Trog Daniela, Yeghiazaryan Kristina, Fountoulakis Michael, Friedlein Arno, Moenkemann Heike, Haertel Nicolai, Schueller Heinrich, Breipohl Winrich, Schild Hans, Leppert David, Golubnitschaja Olga
Department of Radiology, Friedrich-Wilhelms-University of Bonn, D-53105 Germany.
Eur J Pharmacol. 2006 Aug 7;542(1-3):8-15. doi: 10.1016/j.ejphar.2006.05.026. Epub 2006 May 24.
The current chemotherapeutic treatment of glioblastoma patients has minor success. Little is known about the molecular and cellular mechanisms of the resistance of gliomas towards current therapies. This study investigated both suppressive cellular effects and regulation of extracellular matrix remodeling proteins with pro-invasive activity in surviving human glioblastoma cells under clinically relevant treatments. All cellular and molecular biological investigations were performed on the genetically well-defined and clinically relevant p53-wild type U87Mg glioma cells. Malignant glioma cells underwent either radiation or temozolomide treatments alone, or combined chemo/radio treatment. Protein expression patterns were investigated by two-dimensional polyacrylamide gel electrophoresis followed by protein spot identification using tandem mass spectrometry analysis. Specific expression levels were quantified by Western-blotting. Extracellular gelatinase activities for both metalloproteinases MMP-2 and MMP-9 were determined by zymogramms. Survival curves indicated no effective suppression of glioma cells under all treatment conditions tested. Morphological changes demonstrated sub-lethal effect of both temozolomide and combined treatment. Expression of MMP-2, MMP-9, and membrane type 1 matrix metalloproteinases (MT1-MMP) was differentially up-regulated by increasing cellular density and treatment conditions. A significantly enhanced extracellular degrading activity under all treatment conditions tested was demonstrated for MMP-2 only. Being a marker for brain tumour progression and angiogenesis, lysozyme c was highly up-regulated under the combined chemo/radio treatment. The activation of proteins with pro-invasive activity indicates an increasing malignancy grade of surviving glioma cells under treatment conditions tested correlating well with more aggressive tumour phenotypes observed clinically in recurrences of treated glioblastomas.
目前胶质母细胞瘤患者的化疗治疗成效甚微。关于胶质瘤对当前疗法产生耐药性的分子和细胞机制，我们知之甚少。本研究调查了在临床相关治疗下，存活的人胶质母细胞瘤细胞中具有促侵袭活性的细胞抑制作用和细胞外基质重塑蛋白的调控情况。所有细胞和分子生物学研究均在基因明确且临床相关的p53野生型U87Mg胶质瘤细胞上进行。恶性胶质瘤细胞分别接受单独的放射治疗或替莫唑胺治疗，或联合放化疗。通过二维聚丙烯酰胺凝胶电泳研究蛋白质表达模式，随后使用串联质谱分析进行蛋白质斑点鉴定。通过蛋白质印迹法定量特定表达水平。通过酶谱法测定金属蛋白酶MMP-2和MMP-9的细胞外明胶酶活性。生存曲线表明，在所有测试的治疗条件下，胶质瘤细胞均未得到有效抑制。形态学变化表明替莫唑胺和联合治疗均有亚致死效应。MMP-2、MMP-9和膜型1基质金属蛋白酶（MT1-MMP）的表达随着细胞密度和治疗条件的增加而有差异地上调。仅在所有测试的治疗条件下，MMP-2的细胞外降解活性显著增强。作为脑肿瘤进展和血管生成的标志物，溶菌酶c在联合放化疗下高度上调。具有促侵袭活性的蛋白质的激活表明，在测试的治疗条件下，存活的胶质瘤细胞的恶性程度增加，这与在接受治疗的胶质母细胞瘤复发中临床观察到的更具侵袭性的肿瘤表型密切相关。
