Periplocin suppresses osteosarcoma progression by triggering necroptosis and pyroptosis through the “4EBP1-eIF4E” pathway

Osteosarcoma (OS) is an aggressive bone tumor with poor prognosis, highlighting the need for novel therapeutic strategies. Periplocin, a cardiotonic glycoside derived from traditional Chinese medicine, has shown antitumor effects in various cancers. However, its role in OS remains unclear. This study aims to investigate whether Periplocin exerts anticancer effects by inducing necroptosis and pyroptosis in OS cells and to explore the underlying regulatory mechanisms. Human OS and U2 OS cell lines were treated with Periplocin to assess cell viability, migration, proliferation, and cell death using Cell Counting Kit-8, transwell, wound healing, colony formation, and flow cytometry assays. The involvement of necroptosis and pyroptosis was evaluated using specific inhibitors (necrostatin-1 [Nec-1] and disulfiram [DSF]) and Western blot analysis of key markers (phosphorylated receptor-interacting protein kinase 3 [p-RIP3], phosphorylated mixed lineage kinase domain-like protein [p-MLKL], apoptosis-associated speck-like protein containing a CARD [ASC], caspase-1, gasdermin D [GSDMD], interleukin-1 [IL-1], and NOD-like receptor family pyrin domain-containing 3 [NLRP3]). The role of the 4EBP1-eIF4E pathway was examined through eIF4E siRNA knockdown, pharmacological inhibition (4EGI-1), phosphomimetic mutant eIF4E overexpression, and 4EBP1 overexpression. Co-immunoprecipitation (Co-IP) was used to verify changes in the interaction between 4EBP1 and eIF4E during Periplocin-induced anti-OS effects. In vivo antitumor effects were validated using a xenograft mouse model. Periplocin significantly inhibited OS cell proliferation, migration, and colony formation in a dose- and time-dependent manner. In addition, Periplocin induced both necroptosis and pyroptosis, as evidenced by increased expression of p-RIP3, p-MLKL, ASC, Caspase-1, GSDMD, IL-1, and NLRP3, along with elevated OS cell death. These effects were attenuated upon treatment with Nec-1 or DSF. Furthermore, eIF4E knockdown and inhibition, as well as 4EBP1 overexpression, reduced Periplocin-induced OS cell death, and promoted OS cell proliferation, migration, and colony formation. Overexpression of the phosphomimetic mutant eIF4E partially reversed the tumor-promoting effects of eIF4E knockdown. The Co-IP results showed that the eIF4E-4EBP1 interaction significantly decreased after Periplocin treatment. In vivo, Periplocin suppressed tumor growth and enhanced necroptotic and pyroptotic markers in tumor tissues; eIF4E inhibition partially abolished these antitumor effects. This study reveals a novel mechanism by which Periplocin suppresses OS progression through downregulation of 4EBP1, resulting in eIF4E activation and subsequent induction of necroptosis and pyroptosis. These findings highlight the therapeutic potential of Periplocin and the 4EBP1-eIF4E pathway as promising targets for OS treatment.