Figure 1.
Stable cell line A549R responses to both IL-6 and PD-180970.
Luciferase (A) and MTT (B) assays of A549R cells treated with PD-180970 (250 nM) and IL-6 (250 ng/ml) respectively. A549R cells were plated in 96-well plates at a density of 1×104 (100 µl/well in DMEM with 10% FBS). Twelve hours later, media were removed and replaced with fresh media in the presence of IL-6 or PD-180970. Bars show the standard deviation (±SD) (three independent repeats, n = 5 in each repeat). ***p<0.001, **p<0.01,*p<0.05, significant relative to vehicle control. NS, no statistical significance. (C) PD-180970 inhibits STAT3 activity in A549R cells. A549R cells were plated and cultured in 100-mm dishes at 70% confluence. Then cells were treated with PD-180970 (250 nM) for 24 h. DMSO was used as control.
Figure 2.
Brevilin A inhibits STAT3 signaling in a dose dependent manner.
(A) Structure of Brevilin A. (B) Brevilin A inhibits STAT3 phosphorylation in A549R cells. A549R cells were plated and cultured in 100 mm dishes at 70% confluence. Cells were then treated with Brevilin A (12.5 µM and 25 µM ) for 24 h. DMSO was used as control. (C) STAT3 signaling was inhibited by Brevilin A in a dose dependent manner. A549R cells were plated in 96-well plates at a density of 1×104 (100 µl/well in DMEM with 10% FBS). Twelve hours later, media was removed and replaced with fresh media in the presence of Brevilin A at different concentrations for another 24 h (20 µM, 17.5 µM, 15 µM, 12.5 µM, 10 µM, 7.5 µM, 5 µM, 2.5 µM and 1 µM). Luciferase and MTT assays were performed then. Bars show the standard deviation (±SD) (3 independent repeats, n = 5 in each repeat).
Figure 3.
Brevilin A inhibits constitutive activated STAT3 in DU145 and MDA-MB-468 cells.
STAT3 tyrosine 705 phosphorylation was inhibited by Brevilin A in dose (5 µM, 10 µM and 15 µM for 2 h) and time (10 µM for 30 min, 60 min and 120 min) dependent manners in both DU145 (A) and MDA-MB-468 (B) cells, while phosphorylation of p65-Ser536 (A and B), AKT-Ser473 and GSK-3β-Ser9 (C) was not affected correspondingly (10 µM, 2 h). (D) c-Myc and CyclinD1 decreased after 24 h and 48 h treatment with Brevilin A (10 µM). Cells were plated and cultured in 100 mm dishes for 12 h, then were treated with Brevilin A as described. (E) Cleaved PARP increased in DU145 and MDA-MB-468 cells with Brevilin A (10 µM) treatment for 24 h. DMSO was used as control.
Figure 4.
Brevilin A selectively inhibits cell growth of DU145 and MDA-MB-468 cells.
(A) STAT3 tyrosine 705 phosphorylation was detected in hTERT-BJ, DU145 and MDA-MB-468 cells. (B) Left and middle diagrams, hTERT-BJ, DU145 and MDA-MB-468 cells were plated in 96-well plates at a density of 8×103 (100 µl/well in DMEM with 10% FBS). Twelve hours later, media were removed and replaced with fresh media in the presence of Brevilin A (5 µM and 10 µM) for 24 h, 48 h and 72 h, cell viability was measured by MTT assay. Right histogram, 12 hours later cells were plated, media were removed and replaced with fresh media in the presence of Brevilin A (10 µM), AG490 (100 µM), Doxorubicin (1 µM) and Staurosporine (100 nM and 500 nM) for 24 h. Dox, Doxorubicin. Sta, Staurosporine. Bars show the standard deviation (±SD) (3 independent repeats, n = 3 in each repeat). ***p<0.001, **p<0.01, *p<0.05. NS, no statistical significance. (C) Cells were plated in 24-well plates. Twelve hours later, media was removed and replaced with fresh media in the presence of 10 µM Brevilin A for 24 h. DMSO was used as control. Cells were then subjected to an Annexin-V-PI dual staining process. Same exposure program was used at each wavelength.
Figure 5.
Brevilin A blocks STATs activity upon cytokine treatments.
Cells were plated and cultured in 100-mm dishes for 12 h, then media were removed and replaced with fresh media without serum for another 12 h. Brevilin A (10 µM) was added for 30 min pretreatment, then cells were treated with IL-6 (250 ng/ml), IFNα (5000 U/ml) and IFNγ (1500 U/ml) for 2 h. STAT3 phosphorylation were then analyzed by Western-Blot (A and B). (C) Cells were plated and cultured in 100-mm dishes for 12 h, then media was removed and replaced with fresh media without serum for another 12 h. Brevilin A was added for 30 min pretreatment (HEK293T, 10 µM; Hela and HepG2, 15 µM ), then cells were treated with IL-6 (250 ng/ml), IFNα (5000 U/ml) and IFNγ (1500 U/ml) for 4 h. Socs3 mRNA were analyzed by RT-PCR. (D) Real-time qPCR analysis of socs3 mRNA in HEK293T cells treated with Brevilin A in the presence of IL-6 (250 ng/ml). (E) Brevilin A inhibits IFNα and IFNγ induced Tyk2 and JAK2 phosphorylation respectively, as well as STAT1 tyrosine phosphorylation in Hela cells as treated in (B). (F) IRF mRNA were analyzed by RT-PCR in Hela cells in the presence of IFNα as treatments described above.
Figure 6.
Brevilin A blocks JAKs-JH1 tyrosine kinase domain over-expression induced protein phosphorylation.
(A) HEK293T cells expressing JAK2-JH1 were treated with Brevilin A (10 µM and 15 µM) for 2 h and 4 h. STAT3 tyrosine 705 phosphorylation was then detected. (B) HEK293T cells over-expressing c-Src were treated with Brevilin A (15 µM) or PD-180970 (250 nM and 500 nM) for 4 h. Then c-Src induced tyrosine phosphorylation was detected with anti-phosphotyrosine antibody. (C) HEK293T cells over-expressing Flag-tagged c-Src were treated with DMSO, Brevilin A (15 µM) and PD-180970 (500 nM) for 4 h separately. Then c-Src was immuno-precipitated with anti-Flag beads for Western-Blot analysis. (D) JH1 domain of JAK1, JAK2, JAK3 and Tyk2. (E) HEK293T cells expressing JAKs-JH1 were treated with Brevilin A (15 µM) for 4 h. JH1 induced total tyrosine phosphorylation, STAT3 and STAT1 phosphorylation were detected with indicated antibodies. Before treatment (A, B, C and E), cells were plated and cultured in 100-mm dishes for 12 h, and then media were removed and replaced with fresh media without serum for another 12 h.
Figure 7.
Brevilin A blocks JAKs-JH1 tyrosine kinase activity in vitro.
(A) Purified hSTAT3 protein and immuno-precipitated Tyk2-JH1 kinase were used for kinase assay, in the presence of Brevilin A concentration series at 10, 20, 40, and 80 µM. Western-Blot showed that Brevilin A inhibited Tyk2-JH1 kinase activity directly (A, left). The right lane in (A) was the Coomassie Brilliant Blue staining of purified hSTAT3 after SDS-PAGE. Antibody against pTyr705-STAT3 was used for detecting STAT3 phosphorylation. (B) IC50 measurements of four JAKs-JH1 truncated kinases. HEK293T cells expressing JAKs-JH1 were plated and cultured in 100-mm dishes for 12 h, then media was removed and replaced with fresh media without serum for another 12 h. Cells were then treated with Brevilin A at different concentrations for 4 h (5 µM, 7.5 µM, 10 µM, 12.5 µM, 15 µM, 17.5 µM and 20 µM). DMSO was used as control.