Low-intensity pulsed ultrasound and oridonin synergistically induce ferroptosis of pancreatic cancer cells by activating PIEZO1 via the Nrf2/HO-1/GPX4 pathway

doi:10.12122/j.issn.1673-4254.2025.10.12

Abstract

Abstract:

Objective To evaluate the inhibitory effect of oridonin against proliferation of pancreatic cancer cells and the mechanism underlying the synergistic effect of low-intensity pulsed ultrasound (LIPUS). Methods PANC-1 cells treated with different concentrations of oridonin were examined for changes in cell proliferation using CCK-8 assay and in MDA, GSH and ATP levels using flow cytometry. The protein expressions of GPX4, Nrf2 and HO-1 in the treated cells were detected with Western blotting. The effect of Fer-1, a ferroptosis inhibitor, on proliferation of oridonin-treated cells were assessed, and the effects of oridonin combined with LIPUS on PIEZO1 protein expression was evalauted using Western blotting. A C57BL/6J mouse model bearing pancreatic cancer cell xenograft was established and treated with oridonin, LIPUS, or both, and the histological changes in the tumor tissues and tumor cell proliferation were examined with HE staining and immunohistochemistry for Ki67; the changes in GPX4 expression in the tumor tissues were detected using Western blotting and immunofluorescence staining. Results In PANC-1 cells, oridonin treatment significantly inhibited cell proliferation, increased intracellular Fe²⁺, ROS, and MDA levels, and decreased GSH and ATP levels. Oridonin also resulted in lowered GPX4 and increased HO-1 and Nrf2 protein expression levels in the cells. The combined treatment with LIPUS signficiantly enhanced the inhibitory effect of oridonin on PANC-1 cell viability in vitro and on xenograft growth in the mouse models, resulting also in more obvious reduction of the intensity of Ki67 staining and GPX4 protein expression and more pronounced increase of PIEZO1 protein expression in the tumor tissues in the mouse models. Conclusion LIPUS enhances the effect of oridonin to promote ferroptosis of pancreatic cancer cells by activating PIEZO1 through the Nrf2/HO-1/GPX4 pathway.

Key words: low-intensity pulsed ultrasound, oridonin, ferroptosis, piezo1, pancreatic cancer

Bihang SUN, Yujun GUO, Yulin QI, Dan YAO, Wenzhi CHEN, Nianzhi CHEN. Low-intensity pulsed ultrasound and oridonin synergistically induce ferroptosis of pancreatic cancer cells by activating PIEZO1 via the Nrf2/HO-1/GPX4 pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2160-2170.

Figures/Tables 13

Fig.1 Effect of different concentrations of oridoni (Ori) on proliferation of PANC-1 cells (n=6).

Fig.2 Effect of Ori on intracellular Fe2+ level in PANC-1 cells (n=3). ***P<0.001 vs Control.

Fig.3 Effect of Ori on intracellular ROS level (n=3). **P<0.01 vs Control.

Fig.4 Effect of Ori combined with Fer-1 on proliferation of PANC-1 cells (n=6). ****P<0.001 vs Control, ***P<0.005 vs Ori Group.

Fig.5 Effect of Ori on intracellular MDA, GSH, and ATP levels in PANC-1 cells (n=3). *P<0.05, ***P<0.001 vs Control.

Fig.6 Effect of Ori on intracellular GPX4, Nrf2, and HO-1 expressions in PANC-1 cells (n=3). **P<0.001, ***P<0.005 vs Control.

Fig.7 Fluorescence intensity of C11-BODIPY labeling in PANC-1 cells after Ori treatment (Original magnification: ×40) (n=3).

Fig.8 Effect of Ori combined with LIPUS on proliferation of PANC-1 cells (n=6). **P<0.01 vs Ori-20 μmol/L.

Fig.9 Effect of Ori combined with LIPUS on intra-tumoral GPX4 expression in the mouse models (n=3). *P<0.05 vs Control,***P<0.005 vs Ori.

Fig.11 Effect of Ori combined with LIPUS on body weight of the mouse models bearing pancreatic cancer xenograft (n=8).

Fig.12 Effect of LIPUS combined with Ori on tumor volume and weight in the tumor-bearing mice (n=8). ****P<0.001 vs Control, **P<0.01 vs Ori.

Fig.14 Effect of LIPUS on intracellular Fe2+ level (n=3). *P<0.05, **P<0.01 vs Control.

Fig.15 Effect of Ori combined with LIPUS on intracellular PIEZO1 expression (n=3). **P<0.005 vs Control; ***P<0.001 vs Ori.

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