Suppression of TNF-α induced NFκB activity by gallic acid and its semi-synthetic esters: Possible role in cancer chemoprevention

Article abstract of DOI:10.1080/14786410903335232

Gallic acid (3,4,5-trihydroxybenzoic acid), found in many plants either in free-form or part of tannins, is known to possess anti-microbial, antioxidant and cytotoxic properties. NFκB regulates the expression of several genes involved in carcinogenesis. These include anti-apoptotic, cytokines and cell cycle-regulatory genes. It is well established that the transcriptional factor NFκB is deregulated in many forms of cancer. Thus, agents that can suppress NFκB activation have the potential of suppressing carcinogenesis. In the present investigation, gallic acid was isolated from Alchornea glandulosa (Euphorbiaceae) and eight esters were synthesised. These compounds were evaluated against TNF-α-induced NFκB activation with stably transfected 293/NFκB-Luc human embryonic kidney cells. Gallates with IC50 values in a range of 10-56 M mediated inhibitory activity higher than gallic acid (IC50 76.0 4.9 M). In addition to inhibiting NFκB activation, gallic acid mediated a modest cytotoxic effect, and some of the gallates affected cell viability at the tested concentrations. Based on these results, suppression of NFκB activation by gallate esters could play a chemopreventive role in carcinogenesis.

Full text of DOI:10.1080/14786410903335232

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Suppression of TNF-α induced NFκB
activity by gallic acid and its semi-
synthetic esters: possible role in cancer
chemoprevention
Mauro C.C. Morais a b , Suaib Luqman a c , Tamara P. Kondratyuk
a , Maicon S. Petronio d , Luis O. Regasini d , Dulce H.S. Silva d
,
Vanderlan S. Bolzani d , Christiane P. Soares c & John M. Pezzuto a
a College of Pharmacy, University of Hawaii , 34 Rainbow Drive,
Hawaii, Hilo 96720, USA
b Department of Clinical Analyses, Cytology and Cellular Biology
Laboratory , School of Pharmaceutical Sciences, Sao Paulo State
University – UNESP , Rua Expedicionários do Brasil, n.1621,
Centro, 14801-902, Araraquara-SP, Brazil
c IUSSTF Fellow, Genetic Resources and Biotechnology Division ,
Central Institute of Medicinal and Aromatic Plants (Council of
Scientific and Industrial Research) , Lucknow 226015, India
d Department of Organic Chemistry, NuBBE-Nucleus of Bioassays,
Biosynthesis and Ecophysiology of Natural Products , Institute
of Chemistry, São Paulo State University – UNESP , Rua Prof.
Francisco Degni, SN, Bairro Quitandinha, 14801–970 CP 355,
Araraquara-SP, Brazil
Published online: 21 Apr 2010.
To cite this article: Mauro C.C. Morais , Suaib Luqman , Tamara P. Kondratyuk , Maicon S. Petronio ,
Luis O. Regasini , Dulce H.S. Silva , Vanderlan S. Bolzani , Christiane P. Soares & John M. Pezzuto
(2010) Suppression of TNF-α induced NFκB activity by gallic acid and its semi-synthetic esters:
possible role in cancer chemoprevention, Natural Product Research: Formerly Natural Product
Letters, 24:18, 1758-1765, DOI: 10.1080/14786410903335232
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Natural Product Research
Vol. 24, No. 18, 10 November 2010, 1758–1765
Suppression of TNF-a induced NFjB activity by gallic acid and its
semi-synthetic esters: possible role in cancer chemoprevention
Mauro C.C. Moraisaby, Suaib Luqmanacy, Tamara P. Kondratyuka,
Maicon S. Petroniod, Luis O. Regasinid, Dulce H.S. Silvad, Vanderlan S. Bolzanid,
c
Christiane P. Soares and John M. Pezzuto
a
*
aCollege of Pharmacy, University of Hawaii, 34 Rainbow Drive, Hawaii, Hilo 96720, USA;
bDepartment of Clinical Analyses, Cytology and Cellular Biology Laboratory, School of
´
Pharmaceutical Sciences, Sao Paulo State University – UNESP, Rua Expedicionarios do
Brasil, n.1621, Centro, 14801-902, Araraquara-SP, Brazil; IUSSTF Fellow, Genetic
c
Resources and Biotechnology Division, Central Institute of Medicinal and Aromatic Plants
(Council of Scientific and Industrial Research), Lucknow 226015, India; Department of
d
Organic Chemistry, NuBBE-Nucleus of Bioassays, Biosynthesis and Ecophysiology of
Natural Products, Institute of Chemistry, Sa˜o Paulo State University – UNESP, Rua Prof.
Francisco Degni, SN, Bairro Quitandinha, 14801–970 CP 355, Araraquara-SP, Brazil
(Received 14 September 2009; final version received 8 October 2009)
Gallic acid (3,4,5-trihydroxybenzoic acid), found in many plants either in
free-form or part of tannins, is known to possess anti-microbial,
antioxidant and cytotoxic properties. NFꢀB regulates the expression of
several genes involved in carcinogenesis. These include anti-apoptotic,
cytokines and cell cycle-regulatory genes. It is well established that the
transcriptional factor NFꢀB is deregulated in many forms of cancer. Thus,
agents that can suppress NFꢀB activation have the potential of suppressing
carcinogenesis. In the present investigation, gallic acid was isolated from
Alchornea glandulosa (Euphorbiaceae) and eight esters were synthesised.
These compounds were evaluated against TNF-ꢁ-induced NFꢀB activation
with stably transfected 293/NFꢀB-Luc human embryonic kidney cells.
Gallates with IC50 values in a range of 10–56 mM mediated inhibitory
activity higher than gallic acid (IC50 76.0 ꢀ 4.9 mM). In addition to
inhibiting NFꢀB activation, gallic acid mediated a modest cytotoxic
effect, and some of the gallates affected cell viability at the tested
concentrations. Based on these results, suppression of NFꢀB activation by
gallate esters could play a chemopreventive role in carcinogenesis.
Keywords: gallic acid; NFꢀB pathway; cytotoxicity; 293/NFꢀB cells;
Alchornea glandulosa; cancer chemoprevention
1. Introduction
Phenolic acids are natural products which possess a wide spectrum of
biological activity (Nazaruk, Czechowska, Markiewicz, & Borawska, 2008).
*Corresponding author. Email: pezzuto@hawaii.edu
yBoth authors contributed equally to this paper.
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903335232
http://www.informaworld.com

Natural Product Research
1759
Gallic acid (3,4,5-trihydroxybenzoic acid), a secondary metabolite accumulates in
nearly all plants, either in free form or part of tannins, is known to possess
antimicrobial, antiviral, anti-inflammatory, antioxidant and cytotoxic properties
(Aruoma, Murcia, Butler, & Halliwell, 1993; Fiuza et al., 2004; Kratz et al., 2008;
Kroes, van den Berg, van Ufford, van Dijk, & Labadie, 1992; Kubo, Fujita, K. Nihei
& A. Nihei, 2004). It is commonly used in the pharmaceutical industry as salts and
esters. Some ointments used to treat psoriasis and external hemorrhoids contain
˜
gallic acid (Garcıa de Hombre & Perez Penate, 2006; Hatton, 2000). Gallic acid has
´ ´
two major functional groups, hydroxyls and a carboxylic acid, which can yield
numerous esters and salt derivatives. Some galloyl compounds are used as
antioxidants in food and can exhibit important roles in prevention of inflammatory
and atherosclerosis responses by blocking activation of NFꢀB (Murase et al., 1999).
NFꢀB consists of homo and heterodimeric protein complexes acting as a
transcription factor, and is found in many animal cell types. It is involved in
regulation of immune response and inflammation, cell lineage development, cell
apoptosis, cell-cycle progression and oncogenesis due to stimuli such as stress,
cytokines, free radicals, ultraviolet irradiation, oxidised LDL and microbial antigens,
and has been shown to regulate the expression of several genes, including bcl-2,
bcl-xl, cIAP, survivin, TRAF, COX-2, MMP-9, iNOS and cell cycle-regulatory
components, whose products are involved in tumourigenesis (Aggarwal, Sethi, Nair,
& Ichikawa, 2006; Chen, Castranova, & Shi, 2001; Melisi & Chiao, 2007; Thanos
& Maniatis, 1995). Most carcinogens, proinflammatory cytokines such as TNF and
IL-1, or tumour promoters, including cigarette smoke, phorbol esters, okadaic acid,
H2O2, have been shown to activate NFꢀB, and resulting tumours have misregulated
NFꢀB activity (Baldwin, 2001). It is now well established that aberrant regulation of
NFꢀB, and the signalling pathways that control its activity, are involved in cancer
development and progression, as well as in drug resistance, especially during
chemotherapy and radiotherapy (Inoue, Gohda, Akiyama, & Semba, 2007).
Blocking NFꢀB can cause tumour cells to stop proliferating, undergo apoptosis,
or to become more sensitive to the action of antitumour agents (Frydrych &
Mlejnek, 2008; Yamamoto & Gaynor, 2001). Thus, agents that can suppress NFꢀB
activation have the potential to suppress carcinogenesis or tumourigenesis.
In the present investigation, gallic acid was isolated from Alchornea glandulosa
Poepp and Endl. (Euphorbiaceae), and eight semi-synthetic esters were prepared. All
the compounds were evaluated for cytotoxicity as well as potential to inhibit
TNF-ꢁ-induced NFꢀB activation with stably transfected 293/NFꢀB-Luc human
embryonic kidney cells.
2. Materials and methods
2.1. Materials
Methanol, n-hexane, ethyl acetate and p-dioxane were of high performance liquid
chromatography (HPLC) grade (JT Baker). Column chromatography was carried
out over silica gel (0.06–0.20 mm, Acros Organics). Gel permeation chromatography
(GPC) was performed using Sephadex LH-20 (Pharmacia Biotech). Preparative
HPLC was carried out using a Varian Prep-Star 400 system and a Phenomenex C-18
(250 mm ꢁ 21.2 mm) column. The monodimensional NMR experiments were

1760
M.C.C. Morais et al.
recorded on a Varian INOVA 500 spectrometer (11.7 T) at 500 MHz (1H) and
125 MHz (13C), using CDCl3 and DMSO-d6 as solvent (Aldrich). Alkyl alcohols and
DCC were purchased from Aldrich. Dimethyl sulphoxide (DMSO) and sulphorho-
damine B (SRB) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO).
Dulbecco’s Modified Eagle’s Medium (DMEM), antibiotic–antimycotic, hygro-
mycin B, and MEM sodium pyruvate were purchased from Invitrogen Co.
(Carlsbad, CA). Reporter Lysis Buffer, Luciferase Assay System was purchased
from Promega (Madison). Tumour Necrosis Factor-ꢁ (TNF-ꢁ) was purchased from
Calbiochem (Gibbstown, NJ). Alcohol was purchased from J.T. Baker (Phillipsburg,
NJ).
2.2. Isolation of gallic acid
The leaves of A. glandulosa were collected in the Biological Reserve and
Experimental Station at Mogi Guac
A voucher specimen (SP319257) has been deposited in the herbarium of the Botanic
Institute (Sao Paulo-SP, Brazil).
˜
¸ u, Sao Paulo State, Brazil, in March 2005.
˜
The shade-dried plant material (1.5 kg) was ground and defatted with n-hexane
(3.5 L ꢁ 3) at room temperature and exhaustively extracted by maceration with
MeOH (4.2 L ꢁ 3). The crude extract was concentrated under reduced pressure to
yield 3.8 g of a syrup residue, which was diluted with MeOH : H2O (4 : 1) and then
successively partitioned with EtOAc and n-BuOH. After evaporation under reduced
pressure, the partition phases yielded 2.5 g and 0.8 g, respectively. The part of EtOAc
residue (2.0 g) was chromatographed by gel permeation over Sephadex LH-20 eluted
with methanol to afford 11 fractions (A1–A11). Fraction A2 (730 mg) was purified
by RP-HPLC [MeOH : H2O : HOAc (7.5 : 92 : 0.5), UV detection at 265 nm; flow rate
15 mL minꢂ1] to give compound 1 (335 mg). Compound 1 was identified as gallic acid
1
based on analysis of the H and 13C NMR data, as well as by comparison with
authentic material in our laboratory (Figure 1).
O
O
O
OH
O
O
O
O
O
O
HO
O
OH
HO
OH
HO
OH
HO
OH
O
HO
OH
OH
5
OH
1
OH
OH
3
OH
4
2
O
O
O
O
O
OH
O
O
HO
OH
HO
OH
HO
OH
O
O
O
OH
OH
OH
O
7
8
9
6
Figure 1. Molecular structures of gallic acid (1) and semi-synthetic esters (2–9).

Natural Product Research
1761
2.3. Synthesis of esters 2–9
After being cooled at 5ꢃC, the mixture of gallic acid (0.2 mmol) and alcohol
(20 mmol) in p-dioxane (6.0 mL) was added to a solution of N,N0-dicyclohexylcar-
bodiimide (DCC, 1.0 mmol) in p-dioxane (3.0 mL). The solution was stirred for 48 h,
and the solvent was removed under reduced pressure. The residue was partitioned
three times with EtOAc and then filtered. The filtrate was washed successively with
saturated aqueous citric acid solution (three times), saturated aqueous NaHCO3
(three times), water (two times), dried over MgSO4, and evaporated under reduced
pressure. The crude products were purified over a silica gel column using an isocratic
system of CHCl3–MeOH (98 : 2) to give gallate esters 2–8. Structures of the semi-
synthetic esters, methyl gallate (2), ethyl gallate (3), n-propyl gallate (4), isopropyl
gallate (5), n-butyl gallate (6), isobutyl gallate (7), n-pentyl gallate (8) and 3,4,
5-triacetoxybenzoic acid (9), were established by 1H and 13C NMR spectral analysis
(Figure 1).
For synthesis of ester 9, gallic acid (20 mmol) was dissolved in dry pyridine
(5.0 mL) and anhydride acetic (5.0 mL) under hydrogen atmosphere. The mixture
was stirred for 48 h at room temperature, carried to dryness under reduced pressure,
and purified by column chromatography with a mixture of CHCl3–MeOH (85 : 15)
to give a product (9). The NMR spectroscopic data of 9 were compatible with 3,4,
5-triacetoxybenzoic acid.
2.4. NFjB luciferase assay
NFꢀB inhibition assay was conducted using a luciferase reporter gene bioassay as
previously described (Homhual et al., 2006). Human embryonic kidney cells (293/
NFꢀB-Luc) were used to monitor alteration of the NFꢀB pathway. This cell line is
stably transfected with a luciferase reporter construct regulated by the NFꢀB
response element. Transcription factor can bind to the response element when
stimulated by TNF-ꢁ, allowing transcription of the luciferase gene. Luciferase reacts
with substrate, emitting light that was detected using a luminometer. Cells were
seeded (105 cells mLꢂ1) on sterile white walled 96-well plates and grown to
approximately 80% confluence by incubating for 48 h. Gallic acid and alkyl gallates
were tested at 20 mg mLꢂ1 following 1 : 3 serial dilutions. After treatment with tested
compounds for 10 min, cells were incubated for an additional 6 h with or without
TNF-ꢁ (5 ng mLꢂ1). The cells were gently washed with PBS and kept at ꢂ80ꢃC with
luciferase lysis buffer (Promega Corporation). The luciferase assay was performed
using the Luc assay system from Promega following manufacturer instructions.
Luciferase activity was monitored (LUMIstar Galaxy BMG), dose-response curves
were constructed, and results were expressed as IC50 values (i.e. concentration
required to inhibit TNF-ꢁ induced NFꢀB activity by 50%).
2.5. Cytotoxicity assay
Cytotoxicity was determined by the method of Skehan et al. (1990). The cells were
fixed with trichloroacetic acid and stained for 30 min with 0.4% (w/v) sulphorho-
damine B (SRB) prepared in 1% acetic acid. Unbound dye was removed by washing
with 1% acetic acid, and protein-bound dye was extracted with 10 mM Tris base

1762
M.C.C. Morais et al.
[tris (hydroxymethyl)aminomethane] for determination of optical density in a
computer-interfaced, 96-well microtiter plate reader.
2.6. Statistical analysis
Statistical analysis was performed using GraphPad PrismÕ Version 5.01 (GraphPad
Software Inc.). Results were subjected to one-way analysis of variance (ANOVA),
and differences between means were located using Tukey’s multiple comparison test.
Differences between means at the 5% (p 5 0.05) level were considered significant.
The data are expressed as means ꢀ standard error.
3. Results and discussion
Herein we report cytotoxic properties and suppression of TNF-ꢁ induced NFꢀB
activation of gallic acid (1) and eight esters (2–9) determined with stably transfected
293/NFꢀB-Luc human embryonic kidney cells. Table 1 shows the concentrations of
gallates 1–9 required to inhibit NFꢀB-induced activation in 50% (IC50). N-Tosyl-
L-phenylalaninyl-chlormethylketone (TPCK) was adopted as positive control,
exhibiting 98.5% ꢀ 0.5% NFꢀB inhibition at 30 mM, with an IC50 value of about
5 mM. At the highest concentration tested (20.0 mg mLꢂ1), gallic acid mediated a
moderate cytotoxic effect (52.9% ꢀ 3.5% survival), but none of the gallate esters
significantly affected cell viability. The most cytotoxic compound evaluated was 6,
with 48.9% ꢀ 3.3% cell survival.
Among inhibitors of NFꢀB, salicylates, aspirin (Kopp & Ghosh, 1994), apigenin
and curcumin (Gerritsen et al., 1995; Singh & Aggarwal, 1996) were reported to
suppress activation of NFꢀB by preventing the degradation of the NFꢀB inhibitor
(IꢀB), and therefore NFꢀB was retained in the cytosol.
The serine protease inhibitor TPCK can cause a complete block to NFꢀB
activation. TPCK inhibits NFꢀB subunits processing and IꢀB degradation (Mellitis,
Hay, & Goodbourn, 1993). It has also been demonstrated that TPCK inhibits DNA
replication by G0–G1 growth arrest and thus promotes apoptosis by other cytotoxic
stimuli (Kirillova, Chaisson, & Fausto, 1999).
Table 1. Inhibition of TNF-ꢁ-induced NFꢀB
activity by gallic acid (1) and gallate esters (2–9).
Compound
IC50 (mM)
1
2
3
4
5
6
7
8
76.0 ꢀ 4.9
10.3 ꢀ 1.1
54.6 ꢀ 5.1
29.7 ꢀ 3.1
31.1 ꢀ 2.0
56.1 ꢀ 5.9
37.2 ꢀ 4.0
38.3 ꢀ 4.1
48.0 ꢀ 5.9
5.09 ꢀ 2.14
9
TPCK

Natural Product Research
1763
The present study provides evidence that some gallates can act as inhibitors of
NFꢀB. Methyl gallate (2) demonstrated the greatest activity. The inhibitory actions
of gallates do not appear to result from non-specific cellular toxicity but rather are
specific for NFꢀB-dependent gene transcription. In another study, it has been
demonstrated that gallates can inhibit TNF-ꢁ-induced nuclear translocation of
NFꢀB by way of a mechanism independent of IꢀB degradation (Murase et al., 1999).
More recently, prevention of atherosclerosis, cancer, anti-plasmodial and
antioxidant activity of a group of galloyl compounds was described, as well as
structure-activity relationships yielding cytotoxic properties (Fiuza et al., 2004;
Jagan, Ramakrishnan, Anandakumar, Kamaraj, & Devaki, 2008; Locatelli et al.,
2008; Murase et al., 1999; Na et al., 2006; Petroˆ nio et al., 2007; Saxena et al., 2008).
Based on these encouraging intracellular responses, we suggest suppression of NFꢀB
activation by gallate esters could play a chemopreventive role in carcinogenesis, and
further investigations related to the actions of gallates, especially in NFꢀB signaling
pathway and in vivo models, may provide new insights into the cancer
chemopreventive ability.
Acknowledgements
This study is supported by a programme project P01 CA48112 awarded by the National
Cancer Institute and project 04/07932-7 of the Biota-Bioprospecta Program funded by Sao
Paulo State Research Foundation (FAPESP), Brazil. M.C.C. Morais is grateful to CAPES for
a research fellowship. S. Luqman acknowledges the Indo-US Science and Technology Forum
(IUSSTF), New Delhi for research fellowship.
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