Secretion of Sphinganine by Drug-Induced Cancer Cells and Modified Mimetic Sphinganine (MMS) as c-Src Kinase Inhibitor

Abstract

Background: Cancer cells exhibit selective metabolic reprogramming to promote proliferation, invasiveness, and metastasis. Sphingolipids such as sphingosine and sphinganine have been reported to modulate cell death processes in cancer cells. However, the potential of extracellular sphinganine and its mimetic compounds as inducers of cancer cell death has not been thoroughly investigated.

Methods: We obtained extracellular conditioned medium from HCT-116 cells treated with the previously reported anticancer composition, goat urine DMSO fraction (GUDF). The extracellular metabolites were purified using a novel and in-house developed vertical tube gel electrophoresis (VTGE) technique and identified through LC-HRMS. Extracellular metabolites such as sphinganine, sphingosine, C16 sphinganine, and phytosphingosine were screened for their inhibitory role against intracellular kinases using molecular docking. Molecular dynamics (MD) simulations were performed to study the inhibitory potential of a novel designed modified mimetic sphinganine (MMS) (Pubchem CID: 162625115) upon c-Src kinase. Furthermore, inhibitory potential and ADME profile of MMS was compared with luteolin, a known c-Src kinase inhibitor.

Results: Data showed accumulation of sphinganine and other sphingolipids such as C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0) in the extracellular compartment of GUDF-treated HCT-116 cells. Molecular docking projected c-Src kinase as an inhibitory target of sphinganine. MD simulations projected MMS with strong (-7.1 kcal/mol) and specific (MET341, ASP404) binding to the inhibitory pocket of c-Src kinase. The projected MMS showed comparable inhibitory role and acceptable ADME profile over known inhibitors.

Conclusion: In summary, our findings highlight the significance of extracellular sphinganine and other sphingolipids, including C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0), in the context of drug-induced cell death in HCT-116 cancer cells. Furthermore, we demonstrated the importance of extracellular sphinganine and its modified mimetic sphinganine (MMS) as a potential inhibitor of c-Src kinase. These findings suggest that MMS holds promise for future applications in targeted and combinatorial anticancer therapy.

Keywords: HCT-116; neoplasm; therapy.

Figure 1

GUDF Induces Proliferative Arrest and Cell Death in HCT-116 Colon Cancer Cells. (A) HCT-116 colon cancer cells were treated by DMSO and GUDF (50 µg/ml) for 72 hr. Routine microscopy was performed at 100X to observe the cell number and cellular morphology. (B) HCT-116 colon cancer cells were treated by DMSO and GUDF (50 µg/ml) for 72 hr. Percentage total cell and viable HCT-116 colon cancer cells estimated by Trypan blue dye exclusion assay. Trypan blue dye exclusion assay was performed to estimate viable and dead cells. Data are represented as mean ± SD. Each experiment was conducted independently three times. The bar graph without an asterisk denotes that there is no significant difference compared to the DMSO control. * Significantly different from DMSO control at the P-value < 0.05. ** Significantly different from DMSO control at P-value < 0.01. *** Significantly different from DMSO control at P-value < 0.001

Figure 2

Sphinganine is Secreted and Accumulated in HCT-116 Colon Cancer Cells Treated by GUDF Positive Mode ESI MS and MS/MS fragment ion spectra of sphinganine in GUDF treated HCT-116 cancer cells. Sphinganine is known as a sphingolipid base

Figure 3

Sphinganine Displays Strong binding to the Inhibitory Site of the c-Src Kinase, while Sphingosine Showed Binding to the Active Site of the c-Src Kinase. Molecular docking and inhibitory interaction by sphinganine and sphingosine upon c-Src kinase with the help of Autodock Vina. (A). A ribbon structure with a full 3D view between sphinganine and c-Src kinase. (B). A ribbon structure with a full 3D view between sphingosine and c-Src kinase. (C). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between sphinganine and c-Src kinase. (D). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between sphingosine and c-Src kinase

Figure 4

Sphinganine and Luteolin, a Known Inhibitor of c-Src Kinase Show Similar Binding to the Inhibitory Site of the c-Src Kinase. Molecular docking and inhibitory interaction by sphinganine and luteolin upon c-Src kinase with the help of Autodock Vina. (A). A ribbon structure with a full 3D view between sphinganine and c-Src kinase. (B). A ribbon structure with a full 3D view between luteolin and c-Src kinase. (C). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between sphinganine and c-Src kinase. (D). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between luteolin and c-Src kinase

Figure 5

MMS and Luteolin, a Known Inhibitor of c-Src Kinase Show Similar Binding to the Inhibitory Site of the c-Src Kinase. Molecular docking and inhibitory interaction by MMS and luteolin upon c-Src kinase with the help of Autodock Vina. (A). A ribbon structure with a full 3D view between MMS and c-Src kinase. (B). A ribbon structure with a full 3D view between luteolin and c-Src kinase. (C). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between MMS and c-Src kinase. (D). Discovery Studio Visualizer assisted 2-D image of docked molecular structure between luteolin and c-Src kinase

Figure 6

MMS and Luteolin, a Known Inhibitor of c-Src Kinase Show Similar and Stable Inhibitory Complexes. MD simulations for 20ns derived Root Mean Square Deviation (RMSD) plot of c-Src kinase in complex with (A) MMS and (B) luteolin

Figure 7

MMS and Luteolin, a Known Inhibitor of c-Src Kinase Display the Least Fluctuation of Ligand-Protein Complexes. MD simulations for 20ns derived Root Mean Square Deviation (RMSD) plot of c-Src kinase in complex with (A) MMS and (B) luteolin

Figure 8

Protein-Ligand Contact Map of MMS and Luteolin, a Known Inhibitor of c-Src Kinase Shows Key Residues of the Regulatory Domain of the c-Src Kinase. MD simulations for 20 ns derived Ligand-Protein contact map of c-Raf kinase in complex with (A) MMS and (B) Luteolin

Figure 9

ADMET Profile of MMS, Luteolin and PPI (an inhibitor of c-Src kinase) Predicts Favorable Attributes on DILI, Cytotoxicity, Carcinogenicity, and MRTD Values. ADMET profile is generated using v-NN/ADMET server. (A) MMS and (B) Luteolin, (C) PPI

Figure 10

A Proposed Model on the Role of Extracellular Secretion of Sphinganine in Drug-Induced Cell Death in Colon Cancer Cells. A proposition on the potential of modified mimetic sphinganine (MMS) as a potential inhibitor of the c-Src kinase