Recombinant Pierisin-5 Induces Apoptosis and Differential Expression of Bcl-2, Bax, and p53 in Human Cancer Cells.

ABSTRACT

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.