Department of Medicine, Graduate Institute of Biochemistry, Kaohsiung Medical University, Taiwan.
Biochem Pharmacol. 2011 Jul 1;82(1):35-42. doi: 10.1016/j.bcp.2011.03.026. Epub 2011 Apr 6.
Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.
吉西他滨（2'-脱氧-2',2'-二氟胞苷；Gem）是一种核苷抗代谢物，常用于治疗包括膀胱癌在内的多种人类癌症。吉西他滨不仅作为自杀核苷类似物起作用，还抑制 DNA 聚合酶活性并导致链延伸终止。通过二维凝胶电泳分析，鉴定出一种由吉西他滨诱导的蛋白为 UBE2M（也称为 UBC12），它是一种 NEDD8 缀合 E2 酶，有助于蛋白质降解。吉西他滨在几种人类癌细胞系中诱导 UBE2M 在 RNA 和蛋白质水平上的表达。吉西他滨诱导 UBE2M 的表达伴随着 p27（Kip1）蛋白水平的降低，这种降低可以通过 siRNA 沉默 UBE2M 表达或用蛋白酶体抑制剂 MG132 处理细胞来恢复，表明 UBE2M 介导吉西他滨诱导的 p27（Kip1）蛋白降解。LY294002（PI3K 抑制剂）可阻止 UBE2M 和 p27（Kip1）的降低。这些结果表明，PI3K 活性是吉西他滨诱导 UBE2M 表达所必需的，UBE2M 促进 p27（Kip1）的降解。值得注意的是，沉默 UBE2M 表达降低了 NTUB1 细胞对吉西他滨的敏感性，表明 UBE2M 通过降解 p27（Kip1）部分介导细胞对吉西他滨的敏感性。对吉西他滨耐药亚系的分析也表明，UBE2M 的丢失和 p27（Kip1）表达的增加与获得耐药性有关。总之，我们的结果表明 UBE2M 在介导吉西他滨对人尿路上皮癌细胞的细胞毒性中起作用，同时也表明 p27（Kip1）在耐药性中可能具有潜在功能。
