新抗肿瘤抗生素NC0604的分离纯化、结构鉴定及生物活性研究

【作者】 陈彩霞；

【导师】 司书毅； 陈汝贤； 何琪杨； 许鸿章；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 博莱霉素是1966年日本的梅泽滨夫等人首先从轮枝链霉菌（Streptomycesverticillus）的发酵产物中发现的。它是一种糖肽类的抗肿瘤抗生素。自被发现以来,已广泛用于鳞状细胞癌、恶性淋巴瘤、睾丸癌及卵巢癌等的联合化疗中。博莱霉素有着独特的结构和性质,能够介导激活分子氧,并序列选择性降解DNA,且不会引起骨髓抑制和免疫抑制,这在抗肿瘤药物中是非常有吸引力的。但是博莱霉素能够剂量依赖性的导致肺纤维化,使其临床应用受到一定的限制。因此,寻找高效低毒的博莱霉素族新化合物成为研究的一个热点。我们在博莱霉素族化合物的筛选过程中,从轮枝链霉菌平阳变种（Streptomycesverticillus var.pingyangensis n.var）-Q835的发酵液中发现了一个新的组分,通过离子交换树脂、大孔吸附树脂和CM-Sephadex C-25葡聚糖凝胶[NH4⁺]型柱层析分离纯化得到此新活性组分纯品;经各种光谱学分析手段（IR、UV、ESI-Q-TOF2-MS、NMR）确定其具有与博莱霉素相同的母核结构,但末端侧链不同,并被确证为amidepropyl spermidine,经查新确定为一博莱霉素族的新化合物,遂将其命名为NC0604。对NC0604的抗菌谱进行了研究,发现其具有广谱抗菌活性,并且对一些杆菌类及革兰氏阴性菌表现出较好的抗菌活性。通过MTT法和克隆形成率法检测了NC0604的体外抗肿瘤活性,发现其抗瘤谱广、抗肿瘤作用好,对人肝癌HepG2细胞、人口腔上皮癌KB细胞、人乳腺癌MCF-7细胞、人大肠癌HCT116细胞、人胃癌BGC-823细胞及人乳腺癌耐药细胞MCF-7/DOX等都表现出了优于博莱霉素的抗肿瘤活性。用雌性Babl/c小鼠皮下接种鼠肝癌H22细胞进行体内抗肿瘤活性实验,结果显示,在10mg·kg^-1、5mg·kg^-1和2mg·kg^-1三个给药剂量下,博莱霉素对鼠肝癌H22的抑制率分别为75.96%、59.01%和47.41%;而NC0604对鼠肝癌H22的抑制率分别为91.45%、79.69%和69.97%。NC0604在10mg·kg^-1、5mg·kg^-1、2mg·kg^-1三个剂量时对鼠肝癌H22的抑制率分别为同剂量博莱霉素的1.20倍、1.35倍和1.48倍。用雄性昆明种小鼠检测了NC0604的肺毒性,通过生化指标检测即肺组织中羟脯氨酸（HYP）含量测定和观察肺组织病理切片的形态学变化评价了NC0604的肺毒性情况,并与博莱霉素族的其他几个化合物进行了比较,证明NC0604的肺毒性比博宁霉素略强,但明显低于博莱霉素、平阳霉素和博安霉素。对NC0604抗肿瘤作用的分子机理进行了初步的研究。发现NC0604能引起HepG2细胞活性氧自由基增加和细胞线粒体膜电位功能障碍,并将细胞周期阻断于G2/M期。NC0604处理过的HepG2细胞通过Hochest 33342染色后在荧光显微镜下能观察到具有凋亡特征的染色质凝集,并通过凋亡相关的蛋白分子检测进一步表明NC0604具有体外诱导肿瘤细胞凋亡的作用。由此提示NC0604抗肿瘤作用的机制可能与其诱导肿瘤细胞ROS增加、线粒体膜电位障碍及进而诱导细胞凋亡有关。博莱霉素族化合物的抗肿瘤作用比较独特,而该族化合物的各成员的抗肿瘤作用及机制也各有特点,对于NC0604作用机制,仍需进行更深入的研究与探索。更多还原

【Abstract】 Bleomycins are a family of glycopeptides originally isolated from Streptomyces verticillus by Umezawa et al in 1966.As a family of antitumor antibiotics,the bleomycins have been widely used clinically in combination with a number of other agents for the treatment of several types of tumors,notably squamous cell carcinoma, malignant lymphoma,testicular carcinomas and ovarian cancer since their discovery. Bleomycins are attractive therapeutics,as they exhibit both low myelosuppression and low immunosuppression with their unique structures and properties in mediating dioxygen activation and sequence selective degradation of DNA.However,the therapeutic efficacy of bleomycins is limited by the development of dose-dependent lung fibrosis.So,more and more research has been focused on the development of a more potent,less toxic compound ofbleomycin group.In the course of a screening program for new compounds of bleomycins,we found a new component from the culture broth of Streptomyces verticillus var.pingyangensis n.var-Q835.The objective component was isolated by ion exchange resins, macroporous resins and CM-Sephadex C-25[NH4⁺]chromatography.And the structure was then elucidated by spectroscopic analyses including IR,UV,ESI-Q-TOF2-MS and NMR.The results showed that it had the same kernel structure as bleomycin,but a different terminal amine moiety determined as amidepropyl spermidine.It was confirmed as a new compound ofbleomycin group and was named as NC0604.The antibacterial spectrum study revealed that NC0604 exhibited antibacterial activity against members of a wide range of bacterial species.Better antibacterial activities were observed against members of Bacillus species and some Gram-negative bacteria.The growth-inhibitory activity in vitro of NC0604 toward various human tumor cells was determined by MTT assay and clonogenic assay.NC0604 showed better activities against a wide range of human tumor species,especially human HepG2,KB, MCF-7,HCT116,BGC-823 and MCF-7/DOX.Tumor inhibitory effect of NC0604 in vivo was evaluated by using the model of subcutaneously transplanted hepatoma H22 in Balb/c female mice.And the tumor inhibitory rates of NC0604 at concentrations of 10.0 mg·kg^-1,5.0 mg·kg^-1 and 2.0 mg·kg^-1 were 91.45%,79.69%and 69.97%,respectively,which were 1.20,1.35 and 1.48 times higher,respectively,than those of bleomycin（75.96%,59.01%and 47.41%, respectively） under the same administration and dosage.The pulmonary toxicity for mice was determined in male Kunming mice.The pulmonary toxicity was evaluated by determining the content of hydroxyproline（HYP） and observing the histopathological changes of the lung tissues at day 28 after administration of the test compounds.Compared with the other members of bleomycins including bleomycin,pingyangrnycin and boanmycin,NC0604 showed reduced pulmonary toxicity but a little bit severe than boningrnycin.We explored primarily the molecular mechanism of action of NC0604 against tumor cells.The results indicated that NC0604 could increase the ROS level,change the mitochondrial transmembrane potential（△ψm） in HepG2 cells and block the cell cycle at G2/M phase.Features of apoptosis such as chromatin condensation in HepG2 cells were observed under florescent microscope following staining with Hochest 33342. Determination of protein molecules related to cell apoptosis further indicated that NC0604 could induce apoptosis of HepG2 cells in vitro.These indicated that inducing increase of the ROS level,interfering with the△ψm and further inducing cell apoptosis may be one antitumor mechanism of NC0604.Since the bleomycin family has special antitumor activations and each member has its strong point,more further research and exploration on the mechanism of action of NC0604 were expected to be continued.更多还原