Temozolomide Drives Ferroptosis via a DMT1-Dependent Pathway in Glioblastoma Cells

Abstract

Purpose: Temozolomide is used in first-line treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, mechanisms for the anti-tumor effects of temozolomide are largely unknown. Ferroptosis is a form of programmed cell death triggered by disturbed redox homeostasis, overloaded iron, and increased lipid peroxidation. The present study was performed to elucidate the involvement of ferroptosis in the anti-tumor mechanisms of temozolomide.

Materials and methods: We utilized the CCK8 assay to evaluate cytotoxicity. Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), iron, and glutathione (GSH) were measured. Flow cytometry and fluorescence microscope were used to detect the production of reactive oxygen species (ROS). Western blotting, RT-PCR and siRNA transfection were used to investigate molecular mechanisms.

Results: Temozolomide increased the levels of LDH, MDA, and iron and reduced GSH levels in TG905 cells. Furthermore, we found that ROS levels and DMT1 expression were elevated in TG905 cells treated with temozolomide and were accompanied by a decrease in the expression of glutathione peroxidase 4, indicating an iron-dependent cell death, ferroptosis. Our results also showed that temozolomide-induced ferroptosis is associated with regulation of the Nrf2/HO-1 pathway. Conversely, DMT1 knockdown by siRNA evidently blocked temozolomide-induced ferroptosis in TG905 cells.

Conclusion: Taken together, our findings indicate that temozolomide may suppress cell growth partly by inducing ferroptosis by targeting DMT1 expression in glioblastoma cells.

Keywords: DMT1; Temozolomide; ferroptosis; glioblastoma; reactive oxygen species.

Fig. 1. TMZ induces ferroptosis in TG905 cells. (A) Cell viability was measured using the CCK8. (B) LDH release was measured. Percentage of LDH released was expressed based on maximum releasable LDH in the cells induced by 1% Triton X-100. (C) DCFH-DA staining was used to evaluate ROS production under a fluorescence microscope. GSH (D) and MDA (E) levels were analyzed after treatment with TMZ for 48 h. Data are presented as the mean±SD of three independent experiments. ^*p<0.05, ^**p<0.01 compared with the control group (t test). (F) Treatment with Fer-1 mitigated TMZ-induced cell death. Data presented as the mean±SD of three independent experiments. ^*p<0.05 compared with the control group, ^†p<0.05 compared with the TMZ-treated group (ANOVA). TMZ, temozolomide; LDH, lactate dehydrogenase; GSH, glutathione; MDA, malondialdehyde; DCFH-DA, dichlorofluorescein diacetate; ROS, eactive oxygen species.

Fig. 2. Effect of TMZ on iron content and iron-related mRNA and protein expression in TG905 cells. Cells were treated with TMZ for 48 h. (A) Iron content, (B) iron-related mRNA, and (C) protein expression were measured. Data are presented as the mean±SD of three independent experiments. ^*p<0.05, ^**p<0.01 compared with the control group (t test). TMZ, temozolomide.

Fig. 3. Effect of DMT1 silencing on TMZ-induced ferroptosis in TG905 cells. (A and B) The expression of DMT1 protein and mRNA was decreased upon DMT1 knockdown by siRNA. Data are presented as the mean±SD of three independent experiments. ^*p<0.05 compared with the control group (t test). (C–G) Cells were transfected with or without si-DMT1 for 6 h and then treated with TMZ (400 µM) for 48 h. Cell viability, LDH levels, GSH levels, MDA levels, and iron content were analyzed. (H and I) DCFH-DA staining was used to evaluate ROS production under a fluorescence microscope and flow cytometry. Data are presented as the mean±SD of three independent experiments. ^*p<0.05 compared with the control group, ^†p<0.05 compared with the TMZ-treated group (ANOVA). TMZ, temozolomide; LDH, lactate dehydrogenase; GSH, glutathione; MDA, malondialdehyde; DCFH-DA, dichlorofluorescein diacetate; ROS, eactive oxygen species.

Fig. 4. Effect of DMT1 silencing on protein expression in TG905 cells. Cells were transfected with or without si-DMT1 for 6 h and then treated with TMZ (400 µM) for 48 h. Protein expression was measured by Western blot assay. Data are presented as the mean±SD of three independent experiments. ^*p<0.05 compared with the control group, ^†p<0.05 compared with the TMZ-treated group (ANOVA). TMZ, temozolomide; GPX4, glutathione peroxidase 4.