INHIBICION OF TXNRD

1. 1
María Alejandra Alvarez Betin
Third semester of Medicine
Universidad Pontificia Bolivariana.
Teacher: Lina Maria Martinez Sanchez.

2. Introduction
NRF2: protein that controls the way in which certain
genes are expressed, acts as the primary cellular barrier
against the harmful effects of oxidative stress by
regulating the expression of cytoprotective genes when
it is next to Protein 1 associated with EACH similar to
Kelch
β- LAPACHONE: is an ortho naphthoquinone, with
potential antineoplastic and radiosensitizing activity, is
bioactivated creating a oxidoreduction that generates
high levels of superoxide.
2

3. Introduction
TXNRD: Thioredoxin reductase is a major
antioxidant and redox regulator in mammalian
cells, it has roles in preventing and
promoting/sustaining cancer.
SOD1: this gene provides instructions for
making the enzyme superoxide dismutase.
This enzyme attaches to molecules of copper
and zinc to break down toxic, charged oxygen
molecules

4. Overall Objective
Determine whether inhibition of TXNRD or SOD1 overcomes NRF2-
mediated resistance to βlapachone
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5. “1.Methods and
Materials
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6. Cell line generation
6
For what?
Used to obtain information on
the relationship between the
activation / inhibition of
different pathways and their
effects on gene expression
Basis
Stable cell lines were
generated via lentiviral
transduction for 6h. Twenty-
four hours after transduction,
puromycin was added to the
growth medium for 72 h to
select for infected cells.
KEEP1 WT
KEEP1 MUT

7. Western Blot
7
For what?
It is used to study proteins,
estimate their size and
confirm the presence of post-
translational modifications.
Basis
Electrophoresis to separate
proteins
they are transferred to the
surface of a membrane and
exposed to an antibody.
Antibody binding is detected
using a radioactive or chemical
marker.

8. Fluorescence
8
For what?
Visualization and study of the
cellular location of biological
structures that have been
previously excited
quantification and
determination of total
antioxidant capacity
Basis
distribution of an antigen in a
group of cells to direct
fluorescent markers to a
target molecule and observe
through microscopy

9. Nuclear isolation
9
For what?
studying processes such as
nuclear-cytoplasmic shuttling,
and protein–chromatin or
nuclear protein–protein
interactions in response to
diverse stimuli
Basis
dissolution of the cytoplasmic
membrane, using a low
concentration of a nonionic
detergent and fast
centrifugation steps.

10. Results
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FIG 1
KEAP1MUT cell lines
displayed uniformly high
NQO1 protein levels,
while protein levels of
NQO1 in KEAP1WT cells
were highly variable
Overexpression of
KEAP1 C273S and
NRF2 T80K led to the
accumulation of NRF2,
which promoted
resistance to β-
lapachone exposure
NRF2 silencing by
shRNAs markedly
reduced resistance to
β-lapachone in H460
cells (KEAP1MUT)(

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Results FIG 2
A time-dependent
accumulation of DNA
damage in H1299 cells
(KEAP1 WT).
KEAP1WT cells
accumulated γ-H2AX
following β-lapachone
exposure

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Results FIG 2
In H1299 cells promoted
resistance to βlapachone-
induced DNA damage and
decreased accumulation
of ROS
shRNA-mediated silencing of
NRF2 in H460 and HCC15 cells
increased γ-H2AX levels
following β-lapachone exposure

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Results FIG 3
Depletion of catalase did not
affect the β-lapachone
sensitivity of NSCLC cells.
auranofin significantly
increased β-lapachone-
induced cell death and DNA
damage in KEAP1MUT cells

14. DISCUSSION
14
Author
D. Zhang, J. Rennhack,
E.R. Andrechek, C.E.
Rockwell, K.T. Liby,
what he said
Aberrant NRF2 activation
promotes resistance to
therapeutics that rely on
the production of ROS.
Or
A.P. Lundberg, et al Isobutyl-
deoxynyboquinone (IB-
DNQ) is being developed
for clinical use
I.S. Harris, et al KEAP1MUT cells were
highly resistant to
auranofin compared to
KEAP1WT cells

15. 15
CONCLUSION
• I think it is a good investigation because it is about helping people
with cancer which is a really degenerative and complicated disease
for the patient and their family
• I consider that with the molecular biology they could realize more
studies with organisms like the B-lapachone that are not as
invasive as chemotherapy

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