(−)-Epigallocatechin-3-gallate inhibits Met signaling, proliferation, and invasiveness in human colon cancer cells

The Met receptor tyrosine kinase is deregulated in a variety of cancers and is correlated with advanced stage and poor prognosis. Thus, Met has been identified as an attractive candidate for targeted therapy. We compared the tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) and a specific Met inhibitor, SU11274, as suppressing agents of Met signaling in HCT116 human colon cancer cells. Treatment with hepatocyte growth factor increased phospho-Met levels, and this was inhibited in a concentration-dependent manner by EGCG and SU11274 (IC₅₀ 3.0 vs. 0.05 μM, respectively). Downstream activation of Erk and Akt signaling pathways also was suppressed. Both compounds at a concentration of 5 μM lowered cell viability and proliferation, with EGCG being more effective than SU11274, and the invasion of colon cancer cells in Matrigel assays was strongly inhibited. These findings are discussed in the context of the pleiotropic effects of tea catechins, their tissue metabolite levels, and the potential to inhibit colon cancer metastasis and invasion.     

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.     

FAPP2 promotes tumor cell growth in human colon cancer through activation of Wnt signaling

Human phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2) is well-known to function as a cytoplasmic lipid transfer protein during vesicle maturation. However, the expression and role of FAPP2 in tumor remain elusive. In this study, data from immunohistochemical assays displayed that FAPP2 was remarkably upregulated (57.8%) in 90 cases of colon cancer samples in contrast to their corresponding adjacent tissues. Disruption of FAPP2 by CRISPR/Cas9 technique in colon cancer cells led to an attenuated effect on cell growth analyzed by CCK8 and colony formation assays. Meanwhile, the tumorigenicity of FAPP2 downregulated cells also decreased in nude mice model. Accordantly, CCK8 assays also indicated that FAPP2 overexpression could promote colon cancer cell growth. In addition, dual luciferase reporter assays and western blot analyses revealed that Wnt/β-catenin signaling was involved in the FAPP2-regulated tumor cell growth. These findings suggest that FAPP2 could act as an oncogene in the regulation of tumor growth and may provide a new therapeutic target for human colon cancer.     

EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Apoptosis induced by activation of peroxisome-proliferator activated receptor-gamma is associated with Bcl-2 and NF-kappaB in human colon cancer

Peroxisome-proliferator activated receptor-gamma (PPARgamma) has been demonstrated to exert an inhibitory effect on cell growth in most cell types studied, but its role in colon cancer is still uncertain. The molecular mechanism between the activation of PPARgamma and its consequence is unknown. In the present report, we show that the expression of PPARgamma was significantly increased in tumor tissues from human colon cancer compared with non-tumor tissues and that PPARgamma ligands, 15-Deoxy-delta(12,14)prostaglandin J2 or ciglitizone, induced apoptosis in HT-29 cells, a human colon cancer cell line. The occurrence of apoptosis induced by PPARgamma ligands was sequentially accompanied by reduced levels of NF-kappaB and Bcl-2. Over-expression of Bcl-2 significantly protected the cells from apoptosis. This study suggested that a PPARgamma-Bcl-2 feedback loop may function to control the life-death continuum in colonic cells and that a deficiency in generation of PPARgamma ligands may precede the development of human colon cancer.     

NSAIDs activate PTEN and other phosphatases in human colon cancer cells: novel mechanism for chemopreventive action of NSAIDs

Studies on chemoprevention of colorectal cancer have generated increasing interest. The mechanisms involved in NSAIDs chemopreventive action are not fully elucidated. In this study, we examined in human colon cancer cells the effect of indomethacin and NS-398 (a pre-clinical selective COX-2 inhibitor) on expression of 96 genes of the EGF/PDGF signaling pathways essential for cell proliferation, migration, and survival. We found that indomethacin and NS-398 treatment significantly upregulated expression of the tumor suppressor gene, PTEN, the MAP kinase phosphatase-3, MKP-3, and the protein tyrosine phosphatase, SHP2. Additionally, NS-398 treatment increased expression of apoptotic genes such as BAD, STAT1, and CASP3. These results suggest that as a consequence of increased expression of phosphatases such as PTEN and the resulting dephosphorylation of kinases, NSAIDs can negatively regulate the EGF/PDGF pathways in colon cancer cells-a novel mechanism for NSAIDs' chemopreventive actions.     

Enhancement of (−)-epigallocatechin-3-gallate and theaflavin-3-3′-digallate induced apoptosis by ascorbic acid in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells via MAPK pathways

Tea polyphenols (−)-epigallocatechin-3-gallate (EGCG) and theaflavin-3-3′-digallate (TF₃) are two prospective compounds in cancer prevention and treatment. Ascorbic acid (Vc) is essential to a healthy diet as well as being a highly effective antioxidant. In this work, the effects of the combination of EGCG or TF₃ with Vc on the apoptosis and caspases-3/9 activities in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells were determined. Furthermore, the role of mitogen-activated protein kinases (MAPK) pathways in the apoptosis induced by TF₃ or EGCG together with Vc were studied using three MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580). Our results showed that Vc could enhance the EGCG and TF₃ induced apoptosis in SPC-A-1 and Eca-109 cells, and this effect involved the activation of caspase-3 and 9. EGCG, TF₃ and Vc could activate MAPK pathways respectively, and each compound activated different MAPK subfamilies in different cells. This may explain the enhancement of EGCG and TF₃ induced apoptosis by Vc in SPC-A-1 and Eca-109 cells, and will ultimately aid the design of more effective anti-cancer treatments.     

A component of green tea, (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K/Akt pathway and Bcl-2 family proteins

Bladder cancer is the fourth most common cancer in men and ninth most common in women. It has a protracted course of progression and is thus an ideal candidate for chemoprevention strategies and trials. This study was conducted to evaluate the chemopreventive/antiproliferative potential of (-)-epigallocatechin gallate (EGCG, the major phytochemical in green tea) against bladder cancer and its mechanism of action. Using the T24 human bladder cancer cell line, we found that EGCG treatment caused dose- and time-dependent inhibition of cellular proliferation and cell viability, and induced apoptosis. Mechanistically, EGCG inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that EGCG may be an important chemoprevention agent for the management of bladder cancer.     

Inhibition of beta-catenin/Tcf activity by white tea,green tea,and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations

Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in white tea and green tea. Recently, it was reported that the addition of EGCG and other tea polyphenols to cell culture media, minus cells, generated significant levels of H(2)O(2), with the corollary that this might represent an "artifact" in cell culture studies which seek to examine the chemopreventive mechanisms of tea. We show here that in cell growth media with and without serum, and in growth media containing human embryonic kidney 293 (HEK293) cells plus serum, physiologically relevant concentrations of EGCG (< or =25 microM) generated H(2)O(2) with a peak concentration of the order of 10-12 microM. However, addition of 20 microM H(2)O(2) directly to HEK293 cells transiently transfected with wild-type or mutant beta-catenin constructs and TCF-4 had no significant effect on beta-catenin/TCF-4 reporter activity or beta-catenin expression levels. In contrast, 2-25 microM EGCG inhibited beta-catenin/TCF-4 reporter activity in a concentration-dependent fashion and there was a concomitant reduction in beta-catenin protein levels in the cell lysates without changes in TCF-4 expression. The inhibition of reporter activity was recapitulated by white tea and green tea, each tested at a 25 microM EGCG equivalent concentration in the assay, and this was unaffected by the addition of exogenous catalase. The results indicate that physiologically relevant concentrations of tea and EGCG inhibit beta-catenin/TCF-4 reporter activity in HEK293 cells due to reduced expression of beta-catenin and that this is unlikely to be an artifact of H(2)O(2) generation under the assay conditions used here. These data are consistent with the findings from in vivo studies, showing the suppression of intestinal polyps by tea, via an apparent down-regulation of beta-catenin and Wnt target genes.     

Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

(−)-Epigallocatechin-3-gallate induces up-regulation of Th1 and Th2 cytokine genes in Jurkat T cells

In the present study, we found that (−)-epigallocatechin-3-gallate (EGCG) significantly up-regulated the mRNA expression of the Th1/Th2 cytokines including IL-2, IFN-γ, IL-5 and IL-13 in Jurkat T cells. The EGCG-induced mRNA up-regulation of IL-2 and IL-5 was predominantly affected by the extracellular signal-regulated protein kinase (ERK) signalling, whereas IL-13 gene expression, the most responsive to the EGCG treatment, was dependent on neither ERK nor c-jun NH₂-terminal kinase (JNK) signalling. IFN-γ gene expression was partially mitigated by both inhibitors of the ERK and JNK pathways. Furthermore, catalase significantly attenuated the intracellular peroxide production, phosphorylation of ERK and JNK, and all cytokine gene expressions induced by EGCG. In addition, physiologically relevant concentrations of both EGCG and H₂O₂-induced up-regulation of IL-5 gene expression. Our findings provide biological evidence that EGCG induces Th1/Th2 cytokine mRNA expression via H₂O₂ production followed by activation of ERK or JNK in Jurkat T cells.     

Enhanced autophagy plays a cardinal role in mitochondrial dysfunction in type 2 diabetic Goto-Kakizaki (GK) rats: ameliorating effects of (-)-epigallocatechin-3-gallate

Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model--the Goto-Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (-)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.