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1
María Alejandra Alvarez Betin
Third semester of Medicine
Universidad Pontificia Bolivariana.
Teacher: Lina Maria Martinez Sanchez.
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
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
Overall Objective
Determine whether inhibition of TXNRD or SOD1 overcomes NRF2-
mediated resistance to βlapachone
4
“1.Methods and
Materials
5
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
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.
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
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.
Results
10
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)(
11
Results FIG 2
A time-dependent
accumulation of DNA
damage in H1299 cells
(KEAP1 WT).
KEAP1WT cells
accumulated γ-H2AX
following β-lapachone
exposure
12
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
13
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
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
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
16

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Seminario Biologia molecular

  • 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 4
  • 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 10 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)(
  • 11. 11 Results FIG 2 A time-dependent accumulation of DNA damage in H1299 cells (KEAP1 WT). KEAP1WT cells accumulated γ-H2AX following β-lapachone exposure
  • 12. 12 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
  • 13. 13 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
  • 16. 16

Editor's Notes

  1. Fundamento y utilidad
  2. Para examinar la influencia de las mutaciones KEAP1 / NRF2 en la respuesta de NSCLC al tratamiento con β-lapachona, evaluamos la eficacia citotóxica de β-lapachone en un panel de dieciséis líneas celulares de NSCLC, siete de las cuales se caracterizan por la activación de mutaciones de KEAP1. Además, incluimos las células InthestudyCalu3, que albergan una variante polimórfica de NQO1 (NQO1 * 3) que da como resultado niveles de enzimas 95% más bajos. F-determinar si la resistencia a β-lapachona dependía de NRF2, las células H1299 (KEAP1WT) se infectaron con un virus que codifica una mutación inactivadora de KEAP1 (C273S) o una mutación de ganancia de función NRF2 (T80K), para promover la activación aberrante de NRF2 De acuerdo con nuestros hallazgos anteriores, la sobreexpresión de KEAP1C273S y NRF2T80K pero no KEAP1WT condujo a la acumulación de NRF2, que promovió la resistencia a la exposición a β-lapachona G-H460 (KEAP1 MUT) infectados con virus que codifican shRNA contra NRF2 (shNRF2) o un control de shRNA, el silenciamiento de NRF2 por shRNA redujo notablemente la resistencia a β-lapachona en células H460 (KEAP1MUT) (Fig. 1G).
  3. A continuación, evaluamos si las células KEAP1MUT estaban protegidas contra el daño del ADN inducido por β-lapachona [27,40]. Controlamos los niveles de H2A.X fosforilado (γ-H2AX), un marcador molecular sensible del daño en el ADN. Observamos una acumulación de daño de ADN dependiente del tiempo en las células H1299 (KEAP1WT), mientras que las células A549 (KEAP1MUT) no exhibieron un aumento de γ-H2AX después del tratamiento de 2 h con β-lapachona 3 μM
  4. E- la expresión ectópica de KEAP1C273S o NRF2T80K en células H1299(WT) también promovió la resistencia al daño del ADN inducido por βlapachona y la disminución de la acumulación de ROS (Fig. 2E, F y S2A). (E) Evaluación del daño en el ADN de las células H1299 (KEAP1 WT) infectadas con un vector vacío (Control) o un virus que codifica la expresión de NRF2 T80K. A la izquierda, las células se expusieron a 2, 3 o 4 μM de β-lapachona. Niveles de proteínas de NRF2, NQO1, H2AX total (control de carga), Tubulina (control de carga) y el marcador de daño de ADNγ-H2AX (pS139) G-
  5. B- Ensayos de supervivencia de un panel de células NSCLC mutantes KEAP1 infectadas con virus que codifican shRNAs contra SOD1 (1, 2) o con un shRNA de control (shCTL). Las células NSCLC se trataron con vehículo (DMSO al 0,012%) o con β-lapachona 3 μM durante 2 h. La viabilidad celular se evaluó 48 h después del tratamiento. Se realizó la prueba estadística ANOVA unidireccional, seguida de la prueba de comparación múltiple de Dunnett. D-