Abstract:
Background
The sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) axis has been widely studied in cancer research due to its role in modulating sphingolipid metabolism, which determines cell survival or death. Furthermore, SphK1 inhibitors prevent catabolism of ceramides, contributing to tumor cell death. In a previous study we demonstrated a synergistic antitumor effect in vitro and in vivo between the antitumor flavonoid 2´nitroflavone (2NF) and safingol in a murine mammary tumor model. In this work, we investigated the molecular mechanisms underlying this interaction.
Methods
Molecular docking was performed with the online docking web server SwissDock. We used LM3 murine mammary adenocarginoma cells as an HER2+ model for all in vitro experiments and HEK-293 cells for SPHK1-GFP (green fluorescent protein) transfection and translocation assays, which were evaluated by fluorescence microscopy. Protein expression and phosphorylation were determined by Western Blot and mRNA expression was examined by RT-PCR. Proliferation assays were performed employing the hexosaminidase method.
Results
Docking analysis revealed 2NF/SPHK1 interaction near Ser225, a phosphorylation site involved in SPHK1 translocation to the cell membrane. Accordingly, 2NF inhibited SPHK1 phosphorylation at Ser225. In cells transfected with SPHK1 fused to GFP, 2NF blocked the translocation of the enzyme to the cell membrane. Additionally, the combination of 2NF and safingol activated p38, JNK, and ERK MAPKs, and induced ER stress by enhancing the expression of ER stress-related genes and proteins. Pharmacological inhibition of these pathways led to reduced antiproliferative activity, indicating their involvement in the antitumor effect triggered by the combination of both drugs.
Conclusions
In summary, results suggest that synergism may arise from inhibition of SPHK1 activity by safingol through its interaction with the enzyme's active site, combined with the blockade of SPHK1 phosphorylation and translocation by 2NF, both essential steps for its catalytic activity. Furthermore, the activation of MAPKs and the induction of ER stress are critical events leading to the cell death caused by this drug combination.
Biography:
Dr. Viviana C. Blank is a biochemist and researcher at the University of Buenos Aires, Argentina. She holds a Ph.D. in Biological Chemistry and is a member of CONICET's Scientific Researcher Career. Her work focuses on flavonoids and synthetic compounds with antitumor activity, particularly studying their efficacy and mechanisms of action. Dr. Blank also teaches Biological Chemistry at undergraduate and postgraduate levels at the School of Pharmacy and Biochemistry, University of Buenos Aires.
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