Penar P L, Khoshyomn S, Bhushan A, Tritton T R
Division of Neurosurgery, University of Vermont College of Medicine, Burlington, USA.
Neurosurgery. 1997 Jan;40(1):141-51. doi: 10.1097/00006123-199701000-00032.
Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro.
Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor.
Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain.
Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.
多形性胶质母细胞瘤是一种恶性原发性脑肿瘤，尽管进行了积极治疗，患者生存期仍较短，部分原因是其易于侵袭脑组织。多形性胶质母细胞瘤也很独特，因为它是唯一一种表皮生长因子受体（EGFR）过度激活一直与肿瘤生长和患者生存期相关的非上皮性人类肿瘤，并且EGFR激活在体外促进多形性胶质母细胞瘤的侵袭。
使用共聚焦显微镜检查人胶质母细胞瘤球体（来自三名不同患者）与胎鼠脑聚集体的共培养物在0至100 μmol/L染料木黄酮（一种酪氨酸激酶（TK）抑制剂）和3 μmol/L tyrphostin A25（一种特异性EGFR-TK抑制剂）存在下的侵袭情况。
染料木黄酮在10 μmol/L时完全抑制了侵袭（而非附着），这是两种细胞系单层生长抑制的IC20。3 μmol/L的tyrphostin A25（单层的IC20）将第三种细胞系的侵袭率从每小时38.8±6.1%侵袭小时（n = 5）降低至每小时2.9±1.2%侵袭小时（n = 6）（P = 0.0002，双侧t检验，93%抑制），以及从每小时0.54±0.065%（斜率）降至每小时0.028±0.018%（P = 0.00001，95%抑制）。最大侵袭百分比从100±0降至胎鼠脑聚集体的7.4±5.6%。通过32P免疫标记在这些剂量下单层中未检测到与EGFR相关的酪氨酸磷酸化变化，这与低浓度TK抑制剂的已知作用一致。蛋白质印迹法显示野生型和截短型EGFR的表达增加。针对EGFR高亲和力配体结合域的单克隆抗体同样能很好地抑制侵袭，而针对非活性域的抗体则不能。
我们的观察结果支持EGFR激活作为胶质母细胞瘤侵袭正常脑组织的决定因素的作用，并且我们表明在比生长抑制所需浓度低得多的TK抑制剂浓度下即可有效抑制侵袭。
