Figures & data
Table 1. Primer sequences used.
Figure 1. Cephaeline induces lung cancer cell death in vitro. (A) The structure of cephaeline. (B) CCK-8 was used to detect the effect of different concentrations of cephaeline on the cell viability of lung cancer cell line H460 and A549 at different time points (24, 48 and 72 h).
Figure 2. Cephaeline induces upregulation of ROS levels and mitochondrial damage. (A) The ROS level was determined by DCFH-DA probe after treatment with cephaeline for 24 h. Scale bars, 50 μm. (B) The mitochondrial morphological changes were detected by transmission electron microscopy after treatment with cephaeline for 24 h. Scale bars, 500 nm. (C) The changes in mitochondrial membrane potential were detected by Mito-Tracker Red CMXRos probe after treatment with cephaeline for 24 h. Scale bars, 20 μm.
Figure 3. Cephaeline induces ferroptosis in lung cancer cells. (A) The reversal effect of apoptosis inhibitors (Z-VAD-FMK, 50 μM), necroptosis inhibitors (Necrostatin-1, 200 nM) and autophagy inhibitors (Chloroquine, 25 μM) on cephaeline induced lung cancer cell death. Mean ± SD, **p < 0.01 vs. the cephaeline treatment group, n = 4. (B) The reversal effect of ferroptosis inhibitors [liproxstatin-1 (100 nM)/Ferrostatin-1 (2 μM)/Deferoxamine (100 μM) ] on cephaeline induced lung cancer cell death, n = 3. Mean ± SD, **p < 0.01 vs. the cephaeline treatment group. (C) The iron level was detected by FerroOrange fluorescent probe after treatment with cephaeline for 24 h. Scale bars, 10 μm. Mean ± SD, **p < 0.01 vs. the control group.
![Figure 3. Cephaeline induces ferroptosis in lung cancer cells. (A) The reversal effect of apoptosis inhibitors (Z-VAD-FMK, 50 μM), necroptosis inhibitors (Necrostatin-1, 200 nM) and autophagy inhibitors (Chloroquine, 25 μM) on cephaeline induced lung cancer cell death. Mean ± SD, **p < 0.01 vs. the cephaeline treatment group, n = 4. (B) The reversal effect of ferroptosis inhibitors [liproxstatin-1 (100 nM)/Ferrostatin-1 (2 μM)/Deferoxamine (100 μM) ] on cephaeline induced lung cancer cell death, n = 3. Mean ± SD, **p < 0.01 vs. the cephaeline treatment group. (C) The iron level was detected by FerroOrange fluorescent probe after treatment with cephaeline for 24 h. Scale bars, 10 μm. Mean ± SD, **p < 0.01 vs. the control group.](/cms/asset/2b2ba9e6-959c-49e3-9ab6-bee4b2536d2c/iphb_a_2309891_f0003_c.jpg)
Figure 4. Cephaeline induces lipid peroxidation in lung cancer cells. (A and B) The lipid peroxidation was measured by C11 BODIPY 581/591 fluorescent ratio-probe in H460 and A549 cells after treatment with cephaeline for 24 h, Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group. Scale bars, 10 μm. (C and D) The indicator of lipid peroxidation LPO, MDA was detected in H460 and A549 cells after treatment with cephaeline for 24 h, n = 3. Mean ± SD, **p < 0.01 vs. the control group. (E) Effect of cephaeline on LDH release was detected after treatment with cephaeline for 24 h, n = 3. Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group. (F) The key antioxidant GSH was detected after treatment with cephaeline for 24 h, n = 3. Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group. (G and H) The reversal effect of antioxidants NAC and GSH on cephaeline-induced cell death, n = 3. Mean ± SD, **p < 0.01 vs. the control group.
Figure 5. Effect of cephaeline on ferroptosis related genes and proteins in lung cancer cells. (A–D) The ferroptosis-related genes GPX4, SLC7A11, SLC40A1 and Transferrin were detected by RT-PCR in H460 and A549 cells after treatment with cephaeline for 24 h, n = 3. Mean ± SD, **p < 0.01 vs. the control group. (E–I) The ferroptosis-related proteins GPX4, SLC7A11, SLC40A1 and Transferrin were detected by western blot in H460 and A549 cells after treatment with cephaeline for 24 h, repeated three times, Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group. (J–L) The key antioxidant regulatory protein NRF2 was detected by western blot in H460 and A549 cells after treatment with cephaeline for 24 h, n = 3, Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group.
Figure 6. Inhibitory effects of cephaeline on lung cancer cells are alleviated by an NRF2 agonist. (A) The CCK-8 was used to detect the reversal effect of TBHQ (10 μM) on cephaeline (100 nM) induced lung cancer cell death, n = 3, Mean ± SD, **p < 0.01 vs. the control group. (B) Using DCFH-DA probe to detect ROS level in control group, cephaeline treatment group and cephaeline combined TBHQ group. Scale bars, 50 μm. (C) The lipid peroxidation was measured by C11 BODIPY 581/591 fluorescent ratio-probe in control group, cephaeline treatment group and cephaeline combined TBHQ group. Scale bars, 20 μm. (D and E) Effect of cephaeline on LDH release and the key antioxidant GSH was detected in control group, cephaeline treatment group and cephaeline combined TBHQ group, n = 3. Mean ± SD, **p < 0.01 vs. the control group. (F and G) The ferroptosis-related proteins GPX4, SLC7A11 were detected by western blot in control group, cephaeline treatment group and cephaeline combined TBHQ group, repeated three times, Mean ± SD, *p < 0.05, **p < 0.01 vs. the control group.
Figure 7. Inhibitory effect of cephaeline on lung cancer cells in vivo. (A) Schematic diagram of the anti-lung cancer effect of cephaeline in vivo. (B) The antitumour effect of erastin (20 mg/kg) and cephaeline (5 and 10 mg/kg) in vivo were studied by using the subcutaneous tumor xenograft model, n = 6. (C and D) The tumor volume and tumor weight were measured both in control group, erastin group and cephaeline treatment group, Mean ± SD, **p < 0.01 vs. the control group. (E) The body weight was measured both in control group, erastin group and cephaeline treatment group, Mean ± SD. (F) The proteins GPX4, SLC7A11 and SLC40A1 associated with ferroptosis were detected in tumor tissues by Western blotting.
Figure 8. Schematic diagram of molecular mechanism of cephaeline’s antitumour effect. Cephaeline inactivates NRF2 and reduces the expression of its downstream genes GPX4, SLC7A11 and SLC40A1. While the downregulation of GPX4-SLC7A1 gene leads to the inhibition of antioxidant system, including the reduction of key antioxidant GSH. In addition, the downregulation of SLC40A1 gene accompanied by upregulation of transferrin, resulting in a significant increase in intracellular iron ions. Finally, the level of lipid ROS increased and cell ferroptosis occurred.
