多靶向联合干预肉瘤的分子生物学机制

Sarcomas are poor prognosis cancers that arise in younger individuals thus posing a major public health concern. Despite aggressive strategy including surgery and multi modal chemotherapy regimen, the overall survival is 40-70% at early stage and 20-25% at metastatic stage. These statistics highlight the inadequacy of current therapies and the need to find new strategies to manage these cancers. An approach optimizing chemotherapy and reducing the metastatic potential disease using targeted therapies seems to be relevant..Autonomous cell growth resulting in tissue invasion and metastases is the defining feature of all malignant neoplasm. Sarcomas do not necessarily arise solely as a result of an accelerated rate of cell proliferation. Rather, they are the consequence of an imbalance between the rate of cell-cycle progression (cell division) and cell growth (cell mass) on one hand, and programmed cell death (apoptosis) on the other. It is now recognized that aberrant cellular signal transduction pathways play a vital role in driving this imbalance and hence malignant transformation. According to Hanahan and Weinberg, cancer development is the result of six acquired capabilities: self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis and evading apoptosis. Based on this conceptualization, we propose to target different hallmark capabilities of cancer, using combination of new therapies and in concert with standard chemotherapies in sarcomas. .We study two sarcoma cell lines: RD18 (rhabdomyosarcoma) and TC 71 (Ewing's sarcoma). Each cell line was exposed to increasing concentrations of vorinostat, 17-DMAG, and sorafenib as monotherapy, then combined 2 by 2 and with doxorubicin. Viability was assessed by MTS assay. The Chou and Talalay combination index (CI) was used to highlight additive, synergistic or antagonistic effect of combinations. Cell cycle analysis and measure of apoptosis by Annexin V and caspase 3/7 were studied for the most potent triple combinations using Fluorescence activated cell sorter analysis. The optimized combination will be used in vitro and vivo tumor. Western blot and Immunohitology chemistry staining will be used to studay molecular and cellular mechanisms .The major goal of this proposal is to initiate a new strategy for the sarcoma treatment based on a combination of targeted therapies, and whether or not these combinations could improve the standard sarcoma chemotherapy efficacy. Moreover, molecular and cellular mechanisms will be studied to understand the obtained synergies between agents.

肉瘤是一种好发于年青人的恶性肿瘤，该病的死亡率极高。肉瘤的发生机制不详，放疗效果差，手术切除易复发，化疗易耐药，这些特点导致肉瘤的预后极差。因此，急需进一步揭示肉瘤的发生机制，探索新的有效治疗方法。目前，临床应用单一靶点药物治疗肿瘤，存在局限性。为此，多靶向联合用药将成为未来肿瘤治疗的方向。我们预实验显示，doxorubincine（临床化疗药）和去乙酰化酶、休克蛋白90及VEGF受体的抑制剂合用，可显著抑制肉瘤细胞生长和促进细胞凋亡。在此基础上，本课题拟用肉瘤细胞系和裸鼠肉瘤接种模型，采用基因芯片、流式细胞仪和其他分子生物学技术，比较单独和联合用药的抑瘤效果，观察用药前后的细胞增殖、凋亡、血管生成、信号传导通路关键蛋白等变化规律，揭示多靶向联合用药的抑癌机理和信号传导通路，有望发现肉瘤的特异性生物标记物，从而为肉瘤的早期发现和多靶向联合治疗提供理论基础和实验依据。

肉瘤的发生机制不详，放疗效果差，手术切除易复发，化疗易耐药预后极差。该课题主要是注重于进一步揭示肉瘤的发生机制，探索新的有效治疗方法。鉴于多靶向联合用药是未来肿瘤治疗的方向，本课题揭示了多靶向联合用药的抑癌机理，为肉瘤的早期发现和多靶向联合治疗提供了理论基础和实验依据。我们对三个小细胞肉瘤细胞系进行了研究:RD18(横纹肌瘤),A204(未分化肉瘤)和TC 71(尤文氏肉瘤)。根据项目设计用不同的药物靶定向引起肿瘤的不同原因，选用多个因子同时与一种常规的化疗联合，观察用药前后的细胞增殖、凋亡、血管生成、信号传导通路关键蛋白等变化规律，揭示多靶向联合用药的抑癌机理和信号传导通路。每个细胞系被用以递增浓度的伏立诺他（vorinostat），格尔德霉素（17-DMAG）、阿巴卡韦或索拉非尼（sorafenib）做为单一阻断剂，然后两俩结合， 再与常规的化疗药阿霉素（doxorubicin）结合用药。在单一药治疗中, 除了阿巴卡韦外，单一的靶向制剂能降低30 -90%细胞生存率。而vorinostat（伏立诺他），索拉非尼与17-DMAG的双靶向联合疗法均具有协同作用。因阿巴卡韦与其他药物拮抗，没有做进一步的研究。阿霉素与vorinostat和17-DMAG联合,杀死了60%的细胞，而单独使用阿霉素仅杀死12% 。索拉非尼与阿霉素联合用药没有协同作用。用vorinostat、 17-DMAG和阿霉素三联疗法没有显示任何协同作用,但三种药物联合与细胞孵育24小时后，通过检测subG1的量，观察到细胞凋亡从30%增加到70%。我们的结果还确定SAHA, sorafenib 和17-DMAG双重靶向治疗中表现出较强协同协效应。其中以vorinostat、17-DMAG加阿霉素对尤文氏肉瘤肉瘤细胞影响最为显著。vorinostat加17-DMAG、17-DMAG加阿霉素的协同作用是通过抑制AKT信号传导路中的AKT的表达及AKT的磷酸化来实现的。 在研究中我们还意外发现：100 mM乙醇能增加阿霉素治疗软骨肉瘤的疗效，通过抑制软骨肉瘤生长和迁移、严重阻滞细胞周期，并促进p53 mRNA的表达。同时，100 mM低浓度的乙醇不仅不影响肝细胞活力还能够保护阿霉素肝细胞损伤。100 mM乙醇与阿霉素联合使用可能成为一种新的有效治疗软骨肉瘤方法。