exact how and from where the autophagosome emerges is unspecified but a
research has identified a site called pre-autophagosomal structure (PAS).
Autophagy-related (ATG) proteins are the main substance involved in the PAS
leading to autophagosomal production. The discovery of ATG proteins in the
1990s greatly advanced the understanding of autophagy and clarified that
autophagy serves important roles in various biological processes. (Shibutani
ST1,2, ). Phagophore, autophagosome and autophagolysosome formation are
regulated by at least 30 ATG proteins. (I. M. Aparicio).
In recent years, the analysis of knockout models of ATG genes in mice
revealed new knowledge about the functions of autophagy in mammalian cell
development and differentiation. Embryos were divided into groups with various
ATG genes deleted e.g. ATG5, ATG7, ATG3, etc…and and it was shown that deletion
of some ATG genes leads to lethality mid-embryonic development, and the mice
that survive postnatal period display some developmental abnormalities
including an atypical lymphocyte differentiation. (Mizushima).
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. )