Fig 2: Anomanolide C inhibits TNBC cell migration in vitro by causing autophagy-dependent ferroptosis. (A-B) MDA-MB-231 cells were treated for 24 hours with AC (1 µM) alone or in conjunction with 3-MA (1 mM). 3-MA was introduced 6 hours prior to the AC treatment. The ability of the cells to migrate was subsequently evaluated utilizing the transwell assay and scratch test. Representative images and date are shown. Scale bar, 100 µm. (C-D) MDA-MB-231 cells were treated for 24 hours with AC (1 µM) alone or in conjunction with Fer-1 (1 µM). Fer-1 was added 6 hours before AC treatment. The ability of the cells to migrate was then evaluated using the transwell assay and scratch test. Representative images and date are shown. Scale bar, 100 µm. (E-F) MDA-MB-231 cells were transfected with negative-control or ATG5 siRNA. The ability of the cells to migrate was evaluated using the scratch assay and transwell assay after co-treatment with or without AC and Fer-1. Representative images and date are shown. Scale bar, 100 µm. (G-H) MDA-MB-231 cells were transfected with negative-control or overexpression of GPX4. The ability of the cells to migrate was evaluated using the scratch assay and transwell assay after co-treatment with or without AC and 3-MA. Representative images and date are shown. Scale bar, 100 µm. Data are expressed as the mean ± SEM. These results are consistent with those of at least three different experiments. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance was compared between the respective AC groups.

Fig 3: Ferroptosis was involved in CAG injury. (A) GPX4 immunofluorescence staining in the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). The bottom images were the larger images of the frame in the top images, and the arrows indicate the location of GPX4-positive cells. (B) The mean density of GPX4-positive cells in Panel (A). (C, D) The expression and analysis of GPX4 and 4-HNE protein levels in the gastric tissue of rats, as determined by Western blotting (n = 4). (E) TEM analysis of the gastric tissue of rats with 15,000 or 25,000 magnification (n = 3). The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. NAG or Con groups. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Fig 4: Anomanolide C reduces GPX4 protein level via directly inducing GPX4 ubiquitination. (A) The predicted binding mode of AC with GPX4. (B) The combination of AC and GPX4 by molecular dynamic simulation. (C) CETSA analysis of GPX4 combined with AC. β-actin was measured as a loading control. Images and quantitation of the percentage of positive ratios are represented. (D) Volcano plot of autophagy-related ferroptosis genes with significant differences expression in MDA-MB-231 cells treated with or without AC (1 µM). (E) Immunoblotting analysis GPX4 expression in the breast cancer cell lines. (F) AC binds to GPX4 protein as shown by surface plasmon resonance (SPR) measurements. The estimated KD is ~24 µM. (G) MDA-MB-231 and BT549 cell lysates were incubated with IgG or anti-Ub antibody at 4 °C for 6 h, then agarose A + G was added, co-incubated at 4 °C overnight on rotary shaker. The samples were washed with PBS and analyzed by immunoblotting assay. (H) AC was added into MDA-MB-231 and BT549 cells and incubated for 24 h. The proteins were collected and incubated GPX4 antibody for 2 h at 4 °C, then agarose G was added and co-incubated at 4 °C overnight. The ubiquitination level of GPX4 was analyzed by immunoblotting assay with anti-Ub antibody. Data are expressed as the mean ± SEM. These results are consistent with those of at least three different experiments. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance was compared between the respective AC groups.

Fig 5: Anomanolide C induces ferroptosis in TNBC cells in vitro and in vivo. (A) KEGG were performed to predict the potential targets. Functional clustering of gene ontology (GO) was carried out on molecular function. (B-C) Immunofluorescence analysis of Fe2+ levels in MDA-MB-231 and BT549 3D spheroids treated with or without AC for 24h. Quantification of immunofluorescence analysis were shown. Scale bar, 20 μm. (D-E) ROS formation in the absence or presence of AC (24 h) was observed by flow cytometry. Quantification of ROS levels and representative images are shown. (F-G) Western blotting analysis expression of FTH1, GPX4, SLC7A11 and ACSL4 treated with or without AC in MDA-MB-231 and BT549 cells. β-actin was used as a loading control. Quantification of FTH1, GPX4, SLC7A11 and ACSL4 levels and representative images are shown. (H) MDA-MB-231-luc treated nake mice tumors were observed by transmission electron microscopy. The arrow represents mitochondria. (I) Immunoblotting analysis expression of FTH1, GPX4, SLC7A11 and ACSL4 treated with AC (25, 50 mg/kg) in nake mice tumor. β-actin was used as a loading control. Quantification of FTH1, GPX4, SLC7A11 and ACSL4 levels and representative images are shown. Data are presented as the mean ± SEM. These results are consistent with those of at least three different experiments. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance was determined relative to the appropriate AC groups.