Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-κB dependent activation of the urokinase plasminogen activator system

Abstract

Introduction: The mechanisms of bacterial lipopolysaccaride (LPS) induced accelerated metastatic tumor growth in murine models are poorly understood. The urokinase plasminogen activator system, comprising urokinase plasminogen activator (u-PA), urokinase plasminogen activator receptor (u-PAR) and plasminogen activator inhibitor 1 (PAI-1), is intimately implicated in tumor cell extracellular matrix interactions fundamental to tumour cell invasion. The aim of this study was determine if LPS directly promotes tumor cell extracellular matrix adhesion and invasion through activation of the urokinase plasminogen activator system and elucidate the pathways involved in this process. Methods: ELISA was used to assess cellular and supernatant u-PA, u-PAR, PAI-1 levels in sw480 and sw620 colorectal cancer cell lines following stimulation with LPS (100 ng/ml, 1000 ng/ml and 10,000 ng/ml) for different time periods (0, 6, 12 18 and 24 hours). Activated u-PA, TLR-4, Iκ-Bα and phospho- Iκ-Bα were evaluated by western blot analysis and u-PA activity by chromogenic assay. Surface u-PAR and TLR-4 expression were examined by FACS analysis, vitronectin adhesion by fluorgenic adhesion assay and in-vitro invasion by ECmatrix invasion chambers. Results: LPS stimulation upregulates u-PA and u-PAR in a dose and time dependent manner. Stimulation of sw480 and sw620 cancer cells with 100ng/ml LPS for 18 hours augmented cell supernatant u-PA concentration and activity by 50% and 32% respectively (*;p>0.01 compared to baseline). This translated into a 27% increase in tumor cell vitronectin adhesion and a 34% increase in in-vitro ECmatrix invasion above baseline (Table 1) (**,p<0.05). Such effects were ameliorated by u-PA and u-PAR inhibition utilising u-PAR and u-PA blocking antibodies and by the novel u-PA inhibitor wxc-340 (*,p>0.01, **,p>0.05;compared to LPS stimulated cells). Inhibition of NF-κB and TLR-4 significantly impairs LPS mediated activation of the u-PA system, tumor cell vitronectin adhesion and in vitro invasion (Table 1) (*,p<0.01, **,p>0.05 compared to LPS stimulated cells). Conclusions: This study demonstrates that LPS promotes tumour cell extracellular matrix adhesion and in vitro invasion through activation of the urokinase plasminogen activator system in a TLR-4 and NF-κB dependent manner.