Effect of stable inhibition of NRF2 on doxorubicin sensitivity in human ovarian carcinoma OV90 cells.
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.
Yeungnam University, Gyeongsan, Korea.
Sarala
S
Manandhar
Sangwhan
S
Lee
Mi-Kyoung
MK
Kwak
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D055785:Gene Knockdown Techniques / Q000379:methods; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D004317:Doxorubicin; D005978:Glutathione