GPX4 Plays a Crucial Role in Fuzheng Kang'ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis

Abstract

Background: Fuzheng Kang'ai decoction (FZKA) has been widely used to treat Non-Small Cell Lung Cancer (NSCLC) patients in China for decades, showing definitively curative effects in clinic. Recently, we found that FZKA could induce NSCLC cell ferroptosis, another type of programmed cell death (PCD), which is totally different from cell apoptosis. Therefore, in the present study, we aim to discover the exact mechanism by which FZKA induces NSCLC cell ferroptosis, which is rarely studied in Traditional Chinese Medicine (TCM). Methods: Cell proliferation assay were performed to detect the cell viability. Cell ferroptosis triggered by FZKA was observed by performing lipid peroxidation assay, Fe²⁺ Ions assay, and mitochondrial ultrastructure by transmission electron microscopy. Ferroptosis inhibitors including liproxstatin-1 and UAMC 3203 were used to block ferroptosis. The ratio of GSH/GSSG was done to measure the alteration of oxidative stress. Western blot and qRT-PCR were carried out to detect the expression of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and glutathione peroxidase 4 (GPX4) at protein and mRNA levels, respectively. Lentivirus transfection was performed to overexpress GPX4 stably. Animal model was done to verify the effect of FZKA-induced ferroptosis in NSCLC in vivo and immunohistochemistry was done to detect the expression of SLC7A11, SLC3A2 and GPX4 at protein level. Results: First of all, in vitro experiments confirmed the inhibition effect of FZKA on NSCLC cell growth. We then, for the first time, found that FZKA induced NSCLC cell ferroptosis by increasing lipid peroxidation and cellular Fe²⁺ Ions. Moreover, characteristic morphological changes of NSCLC cell ferroptosis was observed under transmission electron microscopy. Mechanistically, GPX4, as a key inhibitor of lipid peroxidation, was greatly suppressed by FZKA treatment both at protein and mRNA levels. Furthermore, system xc^- (SLC7A11 and SLC3A2) were found to be suppressed and a decreased GSH/GSSG ratio was observed at the same time when treated with FZKA. Notably, overexpressing GPX4 reversed the effect of FZKA-induced NSCLC cell ferroptosis significantly. Finally, the above effect was validated using animal model in vivo. Conclusion: Our findings conclude that GPX4 plays a crucial role in FZKA-induced NSCLC cell ferroptosis, providing a novel molecular mechanism by which FZKA treats NSCLC.

Keywords: FZKA; GPX4; NSCLC; TCM; ferroptosis.

FIGURE 1

FZKA inhibited the growth of NSCLC cells in vitro. (A), H1650 and H1299 cells were treated with different concentrations of FZKA for up to 72 h. The cells were collected and processed for CCK-8 assay as described in the Materials and Methods section, *p < 0.05. (B), H1650 and H1299 cells were treated with FZKA (1 mg/ml) for 24 h, followed by EdU proliferation assay. (C). Cultured A549 cells were treated with FZKA (1.5 mg/ml), in the presence and absence of ferroptosis inhibitors. Cell were stained with Annexin V and PI and analyzed by flow cytometry, *p < 0.05.

FIGURE 2

FZKA induced ferroptosis in NSCLC cells by FCM and TEM. (A,B), Cells were stained with C11-BODIPY (10 μM) or FerroOrange (1 μM) for 30 min, the level of lipid peroxidation and Fe²⁺ Ions was detected by flow cytometry, *p < 0.05. (C), Transmission electron microscopy of H1650 and H1299 cells treated with DMSO (10 h), erastin (ferroptosis inducer, 10 µM, 10 h), FZKA (1 mg/ml, 10 h). The mitochondria appeared to be changed including swollen, cristae loss and mitochondira vacuolization in H1650 and H1299 cells. Scale bar represents 1 µM and 600 nm.

FIGURE 3

Ferroptosis inhibitors including liproxstatin-1 and UAMC 3203 reversed the effect of FZKA. A, PC9 cells were treated as (A) FZKA (1.5 mg/ml), UAMC3203 (25 nM) or liproxstatin-1 (200 nM) for 24 h, and stained with BODIPY™ 581/591 C11 (10 μM) for 30 min. The level of lipid peroxidation was detected by flow cytometry. Each point represents the mean ± SEM, n = 3, *p <0.05. (B), Cultured NSCLC cells were seeded in 96 well plate, FZKA (1.5 mg/ml in A549 and PC9 cells, 1 mg/ml in H1650 and H1299 cells), with UAMC3203 (25 nM), or liproxstatin-1 (200 nM) for 24 h. Cell viability was detected by CCK-8 assay, *p < 0.05.

FIGURE 4

FZKA downregulated the GPX4 expression in NSCLC cells at both protein and mRNA levels. (A), The protein expression levels of GPX4 were detected by Western blot. (B), NSCLC cells were treated with different concentrations of FZKA for 24 h. The mRNA expression of GPX4 were detected by qPCR. Each point represents the mean ± SEM, n = 3. *p < 0.05.

FIGURE 5

FZKA decreased the ratio of GSH/GSSG and expression of system xc⁻. (A), The protein expression levels of SLC7A11 and SLC3A2 were detected by Western blot. (B), the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. *p < 0.05.

FIGURE 6

Overexpression of GPX4 reversed the efficacy of FZKA. (A), Cultured H1299 and PC9 cells were transfected with negative control and GPX4 lentiviral vectors, then treated with or without FZKA for 24 h. The expression of GPX4 was detected by western blot. (B), lipid peroxidation assay was performed in A549 cell after treatment with FZKA or/and GPX4 lentivirus. (C), Cells were transfected and treated with FZKA, and CCK-8 assay were then conducted. Each point represents the mean ± SEM, n = 3. *p < 0.05.

FIGURE 7

Validation of FZKA-induced NSCLC cell ferroptosis in vivo. (A), Mice tumor photograph and tumor weight was showed. Data represents Mean ± SEM, n = 7. *p < 0.05. (B), Tumor volume in each group was showed. Data represents Mean ± SEM, n = 7. *p < 0.05. (C), Western blot analyses of GPX4, SLC7A11 and SLC3A2 expression from tumor tissues. Data represents Mean ± SEM, n = 7. *p < 0.05. (D), Immunohistochemistry was carried out to measure the expression of GPX4, SLC7A11 and SLC3A2 in mice tumor tissues. Data represents Mean ± SEM, n = 7. *p < 0.05. (E), The diagram showing FZKA induced NSCLC cell ferroptosis through system xc⁻/GSH/GPX4 axis, and, importantly, GPX4 is the crucial molecular in the process. Finally, inhibition of GPX4 by FZKA leads to NSCLC cell ferroptosis.