mechanisms of cell death 2012

8/17/2019 Mechanisms of Cell Death 2012

http://slidepdf.com/reader/full/mechanisms-of-cell-death-2012 1/42

Mechanisms of Mechanisms of

Cell DeathCell Death



Etiology of cell deathEtiology of cell death

Major Factors

Accidental Genetic

Necrosis Apoptosis

Necrosis:Necrosis:The sum of the morphologic changes that follow cell death in a living tissue or organ

Apoptosis: a physiological process that includes specific suicide signals leading to cell death



Cell deathmode

Morphological features Notes

Apoptosis

• Rounding-up of the cell• Retraction of pseudopodes• Reduction of cellular and nuclear volume

(pyknosis)• Nuclear fragmentation (karyorrhexis)• Minor modication of cytoplasmic organelles• Plasma memrane leing• Engulfment y resident phagocytes! in vivo

‘Apoptosis’ is the original term introduced byKerr et al .14 to define a type of cell deathwith specific morphological features. Apoptosis is !T a synonym ofprogrammed cell death or caspaseactivation.

Autophagy

• #ack of chromatin condensation• Massive vacuoli$ation of the cytoplasm• %ccumulation of (doule-memraned) autophagic

vacuoles• #ittle or no uptake y phagocytic cells! in vivo

‘Autophagic cell death’ defines cell deathoccurring with autophagy" though it maymisleadingly suggest a form of deathoccurring by autophagy as this processoften promotes cell survival.1#"1$

Cornifcation

• Elimination of cytosolic organelles• Modications of plasma memrane

• %ccumulation of lipids in & and # granules• Extrusion of lipids in the extracellular space• 'esuamation (loss of corneocytes) y protease

activation

‘%ornified envelope’ formation or‘&eratini'ation’ is specific of the s&in to

create a barrier function. Although apoptosiscan be induced by in(ury in the basalepidermal layer )e.g." *+," cornification ise-clusive of the upper layers )granular layerand stratum corneum,.1"1/

Necrosis

• ytoplasmic s*elling (oncosis)• Rupture of plasma memrane• +*elling of cytoplasmic organelles

• Moderate chromatin condensation

‘ecrosis’ identifies" in a negative fashion"cell death lac&ing the features of apoptosisor autophagy.4 ote that necrosis can occurin a regulated fashion" involving a precise

se0uence of signals.

st nct mo a t es o cedeath

ell 'eath 'i,er .//0! 12(1)3 4511



6he road to necrosis 6he road to necrosis

Homeostatic ‘steady state’

Cellular adaptations

Reversible cell injury

Irreversible cell injury

Cell death Necrosis



Necrosis3Necrosis3

conseuences of irreversile cell in7uryconseuences of irreversile cell in7ury



Pathogenesis of necrosisPathogenesis of necrosis



Necrosis: a pathological response

to cellular in(ury

Apoptosis: a physiological

response to specific suicide signals"

or lac& of survival signals

%hromatin clumps

%hromatin condenses and migrates to nuclearmembrane. nternucleosomal cleavage leadsto laddering of 2A at the nucleosomal repeat

length" ca. 3 bp.

5itochondria swell and rupture %ytoplasm shrin&s without membrane rupture

6lasma membrane lyses 7lebbing of plasma and nuclear membranes

%ell contents spill out%ell contents are pac&aged in membranebounded bodies" internal organelles still

functioning" to be engulfed by neighbours.

8eneral inflammatory response istriggered

9pitopes appear on plasma membranemar&ing cell as a phagocytic target.

o spillage" no inflammation

www.chembio.uoguelph.ca



In embryonic andfetal development:

• Tissue developmental programs which controlsculpting of embryonic form

• 2evelopmental organi'ation of the nervous system• 9limination of self:reactive components of the immune

system

In the adult:• !n stimulation by T:lymphocytes• n response to 2A damage or abnormality" e.g. by

radiation" viral infection or transformation• n certain organs and tissues" on withdrawal of

supporting hormones

n addition" there are often apoptotic centers in tumors" accounting for the parado-

of slow gross enlargement in the face of rapid cell proliferation" and the rare

spontaneous remission.

%P8P68+9+ %+ % P:;+98#8<9%##;%P8P68+9+ %+ % P:;+98#8<9%##;

9MP8R6%N6 PR8E++9MP8R6%N6 PR8E++




%P8P68+9+ in%P8P68+9+ in C.elegansC.elegans

C.elegans genome: 1;;; genes ); seven:pass

transmembrane receptors" 4/ 'inc finger proteins" and41 protein &inases,

The life cycle of C. elegans from egg to se-ual maturity

)and new eggs, is about < days

ced-1, -3, -4, and -9 )Cell death determining, proteins in

C.elegans are closely related to mammalian apoptosis:

regulating genes

The adult hermaphrodite consists of e-actly ;#;

somatic cells of precisely determined lineage and

function. ndividual cells are named and their

relationships to their neighbors are &nown

!verall" the ;#; somatic cells of adult C.elegans arise

from 1; original cells= e-actly 1<1 somatic cells

undergo programmed cell death in the wild type worm

!f the 1; cells" <3 are neurons" and many of the

programmed deaths also lie in the neuronal lineagewww.chembio.uoguelph.ca


http://slidepdf.com/reader/full/mechanisms-of-cell-death-2012 11/42Nature Immunology 4" 41$ : 43< )3<,

Autophagic cell death (type II programmed cell death) > meaning thatthe cytoplasm is actively destroyed long before nuclear changes become

apparent=

Classical apoptotic cell death > meaning that the chromatin marginates

and the cell and nucleus fragment before morphological changes are seen

in intracellular organelles


http://slidepdf.com/reader/full/mechanisms-of-cell-death-2012 12/42Nature Reviews Cancer 2= $4:$#$ )33,

APP!"I"APP!"I"

"I#NA$""I#NA$"

5itochondria:

dependent

apoptosis

%aspase:

dependent

apoptosis

2eath ?eceptor:

dependent

apoptosis

%aspase:

dependent

apoptosis

%aspase:

independent

apoptosis

%aspase:

independent

necrosis



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'i,erentiation (./1.)

l i l i ll l



9n response to multiple intracellular stressconditions (eg! 'N% damage! cytosolic a.?overload)! pro-survival and pro-death signalsare generated and converge to amitochondrion-centered control mechanism@hen lethal signals prevail! mitochondrialouter memrane permeaili$ation (M8MP)

occurs and leads to mitochondrial trans-memrane potential ('cm) dissipation! arrestof mitochondrial %6P synthesis and 'cm-dependent transport activities Moreover! therespiratory chains gets uncoupled! leading togeneration of reactive oxygen species (R8+)!and proteins that are normally conned *ithinthe mitochondrial inter-memrane space (9M+)are released into the cytosol %mong these!cytochrome c (;6) drives 5 together *iththe cytoplasmic adaptor protein %P%&1 andd%6P 5 the assemly of the so-calledapoptosome! a multi-protein complex thattriggers the caspase-0-caspase-4 proteolyticcascade 'irect 9%P-inding protein *ith lo* p9('9%A#8! also kno*n as second mitochondria-derived activator of caspases! +M%) and hightemperature reuirement protein %. (:6R%.)facilitate caspase activation y seuesteringandBor degrading several memers of theinhiitor of apoptosis protein (9%P) family 8nthe contrary! apoptosis-inducing factor (%9&)and endonuclease < (EN'8<) function in acaspase-independent manner y relocating tothe nucleus and mediating large-scale 'N%

fragmentation 8f note! the serine protease:6R%. also contriutes to caspase-

Caspase%dependent and %independent

&intrinsic apoptosis'





Dpon &%+ ligand (&%+#) inding! thecytoplasmic tails of &%+ (also kno*n as'0! a prototypic death receptor) trimersrecruit (among other proteins) &%+-

associated protein *ith a death domain(&%'')! cellular inhiitor of apoptosisproteins (c9%Ps)! c-&#9Ps and pro-caspase-F(or -1/) 6his supramolecular platform!*hich has een dued Gdeath-inducingsignaling complexH ('9+)! controls theactivation of caspase-F (-1/) @ithin the

'9+! c-&#9Ps and c9%Ps exert pro-survivalfunctions :o*ever! *hen lethal signalsprevail! caspase-F gets activated and candirectly trigger the caspase cascade ymediating the proteolytic maturation ofcaspase-4 (in type 9 cells) or stimulatemitochondrial outer memranepermeaili$ation (M8MP) y cleaving the

A:4-only protein A9' (in type 99 cells)Extrinsic apoptosis can also e ignited ydependence receptors like ' or DNA!*hich relay lethal signals in the asence oftheir ligand (netrin-1) 9n the case of 'and DNA! the pro-apoptotic signalingproceeds through the assemly of a 'R%#-

and 6D%N- (or N#RP1-) containingcaspase-0-activating platform or y the-



trinsic Apoptosis: apoptotic cell death that is induced by etracellular stress

signals that are sensed and propagated by specific transmembrane receptors

* l d N i



Dpon tumor necrosis factor a (6N&a)inding! the cytoplasmic tails of 6N&receptor 1 (6N&R1! a prototypic deathreceptor) trimers recruit 6N&R-associateddeath domain (6R%'')! receptor-interactingprotein kinase 1 (R9P1)! cellular inhiitor ofapoptosis 1 (c9%P1)! c9%P.! 6N&R-associatedfactor . (6R%&.) and 6R%& @ithin the so-called complex 9! R9P1 is polyuiuitinatedy c9%Ps! therey providing a docking sitefor the recruitment of transforming gro*th

factor (6<&)-activated kinase 1 (6%I1)! 6%I1-inding protein . (6%A.) and 6%A4(*hich together deliver a pro-survival signaly activating the transcription factor N&-kA)9n some patho-physiological andexperimental settings! and in particular*hen caspase-F is asent or *hen caspasesare inhiited y pharmacological agents!cylindromatosis (;#')-deuiuitinatedR9P1 engage in physical and functionalinteractions *ith its homolog R9P4!ultimately activating the execution ofnecrotic cell death Regulated necrosis canalso e induced y alkylating 'N% damage(possily y the overactivation of poly(%'P-

riose) polymerase 1! P%RP1) 9n some (utnot all) instances! regulated necrosis



*egulated Necrosis:

necrosis can occur in a regulated manner in addition to spontaneous cell death



Mitotic catastrophe:•cell death occurring in mitosis•cases of cell death that are triggered y aerrant mitosis and executed eitherduring mitosis or in the suseuent interphase•might not even constitute a bona fde cell death executioner mechanism! ut an

onco-suppressive path*ay that precedes and is distinct from! yet operatesthrough! cell death or senescence

(a) 9n the asence of chemical and genetic perturations of the mitotic apparatus (includingchromosomes and the molecular machinery that ensures their faithful segregation)! cells progressthrough the di,erent phases of the cell cycle to generate a diploid o,spring 8n the contrary! ifchromosomal defects or prolems a,ecting the mitotic machinery are sensed during the M phase!cells ecome arrested in mitosis due to the activation of mitotic catastrophe (b–d) 6hese cells canundergo di,erent fates3 they can die *ithout exiting mitosis (b)! reach the <1 phase of thesuseuent cell cycle (through a phenomenon that is kno*n as mitotic slippage) and then die ( c)!

or exit mitosis and undergo senescence (d) 9rrespective of this diversity of outcomes! mitoticcatastrophe can e dened as an oncosuppressive mechanism that <allu$$i et al

ell 'eath = 'i,erentiation (./1.) 10! 1/>5



<allu$$i et alell 'eath = 'i,erentiation (./1.) 10! 1/>5

9n response to stress and during development! eukaryotic cells often activate autophagy! amechanism *herey organelles and portion of the cytoplasm are seuestered in doule-memraned vesicles (autophagosomes) that are delivered to lysosomes for degradation +tress-induced autophagy most often exerts cytoprotective functions and favors the re-estalishment ofhomeostasis and survival (a) 9n this setting! pharmacological or genetic inhiition of autophagyaccelerates cell death 8n the contrary! these interventions freuently inhiit developmental cell

death! indicating that autophagy also constitutes a lethal mechanism that mediates Gautophagiccell deathH (b)

Autophagic cell death:instances of cell death that are accompanied

by a massive cytoplasmic vacuolization



The target se0uence for %ed:< and caspases

)Cys catalytic Asp targeting proteases,

consists of a tetrapeptide with %:terminal Asp (+).



5ethods 9n'ymol. 3/=443@1#:/1

C d d h i i hibi



Caspase cascades and their inhibitors

ell 'eath and 'i,erentiation(./11) 1F! 1JJ151JJ0

&ormation ofmulticomponent

complexes triggersinitiator caspase

dimeri$ationsuKcient for their

activation3•'9+3 'eath-inducing signalingcomplex

•%poptosome•(P9'')osome3 p4-induced protein*ith a deathdomain



5ethods 9n'ymol. 3/=443@1#:/1



Nuclear amins" nucleoplasmin" the B? protein K *1" hn?6 %"?A 6ol upstream binding factor" the p#< regulator 5253"p?7" p3 Kip and p31%ip

+NA related 5%5<" ?epair en'ymes including ?ad#1" poly:A26:ribose

polymerase )6A?6," topoisomerase" inhibitor of caspaseactivated 2ase" ) i%A2C2DD4#,

Cytos,eleton actin" gelsolin" spectrin" &eratin

Cytoplasmic E:catenin" 7cl:3

Protein ,inases 2A dependent protein &inase" protein &inase %" %A5&inase" focal adhesion &inase" 5A6 and 9?K &inases" ?af1" A&t1Cprotein &inase 7" ?!%K .

In vivoIn vivo sustrates of e,ector caspasessustrates of e,ector caspases




Mitochondria play a central roleMitochondria play a central role

in mediating the apoptotic signalin mediating the apoptotic signal

5itochondria:free cytoplasm would not induce apoptosis in vitro

%ytochrome c:neutrali'ing antibodies bloc& apoptosis

%ytochrome c is an abundant protein of the mitochondrial inner

membrane" and acts as an electron transport intermediate.

a and b type cytochromes are inaccessible components of large

comple-es" but cytochrome c is monomeric" freely diffusible in the

inner membrane" and in e0uilibrium between inner membrane"

inter:membrane space and cristae.

The events of apoptotic activation lead to alterations inpermeability of the mitochondrial membrane pore proteins and

release of cytochrome c.

nitial release of cytochrome c occurs by a highly specific

process" involving proteins of the -cl%2 family




2eath receptors of the TD? family" as well as

various o-idants" detergents and

chemotherapeutic drugs" induce the release of

active cathepsins from the lysosomalcompartment. These cathepsins cleave 7id"

which can then mediate cathepsin:induced 56T.

2isruption of the cytos&eleton leads to the

release of the 7F< domain>only proteins 7im

and 7mf. 2A damage induced by radiation or

various chemotherapeutic drugs induces the

p#<:mediated transcription of genes encoding

7a-" 7F< domain>only proteins )o-a or 6uma,"

proteins involved in ?!B generation and

cathepsin 2. 9? stress results in the release of

calcium" which may cause direct mitochondrial

damage or activate 7a- through calpain:

mediated cleavage. +arious death stimuli"

mediated through death receptors" trigger the

production of lipid second messengers )such asganglioside )82<," arachidonic acid )AA, and

ceramide, that are involved in 56T and

mitochondrial damage. 2epending on the

stimulus and the type of cell" as well as the

metabolic status of the cell" 56T leads to either

caspase:mediated apoptosis or caspase:

independent 6%2.

Nature Immunology 4" 41$ : 43< )3<,

+ignaling leading to activation of mitochondria-related apoptosis+ignaling leading to activation of mitochondria-related apoptosis



Acl-. family3 Pro-#ife and Pro-'eath factionsAcl-. family3 Pro-#ife and Pro-'eath factions

-cl%2 and its closest relatives -cl%.$" -cl%/ and Ced%0 are α:helical proteins having all four

7F domains and are pro%survival. They suppress cytochrome c release" and are oncogenic

when overe-pressed. Fowever" 7cl:GB" a splice variant of 7cl:G having 7F4 but lac&ing 7F1

and 7F3 is pro:apoptotic.

-a and -a, lac& the 7F4 domain" and are pro%apoptotic. 7a- e-pression is stimulated by

p#<" a mechanism for pro:apoptotic action of p#<. 9ctopic or overe-pression of 7a- induces

cytochrome c release and apoptosis" and addition of 7a- to mitochondria in vitro induces

cytochrome c release.

The -1%only sub group are strongly pro%apoptotic" and include -im" -i, and gl%3" which

only have the 1/:residue 7F< and the transmembrane region" while -ad and -id only have

7F<. The helical 7F< element allows for homo: and heterodimeri'ation between family

members. The non:homologous regions of 7F<:only proteins could provide lin&s to apoptotic

signaling systems.www.chembio.uoguelph.ca



%aspase:;

9ffector caspases


+ertebrate Apaf%3 activation occurs through

cytochrome c binding. 7cl:3 and 7cl:G appearto act by dimeri'ing with pro:apoptotic agonistssuch as 7a- or 7a&.

ormally" the balance is in favor of 7cl:3 or 7cl:G

" but the 7F<:only factors appear to act to

titrate out the 7cl3C7cl:G" tipping the balance in

favor of 7a-C7a&.

7a- can oligomeri'e in the membrane to form apermeability channel able to transportcytochrome c.

7F<:only factors have been reported to induce

reorgani'ation of the cristae. Alternative modelssuggest that 7idC7adC7a&:li&e factors act to openpermeability channels such as the permeabilitytransition pore" by disrupting the membranepotential" and affecting the voltage:dependentanion channel +2A% and AT6CA26 e-changetransporter.

Acl-. family3 Pro-#ife and Pro-'eath factionsAcl-. family3 Pro-#ife and Pro-'eath factions



Mechanisms of mitochondrial outer membrane permeabilization during cell death. AIF: apoptosis inducing factor; ANT: adenine nucleotide translocase; CL: cardiolipin; Cyt c :cytochrome c ; CyD: cyclophilin D; CsA: cyclosporin A; IMM: inner mitochondrial membrane;MT: mitochondrial permeability transition; !MM: outer mitochondrial membrane; "DAC:#oltage$dependent anion channel%

!rrenius et al." Ann ?ev 6harmacol To-icol 3



5itochondrial damage leads to the release of

numerous mitochondrial proteins that mediate

6%2.• ?elease of cytochrome c triggers caspase

activation and classic apoptosis.• "mac )also &nown as 2iablo, and mi assist

cytochrome c >induced caspase activation by

counteracting caspase inhibitory factors )IAPs,.• AI triggers a caspase:independent death

pathway that culminates in 2A fragmentationand chromatin condensation characteristic of

apoptosis:li&e 6%2.• ndo# cleaves 2A and induces chromatin

condensation• The serine protease activity of mi can

mediate caspase:independent cellular rounding

and shrin&age without changes in the nuclear

morphology• Calcium and *" can lead to severe

mitochondrial dysfunction and necrosis:li&e

6%2 either directly or through autophagy of

damaged mitochondria. Autophagy also may be

associated with cathepsin activation and so can

result in apoptosis:li&e 6%2.


Mitochondria permeaility transition can trigger caspase-dependentMitochondria permeaility transition can trigger caspase-dependent

and caspase-independent programmed cell death (P')3and caspase-independent programmed cell death (P')3



+urvival mechanisms do*nstream of cytochrome c+urvival mechanisms do*nstream of cytochrome c

35 "e6uestration by heat shoc, proteins: Apaf1 interacts with heat shoc& proteins hsp and hsp;. Fsp directly se0uesters %A?2" and bloc&s

caspase:; recruitment" and possibly assembly of the oligomeric apoptosome as well. Fsp; also

associates with the monomeric Apaf1" and may represent a significant fraction of the normal autoinhibitedstate. Fsp; appears to compete with cytochrome c for binding" suggesting action at an earlier step than

hsp.

25 +irect inhibition of the caspase catalysis by Inhibitor of Apoptosis Proteins (IAPs):

Inhibitor of apoptosis proteins (IAPs) represent thefinal line of defense against apoptosis" and act by

binding directly to the substrate site of caspases

"mac7+IA-$: the mitochondrial ans/er to IAPs:

5itochondria initiate the apoptosis cascade by

releasing cytochrome c" but this effect could be

nullified if A6 were allowed to maintain their inhibition

of caspases. The apoptotic signal is instead sustained

by the release of "mac7+IA-$ )second mitochon:

drial activator of caspaseCdirect IA6 binding protein of

low p," which binds to and antagoni'es the A6s.




'E%6: REEP68R+3'E%6: REEP68R+3

Path*ays linking external signal receptors to caspase-FPath*ays linking external signal receptors to caspase-F

A variety of cell surface receptors related to !N%* )tumor necrosis

factor receptor, interact with the apoptotic activation system. Theintracellular portion of the receptor carries a specific protein

interaction domain called the death domain8 ++. The 22 is

activated by pro-imity" brought about when bound e-tracellular

ligand induces receptor oligomeri'ation. Activation can also be

induced in absence of ligand by artificial cross:lin&ing of the

receptor.

%lustered receptor 22s recruit a variety of 22:containing

adapters" of which A++" as:associated death domain protein

)also &nown as 5!?T1, bridges to a second protein interaction

domain" ++" or death effector domain. The cluster of DA22:

292s recruits procaspase:/" which also carries 292s at its :

terminus )corresponding to the %A?2s on 6rocaspase:;,.

6rocaspase:/ is activated to %aspase:/ by pro-imity:induced self:

cleavage. 6rocaspase:1 is the only other caspase with 292

bo-es" and may substitute for %aspase:/ in some cases.

n some cells" TD receptors associate with adaptors lin&ed to cell

proliferation or inflammatory signaling pathways" and may induce

anti%apoptotic c:A6s.




*eceptor $igand Adaptor !arget

as7C+097AP%3 Das:CA6!:1 DA22C5!?T1 6rocaspase:/" apoptosis

!N%*3 TDα T?A22HDA22 6rocaspase:/" apoptosis

!N%*3 TDα T?A22H?61HT?AD359KK" IunCAp1" cell proliferation"

KK" D:κ 7" inflammation" c:A6s

!N%*27C+4 TDα T?AD3HT?AD1

59KK" IunCAp1" cell proliferation"

KK" D:κ 7" inflammation" c:A6s

+*7AP% A6!:< DA22J 6rocaspase:/" apoptosis

+*4 T?ACA6!:3 DA22 6rocaspase:/" apoptosis

+*9 T?ACA6!:3 DA22 6rocaspase:/" apoptosis

+c*3 T?ACA6!:3 none decoy receptor" ligandse0uestration

+c*2 T?ACA6!:3 nonedecoy receptor" ligandse0uestration

+c* Das:CA6!:1 nonedecoy receptor" ligandse0uestration

' h i d d d' th t t i d d d t




The death receptor is stimulated by

ligand:induced activation of the receptortrimer. The receptor death domains )22s,

of Das then recruit DA22 and ?61 to the

receptor comple-. After recruitment to

DA22 through interactions between their

death effector domains )292s," caspase:

/ and caspase:1 are activated and

trigger effector caspases" either directly

or through a 7id:mediated mitochondrialpathway )activation of Apaf:1 and

caspase:;,.

DA22 and ?6 initiate a caspase%

independent necrotic path/ay

mediated by the formation of" most

probably" mitochondrion: or c6A3:

derived ?!B. TD?1 signaling differsfrom Das signaling in the following steps@

first" binding of DA22 and ?6 to the

receptor comple- re0uires the adaptor

protein T?A22= and second" the ?61:

mediated necrotic pathway is inhibited by

DA22 and activated caspase:/

'eath receptor5triggered caspase-dependent'eath receptor5triggered caspase-dependent

and caspase-independent path*aysand caspase-independent path*ays



L<entamicin is an aminoglycosideantiiotic *idely used against infections y<ram-negative microorganismsNephrotoxicity is the main limitation to itstherapeutic eKcacy <entamicinnephrotoxicity occurs in 1/5./ oftherapeutic regimes % central aspect ofgentamicin nephrotoxicity is its tuular

e,ect! *hich may range from a mere lossof the rush order in epithelial cells to anovert tuular necrosis

urr 8pin Rheumatol ./1.!



Figure 1. Methods to detect cell

death-related variales No*adays! acornucopia of techniues is availaleto monitor cell death-relatedparameters @ithin thisGmethodologicalaundanceBredundancyH! the choiceof the most appropriate techniues

and the correct interpretation ofresults are critical for the success ofany study dealing *ith cell death:ere! the most common proceduresto detect deadBdying cells areindicated! together *ith the technicalplatforms that are reuired for theirexecution and the types of specimens

on *hich they can e applied Pleasesee the main text for further details'cm! mitochondrial transmemranepotentialC :P#! high-pressure liuidchromatographyC M8MP!mitochondrial outer memranepermeaili$ationC MP6! mitochondrial

permeaility transitionC M+! massspectrometryC NMR! nuclear magnetic



+etection of apoptotic changes in +NA:+etection of apoptotic changes in +NA:

• ucleic acid staining > nuclear morphology

• 2etection of nuclear 2A fragmentation

• T*9 staining)terminal deo-ynucleotidyl transferase>mediated d*T6 nic& end:labeling,

• Bingle:cell electrophoresis )%omet assay,

5 o l e

c u l a r 6 r o b e s "

1 n c .



+etection of changes in cell membrane integrity:+etection of changes in cell membrane integrity:

• 5embrane permeability

• 6hospholipid symmetry )Anne-in + staining,

5 o l e


1 n c .



2etection of apoptotic changes in mitochondria@

%aspase 6rotease Assays )individual caspases,@

5 o l e


1 n c .

;P!;orphology



2etection of pro: and anti:apoptosis proteins" Das:ligands" cyto&ines" etc.

2etecting changes in gene e-pression for pro: and anti:apoptosis genes



L%rtemisinin and its derivatives are currentlyrecommended as rst-line antimalarials in regions*here Plasmodium alciparum is resistant totraditional drugs 6he cytotoxic activity of theseendoperoxides to*ard rapidly dividing humancarcinoma cells and cell lines has een reported! and

it is hypothesi$ed that activation of the endoperoxideridge y an iron(99) species! to form -centeredradicals! is essential for cytotoxicity 6he studiesdescried here have utili$ed artemisinin derivatives!dihydro-artemisinin! 1/-( p-bromophenoxy)dihydroartemisinin, and 1/-( p-uorophenoxy)dihydroartemisinin, to determine thechemistry of endoperoxide ridge activation toreactive intermediates responsile for initiating cell

death and to elucidate the molecular mechanism ofcell death 6hese studies have demonstrated theselective cytotoxic activity of the endoperoxidesto*ard leukemia cell lines (:#-2/ and Ourkat) overuiescent peripheral lood mononuclear cells 'eoxy-1/-( p-uorophenoxy)dihydroartemisinin, hich lac!sthe endoperoxide ridge! *as /- and 14/-fold lessactive in :#-2/ and Ourkat cells! respectively!conrming the importance of this functional group for

cytotoxicity @e have sho*n that chemical activationis responsile for cytotoxicity y using liuidchromatography-mass spectrometry analysis tomonitor endoperoxide activation y measurement of astale rearrangement product of endoperoxide-derived radicals! *hich *as formed in sensitive :#-2/cells ut not in insensitive peripheral loodmononuclear cells 9n :#-2/ cells the endoperoxidesinduce caspase-dependent apoptotic cell death

characteri$ed y concentration- and time-dependentmitochondrial memrane depolari$ation! activation of



L9n :#-2/ cells the endoperoxides induce caspase-dependent apoptotic cell death characteri$ed yconcentration- and time-dependent mitochondrialmemrane depolari$ation! activation of caspases-4and ->! su-</B<1 'N% formation! and attenuation yen$yloxycaronyl-%'-Quoromethyl ketone! a

caspase inhiitor 8verall! these results indicate thatendoperoxide-induced cell death is a conseuence ofactivation of the endoperoxide ridge to radicalspecies! *hich triggers caspase-dependentapoptosis

mechanisms of cell death 2012

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