N-phenethyl-2-phenylacetamide isolated from Xenorhabdus nematophilus induces apoptosis through caspase activation and calpain-mediated Bax cleavage in U937 cells.
The present study was designed to assess the mechanism of N-phenethyl-2-phenylacetamide (NPPA), one of three new compounds isolated from Xenorhabdus nematophilus, on the induction of apoptosis in U937 cells. NPPA displayed strong inhibitory effects on cell proliferation and viability of U937 cells and induced apoptosis. Investigation of the mechanism of NPPA-induced apoptosis revealed that treatment with NPPA produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase. U937 cells treated with NPPA demonstrated cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the pan-caspase inhibitor (z-VAD-fmk) prevented NPPA-induced apoptosis. These results suggested that NPPA induces apoptosis through cytochrome c-dependent caspase-3 activation in U937 cells. In late stage of apoptosis, 18 kDa fragment of Bax was generated with the down-regulation of the expressions of XIAP following NPPA treatment, suggesting that the modulation of Bax and XIAP proteins plays some roles in NPPA-mediated apoptosis. Pretreatments of z-VAD-fmk and the calpain inhibitor, calpeptin, inhibited Bax cleavage. Pretreatment of z-VAD-fmk restored the expression level of XIAP, but pretreatment of calpeptin did not. These results suggest that the elevated caspase activities cleave XIAP in this experiment. And Bcl-2 over-expression attenuates NPPA-induced apoptosis by inhibiting caspase-3 activation, and subsequently inhibits calpain autolysis and Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be caspase-dependent. Taken together, the apoptotic effects of NPPA may be related, in part to the caspase-3 activation, the down-regulation of XIAP, and Bax cleavage mediated by caspase-dependent calpain activation.
Hwang
Seok-Young
SY
Institute for Medical Science, Keimyung University, Daegu 700-712, Korea.
Paik
Seunguk
S
Park
Sun-Ho
SH
Kim
Hyun-Su
HS
Lee
In-Seon
IS
Kim
Sang-Pyo
SP
Baek
Won-Ki
WK
Suh
Min-Ho
MH
Kwon
Taeg Kyu
TK
Park
Jong-Wook
JW
Park
Jae-Bok
JB
Lee
Jung-Jeung
JJ
Suh
Seong-Il
SI
D000081:Acetamides / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D044482:Benzeneacetamides; D002154:Calpain / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D020298:U937 Cells; D051636:X-Linked Inhibitor of Apoptosis Protein; D020593:Xenorhabdus / Q000737:chemistry*; D051028:bcl-2-Associated X Protein
D000081:Acetamides; D000970:Antineoplastic Agents; C494826:BAX protein, human; D044482:Benzeneacetamides; D003574:Cytochrome c Group; C474341:N-phenethyl-2-phenylacetamide; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; D002154:Calpain; D053148:Caspase 3; D020169:Caspases