Apoptosis: Influence of the Xenobiotics and Diseases

conditions

Dr. Naveen Kumar PhD Scholar

Veterinary Pharmacology and ToxicologyCollege of Veterinary and Animal Sciences,

G.B.P.U.A. & T., Pantnagar-263145, Uttarakhand, INDIA

Email: knaveen7v@gmail.com

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

A peculiar well controlled individual cell death that is caspase mediated

Fragmentation of the cell and organelles

Engulfed by macrophages

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

Importance of ApoptosisImportance of Apoptosis

1) Crucial for embryonic developmentErrors in Apoptosis can lead to Birth Defects

2) Important for maintaining homeostasisCell 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

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

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

APOPTOSIS: important in embryogenesis

Morphogenesis (eliminates excess cells):

Selection (eliminates non-functional cells):

APOPTOSIS: important in adultsTissue remodeling (eliminates cells no longer needed):

Virgin mammary gland Late pregnancy, lactation Involution(non-pregnant, non-lactating)

Apoptosis

Apoptosis- Testosterone

Prostate gland

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

Apoptosis in the development of the nervous system

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

How Apoptosis Differs from Necrosis?How Apoptosis Differs from Necrosis?

Cellular changes associated with Cellular changes associated with apoptosisapoptosis

Early: Chromosome condensation, cell body shrink

Later: Blebbing, Nucleus and cytoplasm fragment-Apoptotic bodies

At last: Phagocytosed

Necrosis

Apoptosis

Sequence in ultra structure change in apoptosis (2-6) and necrosis (7-8)

Apoptosis versus Necrosis

Normal white blood cell Apoptotic white blood cell

During apoptosis (programmed cell death) cells bleb and eventually break apart without releasing contents.

Stimuli for Apoptotic Cell Death Stimuli 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

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

Stimuli for Apoptotic Cell Death Stimuli for Apoptotic Cell Death

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””

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

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

Maniati et al. 2008. The molecular basis of apoptosis in mammals. Simplified overview

There are two major pathways of apoptosis

intrinsic pathway

extrinsic pathway

Engulfment of apoptotic cellsEngulfment of apoptotic cells

Binding

Recognition

Phagocytosis

Internalization

Stages of engulfment of apoptotic cells can be divided into 4 stages

DNA-damage and Apoptosis

Radiation or chemotherapy damages DNA

p53 accumulates

Cell cycle arrested at G1 (allows repair)

If repair fails, p53 triggers apoptosis

Eukaryotic Cell Cycle

M phase (mitotic phase)

G1 phase (Gap 1)

S phase (synthesis phase)

G2 phase (Gap 2)

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

Receptor pathway (physiological):

Death receptors:(FAS, TNF-Receptor, etc)

FAS ligand TNF

Deathdomains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase (inactive)Execution caspase (active)

DeathMITOCHONDRIA

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

BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3

BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Several viral proteins

Physiological Intrinsicreceptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)

Signaling DNA -damage to apoptosis

Apoptosis in model systems

Molecular mechanisms of apoptosis in

mammalian cells

mitochondrial pathway (intrinsic)

& cell death receptor pathway (extrinsic).

bcl-2 gene family.

Caspases.

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)

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.

Conformational changes in BCL-2 family Conformational 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

Active site: CysteineCleavage site: Asparatic acid

Cysteine Asparatic acid specific proteaseAps-Xxx

caspase - a proteolytic system

Caspase?

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

Activation of Caspases

www.sciencedirect.com

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

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

1. Intrinsic Pathway

Mitochondria

Cytochrome C

Apoptosome

Complex

Caspases

Cell Death

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

2. Extrinsic Pathway

Death Ligand

Death Receptors

Caspases

Cell Death

The Biology of Cancer (Garland Science 2007)

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

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

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

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.

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

Epigallocatechin gallete (EGCG)

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

Disease associated with induction and inhibition of apoptotic cell death

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 spermatidsand seminiferous tubules,

↔testosteroneDirect 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-2Alteration of Bcl-2/Bax ratio triggers mitochondrial pathway

Lindane↑cyt c, ↑caspase-9, ↑caspase-3,

↑Fas, ↑FasLMitochondrial pathway, Fas-signaling pathway

Polycyclic aromatichydrocarbons

↑Bax, ↑PARP cleveage Mitochondrial pathway

Mathur et al. (2011). Environmental Toxicants and Testicular Apoptosis . The Open Reproductive Science Journal, Volume 3

Role in Diseases:Role in Diseases:

TOO MUCH: Tissue atrophy

NeurodegenerationThin skin

etc

TOO LITTLE: Hyperplasia

CancerAthersclerosis

etc

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