MECHANISMS OF CELL DEATH- APOPTOSIS AND

AUTOPHAGY
INTRODUCTION

• Autophagy and apoptosis are catabolic pathways essential for organismal homeostasis.

• Autophagy and apoptosis control the turnover of organelles and proteins within cells, and of cells within organisms

• Autophagy is a cell-survival pathway involving the degradation and recycling of obsolete, damaged, or harmful
macromolecular assemblies

• Apoptosis is the canonical programmed cell death pathway.

• Both are interconnected by several molecular nodes of crosstalk, enabling the coordinate regulation of degradation
by these pathways
• Autophagy and apoptosis are both tumor suppressor pathways.

• Autophagy fulfils this role as it facilitates the degradation of oncogenic molecules, preventing development of
cancers, while apoptosis prevents the survival of cancer cells.

• defective or inadequate levels of either autophagy or apoptosis can lead to cancer.

•  A comprehensive understanding of the interconnectivity of autophagy and apoptosis is essential for the
development of effective cancer therapeutics.
AUTOPHAGY- definition

• Autophagy is a cell-survival pathway conserved in all eukaryotes.

• It involves the selective degradation of cellular components, including long-lived proteins,

protein aggregates, damaged cytoplasmic organelles intracellular pathogens,

• Resulting in the recycling of nutrients and the generation of energy 

• Type 2 cell death

• Autophagy is upregulated under stress conditions, including extracellular stress such as nutrition
deprivation, hypoxia, and infection and intracellular stress such as that caused by accumulation of
damaged proteins and organelles and high bioenergetic demands.

• in mammals, it is thought to be involved in many physiological and pathophysiological processes,

including antiaging mechanisms, differentiation and development, immunity, and elimination of microorganisms
• Autophagy is a highly regulated process executed by autophagy-related effectors, many of which are called ATG
proteins.

• The first committed step of autophagy is vesicle nucleation in which macromolecular assemblies selected for
degradation are surrounded by isolation membranes called phagophores.
• initiation of the preautophagosomal membranes, which can be derived from the endoplasmic reticulum (ER) , begins
with the activation of the ULK1 kinase complex.

• This complex is activated by cellular stress via mTORC inhibition and/or AMP-activated protein kinase (AMPK)
activation

• ULK1 phosphorylates Atg13 and Fip200 to form an ULK1/2-mAtg13-Fip200 complex that is stabilized by Atg10

• ULK1 activation promotes the recruitment of a multiprotein complex with class III phosphatidylinositol 3-kinase
(PI3K) activity.

• This complex consists of 4 proteins which are scaffolded by Beclin-1, whose role upon release from the antiapoptotic
protein Bcl-2, is to activate the vacuolar sorting protein Vps34.

• Maturation of the growing autophagosome membrane requires the complexes to recruit two ubiquitin-like
conjugation systems.
• Both these systems involve the E1-like Atg7 which initiates the conjugation of LC3 with
phosphatidylethanolamine (LC3/PE) and Atg5 with Atg12.

• Incorporation of these complexes into the autophagosome membrane is an essential

process. Likewise, Atg4, the protease that cleaves and thereby activates LC3, is required
the formation of the LC3-PE complex

• To end the program, the autophagosome fuses with the lysosome to form the
autophagolysosome.

• The SNARE protein Stx17 is essential for this process.

• Upon completion, the contents of the autophagosomes are degraded by the lysosomal
hydrolases producing amino acids and lipids for protein and other macromolecular
synthesis and metabolism.
Apoptosis

• Apoptosis is the morphologic description of a form of programmed cell death critical to physiologic homeostasis 

in almost every organ system
Type 1 cell death

• Apoptosis is mediated by a series of effectors (e.g., caspases) and regulators

proceeds by one of two major pathways:

• the extracellular pathway, triggered by the binding of a ligand to a death receptor;

• the intracellular pathway, triggered by mitochondrial events after stress signals from within the cell.
• The intrinsic/ intracellular pathway is characterized by pro- and antideath signals converging at mitochondrial
membranes.

• These consequently become permeabilized (MOMP—mitochondrial outer membrane permeabilization), leading

to the release of mitochondrial intermembrane proteins including cytochrome c.

• Rapid cell death follows as MOMP triggers both caspase activation on the apoptosome and blocks caspase
inhibitors.

• this starts a cascade of active caspases which cleave hundreds of cellular substrates ending in cellular demise.

• The Bcl-2 family of proteins consists of proapoptotic and prosurvival proteins which together control MOMP.

• Under basal conditions, the prosurvival proteins, Bcl-2, Bcl-XL, and Mcl-1, inhibit MOMP in two ways. First,
they directly bind and inhibit the proapoptotic effector proteins Bax and Bak, which form the pores in the
mitochondrial membrane.

• Second, they bind to BH3-only proteins such as Bim which prevents them from activating Bax
• The extrinsic receptor-mediated apoptosis pathway is triggered by the ligation of death receptors with their cognate
ligands.

• This stimulates receptor clustering resulting in the recruitment of cytoplasmic adapter proteins, important amongst
which is Fadd.

• Fadd then associates with procaspase-8 leading to the formation of a death-inducing signaling complex (DISC).

• This results in the dimerization and catalytic activation of caspase-8, which can then directly cleave and activate
caspase-3

• Both intrinsic and extrinsic pathways result in caspase-3 activation that is linked to the initiation of the execution
phase of apoptosis.

• Crosstalk between the two pathways is mediated by caspase-8 cleavage and activation of BID.

• BID is a BH3-interacting domain death agonist, the product of which (truncated BID; tBID) is required in some cell
types for death receptor-induced apoptosis
• The execution phase of apoptosis involves the activation of a series of caspases.

• The upstream caspase for the intrinsic pathway is caspase 9 while that of the extrinsic pathway is caspase 8.

• The intrinsic and extrinsic pathways converge to caspase 3.

• Caspase 3 then cleaves the inhibitor of the caspase-activated deoxyribonuclease, which is responsible for
nuclear apoptosis

• In addition, downstream caspases induce cleavage of protein kinases, cytoskeletal proteins, DNA repair
proteins and inhibitory subunits of endonucleases family.

• They also have an effect on the cytoskeleton, cell cycle and signalling pathways, which together contribute to
the typical morphological changes in apoptosis 
 Morphological changes in apoptosis

Morphological hallmarks of apoptosis in the nucleus are chromatin condensation and nuclear fragmentation,
which are accompanied by rounding up of the cell, reduction in cellular volume (pyknosis) and retraction of
pseudopodes

Chromatin condensation starts at the periphery of the nuclear membrane, forming a crescent or ring-like
structure.

The chromatin further condenses until it breaks up inside a cell with an intact membrane, a feature described as
karyorrhexis.

The plasma membrane is intact throughout the total process.

At the later stage of apoptosis some
of the morphological features
include membrane blebbing,
ultrastrutural modification of
cytoplasmic organelles and a loss of
membrane integrity.

Usually phagocytic cells engulf

apoptotic cells before apoptotic
bodies occur.

If the remnants of apoptotic cells

are not phagocytosed such as in the
case of an artificial cell culture
environment, they will undergo
degradation that resembles
necrosis and the condition is
termed secondary necrosis 
What happens in cancer cells
Targeting caspases    
Caspase-based drug therapy Apoptin reported to selectively induce Rohn et al, 2004 [100]
apoptosis in malignant but not normal cells

  Small molecules caspase activators reported Philchenkov et al., 2004 [101]

to lower the activation threshold of caspase
or activate caspase, contributing to an
increased drug sensitivity of cancer cells

Caspase-based gene therapy Human caspase-3 gene therapy used in Yamabe et al., 1999 [102]
addition to etoposide treatment in an AH130
liver tumour model reported to induce
extensive apoptosis and reduce tumour
volume

  Gene transfer of constitutively active caspse-3 Cam et al., 2005 [103]

into HuH7 human hepatoma cells reported to
selectively induce apoptosis

  A recombinant adenovirus carrying Li et al., 2007 [104]

immunocaspase 3 reported to exert
anticancer effect in hepatocellular
carcinoma in vitro and in vivo
Current active trials obtained from clinicaltrials.gov search on August 18, 2019 [67].

Tumor Type NCT Number Anticancer Therapy Autophagy Inhibitor Molecular Marker (if applicable)

Advanced Solid Tumor NCT01266057 Vorinostat, sirolimus HCQ

Advanced Solid Tumor NCT01023737 Vorinostat HCQ

Advanced Solid Tumor NCT00813423 Sunitinib HCQ

Advancer Solid Tumor NCT01480154 MK2206 (AKT inhibitor) HCQ

Breast Cancer NCT03774472 Palbociclib, letrozole HCQ Estrogen Receptor +, HER2

Negative
Breast Cancer NCT03400254 Gedatolisib HCQ
Breast Cancer NCT03032406 Everolimus HCQ
Colorectal Cancer NCT02316340 Vorinostat HCQ
Colorectal cancer NCT03215264 Entinostat, regorafenib HCQ

Glioblastoma Multiforme NCT03008148 Temozolomide, radiation Siroquine

Glioblastoma Multiforme NCT03243461 Temozolomide, valproic acid CQ

Stage of autophagy Target Drug Status

ULK1/2 SBI-0206965 Pre-clinical

Initiation ULK1/2 MRT67307 Pre-clinical

ULK1/2 MRT68921 Pre-clinical

VPS34 VPS34-IN1 Pre-clinical

VPS34 SB02024 Pre-clinical

Nucleation
VPS34 PIK-III Pre-clinical

PIK3C3/VPS34 SAR405 Pre-clinical

ATG4B NSC185058 Pre-clinical

Expansion/elongation ATG4B UAMC-2526 Pre-clinical

ATG4B S130 Pre-clinical

Lysosome CQ Clinical trial

Lysosome HCQ Clinical trial

Fusion
Lysosome Lys05 Pre-clinical

Lysosome/mTORC1 DQ661 Pre-clinical

PPT1 HCQ Clinical trial

Degradation PPT1 Lys05 Pre-clinical

PPT1/mTORC1 DQ661 Pre-clinical