In autophagy, ultimately decreasing cell viability, therefore

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In this
study, we used cell-lines one with ATG7 deleted gene (KP-4 ATG7-) and ATG5 one
with deleted gene (KP-4-ATG-5) generated using the LentivirusX CRISPR/Cas9
System- The lentiviruses encoding the components necessary for
CRISPR/Cas9-mediated genome editing for delivery to mammalian cells that are
difficult to transfect. Autophagy-related gene-5 (ATG-5) is one of the key
regulators of autophagic cell death. It has been widely regarded as a
protective molecular mechanism for tumor cells during the course of
chemotherapy and in recent studies on human gastric cancer, upregulation of
this gene was associated with chemoresistance.( Ge, Jie et al. ) Supporting
these facts are studies revealing that down-regulation of Atg5 expression
suppresses cell death and vacuole formation induced by IFN-gamma (Jong-Ok Pyo).

Inhibition of autophagy by ATG5 and ATG7 gene deletion causes an upregulation
of apoptotic markers in response to verapamil, the autophagy inducer used in this
project. In a research done May 2017, it was discovered that cancer cells
treated with verapamil induce an autophagy flux. Moreover, they found that
inhibition of autophagy in their cell-line (Colon cancer cells) via disruption
of the autophagy genes ATG5 and ATG7 caused an upregulation of apoptotic
markers (cleaved PARP and cleaved caspase 3) in response to verapamil.

Conclusively, it was found that this response is related to the activity of
LDHA as inhibition reduce both basal and verapamil-induced autophagy,
ultimately decreasing cell viability, therefore the potential of using
verapamil with an autophagy inhibitor for cancer treatment. (Orzechowski. A. )

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Another essential protein thought
to be involved in autophagy is the Microtubule-associated protein 1A/1B-light
chain 3B (MAP LC3B or simply LC3) from the ATG8 protein family. (Shpilka T).

During autophagy ATG genes, ATG5 and ATG7, among others, arrange a cascade of
reactions leading to lipidation of LC3. (Metlagel Z). When induced by nutrient
starvation a cytosolic form of LC3 (LC3-I) is conjugated to
phosphatidylethanolamine to form the LC3-phosphotadylethanolamine conjugate
(LC3-II). At the last step of autophagy, while the autolysosome is formed and
the structures within it degraded, LC3-II in the autosomal lumen is degraded by
lysosomal hydrolases. (Tanida I). The rate at which LC3-II is degraded and
replaced reflects therefore starvation-induced autophagic activity. Degradation
of LC3-II inside the autolysosome is estimated by measuring the amount of LC3-I
(cytosolic form) relative to LC3-II (membrane-bound lipidated form) and
comparison of two samples with and without lysosomal inhibitor treatment (e.g.

NH4Cl, Chloroquine, Baflomycine). (Mizushima, )

Under conditions in which autophagy is not induced, e.g.

nutrient-rich conditions, LC3-positive puncta barely exist. This is why the
appearance of LC3 positive puncta sugguest the induction of autophagy.

(Gniadek, T. J)

There is, however a downfall in the
interpretation of the increase in the number of LC3-II puncta. The
autophagosome is a transient and dynamic double-membrane structure. LC3-II is
associated with the outer surface of the outer membrane and inner surface of
the inner membrane. When the autophagosome matures into an autolysoeome, the
outer membrane is liberated into the cytosol and so is the membrane bound
LC3II. These LC3II proteins are delipidated through a second cleavage and is
converted back into the LC3-1 form and probably later reused. The inner
membrane with LC3-II remain trapped within the autolysosome and everything is
finally degraded by the lysosomal enzymes. (Ichimura, Y). ) Imagine that if the
maturation process fails to occur for some reason, then the degradation of the
inner membrane and LC3-II does not take place resulting in an increase LC3-II
due to the accumulated autophagosomes, therefore the maturation process must be
further examined to determine wether or not autophagy is induced and if it goes
into completion. (Kabeya, Y). This is why treatment of autophagy-induced cells
with a lysosomal inhibitor block the decrease of LC3-II form, and this analysis
can be used to determine wether autophagic maturation occurs normally and goes
into completion or not. So jf there is no increase in the LC3-II amount when
cells are treated with lysosomal inhibitors e.g. ammonium chloride, then
autophagic maturation is not occurring normally and this interfers with the
study as we want to make sure that autophagy was induced into completion by
e.g. verapamil, so that it could have been inhibited by e.g. SO and not
somewhere along the path. (Shunsuke Kimura,)

This lysosomal LC3-II is therefore considered an
autophagic marker and its turnover can be detected by immunoblotting or
immunofluorescence due to their difference in mobility, which was the method of
choice in this study (Immunoblotting also referred to as Western blotting)
since it has been a reliable method for monitoring autophagy and
autophagy-related processes, including autophagic cell death, termed autosis.

(Tanida I). Thus,
the amount of LC3-II provides a good index of autophagy induction such as with
verapamil, but the rate of increase or expression of these molecules depends on
the cell type. Due to this an agreement has emerged whereby overall levels of
LC3-II are normalized to a loading control such as ?-actin (as in this study) or ?-tubulin. (Kimura S, Fujita N, Noda T,
Yoshimori T. Monitoring autophagy in mammalian cultured cells through the
dynamics of LC3. Methods Enzymol. 2009;452:1–12)

The increase of levels of LC3-II are not measures of
autophagic flux by definition, but can reflect the induction of autophagic
sequestration and/or inhibition of autophagosome clearance. It is not exactly
known what LC3II exact  mechanism in
autophagy, thus is may be possible to execute autophagy in the absence of
LC3-II. (He C, Klionsky DJ. Annu Rev Genet.).

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