Application of In Silico Computational Biology Strategies and Gene Expression Analysis to Demonstrate Mechanism of Oral Cancer Cell Death by a Natural Peptide ^†

Introduction: This research work is designed to identify biomolecules from Earthworm Coelomic Fluid (ECF) of Eudrilus Eugeniae (EE) that can inhibit cancer cellproliferation. This study aims to construct a homology model of the 18 kDa protein from the ECF of EE (18-ECFP) with molecular dynamics simulation (MDS) to enable its molecular docking with pro-apoptotic caspase receptors with a determination of binding energy scores. This study also evaluates the anti-cancer potential of 18-ECFP on SCC-9 cells in vitro by wet lab techniques.

Methods: Following SDS-PAGE and MALDI-TOF/MS-MS sequencing, the 18 kDa protein was subjected to Nano-LCMS-based AA sequencing. Due to the unavailability of a 3D structure in Protein Data Bank (PDB), it had to be modelled via energy-based methods using Prime module—Schrödinger. The MDS of the protein was analyzed followed by Protein–Protein Docking (PPD) using Schrödinger 2020 software. The op 5 poses exhibiting a high PIPER score were subjected to energy calculations. The 18-ECFP was also evaluated by RT-PCR, Western blot and Q-PCR techniques on SCC-9 cells in vitro to further establish its anti-cancer potential.

Results: The homology model of the 18-ECFP was constructed with Schrödinger software with stable molecular dynamics. PPD demonstrated binding affinity of 18-ECFP with the pro-apoptotic genes Caspase-3 and Caspase-8. The MM-GBSA revealed satisfactory binding energy scores. Gene expression studies revealed an upregulation of the apoptotic genes Caspase-3 and Caspase-8 induced by the 18-ECFP, validating the in silico findings.

Conclusions: This is the first report of a homology model with MDS of an anti-cancer protein from an earthworm source docked to human caspase receptors with a determination of binding energy values supported by validation through multiple in vitro gene expression techniques. The current study has provided valuable insights pertaining to the molecular structure of the novel anti-cancer protein of ECF. The findings may contribute to the development of naturally available drugs to combat cancer.