Journal of Chinese Pharmaceutical Sciences
http://www.jcps.ac.cn
559
Cancer prevention by traditional Chinese medicine and plant phytochemicals column
Nrf2-mediated antioxidant and detoxifying enzyme induction by a combination of curcumin and sulforaphane Francisco Fuentes1#, Yury Gomez2#, Ximena Paredes-Gonzalez2, Avantika Barve3, Sujit Nair4, Siwang Yu5, Constance Lay Lay Saw2, Ah-Ng Tony Kong2* 1. Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306-22, Santiago, Chile 2. Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, New Jersey 08854, USA 3. Novartis Institutes for Biomedical Research, East Hanover, NJ-07470, USA 4. Amrita Cancer Discovery Biology Laboratory, Amrita Vishwa Vidyapeetham University, Amritapuri, Clappana P.O., Kollam, Kerala-690525, India 5. School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
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Abstract: The dietary phytochemicals curcumin (CUR) and sulforaphane (SFN) have shown remarkable cancer chemopreventive effects in many model systems. This study was designed to investigate the induction of Nrf2-mediated antioxidant enzymes by combining doses of CUR and SFN and the effect of their combination on the Nrf2-ARE (antioxidant response element) response in HepG2-C8 cells. We hypothesized that the combination of the polyphenol CUR and the isothiocyanate SFN could enhance the induction of AREs and Nrf2-target enzymes. HepG2-C8 cells were treated with a combination of low doses of CUR, SFN or both. The induction of Nrf2-mediated antioxidant and phase II detoxifying enzymes–heme oxygenase-1 (HO-1) and UDP-glucuronosyltransferase-1A (UGT1A)–was measured by real-time RT-PCR and western blotting. ARE-luciferase activity was also quantified. Low doses of CUR (10 µM) and SFN (12.5 µM) significantly induced the expression of HO-1 and UGT1A1 proteins. Through the use of chemical inhibitors of mRNA and protein synthesis, the combination of CUR and SFN was shown to affect the transcriptional regulation of both HO-1 and UGT1A1. Additionally, the combination of CUR and SFN synergistically induced the expression of Nrf2- and ARE-luciferase activity in HepG2-C8 cells. Thus, CUR and SFN at low concentrations augment therapeutic effects in HepG2-C8 cells. The enhanced ARE-luciferase activity of combined CUR and SFN treatment could partly explain the significant induction of the Nrf2-target enzymes HO-1 and UGT1A1. Taken together, our results suggest that combining low doses of CUR and SNF could be a promising strategy for cancer chemoprevention in humans. Keywords: Antioxidant response element, Curcumin, HepG2-C8 cells, HO-1, Nrf2, Sulforaphane, UGT1A1
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CLC number: R962
1. Introduction
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Document code: A
Increasing evidence supports an inverse relationship between the consumption of cruciferous vegetables and the incidence of several types of cancer, including prostate cancer[1], lung cancer[2], bladder cancer[3], colon cancer[4], and pancreatic cancer[5]. Numerous studies have shown that phytochemicals in cruciferous vegetables up-regulate detoxifying enzyme systems such as phase II
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Article ID: 1003–1057(2016)8–559–11
detoxifying enzymes, including NAD-(P)H: quinone reductase (NQO1), glutathione transferases (GSTs), UDP-glucoronosyltransferase (UGT), epoxide hydrolase, and γ-glutamine cysteine synthetase (γ-GCS), as well as antioxidant enzyme systems such as heme oxygenase-1 (HO-1)[6–9]. Furthermore, the regulation of the basal and inducible expression of these phase II detoxifying and antioxidant enzymes has been shown to be mediated by the antioxidant response element (ARE), which is
Received: 2016-03-15, Revised: 2016-04-10, Accepted: 2016-04-24. Foundation items: Part by Institutional Funds and by R01-CA118947, R01-CA152826, from the National Cancer Institute (NCI), R01AT007065 from the National Center for Complementary and Alternative Medicines (NCCAM) and the Office of Dietary Supplements (ODS). # Francisco Fuentes and Yury Gomez contributed equally to this work. * Corresponding author. Tel.: +848-455-6369, Fax: +732-455-3134, E-mail:
[email protected]
a cis-acting sequence found in the 5‟-flanking region
http://dx.doi.org/10.5246/jcps.2016.08.062
cells and tissues against the toxic effects of reactive
of genes encoding many phase II and antioxidant enzymes[10,11]. ARE-mediated gene expression, and therefore the induction of phase II/antioxidant enzymes, has been linked to important protective mechanisms in
Copyright © 2016 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
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Francisco Fuentes et al. / J. Chin. Pharm. Sci. 2016, 25 (8), 559–569
oxygen species (ROS), as well as endogenous or
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[12–14]
exogenous carcinogenic intermediates
. Additionally,
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(A)
the coordinated induction of these phase II detoxifying
HO
enzymes is under the transcriptional control of the
OH OCH3
OCH3
nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 (B)
or Nrf2), a member of the cap „n‟ collar (CNC) family of basic leucine zipper (bZIP) proteins, which is an essential component of the ARE-binding transcriptional
H3C
N
S
S O
Figure 1. Chemical structures of the chemopreventive agents used in this study. (A) Curcumin; (B) Sulforaphane.
machinery[12,15,16]. Briefly, under normal basal conditions, Nrf2 is tethered to a cytosolic repressor protein called Kelch-like ECH-associated protein 1 (Keap1)[15,17]. The
ITCs[26,27]. Sulforaphane (SFN, Fig. 1B), an isothiocyanate
molecular cascade leading to ARE activation involves
compound found at high levels in broccoli and broccoli
the dissociation of Nrf2 from Keap1, followed by Nrf2
sprouts, contributes to the induction of phase II detoxifying
nuclear translocation, heterodimerization with small
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enzymes[28–30]. Its high potency to inhibit tumorigenesis
Maf (sMaf) protein, binding of the Nrf2/sMaf complex
in animal models has been previously demonstrated[31,32].
to AREs and ultimately the initiation of ARE-driven
In the past, we reported that SFN results in the induction of
gene expression[11,18].
hemeoxygenase-1 (HO-1) via activation of AREs and
Curcumin (CUR, Fig. 1A), a yellow-colored constituent
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of the spice turmeric, has been shown to protect against many types of cancer and to suppress angiogenesis and
metastasis in different animal models[19,20]. Moreover, the chemopreventive efficacy of CUR has been demonstrated
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through its ability to inhibit carcinogen bioactivation via the suppression of specific cytochrome P450 enzymes or the induction of phase II carcinogen detoxifying
enzymes[21,22]. Though the precise mechanism of action has yet to be fully elucidated, the literature suggests that mitochondrial hyperpolarization, resulting from ROS generation, is a prerequisite for CUR-induced apoptosis, and the presence of mitochondrial DNA damage may be a probable mechanism for CUR to induce the apoptosis of HepG2 cells and serve as the initial event triggering a chain of events leading to cell death[23,24]. Isothiocyanates (ITCs) are a class of glucosinolate compounds generated from the metabolic biotransfor-
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through the induction of Nrf2 protein in HepG2-C8 cells[15].
Because numerous dietary chemopreventive agents
show anti-carcinogenic activities when tested in vitro but nonetheless fail to demonstrate comparable efficacy in vivo, usually requiring difficult-to-achieve higher doses, determining whether combinatorial treatments involving lower doses of natural dietary agents that differ in their modes of action may be a more effective cancer chemopreventive strategy is of considerable interest[33–35]. The objectives of the present study were to investigate whether combined treatment of low doses of CUR and SFN could modulate the induction of antioxidant and phase II detoxifying enzymes such as HO-1 or UGT1A, to determine the nature of such interaction in HepG2-C8 cells and to examine whether Nrf2 factors and ARE elements are responsible for the up-regulation of these antioxidant and phase II detoxifying enzymes.
mation of naturally occurring cruciferous vegetables[25]. This biotransformation is believed to occur via the hydrolysis of secondary metabolites (glucosinolates) by the enzyme murosinase during the process of mastication
2. Materials and methods 2.1. Reagents and cell culture
or with the aid of resident microflora in the intestines,
The CUR used in this study was 70% pure as deter-
which promote the hydrolysis of glucosinolates into
mined by HPLC (Sigma Aldrich, St. Louis, MO) (the
Copyright © 2016 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
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Francisco Fuentes et al. / J. Chin. Pharm. Sci. 2016, 25 (8), 559–569
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remaining 30% comprises demethoxycurcumin and
using the RNeasy Mini Kit (Qiagen, Valencia, CA).
bidemethoxycurcumin). Sulforaphane (SFN) was purchased
Actinomycin D (2 µg/mL) was used in the mechanistic
from LKT Laboratories, Inc. (St. Paul, MN) and its
investigation. First-strand cDNA was synthesized from
purity was >99%. HepG2-C8 cells generated in our
1 μg of total RNA using the SuperScript III First-Strand
laboratory by stable transfection of an ARE-luciferase
Synthesis System for RT-PCR (Invitrogen, Carlsbad,
[36]
were cultured and maintained in Dulbecco‟s
CA) according to the manufacturer‟s instructions. The
Modified Eagle‟s Medium (DMEM) supplemented
cDNA was used as the template for real-time PCR
with 10% fetal bovine serum (FBS) and antibiotics
(Applied Biosystems® ViiA™ 7 Real-Time PCR System).
(100 Units/mL penicillin G and 100 Lg/mL streptomycin)
The sequences of the primers used for cDNA amplifi-
at 37 °C in a humidified incubator with 5% CO2. Cells
cation were designed using Primer Express 3.0 software
were allowed to grow up for 24 h to reach 70% confluency.
(Applied Biosystems, Foster City, CA). The 5‟ and 3‟
To treat the cells, CUR and SFN, either alone or in
primers used for amplifying HO-1 were 5‟-TTCAAG-
combination, were prepared in medium supplemented
CAGCTCTACCGCTC-3‟ and 5‟-GGGGGCAGAAT-
with 1% FBS. Cyclohexamide, an inhibitor of protein
CTTGCACTT-3‟. The 5‟ and 3‟ primers used for
synthesis, and Actinomycin D, an RNA synthesis
amplifying UGT1A were 5‟-ATGACCCGTGCCTTT-
construct
inhibitor, were purchased from Sigma Aldrich (St.
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ATCACCCAT-3‟ and 5‟-AGTCTCCATGCGCTTTGCATTGTC-3‟. The 5‟ and 3‟ primers used for amplifying
Louis, MO).
Nrf2 were 5‟-GCGACGGAAAGAGTATGAGC-3‟ and
2.2. MTS assay
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HepG2-C8 cells were cultured in 96-well plates overnight at the density of 1×104 cells/well in 100 µL
of DMEM medium containing 10% FBS and allowed to
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adhere to the plates. After 24 h, the cells were treated
5‟-ACGTAGCCGAAGAAACCTCA-3‟. GAPDH was used as an internal control and was amplified with the 5‟ and 3‟ primers 5‟-AATGGGCAGCCGTTAGGAAA-3‟ and 5‟-ACATGTAAACCATGTAGTTGAGGT-3‟. 2.4. Western blotting
with DMEM/1% FBS and various concentrations of
CUR or SFN alone or in combination using 0.1% DMSO
HepG2-C8 cells were treated with either CUR or
as a control. The cytotoxicity of the treatments was
SFN (10 µM or 12.5 µM, respectively) alone or in
tested using the CellTiter 96 aqueous nonradioactive
combination. Cyclohexamide (100 µg/mL) was also
cell proliferation MTS assay (3-(4,5-dimethylthiazol-2-
used in the mechanistic investigation. After treatment,
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
cells were washed with ice-cold phosphate-buffered saline
tetrazolium, inner salt; MTS) (Promega, Madison, WI).
(PBS, pH 7.4) containing 10 mM Tris-HCl, 50 mM sodium
After 24-h treatment, the HepG2-C8 cells were treated
chloride, 30 mM sodium pyrophosphate, 50 mM sodium
with the MTS solution for 1 h at 37 °C in a humidified
fluoride, 100 µM sodium orthovanadate, 2 mM iodoacetic
5% CO2 atmosphere. Absorbance of the formazan
acid, 5 mM ZnCl2, 1 mM phenylmethylsulfonyl fluoride,
product was read at 490 nm using a μQuant Biomolecular
and 0.5% Triton-X 100. Cell lysates were vigorously
Spectrophotometer (Bio-Tek Instruments Inc., Winooski,
vortexed, homogenized in an ultrasonicator for 10 s and
VT), and cell viability was calculated relative to the
left on ice for 30 min. The homogenates were centrifuged
DMSO control.
at 13 000 r/min for 15 min at 4 °C. The supernatants
2.3. RNA isolation and reverse-transcription PCR
were collected, and equal amounts of total protein from each sample, as determined using the BCA protein assay
Total RNA was extracted from HepG2-C8 cells
(Pierce, IL), were mixed with 4× loading buffer and
subjected to treatment with either CUR or SFN (10 µM
heated at 95 °C for 5 min. The samples were then sepa-
or 12.5 µM, respectively), either alone or in combination,
rated on a 10% Criterion Tris-HCl precast gel at 200 V
Copyright © 2016 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University
http://www.jcps.ac.cn
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Francisco Fuentes et al. / J. Chin. Pharm. Sci. 2016, 25 (8), 559–569
and transferred onto polyvinylidene difluoride (PVDF)
with similar results. Values are expressed as means±SD.
membranes (Immobilon-P, Millipore, Bedford, MA) at
Differences in mRNA and protein expression as well
130 mA for 1.5 h using a semidry transfer system
as luciferase activity were evaluated using Student‟s
(Fisher, PA). The membranes were then blocked over-
t-test. All P values were two-sided, and a P value of
