【作者】 李雪萍；

【导师】 顾谦群； 李静；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏， 2009， 博士

【摘要】 灵菌红素是一类发现于多种细菌、放线菌中的红色抗生素，其骨架特征是含有一个三吡咯环的大共轭体系；该类化合物具有显著的抗肿瘤、抗菌、抗原虫以及免疫抑制等多种活性，备受国内外学者的关注。本实验室近年开展了海洋微生物中抗肿瘤活性次级代谢产物的研究，发现源于海洋羽毛山海绵Mycale plumose的放线菌糖多孢菌Saccharopolyspora sp．（SP2-10）产十一烷基灵菌红素Metacycloprodigiosin（MP）和Undecylprodigiosin（UP）。本论文对该菌株进行优化培养，分离制备MP，重点对MP的抗肿瘤活性及作用机制进行了研究。根据十一烷基灵菌红素的生物合成途径，对海洋放线菌SP2-10进行单因素优化实验。实验选取了不同浓度梯度的乙酸钠（NaAc）、脯氨酸、丝氨酸、甘氨酸、缬氨酸四种氨基酸以及碘乙酰胺（IDAA）作为前体添加物，改变培养基成分，对菌株SP2-10进行优化培养。结果发现，终浓度为10mmol／L的乙酸钠和缬氨酸均能使十一烷基灵菌红素的产量提高3倍，终浓度为0．2mmol／L的碘乙酰胺能较大幅度的提高MP在色素总产量中的比例。继而，对该菌株进行大量发酵，并运用有机溶剂萃取，正相、反相硅胶柱色谱及重结晶法等化学的分离纯化手段对从发酵产物中分离得到单体化合物MP和UP，并采用高效液相色谱与标准品对照测定其纯度，¹HNMR和MS确定其化学结构。首先采用MTT法观察测定了MP对小鼠白血病细胞株P388、人肝癌细胞株BEL-7402细胞生长的影响；采用流式细胞术，检测了MP对P388、BEL7402细胞周期变化的影响；采用Western Blot法检测PARP的裂解情况，判断MP诱导两个细胞系凋亡活性。研究结果表明，MP浓度在10^-5M，10^-6M，10^-7M时，能明显抑制P388、BEL7402肿瘤细胞生长；MP能将细胞周期阻滞于G₂／M期；MP导致PARP的剪切。为了阐明MP诱导肿瘤细胞凋亡的途径和对肿瘤细胞死亡的机制，进一步观察了MP对P388细胞线粒体释放情况的影响，发现此凋亡伴随线粒体的释放，由此判断是线粒体途径的凋亡；采用Western Blot法对上游的细胞分子进行观察，发现MP对AKT激活无影响，但可以引起HSP90的蛋白剪切，进一步采用流式细胞仪法测定细胞内活性氧水平的变化，发现MP可以明显增加P388细胞内活性氧的含量。为明确活性氧信号传导可以引起MAPK通路的变化，采用Western Blot法，观察信号通路的变化，发现MP能明显促进信号分子ERK、P38和JNK的活化，又引入ERK、P38和JNK的抑制剂，发现加ERK抑制剂对MP导致的P388周期阻滞无明显影响，而加P38和JNK抑制剂后能明显逆转MP引起的细胞死亡，保护细胞免受MP损伤。此外还对MP拮抗肿瘤细胞转移作用进行了初步研究，采用MTT法，观察MP对人脐静脉血管内皮细胞株HUEVC细胞的影响，发现MP在低于细胞毒作用剂量时能明显抑制HUEVC细胞管腔的形成。采用划痕法观察MP对高转移小鼠乳腺癌细胞株4TO7细胞迁移的影响，结果显示MP在10^-6M、10^-7M和10^-8M时，能明显抑制4TO7的迁移；Transwell小室法确定MP对肿瘤细胞侵袭的影响，结果显示MP可明显抑制4TO7细胞侵袭。本文首次对十一烷基灵菌红素Metacycloprodigiosin抗肿瘤活性及其作用机制进行了研究。研究发现MP体外能明显抑制多种肿瘤细胞的生长，将细胞阻断于G₂／M期并诱导凋亡；MP诱导活性氧是其导致细胞生长抑制的重要机制，并与其诱导的P38、JNK活化密切相关，而与ERK1／2活化无关；并且初步发现MP还能够抑制肿瘤转移的多个环节，可能对肿瘤转移具有抑制作用。上述研究结果为今后进一步将MP开发成为抗肿瘤药物奠定了基础。更多还原

【Abstract】 Prodiginines are a large family of pigmented tripyrrole secondary metabolites with medicinal potential as antifungal, antibacterial, immunosuppressants and antitumour agents that are produced by several actinomycetes and other bacteria. Because of its multiple bioactivities, prodiginines were attracted a great deal of attention in recent yearsIn our former study, a marine actinomycetes strain from a sponge-derived actinomycete Saccharopolyspora sp. nov SP2-10 with metacycloprodigiosin （MP） and undecylprodigiosin （UP） producing ability has been found. In this dissertation, single factor test was employed to improve MP yields from SP2-10 strain, MP and UP were isolated from the rude ethyl acetate extract, furthermore, the anticancer activities and molecule action mechanism of MP were studied.According to undecylprodigiosin biosynthesis pathway, sodium acetate, several amino acids and iodine acetamide were selected as precursors adding into the actinomycetes medium with four different concentration gradients.It was found that the addition of sodium acetate and Val to the final concentration of 10mmolL^-1 either enhanced MP an UP production by 3 fold. Moreover, iodine acetamide with final concentration of 0.2 mmolL^-1 obviously raised MP proportion in the total pigment production.According to the optimal condition, large-scale fermentation and preparation of the active fractions were performed. MP and UP were isolated and purified using solvent extraction, silica gel column, Sephadex LH20 and etc. The structures were identified by MS and ¹HNMR.Firstly, we investigated the effects of MP on the proliferation and cell cycle progression in cancer cells of murine leukemia cells（P388） and human hepatocellular carcinoma cells（Bel-7402） using MTT assay and flow cytometry. The inhibitive effect could reach about 50% when cells were treated with 1uM MP for 24h. We found that MP induced a significant blockage in the G₂/M phase in P388 cancer cells, with the immunofluorence staining, cells were fixed and immunolabeled for cell nucleus with PI, we found that the nucleus showed morphology characteristic of apoptotic cells, furthermore, we evaluated the PARP cleavage and Cytochrom C release after MP treatment by Western blot, the results revealed that MP treatment could promote the cleavage of poly（ADP-ribose） polymerase protein and the Cytochrom C release from mitochondrion companioned by the cytochrome C increase in cytoplasm, these result indicate that MP can induce cancer cells apoptosis and demonstrate the apoptosis is involved in mitochondrial apoptotic pathway. In addition, with Confocal, the cytoplasm acidification, the other character of apoptosis was also being noticed.In order to further elucidate the mechanisms underlying apoptosis induced by MP treatment, PI-3K/AKT signal pathway was first studied, but no distinct effect on AKT phosphorylation was found, however, we notified that MP could promote cleavage of HSP90 protein according to Western blot, this result suggested us that MP could enhance the concentration of the reactive oxygen species （ROS） within cells and the flow cytometry assay revealed that MP could increase the ROS in P388 cells.Finally, we examined the role of ROS in deterring whether MP triggered phosphorylation of ERK, JNK, P38, the three important signal molecule of MAPK signal pathway. We found that MP induced phosphorylation of P38, ERK and JNK remarkably. Selective inhibitor of P38 and JNK blocked its phosphorylation and prevented the p388 cancer cells from injuring induced by MP treatment except for ERK.Furthermore, in the following study in our paper, we investigated the function of MP in antimetastatic and anticancer effect. According to wound healing assay and the transwell assay, migration and invasion was decreased in 4TO7 cells, treated with 0.001uM MP. With tube formation assay, MP was found to inhibit tube formation of the human umbilical vein endothelial cells, which is an important step for angiogenesis. In conclusion, the systematic research of MP anticancer effect revealed that MP could decrease the proliferation of multiple cancer cell lines and induce blockage in the G₂/M phase. MP induced apoptosis was via mitochondrial pathway. ROS induced by MP was the important mechanism of this action which correlated with phosphorylation of P38 and JNK other than ERK. Besides, MP could exert antimetastatic effect. Therefore, MP appears to be potential candidate for pharmaceutical development as anticancer agents.更多还原