Isolation and Mechanistic Exploration of FS-1: A Pro-Apoptotic Protein from Gloydius brevicaudus Venom in Tumor Cells

Abstract

Background/Objectives: Components potentially inducing tumor cell apoptosis were isolated from Gloydius brevicaudus venom, and their apoptotic mechanisms were investigated.Materials and Methods: The lyophilized venom was fractionated using anion exchange chromatography (DEAE-Sepharose Fast Flow), cation exchange chromatography (Sp-Sepharose Fast Flow), and gel filtration chromatography (Sephadex G-75), leading to the isolation of a component (FS-1) that appeared to inhibit tumor proliferation. SDS-PAGE analysis indicated a single band with an approximate molecular weight of 25 kDa.Results: FS-1 was observed to inhibit the proliferation of HeLa and Hep G2 cell lines in a dose-dependent manner. A time-dependent decrease in IC50 values was noted, with the most pronounced effect observed at 48 hr. Evidence suggested FS-1 induced tumor cell apoptosis: fluorescence staining indicated PS externalization from the inner to the outer leaflet of the cell membrane after 6 hr of treatment. ELISA experiments detected an increase in BAD expression at 12 hr. By 24 hr, an increase in cytosolic Cyt C concentration was observed, accompanied by a decrease in mitochondrial Cyt C concentration. Data suggested enhanced expression of BAX, while reduced expression of Bcl-2 and Bcl-xl was detected. Fluorescence staining indicated the opening of the mitochondrial MPTP. At 36 hr, increased activities of caspase-8 and caspase-9 were measured. By 48 hr, enhanced activities of caspase-3 and caspase-6 were also detected, and DNA damage was observed via agarose gel electrophoresis. Western blot analysis of Bcl-2, Bcl-xl, caspase-3, and caspase-6 supported these observations. Additionally, AIF was detected in the cytoplasm, showing increased concentration and the presence of cleaved bands.Conclusion: Collectively, these findings suggest the successful isolation of anti-tumor protein from Gloydius brevicaudus venom that induces tumor cell apoptosis, mediated through mechanisms involving mitochondrial dysfunction and caspase activation.

Keywords: Gloydius brevicaudus; Venom; Apoptosis; Hep G2; HeLa