Intracellular Ellagic Acid Derived from Goat Urine DMSO Fraction (GUDF) Predicted as an Inhibitor of c-Raf Kinase

Abstract

Background: Dietary chemicals and their gut-metabolized products are explored for their anti-proliferative and pro-cell death effects. Dietary and metabolized chemicals are different from ruminants such as goats over humans. Methods: Loss of cell viability and induction of death due to goat urine DMSO fraction (GUDF) derived chemicals were assessed by routine in vitro assays upon MCF-7 breast cancer cells. Intracellular metabolite profiling of MCF-7 cells treated with goat urine DMSO fraction (GUDF) was performed using an in-house designed vertical tube gel electrophoresis (VTGE) assisted methodology, followed by LC-HRMS. Next, identified intracellular dietary chemicals such as ellagic acid were evaluated for their inhibitory effects against transducers of the c-Raf signaling pathway employing molecular docking and molecular dynamics (MD) simulation. Results: GUDF treatment upon MCF-7 cells displayed significant loss of cell viability and induction of cell death. A set of dietary and metabolized chemicals in the intracellular compartment of MCF-7 cells, such as ellagic acid, 2-hydroxymyristic acid, artelinic acid, 10-amino-decanoic acid, nervonic acid, 2,4-dimethyl-2-eicosenoic acid, 2,3,4'-Trihydroxy,4-Methoxybenzophenone and 9-amino-nonanoic acid were identified. Among intracellular dietary chemicals, ellagic acid displayed a strong inhibitory affinity (-8.7 kcal/mol) against c-Raf kinase. The inhibitory potential of ellagic acid was found to be significantly comparable with a known c-Raf kinase inhibitor sorafenib with overlapping inhibitory site residues (ARG450, GLU425, TRP423, VA403). Conclusion: Intracellular dietary-derived chemicals such as ellagic acid are suggested for the induction of cell death in MCF-7 cells. Ellagic acid is predicted as an inhibitor of c-Raf kinase and could be explored as an anti-cancer drug.

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

“…In brief, GUDF was prepared in DMSO solvent by using standardized steps including vortexing, centrifugation, and filtration by 0.45-micron syringe filter membrane to ensure that GUDF is sterile and compatible with cell culture. This was adopted from a previously published procedure by our lab [43][44][45][46]. Next, treatment by GUDF and DMSO control was allowed for 72 h. At the end of incubation, HCT-116 cells were harvested with the help of a routine procedure and evaluated for loss of proliferation and viability due to GUDF by using a Trypan blue dye exclusion assay.…”

“…However, the importance of this paper is an extension to the previous observations by exploring metabolites in the extracellular conditioned medium of HCT-116 cells treated by GUDF. Therefore, the extracellular conditioned medium of GUDF and DMSO-treated HCT-116 cells were collected in separate sterile tubes and processed by centrifugation to ensure that viable and dead cells are not left in the tube so that only the extracellular nature of metabolites will be analyzed [43][44][45][46].…”

“…The fractionated extracellular metabolites were collected in the running buffer (96 mM glycine of pH at 8.3). The detailed procedure was adopted from previously published inhouse VTGE-assisted purification of metabolites [44,43,45,46]. Furthermore, LC-HRMS analyses of VTGEpurified extracellular metabolite elutes were performed by Agilent TOF/Q-TOF Mass Spectrometer station Dual AJS ESI ion source.…”

“…During LC separation, RPC18 Hypersil GOLD C18 100 x 2.1 mm-3 µm and mobile phase of 100% Water (0.1% FA in water) and 100% Acetonitrile (90% ACN +10% H2O+ 0.1% FA) were used in the proportion of 95% and 5% [39]. Mass spectrometry was performed in a positive mode and analyzed as per the procedure adopted from previously reported methodology [43,46].…”

“…Throughout the equilibrations, systems were coupled with the Martyna-Tobias-Klein barostat method for controlling pressure at 1 atm, and the temperature was regulated by using velocity rescaling Nose Hoover chain thermostat method at 300 K. The M-SHAKE algorithm was used to constrain the bond length of hydrogen atoms. Rest steps were adopted as per previously published procedure [43][44][45][46][47][48].…”