Hirota Fujiki

Green tea: cancer preventive beverage and/or drug

Green tea and (-)-epigallocatechin gallate (EGCG) are now acknowledged cancer preventives in Japan and has made it possible for us to establish the concept of a cancer preventive beverage. For the general population, we recommend 10 cups of green tea daily supplemented with green tea tablets. For cancer patients following treatment, we here present new evidence that green tea and a cancer preventive drug, sulindac, have synergistic preventive effects. An approach to develop green tea capsules as a cancer preventive drug in the US is discussed, aiming at taking full advantage of this cancer preventive beverage.

DNA and RNA as New Binding Targets of Green Tea Catechins

The significance of catechins, the main constituent of green tea, is being increasingly recognized with regard to cancer prevention. Catechins have been studied for interactions with various proteins, but the mechanisms of the various catechins are not yet elucidated. Based on our previous observation that nucleic acids extracted from catechin-treated cells are colored, we studied whether catechins directly interact with nucleic acids using surface plasmon resonance assay (Biacore) and cold spray ionization-mass spectrometry. These two methods clearly showed that (-)-epigallocatechin gallate (EGCG) binds to both DNA and RNA molecules: the Biacore assay indicated that four catechins bound to DNA oligomers, and cold spray ionization-mass spectrometry analysis showed one to three EGCG molecules bound to single strand 18 mers of DNA and RNA. Moreover, one or two molecules of EGCG bound to double-stranded (AG-CT) oligomers of various nucleotide lengths. These results suggest that multiple binding sites of EGCG are present in DNA and RNA oligomers. Double-stranded DNA (dsDNA) oligomers were detected only as EGCG-bound forms at high temperature, whereas at low temperature both the free and bound forms were detected, suggesting that EGCG protects dsDNA oligomers from dsDNA melting to single-stranded DNA. Because both galloyl and catechol groups of EGCG are essential for DNA binding, both groups seem to hold strands of DNA via their branching structure. These findings reveal for the first time the link between catechins and polynucleotides and will intensify our understanding of the effects of catechins on DNA in terms of cancer prevention.

New cancer treatment strategy using combination of green tea catechins and anticancer drugs

Green tea is now recognized as the most effective cancer preventive beverage. In one study, 10 Japanese‐size cups of green tea daily supplemented with tablets of green tea extract limited the recurrence of colorectal polyps in humans to 50%. Thus, cancer patients who consume green tea and take anticancer drugs will have double prevention. We studied the effects of combining (−)‐epigallocatechin gallate (EGCG) and anticancer drugs, focusing on inhibition of cell growth and induction of apoptosis. Numerous anticancer drugs, such as tamoxifen, COX‐2 inhibitors, and retinoids were used for the experiments, and the combination of EGCG and COX‐2 inhibitors consistently induced the enhancement of apoptosis. To study the mechanism of the enhancement, we paid special attention to the enhanced expressions of DDIT3 (growth arrest and DNA damage‐inducible 153, GADD153), GADD45A, and CDKN1A (p21/WAF1/CIP1) genes, based on our previous evidence that a combination of EGCG and sulindac specifically induced upregulated expression of GADD153 and p21 genes in PC‐9 lung cancer cells. The synergistic enhancements of apoptosis and GADD153 gene expression in human non‐small cell lung cancer cells by the combination of EGCG and celecoxib were mediated through the activation of the MAPK signaling pathway. This article reviews the synergistic enhancement of apoptosis, gene expression, and anticancer effects using various combinations of EGCG and anticancer drugs, including the combination of (−)‐epicatechin (EC) and curcumin. Based on the evidence, we present a new concept: green tea catechins as synergists with anticancer drugs. (Cancer Sci 2011; 102: 317–323)

New TNF-α releasing inhibitors as cancer preventive agents from traditional herbal medicine and combination cancer prevention study with EGCG and sulindac or tamoxifen

Herbal medicines are now attracting attention as potential sources of cancer preventive agents. Using inhibition of tumor necrosis factor-alpha (TNF-alpha) release assay, we studied Acer nikoense, Megusurino-ki in Japanese. Inhibitory potential was found in the leaf extract, and the main active principles were identified as geraniin and corilagin. The IC(50) values for TNF-alpha release inhibition were 43 microM for geraniin and 76 microM for corilagin, whereas that for (-)-epigallocatechin gallate (EGCG), the green tea polyphenol, as control was 26 microM. Furthermore, treatment with geraniin inhibited okadaic acid tumor promotion in a two-stage carcinogenesis experiment on mouse skin. Geraniin and corilagin are present in another well-known Japanese traditional herb, Geranium thunbergii, Genno-shoko in Japanese. Considering seasonal variations of the agents and sites of cultivation of herbs, this paper reviews the significance of geraniin as a new cancer preventive agent. In addition, based on accumulated results of green tea as a cancer preventive, we review two important results with EGCG: the synergistic effects of EGCG with sulindac or tamoxifen on cancer preventive activity in PC-9 cells, and cancer prevention of intestinal tumor development in multiple intestinal neoplasia (Min) mice by cotreatment using EGCG with sulindac. We report here new findings on additional gene expression resulting from cotreatment with EGCG and sulindac in PC-9 cells compared with gene expression by EGCG alone or sulindac alone. Overall, our results indicate that, with the continuing spread of cancer chemoprevention as a fundamental medical strategy, both clinicians and researchers should take a closer look at herbal medicine.

Higher cell stiffness indicating lower metastatic potential in B16 melanoma cell variants and in (−)-epigallocatechin gallate-treated cells

PurposeTo understand how nanomechanical stiffness affects metastatic potential, we studied the relationship between cell migration, a characteristic of metastasis, and cell stiffness using atomic force microscopy (AFM), which can measure stiffness (elasticity) of individual living cells.MethodsMigration and cell stiffness of three metastatic B16 melanoma variants (B16-F10, B16-BL6, and B16-F1 cells), and also effects of (−)-epigallocatechin gallate (EGCG), were studied using Transwell assay and AFM.ResultsMigration of B16-F10 and B16-BL6 cells was 3 and 2xa0times higher than that of B16-F1 cells in Transwell assay, and cell stiffness determined by AFM was also different among the three variants, although they have similar morphologies and the same growth rates: Means of Young’s modulus were 350.8xa0±xa04.8xa0Pa for B16-F10 cells, 661.9xa0±xa016.5xa0Pa for B16-BL6 cells, and 727.2xa0±xa013.0xa0Pa for B16-F1 cells. AFM measurements revealed that highly motile B16-F10 cells have low cell stiffness, and low motile and metastatic B16-F1 cells have high cell stiffness: Nanomechanical stiffness is inversely correlated with migration potential. Treatment of highly motile B16-F10 cells with EGCG increased cell stiffness 2-fold and inhibited migration of the cells.ConclusionsOur study with AFM clearly demonstrates that cell stiffness is a reliable quantitative indicator of migration potential, and very likely metastatic potential, even in morphologically similar cells. And increased cell stiffness may be a key nanomechanical feature in inhibition of metastasis.

TNF-α-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells

TNF‐α inducing protein (Tipα) is secreted from Helicobacter pylori (H. pylori): it is a potent inducer of TNF‐α and chemokine genes, mediated through NF‐κB activation, and it also induces tumor‐promoting activity in Bhas 42 cells. To investigate the carcinogenic mechanisms of H. pylori with Tipα, we first examined how Tipα acts on gastric epithelial cells. We found that fluorescent‐Tipα specifically bound to, and then entered, the cells in a dose‐ and temperature‐dependent manner, whereas deletion mutant of Tipα (del‐Tipα), an inactive form, neither bound to nor entered the cells, suggesting the presence of a specific binding molecule. Mutagenesis analysis of Tipα revealed that a dimer formation of Tipα with a disulfide bond is required for both specific binding and induction of TNF‐α gene expression. A confocal laser scanning microscope revealed some Tipα in the nuclei, but del‐Tipα was not present, which indicated that an active form of Tipα can penetrate the nucleus and may be involved in the induction of TNF‐α gene expression. Examination of Tipα production and secretion in 28 clinical isolates revealed that H. pylori obtained from gastric cancer patients secreted Tipα in significantly higher amounts than did H. pylori from patients with chronic gastritis, suggesting that Tipα is an essential factor in H. pylori inflammation and cancer microenvironment in the human stomach. Tipα is thus a new carcinogenic factor of H. pylori that can enter the nucleus through a specific binding molecule, and its mechanism of action is completely different from that of CagA.

Green tea polyphenol stimulates cancer preventive effects of celecoxib in human lung cancer cells by upregulation of GADD153 gene

To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (−)‐epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase‐2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage‐inducible 153 (GADD153) gene, in human lung cancer cell line PC‐9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC‐9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21WAF1 and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC‐9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K‐1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC‐9 cells in combination with N‐(4‐hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen‐activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC‐9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG.

New tumor necrosis factor-α-inducing protein released from Helicobacter pylori for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor-α (TNF-α)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF-α-inducing protein (Tipα) gene from genome sequence of H. pylori strain 26695. Using recombinant Tipα (rTipα) and deleted Tipα (rdel-Tipα) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF-α gene expression and NF-κB activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tipα protein as a homodimer form (38xa0kDa) was found in both extracts and culture medium of various H. pylori strains. rTipα significantly induced TNF-α gene expression and NF-κB activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tipα did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-κB p65, and abrogated TNF-α induction induced by Tipα protein.Conclusion Tipα is a new carcinogenic factor released from H. pylori mediated through NF-κB activation.

Carcinogenic Aspects of Protein Phosphatase 1 and 2A Inhibitors

Okadaic acid is functionally a potent tumor promoter working through inhibition of protein phosphatases 1 and 2A (PP1 and PP2A), resulting in sustained phosphorylation of proteins in cells. The mechanism of tumor promotion with okadaic acid is thus completely different from that of the classic tumor promoter phorbol ester. Other potent inhibitors of PP1 and PP2A - such as dinophysistoxin-1, calyculins A-H, microcystin-LR and its derivatives, and nodularin - were isolated from marine organisms, and their structural features including the crystal structure of the PP1-inhibitor complex, tumor promoting activities, and biochemical and biological effects, are here reviewed. The compounds induced tumor promoting activity in three different organs, including mouse skin, rat glandular stomach and rat liver, initiated with three different carcinogens. The results indicate that inhibition of PP1 and PP2A is a general mechanism of tumor promotion applicable to various organs. This study supports the concept of endogenous tumor promoters in human cancer development.

Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori

PurposeTumor necrosis factor-α inducing protein (Tipα) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipα specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-α and chemokine genes mediated through NF-κB activation, resulting in tumor development. To elucidate mechanism of action of Tipα, we studied a binding protein of Tipα in gastric epithelial cells.MethodsTipα binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipα into the cells was determined by Western blotting and expression of TNF-α gene was quantified by RT-PCR.ResultsNucleolin was co-precipitated with Tipα-FLAG, but not with del-Tipα-FLAG (an inactive mutant). After treatment with Tipα-FLAG, incorporated Tipα was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipα to recombinant His-tagged nucleolin fragment (284–710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipα-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipα and expression of TNF-α gene.ConclusionsAll the results indicate that nucleolin acts as a receptor for Tipα and shuttles Tipα from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipα.