γ‐T3 Increases the Sensitization of Gastric Cancer Cells to OXA by a Regulation of ARHGAP29

ABSTRACT

γ‐Tocotrienol (γ‐T3) exhibits potent anti‐cancer effects in vitro and in vivo. Our previous study demonstrated that γ‐T3 modulates autophagy activity in gastric cancer (GC) cells via the GSK‐3β/β‐Catenin pathway. In this study, we further explored whether γ‐T3 also enhances the chemosensitivity of GC cells to oxaliplatin (OXA) by regulating ARHGAP29, and verified the direct binding between γ‐T3 and ARHGAP29 as well as the physical interaction of ARHGAP29 with GSK‐3β. We employed cell viability assays, flow cytometry, tumor cell spheroid formation assays, RT‐qPCR, Western blot analysis, and stable overexpression transfection. Additionally, cellular thermal shift assay (CETSA), co‐immunoprecipitation (Co‐IP), and chloroquine (CQ) rescue CCK‐8 assays were performed for molecular interaction and autophagy function verification; we established a xenografted tumor model using MKN45 cells in BALB/c nude mice (nu/nu). γ‐T3 and OXA inhibited GC cell proliferation in vitro and in vivo, with a synergistic effect when combined (CI = 0.55 in MKN45 cells). In a mouse model, intraperitoneal injection of γ‐T3 (20 mg/kg b.w.) and OXA (2.0 mg/kg b.w.) suppressed MKN45 cell xenograft growth. This was related to cell cycle arrest and inhibition of cancer stem cells (CSCs). γ‐T3 alone or combined with OXA altered autophagy‐related protein levels after 48 h treatment, with elevated LC3‐II/LC3‐I ratio and Beclin1 expression, and reduced p62 abundance, functional rescue with autophagy inhibitor CQ confirmed that the changed autophagy contributes to drug‐induced cell death, and this phenotypic change was accompanied by suppressed GSK‐3β/β‐Catenin pathway activity. ARHGAP29 was inversely correlated with GC prognosis; CETSA results validated the direct intracellular binding between γ‐T3 and ARHGAP29, and Co‐IP assays confirmed physical interaction between ARHGAP29 and GSK‐3β. γ‐T3 inhibited the expression of ARHGAP29 at protein and mRNA levels in a time‐ and dose‐dependent manner, whether alone or with OXA. Overexpression of ARHGAP29 reversed the synergistic effects of γ‐T3 and OXA in GC cells, further supporting the regulatory linkage between ARHGAP29 and the GSK‐3β/β‐Catenin axis. The combination of γ‐T3 and OXA represents a promising therapeutic strategy to elevate oxaliplatin sensitivity and alter autophagy‐related marker expression in GC, at least partially via the ARHGAP29‐associated modulation of GSK‐3β/β‐Catenin signaling.