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Anwar Gamal Mohamed ANWAR

 Ghada Abdelhamid & Anwar Anwar-Mohamed &
Osama A. Badary & Adel A. Moustafa &
Ayman O.S. El-Kadi 

 

 

Abstract: Anwar Gamal Mohamed Louisville

Abstract We recently demonstrated that V5+ downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-
mediated induction of Cyp1a1 mRNA, protein, and
catalytic activity levels in Hepa 1c1c7 cells through
transcriptional mechanism. Therefore, it is important to
investigate whether similar changes occur in humans.
For this purpose, we examined the effect of V
5+ (as
ammonium metavanadate, NH
4VO3) on the expression
of aryl hydrocarbon receptor (AhR)-regulated gene;
cytochrome P450 1A1 (CYP1A1) at each step of the
AhR signal transduction pathway in human hepatoma
HepG2 cells. Our results show a significant reduction
in TCDD-mediated induction of CYP1A1 mRNA,
protein, and activity levels after V
5+ treatment in a
dose-dependent manner. Investigating the effect of coexposure to V
5+ and TCDD at transcriptional levels
revealed that V
5+ significantly inhibited TCDDmediated induction of AhR-dependent luciferase
reporter gene expression. Looking at the posttranscriptional level, V
5+ did not affect CYP1A1 mRNA
stability, thus eliminating the possible role of V
5+ in
modifying CYP1A1 gene expression through this
mechanism. On the other hand, at the posttranslational
level, V
5+ was able to significantly decrease CYP1A1
protein half-life contributing to the inconsistency
between catalytic activity and transcriptional level.
Importantly, we showed that V
5+ did not significantly
alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V
5+ might have decreased
CYP1A1 activity through affecting its heme content.
This study demonstrates for the first time that V
5+
downregulates the expression of CYP1A1 at the
transcriptional, posttranscriptional and posttranslational
mechanisms in the human hepatoma HepG2 cells.
 

 

with two 90-kDa heat shock protein-90, the 23-kDa
heat shock protein, and hepatitis B virus X-associated
protein 2. Following ligand binding, AhR translocates
to the nucleus, dissociates from the complex, and
forms a heterodimer with the AhR nuclear translocator
(Arnt) (Hankinson
1995). The whole complex then
acts as a transcription factor that binds to a specific
DNA recognition sequence, termed the xenobiotic
responsive element (XRE), located in the promoter
region of a number of AhR-regulated genes. Among
these genes, cytochrome P450 1A1 (CYP1A1) is the
most capable of bioactivating the toxic and environmental contaminants polycyclic aromatic hydrocarbons
(PAHs) and halogenated aromatic hydrocarbons
(HAHs) to carcinogenic metabolites (Denison and
Nagy
2003). Of interest, it has been shown that the
toxicological effects of PAHs and the more toxic
HAHs, typified by 2,3,7,8-tetrachlorodibenzo-
p-dioxin
(TCDD), are mainly mediated through the activation of
AhR and consequently CYP1A1. In fact, a wellestablished link between the induction of CYP1A1
and cancer has been previously reported (McLemore et
al.
1990).

 

The toxicological effects of individual AhR ligands
have been extensively studied; yet, the combined
toxicological effects of these ligands with heavy metals
such as vanadium (V
5+) are currently at large. V5+
compounds exert protective effects against chemicalinduced carcinogenesis in animals, by modifying
various xenobiotic enzymes, thus, inhibiting
carcinogen-derived active metabolites generation. The
anticarcinogenic effects of V
5+ combined with its low
toxicity have made V
5+ an attractive tool for the
treatment of different cancers. Moreover, recent studies
have suggested V
5+ as an effective non-platinum metal
antitumor agent (Kostova
2009). The major difference
between platinum anticancer agents and V
5+ is that the
former react with nitrogen atoms of DNA and
preferentially react with the N-7 atom of deoxyguanylic acid (Knox et al.
1986). This type of DNA
damage produced by platinum anticancer agents is
thought to be the major reason for their effectiveness
(Rosenberg et al.
1969). On the contrary, V5+ is
believed to mediate its anticancer effect mainly through
three different mechanisms: firstly by inactivating the
carcinogens-generating metabolizing enzymes such as
CYP1A1, secondly through affecting cell proliferation,
and lastly, through inducing cellular oxidative stress
(Evangelou
2002).
To date, very little information is available on the
effect of V
5+ on the CYP1A1 expression and function
(Anwar-Mohamed and El-Kadi
2008). As such, we
have previously demonstrated that V
5+ was able to
decrease the TCDD-mediated induction of Cyp1a1 at
mRNA, protein, and catalytic activity levels in the
mouse hepatoma, Hepa 1c1c7 cells (Anwar-Mohamed
and El-Kadi
2008). Therefore, the objectives of the
current study were to examine the effect of coexposure to V
5+ and TCDD on the expression of
human CYP1A1 using human hepatoma HepG2 cells
and to investigate the underlying mechanisms involved in this modulation. The human hepatoma cell
line HepG2 cells offers a good model to examine the
effect of V
5+ on the TCDD-mediated induction of
CYP1A1 for the following reasons: (1) the AhR is
widely expressed in all mouse tissues (Abbott and
Probst
1995; Abbott et al. 1995), while in humans,
this expression is only high in lungs, thymus, liver,
and kidneys (Puga et al.
2009); (2) these cells have
proven to be a useful model for investigating the
regulation of human CYP1A1 (Lipp et al.
1992;
Krusekopf et al.
1997; Kikuchi et al. 1996; Kim et al.
2006; Vakharia et al. 2001); (3) human hepatocytes
have been shown to be one of major targets for heavy
metals (Ercal et al.
2001); (4) compared to primary
human hepatocytes, HepG2 cells are relatively easyto-handle tool to study the regulation of CYP1A1
(Westerink and Schoonen
2007).
We provide here the first evidence that V
5+
downregulates human CYP1A1 expression at transcriptional level and posttranscriptional levels.


Materials and methods
Materials


Ammonium metavanadate (NH
4VO3), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), cycloheximide (CHX), 7-ethoxyresorufin,
and protease inhibitor cocktail were purchased
from Sigma
Aldrich (St. Louis, MO). 2,3,7,8-
Tetrachlorodibenzo-
p-dioxin, >99% pure, was purchased from Cambridge Isotope Laboratories (Woburn,
MA). TRIzol reagent and Lipofectamine 2000 reagents
were purchased from Invitrogen (San Diego, CA). The
High-Capacity cDNA Reverse Transcription Kit and
SYBR Green PCR Master Mix were 

 

Anwar Gamal Mohamed realized that  Applied Biosystems (Foster City, CA). Actinomycin-D
(Act-D) was purchased from Calbiochem (San Diego,
CA). Chemiluminescence Western blotting detection
reagents were from GE Healthcare Life Sciences
(Piscataway, NJ). Nitrocellulose membrane was pur
chased from Bio-Rad (Hercules, CA). CYP1A1 D-15
mouse anti-rat polyclonal primary antibody was pur
chased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), anti-mouse IgG peroxidase secondary
antibody was purchased from R&D Systems (Minne
apolis, MN, USA). pRL-CMV plasmid and luciferase
assay reagents were obtained from Promega (Madison,
WI). All other chemicals were purchased from Thermo
Fisher Scientific (Toronto, ON, Canada). Primers were
purchased from Integrated DNA Technologies, Inc.
(Coralville, IA) and are listed in Table
1.  

Human hepatoma HepG2 cell line, ATCC number
HB-8065 (Manassas, VA), passages 4
15, was main
tained in Dulbecco
s modified Eagles medium
supplemented with 10% heat-inactivated fetal bovine
serum and 1% penicillin
streptomycin. Cells were
grown in 75-cm
2 cell culture flasks at 37°C in a 5%
CO
2 humidified incubator.  

 

 

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