A Phenylbutyrate-Derived Nitric Oxide Donor Induces Pancreatic Cancer Cell Death Accompanied by Impairment of Autophagy-Related Pathways and HIF-1α Reduction

Abstract

4-[4-(Bis(2-(nitrooxy)ethyl)amino)phenyl]butanoic acid (NPB), a phenylbutyrate-derived nitric oxide (NO) donor, has been developed as a potential anticancer agent for pancreatic cancer. In the present study, we investigated the cytotoxic effects of NPB under cellular stress conditions and examined its effects on autophagy-related pathways and hypoxia-inducible factor-1α (HIF-1α) signaling. NPB-induced cell death was enhanced under nutrient-deprived conditions in PANC-1 cells. In addition, NPB induced greater cell death under hypoxic conditions than under normoxic conditions in PANC-1 cells, whereas in BxPC-3 cells, NPB-induced cell death was slightly but significantly lower under hypoxic conditions than under normoxic conditions. Using GFP-LC3-RFP-LC3ΔG reporter cells, NPB suppressed starvation-induced autophagic flux. In pancreatic cancer cells, NPB decreased DAPGreen fluorescence, an indicator of autophagy-related vesicular activity, and increased propidium iodide-positive cells under hypoxic conditions. Western blot analysis showed that NPB induced the accumulation of p62 and LC3 under both normoxic and hypoxic conditions. Under hypoxic conditions, NPB also reduced HIF-1α expression. Under cobalt chloride (CoCl₂)-induced HIF-1α-accumulating conditions, NPB and the NO donor NONOate suppressed HIF-1α expression, whereas OH-PB, a non-NO-releasing analog, showed little effect. Furthermore, the proteasome inhibitor MG132 restored HIF-1α accumulation in NPB-treated cells. Time-course analysis under CoCl₂-treated conditions showed that NPB reduced HIF-1α expression concomitantly with p62 accumulation. These findings suggest that NPB induces pancreatic cancer cell death, particularly under nutrient-deprived and hypoxic conditions, accompanied by impairment of autophagy-related pathways and NO-dependent, proteasome-associated reduction of HIF-1α.