lipoteichoic-acid and Colonic-Neoplasms

Type 2 Toll-like receptors (TLR2s) are expressed in cell membranes and recognize a wide range of pathogen-associated molecular patterns derived from bacteria, such as lipoteichoic acid (LTA). The aim of this study was to evaluate the effect of TLR2 activation by LTA on the activity of type 1 Na(+)/H(+) exchanger (NHE) in T84 intestinal epithelial cells. Short-term (0.5 hour) and long-term (18 hours) TLR2 activation significantly inhibited NHE1 activity in a concentration-dependent manner (0.01-100 µg/ml; -7 ± 3 to -21 ± 3% and 3 ± 3 to -21 ± 3% of control values, respectively). S3226 [3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride], an NHE3-selective inhibitor, did not affect the inhibitory effect on NHE activity. LTA-induced NHE inhibition did not occur in the presence ofethylisopropylamiloride (an NHE1 inhibitor). Long-term TLR2 activation decreased NHE1 affinity for Na(+) (Km= 64.98 ± 1.67 mM) compared with control (Km= 20.44 ± 0.54 mM) without changes in Vmax values. After TLR2 activation, we observed tyrosine-protein kinase (SRC) activation, phosphatidylinositol 3-kinase (PI3K) recruitment, and adenylyl cyclase (AC3) phosphorylation. The total amount of AC3 increased (23 ± 8% of control) after long-term treatment with LTA. Anti-AC3 small interfering RNA prevented LTA-induced NHE1 inhibition, similar to that observed with the AC3 inhibitor KH7 [(±)-2-(1H-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide]. A significant increase in cAMP levels (32 ± 3% and 14 ± 2% after short- and long-term stimulation, respectively) was detected, and inhibition of protein kinase A (PKA), phospholipase C (PLC), and downregulation of protein kinase C (PKC) prevented NHE1 inhibition. Inhibition of nuclear factor-κΒ (NF-κB) failed to revert NHE1 inhibition. We concluded that activation of TLR2 reduces NHE1 activity in epithelial cells through an alternative pathway that is unrelated to NF-κB, which involves SCR, PI3K, AC3, PKA, PLC, and PKC.

Topics: Adenylyl Cyclases; Animals; Biotinylation; Blotting, Western; Cation Transport Proteins; Cell Line; Colonic Neoplasms; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Humans; Hydrogen-Ion Concentration; Kinetics; Lipopolysaccharides; Mice; NF-kappa B; Protein Kinase C; Rats; Signal Transduction; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Teichoic Acids; Toll-Like Receptor 2; Type C Phospholipases

The aim of this study is designed to explore the anti-tumor effect of lipoteichoic acid (LTA) of Bifidobacterium on the expression of survivin in colon cancer LoVo cells and its possible regulatory mechanism.. The changes of survivin mRNA and protein in LoVo cells treated with LTA of Bifidobacterium were detected by RT-PCR and Western blot. Meanwhile, the expressions of pAKT (the key protein kinase in P13K/AKT signal transduction pathway), p53 and PTEN were measured by Western blot.. There were overexpressions of survivin mRNA and protein in LoVo cells. After treated with different dose of LTA of Bifidobacterium, the expressions of survivin mRNA and protein were markedly decreased in a dose-dependent manner (P < 0.01). Besides, the activity of pAKT was decreased significantly (P < 0.01) and the expression of p53 and PTEN was increased (P < 0.01).. LTA of Bifidobacterium can down-regulate the expression of survivin in LoVo cells through inhibiting the activity of PI3K/AKT signal transduction pathway and up-regulate the expression of p53. Accordingly, the activity of caspases is increased, and apoptosis of LoVo cells occurs ultimately.

Topics: Apoptosis; Bifidobacterium; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Dose-Response Relationship, Drug; Down-Regulation; Humans; Inhibitor of Apoptosis Proteins; Lipopolysaccharides; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Survivin; Teichoic Acids; Tumor Suppressor Protein p53

Despite the ability to participate in immune responses and the continuous presence of bacteria and bacterial products, functional responses of intestinal epithelial cells (IEC) seem to be muted. Previously, tolerance to Toll-like receptors (TLRs) ligands has been described in monocytic cells. However, mechanisms in the intestine are unknown.. The effect of purified lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on expression and function of TLRs in intestinal epithelial cells (Colo205, SW480, T84) was assessed by Northern and Western blot and FACS analysis, kinase activity assays, immunohistochemistry, and ELISA.. Expression of TLRs except 10 was detected in primary IEC and TLR1-10 in the cultured cells. Short-term stimulation with LPS or LTA activated proinflammatory signaling cascades in IEC, including phosphorylation of IRAK and MAP kinases and increased IL-8 secretion, whereas prolonged incubation resulted in a state of hyporesponsiveness with no reactivation of the cells by a second challenge with either substance detected. The cells remained responsive to tumor necrosis factor (TNF). Hyporesponsive cells showed no alteration in expression of TLR or signaling molecules but revealed a decrease in TLR surface expression and IRAK activity. Toll-interacting protein (Tollip) mRNA and protein expression were increased in hyporesponsive cells, and overexpression of Tollip in IEC resulted in a significantly decreased proinflammatory response.. Continuous presence of specific bacterial components results in a status of hyporesponsiveness in otherwise reactive IEC. Down-regulation of TLR surface expression and up-regulation of inhibitory Tollip with decreased phosphorylation of IRAK might all contribute to this hyporesponsiveness.

Topics: Bacteria; Bacterial Adhesion; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Epithelial Cells; Gene Expression; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Ligands; Lipopolysaccharides; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Protein Kinases; Receptors, Cell Surface; Signal Transduction; Teichoic Acids; Toll-Like Receptor 1; Toll-Like Receptors