Gabor F, Wollmann K, Theyer G, Haberl I, Hamilton G
Institute of Pharmaceutical Technology, University of Vienna, Austria.
Anticancer Res. 1994 Sep-Oct;14(5A):1943-50.
The 3-5 year survival rates of patients with disseminated Ewing's sarcoma (ES) or the closely related peripheral primitive neuroectodermal tumors (PNET) remain low, even under aggressive treatment involving highly toxic multidrug chemotherapeutic regimens. ES and PNET are sensitive to doxorubicin, but may escape treatment by expression of the multidrug-resistant phenotype and/or other mechanisms. In this study, we have identified albumin as growth supporting factor for ES and PNET cells in IGF-I-supplemented serum-free tissue culture medium. To investigate the specificity and toxicity of albumin-based drug conjugates, doxorubicin was coupled to bovine serum albumin (BSA) by either a two step glutaraldehyde or carbodiimide-C4-spacer technique, yielding monomeric DOX-albumin conjugates with conjugation numbers ranging from 3-20 moles DOX/mole BSA. Cellular uptake of fluorescein-isothiocyanate-(FITC)-labeled albumin and DOX-albumin conjugates could be demonstrated by flow cytometric measurements of cell-associated fluorescence and confocal microscopy. The cytostatic activity of these conjugates against ES/PNET cell lines, a neuroblastoma (LAN-1) and prostate cancer carcinoma cell line (PC-3) and normal lymphoblasts was tested in short-term proliferation assays (48 h). The results show a high selectivity of the DOX-albumin conjugates for ES/PNET cell lines, with highest growth inhibition by conjugates with low DOX conjugation numbers (n = 3) in serum-supplemented medium (17-32 fold loss of activity compared to free DOX), followed by 20-DOX-C4-albumin in serum-free medium and low activity of the other conjugates. In conclusion, DOX-albumin conjugates inhibit the growth of ES/PNET cell lines selectively, showing low activity against the unrelated carcinoma line PC-3 and sparing normal lymphoblasts. The inverse correlation of activity and conjugation number demonstrates a low cytotoxic activity of DOX in acid-stable binding to monomeric albumin, pointing to a selective cytostatic activity of the modified albumin against ES and PNET cells, even in the presence of a 100 fold excess of unmodified serum albumin.
即使采用包含高毒性多药化疗方案的积极治疗，播散性尤因肉瘤（ES）或密切相关的外周原始神经外胚层肿瘤（PNET）患者的3至5年生存率仍然很低。ES和PNET对多柔比星敏感，但可能通过多药耐药表型的表达和/或其他机制逃避治疗。在本研究中，我们已确定白蛋白是在补充胰岛素样生长因子I（IGF-I）的无血清组织培养基中支持ES和PNET细胞生长的因子。为了研究基于白蛋白的药物偶联物的特异性和毒性，通过两步戊二醛法或碳二亚胺-C4-间隔技术将多柔比星与牛血清白蛋白（BSA）偶联，得到结合数范围为3至20摩尔多柔比星/摩尔BSA的单体多柔比星-白蛋白偶联物。通过流式细胞术测量细胞相关荧光和共聚焦显微镜可证明异硫氰酸荧光素（FITC）标记的白蛋白和多柔比星-白蛋白偶联物的细胞摄取。在短期增殖试验（48小时）中测试了这些偶联物对ES/PNET细胞系、神经母细胞瘤（LAN-1）和前列腺癌细胞系（PC-3）以及正常淋巴细胞的细胞生长抑制活性。结果显示多柔比星-白蛋白偶联物对ES/PNET细胞系具有高选择性，在补充血清的培养基中，结合数低（n = 3）的偶联物具有最高的生长抑制作用（与游离多柔比星相比活性损失17至32倍），其次是无血清培养基中的20-DOX-C4-白蛋白，其他偶联物活性较低。总之，多柔比星-白蛋白偶联物选择性抑制ES/PNET细胞系的生长，对不相关的癌细胞系PC-3活性低，对正常淋巴细胞无损害。活性与结合数的负相关表明多柔比星与单体白蛋白的酸稳定结合具有低细胞毒性活性，表明修饰的白蛋白对ES和PNET细胞具有选择性细胞生长抑制活性，即使存在100倍过量的未修饰血清白蛋白。
