumoral effect of endostatin and angiostatin is associated wit h a down-reguration of vascular endothelial growth factor expression in tumor cells[J].FA S EB J,2002,16(13):1802-1804.
6.Rehn M,Veikkola T,Valdre K E,et al.Interaction of endostatin wit h integrins implicated in angiogenesis[J].Proc N at 1Acad Sci US A,2001,98(3):1024-1029.
7.Dixelius J,Larsson H,Sasaki T,et al.Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endot belial cell apoptosis[J].Blood,2000,95(11):3403-3411.
8. Dhanabal M ,Ramchandran R ,Waterman M J ,et al . Endostatin induces endothelial cell apoptosis [J ]. J B iol Chem, 1999,274 (17):11721-11726.
9. Kang H Y,Shim D ,Kang S S ,et al . Protein kinase B inhibits endostatin-induced apoptosis in HUVECs[J ]. J Biochem MolBiol, 2006,39 (1) :97-104.
10. MacDonald N J ,Shivers W Y,Narum D L , et al. Endostatin binds tropomyosin. A potential modulator of t he antitumor activity of endostatin [J]. J Biol Chem, 2001, 276 (27):25190-25196.
11. Hanai J, Dhanabal M, Karumanchi S A, et al. Endostatin causes G1 arrest of endothelial cells through inhibition of cyclinDl [J]. J B iol Chem, 2002,277 (19):16464-16469.
12. Y. Sun, J. Wang, Y. Liu, et al. Results of phase III trial of rh-endostatin (YH-16) in advanced non-small cell lung cancer (NSCLC) patients, J. Clin.Oncol, 2005,23
13. Jing Q, Xinyu Q, Rongcheng L. Recombinant Human Endostatin-associated Acute Left Heart Failure [J]. Clin Oncol, 2008, 20 (3): 268-268.
14. Force T, Krause DS, Van Etten RA. Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition [J].Nat Rev Cancer, 2007, 7(5):332-344.
15. Kerkela R, Grazette L, Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[J]. Nat Med,2006,12(8):908-916.
16. Seidman A, Hudis C, Pierri MK, et al. Cardiac dysfunction in the trastuzumab clinical trials experience [J].J Clin Oncol, 2002, 20(5): 1215-1221.
17. Ewer MS, Vooletich MT, Durand JB. Reversibility of trastuzumab-related cardiotoxicity: new insights based on clinical course and response to medical treatment [J]. J Clin Oncol. 2005, 23(31):7820-7826.
18. Grazette LP, Boecker W, Matsui T, et al. Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy [J]. J Am Coll Cardiol, 2004 Dec 7; 44(11):2231-2238.
19. Yeh ET, Tong AT, Lenihan DJ, et al. Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management [J].Circulation, 2004, 109(25):3122-3131.
1 O'Reilly MS,Boehm T,Shing Y,et al.Endostatin:an endogenous inhibitor of angiogenesis and tumor growth[J].Ceil,1997,88(2):277-285.
2 王金万,孙燕,刘永煜,等.重组人血管内皮抑素联合NP方案治疗晚期NSCLC随机、双盲、对照、多中Ⅲ期临床研究[J].中国肺癌杂志,2005,8(4):283-290.
3 杨林,王金万,孙燕,等.重组人血管内皮抑制素YH-16治疗晚期非小细胞肺癌的临床研究[J].中华肿瘤杂志,2006,28(2):138-141.
4 Force T,Krause DS,Van Etten RA.Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition[J].Nat Rev Cancer,2007,7(5):332-344.
5 Ewer MS,Vooletich MT,Durand JB,et al.Cardiac toxicity of trastuzumab in metastatic breast cancer patients previously treated with high-dose chemotherapy:a retrospective study[J].Br J Cancer,2006,94(7):1016-1020.
6 Perez EA,Rodeheffer R.Clinical cardiac tolerability of trastuzumab[J].J Clin Oncol,2004,22(2):322-329.
7 Michael S.Ewer,Mary Y.Vooletich,Jean-Bernard Durand,et al.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol,2005,23(31):7820-7826.
8 Slamon DJ,Leyland-Jones B,Shak S,et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[J].N Engl J Med,2001,344(11):783-792.
9 Seidman A,Hudis C,Pierri MK,et al.Cardiac dysfunction in the trastuzumab clinical trials experience[J].J Clin Oncol,2002,20(5):1215-1221.
10 Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[J].Nat Meal,2006,12(8):908-916.
1.Taniguchi R,Sato Y,Yamada T,et al.Combined measurements of cardiac troponin T and N-terminal pro-brain natriuretic peptide in patients with heart failure[J].Circ J,2004,68(12):1160-1164.
2.Tsutamoto T,Sakai H,Ishikawa C,et al.Direct comparison of transcardiac difference between brain natriuretic peptide(BNP) and N-terminal NT-proBNP in patients with chronic heart failure[J].Eur J Heart Fail,2007,9(6-7):667-673.
3.Koc M,Bozkurt A,Acart(u|¨)rk E,et al.Usefulness of N-Terminal Pro-B-Type Natriuretic Peptide Increase With Exercise for Predicting Cardiovascular Mortality in Patients With Heart Failure[J].Am J Cardiol,2008,101(8):1157-1162.
4.Yeh ET,Tong AT,Lenihan D J,et al.Cardiovascular complications of cancer therapy:diagnosis,pathogenesis,and management[J].Circulation,2004,109(25):3122-3131.
5.Slamon DJ,Leyland-Jones B,Shak S,et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overe0xpresses HER2[J].N Engl J Med,2001,344(11):783-792.
6.Park YH,Park H J,Kim BS,et al.BNP as a marker of the heart failure in the treatment of imatinib mesyiate [J]. Cancer Lett, 2006, 243(1):16-22.
7. Perik PJ, RikhofB, de Jong FA, et al. Results of plasma N-terminal pro B-type natriuretic peptide and cardiac troponin monitoring in GIST patients do not support the existence of imatinib -induced cardiotoxicity [J]. Ann. Oncol, 2008, 19(2):359-361.
8. Knobloch K, Tepe J, Lichtinghagen R, et al. Monitoring of cardiotoxicity during immunotherapy with Herceptin using simultaneous continuous wave Doppler depending on N-terminal pro-brain natriuretic peptide[J]. Clin Med, 2007, 7(1):88-89.
1.丁彦青.实用病理学七年制研究生试用教材[M](第一版).广州:第一军医大学出版.1999:29-30.
2.Weidner N,Semple JR Welch WR,et al.Tumor angiogenesis andmetastasis-correlation in invasive breast carcinoma[J].New Eng J Med,1991,324(1):1-8
3.Tanaka M,Ito H,Adachi S,et al.Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes[J].Circ Res,1994,75(3):426-433.
4.Abbate A,Biondi-Zoccai GG,Bussani R,et al.Increased myocardial apoptosis in patients with unfavorable left ventricular remodeling and early symptomatic post-infarction heart failure[J].J Am Coil Cardiol,2003,41(5):753-760.
5.Stoneman VE.Bennett MR.Role of apoptosis in atherosclerosis and its therapeutic implications[J].Clin Sci(Lond),2004,107(4):343354.
6.Gomez L,Chavanis N,Arqaud L,et al.Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia reperfusion[J].Am J Physiol Heart Circ Physiol,2005,289(5):H2153-H2158
7.Nakamura T,Ueda Y,Juan Y,et al.Fas-mediated apoptos is in adriamycin-induced cardiomyopathy in rats:in vivo s tudy[J].Circulation.2000,102(5):572-578.
8.Ewer MS,Vooletich MT,Durand JB.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol.2005,23(31):7820-7826.
9.Leri A,Glaudio PP,Li Q.Stretch myocyte apoptos is by activating P53 that enhances the local rennin-angiotens in system and decrease the Bcl-2-to-Bax Protein ratio in the cel[J]I.J Clin Invest,1998,101(7):1326-1342.
1.司徒镇强,吴军正.细胞培养[M].第二版.西安:世界图书出版公司 2007:217-219.
2.Williams GT.Programmed cell death:apoptosis and oncogenesis[J].Cell,1991,65(7):1097-1098.
3.Kerr JF,Wyllie AH.Currie AR.Apoptosis:a basic biological phenomenon with deranging implications in tissue kinetics[J].Br J Cancer,1972,26(4):239-257.
4.Gottlieb RA,Burleson KO,Kloner RA,et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes[J].J Clin Invest,1994,94(4):1621-1628.
5.Kawano H,Okada R,Kawano Y,et al.Apoptosis in acute and chronic myocarditis[J].Jpn Heart J,1994,35(6):745-750.
6.Mallat Z,Tedgui A,Fontaliran F,et al.Evidence of apoptosis in arrhythmogenic right ventricular dysplasia[J].N Engl J Med.1996,335(16):1190-1196.
7.Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[.l].Nat Med,2006,12(8):908-916.
8.Grazette LP,Boecker W,Matsui T,et al.Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes:implications for herceptin-induced cardiomyopathy[J].J Am Coll Cardiol,2004 Dec 7;44(11):2231-2238.
1.A shkenazi A,Dixit VM,Death receptors:signaling and modulation[J].Science,1998,281:1305-1308.
2.Crow MT,Mani K,Nam Y J,et al.The mitochondrial death pathway and cardiac myocyte apoptosis[J].Circ Res.2004,95(10):957-970.
3.Dumont EA,Hofstra L,van Heerde WL,et al.Cardiomyocyte death induced by myocardial ischemia and reperfusion:measurement with recombinant human Annexin-Ⅴ in a mouse model [J]. Circulation. 2000. 102: 1564 -1568.
4. Susin S A, Lorenzo H K, Zamzami N, et al. Mitochondrial release of caspase-2 and-9 during the apoptotic process [J]. J Exp Med, 1999, 189 (2):381-394.
5. Richter C, Schweizer M, Cossarizza A, et al. Control of apoptosis by the cellular ATP level [J]. FEBS Letters, 1996,378 (1): 107-110.
6. Kerkela R, Grazette L, Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate [J]. Nat Med, 2006, 12(8):908-916.
7. Grazette LP, Boecker W, Matsui T, et al. Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy [J]. J Am Coll Cardiol, 2004, 44(11 ):2231-2238.
1.Ferri KF,Kroemer G.Organelle-specific initiation of cell death pathways[J].Nat Cell Biol,2001,3(11):E255-E263.
2.Mehmet H.Caspases find a new place to hide[J].Nature,2000,403(6765):29-30.
3.Nakagawa T,Zhu H,Morishima N,et al.Caspase-12 mediates endoplasmic-reticulumspecific apoptosis and cytotoxicity by amyloidbeta[J].Nature,2000,403(6765):98-103.
4.Nakamura K,Bossy-Wetzel E,Burns K,et al.Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis[J].J Cell Biol,2000,150(4):731-740.
5.Kaufman R.J.Orchestrating the unfolded protein response in health and disease[J].J Clin Invest,2002,110(10):1389-1398.
6.Fon NC,Man BY,Ma X,et al.Celluler vacuolization and apoptosis induced by HBV large surface protein[J].Hepatolo,2002,36(6):1400-1407.
7.Tardif KD,Moil K,Siddiqui A.Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intercellular signaling pathway[J].J Virol.2002.76(15): 745-7459.
8. Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice [J]. Gastroenterology,2003, 124(5):1488-1499.
9. Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle[J]. Cell Calcium, 2002, 32: 235-249.
10. Szegezdi E, Duffy A, O'Mahoney ME, et al. ER stress contributes to ischemia-induced cardiomyocyte apoptosis [J]. Biochem Biophys Res Commun, 2006, 349(4):1406-1411.
11. Toth A, Nickson P, Mandl A, et al.Endoplasmic reticulum stress as a novel therapeutic target in heart diseases[J].Cardiovasc Hematol Disord Drug Targets,2007, 7(3):205-218.
12. Qi X, Vallentin A, Churchill E,et al.deltaPKC participates in the endoplasmic reticulum stress-induced response in cultured cardiac myocytes and ischemic heart[J].J Mol Cell Cardiol, 2007, 43(4):420-428.
13. Thuerauf DJ, Marcinko M, Gude N,et al. Activation of the unfolded protein response in infarcted mouse heart and hypoxic cultured cardiac myocytes[J].Circ Res. 2006,99(3): 275-282.
14. Martindale JJ, Fernandez R, Thuerauf D, et al. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6[J].Circ Res,2006,98(9):1186-1193.
15. Terai K, Hiramoto Y, Masaki M, et al.AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress[J].Mol Cell Biol,2005,25(21):9554-9575.
16. Nickson P, Toth A, Erhardt P. PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress[J].Cardiovasc Res, 2007,73(1):48-56.
17. Patil C,Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus the unfolded protein response in yeast and mammals. Curt Opin Cell Biol, 2001,13(3):349-355.
18. DeGracia D,Kumar R.Owen CR.et al. Molecular pathways of protein synthesis inhibition during brain reperfusion: implications for neuronal survival or death. J Cereb Blood Flow Metab,2002.22(2): 127-141.
19. lwawaki T, Hosoda A, Okuda T, etal. Translational control by the ER transmembrane kinase/ribonuclease IREI under ER stress. Nat Cell Biol, 2001, 3(2): 158-164.
20. Oyadomari S, Moil M. Roles of CHOP/GADDl 53 in endoplasmic reticulum stress. Cell Death Difer, 2004,11 (4):381-389.
21. Ruwhof C, vander Laarse A. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways [J],Cardiovasc Res ,2000,47 (1) :23-37.
22. Feuerstein GZ, Young PR. Apoptosis in cardiac diseases: stress and mitogen activated signaling pathways [J]. Cardiovasc Res ,2000,45 (3) :560
23. Sugden PH, Clerk A."Stress-responsive"mitogen-activated protein kinases(c- Jun N-termimal kinases and p38 mitogen-activated protein kinases) in the myocardium [J]. Circ Res, 1998; 83:345-352.
24. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation [J]. Sciense, 1998, 281:1305-1308.
25. Takemoto Y, Yoshiyama M,Takeuchi K. Increased JNK, AP-1 and NF-kB DNA binding activities in isoproterenol-induced cardiac remodeling[J]. J Mol Cell Cardiol. 1999, 31:2017-2030.
26. Kerkela R, Grazette L. Yacobi R, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate [J]. Nat Med, 2006, 12(8):908-916.
27. Rizzuto R, Pinton P, Carrington W, et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca~(2+) responses[J].Science,1998.280(5370):1763-1766.
1.Folkman J.Antiangiogenesis in cancer therapyendostatin and its mechanisms of action [J].Exp Cell Res,2006,312(5):594-607.
2.Shi H,Huang Y,Zhou H,et al.Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin[J].Blood,2007,110(8):2899-2906.
3.Ginisty H,Sicard H,Roger B,et al.Structure and functions of nucleolin[J].J Cell Sci,1999,112(Pt 6):761-772.
4.Andersen JS,Lyon CE,Fox AH,et al.Directed proteomic analysis of the human nucleolus [J].CUlT Biol,2002,12(1):1-11.
5.Mi Y,Thomas SD,Xu X,et al.Apoptosis in leukemia cells is accompanied by alterations in the levels and localization ofnucleolin[J].J Biol Chem,2003,278(10):8572-8579.
6.Kito S,Shimizu K,Okamura H,et al.Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells[J].Biochem Biophys Res Commun , 2003 , 300(4): 950-956.
7. Soundararajan S, Chen W, Spicer EK, et al. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells [J].Cancer Res. 2008, 68(7):2358-2365.
8. Zhang J, Tsaprailis G, Bowden GT.Nucleolin stabilizes Bcl-X L messenger RNA in response to UVA irradiation [J].Cancer Res. 2008,68(4): 1046-1054.
9. Tsou JH, Chang KY, Wang WC, et al. Nucleolin regulates c-Jun/Sp1-dependent transcriptional activation of cPLA-alpha in phorbol ester-treated non-small cell lung cancer A549 cells [J].Nucleic Acids Res. 2008,36(1):217-227.
10. Hirano K, Miki Y, Hirai Y,et al.A multifunctional shuttling protein nucleolin is a macrophage receptor for apoptotic cells[J].J Biol Chem. 2005,280(47):39284-39293.
11. Schulz M, Schneider S, Lottspeich F, et al. Identification of nucleolin as a glucocorticoid receptor interacting protein [J]. Biochem Biophys Res Commun. 2001,280(2):476-480.
12. Hovanessian AG, Puvion-Dutilleul F, Nisole S, et al. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton [J ]. Exp Cell Res, 2000, 261 (2):312-328.
13. Joo EJ, ten Dam GB, van Kuppevelt TH, et al.Nucleolin: acharan sulfate-binding protein on the surface of cancer cells [J].Glycobiology. 2005, 15(1): 1-9.
14. Chen X, Kube DM, Cooper MJ, et al. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA [J].Mol Ther. 2008, 16(2):333-342
15. Alete DE, Weeks ME, Hovanession AG, et al.Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-sigma [J]. FEBS J, 2006, 273(20):4668-4681.
16. Hovanessian AG. Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin [J].Cell Res. 2006, 16(2): 174-181.
17. Wang KK, Jiang L, Yi YX, et al. Effect of heat shock response on the cleavage of nucleolin induced by oxidative stress Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2004, 29(5):504-508.
18.王慷慨,方立,蒋碧梅,等.核仁素在不同细胞膜表面的表达[J].中国动脉硬化杂志,2007,15(1):63.
[1]Kerkela R,Grazette L,Yacobi R,et al.Cardiotoxicity of the cancer therapeutic agent imatinib mesylate[.1].Nat Med,2006,12(8):908-916.
[2]Jens Voortman,Giuseppe Giaccone.Severe reversible cardiac failure after bortezomib treatment combined with chemotherapy in a non-small cell lung cancer patient:a case report [J].BMC Cancer,2006,6:129.
[3]Seidman A,Hudis C,Pierri MK,et al.Cardiac dysfunction in the trastuzumab clinical trials experience[J].J Clin Oncol,2002,20(5):1215-1221.
[4]Ewer MS,Vooletich MT,Durand JB.Reversibility of trastuzumab-related cardiotoxicity:new insights based on clinical course and response to medical treatment[J].J Clin Oncol.2005,23(31):7820-7826.
[5]Grazette LP,Boecker W,Matsui T,et al.Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes:implications for herceptin-induced cardiomyopathy[J].J Am Coll Cardiol,2004 Dec 7;44(11):2231-2238.
[6]徐扬,何斌.王雅杰,等.Herceptin与阿霉素联合应用对大鼠心脏毒性的影响[J].癌症,2004,23(4):367-371.
[7]Gordon M.S,Matei D,Aghajanianet C,et al.Clinical Activity of Pertuzumab(rhuMAb 2C4),a HER Dimerization Inhibitor,in Advanced Ovarian Cancer:potential predictive relationship with tumor HER2 activation status[J].J Clin Oncol.2006,24(26):4324-4332.
[8]Kaur A,Yu SS,Lee A J,et al.Thalidomide-induced sinus bradycardia[J].Ann Pharmacother,2003,37(7-8):1040-1043.
[9]Zhu X,Wu S,Dahut WL,et al.Risks of proteinuria and hypertension with bevacizumab,an antibody against vascular endothelial growth factor:systematic review and meta-analysis[J].Am J Kidney Dis,2007,49(2):186-193.
[10]Dincer M,A ltundag K.Angiotensin-converting enzyme inhibitors for bevacizumab induced hypertension[J].Ann Pharmacother,2006,40(12):2278-2279.
[11]Escudier B,Eisen T,Stadler WM,et al.Sorafenib in advanced clear-cell renal-cell carcinoma[J]._N Engl J Med,2007,356(2):125-134.
[12]Hahn O,Stadler W.Sorafenib[J].Curr Opin Oncol,2006,18(6):615-621.
[13]Maria Luisa Veronese,Ari Mosenkis,Keith T.Flaherty,et al.Mechanisms of Hypertension Associated With BAY 43-9006[J].J Clin Oncol,2006,24(9):1363-1369
[14]Park YH,Park HJ,Kim BS,et al.BNP as a marker of the heart failure in the treatment of imatinib mesylate[J].Cancer Lett.2006 Nov 8;243(1):16-22.
[15]Strumberg D,Awada A,Hirte H,el al.Pooled safety analysis of BAY 43-9006(sorafenib) monotherapy in patients with advanced solid tumours: Is rash associated with treatment outcome [J]. Eur J Cancer, 2006,42(4):548-556.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |