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Mechanisms of tumor cells ability to avoid apoptosis

Miroslawa Cichorek, PhD Department of Embriology, Medical University of Gdansk head: Miroslawa Cichorek, PhD
or more than twenty years death of tumour cells by the apoptosis pathway has been one of the most vigorously researched issues in neoplasm biology. Scientists still search for factors, which selectively induce death of neoplastic cells along this pathway. At the same time, acknowledging reports claiming that tumour cells are able to effectively defend themselves against destruction on the apoptotic pathway means that research projects are being continued to find out changes in the natural course of apoptosis within cells of various neoplasms. Finding modifications responsible for reduction of tumour cells ability to apoptosis can allow introduction of new therapeutic methods in treatment of neoplasms, which will be based upon unblocking of apoptotic process in tumour cells. Such action could lead to improved therapeutic effectiveness of traditional modalities, chemo- or radiotherapy.

General characteristics of apoptotic process

The history of research on cell death started in 19th century. The term apoptosis was introduced in 1971 by Australian scientist John Kerr [1]. He described characteristic morphology of spontaneously dying cell: it was losing water content, organelles did not change and chromatin was undergoing condensation. A few years later degradation of DNA by endonucleases activated during apoptosis was described. Formed as a result of a aforementioned activation cellular pattern of so called apoptotic ladder constituted the basic marker of apoptosis for many years [2]. The breakthrough in research on cell death came when it was discovered, that it is process regulated by genes the existence of death programmed cells of Caenorhabditis elegans during the development phase of this nematode [3]. Molecular mechanisms of programmed death are evolutionary conservative and progress similarly in developed

animals, including human. Data from recent years show that it also occurs in plant cells [4]. It is now widely acknowledged, that along the path of normal cell transformation into a neoplastic one specific mechanisms develop within the cell which diminish its ability to apoptosis [5,6]. Deregulation of cell death programme can form a basis for numerous pathological processes, including induction of neoplasm development [7-9]. Apoptotic cell sends signals about its death to other cells, for example through externalisation of phosphatidylserine or calreticuline, which leads to activation of scavenger cells, too [10]. Sequence of molecular events taking place during cell apoptosis depends upon cells type and a kind of factor leading to death, this rule regards both normal and tumour cells [11]. Apoptosis is a complex process, where important role is played by epigenetic mechanisms responsible for regulation of activity of proteins participating in this event [12]. The presence of various forms of a given protein (as a result of alternative splicing) and post translation modification of protein molecules (phosphorylation, dephosphorylation, proteolytic cleavage) means that each protein can function 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 best known are: TNFR1, Fas, TRAILR1/R2 (figure 1). Activation of these receptors by corresponding ligands: TNF-, 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 caspase 8 activates effector procaspases, mainly procaspase 3, which in turn leads to activation of further factors en-

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gaged in cell self destruction [13-15]. In case of intrinsic pathway factor inducing apoptosis comes from within the cell itself, it can be for example DNA damage, excessive amount of misfolded proteins accumulated SUMMARY within endoplasmatic reticulum (ER). This can Key words: apoptosis, tumor cell, Bcl-2, IAP result in calcium ions release from ER [16-18] Investigations on tumor development indicate that tumor growth is (figure 1). Mitochondria play a key role [18-20] the result of tumor cells ability to proliferate and die. The decrease on this pathway because apoptogenic factors ability of tumor cells to undergo apoptosis is well documented. The are released from their intramembrane most important elements which inhibit tumor cell s apoptosis are: - in the extrinsic way: decreased expression of death receptors e.g. space into cell cytoplasm including cytochroFas, TNFR1, TRAILR1/R2; decreased content of caspase 8; high me c and AIF Apoptosis Inducing Factor. level of FLIP protein, Proteins from Bcl-2 family are of utmost im- in the intrinsic way: high content of antiapoptotic Bcl-2 proteins; portance in the process of releasing apoptoAPAF inactivation, genic factors from mitochondria [21,22]. - in both ways: high content of IAP proteins, The main control points in apoptosis of tumor cell are: Released cytochrome c together with cyto- regulation of cytochrome c releasing from mitochondria and Ca2+ plasmic protein Apaf-1 form a complex called from endoplasmatic reticulum by pro- and antiapoptotic proteins APOPTOSOME, where procaspase 9 underfrom Bcl-2 family, goes activation, which is the main initiator ca- activity of caspases regulated by IAP proteins. spase of mitochondrial pathway. Caspases 9 Bcl-2 proteins structurally and functionally divide into: - antiapoptotic proteins e.g. Bcl-2, Bcl-XL, Mcl-1; have four BH then activates effector procaspases and a furdomains; many tumor cells have increased content of these prother course of cell damage course is similar to teins, the receptor pathway [14,23,24]. Effector pro- proapoptotic multidomain proteins e.g. Bax, Bak; have three caspases, including among others procaspaBH domains; increase permeability of the outer mitochondrial se 3, undergo activation within cytoplasm lemembrane, - proapoptotic BH3 only proteins e.g. Bid, Bad, Noxa, Puma; have ading to proteolysis of proteins important for only BH3 domain; cooperate with two other Bcl-2 family proteins. the cell life, for example poly-(ADP-ribose) poIAP proteins inhibit caspases mainly by: lymerase (PARP) and cytoskeleton proteins or - direct inhibition of caspase activity probably by masking the activation of DNA cutting endonuclease into active site in the caspase, oligonucleosomal fragments [25-29]. This in - decreasing the content of capases RING domain ubiquitylates caspase and such marked caspase is degradated by proteasome turn leads to cell desintegration into fragsystem. ments surrounded by membrane remnants The increasing knowledge about mechanisms of tumor cells called apoptotic bodies, which are then cledecreased ability to apoptosis gives opportunity to improve effecared by phagocytes [10,14]. Apoptosis is witiveness of chemio- or radiotherapy. dely regarded as so called silent cell death because it does not cause inflammatory reaction of the immune system [10,14]. factors from these organelle, for example cytochrome c (reMany proteins were found to be caspases substrates leased mainly as a result of activation of intrinsic apoptotic displaying characteristic fragments formed as the conse- pathway) (figure 1). Thus Bid protein enables coordination quence of proteolysis with participation of activated effector of both apoptotic pathways [31,32]. caspases allowing thus to define apoptotic changes within In the course of apoptosis caspases, cysteinyl protethe cell. For example, presence of PARP fragment, with mo- ases are key elements both during initiation phase of this lecular weight of 89 kDa inside the cell reflects activation process and during final stages leading to cells death of procaspase 3, while typical apoptotic ladder confirms [25,26,29,30]. activation of endonuclease CAD (Caspase Activated DnaHowever, the cell can control activation of caspases se) [25,29,30]. using various proteins, including mostly IAP proteins family Some cells following activation of caspases 8 (initiator (Inhibitors of Apoptosis Proteins) e.g. survivin, X-IAP , MLcaspase of exstrinsic pathway) are able to activate Bid pro- -IAP; cytoplasmic FLIP protein, Smac/DIABLO protein reletein (proapoptotic protein from Bcl-2 family belonging to the ased from mitochondria and Heat Shock Proteins (HSP) group BH3-only proteins), which in interaction with external [33-38]. mitochondrial membrane cause release of apoptogenic The cell can also trigger apoptosis without caspases ac-

Mechanisms of tumor cells ability to avoid apoptosis

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tivation, for example through AIF (Apoptosis Inducing Factor). Many authors stress the particularly important part played by AIF, because this protein is transferred from mitochondria into nucleus and there activates endonucleases, thus bypassing caspases, what could in turn explain why some cells undergo apoptosis without caspases participation [39,40]. Simultaneously with the changes within mitochondrial membranes ATP synthesis is stopped, oxidation and reduction processes are disturbed and reactive oxygen species are formed, which could also influence the course of apoptosis [20,41]. A lot of attention in research on apoptosis is concentrated upon proteins regulating this process, mainly from Bcl-2 family, among which are both proteins inhibiting apoptosis e.g. Bcl-2, Bcl-XL, and promoting this process e.g. Bax, Bad, Bid, Puma, Noxa. These proteins act by influencing the permeability of mitochondrial membranes and thus release of cytochrome c, AIF and other factors accumulated within intra-membrane mitochondrial space [41-43]. The most important stages of apoptosis are quite well described, but numerous molecular aspects of this process are still unexplained, for example the exact mechanism leading to release of apoptogenic factors from mitochondrial intramembrane space and the role of other organelles like endoplasmic reticulum.

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Main changes occurring in the course of apoptosis within tumour cells

Since the very beginning of the research on apoptosis the possible role of this process in development of neoplasms has been raised. Already, more than thirty years ago, Kerr author of the term apoptosis explained slowing rate of the growth of some tumours as a result of spontaneous apoptosis of neoplastic cells [1]. In the following years inc-rease of apoptosis in tumour cells following radiotherapy and chemotherapy was described [44,45]. It has been proven, that apoptosis constitutes the main pathway of tumour cell destruction in aforementioned therapeutic modalities. It has also

been disclosed that cells of numerous types of tumours are resistant to induction of apoptosis [6-9,46-49]. A question appeared: why? The continuing years of research on mechanisms of apoptosis has proven that tumour cells display a number of modifications, which enable those cells to avoid suicidal death [8,24,47,50]. The above thesis has even been later well documented and so in 2000 Hanahan and Weinberg recognised reduction of ability to apoptotic death as one of six characteristic features of neoplastic cell physiology (the remaining features suggested by the authors are: self-sufficiency with regard to production of growth factors, insensitivity to growth inhibiting factors, limitless replicative potential, ability to induce angiogenesis, ability of cells to invade adjoining tissues and create distant metastases) [5]. It is also generally stressed, that protein p53 is the factor playing major role in the process of tumour cell apoptosis because it recognises damaged DNA. Gen p53 acting as genome guardian, triggers cell apoptosis in cases of irreparable lesions in DNA structure. If it is assumed then that DNA structure alterations form the basis of carcinogenesis process then one could expect to confirm inactivation of protein p53, which normally initiates DNA repair mechanisms, in neoplastic cells and it has really been confirmed in over 50% of tumours, where gene p53 mutations were identified. In the remaining neoplasms inactivation of protein p53 can be effected by other pathways, for example by its posttranslational modifications and

Fig. 1 The extrinsic and intrinsic ways of cells apoptosis.

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I Bcl-2 proteins family Bcl-2 proteins family plays the main role in regulation of mitochondrial apoptotic pathway. Proteins from Bcl-2 family have common at least one out of four regions called Bcl-2 homology domains (BH): BH1, BH2, BH3, BH4. They are present in cytoplasm and bind to external mitochondrial membrane, membranes of endoplasmic reticulum, nuclear membrane [21,22,43]. Protein family is classified into three groups depending upon molecular structure and function: - antiapoptotic, for example: Bcl-2, Bcl-XL, Mcl-1; they contain all four domains; inhibit actions of proapoptic proteins; protect cells against action of numerous factors inducing apoptosis; cells of many neoplasms are characterised by their higher content, Fig. 2 - proapoptotic multidomain, for example: Bax, Bak; they have three Main mechanisms decreasing tumor cells ability to apoptosis.

changes in activity of other proteins regulating p53 activity [51-53]. The further element of the process of apoptosis, which commonly becomes altered in tumour cells, is inactivation of receptor apoptotic pathway. Inactivation of extrinsic pathway of apoptosis is the result of changes in the following receptors: Fas and TRAILR1/R2 [50,54,55], reduced content of procaspase 8 [56] as well as increased content of an inhibitor of this caspase -FLIP protein [57,58]. Other changes occurring in tumour cells and regarding apoptosis are presented in Table 1, the most important among them are the following: - changes of content of proapoptotic and antiapoptotic proteins from Bcl-2 family (the most commonly observed was elevation of antiapoptotic proteins and reduction of proapoptic 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 reduction of cells ability to undergo apoptosis can be the result of modifications taking place at almost any stages along the course of apoptosis. What are then the main mechanisms defending neoplastic cell against apoptosis? Neoplastic cell defends itself against 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; elevation of FLIP protein, - mitochondrial pathway through increase of the level of antiapoptotic proteins from Bcl-2 family; inactivation of APAF, - both pathways due to increased level of IAP proteins. Sites of action of the above mentioned elements in the course of apoptosis are illustrated in figure 2.

Characteristics of the most important proteins decreasibg ability of tumour cells to apoptosis
I FLIP Protein (FLICE/caspase 8 inhibitor protein) Cytoplasmic protein FLIP binds to the forming DISC activating complex instead of adaptor protein FADD, which prevents the activation of procaspase 8 the main initiator procaspase in extrinsic way of apoptosis [34,58] (figure 3). FLIP protein is regarded as the main element inhibiting activation of extrinsic apoptotic pathway thus protecting neoplastic cells against factors secreted by the immune system cells, for example FasL, TNF- [59,60].

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domains; they are responsible mainly for increased permeability of external mitochondrial membrane, - proapoptotic BH3-only proteins for example: Bid, Bad, Noxa, Puma; display only BH3 domain, which enables binding with proteins from the remaining two groups of the Bcl-2 family and controls their actions. Bcl-2 proteins family influence permeability of intracellular 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 external mitochondrial membrane or influence opening of mitochondrial chanales existing within membrane [21,22, 42]. Increase of external mitochondrial membrane permeability results in release of factors present in intramembrane space, for example: cytochrome c, which as an element of apoptosome participates in activation of procaspase 9 [21, 22,42]. The importance of proteins from Bcl-2 family in regulation of release of Ca2+ from endoplasmic reticulum into cytosol in response to some apoptosis inducing factors has been highlighted in recent years [16]. Released calcium

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ions act as secondary messengers and influence numerous processes occurring within the cell during apoptosis, for example: proteases and endonucleases activation. These ions are also taken up by mitochondria leading to alteration of membrane permeability of these organelle [16]. Action of multidomain proapoptic proteins is modified by antiapoptotic proteins, for example: Bcl-2 and BH3-only proteins [21,22,42,43]. Antiapoptotic proteins inhibit outflow of cytochrome c from mitochondria and Ca 2+ ions from endoplasmic reticulum. The exact mechanism of apoptosis regulation by Bcl-2 protein family is still unclear, but there are three suggested pathways of mutual interactions between three groups of proteins within Bcl-2 family: - multidomain proapoptotic proteins are stored in an inactive state through formation of complexes with antiapoptotic proteins; following induction of apoptosis activated. BH3-only proteins bind to antiapoptotic Bcl-2 proteins thus releasing proapoptotic multidomain proteins e.g. Bax, Bak,

Table 1 [1-22]* Changes in the course of apoptosis in various neoplastic cells Gene/protein
p53

Change in gene/protein
Mutations in 50-70% [1]

Observed alteration in apoptosis course

Changes in the course of apoptosis caused by influence upon the expression of genes encoding proteins participating in apoptosis, for example Fas, Bax, Noxa, Puma Blocking of mitochondrial pathway

Antiapoptotic proteins: Bcl-2, Bcl-XL, Mcl-1 Proapoptotic proteins: Bax, Bak Bad Apaf-1 Caspase-9 Caspase-8 Caspase-3 Proteins inhibiting caspases activation: Survivin ML-IAP X-IAP FLIP Fas TRAIL R1/R2 Cytochrome c Heat Shock Protein (HSP)

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]

Blocking of mitochondrial pathway Blocking of caspases activation Reduction of caspases activity Blocking of receptor pathway Blocking of effector phase

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]

Resistance to induction of apoptosis Inhibition of caspases: 3, 6, 7, 9 Inhibition of caspases: 3, 6, 7, 9 Blocking of receptor pathway Blocking of receptor pathway Blocking of receptor pathway Blocking of mitochondrial pathway Inhibition of caspases activation and translocation of AIF into nucleus

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- through binding to the caspase active centre IAPs inhibit activity of these proteases, - thanks to RING domain they participate in binding ubiquitin to caspases, thus these proteins undergo degradation within apoptosome. The exact mechanism of interaction between caspases and IAPs is still being investigated [61,64,65]. Two proteins from IAP family, survivin and livin, both of which were observed to reach higher levels in cells of numerous neoplasms merit special attention [33,35,66]. The presence of survivin or livin in tumour cells is associated with: - more aggressive phenotype [66,67], - shorter survival time [66,67], Fig. 3 - reduced response to chemoPlaces of antiapoptotic proteins action in tumor cells apoptosis. therapy [66,67]. IAP proteins inhibit apoptosis at - after induction of apoptosis activated BH3-only prote- the stage of caspases activation and because this is the criins e.g. tBid, directly activate multidomain proapoptotic tical point for cell future there are also two additional proteproteins, for example Bax, ins inhibiting IAPs, so called IAP inhibitors. - antiapoptotic proteins bind to BH3-only protein which I IAP Inhibitors prevents activation of multidomain proapoptotic proteins. The best known protein of this group is Smac/DIABLO The results of research from recent years stress the particular role of BH3-only proteins in regulation of apoptosis (Second Mitochondrial-derived Activator of Caspase/ Direct IAP Binding protein with Low PI; protein discovered in 2000) [21,22,31,32]. Continuation or blocking of apoptosis is dependent released from mitochondria together with cytochrome upon coactions of the above described proteins from c [36,68]. This protein binds intracellular IAPs (mainly X-IAP) Bcl-2 family, which regulate outflow of cytochrome c from and thus protects activated caspases against inactivation mitochondria and Ca2+ ions from endoplasmic reticulum by IAPs (figure 3). Smac/DIABLO thus acts as an proapop(figure 3). totic factor. Other inhibitors are: Omi/HtrA2, XAF1 (X-IAP-associated factor 1) [20,69]. Cells of numerous neoplasms I IAP Proteins (Inhibitors of Apoptosis Proteins) are characterised by reduced content of these inhibitors IAPs include approximately 10 proteins, for example [68,69]. ML-IAP (Melanoma IAP , livin), survivin, X-IAP (X-linked mammalian IAP), which within their structure contain one or even three BIR domains (Baculoviral IAP Repeat motif) The most important control points in the course of apopwhile some have also RING domain participating in the process of binding ubiquitin to proteins, which undergo degra- tosis, both in normal cells and tumour cells include: - regulation of release of cytochrome c from mitochondation in apoptosome [38,61,62,63]. IAPs inhibit initiatior (caspase 9) and effector (caspases: dria and Ca2+ from endoplasmic reticulum by proapoptotic 3,7) caspases and thus can block both apoptotic path- and antiapoptotic Bcl-2 proteins family, - inhibition of activated caspases by IAPs and their ways: intrinsic and extrinsic (figure 3). Results of the research point out that IAPs can inactiva- inhibitors. Cells of numerous neoplasms can inhibit the course of te caspases in two ways:

Summary

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process of apoptosis thanks to the increased level of antiapoptotic proteins from Bcl-2 family or augmentation of IAP proteins content. Research into mechanisms defending tumour cells

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against apoptosis allows to attempt reactivation of this process in order to obtain better effectiveness of chemoand radiotherapy.

Translation: Kornel Zdanowski, MD

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Adres do korespondencji: Mirosawa Cichorek Zakad Embriologii, Akademia Medyczna w Gdasku ul. Dbinki 1, 80-210 Gdask e-mail: cichorek@amg.gda.pl

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