Isoeugenol Inhibits Sphingosine Kinase 1 (SphK1) and Induces Cytotoxicity in Breast Cancer Cells

Background: Sphingosine kinase 1 (SphK1) is considered as a critical factor as it controls sphingolipid metabolism and promotes cell survival. Targeting SphK1 with small natural compounds presents a promising therapeutic approach for breast cancer, as these compounds may have the ability to inhibit SphK1 activity, reduce tumor growth, and enhances cytotoxicity, potentially paving the way for new treatment approaches in cancer. Methods: This study employed in-silico and in-vitro methods to identify the potent inhibitor of SphK1. Molecular docking was used to determine the binding affinity and molecular interactions of protein-ligand complex. Other in-silico methods namely SwissTargetPrediction and PASS analysis were used to determine the pharmacological potential of the lead compound. The MTT assay was conducted to evaluate the cytotoxic effects of isoeugenol, as well as its impact on the expression, protein levels, and activity of SphK1 in MCF-7 breast cancer cells. Results: Isoeugenol was found as a potent small molecule with highest binding affinity among resveratrol and its structural analogues with SphK1. Results of the molecular docking showed binding affinity of-9.3 kcal/mol of isoeugenol with SphK1, Analysis of the docked isoeugenol-SphK1 complex revealed stable interactions ligand and target protein. Further, results of SwissTargetPrediction analysis showed that isoeugenol has broad pharmacological associations with targeting different proteins and enzymes, which are considered as key factors in various biochemical and molecular pathways, including associated with anticancer processes. MTT assay revealed a significant, dose-and time-dependent decrease in cell viability (both p<0.001). Viability declined progressively at 24, 36, and 48 h, with the effect persisting without further significant change at 60 and 72 h. Further, the IC₅₀ dose of isoeugenol was found to be 102 µM, this concentration was used for further experiments. MCF-7 cells exposed to IC₅₀ dose of isoeugenol showed significantly decreased SphK1 mRNA expression, SphK1 protein levels, and SphK1 kinase activity compared to untreated cells. Conclusion: The findings of the current study reflect the therapeutic potential of isoeugenol and suggest that inhibition of SphK1 by this small molecule may pave the way for cancer therapeutics, including breast cancer.