Functional Genome Analysis, German Cancer Research Centre (DKFZ), Heidelberg, Germany.
Br J Pharmacol. 2011 Mar;162(5):1045-59. doi: 10.1111/j.1476-5381.2010.01125.x.
Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed.
Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down.
Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death.
Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s).
由于化疗耐药性高和转移速度快，胰腺癌是癌症相关死亡的主要原因之一。Nemorosone 是一种多环聚异戊二烯基酰基间苯三酚，最近被确定为一种有前途的抗癌药物。在这里，我们研究了它对胰腺癌细胞的生长抑制作用。基于转录谱分析，提出了一种作用模式。
用 Resazurin 增殖测定法评估 Nemorosone 对胰腺癌细胞和成纤维细胞的细胞毒性。通过 Annexin V/碘化丙啶染色以及细胞色素 c 和半胱天冬酶激活测定来确定细胞凋亡。使用电压依赖性染料 JC-1 和荧光显微镜来检测对线粒体膜电位的影响。从处理的细胞系中分离总 RNA，并进行微阵列分析、随后的途径鉴定和建模。通过定量聚合酶链反应和 siRNA 介导的基因敲低来验证基因表达数据。
Nemorosone 显著抑制癌细胞生长，诱导细胞色素 c 释放和随后的半胱天冬酶依赖性细胞凋亡，迅速消除线粒体膜电位并增加细胞浆钙水平，而成纤维细胞则基本不受影响。表达谱分析显示 336 个基因受 Nemorosone 影响。共有 75 个基因在所有三种细胞系中都发生了改变，其中许多基因都在未折叠蛋白反应 (UPR) 网络内。发现 DNA 损伤诱导转录本 3 是 UPR 介导的细胞死亡中的关键调节因子。
Nemorosone 可能是开发新型抗癌药物的先导化合物，该药物可增强实体瘤中已经升高的 UPR 水平，从而将其诱导进入凋亡。本研究为进一步鉴定 Nemorosone 的直接分子靶标奠定了基础。
