TRAIL predisposes non-small cell lung cancer to ferroptosis by regulating ASK-1/JNK1 pathway

Abstract

Objective: Our current study aimed to assess the relationship between TNF-related apoptosis-inducing ligand (TRAIL) and ferroptosis in non-small cell lung cancer (NSCLC) development.

Methods: The expression of TRAIL was detected by western blot, RT-qRCR and immunohistochemistry. The viability of NSCLC cells was analyzed by CCK-8 kit. The migration and invasion of NSCLC cells were detected by wound healing assay and transwell assay, respectively. Labile iron pool (LIP) was detected based on the calcein-acetoxymethyl ester method. Ferrous iron (Fe²⁺) and iron levels were assessed by detection kits. The levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured using corresponding detection kits. Mice tumor xenograft models were used for the in vivo research.

Results: The expression of TRAIL was reduced in H1299, NCL-H1395, and A549 cells compared with BEAS-2B cells. The up-regulation of TRAIL expression significantly reduced cell viability, invasion, and migration of H1299 and A549 cells. TRAIL reduced the expression of ferroptosis-related genes (FTH1, GPX4, and SLC7A11), increased the levels of LIP, iron, and Fe²⁺, and promoted lipid peroxidation, thereby predisposing NSCLC cells to ferroptosis. TRAIL up-regulated the expression of phosphate modification of ASK-1 and JNK. ASKI-1 inhibitor GS-4977 attenuated the effects of TRAIL on the viability, migration, invasion, and ferroptosis of H1299 cells. Furthermore, TRAIL further suppressed tumor growth and ferroptosis in mice tumor xenograft models.

Conclusion: We indicated that overexpression of TRAIL induced ferroptosis in NSCLC cells and exerted anti-tumor effects. Mechanistically, TRAIL promoted ferroptosis by the activation of the ASK-1/JNK1 pathway. Our results may provide new therapeutic strategies for NSCLC.

Keywords: ASK-1/JNK1 pathway; Ferroptosis; NSCLC; TRAIL.

Fig. 1

TRAIL is down-regulated in NSCLC cells. (A) TRAIL expression was determined in NSCLC cell lines by Western blot. (B) The relative protein level of TRAIL. ^**P < 0.01 versus BEAS-2B. Data were presented as the mean ± SD, and all the experiments were performed at least three times independently

Fig. 2

TRAIL overexpression inhibits cell viability, migration, and invasion of NSCLC cells. (A) TRAIL expression was detected by RT-qPCR. (B) TRAIL protein expression was determined by Western blot. (C) Cell viability was measured by CCK-8 assay. (D) The invasion of A549 and H1299 cells were detected by transwell assay. (E) The migration of A549 and H1299 cells were detected by wound healing assay. ^**P < 0.01, ^***P < 0.001 versus pcDNA3.1-NC. Data were presented as the mean ± SD, and all the experiments were performed at least three times independently

Fig. 3

TRAIL overexpression predisposes ferroptosis of NSCLC cells. A-B The levels of Fe²⁺ and iron were determined by detection kits. C LIP level was determined by calcein-acetoxymethyl ester method. D The levels of ferroptosis-related genes were determined by RT-qPCR. E The levels of SOD, MDA, and CAT were determined by the appropriate kit. ^**P < 0.01 versus pcDNA3.1-NC. Data were presented as the mean ± SD, and all the experiments were performed at least three times independently

Fig. 4

TRAIL interacts with the ASK-1/JNK1 pathway. (A) The expression of ASK-1, JNK1, p-ASK-1, and p-JNK1 was detected by western blot. (B) Cell viability was detected by MTT assay. (C) Wound healing was used to determine cell migration of H1299 cells. (D) The invasion of H1299 cells were detected by transwell assay. ^**P < 0.01, ^***P < 0.001 versus pcDNA3.1-NC. ^#P < 0.05, ^##P < 0.01 versus pcDNA3.1-TRAIL. Data were presented as the mean ± SD, and all the experiments were performed at least three times independently

Fig. 5

GS-4977 attenuates the effects of TRAIL overexpression on ferroptosis in H1299 cells. A-B. The levels of Fe²⁺ and iron were determined by detection kits. C. LIP level was determined by calcein-acetoxymethyl ester method. D. The expression of ferroptosis-related genes was determined by RT-qPCR. E. The levels of SOD, MDA, and CAT were determined by the appropriate kit. ^**P < 0.01 versus pcDNA3.1-NC. ^#P < 0.05, ^##P < 0.01 versus pcDNA3.1-TRAIL. Data were presented as the mean ± SD, and all the experiments were performed at least three times independently

Fig. 6

TRAIL overexpression inhibits tumor growth and promotes ferroptosis in vivo. (A) Tumor volume and weight were measured in different groups (n = 6 for each condition). (B) The relative mRNA expression level of TRAIL was detected through RT-qPCR. (C) IHC analysis of TRAIL for tissues of xenograft tumors. Scale bar: 100 μm. (D) The expression of ferroptosis-related genes was determined by RT-qPCR. (E) The levels of SOD, MDA, and CAT were detected by the appropriate kit. ^**P < 0.01, ^***P < 0.001 versus pcDNA3.1-NC. Data were presented as the mean ± SD.