Glioma cell integrin expression and their interactions with integrin antagonists: Research Article.
Glioma cell integrin expression and their interactions with integrin antagonists: Research Article.
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发表时间：
2005
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通讯作者：
R. Mattern;S. B. Read;M. Pierschbacher;C. Sze;B. Eliceiri;C. Kruse
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作者：
R. Mattern;S. B. Read;M. Pierschbacher;C. Sze;B. Eliceiri;C. Kruse
使用α（ν）β-特异性抗体通过流式细胞术筛选一组人神经胶质瘤细胞外植体的整联蛋白表达。较低百分比的胶质瘤细胞对α（ν）β3（平均阳性%= 20.8%）整联蛋白呈阳性，而较高百分比的胶质瘤细胞对ανβ5（平均阳性%= 72.7%）、VLA 5 α（平均阳性%= 87%）和VLAβ1（平均阳性%= 41.7%）整联蛋白呈阳性。设计、合成并测试一系列RGD肽与整联蛋白受体的结合。基于与分离的整联蛋白受体的结合和整联蛋白在神经胶质瘤细胞系上的表达的结果，选择与α（ν）β3、α（ν）β5和α（ν）β（1）有效结合的肽用于进一步研究其对神经胶质瘤细胞的作用。该肽Ac-c[（Pen）-Tyr（Me）-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH（2）（RGD肽）在置于人工脑脊液中的大鼠脑细胞匀浆中具有良好的稳定性，因此在临床颅内给药中具有很高的潜力。使用HPLC方法定量大鼠脑细胞匀浆中的肽，我们可以证明的RGD肽的半衰期约为20 hr. Relative乱序肽对照（非RGD序列，相同的氨基酸），实验的RGD肽显着降低胶质瘤细胞增殖的大鼠和人胶质瘤细胞测试的整个面板。最近传代的胶质瘤细胞的胶质瘤衍生的细胞外基质蛋白包被的板的粘附被显著抑制的RGD肽。该肽还逆转了接种的神经胶质瘤细胞的附着。RGD肽引起了一些，但不是实质性的，胶质瘤细胞损伤，证明了在体外核DNA形态学定量分析和流式细胞术分析采用7-氨基放线菌素D（7AAD）。我们在组织学上监测了不同剂量的RGD肽重复注入正常插管大鼠脑中所引起的毒性。在安全剂量下，实验性RGD肽处理的脑与输注乱序肽或缓冲液处理的对照组没有显示出显著差异。在携带肿瘤的脑中，相对于乱序肽处理的对照，在RGD肽处理的脑中测量到略小的肿瘤面积，具有更高的坏死-肿瘤指数。这通过颅内肽给药或颅内和腹膜内联合注射获得。从该体外工作中，我们得出结论，所测试的RGD肽的抗神经胶质瘤作用是由于神经胶质瘤增殖和粘附/移动性降低，而不是由于在所分析的时间范围内的显著神经胶质瘤细胞损伤。虽然从我们有限的组织病理学观察中没有发现的其他机制可能是可操作的，但从我们的体内工作中，我们得出结论，重复施用RGD肽到脑中是安全的，但必须更好地将肽递送到浸润的肿瘤细胞。
A panel of human glioma cell explants was screened for integrin expression by flow cytometry using α(ν)β-specific antibodies. A lower percentage of the glioma cells were positive for the α(ν)β3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανβ5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAβ1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the α(ν)β3, α(ν)β5 and α(5)β(1) was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH(2) (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD peptide-treated brains did not show significant differences from those infused with scrambled peptide or buffer-treated controls. In tumor-bearing brains, slightly smaller tumor areas were measured with a higher necrotic-to-tumor index in the RGD peptide treated relative to the scrambled peptide-treated controls. This was obtained with intracranial peptide administrations or combined intracranial and intraperitoneal injections. From this in vitro work, we conclude that the anti-glioma effects of the RGD peptide tested resulted from lowered glioma proliferation and adhesion/mobility, rather than from significant glioma cell injury in the timeframe analyzed. Although other mechanisms not discerned from our limited histopathological observations may be operational, from our in vivo work, we conclude that repeated administration of RGD peptide into brain is safe but that better delivery of the peptides to infiltrating tumor cells is necessary.