TARGETING NQO1 IN COLORECTAL CANCER
Chaithanya Ganji
BACKGROUND MATERIALS AND METHODS
Colorectal cancer (CRC) is the third most common cancer in Materials:
the U.S.A. and is an aggressive malignancy (Zheng et al., 5 ml of XTT reagent
100 µl of Activation reagent
2021). In patients with CRC, studies have indicated an Water bath at 37 °C
elevated level of Nicotinamide Adenine Dinucleotide 96 well microplate
Phosphate Hydrogen (NAD(P)H): Quinone Oxidoreductase 1 ELISA Microplate Reader (450nm; 660nm)
Multichannel Pipettes (100 µl and 50 µl)
(NQO1) which is involved with the electron transport chain
75% Ethanol
and detoxification (Belinsky et al., 1993). An elevated level Trypsin-EDTA
of NQO1 could indicate CRC and help with early detection Sterile 1XPBS from the Sigma company
which would greatly improve the survival rate (Nebert et al., HCT-116 and RKO cell lines (ATCC)
Eagle's Minimum Essential Medium (EMEM) with
2002). The current study is to evaluate the association
10% Fetal Bovine Serum (FBS)
between NQO1 polymorphisms and CRC risk. Furthermore, McCoy's 5A Medium with 10% fetal Bovine
the study explores the binding capacity and efficacy of BBI SerumNQO1 Antibody (Cell signaling Technology)
608 against NQO1 in CRC through molecular docking. The Anti-Rabbit antibody (Cell signaling Technology)
Pierce™ BCA Protein Assay Kit (Thermo Scientific) studies unlike the pooled analysis (Figure 1).
inhibitor will then be tested in vitro using XTT assay for  iBlot 2 Dry Blotting System, Western Blot kit
CRC cell line proliferation. A Western Blot analysis will be (Thermo Scientific)
done to further determine the effects of BBI 608 on NQO1. ECL reagent (Thermo Scientific)
Sample buffer (Bio-rad)
EXPERIMENTAL DESIGN 2-mercaptoethanol (Sigma-Aldrich)
Actin antibody (Cell signaling Technology)
6 well plate
Meta-analysis  RIPA buffer (Cell sinaling Technology)
 Protease and Phosphatase inhibitor cocktail (Sigma-
Aldrich)
LICOR Imaging System (western blot)
 Gel electrophoresis kit (4-20% gradient gel from
BIORAD)
Methods:
Meta-analysis (Revman software), Molecular docking
AutoDock 4.2 tool), Cell proliferation (XTT assay) and
Western blotting (iBlot transfer).
Statistical analysis:
Control: C609T Result acquired from experimental groups were
Polymorphism evaluated using one-way ANOVA by Neumann-Keuls
Independent Variables: test. Data were shown as mean ± SD, that were attained
Genotype and Race from 12 replicates.  Data analysis was performed using
Dependent Variable: CRC GraphPad Prism software (La Jolla, CA).  
Molecular Docking Risk
All Charts and Images were made
by C. Ganji
OBSERVATIONS
Control: Cell lines, BBI
608
Independent Variable:
Dosage
Dependent Variable:
Cell Proliferation
Figure. 1. A Forrest plot for the meta-analysis performed on the
CRC cell line Western Blot relationship between the C609T polymorphism of NQO1 and
proliferation Protocol CRC.
Figure. 2. Funnel plot which shows publication bias at 95%
confidence interval. Most of the studies are within the pyramid
which shows there is minimal publication bias.
Figure. 3. Sensitivity Analysis (forest plot). It shows the effects of individual
. visualizers like LigPlot+ tool. The interactions indicated
Figure 4. Intermolecular hydrogen bonding, and electrostatic interactions
formed between NQO1- Napabucasin complex, the images are drawn by
LigPlot+ tool.
Figure 5. Functions of BBI 608 interacted amino acids (Leu, Leucine; Thr,
Threonine; Gly, Glycine; Tyr, Tyrosine) for mTOR (Mammalian target of
rapamycin) signaling and colorectal cancer (CRC) growth and metastasis.
Dose-dependent BBI 608 on cell proliferation
Figure 6. Dose dependent effect of BBI 608 on HCT 116 (left) and
RKO (right) CRC cell lines. One way ANOVA (***p<0.001). Data
were shown as mean ± SD.
Figure 7. Western blot analysis showed that BBI 608 decreased
the expression of NQO1 in both CRC cell lines. β-actin acted as
a loading control.
RESULTS
• Figure 1 shows that the TT & CT vs CC has an odds
ratio of 1.19 which is greater than 1 showing that there is
a higher risk of CRC in people with the T allele.
• Figure 2 is proof that there was minimal publication
bias.
• Figure 3 shows the odds ratio of each individual study
and is a sensitivity analysis.
• A docking of BBI 608 with NQO1 was performed using
ADT. The most efficient binding energy identified for
complex NQO1- BBI 608 was -7.04 kcal/mol as
depicted in figure 4. Intermolecular interactions between
NQO1 and BBI 608 were investigated through structure
that BBI 608 binds to the surface of target protein
NQO1, through six H-bonds that is constituted of amino
acids Leu103, Thr147, Gly149 (2 H-bonds), Gly150, and
Tyr155, which are at an average atomic distance of
~3.05Å. Furthermore, an MD (Molecular Dynamics)
simulation could be performed, and an analysis could be
done of NQO1-BBI 608 complex for a better
understanding of the possible mode for binding of ligand
with target protein and its stability.
• A XTT viability assay was performed to evaluate anti-
proliferative effect of BBI 608 on CRC cell lines. CRC
(HCT 116 and RKO) cell lines were exposed to varied
concentrations of BBI 608 (1, 2 and 3 µM) for 24h. The
half maximal inhibitory concentrations for both CRC
cell lines were 3µM for BBI 608 (Figure 6).
• Western blot analysis showed a clear decrease in
NQO1 due to BBI 608 in both CRC cell lines (Figure 7).
CONCLUSIONS
• NQO1 is a viable biomarker and targeted molecule in
CRC. The C609T polymorphism which is associated with
the activity level of NQO1, is shown to increase risk of
CRC in Asians and Caucasians. Computational analysis
showed that BBI 608 potentially interacted with NQO1
and can be used as a therapeutic drug. Furthermore, in
vitro results showed that inhibiting NQO1 with the drug
BBI 608 decreased cell proliferation in both CRC cell
lines. Western Blot analysis showed BBI 608 decreased
NQO1 in both CRC cell lines, indicating that BBI 608
could possibly be used as a therapeutic drug.
• BBI608 and the combination of chemotherapy (5-
Fluorouracil) could be a possible approach for CRC
therapy. Knockout or overexpression or site directed
mutagenesis is essential for a better understanding about
BBI 608 targeted NQO1 and its amino acids. These
observations increase the understanding of the function of
NQO1 in CRC. Clinical trials for humans can be
performed to analyze any possible side effects.
REFERENCES
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Nebert, D. W., Roe, A. L., Vandale, S. E., Bingham, E., & Oakley, G. G. (2002).
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benzene, and predisposition to disease: a HuGE review. Genetics in Medicine,
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Zheng, Y., & Wang, Z. (2021). Interpretation of global colorectal cancer statistics.
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