NIH3T3 serum stimulation and cordycepin treatment (Agilent mouse 60K microarray). NIH3T3 serum stimulation and cordycepin treatment (Agilent mouse 60K microarray)

3’deoxyadenosine, also known as cordycepin, has been widely researched as a potential treatment for cancer, yielding promising results in tissue culture as well as in pre-clinical models. A wide variety of mechanisms of action have been proposed, with little agreement between different studies. Here, we confirm that cordycepin triphosphate is likely to be the active metabolite of cordycepin. Data from single and high throughput experiments showed that cordycepin represses growth factor induced gene expression. Bioinformatic analysis, quantitative PCR and western blotting confirmed that cordycepin blocks the PI3K/AKT/mTOR and/or MEK/ERK pathways in 6 cell lines. Effects of cordycepin on translation through mTOR pathway repression were detectable within 30 minutes, indicating a rapid process. Our data show that cordycepin has a broadly similar mechanism of action in all cell lines studied and indicate that its therapeutic target is a cordycepin triphosphate sensitive molecule that is required for growth factor signal transduction. Overall design: NIH3T3 cells were serum starved for 24 hours, treated with vehicle (DMSO) or vehicle plus 20 μM cordycepin for 1.5 hour and stimulated with serum for 30 minutes, in independent triplicate. Total RNA was isolated and used for microarray analysis.3'-脱氧腺苷（3’deoxyadenosine，又称虫草素（cordycepin））作为癌症潜在治疗药物已被广泛研究，并在组织培养体系与临床前模型中取得了颇具前景的研究成果。目前已有多种作用机制被提出，但不同研究之间尚未达成共识。本研究证实，三磷酸虫草素（cordycepin triphosphate）大概率是虫草素的活性代谢产物。单样本与高通量实验数据显示，虫草素可抑制生长因子诱导的基因表达。生物信息学分析、定量PCR（quantitative PCR）与蛋白质免疫印迹（western blotting）实验证实，虫草素可在6种细胞系中阻断PI3K/AKT/mTOR及/或MEK/ERK信号通路。通过抑制mTOR通路对翻译过程产生的影响可在30分钟内被检测到，表明该过程起效迅速。本研究数据表明，虫草素在所有被研究的细胞系中具有大体相似的作用机制，并指出其治疗靶点为一种对三磷酸虫草素敏感、且参与生长因子信号转导的分子。 整体实验设计：将NIH3T3细胞进行24小时血清饥饿处理，分别以溶剂（二甲基亚砜，DMSO）或含20μM虫草素的溶剂处理1.5小时，随后用血清刺激30分钟，每组设置独立的三重生物学重复。提取总RNA并用于微阵列（microarray）分析。