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Enhancement of neutral endopeptidase activity in SK-N-SH cells by green tea extract.


Green tea extract (EFLA [R]85942) is able to induce specifically the neutral endopeptidase (NEP) activity and to inhibit the proliferation of SK-N-SH cells; the angiotensin-converting enzyme (ACE) activity is not influenced under the same conditions. The treatment of the cells with arabinosylcytosine and green tea extract results in a strong enhancement of cellular NEP activity whereas cellular ACE activity was not changed significantly, indicating a green tea extract-specific regulation of NEP expression. Because of its role in the degradation of amyloid beta peptides this enzyme induction of NEP by long term treatment with green tea extract may have a beneficial effect regarding the prevention of forming amyloid plaques.

Key words: green tea, neutral endopeptidase, angiotensin-converting enzyme, enzyme induction

* Introduction

Green tea is one of the most frequently consumed popular beverages in the world. Epidemiological studies have associated the consumption of green tea with a lower risk of several types of cancers (Weisburger and Chung, 2002) and green tea also shows a neuroprotective effect (Hong et al. 2000). In the last years a lot of investigations were performed to clarify the mechanisms responsible for the pharmacological efficiency. Beside studies to investigate direct and acute effects of green tea and its constituents on cellular systems also experiments were done to elucidate the role of green tea in regulation of gene expression and enzyme induction (Embola et al. 2002, Chen et al. 2000). These investigations are focused on the long term effects of green tea which seem to play an important role in its protective activity. The regulation of expression of enzymes included in the metabolism of neuropeptides by daily intake of green tea might give an answer to the question why the constant use of this beverage has beneficial effects.

In our experiments we investigated two zinc-endopeptidases, neutral endopeptidase (EC, NEP) and angiotensin-converting enzyme (EC, ACE), acting as ectoenzymes on the outer surface of different cells and included in the metabolism of regulatory peptides, like substance P, bradykinin, enkephalins, natriuretic peptides and other vasoactive hormonal peptides (Hooper, 1996). In order to see if green tea influences the expression of neuropeptidases we used the neuroblastoma cell line SK-N-SH and studied changes in the specific cellular enzyme activity of NEP and ACE after long term treatment with green tea extract.

* Materials and Methods


L-Leucine-p-nitroanilide, Suc-L-Ala-L-Ala-L-Phe-7-amido-3-methylcoumarin (SAAP-AMC), Aminopeptidase N (APN), arabinosylcytosine, epigallocatechin gallate and phosphoramidon were obtained from Sigma; Hip-L-His-L-Leu was purchased from Bachem; lisinopril was a gift of Schering & Plough (USA); the cell culture media and fetal calf serum (FCS) were obtained from Biochrom; green tea extract (EFLA[R]85942) was obtained from Emil Flachsmann AG (Switzerland). This special extract from Camellia sinensis Kunt. contains according to the specification of the manufacturer 47.5-52.5% polyphenols (EGCG app. 61%), 5-10% caffeine and 0.3-1.2% theobromine analysed by HPLC.

Cell culture

SK-N-SH cells, human neuroblastoma cells, were obtained from ATCC (No. HTB-11) and cultivated in Minimal Essential Medium (MEM with Earls salts) with sodium pyruvat and non-essential amino acids plus 10% foetal calf serum at 37 [degrees]C in a humidified atmosphere with 5% C[O.sub.2] according to ATCC instruction manual (Rockville, USA). Subcultivation was performed in 70 [cm.sup.2] culture flasks until confluence. Then cells were seeded for the enzymatic experiments in 24-well plates (inoculum 100.000 cells/well).

For investigating the acute effect of test compound the cells were cultivated for 5 days until confluence in the 24-well plates. The medium was then removed and replaced by NEP-or ACE-assay solution plus the compound to be tested.

For the long term experiments, 24 h after plating, the cells were incubated with the indicated concentration of the test compound and cultivated for further 4 days. Then the medium was removed and replaced by NEP- or ACE-assay solution.

Determination of ectoenzyme activities

* NEP activity: The assay was performed according to Bormann and Melzig (2000). Briefly, 50 [micro]l of SAAP-AMC-solution (50 [micro]M) and 400 [micro]l of HEPES-buffer (50 mM + 154 mM NaCl, pH 7,4) were added to the intact cell layer after having removed the growth medium. To investigate the acute inhibitory activity of a test substance this compound was simultaneously added. The cells with the assay solution were incubated for 60 min at 37 [degrees]C. The NEP reaction was stopped by addition of 50 [micro]l phosphoramidon (50 [micro]M). 400 [micro]l of the incubation mixture of each well were transferred in an Eppendorf tube, and 20 [micro]l of an APN-solution (1:235 diluted with water) were added and incubated again for 60 min at 56 [degrees]C. The reaction was terminated by addition of 800 [micro]l acetone. The fluorescence of the released AMC was measured (excitation 367 nm, emission 440 nm). A calibration curve with AMC was determined to calculate the enzyme activity.

* ACE activity: The assay was performed according to Bormann and Melzig (2000). Briefly, 20 [micro]l substrate solution (24 mM Hip-L-His-L-Leu in water) plus 260 [micro]l phosphate buffer (83 mM [K.sub.2]HP[O.sub.4] x 3 [H.sub.2]O + 326 mM NaCl, pH 8.3) were added to the intact cell layer after having removed the growth medium. To investigate the acute inhibitory activity of a test substance this compound was simultaneously added. The cells with the assay solution were incubated for 20 min at 37 [degrees]C. 250 [micro]l of the incubation mixture of each well were transferred in an Eppendorf tube and the reaction was stopped by addition of 1 ml 0.4 N NaOH plus 100 [micro]l ophthalaldehyde solution (2% in methanol, fresh prepared). Under exclusion of light this mixture was incubated for 10 min at room temperature and terminated by addition of 300 [micro]l 2 N HCl. The fluorescence of the formed product was measured (excitation 360 nm, emission 500 nm). A calibration curve with L-His-L-Leu was determined to calculate the enzyme activity.

The inhibition rates were calculated in the enzymatic assays in comparison to controls without inhibitor during the enzymatic reaction, considering the absorbance of fluorescence light by test compounds. I[C.sub.50]-values were determined from dose-effect curves by linear regression. The enzyme activities were calculated in nmol/min per 100.000 cells for ACE and NEE

Cell counting

The cells were dissociated with trypsin/EDTA (0.25%/0.02%) and counted with the cell analyzer system CASY (SCHARFE System, Germany). The cell number values represent the mean of at least three independent experiments with two parallel samples.


All values in the tables and figures are expressed as mean [+ or -] standard error of the mean of at least 3 independent experiments with 4 to 5 parallel samples. Wilcoxon's U-test was used to test significance (p < 0.05).

* Results

Effects of green tea extract on cellular enzyme activity

On their cell surface SK-N-SH cells express NEP and ACE as ecto-enzymes actively degrading a variety of neuropeptides (Melzig and Escher, 2002). We measured a basal activity of 10.3 [+ or -] 3 nmol/min per [10.sup.5] cells for ACE (n = 8) and 22 [+ or -] 7 nmol/min per [10.sup.5] cells for NEP (n = 17), respectively.

The long term incubation of the cells with rather low concentrations of green tea extract induced the specific cellular NEP activity (Table 1) and did not influence the cellular ACE activity. Table 1 also shows that the treatment with green tea extract led to an inhibition of cell proliferation. These results indicate that green tea extract specifically up-regulated the NEP activity in connection with a decrease in cellular proliferation.

Effects of green tea extract on cells with blocked DNA synthesis

In further experiments we investigated the effect of green tea extract on SK-N-SH cells with blocked DNA synthesis to refine the knowledge about the mode of action. For this purpose we treated the cells with arabinosylcytosine and different concentrations of green tea extract. Table 2 demonstrates that again only the cellular NEP activity was strongly induced whereas ACE was not significantly affected. As a selective inhibitor of DNA synthesis arabinosylcytosine alone induced also the cellular NEP activity (Table 2) in connection with a strong inhibition of cell proliferation (42 [+ or -] 5%, n = 14). The treatment of the DNA synthesis-blocked cells by green tea extract led to an additional increase in cellular NEP induction demonstrating that the NEP induction was independent from inhibition of cell proliferation.

* Discussion

Green tea extract inhibited the proliferation of SK-N-SH cells, a human neuroblastoma cell line with intact ecto-neuropeptidases. Despite the high similarity between both enzymes ACE and NEP green tea extract only influenced the expression of NEP demonstrating a specific interaction between constituents of the extract and the SK-N-SH cells.

One possible explanation for the observed enzyme induction of NEP might be based on the relation between cellular differentiation and proliferation. In our investigations the inhibition of proliferation was associated with an increase in cellular enzyme activity of NEP but not ACE.

The processes of differentiation and proliferation are mutually exclusive in all cell types. In most cases a non-toxic inhibition of cell proliferation enhances the cellular differentiation state (Iatropoulos and Williams, 1996). This was observed investigating the differentiating effect of retinoic acid on matrix metalloproteinase-2 (MMP-2) expression. The proliferative activity was inversely correlated to MMP-2 expression in SK-N-SH cells (Thier et al., 2000). In contrast to recently published experiments with different components from red wine extract (Melzig and Escher, 2002) which demonstrated that the induction of both enzyme activities is equate with an enhancement of cellular differentiation the green tea extract acts presumably more specific.

For NEP it has been reported that in different cell types the up-regulation of the cellular enzymatic activity was correlated as well as with an enhanced differentiation state and the inhibition of cellular proliferation (Uehara et al. 2001). By the same time the enhanced cellular enzyme activities are a proof for the assumption that the inhibition of cell proliferation was not the result of a cytotoxic effect. For ACE the enzyme induction seems to be coupled with the mitogen-activated protein kinase and early growth response 1 transcription factor (Day et al. 2001). Obviously, these elements of the signal transduction chain are not activated by the treatment of SK-N-SH cells with green tea extract.

Arabinosylcytosine is the most potent selective inhibitor of DNA synthesis (Beranek, 1986) and induced only the cellular NEP activity, not the ACE activity. The additional enhancing effect of green tea extract on cellular NEP activity (Table 2) was not accompanied with an additional decrease in cell proliferation, demonstrating that the green tea extract induced the enzyme independently from the differentiation improvement caused by arabinosylcytosine via inhibition of cell proliferation. Experiments with the combination of arabinosylcytosine and epigallocatechin gallate (EGCG) did not result in differentiation enhancement in comparison to the described effect with green tea extract (data not shown), indicating that EGCG was not the constituent responsible for the observed improvement of cell differentiation. It is known that purinalkaloids like caffeine are able to enhance the differentiation of neuroblastoma cells via cAMP-dependent histone H1 phosphorylation induced by inhibition of phosphodiesterase activity (Ajiro et al. 1990). The concentration of caffeine necessary to induce this effect is much higher than present in the investigated extract, 1 or 2 mM is the inducing concentration, in the extract are only 25 [micro]M caffeine. Theophylline is not a constituent of the extract used in the experiments. It can not be excluded that purinalkaloids contribute to the general effect observed in the experiments but they are not the main constituents responsible for the enzyme induction.

The possible physiological importance of the described effects of green tea extract at the neuroblastoma cell line SK-N-SH should be discussed in relation to the physiological role of NEP and its distribution in different structures of the brain. The enhancement of the differentiation state of neuronal cells results in the improvement of their functional properties. The increase in cellular NEP activity might be associated with an increased neuropeptide metabolism which is necessary to maintain the functional integrity of the brain.

Beside the metabolisms of neuropeptides, NEP is also included in the degradation of amyloid beta peptides and by that linked with pathogenesis of Alzheimer's disease (Shirotani et al. 2001). Regarding this aspect the up-regulation of NEP by green tea extract can be discussed as neuroprotective effect, because an enhanced degradation of the amyloid beta peptides may affect the susceptibility to Alzheimer's disease and prevent the accumulation of amyloid plaques in vivo (Iwata et al. 2000). Recent studies have shown that green tea polyphenols reduce free radical-induced lipid peroxidation and have protective effects against amyloid beta peptides-induced neuronal apoptosis through scavenging reactive oxygen species, which might be beneficial for the prevention of Alzheimer's disease (Choi et al. 2001). In these investigations especially EGCG was the active component of green tea extract. Taking into account our studies green tea extract seems to induce a broad spectrum of biological activities in the brain resulting in a general protecting effect against a variety of damaging influences.

* Conclusions

Beside its established antioxidant properties green tea extract seems to influence also the expression of the neuropeptidase NEP included in the degradation of amyloid peptides and it also seems to diminish the risk of accumulation of plaque forming peptides. This effect might be correlated with the preventive activity of green tea in some lifestyle-related diseases shown by epidemiological data (Sueoka et al. 2001). The possibility, that daily consumption of green tea can also diminish risk for Alzheimer's disease and slow the normal age-related decline in mental acuity merits consideration.
Table 1. Influence of green tea extract on specific enzyme
activity and cell proliferation.

concentration specific cellular enzyme cell proli-
of extract activity in % feration (%)


control 100 [+ or -] 8 100 [+ or -] 3 100 [+ or -] 5
 5 107 [+ or -] 10 130 [+ or -] 19 * 83 [+ or -] 4 **
10 107 [+ or -] 21 133 [+ or -] 10 * 80 [+ or -] 4 **
25 101 [+ or -] 10 173 [+ or -] 12 * 63 [+ or -] 6 **
50 102 [+ or -] 15 220 [+ or -] 61 * 41 [+ or -] 6 **

* Significant difference to the specific enzymatic activity of the

** Significant difference to cell proliferation of the controls

Table 2. Effect of the combination of green tea extract with
arabinosylcytosine (AC) on specific enzyme activity and cell number.

tration specific cellular enzyme relative
of extract activity in % of the controls cell
and AC number


12.5 ng/ml 114 [+ or -] 29 241 [+ or -] 50 100 [+ or -] 5

12.5 ng/ml 106 [+ or -] 26 340 [+ or -] 30 * 97 [+ or -] 8
AC + 5

12.5 ng/ml 112 [+ or -] 12 400 [+ or -] 40 * 91 [+ or -] 7
AC + 10

12.5 ng/ml 83 [+ or -] 9 469 [+ or -] 96 * 82 [+ or -] 8 **
AC + 25

12.5 ng/ml 78 [+ or -] 8 * 240 [+ or -] 40 41 [+ or -] 6 **
AC + 50

* Significant difference to the specific enzymatic activity of cells
treated only with 12.5 ng/ml arabinosylcytosine (control)

** Significant difference to the relative cell number of cells treated
only with 12.5 ng/ml arabinosylcytosine (controls) The treatment with
arabinosylcytosine alone strongly inhibited cell proliferation: 42
[+ or -] 5% to the control without any treatment, n = 14. This value
was defined for the combination experiments as control (100%) and any
additional inhibition of cell proliferation by green tea extract
treatment was calculated on the base of proliferation inhibition by
arabinosylcytosine alone.

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Hooper NM (1996) The biological roles of zinc and families of zinc metalloproteases. In: Zinc metalloproteases in health and disease (ed Hooper BM): 1-21, Tayler & Francis Ltd, London

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Iwata N, Tsubuki S, Takaki Y, Watanabe K, Sekiguchi M, Hosoki E, Kawashima-Morishima M, Lee HJ, Hama E, Sekine-Aizawa Y, Saido TC (2000) Identification of the major Abeta 1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nat Med 6: 143-150

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Shirotani K, Tsubuki S, Iwata N, Takaki Y, Harigaya W, Maruyama K, Kiryu-Seo S, Kiyama H, Iwata H. Tomita T, Iwatsubo T, Saido TC (2001) Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan-and phosphoramidon-sensitive endopeptidases. J Biol Chem 276: 21895-21901

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* Address

Matthias F. Melzig, Free University Berlin, Institute of Pharmacy, Pharmaceutical Biology, Konigin-Luise-Str. 2+4, D-14195 Berlin, Germany

Tel.: ++49-30-838 51451; Fax: ++49-30-838 51461; e-mail:

M.F. Melzig (1) and M. Janka (2)

(1) Institut fur Pharmazie, Freie Universitat Berlin, Germany

(2) Institut fur Biologie, Humboldt-Universitat zu Berlin, Germany
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Author:Melzig, M.F.; Janka, M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Jul 1, 2003
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