ABSTRACT
Objective To observe the influence of p53-upregulated modulator of apoptosis (PUMA) on the growth of human brain glioma cell lines U251 (p53 mutant) and SHG-44 (p53 wild type),and to explore its possible mechanism.Methods Construct the adenovirus PUMA (Ad-PUMA) and vector of adenovirus (AdDsRed) which were respectively transfected into glioma cell lines U251 and SHG-44.Cells proliferation rates were measured with cell counting kit-8 (CCK-8).The apoptotic ratios were detected by flow cytometry.The expression of PUMA and apoptosis associated proteins (bcl-2,Bax) were determined with Western blot analysis.Caspase-3,Caspase-8,Caspase-9 activity were measured by Caspase activity assay kit.Results Compared with vector group and blank control group,Ad-PUMA transfected group showed strong cell proliferating inhibition effects [the inhibition rates were (50.89±4.73) ％ and (44.45±5.33) ％ respectively,P ＜0.05] and pro-apoptotic effects [apoptotic rates were (44.89±5.08) ％ and (31.67±7.32) ％,P ＜ 0.05] in different p53 glioma cell lines U251 and SHG-44.Western blot analysis showed that PUMA protein expression increased after Ad-PUMA transfection,accompanied by the reduced expression of the anti-apoptotic protein bcl-2 and the increased expression of pro-apoptotic protein Bax.The activity of Caspase testing results showed that the Caspase-3,Caspase-9 activity increased significantly,while the Caspase-8 activity changed little.Conclusion No matter how p53 phenotype,PUMA can inhibit glioma proliferation,promote apoptosis,and its mechanism may be through the mitochondrial apoptotic pathways,upregulation of Bax and inhibition of bcl-2 expression,which activated Caspase-9.Ad-PUMA is expected to become a new target for gene therapy of gliomas.
ABSTRACT
Objective To investigate the expression of nuclear factor-κB (NF-κB) and p53 up-regulated modulator of apoptosis (PUMA) in acute lung injury (ALI) induced by severe acute pancreatitis (SAP), and the therapeutic role of proline dithiocarbamate (PDTC). Method SD rats weighed 200～ 250 g were randomly(random number) divided into sham operation group (A group, n = 18), ALI group (B group, n = 18) and PDTC treatment group (C group, n = 18). The model of SAP was eastablished by injecting 1 mL/kg of sodium tauarocholate into the pancreatic capsule of the rats in B group and C group. The model rats in C group were treated with PDTC one hour after modeling. Six rats of each group were sacrificed 6 h,12 h, and 24 hours after modeling. The histopathological changes in lung and pancreas were observed. The levels of NF-κB p65 and PUMA in lung were detected by using Western blotting, and the expressions of bcl-2, bax and caspase-3 mRNA in the lung were detected by using RT-PCR. The lung tissue was taken for examination under transmission electron microscope. TUNEL was used for detection of apoptotic alveolar epithelial cells. Results Six to 24 hours after modeling, the pathological scores in lung of ALI group were significantly higher than those of control group and PDTC group after sodium taurocholate injection ( P ＜ 0.05). The levels of NF-κB p65 and PUMA, and the expressions of bax and caspase3 mRNA in ALI group at different intervals were higher than those in control group and PDTC group ( P ＜ 0.05),whereas the expression of bcl-2 mRNA in ALI group was lower than that in control group and PDTC group ( P ＜0.05). The NF-κB p65 was correlated closely and positively with PUMA ( r= 0.987, P ＜ 0.01). Higher activity of caspase-3 acrtive units was seen in ALI group than that in control group and PDTC group ( P ＜ 0.05). The microvilli disappeared in ALI group 24 hours later. The apoptosis index in ALI group was higher than that in control group and PDTC group ( P ＜ 0.05). Conclusions The apoptosis of alveolar epithelial cells of rats in ALI group is caused by PUMA activated by NF-κB. PDTC treatment can inhibit apoptosis of alveolar epithelial cells of rats in ALI group by inhibiting the activation of NF-κB.