73-Deoxychondropsin A: A specific inhibitor of resorption and osteosarcoma?

Long/Grigoriadis实验室之前的工作已经确定了73-脱氧软骨素a （73-DOC）在骨生物学中的新作用。Paul Long与澳大利亚海洋科学研究所（AIMS）合作，从大堡礁海绵I. ramosa中分离出73-DOC，并对其进行纯化。73-DOC对哺乳动物V-ATPases具有强抑制活性。v - atp酶通过调节细胞内/细胞外ph值在基本的细胞生理过程中发挥作用。此外，对于骨组织，v - atp酶在骨吸收破骨细胞中至关重要，破骨细胞需要v - atp酶来维持正常功能。Long/Grigoriadis实验室的初步和尚未发表的结果已经确定了73-DOC在靶向破骨细胞中的独特作用，尽管由于v - atp酶蛋白复合物的复杂性，73-DOC的精确靶点及其作用机制尚不清楚。该项目将研究73-DOC对破骨细胞吸收的具体影响，并将其扩展到破骨细胞参与肿瘤进展的骨癌模型中。本项目假设73-DOC通过靶向破骨细胞与骨肉瘤等肿瘤共有的v - atp酶亚基亚型，特异性抑制破骨细胞吸收，而不影响骨重塑，从而有效抑制破骨细胞吸收和肿瘤发病。总之，73-DOC给药对破骨细胞吸收和骨肉瘤的影响将采用表型、生化和蛋白质组学相结合的方法进行综合表征。具体目的：表征73-DOC给药对破骨细胞和骨肉瘤细胞表型和功能的影响。确定73-DOC是否通过靶向特定亚基亚型抑制V-ATPase活性，并确定73-DOC介导V-ATPase抑制的分子机制。评估73-DOC介导的特异性V-ATPases亚型抑制是否是治疗溶骨性疾病和骨肉瘤的可行方法。工作计划将遵循新的73-DOC化合物的纯化，造血干细胞破骨细胞分化的体外分析，功能吸收分析和表面轮廓测定法定量，骨肉瘤细胞生长和活性，细胞骨架分析/共聚焦成像，质谱/蛋白质组学分析，以及体内肿瘤建模的逻辑进展。因此，该项目将为学生提供各种多样化和互补的技能，包括细胞生物学和哺乳动物细胞培养，分子分析（qPCR， RNAi策略），蛋白质生物化学/质谱，共聚焦显微镜，以及肿瘤发展的体内模型，包括组织学和免疫定位分析。这些研究方法将确保在博士学位的时间框架内达到指定的目标。

Previous work by the Long/Grigoriadis labs have identified a novel role for 73-deoxychondropsin A (73-DOC) in bone biology. 73-DOC has been isolated from the Great Barrier Reef sponge, I. ramosa, and purified by Paul Long in collaboration with the Australian Institute of Marine Sciences (AIMS). 73-DOC exhibits potent inhibitory activity to mammalian V-ATPases. V-ATPases have a role in fundamental cellular physiology processes through regulation of intra/extracellular pH. Additionally, for bone tissue, V-ATPases are critically important in bone-resorbing osteoclasts which require V-ATPases for normal function. Preliminary and yet unpublished results from the Long/Grigoriadis labs have established a unique role for 73-DOC in targeting osteoclasts, although due to the complexity of the V-ATPase protein complex, the precise targets of 73-DOC and its mechanism of action are not yet known. This project will investigate the specific effects of 73-DOC on osteoclastic resorption and extend this to a bone cancer model in which osteoclasts are implicated in tumour progression. The hypothesis of this project is that 73-DOC specifically inhibits osteoclast resorption, without affecting bone remodeling by targeting V-ATPase subunit isoforms shared between osteoclasts and tumors such as osteosarcoma and thus should effectively inhibit resorption and tumor pathogenesis. Overall, the effects of 73-DOC administration on osteoclast resorption and osteosarcoma will be characterized comprehensively using a combined phenotypic, biochemical, and proteomic approach. Specific aims: Characterize the effect of 73-DOC administration on the phenotype and functioning of osteoclast and osteosarcoma cells. Determine whether 73-DOC inhibits V-ATPase activity by targeting specific subunit isoforms and determine the molecular mechanics of 73-DOC mediated V-ATPase inhibition. Assess whether 73-DOC mediated inhibition of specific V-ATPases isoforms is a viable approach to therapeutically managing osteolytic diseases and osteosarcoma.The plan of work will follow a logical progression from purification of new 73-DOC compound, in vitro assays on osteoclast differentiation from haematopoietic stem cells, functional resorption assays and quantification by surface profilometry, osteosarcoma cell growth and activity, cytoskeletal analysis/confocal imaging, mass spec/proteomics analysis, and in vivo tumour modelling. The project therefore will provide the student with a variety of diverse and complementary skills, including cell biology and mammalian cell culture, molecular analyses (qPCR, RNAi strategies), protein biochemistry/mass spec, confocal microscopy, and in vivo models of tumour development, including histological and immunolocalisation analyses. These research methods will ensure that the specified aims are met within the time-frame of the PhD.