Gurbuz Venhar, Konac Ece, Varol Nuray, Yilmaz Akin, Gurocak Serhat, Menevse Sevda, Sozen Sinan
Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara 06500, Turkey ; Department of Pediatric Infectious Diseases, Faculty of Medicine, Hacettepe University Ankara, Ankara 06100, Turkey.
Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara 06500, Turkey.
Oncol Lett. 2014 Mar;7(3):755-763. doi: 10.3892/ol.2014.1795. Epub 2014 Jan 14.
The aim of the present study was to analyze the molecular mechanisms involved in blocking the signaling pathway and the effects of this on the progression of prostate cancer (CaP) cells . LNCaP human CaP cell line was stimulated with interleukin-6 (IL-6) in the presence/absence of Janus kinase (JAK) 2 (AG490), signal transducer and activator of transcription 3 [(STAT3) S3I-201] inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cytotoxic activity, the activation of phosphorylated (p)-STAT3 protein, caspase (CASP) 3 activity at protein level, vascular endothelial growth factor (VEGF) A, VEGFC, vascular endothelial growth factor receptor 2, STAT3, matrix metalloproteinase-2, myeloid cell leukemia sequence 1 (MCL-1), CASP8 and CASP9 messenger RNA (mRNA) levels were determined. Morphology and apoptosis were confirmed by DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. IL-6 rapidly induced the phosphorylation of STAT3 in a dose- and time-dependent manner with a peak expression at 3 h at a concentration of 25 ng/ml. In addition, AG490 (50 μM) and S3I-201 (300 μM) inhibited STAT3 activation. Western blotting results revealed that p-STAT3 protein expression decreased significantly with AG490 and S3I-201 treatment in LNCaP cells. AG490 and S3I-201 induced the downregulation of VEGFA, MCL-1 and STAT3 and the upregulation of CASP8 and CASP9 mRNA transcription levels. In addition, the inhibitors increased the level of CASP3 protein. Combinations of AG490- and S3I-201-TRAIL did not result in an increase in this effect. Parallel results were found by DAPI staining and TUNEL assay. To the best of our knowledge, this is the first study to investigate the possible clinical use of AG490 or S3I-201, together with the reduced use of chemotherapeutic agents with high cytotoxicity, for their ability to exert an apoptotic effect, targeting the JAK/STAT3 pathway.
本研究的目的是分析参与阻断信号通路的分子机制及其对前列腺癌细胞（CaP）进展的影响。在存在/不存在Janus激酶（JAK）2（AG490）、信号转导和转录激活因子3 [（STAT3）S3I-201]抑制剂以及肿瘤坏死因子相关凋亡诱导配体（TRAIL）的情况下，用白细胞介素-6（IL-6）刺激LNCaP人CaP细胞系。测定细胞毒性活性、磷酸化（p）-STAT3蛋白的激活、蛋白水平的半胱天冬酶（CASP）3活性、血管内皮生长因子（VEGF）A、VEGFC、血管内皮生长因子受体2、STAT3、基质金属蛋白酶-2、髓样细胞白血病序列1（MCL-1）、CASP8和CASP9信使核糖核酸（mRNA）水平。通过4',6-二脒基-2-苯基吲哚（DAPI）染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）试验确认形态和凋亡情况。IL-6以剂量和时间依赖性方式迅速诱导STAT3磷酸化，在浓度为25 ng/ml时3小时达到峰值表达。此外，AG490（50 μM）和S3I-201（300 μM）抑制STAT3激活。蛋白质印迹结果显示，在LNCaP细胞中，AG490和S3I-201处理后p-STAT3蛋白表达显著降低。AG490和S3I-201诱导VEGFA、MCL-1和STAT转录水平下调以及CASP8和CASP9 mRNA转录水平上调。此外，抑制剂增加了CASP3蛋白水平。AG490、S3I-201和TRAIL联合使用并未增强这种效应。DAPI染色和TUNEL试验得到了类似结果。据我们所知，这是第一项研究AG490或S3I-201可能的临床应用以及减少高细胞毒性化疗药物使用的研究，因其能够通过靶向JAK/STAT3途径发挥凋亡作用。
