Figure 1.
The structures of MPG, a novel compound isolated and purified from P. granatum leaves (A) and MPTAG, the most active laboratory synthesized derivative of MPG (B).
Figure 2.
MPTAG prevents the TNF-α-induced NF-κB transcription and activation in human endothelial cells.
(A) The cells were pretreated with or without 400 µM of MPG before induction with TNF-α (10 ng/ml) for 4 h. The total RNA of the cells was isolated and analysed by RT-PCR. The intensity of transcripts was normalized with that of β-Actin levels expressed under similar conditions. (B) The cells were pretreated with MPTAG at varying concentrations and then induced with TNF-α. The cytoplasmic (CE) and nuclear (NE) extracts were prepared from untreated and MPTAG-treated TNF-α-stimulated cells (see “Methods”). The nuclear extracts were analyzed for NF-κB activation by EMSA. (C) The nuclear extracts from unstimulated or TNF-α-stimulated HUVECs were incubated with the indicated antibodies and analyzed for NF-κB activation by EMSA (see “Methods”). (D) The cells were transiently transfected by electroporation with a NF-κB-containing luciferase reporter gene followed by treatment with 400 µM MPTAG and TNF-α stimulation. The supernatants after cell lysis were assayed for luciferase activity. The mean value for cells treated with no MPTAG and no TNF-α was set to 1, and -fold differences were determined by comparing values against this set value. *p<0.005 vs. uninduced cells; **p<0.01 vs. TNF-α-induced cells, statistical difference was set at p<0.05.
Figure 3.
MPTAG prevents the TNF-α-induced NF-κB translocation in human endothelial cells.
(A) The cells were pretreated with MPTAG (400 µM) and induced with TNF-α. The cytoplasmic (CE) and nuclear (NE) extracts were prepared and processed for western blot analysis. (B) The intensity of bands were densitometrically scanned and normalized with that of α-tubulin and Lamin B1 in CE and NE extracts, respectively. The values presented are mean ± SEM. *p<0.005 vs. uninduced cells; **p<0.01 vs. TNF-α-induced cells, statistical significance was set at p<0.05. (C–D) The cells were treated and induced as described in ‘A’ and subjected to immunocytochemical analysis using anti-NF-κBp65 and FITC-labeled anti-rabbit antibodies. DAPI was used for staining the nucleus. The mean intensity levels of NF-κB conjugated to FITC, both in the cytoplasm and nucleus, were quantitated and plotted as mean intensity ± SEM. *,#p<0.05 vs. uninduced cells; **,##p<0.05 vs. TNF-α-induced cells, statistical difference was set at p<0.05. The scale bars represent 50 µm (E–F) MPTAG inhibits TNF-α-induced p65 phosphorylation in endothelial cells. The cells were treated and induced followed by western blot analysis as stated in ‘A’. The intensity of bands were densitometrically scanned and normalized with that of α-actin levels. The values presented are mean ± SEM. *p<0.05 vs. uninduced cells; **p<0.02 vs. TNF-α-induced cells, statistical difference was set at p<0.05.
Figure 4.
MPTAG prevents TNF-α-induced IκBα degradation by inhibiting the activation of IKK-β.
(A–B) The cells were treated with MPTAG (400 µM) followed by induction with TNFα for 30 mins and total cell extracts were processed for western blot analysis using antibodies against IκBα, phosphoIκBα and α-tubulin. (C) The intensity of bands were densitometrically scanned and normalized with that of α-tubulin in panels (i) and (ii). The values presented are mean ± SEM. *p<0.005 vs. uninduced cells; **p<0.01 vs. TNF-α-induced cells, statistical significance was set at p<0.05. (D) The cells were treated with MG-132 (a proteosome inhibitor; 50 µg/ml) for 30 mins and then exposed to TNF-α (10 ng/ml) for the indicated times. Total cell extracts were prepared and immunoprecipitated with anti-IKK-β antibody followed by kinase assay using GST-IκBα as a substrate. The extracts were also subjected to western blot analysis using anti-IKK-β antibody. (E) The cells were treated with MPTAG and MG-132 and then induced with TNF-α for 30 mins followed by kinase assay and western blot analysis as stated above. (F) The total cell extracts were prepared from cells in absence and presence of TNF-α induction (10 ng/ml) and were immunoprecipitated with anti-IKK-β antibody. The kinase assay was performed in the absence or presence of the indicated concentrations of MPTAG. The extracts were also subjected to western blot analysis using anti-IKK-β antibody.
Figure 5.
MPTAG inhibits the TNF-α-induced Akt activation and its association with IKK-β.
(A) The cells were induced with TNF-α (10 ng/ml) for the indicated times. The total cell extracts were prepared and subjected to western blot analysis using anti-phosphoAkt, for both Ser473 and Thr308 residues, and anti-Akt antibodies. (B) The cells were treated with MPTAG (400 µM) and induced with TNF-α for 30 mins followed by western blot analysis as stated above. (C) The intensity of bands were densitometrically scanned and normalized with total Akt levels. The values presented are mean ± SEM. *p<0.05 vs. uninduced cells; **p<0.05 vs. TNF-α-induced cells, statistical significance was set at p<0.05. (D) The cells were induced with TNF-α (10 ng/ml) for the indicated times. The total cell extracts were prepared and immunoprecipitated with anti-IKK-β antibody followed by western blot analysis with anti-Akt and anti-IKK-β antibodies. (E) The cells were pretreated with MPTAG at varying concentrations and induced with TNF-α for 30 mins. The total cell extracts were prepared and processed as stated above and analyzed for western blot as stated above. (F) The intensity of bands were densitometrically scanned and normalized with IKK-β levels. (G) The cells were pretreated with 400 µM MPTAG and then stimulated with 10 ng/ml TNF-α for the indicated times. The total cell extracts were prepared, immunoprecipitated with anti-IKK-β antibody and analyzed by western blot using anti-Akt and anti-IKK-β antibodies. (H) Effect of MPTAG on TNF-α-induced p38 MAPK and ERK1/2 activation. The cells were treated with MPTAG and induced with TNF-α as stated above. The total cell extracts were prepared and analyzed by western blot using anti-phosphospecific p38 MAPK and ERK1/2 antibodies. The same membrane was reblotted with anti-p38 MAPK, ERK 1/2 and β-actin antibodies. The values presented are mean ± SEM. *p<0.05 vs. uninduced cells; **p<0.05 vs. TNF-α-induced cells, statistical significance was set at p<0.05.
Figure 6.
MPTAG blocks Akt activation and NF-κB activation in a PI-3K independent and PKA-dependent manner in TNF-α-stimulated HUVECs.
(A) The cells were either preincubated with wortmannin (100 µM) or H-89 (20 µM) for 30 mins and then induced with TNF-α (10 ng/ml) for additional 30 mins. The total cell extracts were subjected to western blot analysis using anti-phospho-Akt (Thr308) and anti-Akt antibodies. (A; lower panel) The intensity of bands were densitometrically scanned and normalized with Akt levels. The values presented are mean ± SEM. *p<0.02 vs. uninduced cells; **p<0.05 vs. TNFα-induced cells, statistical difference was set at p<0.05. (B) The cells were pretreated with wortmannin in absence and presence of MPTAG before induction with TNF-α. The total cell extracts were subjected to PI3K assay (see “Methods”). The mean value (in pmol) for cells treated with neither MPTAG nor TNF-α (control) was set to 1, and -fold changes were determined by comparing values against this set value. The values presented are mean ± SEM. *p<0.002 vs. uninduced cells; **p<0.005 vs. TNF-α-induced cells, statistical significance was set at p<0.05. (C) The cells were treated and induced as described in (A). The nuclear extracts were assessed for NF-κB activation by EMSA. Effect of MPTAG on Akt phosphorylation in absence and presence of wortmannin and H-89 in TNF-α-stimulated HUVECs. (D; panels (i–iii)). For this, the cells were incubated without or with either wortmannin or H-89 before treatment with different concentrations of MPTAG followed by induction with TNF-α. The total cell extracts were subjected to western blot analysis using anti-phospho-Akt (Thr308) and anti-Akt antibodies. Effect of MPTAG on NF-κB activation in absence and presence of wortmannin and H-89 in TNFα-stimulated HUVECs. (E–F) The cells were treated and induced as stated in (D). The nuclear extracts were prepared and assessed for NF-κB activation by EMSA.
Figure 7.
MPTAG restores PKA activity in TNFα-stimulated HUVECs.
(A) The cells were treated with media (control), MPTAG, SQ 22536, H-89 or their indicated combinations in absence and presence of TNF-α stimulation. PKA activity was assessed in the cell lysates. Results are expressed as mean ± sem of three independent experiments. *p<0.05 vs. control. Proposed model of MPTAG action in TNF-α-stimulated HUVECs. (B) In TNF-α-stimulated HUVECs (without MPTAG treatment), PI-3K-regulated Akt is activated (indicated by solid line arrow) resulting in NF-κB activation. On the other hand, PKA activity remained repressed (indicated by broken line arrow) under these conditions. (C) MPTAG pretreatment to TNF-α-stimulated HUVECs restored the repressed activity of PKA. Thus, derepression of PKA activity resulted in inhibition of Akt and overall inhibition of NF-κB in these cells.