ggggggggggggggg Faculty of Pharmacy

Mansoura University

Prepared by

Muhannad Soliman

2016

Biochemistry Department

Apoptosis & Necrosis and their

relationship with Cancer

Index 1- Introduction 2-3

2- Apoptosis 4- 6 A – Causes 4

B - Mechanism 5-6

3- Necrosis 7-8 A - Mechanism

4- Relationship with cancer 9-10

5- References 11

Introduction

There are two ways in which cells die:-They are killed by injurious agents or induced to commit suicide.

Death by injury Necrosis Cells that are damaged by injury, such as by

mechanical damage exposure to toxic

chemicals undergo a characteristic series of changes:

They (and their organelles like mitochondria) swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted).

The cell contents leak out, leading to

inflammation of surrounding tissues.

Death by suicide Apoptosis Cells that are induced to commit suicide:

shrink; develop bubble-like blebs on their surface; have the chromatin (DNA and protein) in their nucleus degraded; have their mitochondria break down with the release of cytochrome c; break into small, membrane-wrapped, fragments; release (at least in mammalian cells) ATP and UTP.

These nucleotides bind to receptors on wandering phagocytic cells like macrophages and dendritic cells and attract them to the dying cells (a "find-me" signal").

The phospholipid phosphatidylserine, which is normally hidden in the inner layer of the plasma membrane, is exposed on the surface.

This "eat me" signal is bound by other receptors on the phagocytes which then engulf the cell fragments. The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)

The pattern of events in death by suicide is so orderly that the process is often called programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death is also called apoptosis.

Apoptosis Why should a cell commit suicide? There are two different reasons. 1. Programmed cell death is as needed for proper development as mitosis is. Examples:

The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue

between them. The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation

occurs by apoptosis. The formation of the proper connections (synapses) between neurons in the brain requires that

surplus cells be eliminated by apoptosis. The elimination of T cells that might otherwise mount an autoimmune attack on the body occurs by

apoptosis. During the pupal stage of insects that undergo complete metamorphosis, most of the cells of the

larva die by apoptosis thus providing the nutrients for the development of the structures of the adult.

2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism. Examples: Cells infected with viruses One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis [diagram of the mechanism]. (And some viruses mount countermeasures to thwart it) Cells of the immune system As cell-mediated immune responses wane, the effector cells must be removed to prevent them from attacking body constituents. CTLs induce apoptosis in each other and even in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Cells with DNA damage Damage to its genome can cause a cell to disrupt proper embryonic development leading to birth defects to become cancerous. Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis. Is it any wonder that mutations in the p53 gene, producing a defective protein, are so often found in cancer cells (that represent a lethal threat to the organism if permitted to live) Cancer cells Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells.

What makes a cell decide to commit suicide? The balance between: the withdrawal of positive signals; that is, signals needed for continued survival, and the receipt of negative signals.

Withdrawal of positive signals The continued survival of most cells requires that they receive

continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals:

growth factors for neurons Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes

Receipt of negative signals

increased levels of oxidants within the cell damage to DNA by these oxidants or other agents like ultraviolet light , X-rays , chemotherapeutic drugs

accumulation of proteins that failed to fold properly into their proper tertiary structure molecules that bind to specific receptors on the cell surface and signal the cell to begin the

apoptosis program. These death activators include: o Tumor necrosis factor-alpha (TNF-α) that binds to the TNF receptor; o Lymphotoxin (also known as TNF-β) that also binds to the TNF receptor; o Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called

CD95).

The Mechanisms of Apoptosis There are 3 different mechanisms by which a cell commits suicide by apoptosis.

1. One generated by signals arising within the cell; 2. another triggered by death activators binding to receptors at the cell surface: TNF-α ,

Lymphotoxin,Fas ligand (FasL). 3. A third that may be triggered by dangerous reactive oxygen species.

1. Apoptosis triggered by internal signals: the intrinsic or mitochondrial pathway In a healthy cell, the outer membranes of its

mitochondria display the protein Bcl-2 on their surface. Bcl-2 inhibits apoptosis.

Internal damage to the cell o causes a related protein, Bax, to migrate to the

surface of the mitochondrion where it inhibits the protective effect of Bcl-2 and inserts itself into the outer mitochondrial membrane punching holes in it and causing cytochrome c to leak out.

The released cytochrome c binds to the protein Apaf-1 ("apoptotic protease activating factor-1").

Using the energy provided by ATP, these complexes aggregate to form

apoptosomes. The apoptosomes bind to and activate caspase-9. Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get their name

because they cleave proteins — mostly each other — at aspartic acid (Asp) residues). Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7). The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity

(rather like that in blood clotting and complement activation) which leads to digestion of structural proteins in the cytoplasm,degradation of chromosomal DNA, and phagocytosis of the cell.

2. Apoptosis triggered by external signals: the extrinsic or death receptor pathway Fas and the TNF receptor are integral membrane proteins with

their receptor domains exposed at the surface of the cell binding of the complementary death activator (FasL and TNF

respectively) transmits a signal to the cytoplasm that leads to activation of caspase 8 caspase 8 (like caspase 9) initiates a cascade of caspase activation

leading to phagocytosis of the cell.

Example (right): When cytotoxic T cells recognize (bind to) their target, they produce more FasL at their surface. This binds with the Fas on the surface of the target cell leading

to its death by apoptosis. The early steps in apoptosis are reversible — at least in C. elegans. In some cases, final destruction of the cell is guaranteed only with its engulfment by a phagocyte.

3. Apoptosis-Inducing Factor (AIF) Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases. Apoptosis-inducing factor (AIF) is a protein that is normally located in the intermembrane space of mitochondria. When the cell receives a signal telling it that it is time to die, AIF

is released from the mitochondria (like the release of cytochrome c in the first pathway); migrates into the nucleus; binds to DNA, which triggers the destruction of the DNA and cell death.

Apoptosis and Cancer

Some viruses associated with cancers use tricks to prevent apoptosis of the cells they have transformed.

Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53.

Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas o produces a protein similar to Bcl-2 o produces another protein that causes the cell to increase its own production of Bcl-2. Both

these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).

Even cancer cells produced without the participation of viruses may have tricks to avoid apoptosis.

Some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production.

Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.

Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells .

Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).

Necrosis is the prominent mode of cell death that occurs in various neurodegenerative

conditions and as a consequence to ischemic injury in various organs including the brain and heart. Although progress has been made in the last decade in understanding the molecular mechanisms of apoptosis, the biochemical pathways leading to necrotic cell death remain poorly understood. Necrosis has long been thought to be a “passive” process occurring as a consequence of acute ATP depletion. Several ATP-dependent ion channels become ineffective, leading to ion dyshomeostasis, disruption of the actin cytoskeleton, cell swelling, membrane blebbing, and eventual collapse of the cell . Recent reports suggest that in addition to the passive mechanisms, “active” mechanisms, such as Na+ overloading, Ca2+ accumulation, and changes in mitochondrial permeability, may also participate in the necrotic process

Sequence of biochemical events that may lead to necrotic cell death 1- In ischemic or hypoxic injury, energy depletion occurs by defective ATP production combined with the rapid consumption of ATP by ion pumps and through hydrolysis and leakage. The necrotic volume increase associated with necrotic cell death is initiated by an influx of Na+ and release of ATP due to membrane leakage. The increased Na+ level in the cytosol activates Na+-K+-ATPase, resulting in dissipation of ATP. In the beginning stages of the injury, a simultaneous efflux of K+ maintains ion homeostasis. Severe depletion of ATP leads to failure of the pump-leak balance mechanism, leading to an influx of Na+ and water that results in swelling and collapse of the cell. Thus the overload of Na+ concomitant with severe ATP depletion seems to be the major determinant of a necrotic outcome. 2- Cytosolic Ca2+ plays a role in linking ATP depletion and necrosis in some cell types, but several other cell types including hepatocytes and renal tubules can undergo necrotic cell death in its absence. The reactive oxygen species-mediated necrotic volume increase and Na+ influx are suggested to be initiated by the binding of the free radicals to ion channels including nonselective Ca2+ channels. The increased levels of Na+ activate Na+-K+-ATPase and consume ATP, which, in turn, activates nonselective Ca2+ channels, resulting in massive cytosolic Ca2+ accumulation. High levels of Ca2+ can participate in ATP depletion by activating Ca2+-ATPase and mitochondrial depolarization. The increased levels of Ca2+ activate endonucleases to degrade DNA and activate cellular proteases such as calpain to degrade several structural and signaling proteins.

3-Mitochondria participate in necrotic as well as apoptotic cell death by opening the mitochondrial permeability transition pore. an essential role of the mitochondrial permeability transition (MPT) in the release of cytochrome c and initiation of apoptosis in many models, including hepatocytes exposed to TNF-α and Fas ligation.

Several second messengers and proapoptotic proteins including Bcl-2 family members can induce the permeabilization of the MPT pore.BNIP3 is a member of the Bcl-2 family that is loosely associated with mitochondria in the normal state but gets fully integrated into the mitochondrial outer membrane after a death stimulus. BNIP3-transfected cells are found to undergo cell death independently of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. The cells exhibited morphology typical of the necrotic form of cell death with plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy.

It is proposed that BNIP3 can mediate necrosislike cell death through mitochondrial permeability transition pore opening and mitochondrial dysfunction.The expression of BNIP3 is shown to be induced in several cell lines in response to hypoxic injury. Overexpression of the hypoxia-inducible factor-1α has also been shown to induce the expression of BNIP3, resulting in a necrotic form of cell death.Moreover, although ischemia-reperfusion is usually associated with necrotic cell death,more recent studies have shown that apoptosis also occurs after ischemia-reperfusion in cells from liver and other organs.

Necrosis in tumorigenesis

Tumor cell necrosis can provoke an inflammatory response, and stimulate an immune response towards

potentially malignant cells. In this case, necrosis might prevent tumor development. Experiments with

TNFa support this notion.

TNFa is originally isolated as an anti-cancer cytokine, able to kill tumor cells and to induce tumor

regression in mice. On the other hand, mice with impaired TNFa signaling, such as TNFa-/- and TNFR1-/-

mice, are less prone to develop tumors in inducible tumor mouse models. It thus follows that TNFa can

also promote tumorigenesis.

It has been proposed that chronic inflammation, in contrast to an acute inflammatory response, can

promote tumor development.

In agreement with the latter, patients with chronic inflammatory bowel diseases (IBD) have an increased

risk of cancer development,and patients with the familial adenomatous polyposis (FAP) syndrome show a

significant reduction in the number and size of colorectal adenomas upon treatment with the anti-

inflammatory drugs celecoxib. Since necrosis can lead to inflammation and a sustained inflammatory

response can stimulate tumor development, these data provide some indirect evidence for a role of

necrosis in tumor development.

Necrosis in cancer treatment

-necrosis is induced by anti-cancer drugs, particularly by DNA-alkylating drugs. DNA-alkylating agents have

shown to cause necrotic cell death via activation of PARP-1. This necrosis occurs with equal effectiveness in

cells with or without functional apoptosis.

Interestingly, especially cells using aerobic glycolysis are shown to be sensitive for this PARP-mediated

necrosis.

Since many tumor cells depend on aerobic glycolysis, this observation suggests that tumor cells in

particular might be killed through necrosis upon treatment with alkylating agents.

- chemotherapy induces more necrotic than apoptotic cell death in breast cancer patients, and this

necrotic response is associated with a better survival.

- PARP-dependent necrosis is also associated with TNF receptor–independent activation of RIPK1, TRAF2, and downstream effector c-Jun NH2-terminal kinase. The bioenergetic crisis that occurs with acute NAD+ and ATP depletion in glycolytic cells is associated with the accumulation of high concentrations of intracellular calcium and ROS , which in turn cooperate to activate Ca2+-dependent cytosolic proteases calpains. Activated calpains cleave Ca2+extrusion channels and permeabilize lysosomes, leaking executioner cathepsins. High intracellular calcium concentrations and ROS also activate phospholipase A2. Proteolysis and lipid peroxidation ensues, leading to widespread membrane permeabilization and irreversible necrotic cell death.

References

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2.Carswell EA, Old LJ, Kassel RL, et al . An endotoxin-induced

serum factor that causes necrosis of tumors. Proc Natl Acad Sci

USA 1975; 72: 3666–3670.

3. Green S, Dobrjansky A, Carswell EA, et al . Partial purification of

a serum factor that causes necrosis of tumors. Proc Natl Acad SciUSA

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4. http://www.biology-pages.info/A/Apoptosis.html

5. Proskuryov SY, et al. Necrosis: a specific form of programmed cell

death? Exp. Cell Res. 2003;283:1–16. [PubMed]

6. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC420517/