Division of Toxicology, Bloomberg School of Public Health, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA.
Mol Cancer Ther. 2010 Feb;9(2):336-46. doi: 10.1158/1535-7163.MCT-09-0589. Epub 2010 Feb 2.
Loss-of-function mutations in the nuclear factor erythroid-2-related factor 2 (Nrf2) inhibitor Kelch-like ECH-associated protein 1 (Keap1) result in increased Nrf2 activity in non-small cell lung cancer and confer therapeutic resistance. We detected point mutations in Keap1 gene, leading to nonconservative amino acid substitutions in prostate cancer cells. We found novel transcriptional and posttranscriptional mechanisms of Keap1 inactivation, such as promoter CpG island hypermethylation and aberrant splicing of Keap1, in DU-145 cells. Very low levels of Keap1 mRNA were detected in DU-145 cells, which significantly increased by treatment with DNA methyltransferase inhibitor 5-aza-deoxycytidine. The loss of Keap1 function led to an enhanced activity of Nrf2 and its downstream electrophile/drug detoxification pathway. Inhibition of Nrf2 expression in DU-145 cells by RNA interference attenuated the expression of glutathione, thioredoxin, and the drug efflux pathways involved in counteracting electrophiles, oxidative stress, and detoxification of a broad spectrum of drugs. DU-145 cells constitutively expressing Nrf2 short hairpin RNA had lower levels of total glutathione and higher levels of intracellular reactive oxygen species. Attenuation of Nrf2 function in DU-145 cells enhanced sensitivity to chemotherapeutic drugs and radiation-induced cell death. In addition, inhibition of Nrf2 greatly suppressed in vitro and in vivo tumor growth of DU-145 prostate cancer cells. Thus, targeting the Nrf2 pathway in prostate cancer cells may provide a novel strategy to enhance chemotherapy and radiotherapy responsiveness and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.
核因子红细胞 2 相关因子 2（Nrf2）抑制剂 Kelch 样 ECH 相关蛋白 1（Keap1）的功能丧失性突变导致非小细胞肺癌中 Nrf2 活性增加，并导致治疗耐药。我们在前列腺癌细胞中检测到 Keap1 基因的点突变，导致非保守氨基酸取代。我们发现了 Keap1 失活的新的转录和转录后机制，如启动子 CpG 岛超甲基化和 Keap1 的异常剪接，在 DU-145 细胞中。在 DU-145 细胞中检测到极低水平的 Keap1 mRNA，用 DNA 甲基转移酶抑制剂 5-aza-脱氧胞苷处理后显著增加。Keap1 功能丧失导致 Nrf2 及其下游亲电体/药物解毒途径的活性增强。用 RNA 干扰抑制 DU-145 细胞中的 Nrf2 表达减弱了与亲电体、氧化应激和广泛药物解毒相关的谷胱甘肽、硫氧还蛋白和药物外排途径的表达。持续表达 Nrf2 短发夹 RNA 的 DU-145 细胞中的总谷胱甘肽水平较低，细胞内活性氧水平较高。在 DU-145 细胞中减弱 Nrf2 功能可提高对化疗药物和辐射诱导的细胞死亡的敏感性。此外，抑制 Nrf2 大大抑制了 DU-145 前列腺癌细胞的体外和体内肿瘤生长。因此，针对前列腺癌细胞中的 Nrf2 途径可能为增强化疗和放疗反应性、改善生长和致瘤性提供一种新策略，从而改善临床结局。
