Epigallocatechin-3-O-gallate inhibits fibroblast contraction of floating collagen gel: interaction between epigallocatechin-3-O-gallate and platelet derived growth factor

The effects of (-)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts were investigated. EGCG inhibited collagen gel contraction dose-dependently. On the basis of the fact that platelet-derived growth factor (PDGF) is one of the serum components with stimulatory activity in collagen gel contraction, we examined the possibility that interaction between EGCG and PDGF may be involved in this inhibition mechanism. We confirmed this by recombinant PDGF-BB in the present system and we found that EGCG inhibited PDGF-stimulated collagen gel contraction. The results of affinity chromatography indicated that PDGF was bound by EGCG immobilized on agarose gel as detected by enzyme-linked immunoassay and Western blotting. These findings suggest that binding of EGCG to PDGF is at least partly involved in the mechanism of inhibition of collagen gel contraction by EGCG.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.     

Time-dependent intracellular trafficking of FITC-conjugated epigallocatechin-3-O-gallate in L-929 cells

Many in vitro studies about green tea polyphenol, (-)-epigallocatechin-3-O-gallate (EGCG) focused on its pro-apoptotic and anti-proliferative effects on various types of cancer cells, while less attention has been paid to its incorporation into the cytoplasm and nuclear translocation. This study concentrated on the time-dependent intracellular trafficking of EGCG in L-929 cells. EGCG was conjugated with fluorescein-4-isothiocyanate (FITC) via the 3'-OH or 5'-OH group, as confirmed by NMR analysis, and then treated to either suspended or cultured cells. Confocal microscopic observations revealed that FITC-EGCG was clearly seen onto the membrane of suspended cells as well as into the cytoplasm and nucleus within 1h. As an increase in treatment time, it concentrated on the nucleus and then was located at any places of the cells. The cellular uptake of FITC-EGCG in cultured cells was not observed until 1h of culture, but started to be observed after at least 2h. These results imply that although the cellular sensitivity and response to EGCG would be different from those of FITC-EGCG, it would be incorporated into the cytoplasm of cells and further be translocated into the nucleus in a time-dependent manner.     

A lipid raft-associated 67kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression

(-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcepsilonRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcepsilonRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcepsilonRI-suppressive action of EGCG.     

Inhibitory effects of epigallocatechin-3-O-gallate on serum-stimulated rat aortic smooth muscle cells via nuclear factor-kappaB down-modulation

The abnormal growth of vascular smooth muscle cells (VSMCs) plays an important role in vascular diseases, including atherosclerosis and restenosis after angioplasty. Although (-)-epigallocatechin-3-O-gallate (EGCG) has antiproliferative effects on various cells, relatively a little is known about precise mechanisms of the inhibitory effects of EGCG on SMCs. In this study, the inhibitory effects of EGCG on attachment, proliferation, migration, and cell cycle of rat aortic SMCs (RASMCs) with serum stimulation were investigated. Also, the involvement of nuclear factor-kappaB (NF-kappaB) during these inhibitions by EGCG was examined. EGCG treatment resulted in significant (p<0.05) inhibition in attachment and proliferation of RASMCs induced by serum. While non-treated RASMCs migrated into denuded area in response to serum and showed essentially complete closure after 36 h, EGCG-treated cells covered only 31% of the area even after 48 h of incubation. Furthermore, EGCG treatment resulted in an appreciable cell cycle arrest at both G0/G1- and G2/M-phases. The immunoblot analysis revealed that the constitutive expression of NF-kappaB/p65 nuclear protein in RASMCs was lowered by EGCG in both the cytosol and the nucleus in a dose-dependent manner. These results suggest that the EGCG-caused inhibitory effects on RASMCs may be mediated through NF-kappaB down-modulation.     

Nonchelational cell growth inhibition by EDTA

The ability of the dinitrilotetraacetates EDTA, CDTA, and EGTA to inhibit the growth of rat C6 glioma cells was not proportional to their chelational stability constants, suggesting a nonchelational mechanism of action. Ion antagonization studies supported this hypothesis. Growth inhibition did not appear to be caused by an EDTA-ion coordination complex. The chemical structure of the DNTAs suggests that they might act by: increasing zeta potential electronegativity and altering local physical properties; macromolecular crosslinking; and disrupting hydrogen bonds and hydrophobic interactions. Interestingly, their ability to inhibit growth paralleled their hydrophobic surface areas.     

Modulation of the cell cycle during reversible growth inhibition of 3T3 and SV40-3T3 cells with succinylated concanavalin A

Succinylated concanavalin A (ConA), a non-toxic, non-agglutinating derivative of the jack bean lectin ConA inhibits the growth of both normal and SV40-transformed 3T3 cells. The quantitative but not the qualitative growth inhibitory effect of succinyl-ConA can be modulated by the composition of the growth medium. Succinyl-ConA inhibited cells show a low rate of DNA synthesis and accumulate in the G0/G1 phase of the cell cycle. Upon removal of the succinyl-ConA, inhibited cells re-enter the cell cycle synchronously.     

Medulloblastoma cell invasion is inhibited by green tea (-)epigallocatechin-3-gallate

Epigallocatechin-3-gallate (EGCG), the major green tea polyphenol, can reach the brain following oral intake and could thus act as an anti-tumoral agent targeting several key steps of brain cancer cells invasive activity. Because integrin-mediated extracellular matrix recognition is crucial during the cell adhesion processes involved in carcinogenesis, we have investigated the effects of EGCG on different cellular integrins of the pediatric brain tumor-derived medulloblastoma cell line DAOY. Using flow cytometry, we report the levels of expression of several cell surface integrins in DAOY. These include high expression of alpha2, alpha3, and beta1 integrins, as well as alphav and beta3 integrins. Moreover, we provide evidence that EGCG can antagonize DAOY cell migration specifically on collagen by increasing cell adhesive ability through specific gene and protein upregulation of the beta1 integrin subunit. Our results suggest that this naturally occurring green tea polyphenol may thus be used as a nutraceutical therapeutic agent in targeting the invasive character of medulloblastomas.     

Human hair growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG)

Green tea is a popular worldwide beverage, and its potential beneficial effects such as anti-cancer and anti-oxidant properties are believed to be mediated by epigallocatechin-3-gallate (EGCG), a major constituent of polyphenols. Recently, it was reported that EGCG might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5alpha-reductase activity. However, no report has been issued to date on the effect of EGCG on human hair growth. This study was undertaken to measure the effect of EGCG on hair growth in vitro and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. EGCG promoted hair growth in hair follicles ex vivo culture and the proliferation of cultured DPCs. The growth stimulation of DPCs by EGCG in vitro may be mediated through the upregulations of phosphorylated Erk and Akt and by an increase in the ratio of Bcl-2/Bax ratio. Similar results were also obtained in in vivo dermal papillae of human scalps. Thus, we suggest that EGCG stimulates human hair growth through these dual proliferative and anti-apoptotic effects on DPCs.     

Studies of reversible inhibition,irreversible inhibition,and activation of alkaline phosphatase by capillary electrophoresis

Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were based on electrophoretic mixing of inhibitor and enzyme zones in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined, and reversible, noncompetitive inhibition by theophylline has been studied. The K(i) values determined for these reversible inhibitors using capillary electrophoresis are within the range of values reported in the literature for the same enzyme-inhibitor combinations. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0mM and above has been observed. Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400 microM.     

Osteosarcoma cell line growth inhibition by zoledronate-stimulated effector cells

Bisphosphonates have a profound effect on bone resorption and are widely used in the treatment of osteoclast-mediated bone diseases. Zoledronic acid (ZA), a third-generation biphosphonate, has a potent antitumor activity and expands gammadelta (γδ) T cells endowed of major histocompatibility complex-unrestricted lytic activity. Many solid tumors express tumor-specific antigens on their surface, representing targets for immune effector T cells. Nevertheless, the immune surveillance against clinically manifested tumors is relatively inefficient. Therefore, we investigated the hitherto unknown effects of ZA activated γδ T cells of normal donors on osteosarcoma cell lines. γδ T cells were stimulated with ZA and low doses of interleukin-2, and then analyzed for proliferation and generation of effector activity against osteosarcoma cell lines. Our results show the potent anti-tumor activity of ZA-stimulated γδ T cells and the enhanced immunosensitivity of osteosarcoma cell lines to γδ T cells suggesting that osteosarcoma is another γδ T cell susceptible tumor type.     

Effect of ras-gene transformation on the inhibition of NIH3T3 cell growth by pertussis toxin

To investigate the relationship between the effects of a pertussis toxin-inhibitable class of G-proteins and the ras family of protooncogenes on cell growth, we isolated multiple cell lines transformed by oncogenic Hras or Nras genes and measured the ability of pertussis toxin to inhibit their growth rate. Although all of the cell lines were morphologically transformed and could grow in agar suspension, there was considerable variability in their resistance to pertussis toxin, ranging from cell lines completely resistant to pertussis toxin to cell lines as sensitive to pertussis toxin as the parental cells from which they derived. For those lines resistant to pertussis toxin, this resistance is not due to an inability of pertussis toxin to reach or react with its intracellular target; pertussis toxin could be shown to ADP-ribosylate the endogenous G-proteins of all lines tested regardless of whether it affected their growth rate. There was a strong correlation between the level of active ras protein expressed in the different lines and the degree of resistance to pertussis toxin (r = 0.80). Although the Hras-transformed cell lines were more resistant to pertussis toxin as a group than the Nras-transformed cell lines, we believe that this is not a primary difference between Nras and Hras, but, rather, is due to a higher average level of expression of ras in the cell lines receiving Hras. We suggest that the consequences of ras transformation vary with the concentration of oncogenic ras present in the cell, and that different assays or different properties of transformation show different sensitivities to the level of ras expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition of malignant cell growth by ketone bodies

The effect of ketone bodies on the growth, in culture, of transformed lymphoblasts (Raji cells) was investigated. Cell growth was inhibited and this effect was reversible, non-toxic, and proportional to the concentration of D-beta-hydroxybutyrate up to 20mM. The total glucose utilisation and the total lactate production were reduced in proportion to the inhibition of cell proliferation. D-beta-hydroxybutyrate was not metabolised by the cells. Other glycolytic inhibitors and chemical analogues of D-beta-hydroxybutyrate either did not inhibit or proved to be too toxic for cell growth. D-beta-hydroxybutyrate also inhibited the growth of rabbit kidney (RK13), HeLa, mouse melanoma (B16), fibroblast and trypsin-dispersed human thyroid and beef testis cells. Moreover, in vivo dietary-induced ketosis reduced the number of B16 melanoma deposits in the lungs of C57BL/6 mice by two-thirds. The significance of these results in the clinical management of cancer cachexia is discussed.     

Phenotypes of HeLa S3 variant cell lines resistant to growth inhibition by sodium butyrate

Summary HeLa cell variants capable of multiplying in the presence of sodium butyrate were used to study the relationship of cell cycle position to human chorionic gonadotropin (hCG) production and regulation of the genes encoding hCG α- and β-subunits. The butyrate-resistant variants exhibit several different stable phenotypes. In wild-type HeLa cells, butyrate arrests cell division and modulates synthesis of α- and β-subunits of glycoprotein hormones by coordinately regulating steady-state levels of their respective mRNAs. Because the variant cell lines replicate, in addition to producing hCG subunits in the presence of butyrate, cell cycle arrest does not seem to be a requirement for expression of glycoprotein hormone genes. Studies of histone modification suggest that neither hyperacetylation of histones H3 and H4 nor dephosphorylation of histones H1 and H2A mediates inhibition of cell replication. In the variants, α-subunit and hCGβ levels are independently regulated, as a consequence of independent regulation of α- and β-hCG mRNA levels. Long-term effects of butyrate include derepression of some genes (hCGβ in the variant AO) and repression of others (hCGα in variant AO). Moreover, hormone production correlates with the steady-state levels of mRNA for each of the subunits, suggesting that regulation occurs before translation. These findings indicate that the butyrate-resistant variant cell lines are valuable for studies of the molecular mechanisms involved in regulation of expression of ectopic hormones.     

Metabolic changes during cell growth inhibition by p27 overexpression

The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.