Abstract: Objective To investigate the effects of Spautin-1, an inhibitor of ubiquitin-specific peptidase 10 (USP10), on the proliferation, migration, autophagy, and apoptosis of human pancreatic cancer cell line PaTu 8988t, and to explore the underlying mechanisms. Methods Analysis of standardized TCGA/GTEx pancreatic ductal adenocarcinoma (PAAD) data from UCSC Xena was performed with GEPIA 2 to assess USP10 expression, its prognostic value and its potential correlation with autophagy genes (MAP1LC3B and SQSTM1). Validation and visualization were conducted with R language on the GEO dataset. PaTu 8988t cells were incubated with Spautin-1 (0~200 μmol/L). Cytotoxicity was determined by CCK-8 assay, and 20 μmol/L was selected for subsequent experiments. Cell migration and invasion were evaluated by wound healing and Transwell assays, respectively. Apoptosis was detected by flow cytometry (annexin V-FITC/PI double staining). Intracellular reactive oxygen species (ROS) level was measured with the DCFH-DA probe. Protein expression of USP10, LC3-Ⅱ and p62 was measured by Western blot. All experiments were independently repeated at least three times. Results 1)GEPIA 2 USP10 was up-regulated in PAAD accompanied by a poor prognosis/The expression was positively correlated with the autophagy genes MAP1LC3B and SQSTM1. Validation with GSE16515 dataset confirmed this expression trend, and clustering analysis suggested USP10's potential involvement in autophagy regulation. 2)Spautin-1 dose-dependently inhibited cell viability. 3)Spautin-1(20 μmol/L) significantly suppressed scratch healing rates and Transwell invasion (P＜0.05). 4)Spautin-1 down regulated LC3-Ⅱ and USP10 protein levels while up-regulating p62, indicating impaired autophagy flux(P＜0.05). 5)Spautin-1 significantly increased intracellular ROS level and apoptosis rates(P＜0.05). Conclusions Spautin-1 effectively inhibits proliferation and migration/invasion of PaTu 8988t cells. Its effects are accompanied by a reduced USP10 expression, blocked autophagy flux, ROS accumulation and increased apoptosis which suggest a potential mechanism involving USP10 inhibition, autophagy disruption and ROS-dependent apoptosis.

Key words: pancreatic cancer, ubiquitin-specific peptidase 10, autophagy inhibitor, apoptosis, reactive oxygen species