mechanisms of cell death and role in cancer thrapy

1. Cell Death – Mechanisms
K Siva Prasad

2. • The balance between cell viability and cell
death is critical for all organisms, and involves
a network of proteins and communication
amongst several signalling pathways.

3. Why ??
• Interesting Fact!
Without apoptosis, 2 tons of bone
marrow and lymph nodes and a 16-km
intestine would probably accumulate in a
human by the age of 80

4. • Impaired cell death is associated with various
human pathologies, including
1. Immunodeficiency
2. Autoimmunity
3. neurodegenerative diseases
4. and cancer
• Similar to proliferation, cell death is also essential
for embryonic and postnatal development and
for tissue homeostasis.

5. What is Death ?
In the absence of a clearly defined biochemical
event that can be considered as the point-of no-
return,
the NCCD proposes that a cell should be
considered dead when any one of the following
molecular or morphological criteria is met:
(1) the cell has lost the integrity of its plasma
membrane, as defined by the incorporation of
vital dyes (e.g., PI) in vitro;

6. (2) the cell, including its nucleus, has undergone
complete fragmentation into discrete bodies
(which are frequently referred to as ‘apoptotic
bodies’); and/or
(3) its corpse (or its fragments) has been
engulfed by an adjacent cell in vivo.

7. Cell death can be classified according to its:
1. Morphological Appearance (which may be
apoptotic, necrotic, autophagic or associated
with mitosis),
2. Enzymological Criteria (with and without the
involvement of nucleases or of distinct classes of
proteases, such as caspases, calpains, cathepsins
and transglutaminases),
3. Functional Aspects (programmed or accidental,
physiological or pathological)
4. Immunological Characteristics (immunogenic or
nonimmunogenic).

8. Three Pathways of Cell
Death
APOPTOSIS
NECROSIS
AUTOPHAGY

9. Life and Death Matter
• Autophagy, which has been proposed as a third mode
of cell death, is a process in which cells generate
energy and metabolites by digesting their own
organelles and macromolecules
• Autophagy allows a starving cell, or a cell that is
deprived of growth factors, to survive.
• However, cells that do not receive nutrients for
extended periods ultimately digest all available
substrates and die (autophagy-associated cell death)

11. • While initially most of the focus has been
placed on apoptosis, which is a highly
regulated mechanism involving interplay
among a large number of pro and
antiapoptotic proteins,
• mounting evidence has emerged to support
the genetic regulation of necrosis

15. Apoptosis
• Apoptosis (or type I programmed cell death) is
a genetic pathway for rapid and efficient
killing of unnecessary or damaged cells that
was initially described by Vogt (1842)
• Apoptosis (commonly pronounced ap-a-tow' -
sis), coined from the Greek apo or from, and
ptosis or falling, to make the analogy of leaves
falling off a tree.

18. Caenorhabditis elegans

20. Triggers ....
• Apoptosis is triggered in mammalian cells in
response to
• endogenous stimuli (eg, growth factor
deprivation) or exogenous stimuli (eg, irradiation
or genotoxic chemotherapeutic drugs).
• Apoptosis is also induced in response to
inadequate cell–matrix interactions and this
specific type of apoptosis is known as anoikis.
• Physiological during development

21. BCL-2 Family Members and Their Roles in
Apoptosis
• The mammalian Bcl-2 gene, a homolog of the
C. elegans Ced-9, is a prototypical member of
the BCL-2 family that includes
• antiapoptotic proteins such as BCL-2, BCL-XL,
• BCL-W, A1, and MCL-1, and
• proapoptotic proteins, such as BAX, BCL-XS,
BAK, BAD, BIK, BIM, BID, and PUMA

22. BAX
BCL-XS
BAK
BAD
BIK
BIM
BID, and PUMA
• BCL-2
• BCL-XL
• BCL-W
• A1
• MCL-1

23. • “BCL-2 homology” (BH) regions including BH1,
BH2, BH3, and BH4
• These domains allow the formation of homo-
and heterodimers between BCL-2 family
members and are essential for the function of
these proteins.

25. • Overexpression of BCL-2 also prolongs the
survival of cells and increases their resistance
to a variety of apoptotic stimuli including
1. glucocorticoids,
2. phorbol esters,
3. ionomycin,
4. anti-CD3 monoclonal antibodies,
5. irradiation,
6. and chemotherapeutic agents.

26. Caspases
• “cysteine-dependent aspartate specific
proteases,” are the orthologs of the C. elegans
protein Ced-3.
• The first caspase (ICE or CASP1) was identified
in 1993 on the basis of its similarity to Ced-3.
• More than 14 mammalian caspases have since
been cloned

30. • Although caspases are primarily known for
their apoptotic function,
• caspases such as CASP1 and CASP11 play
important roles in inflammation.
• Mounting evidence supports that CASP8 also
possesses nonapoptotic functions.

31. • “initiators” (eg, CASP8, CASP9 and CASP10) or
“executioners” of apoptosis (eg, CASP3,
CASP6, and CASP7).
• Initiator procaspases contain large
prodomains,
• whereas executioner procaspases contain
small prodomains.

32. • Hundreds of caspase substrates have been
identified
1. cytoskeleton proteins (eg, Actin and Gelsolin),
2. nuclear proteins (eg, Lamin A and B),
3. proteins involved in DNA damage repair (eg,
PARP, RAD51, and DNA-PKcs),
4. cell-cycle proteins (eg, p21, p27, CDC27, and
RB),
5. cytokines (eg, IL-1β and IL-18)
6. apoptotic proteins (eg, Caspases, BCL-2, BCL-XL,
BID, BAX, and ICAD)

33. • The caspase-activated DNase (CAD) is inactive
when associated with its inhibitor ICAD (also
known as DNA fragmentation factor [DFF]).
• In response to apoptotic stimuli, ICAD is
cleaved by caspases allowing the release of
the active endonuclease CAD to cleave DNA of
apoptotic cells to produce internucleosomal
DNA cleavage, a characteristic of apoptosis.

34. The Death Receptor
Apoptotic Pathway
• The death receptor or extrinsic apoptotic
pathway is initiated by the interaction of the
death receptors, members of the tumor
necrosis factor (TNF) receptor superfamily,
with their ligands

35. • The death receptors share the presence of an
approximately 80-amino-acid motif known as
the death domain (DD) in their cytoplasmic
tails.
• Six human death receptors have been
identified and include FAS (also known as
CD95 or APO-1), TNFR-1, death receptor (DR)
3, DR4 (also known as TRAILR1), DR5 (also
known as TRAILR2), and DR6.

36. • Cellular FLICE-like inhibitory protein (cFLIP), a
pseudo-CASP8 protein with a nonfunctional
catalytic domain, inhibits the death receptor
apoptotic pathway by precluding the
recruitment of CASP8 to the DISC.

40. The Mitochondrial
Apoptotic Pathway
• Intrinsic apoptotic pathway, activate BAX and
BAK and lead to mitochondrial outer
membrane permeabilization and the release
of a number of proteins important for
apoptosis (eg, Cytochrome C, SMAC, and OMI)

42. • 8 human IAPs have been identified, including
the X-linked IAP (XIAP), Survivin, and cellular
IAP1 and -2 (c-IAP1 and c-IAP2).
• Several IAPs bind directly to caspases such as
CASP3, -7, and -9 and inhibit their functions.
• This IAPs-mediated inhibition of caspases can
be antagonized by the proteins SMAC and
OMI

43. • When SMAC and OMI are released from the
mitochondrial intermembrane space into the
cytosol, they bind directly to XIAP and
suppress its inhibition of caspases.

45. Cross Talk
• Communication between the death receptor
and mitochondrial apoptotic pathways is best
demonstrated by CASP8 cleavage of BID.
• This cleavage generates a truncated form of
BID (tBID) that cooperates with BAX to form
openings in the outer mitochondrial
membrane, leading to release of proteins
including Cytochrome C from the
mitochondrial intermembrane space

47. Animal Models
• Bcl-2−/− mice die by 6 weeks of age.
• Inactivation of Bcl-XL in mice results in
embryonic lethality by day 14 of gestation
• Bax−/− mice display male sterility and mild
lymphoid Hyperplasia
• perinatal lethality was observed in
Bax−/−Bak−/− mice.

48. • The surviving Bax−/−Bak−/− mice also
accumulate cells of the central nervous
• system and fail to remove their interdigital
webs.

49. • Casp3−/− and casp9−/− mice exhibit a similar
brain defect characterized by ectopic masses
of supernumerary cells that escape apoptosis
during brain development
• Autoimmune lymphoproliferative syndrome
(ALPS), mutations of FAS or FASL

50. Necrosis
• Necrosis is derived from the Greek word
nekros for corpse, and it involves rapid
swelling of the cell, loss of plasma membrane
integrity, and release of the cellular contents
into the extracellular environment, resulting in
an acute inflammatory response.

51. • a type of programmed necrotic cell death, called
necroptosis has been identified as induced by
interaction of death domain receptors with their
respective ligands under conditions of defective o
inhibited downstream apoptotic machinery.
• Although necrosis was thought initially to be a
nonregulated process, recent studies
demonstrate that necroptosis, a programmed
type of necrosis, is highly regulated by a network
of proteins

53. Role in Cancer