Role of Bcl-2 family members in caspase-3/9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells.
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on monocyte, we infected human U937 cells with the P. aeruginosa strain in vitro. To explore the expression of Bcl-2 and Bax as well as caspase-3/9 activation in the apoptosis of human U937 cells induced by P. aeruginosa, Hoechst 33258 staining and Giemsa staining as well as Flow cytometry analysis were used to determine the rate of apoptosis, and the expressions of Bcl-2 and Bax were assayed by RT-PCR and Western blotting respectively. Bax protein conformation change was assayed by immunoprecipitation. Cytochrome c release was measured by Western blotting. Moreover, exposure of U937 cells to P. aeruginosa measured caspase-3/9 activity. It was found that the apoptosis of human U937 cells could be induced by Pseudomonas aeruginosa in a dose- and time-dependent manner. Also, there were a tendency of alterations with an increased expression level of Bax and a reduced expression level of Bcl-2, increased levels of cytochrome c release, and also with an increased activation of caspase-3/9 and Bax protein conformation change. For the evaluation of the role of caspases, caspase-3/9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK respectively were used. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked P. aeruginosa-induced U937 apoptosis. It is concluded that P. aeruginosa can induce apoptosis with an up-regulated expression of Bax and a down-regulated expression of Bcl-2, which resulted in increased levels of cytochrome c release and increased caspase-3 and -9 in human U937 cells.
Chai
W S
WS
Department of Infectious Diseases, The First Affiliated Hospital, China Medical University, No.155, Nanjing Northern Street, Heping Ward, Shenyang 110001, China.
Zhu
X M
XM
Li
S H
SH
Fan
J X
JX
Chen
B Y
BY
D017209:Apoptosis / Q000187:drug effects*; D001399:Azure Stains; D006690:Bisbenzimidazole; D053148:Caspase 3 / Q000502:physiology*; D053453:Caspase 9 / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D011552:Pseudomonas Infections / Q000473:pathology*; D011550:Pseudomonas aeruginosa; D020298:U937 Cells; D051028:bcl-2-Associated X Protein / Q000502:physiology*
D001399:Azure Stains; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9; D006690:Bisbenzimidazole