Ryhänen Tuomas, Mannermaa Eliisa, Oksala Niku, Viiri Johanna, Paimela Tuomas, Salminen Antero, Atalay Mustafa, Kaarniranta Kai
Department of Ophthalmology, University of Kuopio, Kuopio, Finland.
Eur J Pharmacol. 2008 Apr 28;584(2-3):229-36. doi: 10.1016/j.ejphar.2008.02.010. Epub 2008 Feb 14.
Drug delivery to retinal cells has represented a major challenge for ophthalmologists for many decades. However, drug targeting to the retina is essential in therapies against retinal diseases such as age-related macular degeneration, the most common reason of blindness in the developed countries. Retinal cells are chronically exposed to oxidative stress that contributes to cellular senescence and may cause neovascularization in the most severe age-related macular degeneration cases. Various pre- and clinical studies have revealed that heat shock protein 90 (HSP90) inhibitors, such as geldanamycin and radicicol, are promising drugs in the treatment of different malignant processes. In this study, our goal was to compare the effects of 0.1 microM, 1 microM or 5 microM geldanamycin or radicicol on the oxidative stress response, cytotoxicity, and efflux protein activity (a protein pump which removes drugs from cells) in ARPE-19 (human retinal pigment epithelial, RPE) cells. Our findings indicate that geldanamycin and radicicol increased HSP70 and HSP27 expression analyzed by western blotting. Cellular levels of protein carbonyls were increased in response to 0.1 microM (P=0.048 for 24 h, P=0.018 for 48 h) or 5 microM (P=0.030 for 24 h, P=0.046 for 48 h) radicicol but not to geldanamycin analyzed by ELISA assay. In addition, HNE-protein adducts were accumulated in the RPE cells exposed to 0.1 microM or 5 microM radicicol but not to geldanamycin analyzed by western blotting. However, MTT assay revealed that 5 microM geldanamycin reduced cellular viability 20-30% (P<0.05 for 24 h, P<0.01 for 48 h), but this was not observed at any radicicol concentration in RPE cells. Interestingly, the increased oxidative stress response was associated with efflux protein inhibition (20-30%) when the cells were exposed to 1 microM or 5 microM (P<0.05) radicicol, but not in geldanamycin-treated RPE cells. These novel findings help in understanding the influence of HSP90 inhibition and regulatory mechanisms of drug delivery to retinal cells.
几十年来，向视网膜细胞给药一直是眼科医生面临的一项重大挑战。然而，药物靶向视网膜对于治疗视网膜疾病至关重要，比如年龄相关性黄斑变性，这是发达国家失明的最常见原因。视网膜细胞长期暴露于氧化应激之下，氧化应激会导致细胞衰老，并可能在最严重的年龄相关性黄斑变性病例中引发新生血管形成。各种临床前和临床研究表明，热休克蛋白90（HSP90）抑制剂，如格尔德霉素和萝卜硫素，在治疗不同的恶性病变过程中是很有前景的药物。在本研究中，我们的目标是比较0.1微摩尔/升、1微摩尔/升或5微摩尔/升的格尔德霉素或萝卜硫素对ARPE-19（人视网膜色素上皮，RPE）细胞氧化应激反应、细胞毒性和外排蛋白活性（一种将药物从细胞中清除的蛋白泵）的影响。我们的研究结果表明，通过蛋白质印迹分析，格尔德霉素和萝卜硫素可增加HSP70和HSP27的表达。通过酶联免疫吸附测定法分析，响应于0.1微摩尔/升（24小时时P = 0.048，48小时时P = 0.018）或5微摩尔/升（24小时时P = 0.030，48小时时P = 0.046）的萝卜硫素，蛋白质羰基的细胞水平会升高，但对格尔德霉素则无此现象。此外，通过蛋白质印迹分析，在暴露于0.1微摩尔/升或5微摩尔/升萝卜硫素的RPE细胞中会积累HNE-蛋白质加合物，但对格尔德霉素则无此现象。然而，MTT试验表明，5微摩尔/升的格尔德霉素会使细胞活力降低20 - 30%（24小时时P < 0.05，48小时时P < 0.01），但在RPE细胞的任何萝卜硫素浓度下均未观察到这种情况。有趣的是，当细胞暴露于1微摩尔/升或5微摩尔/升（P < 0.05）萝卜硫素时，氧化应激反应的增加与外排蛋白抑制（20 - 30%）相关，但在格尔德霉素处理的RPE细胞中则不然。这些新发现有助于理解HSP90抑制的影响以及药物向视网膜细胞递送的调节机制。
