Anti-proliferative activity of earthworm coelomic fluid using oral squamous carcinoma KB 3-1 cells: An In vitro study with serine protease analysis

Abstract

Background: Naturally available extracts have gained importance as adjunctive pharmacologic modalities. The earthworm coelomic fluid (ECF) has shown promising in vitro cytotoxic effect in hepatocellular, colorectal, and breast cancers. Few in vitro studies have been performed pertaining to oral cancer. Objective: The objective of this study was to evaluate the time- and dose-dependent antiproliferative effect of ECF of Eudrilus eugeniae (EE), Eisenia fetida (EF), and Perionyx excavatus (PE) on oral cancer cell line KB 3–1. The chemical nature of the ECF was analyzed as well. Materials and Methods: The ECF was collected employing the cold shock method. Lowry's protein estimation was used for determining the total protein content. ECF of EE, EF, and PE at concentration of 6.25, 12.5, 25, and 50 μg/mL was tested on KB 3–1 cells in vitro. The 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide antiproliferative assay was used to determine the time- and dose-dependent antiproliferative effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to separate and observe prominent protein bands in the three extracted fluids. The chemical nature of the band was analyzed through an enzyme zymography. Results: ECF of PE showed a maximum inhibitory percentage of 94.29% at 50 μg/mL, followed by ECF of EE and EF with values of 89.23% and 79.44%, respectively. ECF of EE showed a superior IC50 value of 3.70 μg/mL as compared to EF and PE at 48 h. Conclusion: ECF of EE has a promising antiproliferative effect in a time- and dose-dependent manner on oral squamous carcinoma KB 3–1 cells in vitro. The serine protease presence in ECF of EE was demonstrated. Abbreviations used: EFE: Earthworm fibrinolytic enzyme; BSA: Bovine serum albumin; DMSO: Dimethyl sulphoxide; ECF: Earthworm coelomic fluid; EE: Eudrilus eugeniae; EF: Eisenia fetida; PE: Perionyx excavatus; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; IC: Inhibitory concentration; MEM: Minimal essential medium; MTT: 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide; NCCS: National Centre for Cell Science; NCI: National Cancer Institute; OD: Optical density; PBS: Phosphate-buffered saline; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEMED: Tetramethylethylenediamine.