Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis

Abstract

Ferroptosis, a novel form of programmed cell death, is characterized by iron-dependent lipid peroxidation and has been shown to be involved in multiple diseases, including cancer. Stimulating ferroptosis in cancer cells may be a potential strategy for cancer therapy. Therefore, ferroptosis-inducing drugs are attracting more attention for cancer treatment. Here, we showed that erianin, a natural product isolated from Dendrobium chrysotoxum Lindl, exerted its anticancer activity by inducing cell death and inhibiting cell migration in lung cancer cells. Subsequently, we demonstrated for the first time that erianin induced ferroptotic cell death in lung cancer cells, which was accompanied by ROS accumulation, lipid peroxidation, and GSH depletion. The ferroptosis inhibitors Fer-1 and Lip-1 but not Z-VAD-FMK, CQ, or necrostatin-1 rescued erianin-induced cell death, indicating that ferroptosis contributed to erianin-induced cell death. Furthermore, we demonstrated that Ca²⁺/CaM signaling was a critical mediator of erianin-induced ferroptosis and that blockade of this signaling significantly rescued cell death induced by erianin treatment by suppressing ferroptosis. Taken together, our data suggest that the natural product erianin exerts its anticancer effects by inducing Ca²⁺/CaM-dependent ferroptosis and inhibiting cell migration, and erianin will hopefully serve as a prospective compound for lung cancer treatment.

Fig. 1

Erianin inhibits cell proliferation and triggers cell death and G2/M cell cycle arrest in lung cancer cells. a Cell viability was measured using the CCK-8 assay. b Representative cell morphological changes are shown. c, d Representative results of annexin V/FITC/PI staining and quantitative analysis, **p < 0.01. e Monolayer culture; quantitative analyses of colony numbers are shown (f, g), *p < 0.05, **p < 0.01. h Representative results of cell cycle and quantitative analyses (i, j), *p < 0.05, **p < 0.01

Fig. 2

Erianin suppresses the migration of lung cancer cells. a Representative results of wound-healing assays. b Transwell migration assay with the 24-well Transwell system and quantitative analysis (original magnification: ×100). c The numbers of migrated cells were counted in five representative high-power fields per Transwell plate, **p < 0.01. d The expression of several key cell metastatic signal regulators, vimentin, E-cadherin, N-cadherin, slug, snail, and MMP-9, were examined by western blotting after treatment with erianin for 24 h

Fig. 3

The effect of erianin alone or in combination with other cell death inhibitors on the inhibition of growth of lung cancer cells. a H460 and H1299 cells were treated with erianin with or without Z-VAD-FMK for 24 h, and the inhibition of growth was assayed. b H460 and H1299 cells were treated with erianin with or without CQ for 24 h, and the inhibition of growth was assayed. c H460 and H1299 cells were treated with erianin with or without necrostatin-1 for 24 h, and the inhibition of growth was assayed. d Heatmap showing the mRNA and KEGG pathway enrichment analyses in H460 cells. e Heatmap showing the mRNA and KEGG pathway enrichment analyses in H1299 cells

Fig. 4

Ferroptosis contributes to erianin-induced cell death in lung cancer cells. a The cellular ROS level was analyzed by a flow cytometer, *p < 0.05, **p < 0.01. b Intracellular GSH levels in H460 and H1299 cells treated with erianin, *p < 0.05, **p < 0.01. c Intracellular MDA levels in H460 and H1299 cells treated with erianin, *p < 0.05, **p < 0.01. d H460 and H1299 cells were treated with erianin with or without the ROS scavenger NAC for 24 h, and cell viability was assayed, **p < 0.01. e H460 and H1299 cells were treated with erianin with or without the ROS scavenger GSH for 24 h, and cell viability was assayed, **p < 0.01. f Intracellular chelatable iron in H460 and H1299 cells treated with erianin was determined using the fluorescent indicator Phen Green SK (green). g Transmission electron microscopy (TEM) was used to observe ferroptosis in H460 and H1299 cells (original magnification: ×100). h The expression of several key ferroptosis regulators was examined by western blotting. i H460 and H1299 cells were treated with erianin with or without ferroptosis inhibitors for 24 h, and the inhibition of growth was assayed, *p < 0.05

Fig. 5

Calcium/calmodulin signaling contributes to erianin-induced ferroptosis in lung cancer cells. a The most likely predicted targets of erianin. b Heatmap showing the mRNA analysis of Ca²⁺/CaM signaling. c The intracellular Ca²⁺ concentration in H460 and H1299 cells treated with erianin was detected according to the fluorescence intensity of the calcium indicator Fluo-3/AM with fluorescence microscopy. d The intracellular Ca²⁺ concentration in H460 and H1299 cells treated with erianin was detected by measuring the fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. e The expression of CaM was examined by western blotting after treatment with erianin for 24 h. f The effect of blocking Ca²⁺/CaM signaling with ruthenium red on erianin-induced ferroptosis in lung cancer cells after treatment for 24 h. g The effect of blocking Ca²⁺/CaM signaling with ruthenium red on erianin-induced inhibition of growth of lung cancer, *p < 0.05

Fig. 6

CaM inhibition by calmidazolium rescued erianin-induced ferroptosis. a The effect of blocking Ca²⁺/CaM signaling with calmidazolium on erianin-induced inhibition of growth in H460 cells, **p < 0.01. b The effect of blocking Ca²⁺/CaM signaling with calmidazolium on erianin-induced inhibition of growth in H1299 cells, **p < 0.01. c Intracellular chelatable iron in H460 and H1299 cells after treatment with a combination of calmidazolium and erianin was determined using the fluorescent indicator Phen Green SK (green). d The intracellular Ca²⁺ concentration in H460 and H1299 cells after treatment with a combination of calmidazolium and erianin was detected by measuring the fluorescence intensity of the calcium indicator Fluo-3/AM with fluorescence microscopy. e Western blotting showed the effect of blocking Ca²⁺/CaM signaling with calmidazolium treatment for 24 h on erianin-induced ferroptosis in lung cancer

Fig. 7

Erianin exerts its antitumor effects in vivo. a Representative image of H460 xenograft tumors after erianin treatment. b Tumor volume in each group. Data are expressed as the means ± standard deviations (SDs). c Scheme of tumor inoculation and systemic injection. d Bioluminescent imaging of disseminated H460-luc orthotopic xenograft tumors at different time points posttreatment, and representative images of H460-luc orthotopic xenograft lung tumors after erianin treatment. e Fold change in the average radiance per mouse after normalization to the day 0 tumor burden as determined by bioluminescent imaging, **p < 0.01. f Fold change in the average radiance per mouse at the experimental endpoint (day 12) for each treatment group (mean ± SEM), **p < 0.01

Fig. 8

H&E and immunohistochemical staining of xenograft tumor sections. a H&E staining of tissue sections of major organs was analyzed 3 days after the last injection of erianin. b Immunohistochemical staining of several ferroptotic and metastatic proteins

Fig. 9

Scheme showing the central role of erianin in ferroptosis induction and inhibition of cell migration