Benzyl Isothiocyanate (BITC) Inhibits Migration and Invasion of Human Colon Cancer HT29 Cells by Inhibiting Matrix Metalloproteinase-2/-9 and Urokinase Plasminogen (uPA) through PKC and MAPK Signaling Pathway

Abstract

Benzyl isothiocyanate (BITC), a component of dietary cruciferous vegetables, has antioxidant and anticancer properties. In this study, we show for the first time the antimetastatic effects of BITC in human colon cancer HT29 cells. BITC had an inhibitory effect on cell migration and invasion. Protein levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-plasminogen activator (u-PA) were reduced by BITC in a concentration-dependent manner. BITC also exerted an inhibitory effect on phosphorylation of c-Jun N-terminal kinase 1 and 2 (JNK1/2), extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) that are upstream of nuclear factor kappa B (NF-kappaB). BITC inhibited DNA binding activity of NF-kappaB. Moreover, BITC decreased the levels of c-Fos, c-Jun, Ras, FAK, PI3K and GRB2 in HT29 cells. Reductions in the enzyme activity, protein and mRNA (mRNA) levels of MMP-2 were observed in BITC-treated HT29 cells. BITC also inhibited mRNA levels of MMP-2, -7, and -9 in HT29 cells. Results from zymography showed that BITC treatment decreased MMP-2 expression in a concentration-dependent manner. BITC inhibited PKCdelta activity in HT29 cells. Furthermore, inhibitors specific for JNK (SP600125) reduced expression of MMP-2, MMP-9, and u-PA. These results demonstrated that BITC could alter HT29 cell metastasis by reduction of MMP-2, MMP-9, and u-PA expression through the suppression of a PKC, MAPK signaling pathway and inhibition of NF-kappaB levels. These findings suggest that BITC has potential as an antimetastatic agent.

Gallic acid suppresses the migration and invasion of PC-3 human prostate cancer cells via inhibition of matrix metalloproteinase-2 and -9 signaling pathways

“…Approximately 2x10 5 cells/well of pc-3 cells were cultured in a 12-well plate for 24 h wounding by scratching with a pipet tip, and cells in the each well were incubated with serum-free RPMI-1640 medium and treated with or without GA (0, 25 and 50 µM) for 24 h. Cells were photographed using a phase-contrast microscope (x200) as previously described (26,27).…”

“…All cells in each treatment were incubated for 24 or 48 h at 37˚C in a humidified atmosphere with 95% air and 5% CO 2 . At the end of incubation, a cotton swab was used to remove the non-invasive cells maintained in the upper chamber and the invasive cells were fixed with 4% formaldehyde in PBS and stained with 2% crystal violet in 2% ethanol and cells were counted and photographed under a light microscope at x200 magnification (27,28).…”

“…the collected individual medium was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) containing 0.1% gelatin (Sigma-Aldrich Corp.). After electrophoresis, the gels were soaked in 2.5% Triton X-100 in dH 2 O twice for a total of 60 min at 25˚C, then were incubated in substrate buffer (pH 7.6, 50 mM Tris, 10 mM cacl 2 , 50 mM and 0.05% Brij 35) at 37˚C for 18 h. Bands corresponding to activity of MMP-2 and -9 were visualized by negative staining using 0.3% Coomassie Brilliant Blue in 50% methanol and 10% acetic acid as described elsewhere (27). Quantification of the data from the band density was performed by NIH ImageJ software.…”

“…Quantitative PCR conditions were: 2 min at 50˚C, 10 min at 95˚C and 40 cycles of 15 sec at 95˚C; 1 min at 60˚C using 1 µl of the cDNA reverse-transcribed as described above, 2X SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of forward and reverse primers. Applied Biosystems 7300 Real-Time PCR system was used for each assay in triplicate and expression fold-changes were derived using the comparative CT method (25,27).…”

Possible contribution of aminopeptidase N (APN/CD13) to migration and invasion of human osteosarcoma cell lines

“…cells (1x10 6 cells/well) were plated in 12-well tissue culture plates and incubated in serum-free McCoy's 5A medium in the presence of 1 nM IL-6 or 100 µM bestatin for 24 and 48 h. the conditioned medium was then collected and separated by electrophoresis on 10% SDS-PAgE containing 0.2% gelatin (Sigma). At the end of electrophoresis, the gels were soaked twice in 2.5% Triton X-100 in dH 2 O at 25˚C for a total of 60 min, then incubated in substrate buffer (50 mM Tris HCl, 5 mM CaCl 2 , 0.02% nan 3 and 1% triton X-100, pH 8.0) at 37˚C for 18 h. Bands corresponding to MMP-2 and -9 activity were visualized by negative staining using 0.2% coomassie Blue in 50% methanol and 10% acetic acid as described elsewhere (43,44). Bands were quantified using NIH imagej software (NIH, Bethesda, MD, uSA) as previously described (45,46).…”