Wrześniok Dorota, Surazyński Arkadiusz, Karna Ewa, Buszman Ewa, Pałka Jerzy
Department of Pharmaceutical Chemistry, University Medical School of Silesia, Jagiellońska 4, 41-200 Sosnowiec, Poland.
Life Sci. 2005 Jun 17;77(5):528-38. doi: 10.1016/j.lfs.2004.10.070. Epub 2005 Feb 12.
Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K1 = 1.84 x 10(6) M(-1), and the second class of weak binding sites with the association constant K2 = 5.26 x 10(3) M(-1). The number of total binding sites were n1 = 0.1260 and n2 = 0.2861 mumol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC50 approximately 2 microM). Melanin at 100 microg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to puromycin - treated cells (2 microM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.
嘌呤霉素是一种通过抑制蛋白质合成发挥作用的实验性抗肿瘤抗生素。由于其不良副作用（如内耳和肾脏损伤），该抗生素未被批准用于临床试验。副作用的器官特异性背后的机制尚不清楚。鉴于许多药物会与黑色素形成影响其药理活性的复合物，我们确定了嘌呤霉素是否与黑色素相互作用以及该过程如何影响培养的人皮肤成纤维细胞中胶原蛋白的生物合成。我们的结果表明，嘌呤霉素与黑色素形成复合物。与黑色素结合的嘌呤霉素量随着初始药物浓度的增加而增加。对药物与黑色素结合的Scatchard图分析表明，嘌呤霉素 - 黑色素复合物形成中至少涉及两类独立的结合位点：一类是强结合位点，缔合常数K1 = 1.84 x 10(6) M(-1)，另一类是弱结合位点，缔合常数K2 = 5.26 x 10(3) M(-1)。每1 mg黑色素的总结合位点数量分别为n1 = 0.1260和n2 = 0.2861 μmol嘌呤霉素。我们发现嘌呤霉素诱导胶原蛋白和DNA生物合成的抑制（IC50约为2 μM）。100 μg/ml的黑色素对DNA合成产生约20%的抑制，但对培养的成纤维细胞中的胶原蛋白生物合成没有影响。然而，向用嘌呤霉素处理的细胞（2 μM）中添加黑色素（100 μg/ml）消除了嘌呤霉素对胶原蛋白和DNA生物合成的抑制作用。我们认为，参与胶原蛋白代谢的IGF - I受体表达可能是嘌呤霉素诱导胶原蛋白生物合成抑制的靶点之一。通过Western免疫印迹分析发现，黑色素消除了嘌呤霉素诱导的IGF - I受体表达以及MAP激酶ERK1和ERK2表达的降低。这些数据表明，嘌呤霉素的组织特异性药理活性可能取决于组织中黑色素的丰度。
