Timosaponin B-II suppresses gastric cancer cell proliferation and induces apoptosis via the Nrf2/miR-455-3p/KLF6 pathway.

BACKGROUND: Gastric cancer (GC), known for its aggressive growth and metastasis, remains a leading cause of cancer-related mortality. Although Timosaponin B-II (TB-II) from Anemarrhena asphodeloides has shown anticancer potential, its underlying mechanisms in GC are not yet fully understood. METHODS: Our study investigates and compares the expression patterns of Kruppel-Like Factor 6(KLF6) in GC tissues across different TNM stages, while exploring its upstream regulatory factors. GC cells were treated with Timosaponin B-II (TB-II), and alterations in cell proliferation and apoptosis rates were evaluated using CCK-8 and TUNEL assays. Furthermore, the expression levels of nuclear factor erythroid 2-related factor 2(Nrf2), miR-455-3p, and KLF6 were quantified to elucidate the mechanisms underlying TB-II's effects. To confirm the interaction between miR-455-3p and KLF6, as well as Nrf2 and miR-455-3p, bioinformatics analysis, luciferase assays, and ChIP-PCR were conducted. Finally, protein synthesis and degradation assays were performed to explore the mechanism by which TB-II regulates the expression and activity of Nrf2. RESULTS: Both mRNA and protein expression levels of KLF6 were significantly lower in GC tissues compared to adjacent normal tissues. Notably, only KLF6 protein expression exhibited a decline in GC tissues from stages I to III of GC, whereas its upstream regulator, miR-455-3p, displayed the opposite trend. Treatment with TB-II markedly inhibited GC cells proliferation and induced apoptosis in a dose-dependent manner. Mechanistically, TB-II treatment upregulated the expression of Kelch-like ECH-associated protein 1(Keap1) protein, facilitating the formation of the Keap1/Nrf2 complex, which enhanced the ubiquitin-mediated degradation of Nrf2 in GC cells. Consequently, the transcriptional activation of miR-455-3p by Nrf2 was suppressed, resulting in the upregulation of the tumor suppressor KLF6. Silencing KLF6 can counteract the effects of TB-II in inhibiting proliferation and promoting apoptosis in GC cells. CONCLUSION: TB-II suppresses GC cell proliferation and induces apoptosis via the Nrf2/miR-455-3p/KLF6 pathway.