Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo
Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo
复制标题
DOI：
10.1016/s0002-9440(10)62555-2
复制
发表时间：
2002-04-01
影响因子：
6
通讯作者：
Martin, PM
中科院分区：
文献类型：
--
作者：
Ouafik, L;Sauze, S;Martin, PM
胶质母细胞瘤是最常见的恶性脑肿瘤，目前尚无有效的治疗方法。生长因子是治疗策略的潜在靶点，因为它们对肿瘤生长和进展至关重要。肽酰甘氨酸a-酰胺化单加氧酶是从其无活性的甘氨酸延伸前体产生a-酰胺化生物活性肽的酶。成胶质细胞瘤和神经胶质瘤细胞系中肽酰甘氨酸α-酰胺化单加氧酶mRNA的高表达表明α-酰胺化肽参与脑中的致瘤生长过程。在筛选酰胺化肽后，发现人胶质母细胞瘤细胞系表达高水平的肾上腺髓质素（AM）mRNA，并且免疫反应性AM被释放到培养基中。AM是一种多功能调节肽，具有促有丝分裂和血管生成等能力。实时定量逆转录-聚合酶链反应分析显示AM mRNA与肿瘤类型和分级相关，在所有分析的胶质母细胞瘤中均呈高表达，而在间变性星形细胞瘤中呈低表达，在低级别星形细胞瘤和少突胶质细胞瘤中几乎检测不到。在本研究中，我们还证明了存在的mRNA编码的假定AM受体，降钙素受体样受体/受体活性修饰蛋白-2和-3（CRLR/RAMP 2; CRLR/RAMP 3）在胶质瘤组织和胶质母细胞瘤细胞系，并进一步表明，外源性添加AM可以刺激这些胶质母细胞瘤细胞在体外的生长。这些结果表明，AM可能作为胶质母细胞瘤细胞的自分泌生长因子。检验自分泌假说的一种方法是中断内源性AM的功能。在此，我们证明了特异于AM的多克隆抗体阻断了激素与其细胞受体的结合，并在体外使U87胶质母细胞瘤细胞的生长降低了33%（P < 0.001）。肿瘤内施用抗AM抗体导致治疗后21天皮下U87异种移植物重量减少70%（P < 0.001）。此外，在抗体处理的肿瘤中血管密度降低。这些发现支持AM可能作为人胶质母细胞瘤的有效自分泌/旁分泌生长因子发挥作用，并证明抑制AM（由肿瘤细胞产生）的作用可能抑制体内肿瘤生长。
Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Growth factors are potential targets for therapeutic strategies because they are essential for tumor growth and progression. Peptidylglycine a-amidating monooxygenase is the enzyme producing a-amidated bioactive peptides from their inactive glycine-extended precursors. The high expression of peptidylglycine a-amidating monooxygenase mRNA in glioblastoma and glioma cell lines points to the involvement of a-amidated peptides in tumorigenic growth processes in the brain. After screening of amidated peptides, it was found that human glioblastoma cell lines express high levels of adrenomedullin (AM) mRNA, and that inummoreactive AM is released into the culture medium. AM is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities among others. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that AM mRNA was correlated to the tumor type and grade, with high expression in all glioblastomas analyzed, whereas a low expression was found in anaplastic astrocytomas and barely detectable levels in low-grade astrocytomas and oligodendrogliomas. In the present study we also demonstrate the presence of mRNA encoding the putative AM receptors, calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) in both glioma tissues and glioblastoma cell lines and further show that exogenously added AM can stimulate the growth of these glioblastoma cells in vitro. These findings suggest that AM may function as an autocrine growth factor for glioblastoma cells. One way to test the autocrine hypothesis is to interrupt the function of the endogenously produced AM. Herein, we demonstrate that a polyclonal antibody specific to AM, blocks the binding of the hormone to its cellular receptors and decreases by 33% (P < 0.001) the growth of U87 glioblastoma cells in vitro. Intratumoral administration of the anti-AM antibody resulted in a 70% (P < 0.001) reduction in subcutaneous U87 xenograft weight 21 days after treatment. Furthermore, the density of vessels was decreased in the antibody-treated tumors. These findings support that AM may function as a potent autocrine/paracrine growth factor for human glioblastomas and demonstrate that inhibition of the action of AM (produced by tumor cells) may suppress tumor growth in vivo.