Yeungnam University, Gyeongsan, Korea.
Arch Pharm Res. 2010 May;33(5):717-26. doi: 10.1007/s12272-010-0511-z. Epub 2010 May 29.
The transcription factor NRF2 defends the cell from oxidative stress by up-regulating a large number of antioxidant genes through its binding with antioxidant response element on gene promoters. Cancer cells are known to possess high levels of antioxidant genes that increases survival in cancer microenvironment of oxidative stress, particularly in the treatment with anticancer agents. In the current study we have examined the role of the NRF2 in doxorubicin sensitivity and tumor growth by establishing stable cell line expressing NRF2 shRNA in the human ovarian carcinoma cell line OV90. On knockdown of NRF2 through NRF2-specific shNRF2 expressing lentiviral plasmid, antioxidant response element-driven luciferase activity as well as the expression of NRF2-target genes were significantly suppressed compared to nonspecific scrambled RNA (scRNA) expressing cells. In addition, shNRF2 expressing OV90-shNRF2 cells showed a reduction in total GSH levels by 82% and cell growth was observed to be significantly retarded compared to scRNA control cells. Furthermore, stable inhibition of NRF2 sensitized OV90 cells were seen following doxorubicin treatment as shown by the analysis with MTT assay and propidium iodide-fluorescence-activated cell sorting. OV90-shNRF2 cells showed higher levels of cell death and apoptosis in response to doxorubicin than OV90-scRNA cells. While, when BALBc (nu/nu) mice with OV90 tumor xenograft in the flanks were injected with NRF2 shRNA containing viral particles and treated with doxorubicin a pattern of retardation in tumor growth was seen in shRNA group compared to scRNA group, but this difference was not statistically significant. In conclusion, we propose that the NRF2 signaling might be a molecular target to repress tumor growth and enhance cytotoxic effects of anticancer agent in cancer cells.
转录因子 NRF2 通过其与基因启动子上的抗氧化反应元件结合，上调大量抗氧化基因，从而抵抗细胞氧化应激。众所周知，癌细胞具有高水平的抗氧化基因，这增加了它们在氧化应激的癌症微环境中的存活能力，尤其是在接受抗癌药物治疗时。在本研究中，我们通过在人卵巢癌细胞系 OV90 中建立稳定表达 NRF2 shRNA 的细胞系，研究了 NRF2 在阿霉素敏感性和肿瘤生长中的作用。通过 NRF2 特异性 shNRF2 表达慢病毒质粒敲低 NRF2 后，与非特异性 scrambled RNA（scRNA）表达细胞相比，抗氧化反应元件驱动的荧光素酶活性以及 NRF2 靶基因的表达均显著受到抑制。此外，与 scRNA 对照细胞相比，shNRF2 表达的 OV90-shNRF2 细胞的总 GSH 水平降低了 82%，并且观察到细胞生长明显受到抑制。此外，如 MTT 分析和碘化丙啶-荧光激活细胞分选所示，稳定抑制 NRF2 可使 OV90 细胞对阿霉素敏感。与 OV90-scRNA 细胞相比，OV90-shNRF2 细胞对阿霉素的细胞死亡和凋亡水平更高。然而，当侧翼带有 OV90 肿瘤异种移植物的 BALBc（nu/nu）小鼠被注射含有 NRF2 shRNA 的病毒颗粒并接受阿霉素治疗时，与 scRNA 组相比，shRNA 组的肿瘤生长速度明显减慢，但这种差异没有统计学意义。总之，我们提出 NRF2 信号可能是抑制肿瘤生长和增强癌细胞中抗癌药物细胞毒性作用的分子靶点。
