Cancer cell selective probe by mimicking EGCG.

Targeting proteins that are overexpressed in cancer cells is the major strategy of molecular imaging and drug delivery systems. The 67-kDa laminin receptor (67LR), also known as oncofetal antigen, is overexpressed in several types of cancer, including melanoma, multiple myeloma, cervical cancer and bile duct carcinoma. 67LR is involved in tumour growth, tumour metastasis and drug resistance. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) directly binds to cell-surface 67LR and induces apoptosis through the protein kinase B (Akt)/endothelial nitric oxide synthase/nitric oxide/cyclic GMP (cGMP) axis. Here we report the optimum hydroxyl group for the utilization of EGCG as a novel fluorescent EGCG-mimic imaging probe based on 67LR agonist characters, including Akt activation and inhibitory effect on viable cell number in cancer cells. 67LR specific targeting is unambiguously confirmed with the use of a non-labelled EGCG competitive assay and 67LR knockdown. Importantly, this probe strongly binds to multiple myeloma cells compared with its binding to normal cells.

Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.

INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor.

Understanding ß cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic ß cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR + GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion.

Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis.

(-)-Epigallocatechin-3-O-gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG-induced anti-AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well-known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti-erectile dysfunction drug, synergistically enhanced the anti-AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67-kDa laminin receptors.

Development of laminin receptor agonists: identification of important functional residues by alanine scanning.

An antagonist of cellular adhesion and motility, acetyl-C-[S-Acm]-VIGYSGDRC-[S-Acm]-NH(2) (mEGF(33-42)), shares homology with the agonist sequence CDPGYIGSR-NH(2). It has been proposed that the latter peptide binds to the high affinity 67 kDa laminin receptor. Both peptides have equal affinities for the receptor and similar conformations have been derived for both. We have examined the importance of individual non-homologous residues with respect to receptor binding and antagonistic properties of mEGF(33-42). Alanine scanning of non-conserved residues in the N-terminal half of mEGF(33-42) caused loss of biological activity with respect to cell attachment, receptor binding and migratory response. Substitution of alanine for serine (position 6) caused loss of laminin-specific cell attachment and receptor binding activities. However, the peptide did stimulate migration suggesting that this peptide may be a non-specific stimulator of migration. In contrast, alanine substitution for the C-terminal Cys-S-Acm had no apparent effect on the attachment or receptor binding activities of the peptide but generated an agonist from the antagonist parent. Comparison of the modelled folds of the alanine containing peptides revealed the presence of significant helical content in those peptides capable of stimulating migration and suggests that a reduction in bulk in the N-terminal residues is not conducive to adopting a productive binding conformation.

Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts.

The 67-kD elastin-laminin receptor (ELR) subunit which carries the recognition site for elastin peptides (EP) is a lectin. Its binding with galactosides can modulate the kinetics of its interaction with its ligand, EP. In this study the biosynthesis of proteins, collagen and fibronectin were evaluated in the presence of agonists and antagonists of the receptor on human skin fibroblasts. The biosynthesis of total proteins determined by 3H-proline incorporation and of fibronectin (by immunoprecipitation) were shown to increase with passage number. The presence of 1 microg/ml kappa-elastin (EP) in the culture medium increased both total proteins and fibronectin biosynthesis. Melibiose, an agonist of the receptor at 5 microg/ml (140 microM), decreased both proteins and fibronectin biosynthesis in the culture medium of human skin fibroblast at the 10th and 15th passage. These results show that the ELR can control the biosynthetic mechanisms of some of the macromolecular constituents of extracellular matrix such as fibronectin and moderate its age and passage-dependent upregulation.