NF-κB; IκB-α (IC50 = 10 μM)

BAY 11-7085 inhibits TNFa-induced surface expression of E-selectin, VCAM-1, and ICAM-1with IC50 values in the range of 5-10 μM. With an IC50 value of roughly 10 μM, BAY 11-7085 dose-dependently stabilizes IκBα . There is a direct correlation between the drug concentration that stabilized IκBα, the concentration that inhibits nuclear NF-kB levels, and the concentration that inhibits adhesion molecule expression[1].
It has been demonstrated that BAY 11-7085 causes a variety of cells to undergo apoptosis and inhibits cell proliferation. Cell proliferation and DNA synthesis in ovarian endometriotic cyst stromal cells are significantly reduced by BAY 11-7085 (ECSCs), which also causes apoptosis and the arrest of these cells' cell cycle in the G0/G1 phase. By suppressing antiapoptotic proteins, BAY 11-7085 causes ECSCs to undergo apoptosis, and caspase-3, -8, and -9-mediated cascades are involved in this mechanism[2].

---

Identification of Novel Inhibitors of ICAM-1, VCAM-1, and E-selectin Surface Expression [1]
Two structurally related compounds, compound 1 and compound 2/BAY 11-7085 (Fig. 1), were identified as inhibitors of cytokine-induced surface expression of ICAM-1 as measured by fluorescence immunoassay as described by Gerritsen et al. (42). Drug effects on TNFα-induced surface expression of E-selectin, VCAM-1, and ICAM-1 were determined by fluorescence immunoassay as described under “Experimental Procedures.” Compound 1 and compound 2 inhibited the surface expression of all three adhesion molecules with IC50 values in the range of 5–10 μm (Fig. 2). To determine whether the effects of the test compounds were reversible, we compared E-selectin levels in cells stimulated with TNFα in the presence of compound 1 (10 μm) with the levels obtained when the cells were pretreated with compound 1 followed by a 1 h “washout” period and then a 3-h stimulation with TNFα in the absence of test drug. Treatment of HUVEC with 10 μm compound inhibited E-selectin expression by 57% and a similar level of inhibition was seen when the drug was “washed out” prior to TNFα treatment (Fig. 3). Thus, compound 1 irreversibly inhibits surface expression of E-selectin. Other effects of compound 1 were reversible (see below) suggesting that the inability to reverse the inhibition of TNFα-induced E-selectin expression was not due to retention of the compound by the cell. There was no detectable cytotoxicity as measured by MTT assay even after 16 h treatment of cells with this dose (10 μm) of the test compound (IC50 in the MTT assay ranged from 25–38 μm). Thus, it is likely that the drug irreversibly modifies a cellular target.

---

Inhibition of Nuclear NF-κB [1]
The TNFα-induced expression of adhesion molecules E-selectin, VCAM-1, and ICAM-1 requires the transcription factor NF-κB (5, 6, 7, 8, 9, 10, 11, 12). Therefore, we evaluated the test compounds for effects on nuclear translocation of NF-κB. We carried out electrophoretic mobility shift assay to determine the levels of NF-κB in nuclear extracts from HUVEC treated with TNFα in the presence of compound 1 or compound 2/BAY 11-7085. As previously observed, TNFα-induced nuclear translocation of NF-κB occurs within 15 min in the absence of test compound (Fig. 4 A, lane 2 and Fig. 4 B, lane 2). At 20 μm, both test compounds completely inhibited nuclear NF-κB (Fig. 4 A, lane 4, Fig. 4 B, lane 3). A lower dose of compound 1 (10 μm) also reduced nuclear NF-κB (Fig. 4 A, lane 3).

---

Most of the current medical treatments for endometriosis aim to downregulate estrogen activity. However, a high recurrence rate after medical treatment has been the most significant problem. BAY 11-7085, a soluble inhibitor of NK-kappaB activation, has been shown to inhibit cell proliferation and induce apoptosis of a variety of cells. To examine the potential application of BAY 11-7085 in the treatment of endometriosis, we investigated the effects of this agent on the cell proliferation and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSCs) by a modified methylthiazole tetrazolium assay, a 5-bromo-2'-deoxyuridine incorporation assay, and internucleosomal DNA fragmentation assays. The effect of BAY 11-7085 on the cell cycle of ECSCs was also determined by flow cytometry. The expression of apoptosis-related molecules was examined in ECSCs with Western blot analysis. BAY 11-7085 significantly inhibited the cell proliferation and DNA synthesis of ECSCs and induced apoptosis and the G0/G1 phase cell cycle arrest of these cells. Additionally, downregulation of the B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-X(L) expression with simultaneous activation of caspase-3, -8, and -9 was observed in ECSCs after treatment with BAY 11-7085. These results suggest that BAY 11-7085 induces apoptosis of ECSCs by suppressing antiapoptotic proteins, and that caspase-3-, -8-, and -9-mediated cascades are involved in this mechanism. Therefore, BAY 11-7085 could be used as a therapeutic agent for the treatment of endometriosis [2].

---

Effects of BAY 11-7085 on cell proliferation and cell viability of ECSCs and NESCs. [2]
The effects of BAY 11-7085 on the cell proliferation and cell viability of ECSCs and NESCs were investigated by modified MTT assay. As shown in Fig. 1A, the number of viable ECSCs and NESCs was significantly decreased by the addition of increasing amounts of BAY 11-7085 (66.1 and 54.7% decreases, respectively, at a concentration of 10 μM). BAY 11-7085 showed a stronger inhibitory effect on the cell viability of ECSCs than on that of NESCs (Fig. 1B).

To further assess the effects of BAY 11-7085 on cell proliferation, the DNA synthesis of ECSCs and NESCs after BAY 11-7085 treatment was evaluated by BrdU incorporation assay. As shown in Fig. 2, BAY 11-7085 treatment resulted in a significant inhibition of the BrdU incorporation of ECSCs in a dose-dependent manner (53.2% decrease at a concentration of 10 μM), whereas BAY 11-7085 was associated with only a weak inhibitory effect on the BrdU incorporation of NESCs (38.2% decrease at a concentration of 10 μM).

---

Effects of BAY 11-7085 on apoptosis of ECSCs and NESCs. [2]
The apoptotic effects of BAY 11-7085 on ECSCs and NESCs were assessed by evaluating the presence of internucleosomal DNA fragmentation. BAY 11-7085 induced the fragmentation of internucleosomal DNA in ECSCs (Figs. 3 and 4). A DNA ladder was detected by electrophoresis in BAY 11-7085-treated ECSCs, suggesting the presence of apoptotic cells (Fig. 3). The apoptosis of both ECSCs and NESCs was significantly induced by the addition of increasing amounts of BAY 11-7085 (725.1 and 368.2% increases, respectively, at a concentration of 10 μM; Fig. 4A). BAY 11-7085 showed a stronger stimulatory effect on the apoptosis of ECSCs than that of NESC (Fig. 4B).

---

Effects of BAY 11-7085 on the cell cycle of ECSCs. [2]
The effect of BAY 11-7085 on the cell cycle of ECSCs was determined by flow cytometry. As shown in Fig. 5, the culturing of ECSCs cultured for 2 days in the presence of BAY 11-7085 (10 μM) resulted in an accumulation of these cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the proportion of those in the S-phase. Similar results were obtained in all repeated experiments.

---

Effects of BAY 11-7085 on expression of apoptosis-related proteins in ECSCs. [2]
To analyze the underlying mechanisms of the above-mentioned findings, we evaluated the expression of apoptosis-related proteins in ECSCs. As shown in Fig. 6, BAY 11-7085 downregulated the expression of Bcl-2 and Bcl-XL proteins and upregulated the expression of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 proteins in ECSCs. The levels of Bax, Fas, and Fas ligand protein in ECSCs remained unchanged. Similar results were obtained in all repeated experiments.

In the rat carrageenan paw model and the rat adjuvant arthritis model, BAY 11-7085 acts as an anti-inflammatory drug. The rat carrageenan paw model exhibits a dose-dependent decrease in swelling[1].

---

Anti-inflammatory Actions of Compound 2/BAY 11-7085 [2]
Compound 2/BAY 11-7085 was evaluated in two in vivo models of inflammation. As shown in Fig. 10, compound 2 demonstrated a dose-dependent reduction in swelling in the rat carrageenan paw model. Compound 2 was also evaluated in established rat adjuvant arthritis (Fig. 11). In the vehicle-treated control group, the mean volume of both hind paws increased by 0.39 ± 0.15 ml. Compound 2, given intraperitoneally at 20 mg/kg, but not at 5 mg/kg, significantly reduced the mean paw edema of the rats, to levels similar to those observed with the positive control, dexamethasone, at 1 mg/kg intraperitoneally. Thus, this compound acted as an anti-inflammatory agent in both the rat carrageenan paw and the rat adjuvant arthritis model.

The procedures for the gel kinase assay are described below for determining which proteins phosphorylate IκB-α . From HUVEC that have been exposed to TNFα (100 units/ml) for 15 minutes in the presence or absence of the inhibitor (20 μM, pretreatment for 1 hour), as indicated, whole cell extracts are made. A 10% SDS gel with 0.5 mg/ml HIS-IκB-α is used to separate proteins. Gels are washed two times in 20% propanol, 50 mM Hepes, pH 7.6, for 30 min and two times in buffer A (50 mM Hepes, pH 7.6, 5 mM 2-mercaptoethanol) for 30 min, followed by a 1-h incubation with buffer A containing 6 M urea, 1 h each in 3, 1.5, and 0.75 M urea in buffer A and 0.05% Tween 20 and 1 h in buffer A with 0.05% Tween 20. The kinase assay is conducted for 1 hour at 30 °C with 50 μM ATP, 5 Ci/ml [32P]ATP, 20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM sodium vanadate, and 2 mM dithiothreitol present. The gel is dried, exposed to film, and then washed with 5% trichloroacetic acid and 1% sodium pyrophosphate. A different gel devoid of HIS-IκB-α is tested as a control.

---

Immune Complex Kinase Assays [1]
Extracts were prepared from control and TNFα-treated HUVEC. Cells were solubilized with Triton lysis buffer (TLB, 20 mm Tris, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mm NaCl, 2 mm EDTA, 25 mm β-glycerophosphate, 1 mm sodium orthovanadate, 2 mm pyrophosphate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin). Extracts were centrifuged at 14,000 × g for 15 min at 4 °C. The JNK, p38, or ERK protein kinases were immunoprecipitated by incubation for 1 h at 4 °C with specific rabbit polyclonal antibodies bound to protein-A Sepharose. The rabbit polyclonal JNK-1 and p38 antibodies have been described. The immunoprecipitates were washed twice with TLB and twice with kinase buffer (20 mm Hepes, pH 7.4, 20 mmβ-glycerophosphate, 20 mm MgCl2, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate). The kinase assays were initiated by the addition of 1 μg of substrate protein and 50 μm[γ-32P]ATP) (10 Ci/mmol) in a final volume of 25 μl. The reactions were terminated after 15 min at 30 °C by addition of Laemmli sample buffer. Control experiments demonstrated that the phosphorylation reaction was linear with time for at least 30 min under these conditions. The phosphorylation of the substrate proteins was examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography.

---

In Gel Kinase Assay [1]
In gel kinase assay for the proteins that phosphorylate IκB-α was carried out according to the method of Hibi et al. and as detailed below. Whole cell extracts were prepared from HUVEC treated with TNFα (100 units/ml) for 15 min in the presence or absence of compound 1 (20 μm, pretreatment for 1 h) as indicated. Proteins were separated on a 10% SDS gel containing 0.5 mg/ml HIS-IκB-α. Gels were washed two times in 20% propanol, 50 mm Hepes, pH 7.6, for 30 min and two times in buffer A (50 mm Hepes, pH 7.6, 5 mm 2-mercaptoethanol) for 30 min, followed by a 1-h incubation with buffer A containing 6 m urea, 1 h each in 3, 1.5, and 0.75 m urea in buffer A and 0.05% Tween 20 and 1 h in buffer A with 0.05% Tween 20. The kinase assay was carried out for 1 h at 30 °C in the presence of 50 μm ATP, 5 μCi/ml [32P]ATP, 20 mm Hepes, pH 7.6, 20 mm MgCl2, 20 mm β-glycerophosphate, 20 mm p-nitrophenyl phosphate, 1 mm sodium vanadate, 2 mm dithiothreitol. The gel was washed with 5% trichloroacetic acid and 1% sodium pyrophosphate, dried, and exposed to film. A separate gel with no HIS-IκB-α was assayed as a control.

ECSCs cells are incubated with BAY 11-7085 (0.01 to 10 μM) for 48 hours. Then, each well receives 20 μL of WST-1 dye, and the cells are incubated for an additional 4 hours. Every experiment is carried out with 10% FBS present. Absorbance at 540 nm is used to assess cell proliferation[2].

---

Cell Surface Fluorescent Immunoassay [1]
Cell surface binding assays were performed at 4 °C on viable human umbilical vein endothelial cell monolayers in microtiter plates, using saturating concentrations of monoclonal antibody supernatants and a secondary fluorescent-conjugated F(ab′)2 goat anti-murine IgG (Caltag Labs, San Francisco, CA) as previously detailed. Fluorescence intensities were determined using an automated microtiter plate reader.

---

Interleukin-6 and Interleukin-8 Assays [1]
The effects of compounds 1 and 2 on interleukin-6 (IL-6) and interleukin-8 (IL-8) production were evaluated on HUVEC that were grown to confluence on 96-well microtiter plates. The cells were preincubated with the drugs at concentrations of 0, 1, 5, 10, or 25 μm and then incubated with TNFα (10 units/ml) and drug for 16 h. The culture supernatants were removed and assayed for IL-6 and IL-8 content using enzyme-linked immunoassay kits.

---

Electrophoretic Mobility Shift Assay [1]
Nuclear extracts were prepared from test or control HUVEC in the presence of 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1.5 μg/ml pepstatin A, 40 μm ALLN (calpain inhibitor 1), 1 mm sodium orthovanadate, and 1 mm sodium fluoride as described previously. Oligonucleotides were gel-purified, annealed, and end-labeled with [α-32P]dCTP (50 μCi; specific activity of 3000 Ci/mmol) and the Klenow fragment of Escherichia coli DNA polymerase I. Binding reactions were performed in the presence of 10 mm Tris, pH 7.5, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, and 1 μg of poly(dI·dC) and electrophoresis was carried out as described previously . The following oligonucleotides were utilized: VCAM-κB (vNF-WT), 5′ CTGGGTTTCCCCTTGAAGGGATTTCCCTC and the complementary strand. Protein DNA complexes were resolved on 4% polyacrylamide gels.

---

Western Blots [1]
Following experimental treatment of HUVEC, cytosolic and nuclear protein extracts were prepared, subjected to electrophoresis on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose in 25 mm Tris, 192 mm glycine, 5% methanol at 100 V for 1 h as described previously (10, 27). Anti-IκBα and anti-p38 antisera were used at dilutions of 1:1000. Rabbit antisera directed against the phosphorylated p38 (Tyr-182) were used at a 1:1000 dilution. Mouse anti-phosphotyrosine antibodies were used at 1:1000 dilution. Immunoreactive proteins were detected by enhanced chemiluminescent protocol using 1:10,000 horseradish peroxidase-linked donkey anti-rabbit or sheep anti-mouse secondary antiserum. Blots were exposed to film for 1–15 min and then developed.

---

Assessment of cell proliferation and cell viability of ECSCs and NESCs. [2]
The cell proliferation and cell viability of ECSCs and NESCs after BAY 11-7085 treatment were determined in 96-well plates by a modified methylthiazoletetrazolium (MTT) assay using WST-1 following the manufacturer's protocol. We distributed 5 × 104 cells in DMEM supplemented with 10% FBS into each well of a 96-well flat-bottomed microplate and incubated them overnight. The medium was then removed, and the cells were incubated for 48 h with 200 μl of experimental medium containing various concentrations of BAY 11-7085 (0.01–10 μM). Thereafter, 20 μl of WST-1 dye were added to each well, and the cells were further incubated for 4 h. All experiments were performed in the presence of 10% FBS. Cell proliferation was evaluated by measuring absorbance at 540 nm. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.
Cell proliferation of ECSCs and NESCs after BAY 11-7085 treatment was determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation using cell proliferation enzyme-linked immunosorbent assay. We placed 1 × 104 cells in DMEM supplemented with 10% FBS into each well of a 96-well flat-bottomed microplate and incubated them overnight. The medium was then removed, and the cells were incubated for 48 h with 100 μl of experimental medium containing various concentrations of BAY 11-7085 (0.01–10 μM). We then added 10 μl of BrdU (10 mM) to each well and incubated the samples for 2 h. BrdU incorporation was then evaluated according to the manufacturer's protocols. All experiments were performed in the presence of 10% FBS. Cell proliferation was evaluated by measuring absorbance at 450 nm. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.

---

Assessment of internucleosomal DNA fragmentation in ECSCs. [2]
Internucleosomal DNA fragmentation in ECSCs after BAY 11-7085 treatment was evaluated using a Quick Apoptotic DNA Ladder Detection Kit, as previously described. Cells (1 × 107) of ECSCs were plated onto 100-mm culture dishes in 10 ml of DMEM supplemented with 10% heat-inactivated FBS and cultured overnight. The supernatant was then replaced with fresh culture medium (DMEM + 10% FBS) containing BAY 11-7085 (10 μM). Twenty-four hours after stimulation, the DNA was extracted from these cells according to the manufacturer's protocol. DNA fragmentation was analyzed by electrophoresis on an agarose gel (1.2%). The DNA bands were visualized by staining with ethidium bromide and were photographed under ultraviolet light using a transilluminator.

---

Assessment of BAY 11-7085-induced apoptosis in ECSCs and NESCs. [2]
The BAY 11-7085-induced apoptosis of ECSCs and NESCs was quantified by direct determination of nucleosomal DNA fragmentation by Cell Death Detection ELISA (Roche) as previously described. The assay used specific monoclonal antibodies directed against histones from fragmented DNA, allowing the determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Briefly, 1 × 106 cells were plated onto 24-well culture plates in 1 ml of culture medium with 10% heat-inactivated FBS and cultured overnight. The supernatant was replaced with fresh culture medium (DMEM + 10% FBS) containing various amounts of BAY 11-7085 (0.01–10 μM). Twenty-four hours after stimulation, the cells were lysed according to the manufacturer's instructions, followed by centrifugation (200 g, 5 min). The mono- and oligonucleosomes contained in the supernatants were determined using an anti-histone-biotin antibody. The concentration of nucleosomes-antibody was determined photometrically at a wavelength of 405 nm using 2,2′-azino-di-(3-ethylbenzthiazoline-sulfonate) as a substrate. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.

---

Analysis of cell cycle by flow cytometry. [2]
The cell cycle was analyzed by flow cytometry after 2 days of culture with or without BAY 11-7085, as previously described. Briefly, ECSCs were cultured in DMEM supplemented with 10% FBS at <60% confluence for 2 days with or without the presence of BAY 11-7085 (10 μM). They were then trypsinized, washed in phosphate-buffered saline (PBS), fixed in methanol, and incubated for 30 min at 4°C in the dark with a solution of 5 μg/ml propidium iodide, 1 mg/ml RNase, and 0.1% Nonidet P-40. Flow cytometric analysis of the cell cycle was performed immediately after staining using the CellFIT program, in which the S-phase was calculated using an RFit model.

---

Assessment of the expression of apoptosis-related proteins in ECSCs. [2]
The expression of apoptosis-related proteins (Bcl-2, Bcl-XL, Bax, Fas, Fas ligand, caspase-3, caspase-8, and caspase-9) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in ECSCs was investigated by Western blotting analysis. Subconfluent ECSCs were cultured on 100-mm dishes for 24 h with or without the presence of BAY 11-7085 (10 μM).

Rats: Rats receive 0.1 mL of 1% suspension of carrageenan in distilled water as a subplantar injection into the footpad of the right hind paw. Rats receive either a fine suspension of BAY 11-7085/compound 2 (1, 5 or 50 mg/kg) in vehicle or vehicle (polyethylglycol 400 diluted 1:5 in 5% bovine serum albumin/water) intraperitoneally for one hour prior to injection. Rats pretreated with 20 mg/kg of ibuprofen are part of a positive control group that is also present. The injection volume of the paw is measured four hours after carrageenan administration. Edema volume is calculated[1].

---

Carrageenan Paw Edema [1]
Male Harlan Sprague Dawley rats 150–175 g were used. A 1% suspension of carrageenan in distilled water was administered to rats as 0.1 ml subplantar injection into the footpad of the right hind paw as described previously. One h prior to injection rats were treated intraperitoneally with vehicle (polyethylglycol 400 diluted 1:5 in 5% bovine serum albumin/H2O) or a fine suspension of compound 2/BAY 11-7085 (1, 5, or 50 mg/kg) in vehicle. A positive control group was also included in which rats were pretreated with 20 mg/kg ibuprofen. Four hours after carrageenan administration, the volume of the injected paw was measured by means of a water displacement plethysmograph. Edema volumes were determined as the difference between the paw volumes of each rat at time 0 and 4 h. Each group contained five animals. Data were analyzed by a one-way analysis of variance and, if indicated, differences between groups analyzed by Bonferroni's modifiedt test. A p < 0.05 was considered significant.

---

Adjuvant Arthritis [1]
Inbred, male Lewis rats 8–10 weeks of age weighing 250–275 g were used. Five animals per group were used, and the animals were allowed to feed ad libitum on laboratory rat chow and water. Heat-inactivated Mycobacterium butyricum was suspended at 10 mg/ml in mineral oil and administered as 0.1-ml injection (1 mg/animal) at the base of the tail. Paw volumes were determined by a water displacement plethysmograph as described above. Volumes were determined on the indicated dates and values compared with initial time 0 measurements. Vehicle (0.5% methyl cellulose) or drug (compound 2/BAY 11-7085 or dexamethasone at the indicated concentrations) was administered once a day as an intraperitoneal injection (200 μl). Data from these studies are expressed as the mean difference in foot pad volume. At day 20 animals were sacrificed by CO2 inhalation.

[1]. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesionmolecule expression show anti-inflammatory effects in vivo. J Biol Chem. 1997 Aug 22;272(34):21096-103.

[2]. Application of the nuclear factor-kappaB inhibitor BAY 11-7085 for the treatment of endometriosis: an in vitro study. Am J Physiol Endocrinol Metab. 2007 Jul;293(1):E16-23.

BAY 11-7085 is a sulfone that is benzene substituted by [(E)-2-cyanoethenyl]sulfonyl and tert-butyl groups at position 1 and 4, respectively. It is an irreversible inhibitor of IkappaB-alpha phosphorylation in cells (IC50 = 10 muM) and prevents the activation of NF-kappaB. It has a role as an anti-inflammatory agent, a ferroptosis inducer, a NF-kappaB inhibitor, an apoptosis inducer, an autophagy inducer, an antibacterial agent, an EC 2.7.11.10 (IkappaB kinase) inhibitor and an antineoplastic agent. It is a nitrile, a sulfone and a member of benzenes.

---

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-alpha-induced phosphorylation of IkappaB-alpha, resulting in decreased nuclear factor-kappaB and decreased expression of adhesion molecules. The effects on both IkappaB-alpha phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 microM. These agents selectively and irreversibly inhibited the tumor necrosis factor-alpha-inducible phosphorylation of IkappaB-alpha without affecting the constitutive IkappaB-alpha phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130-140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IkappaBalpha phosphorylation exert anti-inflammatory activity in vivo.[1]

---

The anti-inflammatory effects of the test compounds in two animal models are striking and are consistent with the action of other pharmacologic agents that inhibit adhesion molecule expression and leukocyte recruitment. Since these novel compounds inhibit NF-κB signaling, they would be expected to affect κB-dependent expression of many other genes including IL-1, IL-6, tissue factor, and TNFα in lymphoid cells, monocytes, and endothelial cells (reviewed in Ref. 24). Thus, the observed anti-inflammatory action probably reflects not just inhibition of adhesion molecules but also effects on many other important mediators of inflammation in a variety of cell types. Understanding the mechanism by which these agents disrupt the NF-κB/IκB regulatory pathway will be useful in identifying novel anti-inflammatory agents that are both highly specific and effective. Such drugs may be useful as therapeutic agents in disorders involving up-regulation of endothelial adhesion molecules including ischemia, reperfusion injury, asthma, transplantation, inflammatory bowel disease, rheumatoid arthritis, and atherosclerosis. [1]

---

Most of the current medical treatments for endometriosis aim to downregulate estrogen activity. However, a high recurrence rate after medical treatment has been the most significant problem. BAY 11-7085, a soluble inhibitor of NK-kappaB activation, has been shown to inhibit cell proliferation and induce apoptosis of a variety of cells. To examine the potential application of BAY 11-7085 in the treatment of endometriosis, we investigated the effects of this agent on the cell proliferation and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSCs) by a modified methylthiazole tetrazolium assay, a 5-bromo-2'-deoxyuridine incorporation assay, and internucleosomal DNA fragmentation assays. The effect of BAY 11-7085 on the cell cycle of ECSCs was also determined by flow cytometry. The expression of apoptosis-related molecules was examined in ECSCs with Western blot analysis. BAY 11-7085 significantly inhibited the cell proliferation and DNA synthesis of ECSCs and induced apoptosis and the G0/G1 phase cell cycle arrest of these cells. Additionally, downregulation of the B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-X(L) expression with simultaneous activation of caspase-3, -8, and -9 was observed in ECSCs after treatment with BAY 11-7085. These results suggest that BAY 11-7085 induces apoptosis of ECSCs by suppressing antiapoptotic proteins, and that caspase-3-, -8-, and -9-mediated cascades are involved in this mechanism. Therefore, BAY 11-7085 could be used as a therapeutic agent for the treatment of endometriosis.[2]

---

In summary, we have demonstrated that BAY 11-7085 is able to induce apoptosis and the G0/G1-phase cell cycle arrest of ECSCs. Downregulation of Bcl-2 and Bcl-XL expression with simultaneous upregulation of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 expression was induced by BAY 11-7085 treatment, suggesting that BAY 11-7085 may be applicable for the medical treatment of endometriosis as an adjuvant approach in combination with current medical treatment for this disease. Further studies with other inhibitors of NF-κB, JNK, or p38 MAPK on the cell proliferation and apoptosis of endometriotic cells may contribute to the establishment of more effective and sophisticated treatment strategies for endometriosis. [2]

C13H15NO2S

249.33

249.082

C, 62.63; H, 6.06; N, 5.62; O, 12.83; S, 12.86

196309-76-9

5353432

White to off white solid powder

1.1±0.1 g/cm3

407.1±45.0 °C at 760 mmHg

80-82℃

200.0±28.7 °C

0.0±0.9 mmHg at 25°C

1.535

2.51

0

3

3

17

420

0

S(/C=C/C#N)(C1C=CC(=CC=1)C(C)(C)C)(=O)=O

VHKZGNPOHPFPER-ONNFQVAWSA-N

InChI=1S/C13H15NO2S/c1-13(2,3)11-5-7-12(8-6-11)17(15,16)10-4-9-14/h4-8,10H,1-3H3/b10-4+

(E)-3-(4-tert-butylphenyl)sulfonylprop-2-enenitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (8.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (8.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)