Apoptosis and Angiogenesis Flashcards
what are some normal functions of apoptosis
-organism development such as a tadpole developing into a frog must lose its tail through apoptosis, human hand begins as mass of cells then apoptosis carves out fingers
-regulation of aged/damaged cell population
-immune response to infections such as viral infections where cell death is carried out without causing inflammation
what are the hallmarks of apoptosis
-it is programmed cell death
-it is highly regulated
-cells undergo key morphological changes, beginning with an apoptotic signal, which are cell shrinkage and chromatin condensation, blebbing and nuclear fragmentation, formation of apoptotic bodies
what are the differences between apoptosis and necrosis
-in necrosis small blebs form and the structure of the nucleus is changed then the blebs fuse to become larger but no organelle is contained within the blebs, finally the cell lyses and releases its contents and the organelle are no longer functional
-in apoptosis small blebs form and the nucleus, DNA, and organelles break off within these blebs, finally the cell is broken into several apoptotic bodies all containing the functional organelles and these can then be digested by macrophages
what are some triggers of apoptosis and what do they all have in common
radiation, cytotoxic agents, toxins (eg. bacterial), deprivation of growth factor (eg. insulin)/nutrient/oxygen (hypoxia, although some cells such as cancer cells can overcome hypoxia)
what are caspases
-the enzymic mediator molecules of apoptosis key to both intrinsic and extrinsic pathway
-larger inactive form is pro-caspases
-they are cysteine protease that cleaves after aspartic acid residues
-composed of a large and small subunit
-there are initiator and executioner caspases, initiators can be stopped before triggering full apoptosis but once executioners are triggered they cannot be stopped
what are the initiator caspases
caspase 8, 9, and 10
what domains and interactions do the initiator caspases have
-prodomain has DED region in procaspase 8 and 10, and CARD region in procaspase 9
-homophilic interactions between these regions and adaptor proteins (DED-DED or CARD-CARD)
what are the executioner caspases and what pathway are they involved in
caspase 3 and 7, involved in both pathways in fact the activation of executioner caspases is where the intrinsic and extrinsic pathway overlap
what do executioner caspases do, how are they activated, and what do they form
executioner caspases (aka effector) carry out regulated disassembly of the cell, they are activated by proteolytic cleavage which is mediated by initiator caspases, procaspase cleavage leads to a tetramer of small and large heterodimers
how do executioner caspases kill the cell
-disassemble cell structures such as degrading the nuclear lamina by making it more porous
-inactivation of apoptotic inhibitors such as iCAD
-deregulation of other protein activity
what is the intrinsic pathway activated by and what follows
activated by cellular stresses (DNA damage, hypoxia etc), this stress signal is relayed to the mitochondria (intrinsic pathway aka mitochondrial) which leads to mitochondrial outer membrane permeabilization (MOMP) which releases pro-apopotic proteins into the cytoplasm
what are the pro-apoptotic proteins released following MOMP and what are their roles
cytochrome C and Smac
-cytochrome C associates with other pro-apoptotic proteins such as APAF1 to allow formation of the apoptosome
-Smac inactivates a group of anti-apoptotic proteins called IAPs
what is procaspase 9 relationship with cytochrome C and APAF1
procaspase 9 interacting with cytochrome C and APAF1 causes the activation of caspase 9 which leads to the activation of the executioner caspases
what are the members of the Bcl2 protein family
-pro-apoptotic BAX subfamily composed of BAX and BAK which are essential for MOMP
-pro-apoptotic BH3 only subfamily composed of BIM, BAD, NOXA, PUMA, and BID and they activate BAX and BAK
-anti-apoptotic subfamily composed of Bcl2, BclXL, MCL1, and Bclw and are key for cell survival by binding and thus inhibiting the other members in the family
how to BH3 only proteins activate BAX and BAK
either directly by binding (tBID, BIM, and PUMA) or indirectly by binding to their repressors (pro-survival Bcl2 proteins)
how are BH3 only proteins regulated and activated
regulated at transcriptional and post-translational levels and activated in response to stimuli such as UV-BIM, genotoxic damage-Noxa and PUMA, cytokine deprivation-BAD, and death receptors-tBID
what determines whether cytochrome C will be released from the mitochondrion
the relative levels of pro and anti - apoptotic proteins within each channel
what are some IAPs (inhibitors of apoptosis)
8 proteins including cIAP1, cIAP2, XIAP, and survivin
-XIAP inhibits caspase 3 and 9
-cIAP1 ubiquitinates caspase 3 and 7
how do cancer cells manipulate the apoptotic process
-upregulation of anti-apoptotic proteins such as Bcl2 and Mcl1 is common in cancers
-inactivation of pro-apoptotic proteins (BAX inactivated in majority of colon cancers)
how is signalling regulated in the intrinsic pathway
-Akt is a kinase activated by many growth factors, that can inactivate Bad and downregulate Bim and Puma expression through the transcription factor FOXO3a, Akt also prevents cleavage of Bid from extrinsic pathway
-p53 protein levels can increase in normal (not cancer) cells upon cellular stress, p53 induces expression of BAX, PUMA, and NOXA and inactivation of Bcl2
overview of the extrinsic pathway
-induced by external signalling ie. a ligand binding to a specific membrane receptor
-two ways to activate are to increase receptor expression or to increase ligand expression
-death receptors/ligands are members of the TNF family of proteins (TNFalpha, TRAIL, FasL)
what are the ligands and what death receptors do they bind to
-TRAIL binds to DR4 and DR5
-TNF binds to TNFR1
-FasL binds to Fas (APO1/CD95)
what are the key steps in the extrinsic pathway
-ligand/receptor binding, when a ligand binds to its receptor it causes a conformational change such as receptor trimerization (the change being the formation of a trimer)
-recruitment and formation of DISC which then recruits and activates the initiator caspase 8 and sometimes 10, as well as DISC triggering self cleavage and thus activating the caspases
-active initiator caspases then activate the executioner caspases
what does receptor trimerization cause
brings 3 death domains together which allows for the recruitment of FADD which forms DISC
how does DISC (death inducing signalling complex) transform into death
active caspase 8 (initiator) can lead to direct activation of caspase 3 and activation of BID which promotes MOMP thus leading to death
what is anoikis
anchorage independent (unanchored), in normal cells will result in apoptosis as cells need cell-cell interactions for survival (cancer cells evade this), this occurs through lack of stimulation from extracellular matrix turns off pro-survival signalling and stimulates caspase 8 activation leading to death
what is the workflow of FasL leading to apoptosis
-Fas expression increased by p53 activation
-FasL either membrane bound on nearby cell or truncated which is its soluble form
-the FasL on cytotoxic T cell binds to Fas on tumour cell which stimulates the extrinsic pathway in the tumour cell and perforin and granzyme release from the cytotoxic T cell leading to death of the tumour cell
what is the immune response to apoptosis
phagocytic cells are recruited to the area where they then recognise phosphatidylserine on the apoptotic cell surface (apoptotic bodies - small portions of the cell to make it easier to digest), it then engulfs and degrades these
what is angiogenesis
the formation of new blood vessels from existing blood vessels
why is angiogenesis a key process in tumour growth
key process in tumour growth and metastasis as when blood supply is deficient the tumour cant grow and necrosis occurs (tumour bigger than 2mm requires a blood supply), it provides tumour with nutrients and oxygen and removes metabolic products
what is the angiogenic event sequence 1-9
1-HIF1 induction
2-HIF1 upregulates VEGF and MMPs
3-VEGF promotes endothelial cell proliferation and MMPs
4-destruction of basement membrane/support matrix by MMPs
5-MMPs cause release of angiogenic growth factors held in matrix
6-migration and proliferation of endothelial cells
7-expression of integrins by developing endothelium
8-capillary sprout formation
9-vascularization of tumour and formation of capillary loops
what are the details of 1- HIF1 induction
hypoxia induces HIF1 as in normoxia HIF1 is degraded quickly however low oxygen leads to HIF1 activation, HIF1 can also be upregulated by oncogenic signalling eg. by EGFR, HER2, Akt etc
what are the details of 2- HIF1 upregulates VEGF
this occurs in transformed cells, HIF1 is activated by: oncogenic signals, loss of TS function, or dysregulation of HIF1 degradation
what are the details of 3- VEGF promotes endothelial cell proliferation and MMPs
VEGF is released from cancer cells, enters the tissue surrounding the tumour, and moves towards endothelial cells thus VEGF induces MMPs from endothelium
what are the details of 4- destruction of basement membrane by MMPs
MMP1. MMP2, MMP7, and MMP9 all breakdown support and expose the endothelium
what are the details of 5- MMPs cause release of angiogenic growth factors held in the matrix
all the growth factors that were stuck in the matrix can now get through and reach the endothelial cells
6- migration and proliferation of endothelial cells
self explanatory
what are the details of 7- expression of integrins by developing endothelium
signalling via alphaVbeta4 integrins causes: reduced p53 activity, reduced BAX, reduced p21 expression, and increased Bcl2 expression
what are the details of 8- capillary sprout formation
-sprouting is led by the tip cell which can sense their environment to direct migration
-there are then migrating cells, dividing cells, and differentiating cells
-stalk cells are more proliferative
-the canal is sculpted by apoptosis and finally there is basement membrane deposition
what are the details of 9- vascularization of tumour and formation of capillary loops
this involves growth factors recruiting pericytes to support new blood vessels so the tumour now has access to blood supply, however this blood supply/capillary is chaotic which is a trademark of cancer and presents structures such as corkscrew vessels which are chaotic, unreliable, and leaky which can cause internal bleeding from the tumour
what is VEGF and what does it do, what regulates its expression
-an angiogenic factor
-proper name is VEGFA
-it is a secreted homodimeric glycoprotein with endothelial cell mitogenic activity
-VEGF stimulates endothelial cell migration and proliferation
-hypoxia regulates its expression
-there are numerous splice variants, 7 isoforms
what are the VEGF receptors and key roles, what does their signalling activate
-VEGFR 1, 2, 3 (tyrosine kinase receptors)
-VEGFR 2 expression is the key mediator of VEGF-induced angiogenesis
-signalling activates NOS, Akt, STAT3, MAPKs
what are angiopoietins
-angiogenic factors
-there is Ang1 and Ang2
-their receptors are Tie2-RTK
what is Ang1 role
stabilizes vessels, endothelial cell survival signals, and maintains endothelial barrier
what is Ang2 role
-destabilises vessels, mainly produced by endothelial cells, induced by VEGF, PDGFB, and IGF1, stored in granules and exocytosis is promoted by hypoxia
-Ang2 normally functions as an Ang1 antagonist, it promotes the dissociation of pericytes from pre-existing vessels and increases vascular permeability which facilitates the infiltration of proteases, cytokines, and angiogenic myeloid cells
what are the anti-angiogenic factors and their roles
-angiostation which inhibits bFGF-induced migration and proliferation (angiostatin is formed by MMPs cleaving plasminogen)
-endostatin which is internal fragment of collagen XVIII (18)
-IFNalpha and beta which suppress synthesis of bFGF and IL8
-thrombospondin1 which is secreted by different cell types, it inhibits endothelial cell growth and can cause autocrine FasL signalling
what are the other blood vessel recruitment types in cancer besides sprouting angiogenesis
-intussusception where a hole forms and the existing vessel splits in two
-vasculogenesis where a new blood vessel is made from a stem cell
-vessel co-option where cancer cells grow along the existing blood vessel so instead of supplying what it intended to supply the blood vessel instead unwillingly supplies the cancer cells
-vessel mimicry where cancer cells form vessel like structures themselves
-trans-differentiation of cancer cells where cancer cells can transform to act like a different type of cell ie. cancer stem-like cells can differentiate into endothelial cells
what are current anti-angiogenic drugs used in cancer treatments and how do they act on the tumour
-anti-angiogenic drugs dont choke/suffocate a tumour as commonly thought instead they bring normal blood supply to the tumour to eradicate the chaos and this allows drugs it is used in combination with such as chemotherapy or immunotherapy drugs to have better access to the tumour and thus get a better response
- used in treatment of breast, colorectal, ovarian, lung, gastric, and liver cancers
-drugs are: Avastin which is a small monoclonal antibody specific against VEGF by binding to it and stopping VEGF signalling, Ramucirumab similar to former is a small monoclonal antibody but this time is specific against VEGFR, and finally small molecule kinase inhibitors (dirty drugs, many targets-VEGFR, PDGFR, FGFR, c-KIT, can be useful but not specific so likely side effects)
Recap: what are the molecular responses to hypoxia
activates HIF1 which causes oncogenic signal, loss of tumour suppressor, lack of oxygen, and stops degradation process
Recap: what are the key events of angiogenesis
9 events, key goal is to clear a path through the matrix then growth factors can begin growing
Recap: how does angiogenesis contribute to cancer progression
gives the tumour a blood supply and allows for metastasis
Recap: how can angiogenesis be targeted
3 ways: through VEGF antibodies (against growth factors), VEGFR (against receptor), and kinase inhibitors
