Abstract

Objective To explore the potential role and mechanism of platycodin D (PD) overcomes cisplatin (DDP) resistance in gastric cancer cells. Methods DDP-resistant gastric cancer cells AGS/DDP and HGC-27/DDP were constructed. The cells were divided into the following 6 groups, including erastin (10 µg/mL), PD (20 µmol/L), PD+ferrostatin-1 (Fer-1, 50 nmol/L), PD + overexpression negative control (OE-NC, 2 µg/µL), PD + overexpression of Tousled-like kinase 1 (OE-TLK1, 2 µg/µL), PD+ overexpression of DNA methyltransferase 3 alpha (OE-DNMT3A, 2 µg/µL). Cell viability and proliferation were assessed using cell counting kit-8 (CCK-8) or EdU kits. Ferroptosis-related indices including Fe 2+ , malondialdehyde (MDA) and reactive oxygen species (ROS) were measured employing relevant kits. The interactions between TLK1 and ubiquitin-like with PHD and ring finger domains 2 (UHRF2) or UHRF2 and DNMT3A were confirmed utilizing co-immunoprecipitation (Co-IP) assays. The ubiquitination of DNMT3A was analyzed through immunoprecipitation. In vivo , efficacy of PD was evaluated using subcutaneous graft tumors implanted in nude mice. DDP-resistant AGS gastric cancer cells (AGS/DDP, 1 × 10 7 cells) were injected into the subcutaneous of nude mice, 1-week post-injection, DDP (5 mg/kg) was intraperitoneally injected 3 times per week. Male nude mice were randomly divided into 4 groups ( n =6): control (0.9% saline), PD (10 mg/kg, intraperitoneally daily), PD + OE-NC (2 × 10 10 vp/dose, subcutaneous injection) and PD + OE-TLK1 (2 × 10 10 vp/dose, subcutaneous injection). Tumor volume growth curves were recorded and tumor weights were measured at the end of the experiment. Proliferation marker protein KI-67 and glutathione peroxidase 4 (GPX4) expressions were detected using immunohistochemical staining. Western blot analysis was performed to detect TLK1, phosphorylated-UHRF2 (p-UHRF2), UHRF2, DNMT3A, and arachidonate 15-lipoxygenase (ALOX15), ferroptosis-related proteins GPX4 and solute carr