Department of Oncology, Faculty of Medicine Dentistry and Health Sciences, The Institute for Cancer Studies, University of Sheffield, Sheffield S10 2RX, UK.
Br J Cancer. 2011 Mar 29;104(7):1098-105. doi: 10.1038/bjc.2011.56. Epub 2011 Mar 8.
Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU).
Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation.
Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM.
We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.
葡萄膜黑色素瘤（UM）是成人中最常见的原发性眼内肿瘤，常转移至肝脏。肝转移瘤难以治疗，主要对化疗无反应。为了研究为什么 UM 如此具有化疗耐药性，我们研究了丝裂霉素 C（MMC）和顺铂与羟基脲（HU）相比对视交叉连接剂的影响。
采用集落形成试验检测 MMC、顺铂和 HU 在已建立的 UM 细胞系中的敏感性。在短期 UM 原代培养的 MTT 测定中证实了 UM 对 MMC 的反应。通过 Western blot 分析细胞色素 P450 还原酶（CYP450R）的表达，并通过 COMET 分析结合 γ-H2AX 焦点形成评估 DNA 交联。
两种已建立的细胞系和 UM 的原代培养均对交联剂 MMC 具有耐药性（在每种情况下，与对照组相比，Student's t 检验均<0.001）。在两种已建立的 UM 细胞系中，MMC 未诱导 DNA 交联损伤（与对照组相比，在每种情况下，Student's t 检验均<0.05）。总共，在 6 个 UM 中测试的 6 个显示出代谢酶 CYP450R 的表达减少，并且 CYP450R 的瞬时表达增加了 UM 对 MMC 的敏感性。
我们认为，CYP450R 的表达减少导致 UM 对 MMC 的耐药性，这是由于缺乏生物激活所致，通过用 CYP450R 补充 UM 细胞系可以逆转这种情况。
