熊果酸对肝癌细胞与血管内膜粘附的干预作用及其机制研究

Tumor recurrence and metastasis is the leading cause of liver cancer mortality. There is no effective method so far to prevent recurrence and metastasis of tumor. Traditional anti-cancer drugs for the treatment of hepatoma metastasis have high toxic side effects and low safety factor. Our previous study showed that ursolic acid and its derivatives (UAs) could lead to a signi?cant reduction of hetero-adhesion of HepG2 cells to human umbilical vein endothelial cell (HUVEC) in vitro, reduce HepG2 cell migration in a wound-healing assay, inhibit the expression of adhesion molecules on HUVEC. In addition, UAs suppressed experimental lung metastasis of mouse melanoma B16-F10 cells in vivo. Moreover，GeneChip studies showed that UAs had a significant impact on the cellular focal adhesion pathway in HepG2 cells. These results revealed the potential of UAs for hepatoma metastasis chemoprevention. Therefore, we proposed the new idea that UAs may interfere with the initial step of cancer metastatic cascade. In this project, we will further investigate the interference effect of UAs on the adhesion of hepatoma cells with vascular endothelium and reveal its mechanisms on both a molecular, cellular and animal level, which includes: To demonstrate the interaction with UAs, cell adhesion molecules, platelets and cytokines; To explore the interference effect and mechanism of UAs with hetero-adhesion of hepatoma cells to vascular endothelium; To clarify the characteristics and seek the relations of time-effect and dosage-effect by using UAs for restraining hepatoma cells from initial metastatic cascade; Furthermore, the possible mechanisms of UAs inhibits hepatoma metastasis and underlying molecular targets will be revealed. This study will provide an important scientific basis for the intervention effect of UAs in the key step of liver cancer hematogenous metastasis, and explore a new strategy for the early prevention of hepatoma metastasis.

肝癌术后复发转移是癌症患者高死亡率的主要原因，尚无有效防治方法。传统抗肝癌复发转移化疗药物毒副作用大且安全系数低。我们前期研究显示：高效低毒的熊果酸及其衍生物(UAs)体外具有较好抗肝癌细胞粘附迁移能力，并可抑制肝癌细胞和血管内皮细胞表面粘附因子表达；体内可有效减少小鼠黑色素瘤肺转移，提示其具有抗肿瘤转移潜力；进一步基因芯片技术发现其对肝癌细胞粘附信号通路有显著影响。综上本项目提出UAs可干预肝癌转移关键步骤的新设想，拟从分子-细胞-动物水平研究其干预肝癌细胞与血管内膜粘附作用及机制。包括：研究药物对细胞表面粘附分子、血小板和刺激因子等影响；探索药物干预肝癌细胞与血管内膜粘附的作用及机制；阐明药物干预肝癌细胞粘附转移作用特点及其量效-时效关系；揭示药物抗肝癌转移的可能机制及分子靶点。本项目将为UAs在肝癌血行转移源头步骤中的干预作用提供重要科学依据，为肝癌复发转移早期防治探讨新策略。

肝癌是人类最常见的恶性肿瘤之一。术后高复发转移率是肝癌患者高死亡率的主要原因，也是目前提高患者生存率亟待解决的瓶颈问题。传统治疗肝癌复发转移的化疗药物毒副作用大且安全系数低。本项目通过体内外实验研究天然产物熊果酸及其衍生物（UAs）对肝癌复发转移的早期源头步骤——肿瘤细胞与血管内膜粘附的干预作用，并探讨其作用机制及分子靶点。完成了以下工作：UAs体外对不同种类肝癌细胞（HepG2、MHCC-97L 和MHCC-97H）显示不同程度的增殖抑制作用且对正常细胞（HUVEC、LO2）毒性相对较低；在安全有效的浓度范围内，药物可显著抑制人肝癌细胞与细胞外基质/血小板/血管内皮细胞之间的粘附能力，且其抑制效果呈量效依赖关系。体内动物实验结果显示，药物可有效抑制小鼠H22肝癌血道转移模型肺部组织的粘附和着床，并浓度依赖性地抑制肿瘤组织中粘附分子ICAM-1的表达水平，进而干预肝癌的血道转移过程；此外，药物可提高肝损伤小鼠免疫器官的胸腺指数和脾脏指数，且具有较好的保肝护肝效果。分子水平研究显示，UAs可下调细胞粘附分子（ICAM-1、Integrin α6β1、VCAM-1和E-selectin等）mRNA和蛋白的表达水平，并调控血管内皮生长因子（VEGF）和细胞粘附通路相关分子（Src、FAK、paxillin和PTEN等）的信号传导；经siVEGF基因沉默后，药物仍可不同程度地抑制肝癌细胞基质粘附的能力。综上，本研究揭示了UAs干预肿瘤细胞与血管内膜粘附的作用机制是通过下调细胞表面粘附分子的表达，并调控整合素激活FAK介导的信号转导通路，从而干预肿瘤细胞体内外的粘附、侵袭及转移过程。研究结果为UAs作为抗肿瘤转移新药开发，尤其是将其应用于肝癌复发转移的早期防治提供了科学依据。