Bouterfa H L, Sattelmeyer V, Czub S, Vordermark D, Roosen K, Tonn J C
Department of Neurosurgery, University of Würzburg, Germany.
Anticancer Res. 2000 Jul-Aug;20(4):2761-71.
Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation.
In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates.
Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because supplementation of the media with 50-100 microM mevalonate restored all tau eta epsilon effects. Invasion of tumor cells into brain tissue was not effected while migration was reduced beta upsilon about 30-40% in cells treated with high concentrations (> or = 100 microM) of lovastatin. This was surprising because drug treatment at lower concentrations led to a disruption of the actin cytoskeleton, as indicated by Phalloidin staining.
Our data strongly suggest that inhibition of elevated Ras activity by lovastatin effectively targets the MAPK and probably other signaling pathways thus offering a pharmacological based approach for a potential treatment of human astrocytomas.
恶性星形细胞瘤是人类最常见的原发性颅内肿瘤。由于其高增殖能力和弥漫性侵袭脑的能力，所有治疗方法都受到限制。酪氨酸激酶受体及其信号通路的扩增被认为与星形细胞瘤的分子发病机制有关，为治疗干预提供了可能的新靶点。特别是，星形细胞瘤虽然缺乏致癌性Ras突变，但活化Ras水平升高。洛伐他汀是β-羟基-β-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶）的抑制剂，目前用于治疗高胆固醇血症患者。此外，它抑制Ras超家族蛋白质中几个成员的异戊二烯化，因此具有多种细胞效应，包括减少增殖。
在本研究中，我们研究了洛伐他汀对两种人胶质瘤细胞系以及从多形性胶质母细胞瘤（GBM）患者活检中建立的15种原代细胞培养物的影响。通过细胞计数和MTT测定法测定胶质瘤细胞系和原发性肿瘤细胞的增殖。用鬼笔环肽对肌动蛋白丝进行染色来分析细胞形态。使用TUNEL测定法测量细胞凋亡。为了研究这种药物对胶质瘤细胞运动性的影响，使用三维球体解体试验研究肿瘤细胞迁移。此外，通过肿瘤球体与大鼠脑聚集体之间的对抗试验分析肿瘤细胞侵袭。
使用洛伐他汀抑制法尼基生物合成导致Ras介导的信号传导受阻，表现为MAPK活性降低。因此，肿瘤细胞增殖减少高达80%。洛伐他汀似乎通过诱导凋亡降低胶质瘤活力，形态学变化和TUNEL阳性细胞增加表明了这一点。洛伐他汀通过类异戊二烯耗竭起作用，因为用50-100 microM甲羟戊酸补充培养基可恢复所有效应。肿瘤细胞向脑组织的侵袭未受影响，而在高浓度（≥100 microM）洛伐他汀处理的细胞中迁移减少约30-40%。这令人惊讶，因为较低浓度的药物处理导致肌动蛋白细胞骨架破坏，鬼笔环肽染色表明了这一点。
我们的数据强烈表明，洛伐他汀抑制升高的Ras活性有效地靶向MAPK以及可能的其他信号通路，从而为人类星形细胞瘤的潜在治疗提供了一种基于药理学的方法。
