Indole-3-Propionic Acid Disrupts Glycolytic Adaptation and Promotes Oxidative Stress-Associated Injury in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) shows aggressive invasion, frequent recurrence, and marked metabolic adaptability. Microbial tryptophan metabolites are increasingly recognized as potential modulators of cancer-related signaling; however, the direct role of indole-3-propionic acid (IPA) in OSCC remains poorly defined. This study examined whether IPA suppresses malignant fitness in HSC-3 and SCC-25 cells while using OKF6/TERT-1, an immortalized human oral keratinocyte cell line, as a non-malignant comparator where biologically appropriate. HSC-3 and SCC-25 cells were treated with vehicle or IPA. Based on the calculated IC50 values from 72 h viability assays, fixed concentrations of 300 µM IPA for HSC-3 cells and 400 µM IPA for SCC-25 cells were selected for subsequent functional experiments. OKF6/TERT-1 cells were used as a non-malignant comparator for baseline transcriptional analyses. Metabolic activity was assessed by the MTS assay. Cytotoxicity and cell death-related stress were additionally examined using lactate dehydrogenase release and colorimetric caspase-3 activity assays. Migration was determined by a wound-healing assay. Metabolic adaptation was evaluated by glucose consumption, lactate secretion, conditioned-medium pH, intracellular adenosine triphosphate, spectrophotometric intracellular reactive oxygen species measurement, and quantitative real-time polymerase chain reaction. Baseline profiling showed that glycolysis-associated transcripts were consistently higher in HSC-3 and SCC-25 cells than in OKF6/TERT-1 cells, with the strongest malignant shift observed in HSC-3. IPA (300 µM for HSC-3, 400 µM for SCC-25) reduced 72 h viability to 55.3% in HSC-3 and 64.8% in SCC-25 cells. IPA increased intracellular reactive oxygen species by 1.86-fold in HSC-3 and 1.49-fold in SCC-25 cells, increased caspase-3 activity, and elevated lactate dehydrogenase release. In parallel, glucose consumption, lactate secretion, adenosine triphosphate content, and migration decreased, whereas conditioned-medium pH and CDH1 expression increased. IPA may directly suppress OSCC cell survival, motility, and glycolytic adaptation while promoting oxidative stress-associated injury and modulating epithelial–mesenchymal transition markers. This metabolic framework may provide a novel experimental direction for OSCC research.