1. Programmed Cell death Saeb Aliwaini April/2013

2. Introduction Human Body makes 10 billion cells every day. Cell death makes balance : There are various forms of cell death. Amongst these, two well-known pathways are necrosis and apoptosis. Other less described cell death pathways are mitotic catastrophe,autophagy and necroptosis.

3. Apoptosis Apo = from, ptosis = falling ( dropping off) Apoptosis is characterized by specific biochemical and morphological features that culminate in shrinkage of the cell to apoptotic bodies that are engulfed by neighboring macrophages.

4. A- Apoptosis occurs during normal cell turnover, tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance. Major inducers of apoptosis - Irreparable DNA damage - Cell cycle perturbation - Death ligands, e.g., Fas ligand - Growth factor withdrawal - Calcium influx -Free radicals - Radiation therapy - Chemotherapy drugs Other inducers

5. Mechanisms of Apoptosis The molecular mechanisms of apoptosis were highly conserved through evolution

6. Mechanisms of Apoptosis

7. Caspases: The "c" of "caspase" refers to a cysteine protease, while the "aspase" refers to the enzyme's unique property to cleave after aspartic acid residues Their proteolytic activity cleaves their substrate after Asp residues. Caspases are synthesized in cells as catalytically inactive zymogens Their activity is irreversible Tow types of caspases in Apoptosis: Initiator and effector Caspases and Caspases dependent Apoptosis

8. -Is typically activated in the early stages of apoptosis. -It cleaves key cellular components: Such cytoskeleton proteins and nuclear proteins such as DNA repair enzymes. -Nuclear condensation and cell shrinkage. -The cells express (translocation of phosphatidyl serine from the inside of the cell to the outer surface). Caspases and Caspases dependent Apoptosis

9. A- Extrinsic pathway -Is mediated by death receptors -Apoptosis via this mechanism is very rapid Induction of apoptosis by TNF receptor -Cell membrane receptors, which belong to tumor necrosis factor (TNF) family. - Including TNF-receptor 1, Fas, DR3, DR4, DR5, and DR6 -Specific ligands, such as TNF-α, lymphotoxin, Fas ligand -Conformational changes to form death inducing signaling complex. ( DISC) -Followed by recruitment of one of the caspases, typically caspase 8, to the DISC

11. Caspases and Caspases dependent Apoptosis B- Intrinsic pathway -Also called mitochondrial- mediated apoptosis. -Main inducer : DNA damage - The Bcl2 families of proteins are the main mediators of this process. This family has up to 4 conserved domains, known as the Bcl2 homology (BH) domains

12. The Bcl2 family contains both pro-apoptotic members and anti-apoptotic members. The pro-apoptotic members : -Bax family that consist of Bax, Bok and Bak. -BH-3 only proteins, such as Bid, Bad, and Bim. -The anti-apoptotic members of the Bcl2 family contain all 4 conserved domains, such as Bcl2 and Bcl xl. -Bcl2 are found exclusively within intracellular membranes and within the cytosol.

13. How they work? Pro apoptotic proteins : to release apoptotic-signaling factors from the mitochondria through mitochondrial outer membrane permeabilization (MOMP) via opening of the membrane permeability transition pore. Anti-apoptotic: function by preventing apoptosis through heterodimerization with pro-apoptotic proteins and self over-expression.

14. - cytochrome c, Smac/DIABLO, and HtrA2/Omi are capsases dependent - The apoptosome formation and effector caspase activation that causes the token apoptotic events, such as chromatin condensation, plasma membrane asymmetry, and cellular blebbing

17. Caspase-independent apoptosis Various factors are involved: 1- Apoptosis-inducing factor: - The lysosomal protease cathepsin D trigger AIF. -AIF becomes an active cell killer when it is released to the cytosol. -With factor # 2 (endonuclease G) they induce peripheral chromatin condensation. -The lethal effects of AIF are controlled by the anti-apoptotic protein heat shock protein 70 that interacts with AIF and protects against its apoptogenic effects.

19. 3- Endoplasmic reticulum Calcium release and activation of caspase12

20. Inhibitor of apoptosis proteins: -The IAP proteins function as endogenous caspase inhibitors -NAIP, c-IAP1, c-IAP2, XIAP, and Survivin -Survivin prevent caspase-9 activation within a functional apoptosome -Control of mitosis- Survivin can enhance microtubule stability in metaphase spindle formation.

21. Signals involved in Apoptosis Stimulus

22. Cisplatin mechanism of action Necroptosis Autophagy Mitotic catastrophe Necrosis Tamoxifin, Doxorubicin, 5 FU and other chemotherapeutics

24. Apoptosis assays 1. Cytomorphological alterations 2. DNA fragmentation 3. Detection of caspases, cleaved substrates, regulators andinhibitors 4. Membrane alterations 5. Mitochondrial assay

25. Transmission electron microscopy (TEM) Detection of Caspases, Cleaved Substrates. (PARP, H2AX,.. ) Apoptosis PCR microarray is a relatively new methodology that uses real-time PCR to profile the expression of at least 112 genes involved in apoptosis Membrane Alterations externalization of phosphatidylserine residues on the outer plasma membrane of apoptotic cells allows detection via Annexin V Mitochondrial Assays mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway.

26. Cytomorphological alterations

28. 1. Cytomorphological alterations and 2- DNA fragmentation

29. 2. DNA fragmentation

30. 2-DNA fragmentation

31. G0-G1 Typical Cell cycle profile G2-M S SubG1 G1 S G2 M 2n 4n 2n Cell cycle analysis MDA-MB-231 cells Plate 3 X 10 5 cells/well in 6 well plates 48 hours settle Treat with 0.2 µm AJ-5 For 24 and 48 hours Trypsinize and fix with cold ethanol for at least 30min FACS process and analysis A- Cell cycle analysis

32. AJ-5 induces a G1 arrest and apoptosis (sub-G1 peak) in breast cancer cells MDA-MB-231 MCF7

33. 33 Treat cells with 0.2 µm AJ-5 for 24 and 48 hours Annexin V staining MDA-MB-231 cells Plate 3 X 10 5 cells/well in 6 well plates 48 hours settle Trypsinize and stain cells with (Annexin V and PI) Necrosis Late apoptosis Early apoptosis Viable cells FACS analysis 3- Membrane alterations

34. AJ-5 induces apoptosis in breast cancer cells in accordance with the cell cycle analysis.

35. Detection of caspases, cleaved substrates, regulators andinhibitors 0.0 µM AJ-50.2 µM AJ-50.0 µM AJ-50.2 µM AJ-5 - Nuclear fragmentation after treatment with 0.2 µM AJ-5 for 24 h followed by Hoescht staining. MCF7MDA-MB-231 AJ-5 treatment leads to an increase in PARP cleavage level and nuclear fragmentation confirming that it induces apoptosis.

36. 5. Mitochondria assays o Cytochrome C release Methodologies: Apoptosis Assays Gang et. al (2010)

38. 38 Extrinsic pathway Apoptosis Intrinsic pathway Bid, Bad, Puma, Noxa p53 Effector Caspases 3,7 and 6 Caspase 8 Cyto C MAPK p21 Cell cycle arrest BH3 proteins Which apoptosis pathway is induced by AJ-5 ?

39. MCF7MDA-MB-231 apoptosis pathway Markers? Check for intrinsic and extrinsic apoptosis pathways markers AJ-5 activates both intrinsic and extrinsic as early as 1h of the treatment