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[Nrf2 down-regulated cell line H460-N5 with Keap1 over-expression increased sensitivity to anti-cancer drugs]
Li-yan Qu1, Peng Gao, Hong-yan Wang
1Department of Biochemistry and Genetics, College of Medicine, Zhejiang University, Hangzhou, China.
Objective:
To maked a Nrf2 down-regulated cell line by over-expressing Keap1 in H460 cells to study the role of Nrf2 in drug resistance.
Methods:
Transfecting H460 cells with mKeap1-pEGFP and screenig for Keap1 expressing clones by Western blotting with antibodies against Nrf2, HO-1, NQO1 and AKR1C. The cell line with Keap1 over-expression was further confirmed by real-time PCR. The cytotoxicity of H460-N5 to anti-cancer drugs was evaluated by MTS assay.
Result:
MTS assay results showed the enhanced cytotoxicity of anticancer drugs (Oxaliplatin, Doxorubicin and Etopside) to the H460 cell line with keap1 overexpression compared to the control cell line. In H460-N0 cells, the IC(50) values of Oxaliplation and Etopside were 93 micromol/L and 100 micromol/L respectively whereas the IC(50) values of the two drugs were 42 micromol/L and 30 micromol/L correspondingly in H460-N5 cells. A Nrf2 down-regulated cell line H460-N5 and a control cell line with GFP over-expression have been identified.Down-regulation of Nrf2 enhanced the cytotoxicity of Oxaliplatin, Doxorubicin and Etopside. The IC(50) value of Doxorubicin to H460-N0 cell was above 3 mg/L, but that to H460-N5 cell was about 2 mg/L.
Conclusion:
A Nrf2 down-regulated cell line H460-N5 and a control cell line with GFP over-expression have been identified. Down-regulation of Nrf2 enhanced the cytotoxicity of Oxaliplatin, Doxorubicin and Etopside.
Insights
Researchers created a Nrf2-downregulated cell line to study its role in drug resistance. This Nrf2 downregulation enhanced the cytotoxicity of common anticancer drugs, offering new insights into cancer treatment strategies.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- The Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is a key regulator of cellular defense against oxidative stress.
- Nrf2 plays a complex role in cancer, often promoting cell survival and drug resistance.
- Understanding Nrf2's role in drug resistance is crucial for developing more effective cancer therapies.
Purpose of the Study:
- To generate a Nrf2-downregulated cell line by overexpressing Kelch-like ECH-associated protein 1 (Keap1) in H460 lung cancer cells.
- To investigate the impact of Nrf2 downregulation on the sensitivity of H460 cells to various anticancer drugs.
Main Methods:
- H460 cells were transfected with mKeap1-pEGFP to overexpress Keap1.
- Keap1 expression and Nrf2 levels were confirmed using Western blotting and real-time PCR.
- Cytotoxicity assays (MTS assay) were performed to evaluate drug sensitivity.
Main Results:
- A stable Nrf2-downregulated cell line (H460-N5) and a control cell line (H460-N0) were successfully established.
- The Nrf2-downregulated H460-N5 cells exhibited significantly enhanced sensitivity to Oxaliplatin, Doxorubicin, and Etoposide compared to control cells.
- IC50 values for Oxaliplatin and Etoposide were reduced in H460-N5 cells, indicating increased drug-induced cytotoxicity.
Conclusions:
- Downregulation of Nrf2 in H460 cells potentiates the cytotoxic effects of Oxaliplatin, Doxorubicin, and Etoposide.
- These findings highlight the role of Nrf2 in conferring resistance to chemotherapy.
- Targeting the Nrf2 pathway could be a promising strategy to overcome drug resistance in lung cancer.
