Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.
Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating 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 antiapoptotic 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 human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.
Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 614-052, Republic of Korea.
Cheng-Yun
CY
Jin
Dong-Oh
DO
Moon
Yung Hyun
YH
Choi
Jae-Dong
JD
Lee
Gi-Young
GY
Kim
D000364:Agaricus / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D007211:Indoles; D009842:Oligopeptides / Q000494:pharmacology; D051058:Oncogene Protein v-akt / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D020298:U937 Cells
D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C007293:DAPI; D051058:Oncogene Protein v-akt; D053148:Caspase 3; D020169:Caspases