Pierisin-5
protein (pie-5) belongs to a family of
proteins possessing
DNA-dependent
ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous
protein subunits for basic scientific research, in addition to the development and production of diagnostics and
vaccines. In this study, a new method for the in vitro expression of the cytotoxic
protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5
protein (rpie-5) was investigated in different human
cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5
protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5
protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 μg/mL) was more sensitive when compared with HepG2 (IC50 1.9 μg/mL) and AGS (IC50 3.7 μg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in
cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of
lactose dehydrogenase (LDH)
enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating
protein-X), Bad, APAF-1 (
apoptotic protease activating factor-1), Cyt-c, and
caspase-3/9 and the downregulation of Bcl-2 (
B-cell lymphoma 2) in rpie-5-treated
cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.