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 Abstract : Anwar Gamal Mohamed Louisville

Anwar gamal mohemed realized  that  Recent data suggest that vanadium (V5+) compounds exert protective effects against chemical-induced
carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown
that V5+ down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent
mechanism. However, incongruously, there is increasing evidence that V5+ is found in higher amounts in
cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the
possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous
antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P)
H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhRdeficient version, c12, were treated with increasing concentrations of V5+ in the presence of two distinct
Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our
results showed that V5+ inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and
catalytic activity levels. At transcriptional level, V5+ was able to decrease the TCDD- and SUL-induced nuclear
accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting
Nrf2 protein levels. Looking at post-transcriptional level; we found that V5+ did not affect Nqo1 mRNA
transcripts turn-over rates. However, at the post-translational level V5+ increased Nqo1 protein half-life. In
conclusion, the present study demonstrates that V5+ down-regulates Nqo1 at the transcriptional level,
possibly through inhibiting the ATP-dependent activation of Nrf2
 
1-Introduction
 
Anwar Gamal Mohamed reserch show that The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic
flavoenzyme that catalyzes the two-electron reduction of a broad
range of substrates (Korashy and El-Kadi, 2006). NQO1 plays a pivotal
role in detoxifying quinones to their corresponding hydroquinone
derivatives (Lind et al., 1982). Such an effect helps in maintaining
endogenous antioxidants like ubiquinone and vitamin E in their
reduced and active forms, thus protecting tissues from mutagens,
carcinogens, and oxidative stress damage (Ross, 2004).
NQO1 gene expression can be induced through two separate
regulatory elements associated with its 5′-flanking region. The first
pathway includes activation of a cytosolic transcription factor, the aryl
hydrocarbon receptor (AhR). The inactive form of AhR is attached to a
complex of two heat shock proteins 90 (HSP90), hepatitis B virus Xassociated protein (XAP2), and the chaperone protein p23 (Hankinson, 1995; Meyer et al., 1998). Upon ligand binding, the AhR–ligand
complex dissociates from the cytoplasmic complex and translocates to
the nucleus where it associates with the aryl hydrocarbon nuclear
translocator (Arnt) (Whitelaw et al., 1994). The whole complex then
acts as a transcription factor to mediate the induction of NQO1
through activating the xenobiotic responsive element (XRE) located in
its promoter region (Nebert et al., 2004). The second pathway involves
activation of the antioxidant responsive element (ARE). In fact, the
increased expression of NQO1 gene expression in response to
oxidative stress caused by agents such as isothiocyanate sulforaphane
(SUL), tert-butylhydroquinone (t-BHQ) and H2O2 occurs primarily
through this signaling pathway (Itoh et al., 1997). Perturbation in the
redox status of the cell activates the nuclear factor erythroid 2-related
factor-2 (Nrf2), a redox-sensitive member of the cap ‘n’ collar basic
leucine zipper (CNC bZip) family of transcription factors (Itoh et al.,
1997). Subsequently, Nrf2 dissociates from its cytoplasmic tethering
polypeptide, Kelch-like ECH associating protein 1 (Keap1),

2- Materials and methods by Anwar Gamal Mohamed Frankfort


Anwar Gamal Mohamed Archives said that Materials. Ammonium metavanadate (NH4VO3), cycloheximide
(CHX), 2,6-dichlorophenolindophenol, fluoroscamine, anti-goat IgG
peroxidase secondary antibody, protease inhibitor cocktail,
dicoumarol, and isothiocyanate sulforaphane were purchased from
Sigma Chemical Co. (St. Louis, MO). 2,3,7,8-Tetrachlorodibenzo-pdioxin, N99% pure, was purchased from Cambridge Isotope
Laboratories (Woburn, MA). TRIzol reagent was purchased from
Invitrogen (Grand Island, NY). High-Capacity cDNA Reverse
Transcription Kit and SYBR® Green PCR Master Mix were purchased
from 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 purchased
from Bio-Rad Laboratories (Hercules, CA). NAD(P)H:quinone
oxidoreductase 1 (Nqo1) rabbit polyclonal primary antibody was
generously provided by Dr. David Ross (University of Colorado,
Denver, CO). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh)
primary antibody, anti-goat and anti-rabbit IgG peroxidase secondary
antibodies were purchased from Santa Cruz Biotechnology, In. (
 
Anwar Gamal Mohamed Louisville:  dissolved in 1× sample buffer, boiled for 5 min, separated by 10% SDS
PAGE and electrophoretically transferred to a nitrocellulose
membrane. Protein blots were blocked for 24 h at 4 °C in blocking
buffer containing 5% skim milk powder, 2% bovine serum albumin and
0.05% (v/v) Tween-20 in Tris-buffered saline solution (TBS; 0.15 M
sodium chloride, 3 mM potassium chloride, 25 mM Tris–base). After
blocking, the blots were incubated with a primary polyclonal goat
anti-mouse Nqo1 antibody for 2 h at room temperature, or primary
polyclonal goat anti-mouse Gapdh antibody for 24 h at 4 °C in TBS
containing 0.05% (v/v) Tween-20 and 0.02% sodium azide. Incubation
with a peroxidase-conjugated goat anti-rabbit IgG for Nqo1, and rabbit
anti-goat IgG secondary antibody for Gapdh was carried out in
blocking buffer for 2 h at room temperature. The bands were
visualized with the enhanced chemiluminescence method according
to manufacturer's instructions (GE Healthcare Life Sciences,
Piscataway, NJ). The intensity of Nqo1 protein bands were quantified,
relative to the signals obtained for Gapdh protein, using ImageJ
software.
 
Anwar Gamal Mohamed Frankfort  show that Determination of Nqo1 enzymatic activity. After incubation with the
test compounds for the indicated time points, cells were washed
thoroughly twice with PBS containing EDTA, thereafter collected in
500 μl homogenate buffer containing 25 mM Tris–HCl pH 7.4, 250 mM
sucrose, and 5 μM FAD. The cells were then homogenized for 1 min
and then sonicated for 5 s on ice, thereafter the cell homogenates were
centrifuged at 12,000 ×g for 15 min, and the supernatant was
transferred to new microcentrifuge tubes which were kept in −80
freezer till further use. The Nqo1 activity was determined by the
continuous spectrophotometric assay to quantitate the reduction of its
substrate, 2,6-dichlorophenolindophenol (DCPIP) as described
previously (Korashy and El-Kadi, 2006; Preusch et al., 1991). Briefly,
20 μg of cell homogenate protein was incubated with 1 ml of the assay
buffer [40 μM DCPIP, 0.2 mM NADPH, 25 mM Tris–HCl, pH 7.8, 0.1% (v/
v) Tween 20, and 0.7 mg/ml bovine serum albumin, 0 or 30 μM
dicoumarol]. The rate of DCPIP reduction was monitored over 90 s at
600 nm with an extinction coefficient (ɛ) of 2.1 mM−1 cm−1. The
Nqo1 activity was calculated as the decrease in absorbance per min
per mg of total protein of the sample which quantitates the
dicoumarol-inhibitable reduction of DCPIP.

3-Results
 
Acording to Anwar Gamal Mohamed frankfort Concentration-dependent effect of V5+ on TCDD-mediated induction of
Nqo1 mRNA
To examine the ability of V5+ to modulate Nqo1 gene expression,
Hepa 1c1c7 cells were treated with various concentrations of V5+ in
the presence of 1 nM TCDD (Fig. 1A). Thereafter, Nqo1 mRNA was
assessed using real-time PCR. The concentrations of V5+ used hereafter were chosen after determining the ability of wide range of concentrations to modulate the Nqo1 gene expression without
significantly affecting cell viability (Anwar-Mohamed and El-Kadi, 2008). Initially, TCDD alone caused 470% increase in Nqo1 mRNA levels
that was inhibited in a dose-dependent manner by V5+, starting at the lowest concentration tested which is 25 μM (45%), and reaching the
maximum inhibition at the concentration of 250 μM (80%) (Fig. 1A).
Concentration-dependent effect of V5+ on TCDD-mediated induction of Nqo1 protein and catalytic activity
To examine whether the observed inhibitory effect of V5+ on the Nqo1 mRNA is reflected at the protein and catalytic activity levels,
 
 

 

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