Apoptosis: Influence of the Xenobiotics and Diseases conditions.

1. Apoptosis: Influence of the
Xenobiotics and Diseases conditions
Dr. Naveen Kumar
PhD Scholar
Veterinary Pharmacology and Toxicology
College of Veterinary and Animal Sciences,
G.B.P.U.A. & T., Pantnagar-263145,
Uttarakhand, INDIA
Email: knaveen7v@gmail.com

2.  Apoptosis derived from Greek "falling off" (as for autumn leaves)
 A distinct reaction pattern which represents programmed single-cell
suicide
 Cells actually expend energy in order to die
 The physiological way for a cell to die
What is Apoptosis

3. A peculiar well controlled individual cell death that is
caspase mediated
Fragmentation of the cell and organelles
Engulfed by macrophages

4.  The Nobel prize in physiology or medicine 2002 was awarded jointly- for
their discoveries concerning “genetic regulation of organ development and
programmed cell death” .
PRIZED
Sydney Brenner H. Robert Horvitz John E. Sulston

5. Importance of ApoptosisImportance of Apoptosis
1) Crucial for embryonic development
Errors in Apoptosis can lead to Birth Defects
2) Important for maintaining homeostasis
Cell death is balanced with mitosis to regulate cell number.
3) Improper regulation contributes to human disease
- Neurodegenerative diseases
Parkinson’s
Alzheimer’s
- Cancer
- Autoimmune diseases e.g. (diabetes type I)
- Viral diseases

6. CharacteristicsCharacteristics
An active cytological process
It is programmed or controlled by genetic protocol
It may be triggered by intrinsic or extrinsic stimuli
It occurs in almost all living creatures

7. Morphology
• Cell shrinkage (condensation of cytoplasm)
 Breakdown of mitochondria; release of cytochrome C
 Nuclear condensation
 Nuclear fragmentation
 Cell membrane blebbing
 Fragmentation; apoptotic body formation: membrane-bound
cellular fragments, which often lack nuclei
 Phagocytosis

8. APOPTOSIS: important in embryogenesis
Morphogenesis (eliminates excess cells):
Selection (eliminates non-functional cells):

9. APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):
Virgin mammary gland Late pregnancy, lactation Involution
(non-pregnant, non-lactating)
Apoptosis
Apoptosis
- Testosterone
Prostate gland

10. Programmed Cell Death in Neuronal DevelopmentProgrammed Cell Death in Neuronal Development
 The cells in the tadpole tail are induced to undergo apoptosis stimulated by
the increases in thyroid hormone that occurs during metamorphosis
Apoptosis during the metamorphosis of a tadpole into a frog

11. Apoptosis in the development of the nervous system

12. STAGES OF CLASSIC APOPTOSISSTAGES OF CLASSIC APOPTOSIS
Healthy cell
DEATH SIGNAL (extrinsic or intrinsic)
Commitment to die (reversible)
EXECUTION (irreversible)
Dead cell (condensed, crosslinked)
ENGULFMENT (macrophages, neighboring cells)
DEGRADATION

13. How Apoptosis Differs from Necrosis?How Apoptosis Differs from Necrosis?

14. Cellular changes associated with apoptosisCellular changes associated with apoptosis
Early: Chromosome
condensation, cell body shrink
Later: Blebbing, Nucleus and
cytoplasm fragment-Apoptotic
bodies
At last: Phagocytosed

15. Necrosis
Apoptosis
Sequence in ultra structure change in apoptosis (2-6)
and necrosis (7-8)
Apoptosis versus Necrosis

16. Normal white blood cell Apoptotic white blood cell
During apoptosis (programmed cell death) cells bleb
and eventually break apart without releasing contents.

17. Stimuli for Apoptotic Cell DeathStimuli for Apoptotic Cell Death
(in Mammals)(in Mammals)
I. Growth factor deficiencies
II. Ionizing radiation and anticancer drugs damage DNA and
apoptosis follows ( p53 expression)
i. Viral infection (eg.-HIV- infected cells make high levels of
FasL which will induce apoptosis in HIV-uninfected T cells)
ii. Free radical toxicity

18. i. Hypoxia- apoptosis (if mild) or necrosis if the hypoxia is severe
or prolonged
ii. Death receptor activation (such as Fas or CD95
triggering)
iii. Metabolic or cell cycle perturbation
iv. Misfolded proteins

19. Stimuli for Apoptotic Cell DeathStimuli for Apoptotic Cell Death

20. Biochemical Events in ApoptosisBiochemical Events in Apoptosis
Caspases (cysteine proteases) cleave the cytoskeleton and
activate DNAses and other enzymes
DNA breaks into 50- to 300-kilobase pieces; further broken
into multiples of 200 base pairs by endonucleases (Ca++
and Mg++)- demonstrated as a “ladder pattern” on agarose
gel; also proteases.
Phosphatidylserine is exposed and attracts macrophages
with little “collateral damage””

21. Apoptosis - mechanismsApoptosis - mechanisms
Four stages of apoptosis:
i. Committment to death by extracellular or intracellular
triggers/signals
ii. Cell killing (execution) by activation of intracellular
proteases (caspases)
iii. Engulfment of cell corpse by other cells
iv. Degradation of the cell corpse within the lysosomes of
phagocytic cells

23. Extrinsic factors
 E.g. by members of
the TNF family
Intrinsic
mechanisms
 E.g. hormone
withdrawal
The two major pathways for caspase activation in
mammalian cells

24. Maniati et al. 2008. The molecular basis of apoptosis in mammals. Simplified overview
There are two
major pathways
of apoptosis
intrinsic
pathway
extrinsic
pathway

25. Engulfment of apoptotic cellsEngulfment of apoptotic cells
Binding
Recognition
Phagocytosis
Internalization
Stages of
engulfment of
apoptotic cells can
be divided into 4
stages

26. DNA-damage and Apoptosis
Radiation or chemotherapy damages DNA
p53 accumulates
Cell cycle arrested at G1 (allows repair)
If repair fails, p53 triggers apoptosis

27. Eukaryotic Cell Cycle
 M phase
(mitotic phase)
 G1 phase
(Gap 1)
 S phase
(synthesis
phase)
 G2 phase
(Gap 2)

28. Tumor Necrosis Factor and Cytotoxic Lymphocytes
in Apoptosis
 Fas (CD95) –FasL induces apoptosis in lymphocytes that recognize
“self”; Fas/FasL mutations may cause autoimmune disease
 TNF/TNFR1-TRADD-FADD causes caspase activation and
APOPTOSIS; TNF also activates NF-kB which aids cell SURVIVAL and
is antiapoptotic
 Foreign Ag-CTLs- lymphocytes produce PERFORMIN which allows
entry of GRANZYME which activates caspases; CTLs kill target cells

29. Receptor pathway (physiological):
Death receptors:
(FAS, TNF-Receptor, etc)
FAS ligand TNF
Death
domains
Adaptor proteins
Pro-caspase 8 (inactive) Caspase 8 (active)
Pro-execution caspase (inactive)
Execution caspase (active)
DeathMITOCHONDRIA

30. Intrinsic pathway (damage):
Mitochondria
Cytochrome c release
Pro-caspase 9 cleavage
Pro-execution caspase (3) cleavage
Caspase (3) cleavage of cellular proteins,
nuclease activation,
etc.
Death
BAX
BAK
BOK
BCL-Xs
BAD
BID
B IK
BIM
NIP3
BNIP3
BCL-2
BCL-XL
BCL-W
MCL1
BFL1
DIVA
NR-13
Several
viral
proteins

31. Physiological Intrinsic
receptor pathway damage pathway
MITOCHONDRIAL SIGNALS
Caspase cleavage cascade
Orderly cleavage of proteins and DNA
CROSSLINKING OF CELL CORPSES; ENGULFMENT
(no inflammation)

32. Signaling DNA -damage to apoptosis

33. Apoptosis in model systems
 Molecular mechanisms of apoptosis in
mammalian cells
 mitochondrial pathway (intrinsic)
& cell death receptor pathway (extrinsic).
 bcl-2 gene family.
 Caspases.

34. bcl-2 gene family
Bcl-2 Family & IAP Family (Inhibitor of Apoptosis)
are critical regulators of cell death
Bcl-2 Family – Regulate whether MOMPs Occurs
Anti-Apoptotic Factors - Death Inhibitors
 Function to Inhibit MOMPs
by Pro Apoptotic Factors
Pro-Apoptotic Factors- Death Activators
 Bind and inhibit Death Inhibitors
 Directly cause Permeabilization of MOM to Stimulate
Release of Cytochrome C ( BAX and BAK)

35.  The translocated bcl-2 gene is under the control of an active
immunoglobulin promoter that drives high levels of constitutive
expression
 The oncogenic version is formed through a reciprocal chromosomal
translocation in which parts of the chromosome 14 and chromosome 18
are exchanged
 We now know that there are at least 24 Bcl-2-related proteins, 6 are
anti-apoptotic and 18 are pro-apoptotic.

36. Conformational changes in BCL-2 familyConformational changes in BCL-2 family
members during apoptosismembers during apoptosis
o BAX undergoes extensive conformational changes during the
mitochondrial translocation process
o BAX is a member of the Bcl-2 gene family
Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews
Molecular Cell Biology, 9, 47-59

37. Active site: Cysteine
Cleavage site: Asparatic
acid
Cysteine Asparatic
acid specific
protease
Aps-Xxx
caspase - a proteolytic system
Caspase?

38. Single chain of pro-enzymes
Contains an N-terminal domain, a small subunit and
a large subunit (similar to a ribosome)
Apoptotic stimulus Activation Substrate
Cleavage Enzyme

39. Activation of Caspases
www.sciencedirect.com

40. Caspases involved in apoptosis
3 Types of Caspases
 Inflammatory Caspases: -1, -4, and -5
 Initiator Caspases: -2, -8, -9, and -10
 Long N-terminal domain
 Interact with effector caspases
 Effector Caspases: -3, -6, and -7
 Little to no N-terminal domain
 Initiate cell death

41. Main Pathways Regulating Caspase Activation
During Apoptosis
 Two Caspase Pathways:
Intrinsic Pathway & Extrinsic Pathway
I. Intrinsic Pathway-
 Mitochondrial mediated major pathway in mammalian
cells
o Outer Mitochondrial Membrane Permeabilization (MOMP)
o Release of cytochrome C from mitochondrial intermembrane
space into cytosol
o Apoptosome Formation- Activation of Initiator Caspase
o Effector Caspases activated

42. 1. Intrinsic Pathway

Mitochondria

Cytochrome C

Apoptosome
Complex

Caspases

Cell Death

43. II. Extrinsic Pathway-
 Signaling through Death Receptors
o Ligand bound death receptors
o Adaptor protein association
o Initiator caspase recruitment and activation
o Effector caspases activated

44. 2. Extrinsic
Pathway

Death Ligand

Death Receptors

Caspases

Cell Death

45. The Biology of Cancer (Garland Science 2007)

46. IAPs
Inhibitor of Apoptosis
Proteins (IAPs)
 Bind Pro-caspases
prevent activation of
apoptosis
 Bind Caspases
and inhibit Activity
Inhibit one part in the
cascade = failure to
apoptose

47.  Natural phenolic compounds , in herbs and diet play an
important role in cancer prevention and treatment.
Eg., phenolic acids, flavonoids, tannins, stilbenes, curcuminoids,
coumarins, lignans, quinones, etc.
 They are responsible for their chemopreventive properties
like
 anticarcinogenic
 antimutagenic
 antioxidant &
 anti-inflammatory effects
Herbs and Dietary FactorsHerbs and Dietary Factors

48.  Phenolic compounds inducing apoptosis
 arresting cell cycle
 regulating carcinogen metabolism and ontogenesis
expression
 inhibiting DNA binding
 cell adhesion, migration, proliferation or
differentiation &
 blocking signalling pathways.
Wu-Yang Huang and Yi-Zhong Cai .(2010).Natural Phenolic Compounds From Medicinal Herbs and Dietary Plants: Potential Use for
Cancer Prevention. Nutrition and Cancer, 62(1), 1–20

49.  The majority of polyphenols present in food are flavonoids and phenolic
acids that are an integral part of the human diet
 Flavonoids (such as epigallocatechin-3-gallete (EGCG), quercetin, and
curcumin) act by induction of apoptosis
 Certain natural products have been shown to inhibit the activation of nuclear
factor kappa B (NF-κB) and Akt signaling pathways, both of which are known
to maintain a homeostatic balance between cell survival and apoptosis
 Dietary factors affecting apoptosis can influence carcinogenesis
 Activation of apoptosis in pre-cancerous cells is one of the most
important mechanisms of cancer chemoprevention by dietary factors*
* Martin, K.R. (2006) Targeting apoptosis with dietary bioactive agents. Experimental Biolology and Medicine (Maywood), 231, 117-129.

50. Induction of apoptosis by combining natural compounds and anticancer
drugs/radiation (through NF-κB or PI3K/ Akt pathway) in vivo
Epigallocatechin gallete (EGCG)

51. A partial list of the agents that have been reported to
induce or inhibit apoptosis

52. Disease associated with induction and inhibition of
apoptotic cell death

53. Chemicals that induced apoptosis
 Most of the environmental toxin induce apoptosis by oxidative stress
(OS) mediated
Compound Genes/ Proteins Involved Proposed Mechanisms
Bisphenol
↑Fas, ↑FasL, ↑caspase-3
Fas-signaling pathway triggers
Leydig cell apoptosis that then
induces germ cell apoptosis
↑Degradation of late spermatids
and seminiferous tubules,
↔testosterone
Direct cell damage
Ethanol
↓pAkt, ↓pErk1/2, ↓Bad
Suppression of survival-signaling
pathway
↑FasL Fas-signaling pathway
Methoxychlor ↑Fas, FasL, caspase-3, NF-↑ ↑ ↓
κB
Mitochondria and FasL-
mediated signaling pathway
1,3-Dinitrobenzene ↑Bax, ↓Bcl-2
Alteration of Bcl-2/Bax ratio
triggers mitochondrial pathway
Lindane
↑cyt c, ↑caspase-9, ↑caspase-3,
↑Fas, ↑FasL
Mitochondrial pathway, Fas-
signaling pathway
Polycyclic aromatic
hydrocarbons
↑Bax, ↑PARP cleveage Mitochondrial pathway
Mathur et al. (2011). Environmental Toxicants and Testicular Apoptosis . The Open Reproductive Science Journal, Volume 3

54. Role in Diseases:Role in Diseases:
TOO MUCH: Tissue atrophy
Neurodegeneration
Thin skin
etc
TOO LITTLE: Hyperplasia
Cancer
Athersclerosis
etc

55. Problem can be solved:
Activate apoptosis in cancer cells
- if apoptosis is improperly activated or regulated, result may cause
cancer
Finding useful mechanisms
Needs to be selective pathways (trail)
Treatment OptionsTreatment Options