Heme oxygenase-1 leads to cisplatin resistance in nasopharyngeal carcinoma by reducing oxidative stress and ferroptosis

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a malignancy with high a mortality rate. This study investigates the impact of heme oxygenase-1 (HO-1) in cisplatin (CDDP) resistance in NPC.

Methods: The time-dependent effect of CDDP on two NPC cell lines (HK1 and C666-1) were investigated. CDDP-resistant cells were established by exposing the parental cells to increasing concentrations of CDDP. Parental cells received treatment of the HO-1 inducer Hemin while resistant cells received treatment of the HO-1 inhibitor ZnPP to explore the influence of HO-1 activity on CDDP resistance in NPC cell lines. Erastin was used to verify the effect of ferroptosis on CDDP sensitivity in cells. Parallel settings were performed in mouse xenograft tumor models for in vivo validation.

Results: CDDP time-dependently reduced growth and mobility of NPC cells within the initial 48 h, but the cytotoxicity was no longer significantly enhanced afterward. HO-1 was upregulated in cells after CDDP treatment, showing correlation with acquired CDDP resistance. Inducing HO-1 activity in parental cells protected cells from oxidative damage, apoptosis, and ferroptosis, while suppressing HO-1 activity using ZnPP restored the therapeutic efficacy of CDDP in drug resistant cells. Moreover, the Erastin treatment also restored the cytotoxicity of CDDP to the resistant cells. In mice bearing xenograft tumors, treatment with either ZnPP or Erastin weakened the growth and weight of tumors.

Conclusion: This work suggests that HO-1 is pertinent to acquired CDDP resistance in NPC cells by suppressing oxidative stress and ferroptosis.

Keywords: CDDP resistance; Ferroptosis; HO-1; Nasopharyngeal carcinoma; Oxidative stress.

Fig. 1

Tumor suppressive effects of CDDP and the development of drug resistance. HK1 and C666-1 cells were treated with DMSO or 5 µM CDDP for 24, 48, and 72 h respectively. A: CCK-8 assays for NPC cell viability. B: Colony formation assays for NPC cell growth. C: Scratch tests for NPC cell migration. D: Transwell assays for NPC cell invasion. E: TUNEL assays for cell apoptosis. F: Immunofluorescence staining for γ-H2AX expression. Six independent experiments were performed. Data are present as dot and bars, with each dot indicating one independent experiment. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant

Fig. 2

HO-1 is upregulated in CDDP-resistant NPC cells. A-B: Volcano plots and heat map for differentially expressed genes in C666-1 cells after 72 h of CDDP (5 µM) treatment; C-D: qPCR and immunoblot analysis for HO-1 mRNA and protein expression in CDDP-treated NPC cells. Parental NPC cell lines (HK1 and C666-1) were exposed to an ascending series of CDDP (0.1–10 µM) to induce drug-resistant cell lines (HK1/R and C666-1/R). E-F: CCK-8 assay for cell viability at different CDDP concentrations. G: TUNEL assays for apoptosis in parental and resistant NPC cell lines under 5 µM CDDP treatment. H-I: qPCR and immunoblot analysis for HO-1 mRNA and protein expression in parental and resistant NPC cell lines. Six independent experiments were performed. Data are present as dot and bars, with each dot indicating one independent experiment. One-way ANOVA was used for comparisons among multiple groups. Three independent experiments were conducted. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

HO-1 contributes to CDDP resistance in NPC cells. Parental HK1 and C666-1 cells were administered a HO-1 inducer Hemin while HK1/R and C666-1/R cells were administered a HO-1 inhibitor ZnPP. A-B: CCK-8 assay for viability of cells and the IC50 values of CDDP. The cells were subjected to 5 µM CDDP treatment. C: Colony formation assays for NPC cell growth. D: Scratch tests for NPC cell migration. E: Transwell assays for NPC cell invasion. F: TUNEL assays for cell apoptosis. G: Immunofluorescence staining for γ-H2AX expression. H, DCFH-DA staining for ROS production. The conditioned media of these cell cultures were collected for HUVEC incubation. H: Angiogenesis ability of the HUVECs. I, ELISA tests for VEGFA and MMP9 levels in conditioned media. Six independent experiments were performed. Data are present as dot and bars, with each dot indicating one independent experiment. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

HO-1 protects NPC cells from oxidative damage and ferroptosis. A-B, Immunoblot analyses for GPX4 and ACSL4 levels in differentially treated NPC cells. C-D, Biochemical analysis of SOD and MDA protein expression in cells. E, Mitotracker analysis of mitochondrial homeostasis in each group of cells. Six independent experiments were performed. Data are present as dot and bars, with each dot indicating one independent experiment. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Erastin enhances the anti-tumor effects of CDDP on NPC cells. HK1/R and C666-1/R cells received treatments of Erastin and CDDP. A-B: CCK-8 assay for viability of cells and the IC50 values of CDDP. The cells were subjected to 5 µM CDDP treatment. C: Colony formation assays for NPC cell growth. D: Scratch tests for NPC cell migration. E: Transwell assays for NPC cell invasion. F: TUNEL assays for cell apoptosis. G: Immunofluorescence staining for γ-H2AX expression. Six independent experiments were performed. Data are present as dot and bars, with each dot indicating one independent experiment. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

ZnPP or Erastin treatment enhances the tumor-suppressive effects of CDDP on mice. HK1/R and C666-1/R cells were injected into nude mice subcutaneously to establish xenograft tumor models, followed by ZnPP or Erastin treatment. A: Volume of the xenograft tumors. B: Weight of the xenograft tumors. C: IHC assays for the expression of Ki67 and Cleaved Caspase-3 in xenograft tumors. D: TUNEL assay for cell apoptosis detection in tumors. E-F: Biological analyses for SOD activity and MDA content in xenograft tumors. G: IHC assays for the expression of 4-HNE in xenograft tumors. Each group contained 6 mice. Data are present as dot and bars, with each dot indicating data from one mouse. Differences were compared by the ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001