Combined Effects of ATRA and ASA on HeLa Cell Viability, MAPK/AKT-Related Protein Abundance, and Intracellular VEGF: An Integrated In Vitro and In Silico Study

Background/Objectives: All-trans retinoic acid (ATRA) and acetylsalicylic acid (ASA; aspirin) have been reported to influence cancer-associated signaling through distinct molecular mechanisms, but their combined effects on cervical cancer cells remain incompletely characterized. This study evaluated the individual and combined effects of ATRA and ASA on HeLa human cervical cancer cells and used computational analyses as supportive, hypothesis-generating mechanistic contextualization. Methods: Cell viability was assessed by MTT assay after treatment with increasing concentrations of ATRA and ASA for 24, 48, and 72 h. The abundance of total ERK1/2, phospho-AKT (Ser473), total p38 MAPK, and total JNK was assessed by Western blotting, while intracellular HIF-1α and VEGF concentrations were measured in cell lysates by ELISA. Computational analyses included cross-validated response modeling, bootstrap estimation of Bliss excess inhibition, STRING network topology and enrichment, SwissTargetPrediction/SEA consensus target prediction, TCGA-CESC survival analysis, and redocking-validated molecular docking. Results: ATRA and ASA reduced HeLa cell viability in a dose- and time-dependent manner. Treatment was associated with lower total ERK1/2 abundance and lower phospho-AKT (Ser473) abundance, whereas ASA-containing conditions were associated with higher stress-associated MAPK markers, particularly JNK. Intracellular VEGF concentrations were lower after ATRA, ASA, and combined treatment, while intracellular HIF-1α concentrations were not significantly altered under normoxic culture conditions. Computational response modeling showed high predictive accuracy for ATRA and ASA, while bootstrap Bliss analysis indicated dose-dependent interaction behavior rather than uniform synergy. STRING analysis supported pathway-level coherence among retinoid, COX/prostaglandin, MAPK/AKT, hypoxia, and angiogenesis-related modules. TCGA-CESC analyses associated VEGFA, HIF1A, and PTGS2 with adverse survival outcomes in adjusted models. Docking supported ATRA–RARG and salicylate–COX-2 pose plausibility but did not support direct VEGFR2 or AKT1 targeting by ASA or salicylate. Conclusions: These findings suggest that ATRA and ASA reduce HeLa cell viability and are associated with altered MAPK/AKT-related protein abundance and lower intracellular VEGF concentrations in HeLa cervical cancer cells. The computational results provide pathway-level support and target prioritization for future validation but should not be interpreted as proof of direct binding, causal pathway mediation, treatment efficacy, or clinical benefit.