Apoptosis Notes

Apoptosis: Programmed Cell Death

  • Orderly destruction of the cell characterized by:

    • Chromatin condensation: DNA becomes tightly packed.

    • Fragmentation of DNA: DNA is broken down into smaller pieces.

    • Cell shrinkage: The cell decreases in size.

    • Membrane disruption: The cell membrane loses its integrity.

    • Cell “blebbing” protrusions: The formation of bubble-like protrusions on the cell surface.

    • Scattering of cellular debris: The remnants of the cell are dispersed.

  • Necrotic cell death is much messier compared to apoptosis; involves inflammation and damage to surrounding tissues.

Apoptosis: Caspase Cascade

  • Orderly breakdown of cells relies on caspase enzymes.

    • Cysteine-rich and cleave after aspartate amino acids.

    • Produced as inactive zymogens (proenzymes) with very low activity.

    • Upon activation by stimuli, converted to active enzyme.

    • An amplified cascade of reactions follows, rapidly activating downstream enzymes.

Extrinsic Death Receptor Pathway

  • DISC (Death-inducing signaling complex) pathway stimulated by external signals (death factors) binding to cellular receptors (death receptors).

  • Combination of death ligands, death receptors, death adaptor proteins, and initiator caspase; critical for initiating apoptosis from external signals.

Tumor Necrosis Factor (TNF) Signaling

  • TNF binds to receptor, inducing receptor trimerization, causing exposure of DD (death domains) on cytoplasmic tails of receptor.

  • DD binds to TRADD (TNF receptor associated proteins) adaptor proteins.

  • Binding of procaspase-8 via DED (death effector domains) induces self-cleavage, activation of the initiator caspase-8, which activates executioner caspases; this cascade leads to cell disassembly.

FAS Signaling

  • Fas receptor is a member of the TNF receptor family.

  • Fas-mediated cell signaling is important in immune cell responses, including cytotoxic T cell-mediated cell death; plays a key role in regulating immune responses.

  • Cell-associated FAS ligand (CD95L) binds/activates FAS receptor, inducing receptor trimerization, causing exposure of DD (death domains) on cytoplasmic tails of receptor.

  • DD binds to FADD (FAS-Associated death domain proteins) adaptor proteins.

  • Binding of procaspase-8 via DED (death effector domains) induces self-cleavage, activation of the initiator caspase-8, which activates executioner caspases.

Inhibitors

  • C-FLIP inhibits extrinsic apoptotic cascade, binding to DED (death effector domains).

  • It inhibits recruitment and activation of the initiator caspase-8; acts as a crucial regulator of apoptosis.

  • C-FLIP is upregulated in many cancers with poor clinical outcome and is often associated with resistance to radiation chemotherapy and anti-cancer immune response; this upregulation allows cancer cells to evade apoptosis.

Intrinsic Apoptotic Pathway

  • Triggered by intracellular signals such as DNA damage and oxidative damage.

  • Causes release of pro-apoptotic signals from mitochondria and triggers caspase cascade; involves the release of cytochrome C and other mitochondrial proteins.

Bcl-2 Proteins

  • Control permeability of mitochondrial membrane.

    • Some members (e.g., Bcl-2) are anti-apoptotic.

    • Other members (e.g., Bax, Bak) are pro-apoptotic.

  • All members contain one or more Bcl-2 homology (BH) protein domains mediating protein-protein interactions.

    • BH3-only (e.g., Bad, Bid) contain only one BH3 domain. Other members also contain a transmembrane domain for insertion into the mitochondrial membrane.

  • Balance/ratio between pro- and anti-apoptotic proteins determines cell fate; this balance is critical in deciding whether a cell lives or dies.

  • Pro-apoptotic members (e.g., Bax, Bak) form pores in the outer mitochondria membrane, triggering release of contents and triggering the caspase cascade.

  • Anti-apoptotic members (e.g., Bcl-2; Bcl-XL) sequester pro-apoptotic proteins to inhibit cell death.

  • Pro-apoptotic member (Bad) can counter by sequestering Bcl-2; Bcl-xL, allowing Bak/Bax to insert.

Steps

  • Mitochondrial outer membrane permeabilization (MOMP):

    • Cellular damage/stress triggers “transient” binding of pro-apoptotic factors: Bid to Bax (“kiss and run”).

    • Activated Bax inserts within the mitochondrial membrane, resulting in the release of apoptotic regulators from the mitochondrial intermembrane space.

  • Formation of the Apotosome (CIRCLE OF DEATH):

    • Released cytochrome C recruits pro-apoptotic Apaf-1.

    • This interaction increases aggregation of procaspase 9, triggering activation of this initiator caspase.

    • Downstream “executioner” caspases are activated, resulting in cleavage of cellular target proteins.

Interaction Between Intrinsic and Extrinsic Pathways: Bid

  • The pro-apoptotic protein Bid is activated by caspase 8 via the extrinsic pathway and by DNA damage via the intrinsic pathway.

  • Activated Bid promotes MOMP and apoptosis; it serves as a critical link between the two pathways.

Intrinsic Pathway: Regulation

  • Inhibitors of apoptosis proteins (IAPs) inhibit caspase activation, thus decreasing apoptosis.

    • Transcription factor NFkB controls the production of IAPs.

    • Smac/Diablo inhibits IAPs, inhibiting the inhibitors, so it increases apoptosis.

  • Cytotoxic immune cells can release Granzyme B, which can activate caspases, activating Bid (and other caspases, as well).

p53: The Master Guardian/The Cancer Killer
p53-Mediated Cell Death: Transcriptional Dependent Mechanisms

  • Directly alters transcription of pro- and anti-apoptotic proteins.

    • Increases expression of pro-apoptotic proteins (e.g., Bax, PUMA).

    • Decreases expression of anti-apoptotic proteins (e.g., Bcl-2).

  • Activates Extrinsic Death pathway

    • P53 mediates transcription of FAS receptor increasing levels of this death receptor

  • P53 interferes with the pro-survival PI3K pathway

    • Increases transcription of IGF (Insulin-like growth factor) binding protein-3 which sequesters IGF-1 preventing activation of PI3K pathway

  • Indirectly alters transcription of pro-apoptotic proteins by increasing production of FOXO3

    • FOXO3 increases transcription of pro-apoptotic molecules

P53-Mediated Cell Death: Transcriptional Independent Mechanisms

  • Cytoplasmic p53 binds anti-apoptotic Bcl-xL, resulting in pro-apoptotic Bax activation, membrane insertion, MOMP.

  • Cytoplasmic P53 can also activate Bax by direct transient interaction “kiss and run” and insertion of Bax into mitochondrial membrane

  • In addition, P53 upregulation of pro-apoptotic PUMA increases p53“kiss and run” delivery of Bax.

Extrinsic Death Receptor Pathway: Common Mutations

  • Decrease in FAS pathway often found in UV exposure and skin cancer; compromises the cell's ability to undergo apoptosis when damaged.

  • Many cancers have decreased caspase activity, especially initiator caspase, Caspase 8; this reduction impairs the cell's ability to initiate apoptosis.

Apoptosis: Cancer vs Normal Cells

  • While cancer cells have decreased cellular apoptosis, they are actually “poised/primed” to die compared to normal cells.

  • Normal cells do not contain activated caspases, whereas cancer cells contain activated caspases inhibited by upregulated inhibitors (IAPs) (increase in both pro-apoptotic and anti-apoptotic proteins).

  • Cancer cells die because the caspase cascade is already activated and there are high levels of pro-apoptotic proteins.

Some Therapy Options

  • Stimulate/increase extrinsic death pathway.

  • Use substances that inhibit the inhibitors (IAPs) like smac/diablo.

  • Increase intracellular levels of pro-apoptotic/decrease intracellular levels of pro-life.

  • Use substances that mimic the BH3 proteins like Bad and sequester pro-life (BCL2; Bcl-xL) and allow Bax/Bak association.

Extrinsic Death Receptor Pathway: Therapeutics

  • TRAIL and TRAIL receptors (DR4,5) subfamily of TNF receptors specifically found to increase apoptosis in cancer cells.

  • Triggering this pathway therefore will trigger cancer cell death.

  • Recombinant TRAIL ligand and TRAIL receptor agonists currently in clinical trials to trigger death receptors.

    • Dulanemin: recombinant Trail ligand analogue Agonist antibodies against TRAIL-R1 or R2

Apoptosis: Cancer Therapeutics
Inhibit the Inhibitors (IAPs)

  • Several approaches in pre-clinical studies:

    • Small molecule inactivation of IAPS (polyphenol ureas).

    • Drugs that mimic Smac/Diablo and inhibit IAPs.

    • Antisense to decrease expression of IAP proteins.

Bcl-2 Proteins

  • SAHA: HDAC (histone Deacetylase) inhibitor results in epigenetically “turning on” pro-apoptotic proteins (Bid).

    • Drug is approved for non-Hodgkin’s lymphoma.

BH3 Mimetics

  • Small molecule bind to pro- survival proteins (e.g. BCL2; BClxl; MCL1)

  • This increases association of Bax;Bak and result in MOMP and apoptosis

FDA Approved BH3 Mimetics

  • BH3 mimetics: Binds to pro-life proteins to prevent their association with BAX/BAK….basically increases the number of BH3 proteins thereby increasing BAX/BAK association/cell death

  • Navitoclax binds to several pro-life proteins (BCl-2;BCl- Xl;MCL1) to displace proapoptotic BH3 proteins (BAD;BIM:NOXA)

  • Venetoclax specific to BCL2 displace several proapoptic proteins including BAK/BAX Venetoclax highly effective on patients with CLL (chronic lymphocyte leukemia) However, some patients develop resistance to treatment….

BH3 Mimetics Expanded

  • Small molecule (e.g Venetoclax) bind to pro-survival proteins (e.g. BCL2) This releases pro-apoptotic protein (BIM)

  • This increases apoptosis in at least 3 ways:

    1. The removal of the pro-life BCL2 from Bax allows for Bax:Bax interaction

    2. Released pro-apoptotic BIM proteins enable BIM to displace other pro-life proteins (like MCL-1) from Bax.

    3. Released BIM can also activate Bax via “kiss and run” transient binding to Bax.

Cancer Cells “Poised/Primed” to Die

  • Normal cells have low levels of pro-apoptotic factors so Treatment with BH3 mimetics have minimal effect

  • Cancer cells have elevated levels of pro and anti- apoptotic factors so Treatment of cancer cells with BH3 mimetics result in binding of anti-apoptotic and activation of apoptotic …