Vasoactive Intestinal Peptide Induces Autophagy and Cytotoxicity in Renal Cell Carcinoma via SIRT3‐Dependent Signaling Pathway

Renal cell carcinoma (RCC) is a common urologic malignancy with limited treatment options. The neuropeptide vasoactive intestinal peptide (VIP) regulates diverse biological processes by binding to VPAC1 and VPAC2 receptors, but its role in RCC remains unclear. The proliferation of RCC Caki-1 cells was evaluated using the Cell Counting Kit-8 (CCK-8), while lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GPT) release were measured using commercial kits. Reactive oxygen species (ROS) levels were analyzed via dihydroethidium (DHE) staining. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were quantified using commercial assay kits. Autophagy was assessed through monodansylcadaverine (MDC) staining, and the expression levels of related mRNAs and proteins were quantified by RT-PCR and Western blotting, respectively. VIP treatment (0-50 μM) exhibited dose-dependent cytotoxic effects, including reduced cell viability, increased LDH and γ-GPT release, and induction of oxidative stress as evidenced by elevated ROS and MDA levels with concomitant decrease in SOD2 activity. At concentrations of 5-10 μM, VIP induced autophagic responses characterized by enhanced MDC staining, increased microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, upregulated Beclin1 expression, and accelerated p62 degradation. Mechanistically, VIP-mediated autophagy was associated with sirtuin 3 (SIRT3) downregulation and increased Ac-FOXO1 levels, with SIRT3 overexpression effectively counteracting the VIP-induced autophagic effects. Our findings demonstrate that VIP induces both cytotoxic effects and autophagy in VPAC1-expressing RCC cells through modulation of the SIRT3/FOXO1 signaling pathway, highlighting its potential therapeutic value for renal cell carcinoma.