At 80% confluency, cells were treated with increasing concentrations of sulindac (grey), sulindac sulfide (white), and sulindac sulfone (black).

(A) PCR product of RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO or sulindac sulfide. Lane 1, 50 bp DNA ladder; lane 2, MMP25 after sulindac sulfide treatment; lane 3, MMP25 after DMSO treatment; lane 4, Trypsin1 after sulindac sulfide treatment; lane 5, Trypsin1 after DMSO treatment; lane 6, MMP7 after sulindac sulfide treatment; lane 7, MMP7 after DMSO treatment; lane 8 RPL-19 after sulindac sulfide treatment; lane 9, RPL-19 after DMSO treatment. Results demonstrate that only MMP7 expression is affected by sulindac sulfide treatment. (B) Quantitative RT-PCR of MMP25, Trypsin1, and MMP7 after DMSO or sulindac sulfide treatment. The results were obtained using the ΔΔCt method with hRPL-19 as the housekeeping gene. Results indicate quantitative differences in MMP7 expression after sulindac sulfide treatment relative to DMSO treatment. (C) Western blot of secreted fraction of HT-29 cells demonstrating that the protein levels of MMP25 in the membrane fraction are similar between sulindac sulfide and DMSO treated cells.

Secreted proteomes of DMSO-treated and sulindac sulfide-treated cells were analyzed for LTB4 levels using an enzymatic immunoassay.

(A) Real time RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO, sulindac (S), sulindac sulfide (Sd) and sulindac sulfone (Sn). The results were obtained using the ΔΔCt method with hRPL-19 as the housekeeping gene. C_T values between 20.10 and 20.56 across all samples show constant levels of hRPL-19 expression. (B) The expression of hMMP7 after 24 hours of sulindac treatment. The downregulation of hMMP7 was significant at all concentrations. The results were obtained using the ΔΔCt method with hRPL-19 as the housekeeping gene. C_T values between 23.22 and 23.56 across all samples show constant levels of hRPL-19 expression. (C) Cytosolic, membrane, and secreted proteome fractions extracted from DMSO- and sulindac sulfide-treated HT-29 cells were subjected to western blot analysis using an antibody which does not differentiate between active and inactive MMP7 (top), and an antibody that only recognizes the active site (bottom). The expected band for MMP7 appears at 28 kDa, however a band at 45 kDa is often reported, possibly a dimer. Cyto denotes cytosolic fraction; Mem, membrane fraction, Secr, secreted fraction, and aMMP7, active MMP7.

Metallohydrolase activity-based labeling of HT-29 cells secreted proteomes. MMP2 and MMP7 standards were added to cell proteomes (arrows). The band displaying a strong decrease (*) after sulindac sulfide treatment was extracted and analyzed by mass spectrometry. MS/MS spectra of LVVDLTDIDPDVAYSSVPYEK and LTYTAEVSVPK peptides corresponded to amino acids 366–386 and 155–165, respectively, of the LTA4H protein. Protein content across all samples was adjusted to 1 mg/ml and the equivalent of 15 µg of proteome was added per lane. MW denotes molecular weight; STDs, standards, Sd, sulindac sulfide-treated and D, DMSO-treated.