Bufalin Induces Programmed Necroptosis in Triple-Negative Breast Cancer Drug-Resistant Cell Lines through RIP1/ROS-Mediated Pathway

Objective: To explore the effect and mechanism of action of bufalin in triple-negative breast cancer(TNBC) drug-resistant cell lines. Methods: The normal human mammary epithelial cell line, TNBC cell line, TNBC adriamycin-resistant cell line, and TNBC docetaxel-resistant cell line were treated with different doses of bufalin(0–1,000 nmol/L) at different time points(0–72 h). Propidium iodide staining, AV-FITC/PI double staining, Hoechst 33342/PI double staining and transmission electron microscopy(TEM) were used to evaluate the death patterns of the cell lines. Results: Bufalin killed the TNBC cell line and its drug-resistant cell lines in a dose/time-dependent manner(all P<0.01). After treatment with bufalin for 24 h, the adriamycinresistant cell line showed a co-existing pattern of necroptosis and apoptosis. However, at 48 h, necroptosis was the main manifestation. After treatment with bufalin, the expressions of tumor necrosis factor α, phospho-tumor necrosis factor receptor 1, phospho-receptor interacting protein 1 and c-caspase 3 increased(all P<0.01), the killing effect of bufalin could be mostly inhibited by NEC-1, and by z-VAD-fmk(both P <0.01). Besides, the intracellular reactive oxygen species(ROS) levels increased considerably(P<0.01), the antioxidant N-acetyl cysteine or Nec-1 could inhibit the increase of ROS level and the killing effect of bufalin(all P<0.01). The adriamycin-resistant cell line exhibited necroptosis characteristic after 48 h of bufalin treatment under TEM. Conclusions: Bufalin could induce necroptosis through RIP1/ROS-mediated pathway to kill the drug-resistant TNBC cell lines. This finding provides critical experimental data and theoretical basis for the clinical application of bufalin to overcome the difficulties in the treatment of TNBC.