Heimberger Amy B, Wang Enze, McGary Eric C, Hess Kenneth R, Henry Verlene K, Shono Tadahisa, Cohen Zvi, Gumin Joy, Sawaya Raymond, Conrad Charles A, Lang Frederick F
Brain Tumor Center, Department of Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Neuro Oncol. 2005 Jan;7(1):1-11. doi: 10.1215/S1152851704000420.
Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type-1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphatebuffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1alpha in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial.
雷帕霉素此前已被证明对脑内胶质瘤异种移植有效，并对胶质瘤具有细胞生长抑制作用。然而，关于雷帕霉素的作用机制知之甚少。我们研究的目的是进一步研究雷帕霉素的体外和体内作用机制，阐明可能适用于临床试验研究的分子终点，并研究治疗失败的潜在机制。在10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）缺失的胶质瘤细胞系U-87和D-54中，但在少突胶质细胞瘤细胞系HOG（PTEN缺失）中未观察到，IC50剂量的雷帕霉素导致细胞在G1期积累，早在给药后24小时，进入S期的细胞比例相应下降。所有测试的胶质瘤细胞系在用雷帕霉素培养时，即使在低于IC50的剂量下，血管内皮生长因子（VEGF）的产生也显著减少。暴露于雷帕霉素48小时后，胶质瘤细胞系（但不包括HOG细胞）显示膜型1基质金属蛋白酶（MMP）侵袭分子下调。在U-87细胞中，MMP-2下调，在D-54细胞中，雷帕霉素处理后MMP-2和MMP-9均下调。对体内已建立的皮下U-87异种移植瘤进行治疗导致肿瘤明显消退（P < 0.05）。对皮下U-87肿瘤的免疫组织化学研究表明，用雷帕霉素治疗的小鼠中VEGF的产生减少。明胶酶谱分析显示，与用磷酸盐缓冲盐水处理的对照组相比，用雷帕霉素处理的皮下U-87异种移植瘤小鼠中MMP-2明显减少。相比之下，对已建立的脑内U-87异种移植瘤进行治疗，尽管肿瘤内的Akt途径受到抑制，但并未导致中位生存期延长。此外，与我们对皮下肿瘤的研究结果相反，脑内U-87异种移植瘤的免疫组织化学和定量蛋白质印迹分析结果表明，VEGF的产生并不显著，这表明脑内缺氧诱导因子1α可能存在差异调节。这些发现表明，雷帕霉素对胶质瘤的复杂作用机制包括细胞生长抑制、抗VEGF和抗侵袭活性，但这些作用取决于肿瘤在体内的位置，对临床试验的设计具有重要意义。
