Molecular cloning of the Bombus terrestris bumblebee venom protein phospholipase A2 and its anti-leukemia effects on K562 cells

Bee venom has been used for treating various diseases for a long time. However, the bioactive constituents of bee venom and its mechanisms remain poorly understood. In the present study, phospholipase A(2) (Bt-PLA(2)) cDNA was cloned, and a mature form of Bt-PLA2 was purified from bumblebee venom (Bombus terrestris). The differentiation induction and apoptosis induction activities of Bt-PLA(2) on chronic myelogenous leukemia (CML) K562 cells were also evaluated. Bt-PLA(2) cDNA has 540 nucleotides that encode a 180-amino-acid protein. The purified, mature form of Bt-PLA(2) was an 18-kDa protein, and it inhibited K562 cell growth, determined by an IC50 value of 29.5 ng/mu l. Moreover, Bt-PLA(2) induced erythroid differentiation of K562 cells in a dose-dependent manner, and this was supplemented with the upregulation of glycophorin A (GPA) mRNA expression. Bt-PLA(2)-induced apoptosis, analyzed by DAPI staining, was correlated with the result analyzed by AnnexinV-FITC/PI binding. Furthermore, activation of caspase 3 and poly ADP-ribose polymerase (PARP) and inhibition of p-Akt, determined by western blot, further demonstrated that Bt-PLA(2) induced apoptosis mainly through the Akt pathway. The parallel induction of erythroblasts differentiation of K562 cells and apoptosis due to Bt-PLA2 treatment demonstrated the potential use of Bt-PLA(2) as an anti-leukemia drug lead.