Boonjaraspinyo Sirintip, Boonmars Thidarut, Wu Zhiliang, Loilome Watcharin, Sithithaworn Paiboon, Nagano Isao, Pinlaor Somchai, Yongvanit Puangrat, Nielsen Phuangphaka Sadee, Pairojkul Chawalit, Khuntikeo Narong
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.
Tumour Biol. 2012 Oct;33(5):1785-802. doi: 10.1007/s13277-012-0438-8. Epub 2012 Jun 26.
Our previous report showed that platelet-derived growth factor (PDGF) and related genes were upregulated in a Syrian hamster model and could be detected in all human cholangiocarcinoma (CCA) tissues. We therefore hoped that PDGF could be used as a diagnostic and prognostic marker. We analyzed 78 samples of human CCA and adjacent tissues for PDGF and related gene expression, and localized PDGF protein expression. The mechanism of anti-cancer drugs on PDGF and related genes or proteins in CCA cell lines (OCA17, M156, and KKU100) was studied through MTT cell viability assay, quantitative real-time PCR, and immunoblotting. Mutagenesis of the PDGFRA coding region was analyzed. Moreover, the PDGFRA in sera of CCA patients and healthy controls was investigated. PDGFA was found to be upregulated in CCA tissue (84.6 %). Positive PDGFA immunohistochemical staining was significantly correlated with status (P = 0.000), stage of CCA (P = 0.013), metastasis (P = 0.017), and short survival rate (P = 0.005), and the multivariate analysis confirmed that PDGFA positive immunostaining had a higher likelihood of the risk of death (HR = 2.907, P = 0.016). For DNA point mutation of the PDGFRA sequence, silent mutations were found at tyrosine kinase 2 V824V (exon 18) and A603A (exon 13), and a missense mutation in S478P (exon 10); there was only a missense mutation in S478P (29 %) that has significant correlation with the histopathological grading (P = 0.037) and positive immunoreactive PDGFA (P = 0.021). In vitro cell line study by immunowestern blotting found that sunitinib malate had an inhibitory effect on the PDGFA pathway by decreasing p-PDGFRA, AKT, and p-AKT expression. The serum level of PDGFA in CCA patients was significantly higher than those of healthy control by 1.4-fold (P = 0.014). The present results suggest that PDGFA and PDGFRA may be used for CCA prognosis and/or as diagnostic candidate markers.
我们之前的报告显示，血小板衍生生长因子（PDGF）及相关基因在叙利亚仓鼠模型中上调，且在所有人类胆管癌（CCA）组织中均可检测到。因此，我们希望PDGF可作为一种诊断和预后标志物。我们分析了78份人类CCA及相邻组织样本中PDGF及相关基因的表达，并对PDGF蛋白表达进行定位。通过MTT细胞活力测定、定量实时PCR和免疫印迹研究了抗癌药物对CCA细胞系（OCA17、M156和KKU100）中PDGF及相关基因或蛋白的作用机制。分析了PDGFRA编码区的诱变情况。此外，还研究了CCA患者和健康对照血清中的PDGFRA。发现PDGFA在CCA组织中上调（84.6%）。PDGFA免疫组化阳性染色与CCA的状态（P = 0.000）、分期（P = 0.013）、转移（P = 0.017）及短生存率（P = 0.005）显著相关，多因素分析证实PDGFA阳性免疫染色有更高的死亡风险可能性（HR = 2.907，P = 0.016）。对于PDGFRA序列的DNA点突变，在酪氨酸激酶2的V824V（外显子18）和A603A（外显子13）处发现沉默突变，在S478P（外显子10）处发现错义突变；仅S478P处的错义突变（29%）与组织病理学分级（P = 0.037）和PDGFA免疫反应阳性（P = 0.021）有显著相关性。通过免疫印迹法进行的体外细胞系研究发现，苹果酸舒尼替尼通过降低p - PDGFRA、AKT和p - AKT的表达对PDGFA通路有抑制作用。CCA患者血清中PDGFA水平显著高于健康对照，为其1.4倍（P = 0.014）。目前的结果表明，PDGFA和PDGFRA可用于CCA的预后评估和/或作为诊断候选标志物。
