Nrf2 Knockout Affected the Ferroptosis Signaling Pathway against Cisplatin-Induced Hair Cell-Like HEI-OC1 Cell Death

Abstract

Cisplatin is a well-known and widely used anticancer drug with high therapeutic efficacy in solid tumors; however, side effects are common with its use. Because cisplatin can be retained in the cochlea, ototoxicity leading to hearing loss limits its clinical applications. Here, we report that Nrf2 knockout (KO) strongly increased cisplatin resistance in HEI-OC1 cells, which are immortalized cells from the murine organ of Corti. The underlying mechanism of this phenomenon was uncovered, and an important novel therapeutic target for combating cisplatin-induced hearing loss was identified. Preliminary investigations determined that Nrf2 KO markedly decreased TfR1 protein levels and increased GPX4 protein levels. Thus, ferroptosis may protect organisms from cisplatin-induced cell death. Furthermore, Nrf2 KO cells were resistant to the classical ferroptosis inducers RSL3 and erastin, providing solid evidence that Nrf2 KO inhibits ferroptosis and that knocking out Nrf2 may be a new clinical strategy to prevent cisplatin-induced hearing loss.

Figure 1

Cisplatin-induced cell death in the mouse cochlea. (a, top) Mode of cisplatin administration to CBA/J mice. (a, bottom) Phalloidin-labeled hair cells of the organ of Corti. (b) Hair cell quantification and statistical analysis. (c) Statistical analysis of the ABR threshold shift. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 20 μm.

Figure 2

Cisplatin-induced HEI-OC1 cell death. (a) Light field and flow cytometry analysis after cells were treated with 25 μM cisplatin for 24 h. (b) Number of early apoptotic cells in A. (c) Number of dead cells in A. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 100 μm.

Figure 3

Nrf2 participates in cisplatin-induced HEI-OC1 cell death. (a) Western blots of Nrf2, KEAP1, and GAPDH after cisplatin treatment for different lengths of time. (b) Western blot confirming Nrf2 KO in the desired cell line. (c) Control and Nrf2 KO cells were treated with 25 μM tBHQ for 24 h. (d) Control and Nrf2 KO cells treated with varying concentrations of cisplatin for 24 h were analyzed by CCK-8 assays. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4

Nrf2 KO strongly increases cisplatin resistance in HEI-OC1 cells. (a) Images showing control and Nrf2 KO cells after 25 μM cisplatin treatment for 24 h in a light field. (b) LDH assays of the cells in A. (c) Flow cytometry analysis of cell apoptosis of the cells in A. (d) Number of early apoptotic cells in C. (e) DAPI and TUNEL double labeling of apoptotic cells after treatment with 25 μM cisplatin. (f) Image showing control and Nrf2 KO cells after 100 μM cisplatin treatment for 24 h in a light field. (g) LDH assays of the cells in F. ns: no significant difference, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 100 μm.

Figure 5

Nrf2 KO affects ferroptosis signaling pathway-induced cisplatin resistance. (a) Western blots of Nrf2, TfR1, and GPX4 protein expressions in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (b) RT -PCR verification of TfR1 mRNA expression levels in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (c) RT -PCR verification of GPX4 mRNA expression levels in control and Nrf2 KO cells with or without 25 μM cisplatin treatment for 24h. (d) FerroOrange indicated the intracellular Fe²⁺ content after treatment with 25 μM cisplatin for 24h. (e) Relative fluorescence intensity of FerroOrange for the cells in (d). (f) Western blots of Nrf2, CTR1, TfR1, p53, and GPX4 protein expressions in control and Nrf2 KO cells treated with 25 μM cisplatin for different lengths of time. ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001. Scale bar, 20 μm.

Figure 6

Nrf2 KO alleviated mitochondrial ROS and lipid peroxide levels after cisplatin treatment. (a) MitoSOX Red staining indicated mitochondrial ROS accumulation in cells after treatment with 25 μM cisplatin for 24 h. (b) Relative fluorescence intensity of MitoSOX Red in the cells in A. (c) Liperfluo indicated the lipid peroxide levels in cells after treatment with 25 μM cisplatin for 24 h. (d) Relative fluorescence intensity of Liperfluo in the cells in C. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 20 μm.

Figure 7

The Nrf2 KO cell line was resistant to the ferroptosis inducer RSL3. (a) LDH release levels in control and Nrf2 KO cells treated with different concentrations of RSL3 for 24 h. (b) Image showing control and Nrf2 KO cells after 1 μM RSL3 treatment for 24 h in a light field. (c) LDH release levels in B. (d) Western blots of Nrf2, CTR1, TfR1, p53, and GPX4 protein expression in control and Nrf2 KO cells treated with 1 μM RSL3 at different time points. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 100 μm.

Figure 8

Nrf2 KO cells were resistant to the ferroptosis inducer erastin. (a) LDH release levels in control and Nrf2 KO cells treated with different concentrations of erastin for 24 h. (b) Image showing control and Nrf2 KO cells after 3 μM erastin treatment for 24 h in a light field. (c) LDH release levels in B. (d) Western blots of Nrf2, CTR1, TfR1, and GPX4 protein expression in control and Nrf2 KO cells treated with 3 μM erastin at different time points. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar, 100 μm.