Abstract
Purpose: To investigate the mechanism by which pinitol inhibits tumor necrosis factor-α (TNF-α)- induced expression of matrix metalloproteinase-9 (MMP-9) and invasion of prostate cancer LNCaP cells. Methods: Reverse transcription-polymerase chain reaction (RT-PCR) together with Western blot analysis was used to analyze the expression of MMP-9 and nuclear factor-κB (NF-κB) subunits, p65 and p50, in TNF-α- treated LNCaP cells, while 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and DNA fragmentation were used to evaluate cell viability and apoptosis. MMP-9 activity and invasion were measured by gelatin zymography and matrigel invasion assay, respectively. DNA-binding activity of NF-κB and AP-1 was determined by electrophoretic mobility shift assay and luciferase activity. Results: MMP-9 activity significantly increased in response to TNF-α; however, pinitol reduced TNF-α- induced MMP-9 activity without cytotoxicity. Matrigel invasion assay showed that pinitol reduced TNF-α-induced invasion of prostate cancer LNCaP cells. Further, it downregulated the expression of MMP-9 gene induced by TNF-α-treatment. Pinitol suppressed TNF-α-induced NF-κB activity by suppressing nuclear translocation of the NF-κB subunits, p65 and p50. Conclusion: The results indicate that pinitol is a potential anti-invasive agent and acts by suppressing TNF-α-induced cancer cell invasion and specifically inhibiting NF-κB as well as downstream target genes such as MMP-9.
Highlights
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases that are collectively capable of degrading all components of the extracellular matrix
Cell migration was significantly increased by tumor necrosis factor-α (TNF-α) treatment and the effects were inhibited in the presence of pinitol (Fig 1D)
These data indicate that pinitol inhibits TNF-αinduced migration, and that the acquisition of migration is related to tumor invasion without cytotoxicity
Summary
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases that are collectively capable of degrading all components of the extracellular matrix. Little is known about the effects of pinitol on the expression and invasion of MMP-9 in prostate cancer cells. The effects of pinitol on MMP-9 expression and invasion in TNF-α-stimulated prostate cancer LNCaP cells were evaluated. Precoated filters (6.5 mm in diameter, 8 μm poresize, Matrigel 100 μg/cm2) were rehydrated and 5 × 104 cells in medium with or without pinitol or MMP-9 inhibitor I (5 nM) in the presence or absence of TNF-α (20 ng/ml) were seeded into the upper part of each chamber. Synthetic complementary NF-κB-binding oligonucleotides (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) (Santa Cruz Biotechnology) were biotinylated using the biotin 30-end DNA labeling kit (Pierce) according to the manufacturer′s instructions and annealed for 1 h at room temperature. P < 0.05 was adopted as an indication of statistical significance
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