ABS 031: Evaluating the Efficacy of Acetylcholine and C14 Ceramide as Potent Targeted Therapies in Inhibiting Different High-Grade Serous Carcinoma Cell Lines

Rasagna Vuppala ¹ , Lilya Matyunina ¹ , Asha Rao ¹ , Prof. Jeffrey Skolnick ¹

¹ Georgia Institute of Technology

Van Wickle (2025) Volume 1, ABS 031

Introduction: Ovarian Cancer is one of the most lethal gynecologic malignancies, in part driven by its limited treatment options. As a result, there is significant growth in research for innovative approaches to identify new therapeutic targets. The bioinformatics team of the Skolnick Lab optimized computational predictions of biomolecular interactions on AlphaFold3 to identify over fifty target metabolites with the potential to treat Ovarian Cancer. The experimental researchers focused on testing the efficacy of two of those metabolites, Acetylcholine and C14 Ceramide, on inhibiting the growth of different Ovarian Cancer cell lines. Acetylcholine and C14 Ceramide are neurotransmitters and sphingolipids which respectively have an impact on the autocrine-proliferative network and apoptosis pathways in the body. By treating three differing cell lines (OVCAR4 (Epithelial Cells), HEY A8 (Mesenchymal Cells), AGO7307 (Fibroblasts)) with six varying metabolite concentrations, scientists intended to note if the metabolites suppressed cell line growth. While Acetylcholine selectively inhibited only OVCAR4, C14 Ceramide hindered growth across both OVCAR4 and HEY A8 cell lines. Neither of the metabolites induced any change in AGO7307 growth, serving as a model for no damage caused to the normal cells in the human body. Since there was a dramatic difference in metabolite inhibition across differing cell line phenotypes, the results from this experiment emphasize the dire need for discovering phenotypical-based treatments, offering patients with niche therapeutic targets to treat their specific cancer subtype.

Methods: To test for the effects of Acetylcholine and C14 Ceramide on suppressing cancer cell growth, researchers performed a series of cell culture procedures involving defrosting, culturing, plating, adding metabolites, collecting, and counting the cells all under controlled environments. Before beginning each process, they activated the UV light in the Biosafety Hood to eliminate potential contamination and sprayed the hood with ethanol. Any required liquid reagents were placed in a 37°C water bath for warming, with the most significantly used reagent being cell culture media to support the growth and maintenance of cells in vitro. All the experiments entailed following conventional cell culture protocols for contamination prevention such as consistently spraying the BioSafety Hood with ethanol when working with cultures. Each cell culture assay spanned seven days, concluding with cell counting and metabolite inhibition analysis. Data was transformed to determine average cell count, cell viability, and other values of significance.

Results: The results, based on cancer cell viability, revealed differential responses of Ovarian Cancer and control cell lines to metabolite treatments. Acetylcholine had no inhibitory effect on HEY A8 or AGO7307 cells, but demonstrated a borderline trend of inhibition on OVCAR4 cells (p = 0.064), warranting further investigation. In contrast, C14 Ceramide significantly reduced viability in both HEY A8 (p = 0.022) and OVCAR4 (p = 0.000) cell lines, with no effect on AGO7307 cells. C14 Ceramide strongly exhibits the potential as an Ovarian Cancer cell inhibitor across different subtypes, while Acetylcholine’s effects appear more limited and cell-line specific.

Discussion: Acetylcholine slightly selectively inhibited epithelial OVCAR4 cells but had limited effect on the inhibition of mesenchymal HEYA8F8 cells, suggesting its action may be mediated through cholinergic receptors, which are present at higher quantities on epithelial cells than mesenchymal cells. C14 Ceramide inhibited both mesenchymal (HEYA8F8) and epithelial (OVCAR4) ovarian cancer cells, having a broader apoptotic role. Both metabolites did not inhibit fibroblast control cells, indicating their selectivity for specific cancer cells rather than general cytotoxicity. Cancer cell heterogeneity influences drug sensitivity and resistance, accentuating the need for tumor phenotyping to design effective, targeted therapies for Ovarian Cancer patients.