SFN inhibits HIF-1α protein accumulation.

<p>Cell lines were treated as described below, and total cell lysates subjected to immunoblot analysis. A, A549 cells were pretreated with or without SFN and MG132 (20 µM) for 1 hour. B, MCF7 cells were pretreated for 1 hour with indicated concentration of SFN, and then treated with 100µM cobalt chloride (Co⁺²) for an additional 5.5hours. C, 4T1 cells were pretreated for 20 min with 20 µM SFN, followed by treatment with 250µM cobalt chloride or 200µM desferroxamine as indicated for 4hours. D. 293 cells were pretreated for 20 min with indicated concentrations of SFN, followed by stimulation with 250 µM cobalt chloride for an additional 4 hours.</p

SFN dose dependently inhibits HIF-1α and Pol II occupancy at the mPGES-1 promoter.

<p>A549 cells were pretreated with or without SFN and HIF-1α inhibitor 2-Methoxyestradiol (2ME2, 0.2 mM) for 30 minutes. IL1b (1 ng/ml) was either added (left panels) or not (right panels) and cells were cultured for another 4 hours. ChIP assays, coupled with quantitative real time PCR, were performed using HIF-1α (A), Pol II (B) or Egr-1(C) antibodies. The results are expressed as fold change of protein binding to the mPGES-1 promoter relative to untreated cells. (mean ± SEM; n = 3) *, P<0.05, **, P<0.01, ***, P<0.001 (Student's t test).</p

A model for the role of SFN in the regulation of PGE2 production.

<p>SFN inhibits HIF-1α protein abundance, which leads to a reduction of HIF-1α on the promoter of mPGES-1 gene. This results in inhibition of transcription of mPGES-1 and suppression of PGE2 production.</p

SFN suppresses mPGES expression and PGE2 production driven by overexpression of HIF1α.

<p>A549 cells were transfected with indicated amounts of HIF-1α and GFP expression vectors. The total amount of transfected DNA (4 µg) was kept constant by addition of the empty vector. The cells were then left untreated or treated with increasing concentrations of SFN for 40 hours. The total cell extracts were prepared for immunoblot analysis to evaluate protein levels. The cell culture media were used for measuring PGE2 concentration by EIA assay.</p

SFN suppresses the HIF-1–mPGES –PGE2 axis by control of HIF-1α protein expression without altering mRNA levels.

<p>A, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). B&C, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added or not and cells were cultured for another 4 hours. Total cell lysates were subjected to immunoblot analysis (panel B) and total RNA was analyzed by quantitative RT-PCR (panel C).</p

SFN does not inhibit COX-2 expression in A549 cells.

<p>A, A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added (left) or not (right) and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot analysis. B, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). C, A549 cells were treated with 1 ng/ml IL1β either with or without 10 µM SFN for different times. Total cell lysates were subjected to immunoblot analysis.</p

SFN inhibits mPGES-1 expression and PGE2 production in tumors <i>in vivo</i>.

<p>2.5 × 10⁶ A549 cells were injected subcutaneously on the flank of athymic nude mice. After 6 weeks, animals were treated with PBS vehicle (control) or 0.5 mg SFN by intraperitoneal injection and sacrificed 24 hours later. Tumor tissue samples were weighed and frozen for subsequent RT-PCR assay (A), immunoblot analysis (B) and PGE2 measurement (C). The results in A and B are expressed as fold changes relative to PBS treated mice (mean ± SEM; n = 4). *, P<0.05, **, P<0.01, (Student's t test). Four mice in each group (PBS vehicle or SFN treated) were used in this experiment.</p

SFN suppresses mPGES-1 expression by inhibiting mRNA transcription.

<p>A&B. A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot with mPGES-1 antibody. Immunoblot shown (A) is representative of three experiments, that are quantified in panel B, with results expressed as mean fold change ± SEM (n = 3). C&D, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added (C) or not (D) and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as mean fold change ± SEM (n = 3). *, P<0.05, **, P<0.01, ***, P<0.001 (Student's t test).</p

SFN dose dependently inhibits IL1β induced PGE2 production.

<p>A549 cells were pretreated for 30 minutes in the presence or absence of SFN and then cultured with or without 1 ng/ml IL1β for 24 hours. PGE2 in the media was measured using EIA assay. *, p<0.05, **, p<0.01, ***, p<0.001 (Student's t test). Data are presented as mean ± SEM (n = 3).</p