Suppression of Met activation in human colon cancer cells treated with (−)-epigallocatechin-3-gallate: Minor role of hydrogen peroxide

Colorectal cancer is the second leading cause of cancer-related deaths in the U.S. Met, the receptor for hepatocyte growth factor (HGF), is over-expressed in colon tumors and is associated with poor prognosis. Recently, the green tea polyphenol (−)-epigallocatechin gallate (EGCG) was reported to suppress Met activation in breast cancer cells. However, the possible confounding effect of hydrogen peroxide (H₂O₂), produced when EGCG is added to cell culture media, was not assessed. In the present study, the human colon cancer cell lines HCT116 and HT29 were used to examine the relationships between Met activation, EGCG treatment, and H₂O₂ generation. At concentrations of 0.5, 1, and 5 μM, EGCG suppressed markedly the activation of Met in the presence of HGF. Concentrations of 10 μM EGCG and below generated low amounts of H₂O₂ (<1.5 μM), whereas higher H₂O₂ concentrations (>5 μM) were required to directly increase the phosphorylation of Met. Moreover, suppression of Met activation by EGCG occurred in the presence or absence of catalase, suggesting that such effects were not an ‘artifact’ of H₂O₂ generated from EGCG in cell culture media. We conclude that EGCG might be a beneficial therapeutic agent in the colon, inhibiting Met signaling and helping to attenuate tumor spread/metastasis, independent of H₂O₂-related mechanisms.     

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E₂ is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE₂ biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE₂ biosynthesis, namely cytosolic phospholipase (cPL)A₂, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E₂ synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA₂ up to 30 μM and moderately blocked isolated COX-1 (IC₅₀ > 30 μM). However, EGCG efficiently inhibited the transformation of PGH₂ to PGE₂ catalyzed by mPGES-1 (IC₅₀ = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE₂ generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF_1α) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE₂ biosynthesis by EGCG.     

Green tea catechins potentiate the neuritogenic action of brain-derived neurotrophic factor: Role of 67-kDa laminin receptor and hydrogen peroxide

Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 μg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 μM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H₂O₂ in the potentiation. Consistently, exogenous sublethal concentrations of H₂O₂, added as a bolus dose (5 μM) or more effectively through a steady-state generation (1 μM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67LR and H₂O₂, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.     

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells

Objectives: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ‐tocotrienol displays potent anti‐cancer activity that is associated with suppression of HER/ErbB receptor signalling. Experiments were conducted to investigate the effects of γ‐tocotrienol treatment on HGF‐dependent +SA mammary tumour cell proliferation, upon Met activation. Materials and methods: The +SA cells were maintained in serum‐free defined media containing 10 ng/ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining. Results and conclusions: Treatment with γ‐tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF‐dependent +SA cell replication in a dose–responsive manner. Treatment with 4 μm γ‐tocotrienol reduced both total Met levels and HGF‐induced Met autophosphorylation. In contrast, similar treatment with 5.5 μm SU11274 inhibited HGF‐induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ‐tocotrienol (2 μm ) and SU11274 (3 μm ) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of γ‐tocotrienol on Met expression and activation, and strongly suggest that γ‐tocotrienol treatment may provide significant health benefits in prevention and/or treatment of breast cancer, in women with deregulated HGF/Met signalling.     

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC₅₀ concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.     

Mechanism-based inhibition of cancer metastasis with (−)-epigallocatechin gallate

Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (−)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into the lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial–mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 μM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 μM EGCG increased Young’s modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 μM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-β-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.     

Green tea flavonoid epigallocatechin-3-gallate (EGCG) inhibits cardiac hERG potassium channels

The catechin EGCG is the main flavonoid compound of green tea and has received enormous pharmacological attention because of its putative beneficial health effects. This study investigated for the first time the effect of EGCG on hERG channels, the main pharmacological target of drugs that cause acquired long QT syndrome.Cloned hERG channels were expressed in Xenopus oocytes and in HEK293 cells. Heterologous hERG currents were inhibited by EGCG with an IC₅₀ of 6.0 μmol/l in HEK293 cells and an IC₅₀ of 20.5 μmol/l in Xenopus laevis oocytes. Onset of effect was slow and only little recovery from inhibition was observed upon washout. In X. laevis oocytes EGCG inhibited hERG channels in the open and inactivated states, but not in the closed states. The half-maximal activation voltage of hERG currents was shifted by EGCG towards more positive potentials.In conclusion, EGCG is a low-affinity inhibitor of hERG sharing major electrophysiological features with pharmaceutical hERG antagonists.     

SU5416 and EGCG Work Synergistically and Inhibit Angiogenic and Survival Factors and Induce Cell Cycle Arrest to Promote Apoptosis in Human Malignant Neuroblastoma SH-SY5Y and SK-N-BE2 Cells

Malignant neuroblastomas are solid tumors in children. Available therapeutic agents are not highly effective for treatment of malignant neuroblastomas. Therefore, new treatment strategies are urgently needed. We tested the efficacy of combination of SU5416 (SU), an inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2), and (−)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, for controlling growth of human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells. Combination of 20 μM SU and 50 μM EGCG synergistically inhibited cell survival, suppressed expression of VEGFR-2, inhibited cell migration, caused cell cycle arrest, and induced apoptosis. Combination of SU and EGCG effectively blocked angiogenic and survival pathways and modulated expression of cell cycle regulators. Apoptosis was induced by down regulation of Bcl-2, activation of caspase-3, and cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). Taken together, this combination of drugs can be a promising therapeutic strategy for controlling the growth of human malignant neuroblastoma cells.     

Design, synthesis, and biological activity of novel Magmas inhibitors

Magmas (mitochondria associated, granulocyte-macrophage colony stimulating factor signaling molecule), is a highly conserved and essential gene, expressed in all cell types. We designed and synthesized several small molecule Magmas inhibitors (SMMI) and assayed their effects on proliferation in yeast. We found that the most active compound 9 inhibited growth at the 4 μM scale. This compound was shown by fluorometric titration to bind to Magmas with a K_d = 33 μM. Target specificity of the lead compound was established by demonstrating direct binding of the compound to Magmas and by genetic studies. Molecular modeling suggested that the inhibitor bound at the predicted site in Magmas.     

Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC₅₀ of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC₅₀ of ∼ 20 μM. The 2D ¹H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.     

с-Met receptor can be activated by extracellular alkaline medium

Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.     

A broad spectrum anticancer nucleoside with selective toxicity against human colon cells in vitro

2′,3′-Bis-O-tert-butyldimethylsilyl-5′-deoxy-5′-[N-(methylcarbamoyl)amino]-N⁶-(N-phenylcarbamoyl)adenosine, a new member of the N⁶,5′-bis-ureidoadenosine class of anticancer nucleosides, is found to exhibit broad spectrum antiproliferative activity. A majority of the cell lines in the NCI-60 are inhibited with an average GI₅₀ = 3.13 μM. Selective toxicity against human colon cancer cell lines (COLO 205, HCC-2998, HCT-116, HT29, KM12) was also exhibited (LC₅₀’s = 6-10 μM).     

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function

Mice deficient in group 1b phospholipase A₂ have decreased plasma lysophosphatidylcholine and increased hepatic oxidation that is inhibited by intraperitoneal lysophosphatidylcholine injection. This study sought to identify a mechanism for lysophosphatidylcholine-mediated inhibition of hepatic oxidative function. Results showed that in vitro incubation of isolated mitochondria with 40–200 μM lysophosphatidylcholine caused cyclosporine A-resistant swelling in a concentration-dependent manner. However, when mitochondria were challenged with 220 μM CaCl₂, cyclosporine A protected against permeability transition induced by 40 μM, but not 80 μM lysophosphatidylcholine. Incubation with 40–120 μM lysophosphatidylcholine also increased mitochondrial permeability to 75 μM CaCl₂ in a concentration-dependent manner. Interestingly, despite incubation with 80 μM lysophosphatidylcholine, the mitochondrial membrane potential was steady in the presence of succinate, and oxidation rates and respiratory control indices were similar to controls in the presence of succinate, glutamate/malate, and palmitoyl-carnitine. However, mitochondrial oxidation rates were inhibited by 30–50% at 100 μM lysophosphatidylcholine. Finally, while 40 μM lysophosphatidylcholine has no effect on fatty acid oxidation and mitochondria remained impermeable in intact hepatocytes, 100 μM lysophosphatidylcholine inhibited fatty acid stimulated oxidation and caused intracellular mitochondrial permeability. Taken together, these present data demonstrated that LPC concentration dependently modulates mitochondrial microenvironment, with low micromolar concentrations of lysophosphatidylcholine sufficient to change hepatic oxidation rate whereas higher concentrations are required to disrupt mitochondrial integrity.     

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k₅) of the reversible and loose PfENR-EGCG binary complex to a tight [PfENR-EGCG]^∗ or EI^∗ complex was calculated to be 4.0 × 10⁻² s⁻¹. The low dissociation rate constant (k₆) of the [PfENR-EGCG]^∗ complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (K_i^∗) of 7.0 ± 0.8 nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k₆ of the EI∗ complex by 100 times, lowering the overall K_i^∗ of EGCG to 97.5 ± 12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.     

Inhibition of mammalian thioredoxin reductase by black tea and its constituents: Implications for anticancer actions

Black tea is recently reported to have anti-carcinogenic effects through pro-oxidant property, but the underlying mechanisms remain unclear. Mammalian cytosolic thioredoxin reductase (TrxR1) is well -known for its anti-oxidation activity. In this study, we found that black tea extract (BTE) and theaflavins (TFs), the major black tea polyphenols, inhibited the purified TrxR1 with IC₅₀ 44 μg/ml and 21 ± 1 μg/ml, respectively. Kinetics of TFs exhibited a mixed type of competitive and non-competitive inhibition, with K_is 4 ± 1 μg/ml and K_ii 26 ± 5 μg/ml against coenzyme NADPH, and with K_is 12 ± 3 μg/ml and K_ii 27 ± 5 μg/ml against substrate DTNB. In addition, TFs inhibited TrxR1 in a time-dependent manner. In an equilibrium step, a reversible TrxR1-TFs complex (E * I) forms, which is followed by a slow irreversible first-order inactivation step. Rate constant of the inactivation was 0.7 min⁻¹, and dissociation constant of E * I was 51.9 μg/ml. Treatment of NADPH-reduced TrxR1 with TFs decreased 5-(Iodoacetamido) fluorescein incorporation, a fluorescent thiol-reactive reagent, suggesting that Sec/Cys residue(s) in the active site may be involved in the binding of TFs. The inhibitory capacity of TFs depends on their structure. Among the TFs tested, gallated forms had strong inhibitory effects. The interactions between TFs and TrxR1 were investigated by molecular docking, which revealed important features of the binding mechanism of theaflavins. An inhibitory effect of BTE on viability of HeLa cells was observed with IC₅₀ 29 μg/ml. At 33 μg/ml of BTE, TrxR1 activity in HeLa cells was decreased by 73% at 22 h after BTE treatment. TFs inhibited cell viability with IC₅₀ 10 ± 4 μg/ml for HeLa cells and with IC₅₀ 20 ± 5 μg/ml for EAhy926 cells. The cell susceptibility to TFs was inversely correlated to cellular levels of TrxR1. The inhibitory actions of TFs on TrxR1 may be an important mechanism of their anti-cancer properties.     

Inhibition of hepatoma cell invasion beneath mesothelial-cell monolayer by sera from tea- and related component-treated rats and their modes of action

The bioavailability and action of teas on the invasion of a rat ascites hepatoma cell line, AH109A, were determined and their modes of action were by co-culturing the cancer cells with a rat mesentery-derived mesothelial-cell (M-cell) monolayer in the presence of sera from rats orally given teas and their component, (-)-epigallocatechin gallate (EGCG). The rat sera obtained 2 and 5 hr after oral intubation of a low concentration of green, oolong, or black tea, or EGCG significantly inhibited AH109A invasion underneath the M-cell monolayer. These sera showed a time-dependent and significant inhibitory effect on the AH109A invasion. The 2-hr sera and 2.5 μM EDTA in the medium completely eliminated the enhancement of AH109A invasion induced by a reactive oxygen species (ROS)-generating system. These results show that the inhibition of relevant ROS-potentiated invasion of AH109A cells across the M-cell monolayer may be due to the antioxidative action of EGCG, the in vivo metabolites, and tea-induced changes in the endogenous substances. The results suggest that the drinking of tea in daily life may have certain preventive and therapeutic effects against cancer cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Combined effect of epigallocatechin gallate and triclosan on enoyl-ACP reductase of Mycobacterium tuberculosis

Among the various inhibitors known for enoyl-acyl carrier protein (ACP) reductases, triclosan and green tea catechins are two promising candidates. In the present study, we show, for the first time that epigallocatechin gallate (EGCG), a major component of green tea catechins, inhibits InhA, the enoyl-ACP reductase of Mycobacterium tuberculosis with an IC50 of 17.4 μM. EGCG interferes with the binding of NADH to InhA. We also demonstrate that EGCG increased the inhibitory activity of triclosan towards InhA and vice versa. Direct binding assay using [³H]EGCG and fluorescence titration assay support the spectrophotometric/kinetic inhibition data. The biochemical data has been explained by docking simulation studies.