一种新型熊果酸衍生物IUA逆转骨肉瘤耐药的作用及机制研究

Osteosarcoma is the most common malignant bone tumor, which often causes amputation, pulmonary metastasis, or even death. The drug resistance (especially the multiple drug resistance) is the main reason of the failure of clinical treatment, or the recurrence and metastasis of osteosarcoma. There isn't any effective treatment or medicine found for reversing drug resistance of osteosarcoma at present. Thus, to reverse the drug resistance of osteosarcoma becomes very important. JNK signal pathway is bound up with the generation and drug resistance of tumor. The applicant has screen out a new type of ursolic acid derivatives IUA on his preparation work, which is learnt to play a role on antagonize the osteosarcoma through inhibiting the JNK signal pathway. Which is more important, that during the early stage of the experiment, the applicant has discovered the IUA may also increase the affection on killing drug resistant osteosarcoma cells by chemotherapeutic drugs. But we haven't discovered the specific mechanism yet. Thus, we put forward the hypothesis: IUA could be used to reverse the drug resistance of osteosarcoma by inhibiting the JNK signal pathway. This project will study further on the specific role and molecular mechanism of IUA to reverse the drug resistance of osteosarcoma, by a multiple of cells and animal experiments, on the basis of preliminary experiments. We believe that with the launch of this project, it is possible to illuminate the mode of function and specific molecular mechanism for IUA to reverse osteosarcoma drug-resistance, providing theoretical support for treating osteosarcoma by natural small molecular compounds derivatives, particularly IUA. This may solve the problem of drug resistance of osteosarcoma to some extent.

骨肉瘤是最常见的原发性恶性骨肿瘤，常伴随截肢、肺转移、死亡等后果。耐药性的产生（尤其是多重耐药性）正是临床治疗失败及骨肉瘤复发、转移的主要原因，但目前尚无有效逆转骨肉瘤耐药的方法和药物。JNK信号通路和肿瘤的发生及耐药密切相关，申请人在前期工作基础上筛选得到一种新型熊果酸衍生物IUA，发现其可以通过抑制JNK信号通路发挥抗骨肉瘤效应，更重要的是申请人前期实验发现IUA还可以增加化疗药对耐药骨肉瘤细胞的杀伤作用，但具体机制仍然不明。所以我们提出这样的假说：IUA可能通过抑制JNK信号通路来逆转骨肉瘤的耐药性。本项目将在前期实验的基础上利用多种细胞和动物实验深入研究IUA逆转骨肉瘤耐药性的具体作用及分子机制。相信本项目的顺利开展，有望阐明IUA逆转骨肉瘤耐药的作用方式及具体分子机制，为以 IUA 为代表的天然小分子化合物衍生物治疗骨肉瘤提供一定理论支持,在一定程度上解决骨肉瘤耐药难题。

骨肉瘤是最常见的原发性恶性骨肿瘤，好发于儿童和青少年。由于新辅助化疗的出现，骨肉瘤病人的预后有了很大的提高。然而在过去30年里骨肉瘤病人的总体生存率并没有随着抗骨肉瘤治疗技术的提高而明显改变。熊果酸具有多种生物学效应如抗癌、抗病毒、抗细菌、保肝、抗炎等。其中以抗肿瘤作用为主。由于熊果酸生物活性相对较低，很大程度上限制了其进一步研发和使用。我们前期研究发现，自主合成的新型熊果酸衍生物IUA具有抗骨肉瘤作用。然而，IUA对耐药骨肉瘤的效果仍然未知。本课题通过CCK8和细胞克隆形成实验发现，IUA可以增加耐药骨肉瘤细胞KHOSR 和U2OSR对顺铂的敏感性。并通过流式细胞术检测凋亡和western bolt检测凋亡相关蛋白发现IUA可以增加顺铂对耐药骨肉瘤细胞KHOSR 和U2OSR的凋亡。我们继而发现miR-381在耐药骨肉瘤细胞KHOSR 和U2OSR中的表达较正常骨肉瘤细胞KHOS 和U2OS低。而IUA可以提高耐药骨肉瘤细胞KHOSR 和U2OSR中miR-381的表达。通过细胞转染技术使耐药骨肉瘤细胞高表达miR-381发现，提高miR-381的表达可以增加耐药骨肉瘤对顺铂的敏感性。并且，IUA作用协同高表达miR-381与单独IUA作用或者单独高表达miR-381相比，并不能进一步增加耐药骨肉瘤细胞对顺铂的敏感。所以我们可以推断，IUA可以通过提高miR¬-381的表达来逆转耐药骨肉瘤细胞的耐药性。我们通过网站预测发现，HDAC4是miR-381的靶点，通过PCR和western bolt检测发现高表达miR-381可以抑制HDAC4表达。荧光素酶试验确认miR-381可以结合HDAC4。我们通过免疫荧光试验发现IUA作用骨肉瘤细胞后，细胞内HDAC4表达下降。并通过SiRNA干扰耐药骨肉瘤细胞KHOS-R和U2OS¬-R中HDAC4表达发现，降低HDAC4表达也可以逆转骨肉瘤对顺铂的耐药。IUA作用协同干扰HDAC4表达与单独IUA作用或者单独干扰HDAC4表达相比，并不能进一步增加耐药骨肉瘤细胞对顺铂的敏感。所以，IUA可能通过调控miR-381/HDAC4轴来逆转骨肉瘤的耐药。.综上所诉，我们得出结论：IUA可以通过调控miR-381/HDAC4轴来逆转骨肉瘤对顺铂的耐药性。IUA的药理作用及其机制能够在未来为治疗耐药骨肉瘤患者提供新的思路和方案。