Apoptosis

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For more than twenty years death of tumour cells by theapoptosis pathway has been one of the most vigoro-

usly researched issues in neoplasm biology.Scientists still search for factors, which selectively indu-

ce death of neoplastic cells along this pathway. At the sa-me time, acknowledging reports claiming that tumour cellsare able to effectively defend themselves against destruc-tion on the apoptotic pathway means that research projectsare being continued to find out changes in the natural cour-se of apoptosis within cells of various neoplasms. Findingmodifications responsible for reduction of tumour cells abi-lity to apoptosis can allow introduction of new therapeuticmethods in treatment of neoplasms, which will be basedupon unblocking of apoptotic process in tumour cells. Suchaction could lead to improved therapeutic effectiveness oftraditional modalities, chemo- or radiotherapy.

General characteristics of apoptotic process

The history of research on cell death started in 19th cen-tury. The term ”apoptosis” was introduced in 1971 by Au-stralian scientist John Kerr [1]. He described characteristicmorphology of spontaneously dying cell: it was losing wa-ter content, organelles did not change and chromatin wasundergoing condensation. A few years later degradation of DNA by endonucleases activated during apoptosis wasdescribed. Formed as a result of a aforementioned activa-tion cellular pattern of so called “apoptotic ladder” constitu-ted the basic marker of apoptosis for many years [2].

The breakthrough in research on cell death came whenit was discovered, that it is process regulated by genes – the existence of death programmed cells of Caenorhab-ditis elegans during the development phase of this nema-tode [3].

Molecular mechanisms of programmed death are evolu-tionary conservative and progress similarly in developed

animals, including human. Data from recent years showthat it also occurs in plant cells [4].

It is now widely acknowledged, that along the path of normal cell transformation into a neoplastic one specificmechanisms develop within the cell which diminish its abi-lity to apoptosis [5,6].

Deregulation of cell death programme can form a basisfor numerous pathological processes, including inductionof neoplasm development [7-9].

Apoptotic cell sends signals about its death to othercells, for example through externalisation of phosphatidyl-serine or calreticuline, which leads to activation of scaven-ger cells, too [10].

Sequence of molecular events taking place during cellapoptosis depends upon cell’s type and a kind of factor le-ading to death, this rule regards both normal and tumourcells [11].

Apoptosis is a complex process, where important role isplayed by epigenetic mechanisms responsible for regula-tion of activity of proteins participating in this event [12].

The presence of various forms of a given protein (asa result of alternative splicing) and post translation modifi-cation of protein molecules (phosphorylation, dephospho-rylation, proteolytic cleavage) means that each protein canfunction as both proapoptotic and antiapoptotic factor [11].

Basic pathways leading to death of apoptotic cells are:- receptor pathway (extrinsic),- mitochondrial pathway (intrinsic).The extrinsic pathway involves activation of cell surface

receptors, so called death receptors, among which the bestknown are: TNFR1, Fas, TRAILR1/R2 (figure 1). Activationof these receptors by corresponding ligands: TNF-α, FasL,TRAIL secreted by for example cells of the immune system,leads to formation of death-inducing signalling complex(DISC), where activation of procaspase 8 occurs. Then ca-spase 8 activates effector procaspases, mainly procaspa-se 3, which in turn leads to activation of further factors en-

Mechanisms of tumor cell’s abilityto avoid apoptosisMiroslawa Cichorek, PhDDepartment of Embriology, Medical University of Gdanskhead: Miroslawa Cichorek, PhD

gaged in cell self destruction [13-15].In case of intrinsic pathway factor inducing

apoptosis comes from within the cell itself, itcan be for example DNA damage, excessiveamount of misfolded proteins accumulatedwithin endoplasmatic reticulum (ER). This canresult in calcium ions release from ER [16-18](figure 1). Mitochondria play a key role [18-20]on this pathway because apoptogenic factorsare released from their intramembrane space into cell cytoplasm including cytochro-me c and AIF – Apoptosis Inducing Factor.Proteins from Bcl-2 family are of utmost im-portance in the process of releasing apopto-genic factors from mitochondria [21,22].

Released cytochrome c together with cyto-plasmic protein Apaf-1 form a complex calledAPOPTOSOME, where procaspase 9 under-goes activation, which is the main initiator ca-spase of mitochondrial pathway. Caspases 9then activates effector procaspases and a fur-ther course of cell damage course is similar tothe receptor pathway [14,23,24]. Effector pro-caspases, including among others procaspa-se 3, undergo activation within cytoplasm le-ading to proteolysis of proteins important forthe cell life, for example poly-(ADP-ribose) po-lymerase (PARP) and cytoskeleton proteins oractivation of DNA cutting endonuclease intooligonucleosomal fragments [25-29]. This inturn leads to cell desintegration into frag-ments surrounded by membrane remnantscalled apoptotic bodies, which are then cle-ared by phagocytes [10,14]. Apoptosis is wi-dely regarded as so called silent cell deathbecause it does not cause inflammatory reac-tion of the immune system [10,14].

Many proteins were found to be caspases substrates displaying characteristic fragments formed as the conse-quence of proteolysis with participation of activated effectorcaspases allowing thus to define apoptotic changes withinthe cell. For example, presence of PARP fragment, with mo-lecular weight of 89 kDa inside the cell reflects activation of procaspase 3, while typical ”apoptotic ladder” confirmsactivation of endonuclease CAD (Caspase Activated Dna-se) [25,29,30].

Some cells following activation of caspases 8 (initiatorcaspase of exstrinsic pathway) are able to activate Bid pro-tein (proapoptotic protein from Bcl-2 family belonging to thegroup BH3-only proteins), which in interaction with externalmitochondrial membrane cause release of apoptogenic

factors from these organelle, for example cytochrome c (re-leased mainly as a result of activation of intrinsic apoptoticpathway) (figure 1). Thus Bid protein enables coordinationof both apoptotic pathways [31,32].

In the course of apoptosis caspases, cysteinyl prote-ases are key elements both during initiation phase of thisprocess and during final stages leading to cell’s death[25,26,29,30].

However, the cell can control activation of caspasesusing various proteins, including mostly IAP proteins family(Inhibitors of Apoptosis Proteins) e.g. survivin, X-IAP, ML--IAP; cytoplasmic FLIP protein, Smac/DIABLO protein rele-ased from mitochondria and Heat Shock Proteins (HSP)[33-38].

The cell can also trigger apoptosis without caspases ac-

Mechanisms of tumor cell’s abilityto avoid apoptosisSUMMARYKey words: apoptosis, tumor cell, Bcl-2, IAP

Investigations on tumor development indicate that tumor growth isthe result of tumor cells’ ability to proliferate and die. The decreaseability of tumor cells to undergo apoptosis is well documented. Themost important elements which inhibit tumor cell’ s apoptosis are:- in the extrinsic way: decreased expression of death receptors e.g.Fas, TNFR1, TRAILR1/R2; decreased content of caspase 8; highlevel of FLIP protein,- in the intrinsic way: high content of antiapoptotic Bcl-2 proteins;APAF inactivation, - in both ways: high content of IAP proteins,The main control points in apoptosis of tumor cell are:- regulation of cytochrome c releasing from mitochondria and Ca2+

from endoplasmatic reticulum by pro- and antiapoptotic proteinsfrom Bcl-2 family,- activity of caspases regulated by IAP proteins. Bcl-2 proteins structurally and functionally divide into:- antiapoptotic proteins e.g. Bcl-2, Bcl-XL, Mcl-1; have four BHdomains; many tumor cells have increased content of these pro-teins,- proapoptotic multidomain proteins e.g. Bax, Bak; have three BH domains; increase permeability of the outer mitochondrialmembrane,- proapoptotic BH3 only proteins e.g. Bid, Bad, Noxa, Puma; haveonly BH3 domain; cooperate with two other Bcl-2 family proteins. IAP proteins inhibit caspases mainly by:- direct inhibition of caspase activity probably by masking theactive site in the caspase,- decreasing the content of capases – RING domain ubiquitylatescaspase and such marked caspase is degradated by proteasomesystem. The increasing knowledge about mechanisms of tumor cell’sdecreased ability to apoptosis gives opportunity to improve effec-tiveness of chemio- or radiotherapy.

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tivation, for example through AIF (Apoptosis Inducing Fac-tor). Many authors stress the particularly important partplayed by AIF, because this protein is transferred from mi-tochondria into nucleus and there activates endonucle-ases, thus bypassing caspases, what could in turn explainwhy some cells undergo apoptosis without caspases parti-cipation [39,40].

Simultaneously with the changes within mitochondrialmembranes ATP synthesis is stopped, oxidation and re-duction processes are disturbed and reactive oxygen spe-cies are formed, which could also influence the course of apoptosis [20,41].

A lot of attention in research on apoptosis is concentra-ted upon proteins regulating this process, mainly from Bcl--2 family, among which are both proteins inhibiting apopto-sis e.g. Bcl-2, Bcl-XL, and promoting this process e.g. Bax,Bad, Bid, Puma, Noxa. These proteins act by influencingthe permeability of mitochondrial membranes and thus release of cytochrome c, AIF and other factors accumula-ted within intra-membrane mitochondrial space [41-43].

The most important stages of apoptosis are quite welldescribed, but numerous molecular aspects of this processare still unexplained, for example the exact mechanism leading to release of apoptogenic factors from mitochon-drial intramembrane space and the role of other organelleslike endoplasmic reticulum.

Main changes occurring in the course of apoptosis within tumour cells

Since the very beginning of theresearch on apoptosis the possiblerole of this process in developmentof neoplasms has been raised. Al-ready, more than thirty years ago,Kerr – author of the term ”apopto-sis” – explained slowing rate of thegrowth of some tumours as a resultof spontaneous apoptosis of neo-plastic cells [1].

In the following years inc-reaseof apoptosis in tumour cells follo-wing radiotherapy and chemothe-rapy was described [44,45]. It hasbeen proven, that apoptosis consti-tutes the main pathway of tumourcell destruction in aforementionedtherapeutic modalities. It has also

been disclosed that cells of numerous types of tumours areresistant to induction of apoptosis [6-9,46-49].

A question appeared: why? The continuing years of re-search on mechanisms of apoptosis has proven that tumo-ur cells display a number of modifications, which enablethose cells to avoid suicidal death [8,24,47,50]. The abovethesis has even been later well documented and so in 2000Hanahan and Weinberg recognised reduction of ability toapoptotic death as one of six characteristic features of neoplastic cell physiology (the remaining features sug-gested by the authors are: self-sufficiency with regard toproduction of growth factors, insensitivity to growth inhibi-ting factors, limitless replicative potential, ability to induceangiogenesis, ability of cells to invade adjoining tissuesand create distant metastases) [5].

It is also generally stressed, that protein p53 is the fac-tor playing major role in the process of tumour cell apopto-sis because it recognises damaged DNA. Gen p53 actingas ”genome guardian”, triggers cell apoptosis in cases of irreparable lesions in DNA structure.

If it is assumed then that DNA structure alterations formthe basis of carcinogenesis process then one could expectto confirm inactivation of protein p53, which normally initia-tes DNA repair mechanisms, in neoplastic cells and it hasreally been confirmed in over 50% of tumours, where genep53 mutations were identified. In the remaining neoplasmsinactivation of protein p53 can be effected by other path-ways, for example by its posttranslational modifications and

Fig. 1

The extrinsic and intrinsic ways of cell’s apoptosis.

changes in activity of other proteins regulating p53 activity[51-53].

The further element of the process of apoptosis, whichcommonly becomes altered in tumour cells, is inactivationof receptor apoptotic pathway. Inactivation of extrinsic pa-thway of apoptosis is the result of changes in the followingreceptors: Fas and TRAILR1/R2 [50,54,55], reduced con-tent of procaspase 8 [56] as well as increased content of aninhibitor of this caspase -FLIP protein [57,58].

Other changes occurring in tumour cells and regardingapoptosis are presented in Table 1, the most importantamong them are the following:

- changes of content of proapoptotic and antiapoptoticproteins from Bcl-2 family (the most commonly observedwas elevation of antiapoptotic proteins and reduction ofproapoptic proteins level),

- inactivation of Apaf-1 leading in turn to inhibition of procaspase 9 activation,

- increased level of IAP proteins, for example survivin, X-IAP, ML-IAP.

These data published in recent years show that reduc-tion of cells ability to undergo apoptosis can be the result of modifications taking place at almost any stages alongthe course of apoptosis.

What are then the main mechanisms defending neopla-stic cell against apoptosis? Neoplastic cell defends itselfagainst apoptosis induced by:

- receptor pathway through reduction of receptors

expression for apoptosis inducing factors such as FasL,TNF-a, TRAIL; reduction of the level of procaspase 8; ele-vation of FLIP protein,

- mitochondrial pathway through increase of the level of antiapoptotic proteins from Bcl-2 family; inactivation ofAPAF,

- both pathways due to increased level of IAP pro-teins.

Sites of action of the above mentioned elements in thecourse of apoptosis are illustrated in figure 2.

Characteristics of the most importantproteins decreasibg ability of tumour cells to apoptosis

� FLIP Protein (FLICE/caspase 8 inhibitor protein)Cytoplasmic protein FLIP binds to the forming DISC

activating complex instead of adaptor protein FADD, whichprevents the activation of procaspase 8 – the main initiatorprocaspase in extrinsic way of apoptosis [34,58] (figure 3).FLIP protein is regarded as the main element inhibiting ac-tivation of extrinsic apoptotic pathway thus protecting neo-plastic cells against factors secreted by the immune systemcells, for example FasL, TNF-α [59,60].

� Bcl-2 proteins familyBcl-2 proteins family plays the main role in regulation

of mitochondrial apoptotic path-way. Proteins from Bcl-2 family ha-ve common at least one out of fourregions called Bcl-2 homology do-mains (BH): BH1, BH2, BH3, BH4.They are present in cytoplasm andbind to external mitochondrialmembrane, membranes of endo-plasmic reticulum, nuclear mem-brane [21,22,43].

Protein family is classified intothree groups depending upon mo-lecular structure and function:

- antiapoptotic, for example:Bcl-2, Bcl-XL, Mcl-1; they containall four domains; inhibit actions ofproapoptic proteins; protect cellsagainst action of numerous factorsinducing apoptosis; cells of manyneoplasms are characterised bytheir higher content,

- proapoptotic multidomain, forexample: Bax, Bak; they have three

Fig. 2

Main mechanisms decreasing tumor cells’ ability to apoptosis.


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domains; they are responsible mainly for increased perme-ability of external mitochondrial membrane,

- proapoptotic BH3-only proteins for example: Bid, Bad,Noxa, Puma; display only BH3 domain, which enables bin-ding with proteins from the remaining two groups of the Bcl--2 family and controls their actions.

Bcl-2 proteins family influence permeability of intracellu-lar membranes for ions and proteins. It is widely believed,that Bax (present in cytosol) and Bak proteins (present in external mitochondrial membrane) form pores in externalmitochondrial membrane or influence opening of mitochon-drial chanales existing within membrane [21,22, 42]. Incre-ase of external mitochondrial membrane permeability re-sults in release of factors present in intramembrane space,for example: cytochrome c, which as an element of apop-tosome participates in activation of procaspase 9 [21,22,42].

The importance of proteins from Bcl-2 family in regula-tion of release of Ca2+ from endoplasmic reticulum into cy-tosol in response to some apoptosis inducing factors hasbeen highlighted in recent years [16]. Released calcium

ions act as secondary messengers and influence numero-us processes occurring within the cell during apoptosis, forexample: proteases and endonucleases activation. Theseions are also taken up by mitochondria leading to alterationof membrane permeability of these organelle [16].

Action of multidomain proapoptic proteins is modifiedby antiapoptotic proteins, for example: Bcl-2 and BH3-onlyproteins [21,22,42,43].

Antiapoptotic proteins inhibit outflow of cytochromec from mitochondria and Ca 2+ ions from endoplasmic reti-culum.

The exact mechanism of apoptosis regulation by Bcl-2protein family is still unclear, but there are three suggestedpathways of mutual interactions between three groups of proteins within Bcl-2 family:

- multidomain proapoptotic proteins are stored in an in-active state through formation of complexes with antiapop-totic proteins; following induction of apoptosis activated.BH3-only proteins bind to antiapoptotic Bcl-2 prote-ins thus releasing proapoptotic multidomain proteins e.g.Bax, Bak,

Table 1 [1-22]*Changes in the course of apoptosis in various neoplastic cells

p53

Antiapoptotic proteins:Bcl-2, Bcl-XL, Mcl-1

Proapoptotic proteins:Bax, BakBad

Apaf-1

Caspase-9

Caspase-8

Caspase-3

Proteins inhibiting caspases activation:SurvivinML-IAPX-IAPFLIP

Fas

TRAIL R1/R2

Cytochrome c

Heat Shock Protein (HSP)

Gene/protein Observed alteration in apoptosis courseChange in gene/protein

Mutations in 50-70% [1]

Increased level [2,3]

Decreased level [2,3]Hyperphosphorylation [2]

Reduced content, inactivation [4,5]

Reduced content [6]

Hypermethylation, gene loss or mutations [7-10]

Lack of translocation into the nucleus [11], Reduced level [12]

Increased level [13,14]Increased level [13,14]Increased level [14,15]Increased level [15,16]

Decreased expression and mutations [17,18]

Mutations [19]

Lack of release [20], Release defect [21]

Increased level [3,22]

Blocking of mitochondrial pathway


Blocking of caspases activation

Reduction of caspases activity

Blocking of receptor pathway

Blocking of effector phase

Resistance to induction of apoptosisInhibition of caspases: 3, 6, 7, 9Inhibition of caspases: 3, 6, 7, 9Blocking of receptor pathway




Inhibition of caspases activation and transloca-tion of AIF into nucleus

Changes in the course of apoptosis caused by influence upon the expression of genesencoding proteins participating in apoptosis,for example Fas, Bax, Noxa, Puma

- after induction of apoptosis activated BH3-only prote-ins e.g. tBid, directly activate multidomain proapoptoticproteins, for example Bax,

- antiapoptotic proteins bind to BH3-only protein whichprevents activation of multidomain proapoptotic proteins.

The results of research from recent years stress the par-ticular role of BH3-only proteins in regulation of apoptosis[21,22,31,32].

Continuation or blocking of apoptosis is dependentupon coactions of the above described proteins from Bcl-2 family, which regulate outflow of cytochrome c frommitochondria and Ca2+ ions from endoplasmic reticulum(figure 3).

� IAP Proteins (Inhibitors of Apoptosis Proteins)IAPs include approximately 10 proteins, for example

ML-IAP (Melanoma IAP, livin), survivin, X-IAP (X-linked ma-mmalian IAP), which within their structure contain one oreven three BIR domains (Baculoviral IAP Repeat motif) while some have also RING domain participating in the pro-cess of binding ubiquitin to proteins, which undergo degra-dation in apoptosome [38,61,62,63].

IAPs inhibit initiatior (caspase 9) and effector (caspases:3,7) caspases and thus can block both apoptotic path-ways: intrinsic and extrinsic (figure 3).

Results of the research point out that IAPs can inactiva-te caspases in two ways:

- through binding to the caspa-se active centre IAPs inhibit activityof these proteases,

- thanks to RING domain theyparticipate in binding ubiquitin tocaspases, thus these proteins un-dergo degradation within apopto-some.

The exact mechanism of interac-tion between caspases and IAPs isstill being investigated [61,64,65].

Two proteins from IAP family, su-rvivin and livin, both of which wereobserved to reach higher levels incells of numerous neoplasms meritspecial attention [33,35,66].

The presence of survivin or livinin tumour cells is associated with:

- more aggressive phenotype[66,67],

- shorter survival time [66,67],- reduced response to chemo-

therapy [66,67].IAP proteins inhibit apoptosis at

the stage of caspases activation and because this is the cri-tical point for cell future there are also two additional prote-ins inhibiting IAPs, so called IAP inhibitors.

� IAP InhibitorsThe best known protein of this group is Smac/DIABLO

(Second Mitochondrial-derived Activator of Caspase/ DirectIAP Binding protein with Low PI; protein discovered in 2000)released from mitochondria together with cytochromec [36,68]. This protein binds intracellular IAPs (mainly X-IAP)and thus protects activated caspases against inactivationby IAPs (figure 3). Smac/DIABLO thus acts as an proapop-totic factor. Other inhibitors are: Omi/HtrA2, XAF1 (X-IAP--associated factor 1) [20,69]. Cells of numerous neoplasmsare characterised by reduced content of these inhibitors[68,69].

SummaryThe most important control points in the course of apop-

tosis, both in normal cells and tumour cells include: - regulation of release of cytochrome c from mitochon-

dria and Ca2+ from endoplasmic reticulum by proapoptoticand antiapoptotic Bcl-2 proteins family,

- inhibition of activated caspases by IAPs and theirinhibitors.

Cells of numerous neoplasms can inhibit the course of

Fig. 3

Places of antiapoptotic proteins action in tumor cells’ apoptosis.


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process of apoptosis thanks to the increased level of anti-apoptotic proteins from Bcl-2 family or augmentation of IAPproteins content.

Research into mechanisms defending tumour cells

against apoptosis allows to attempt reactivation of thisprocess in order to obtain better effectiveness of chemo-and radiotherapy.

Translation:Kornel Zdanowski, MD

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62. Salvesen G., Duckett C.: IAP proteins: blocking the road to death’s door. Nature Rev Mol Cell Biol, 2002, 3: 401-410.

63. Vaux D., Silke J.: IAPs, RINGs and ubiquitylation. Nat Rev MolCell Biol, 2005, 6: 287-297.

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Adres do korespondencji:Miros∏awa CichorekZak∏ad Embriologii, Akademia Medyczna w Gdaƒskuul. D´binki 1, 80-210 Gdaƒske-mail: [email protected]

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