Arnicolide D Inhibits Oxidative Stress-induced Breast Cancer Cell Growth and Invasion through Apoptosis, Ferroptosis, and Parthanatos

Background: Breast cancer is the most common malignant tumor in women, and its pathogenesis is very complicated. More and more studies have found that Traditional Chinese Medicine plays an important role in tumor prevention. Objective: To investigate the mechanism of arnicolide D isolated from Centipeda minima in breast cancer. Methods: Cell Counting Kit-8 (CCK-8), western blot, RT-qPCR, ELISA, flow cytometry, and Transwell were used to detect the effect of arnicolide D on the biological function of breast cancer cells. Results: Arnicolide D promoted reactive oxygen species (ROS) production and induced a decrease in mitochondrial membrane potential in breast cancer cells, thereby inhibiting cell viability and increasing lactate dehydrogenase (LDH) release. Arnicolide D activated the classical apoptosis pathway to induce cell apoptosis; it significantly promoted PARP-1 expression, enhanced the nuclear translocation of apoptosis-inducing factor (AIF), and reduced the expression of AIF in mitochondria, indicating that it can induce the occurrence of parthanatos in a ROS dependent manner. In addition, arnicolide D down-regulated glutathione peroxidase 4 (GPX4) expression and increased the accumulation of Fe²⁺ and malondialdehyde (MDA), thereby activating ferroptosis. Apoptosis inhibitor, ferroptosis inhibitor, PARP inhibitor, PARP-1 siRNA, AIF siRNA and GPX4 overexpression vector significantly attenuated the inhibitory effect of arnicolide D on cell viability and reduced LDH release, which indicates that arnicolide D inhibits breast cancer cell growth by inducing apoptosis, parthanatos and ferroptosis. Arnicolide D also reduced breast cancer cell invasion and inhibited the expression of matrix metallopeptidase (MMP)-2 and MMP-9. Conclusion: Arnicolide D can activate a variety of cell death modes by inducing oxidative stress, thereby inhibiting the growth and invasion of breast cancer cells, indicating that arnicolide D has a good anti-tumor effect.