Caspase-Independent Cell Death Induced by a Nitric Oxide Donor Derived from Valproic Acid in Human Pancreatic Cancer Cells

Nitric oxide (NO) plays diverse roles in tumor biology, including modulation of blood flow and induction of cell death at high concentrations. In this study, we synthesized a novel NO donor derived from valproic acid (NVA) and investigated its cytotoxic mechanism in human pancreatic cancer cells. NVA released approximately 40% of its total nitrate/nitrite (NO_x) immediately after dissolution in phosphate-buffered saline and then remained almost unchanged for 72 h, indicating a rapid initial NO release followed by stabilization. NVA significantly decreased the viability of BxPC-3 cells, whereas valproic acid (VA) alone had little effect. Flow cytometric analysis using Annexin V revealed that NVA-induced cell death was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. Furthermore, Western blotting showed no cleavage of caspase-3 or poly(ADP-ribose) polymerase (PARP) following NVA exposure, suggesting that apoptosis was not the major pathway. These findings indicate that NVA induces NO-dependent, caspase-independent cell death, distinct from classical apoptosis. The present study provides fundamental insights into the potential use of VA-based NO donors as antitumor agents against pancreatic cancer.