CYC1通过ROS相关通路调节骨肉瘤生物靶向治疗药物TRAIL耐药性的机制研究

Intractable resistance to chemotherapy drug is the main obstacle of treating OS in clinic. And there is need for new agents to treat OS. A kind of new targeted biotherapy drug, TRAIL (Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand), gains excessive attentions for its effective anti-tumor capacity. But when it comes to treat OS in clinic, it faces big obstacle of drug resistance. In the previous research of our project, with SELDI-TOF-MS (Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry) method, we screened out a biomarker in serum, CYC1. And the further research showed that down-regulating CYC1 expression could enhance the apoptosis of osteosarcoma induced by TRAIL. At the same time, the ROS production was also elevated, and pathway of MAPK was abnormally activated. The expression of Cytc and Bax were also up regulated. Based on our previous research and associated reported papers, we hypothesize that: CYC1 can regulate MAPK pathway and Cardiolipin peroxidation by changing ROS production and then influence the apoptosis of osteosarcoma induced by TRAIL. Down-regulating expression of CYC1 can significantly overcome the drug resistance of TRAIL on osteosarcoma. Our research will reconstruct over-expressed and RNAi interfering of CYC1 gene OS models, and will study the relation between CYC1 and ROS production, the pathway regulating apoptosis induced by TRAIL. Doing those to identify the hypothesis mentioned above, and unveil the molecular mechanism of CYC1 regulating TRAIL refractory on osteosarcoma. Through our study, we may find out the mechanism of TRAIL refractory on OS, and the inhibitor of CYC1 could be a enhancer of TRAIL inducing OS apoptosis, which provide a new method to treat OS with this kind of biotherapy targeted drug.

顽固化疗耐药是制约骨肉瘤（OS）临床疗效的突出难题，亟需新的治疗药物。TRAIL是一种对实体肿瘤有高效抑制作用的新型生物靶向药物，但OS对TRAIL显著耐药使其应用受限。课题组前期发现OS血清标志蛋白CYC1，深入研究发现下调CYC1可显著增强TRAIL诱导的OS细胞凋亡；同时活性氧簇（ROS）生成显著提高，MAPK-JNK通路异常活化，Cytc、Bax等表达增加。基于以上发现，我们假设CYC1通过影响ROS生成调节MAPK通路、CL过氧化，进而改变TRAIL诱导的OS凋亡；下调CYC1可显著克服OS对TRAIL的耐药性。本研究拟构建CYC1过表达和沉默的细胞及动物模型，深入研究CYC1和ROS生成关系及其调控TRAIL诱导凋亡的信号通路，验证以上假说。本研究有望阐明CYC1调控OS对TRAIL固有耐药性的分子机制；CYC1抑制剂可作为TRAIL治疗OS的增敏剂，为OS临床治疗提供新途径。

研究背景：骨肉瘤（OS）是骨科临床中最常见的恶性骨肿瘤，最常发生于青少年儿童。病情严重者常需截肢，并可因肿瘤肺转移等严重并发症而死亡。近年来，一种生物治疗药物——肿瘤坏死因子相关凋亡诱导配体（TRAIL）——在实体恶性肿瘤的临床试验中取得了令人瞩目的研究成果。然而研究发现几乎所有骨肉瘤细胞株对TRAIL均具有耐药性, 使TRAIL在骨肉瘤中的临床应用大大受限。课题组前期发现CYC1在骨肉瘤患者血清中的表达显著增加且发现下调CYC1可以抑制OS细胞增殖，同时显著增强TRAIL诱导的OS凋亡，且蛋白芯片提示ROS下游的MAPK通路异常激活，Bax、Cytc表达增高。.研究内容：1、CYC表达对ROS系统的调节作用，具体包括CYC1过表达和CYC1-RNAi 沉默细胞株复苏、细胞株中ROS含量检测、ROS生成干扰因素的检测和排除以及CYC1 基因补偿(Rescue)实验；2、研究并验证ROS调控骨肉瘤对TRAIL耐药性的分子机制，这个部分又分 A、B 两个假说进行研究，分为细胞体外实验和动物体内实验 3、测试A、B两通路协同作用，为临床应用研究打下基础，具体包括在细胞和动物水平测试A、B两通路协同作用。.结果：本研究通过构建CYC1过表达和沉默的细胞及动物模型，深入研究了CYC1和ROS生成关系及其调控TRAIL诱导凋亡的信号通路，验证了：CYC1通过影响ROS生成调节MAPK通路、CL过氧化，进而改变TRAIL诱导的OS凋亡；下调CYC1可显著克服OS对TRAIL的耐药性。.结论及意义：阐明了CYC1调控OS对TRAIL固有耐药性的分子机制，对于探索骨肉瘤耐药的分子机制具有重要意义：CYC1抑制剂可作为TRAIL疗OS的增敏剂，为OS临床治疗提供新途径。