Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes  Greg C. Kaiser, Fang Yan, D.Brent Polk  Gastroenterology  Volume 116, Issue 3, Pages 602-609 (March 1999) DOI: 10.1016/S0016-5085(99)70182-4 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 The antiproliferative effect of high-dose TNF-α is inhibited by mesalamine. YAMC monolayers were serum-starved at nonpermissive conditions for 24 hours before use. Cells were incubated for 24 hours at 37°C with EGF and/or TNF-α in the presence or absence of mesalamine (5-ASA) pretreatment. Cell numbers were determined as described in Materials and Methods. *P < 0.005 compared with control; ΦP < 0.001 compared with 100 ng/mL TNF-α; ωP < 0.001 compared with 10 ng/mL EGF; ΣP < 0.001 compared with 1 ng/mL TNF-α and 10 ng/mL EGF; and ξP < 0.001 compared with 100 ng/mL TNF-α and 10 ng/mL EGF. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 TNF-α–stimulated ERK1/ERK2 activation is blocked by mesalamine. YAMC cells were incubated with EGF for 2 minutes or TNF-α for 15 minutes in the presence or absence of mesalamine (5-ASA) at 37°C. Triton-soluble lysates were obtained, proteins separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane, and Western blot analysis was conducted with an antiactive ERK1/ERK2 antibody (top panel) and an anti–pan ERK1/ERK2 antibody (lower panel) as described in Materials and Methods. Proteins were visualized by enhanced chemiluminescence. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Inhibition of TNF-α–stimulated ERK1/ERK2 and JNK/SAPK activation by mesalamine is both concentration and time dependent. In the top panels, YAMC cells were incubated with various concentrations of mesalamine (5-ASA), then cultured with TNF-α for 15 minutes at 37°C. (A) Antiactive ERK1/ERK2 was detected as described in Figure 2. (B) Antiactive JNK/SAPK was identified by Western blot analysis of Triton-soluble lysates as described in Materials and Methods. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 ERK1/ERK2 activation by ceramide is blocked by mesalamine. YAMC cells were treated with TNF-α or ceramide in dimethyl sulfoxide for 15 minutes at 37°C in the presence or absence of mesalamine (5-ASA). Antiactive ERK1/ERK2 was detected as described in Figure 2. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 TNF-α–stimulated nuclear translocation of NF-κB is inhibited by mesalamine. YAMC cells were grown on collagen-coated Thermanox culture slides at 37°C and received (A) no treatment, (B) 100 ng/mL TNF-α for 30 minutes, (C) 20 mmol/L mesalamine for 30 minutes, or (D) the combination of TNF-α and mesalamine. Cells were then fixed, probed with an anti–NF-κB p65 antibody, and incubated with a secondary antibody linked to Cy3 as described in Materials and Methods. Images were obtained using confocal laser immunofluorescent microscopy. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Iκ-Bα degradation is inhibited by mesalamine. YAMC cells were treated with 100 ng/mL TNF-α for various times at 37°C as indicated in the presence or absence of (A) mesalamine (5-ASA) or (B) treated with TNF-α for 15 minutes in the presence of mesalamine at various concentrations. Cytosolic fractions were prepared; proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane; and Western blot analysis was conducted with an anti–Iκ-Bα antibody as described in Materials and Methods. Proteins were visualized by enhanced chemiluminescence. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 TNF-α–stimulated degradation of IκB and activation of MAP kinase are not affected by pH nor osmolarity. YAMC cells were treated with TNF-α for 15 minutes alone or in combination with a 30-minute pretreatment with mesalamine (5-ASA), equivalent osmotic concentrations of mannitol, equivalent pH-corrected media with butyric acid, or the combined osmotic and pH effects or various concentrations of 4-aminosalicylic acid at 37°C. Cell lysates were prepared for Western blot analysis with anti-IκB or antiactive ERK1/ERK2 or anti-ERK1/ERK2, as indicated. Gastroenterology 1999 116, 602-609DOI: (10.1016/S0016-5085(99)70182-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions