Bcl-2 and Caspase-3 Are Major Regulators in Agaricus blazei-lnduced Human Leukemic U937 Cell Apoptosis through Dephoshorylation of Akt

摘要

Agaricus blazei is a medicinal mushroom that possesses antimetastatic,antitumor,antimutagenic,and im-munostimulating effects.However,the molecular mechanisms involved in A.blazei-mediated apoptosis remain unclear.In the present study,to elucidate the role of the Bcl-2 in A.blazei-mediated apoptosis,U937 cells were transfected with either empty vector(U937/vec)or vector containing cDNA encoding full-length Bcl-2(U937/Bcl-2).As compared with U937/vec,U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2.Treatment of U937/vec with 1.0-4.0 mg/ml of A.blazei extract(ABE)for 24 h resulted in a significant induction of morphologic features indicative of apoptosis.In contrast,U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features.ABE-induced apoptosis was accompanied by downregulation of anti-apoptotic proteins such as X-linked inhibitor of apoptosis protein(XIAP),inhibitor of apoptosis protein(cIAP)-2 and Bcl-2,activation of caspase-3,and cleavage of poly(ADP-ribose)polymerase(PARP).Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins,such as cIAP-2 and Bcl-2,but not XIAP.Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells.Furthermore,treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment.This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage.ABE also triggered the downregulation of Akt,and combined treatment with LY294002(an inhibitor of Akt)significantly decreased cell viability.The results indicated that major regulators of ABE-induced apoptosis in hum an leukemic U937 cells are Bcl-2 and caspase-3,which are associated with de-phosphorylation of the Akt signal pathway.