Figure 1.
Structures of PMS1077 and its structural analogs.
Figure 2.
Effects of PMS1077 and its structural analogs on TNF-α induced NF-κB activation.
(A) DU145-NF-κB-Luciferase cells were treated with PMS1077 and its structural analogs at final concentration of 50 µM. TPCA-1 (2 µM) is a specific inhibitor of NF-κB for positive control and DMSO as vehicle. Then cells were left untreated or exposed to TNF-α (20 ng/ml) for 12 h. Luciferase activity was measured using ONE-Glo® Luciferase Assay System (Promega). (B) DU145 cells were treated with PMS601, PMS1077, PMS1120 and PMS1144 at the indicated concentrations for 12 h. Cell viability was determined using MTT assay. The values are the mean±S.D. for three independent replicates. *P<0.05 versus TNF-α stimulated group.
Figure 3.
PMS1077 inhibited TNF-α induced expression of NF-κB regulated reporter gene.
HEK293T cells were transiently co-transfected with NF-κB-Firefly luciferase and TK-Renilla Luciferase reporter vectors for NF-κB activity assay. (A) NF-κB-dependent reporter gene expression induced by TNF-α. Transfected cells were treated with vehicle or TNF-α (1, 5, 10, 20 and 50 ng/ml) for 12 h; (B) TPCA-1 inhibited the expression of the TNF-α induced NF-κB dependent reporter gene. Transfected cells were treated with vehicle or TPCA-1(1, 2, 3, and 4 µM), then incubated with TNFα (20 ng/ml) for 12 h; (C) PMS1077 inhibited TNF-α induced, NF-κB dependent reporter gene expression. Transfected cells were treated with vehicle or PMS1077 (20, 40, 60 and 80 µM), then incubated with TNF-α (20 ng/ml) for 12 h; (D) PMS601 showed no effect on TNF-α induced NF-κB dependent reporter gene expression. Transfected cells were treated with vehicle or PMS601 (20, 40, 60 and 80 µM), then incubated with TNF-α (20 ng/ml) for 12 h. After treatment, luciferase activity in (A), (B), (C), and (D) were measured using a dual luciferase assay system (Promega). Data shown represent the relative luciferase activity normalized against Renilla luciferase activity. The values are the mean±S.D. for three independent replicates. *P<0.05 versus TNF-α stimulated group; NS, not significant.
Figure 4.
PMS1077 inhibited TNF-α induced IκB-α degradation, IκB-α phosphorylation, p65 phosphorylation.
(A) TNF-α induced IκB-α degradation, IκB-α phosphorylation. HEK293T cells were treated with TNF-α (20 ng/ml) for the indicated periods of time; (B) PMS1077 inhibited TNF-α induced IκB-α degradation. HEK293T cells were treated with vehicle or PMS1077 (20, 40, 60 and 80 µM) or PMS601 (80 µM) and TPCA-1(1 µM), then incubated with TNF-α (20 ng/ml) for 0.5 h; (C) PMS1077 inhibited TNF-α induced IκB-α phosphorylation. HEK293T cells were treated with vehicle or PMS1077 or PMS601 or TPCA-1 as in (B), then incubated with TNF-α (20 ng/ml) for 2 h; (D) DU145 cells were treated with vehicle or PMS1077 (40 and 80 µM) and TPCA-1(1 µM), then cells were either left untreated or exposed to TNF-α (20 ng/ml) for 12 h. After treatment, the whole cell lysates in (A), (B), (C), and (D) were prepared and analyzed by Western blotting with antibodies for IκB-α, phosphorylation-IκB-α (Ser-32), phosphorylation-p65 (Ser-536) and GAPDH. All data shown are representative of three independent experiments.
Figure 5.
PMS1077 inhibited TNF-α-induced NF-κB/P65 nuclear translocation.
(A) DU145 cells were pretreated with PMS1077 (50 µM) or TPCA-1(2 µM) for 6 h and followed by TNF-α (20 ng/ml) stimulation for 0.5 h. Cytoplasmic and nuclear extracts representing equal numbers of cells were analyzed by Western blotting using the indicated antibodies. (B) PC-3 cells were pretreated with PMS1077 (50 µM) for 6 h and followed by TNF-α (20 ng/ml) stimulation for 0.5 h. TPCA-1 (2 µM) and DMSO were used as positive NF-κB inhibitor and negative control, respectively. After treatment, cells were stained with primary anti-p65 antibody and Cy3 fluorescein-conjugated secondary antibody (Red), and then the nucleus was counterstained with DAPI (blue) and examined using Fluorescence microscopy. Images were acquired for each fluorescence channel, using suitable filters with 40×objective. The red and blue images were merged using Image J software. All data shown are representative of three independent experiments.
Figure 6.
The model of PMS1077 binding to IKK-β.
(A) The 3D model of PMS1077 (ball and stick style) interaction with IKK-β kinase domain (residues shown in stick model), hydrogen bonds are shown in dashed line. (B) The 2D diagram of interactions between PMS1077 and IKK-β.
Table 1.
Comparison binding affinity between compounds and IKK-β by 3 different scores.
Figure 7.
PMS1077 sensitized cancer cells to TNF-α induced apoptosis and inhibited the TNF-α induced expression of anti-apoptotic gene.
(A) DU145 cells were treated with vehicle, PMS1077 or PMS601 as the indicated concentration (0–80 µM) and then incubated with or without TNF-α (20 ng/ml) for 24 h. Cell viability was determined by MTT assay. (B) DU145 cells were treated with vehicle or PMS1077 (40 and 80 µM), and then incubated with TNF-α (20 ng/ml) for 24 h. Treated cells were observed by differential interference contrast microscope. (C) DU145 cells were treated as (B) and apoptosis analysis was performed by flow cytometry through annexin-V-FITC and Propidium iodide (PI) staining treated cells. (D) DU145 cells were treated with vehicle or PMS1077 (40 and 80 µM), then incubated with TNF-α (20 ng/ml) for 12 h. After treatment, the whole cell lysates were prepared and analyzed by Western blotting with antibodies as indicated. (E) DU145 cells were incubated with TNF-α (20 ng/ml) for 1 h, with vehicle or PMS1077 pretreatment for 6 h. The mRNA level of NF- κB target genes were examined using RT–PCR. (F) DU145 cells were treated as (D) and whole cell lysates were prepared and analyzed by Western blotting with antibodies for PARP and GAPDH. All data shown are representative of three independent experiments.