Mechanisms of Tumor Cell Necrosis

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Current Pharmaceutical Design, 2010, 16, 56-68

Mechanisms of Tumor Cell Necrosis Sergey Y. Proskuryakov1 and Vladimir L. Gabai2* 1

Medical Radiology Research Center, Obninsk, Russia; 2Boston University Medical School, Boston, MA, USA Abstract: Until recently, necrosis, unlike apoptosis, was considered as passive and unregulated form of cell death. However, during the last decade a number of experimental data demonstrated that, except under extreme conditions, necrosis may be a well-regulated process activated by rather specific physiological and pathological stimuli. In this review, we consider mechanisms and the role of necrosis in tumor cells. It became recently clear that the major player in necrotic cascade is a protein kinase RIP1, which can be activated by number of stumuli including TNF, TRAIL, and LPS, oxidative stress, or DNA damage (via poly-ADP-ribose polymerase). RIP1 kinase directly (or indirectly via another kinase JNK) transduces signal to mitochondria and causes specific damage (mitochondrial permeability transition). Mitochondrial collapse activates various proteases (e.g., calpains, cathepsin) and phospholipases, and eventually leads to plasma membrane destruction, a hallmark of necrotic cell death. Necrosis, in contrast to apoptosis, usually evokes powerful inflammatory response, which may participate in tumor regression during anticancer therapy. On the other hand, excessive spontaneous necrosis during tumor development may lead to more aggressive tumors due to stimulatory role of necrosis-induced inflammation on their growth.

Keywords: Programmed cell death, RIP1 kinase, mitochondria, oxidative stress, inflammation, anti-cancer therapy, tumor progression. INTRODUCTION A few decades ago the discovery of new patterns of cell death led to emergence of the concept of apoptosis. During apoptosis there were remarkably arranged morphological and biochemical events while necrosis was considered as apparently deranged (or accidental) form of cell death. Morphologically, necrosis is quite different from “classical” apoptosis. In the course of apoptosis cells first shrink, their nuclei condense, and then they disintegrate into well-enclosed apoptotic bodies, while during necrosis cells first swell (“oncosis”), then the plasma membrane collapses, and cells are rapidly lysed. Biochemical hallmarks of apoptosis such as activation of specific proteases (caspases) and oligonucleosomal DNA fragmentation are usually absent in necrotic cells. However, improvement of methods of differentiation of apoptosis and necrosis revealed that there are many examples when some biochemical and morphological characteristics of both modes of cell death can be found in the same cell. This indicates that there is a spectrum of suicidal programs in cells, and “classical” necrosis and apoptosis are the extremes of the spectrum (see ref [1] for review). Moreover, during last decade necrotic cell death has brought much more attention and now regarded by many researchers as specific form of programmed cell death (PCD), type III PCD, along with apoptosis (type I PCD), and autophagy (type II PCD) (see ref [1-5] for review). The most common assays for measuring necrosis in vitro is permeability of cell plasma membrane to vital dyes (like trypan blue or propidium iodide), and efflux of cytosolic enzymes (lactate dehydrogenase, creatine kinase). Reduction of tetrazolium salts (e.g., MTT assay) is also often used, but it rather measures mitochondrial activity of cells which can be compromised not only due to necrosis, but to apoptosis or autophagic cell death as well. Of note, however, is that in vitro apoptosis as well as autophagic death finally also leads to plasma membrane permeabilization (“secondary necrosis”), but it does not usually occur in vivo during apoptosis since apoptotic cells are digested by macrophages or surrounding cells before their plasma membrane becomes disrupted. Furthermore, secondary necrosis may also occur due to mitotic catastrophe as a consequence irreversible DNA damage. In this review the term “necrosis” will be mostly attributed to cell *Address correspondence to this author at the Dept Biochemistry, Boston University Medical School, 715 Albany St, Boston, MA 02118, USA; E-mail: [email protected]

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death accompanied by a rapid efflux of cell constituents in extracellular space without activation of caspases or autophagy. We will discuss mechanisms of tumor cell necrosis and its implication in anti-cancer therapy and tumor development. 1. OCCURRENCE OF TUMOR CELL NECROSIS IN VITRO AND IN VIVO Instant killing of cells by extreme conditions such as severe temperature or acids causes unregulated processes of destruction of cell membranes and cytosol. This led to the common assumption that when cell destruction is accompanied by a rapid disruption of the plasma membrane, cytoplasmic structures, and the nucleus, it indicates that the death is passive and unregulated. However, such conclusion disregarded many phenomena where necrotic cell death is a regulated process activated by rather specific physiological and pathological conditions. Of note, a variety of conditions that activate necrosis may activate apoptosis and autophagic cell death as well, dependent on intensity of stimuli and cell type. Among the most common conditions of activation of necrosis are ischemia and hypoxia, which can be simulated in vitro by deprivation of oxygen, glucose and other nutrients (Table 1). These hypoxic and ischemic conditions are often occurs during tumor growth in vivo due to inadequate vascularization. The main cause of necrosis under these conditions is obviously energy deprivation, and to survive this stress cells need either increase energy production or decrease energy consumption. Since in the absence of oxygen and/or nutrients cells have limited capability to boost ATP generation, decreasing ATP consumption is often the only way to avoid necrosis. For instance, protective effect of AG1478, an inhibitor of EGF receptor, from hypoxia-induced necrosis of glioma cells is associated with preservation of ATP [6]. Furthermore, LKB1, serine-threonine kinase which is often mutated in tumors, is important for activity of AMP-activated protein kinase and survival of glucose-deprived tumor cells [7]. Another interesting example of adaptation to avoid hypoxia-induced necrosis has been recently described. Ginouves and co-workers found that, while acute hypoxia (1% 02 ) caused stabilization of hypoxia-induced factors (HIF1 and HIF2), under chronic hypoxia (more than 3 days) these factors are degraded and this degradation is crucial for protection of cells from hypoxia-induced necrosis [8]. Several components of immune system are capable of inducing necrosis, among them TNF, FAS, and TRAIL. Activation of FAS receptors with anti-FAS ligand is also able to provoke necrosis

© 2010 Bentham Science Publishers Ltd.

Mechanisms of Tumor Cell Necrosis

Table 1.

Current Pharmaceutical Design, 2010, Vol. 16, No. 1

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Activators and Inhibitors of Necrosis Activators

Cells

Inhibitors

References

Ischemia

Various cells

Necrostatin-1

[77]

Hypoxia

Glioma

AG1478

[6]

+

MPP

PC-12

-

[61]

Rotenone

PC-12

-

[61]

Antimycin A

Rat-1

-

[63]

Glutamate

HT-22

Necrostatin-1

[174]

TNF

L929, MEF

BHA

[29]

TNF+zVAD

U937, THP-1

GA

[27]

FAS

T-cells

-

[175]

TRAIL (pH6.5)

HT-29

zVAD

[26]

LPS

Macrophages

-

[176]

Anti-CD47

B-lymphocytes, CLL

TPCK

[9]

IL1+IFN

L929

SB203580; sc-514

[56]

ZVAD

L929

Rapamycin

[129]

Ceramide

Jurkat, 3T3

-

[28]

ROS

MEF

SP600125

[38]

RNS

U937, MEF

Rotenone

[39,55]

Photodynamic therapy

HeLa

MitoQ

[177]

As203

HeLa

NAC, PARP inhibitors

[81]

Tamoxifen

MCF7, MDA-MB231

Estrogens, PD98059, BA

[13,14]

MNNG

MEF

PARP inhibitors, SP600125

[10,30]

Imatinib

K562

TPCK

[18]

Honokiol

HL-60, MCF7, HEK293

CsA

[16]

Shikonin

MCF7, HEK293

Necrostatin-1

[17]

Resveratrol

MCF7

-

[15]

Abbreviations and Notes: AG1478 – Inhibitor of EGF receptor; MPP+- 1-Methyl-4-Phenylpyridinium; BHA- butylated hydroxyanisole; GA – geldanamycin; zVAD – caspase inhibitor; TPCK – serine protease inhibitor; SB203580 – p38 MAPK inhibitor; NAC- N-acetylcystein; sc514 – NFkB inhibitor; SP600125 – JNK inhibitor; MNNG – N-methyl- N’nitro- N’ –nitrosoguanidine; PD98059 – ERK1/2 inhibitor; BA – bonkrekic acid; CsA – cyclosporin A.

along with apoptosis, but ligation of CD47 receptors in lymphoid cells induces almost exclusively necrosis [9](Table 1). Various treatments used for anticancer therapy also may cause necrosis of tumor cells. Among them are DNA-alkylating drugs such as nitrogen mustard or MNNG [10], arsenic trioxide [11], photodynamic therapy [12], and tamoxifen [13,14] (Table 1). Resveratrol [15] and some components of traditional Chinese herbal medicine such as honokiol [16] and shikonin [17] are also capable to induce necrosis of tumor cells (Table 1). Importantly, resistance to apoptosis does not prevent killing of tumor cells by these agents. Accordingly, caspase inhibitors failed to prevent necrosis of BCR-ABL-positive human leukemic cells treated with a novel anticancer drug (a protein kinase inhibitor) imatinib (Gleevec) [18](Table 1). Interestingly, genotoxic stresses such as actinomycin D, UV radiation, or cisplatin caused apoptosis in young fibroblasts, but necrosis in old (senescent) human fibroblasts,

which is apparently associated with inability to stabilize tumor suppressor p53 [19]. There are several key events during cancer initiation and progression, and suppression of apoptosis is considered among the most important [20]. Indeed, many oncogenes can activate apoptotic program, and cancer cells often disable tumor suppressors implicated in apoptosis such as p53 or Bax, or overexpress apoptosis inhibitors such as Bcl-2, Bcl-x, or survivin. Furthermore, during in vivo growth, tumor cells may be subjected to ischemia and attack by cytotoxins from immune system, and disabling apoptosis can protect tumor cells from these adverse conditions and promote tumorigenesis. The above findings that apoptosis-resistant cells are still vulnerable to therapy-induced necrosis may have clinical implications. Indeed, Dinnen et al found that p53 Cterminal 22-aa peptide (aa 361-382) linked to truncated 17-aa peptide from Drosophila antennapedia homeobox domain (to

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facilitate cellular uptake) can induce selective necrosis in p53mutant prostate tumor cells [21]. Recent studies from E. White’s lab also demonstrated important role of autophagy in protection of tumor cells from ischemic conditions in vitro and in vivo during tumor development. It was found that apoptosis-resistant cells die via necrosis only when autophagy is suppressed (e.g., by activation of AKT) [22] (see also section 4 below). 2. SIGNALING PATHWAYS AND MODULATORS OF NECROSIS 2.1. Receptors and Protein Kinase Cascades Among the best studied examples of necrotic cell death is that activated by engagement of TNF, FAS, or TRAIL receptors. Normally, activation of these receptors leads to apoptosis via FADD/Caspase-8 signalling; however, when apoptosis is inhibited (genetically or by caspase inhibitors), alternative necrotic pathway is initiated. Critical role in this receptor-activated necrosis is played by protein kinase RIP1, which was demonstrated in several studies, including its knockdown [23] (Fig. 1). Along with activation of necrosis, RIP1 is involved in anti-apoptotic signalling via activation of NF-kB; however, caspase-8 can cleave RIP1 thus promoting apoptosis and preventing necrosis [24]. Activation of RIP1, beside death receptors, can be caused by LPS via toll-like receptors (TLR) TLR3 and TLR4 [25]. As in the case of death receptor activation, LPS normally induce apoptosis in macrophages when NF-kB dependent anti-apoptotic pathway is inhibited, but when apoptosis is suppressed, RIP1-dependent necrosis is initiated. Therefore, switching between apoptosis and necrosis during activation of death and TLR receptors generally depends on activity of caspase-8: when this caspase is disabled, RIP1-dependent necrosis is activated. However, there is interesting exception when at acidic pH 6.5 (which can often occur in tumors due to high glycolysis), TRAIL induce RIP1-dependent necrosis of tumor cells which was accompanied by caspase-8 and caspase-3 activation and prevented, rather than accelerated, by caspase inhibitors [26]. What are downstream targets of RIP1 kinase? In human myelomonocytic U937 and TNP-1 cells treated with TNF or FAS in the presence of caspase inhibitor, necrosis was dependent on RIP1induced inhibition of ATP/ADP translocator in mitochondria , which leads to decrease in ATP levels [27]. RIP1-deficient cells also defective in ceramide accumulation, and prevention of its accumulation protected them from TNF-induced necrosis [28]. Recent study of Kim et al. [29] on L929 murine fibrosarcoma cells demonstrated that RIP1 is also essential for recruitment of NADPH oxidase Nox1 in complex with TRADD (Fig. 1). Nox1 is responsible for generation of ROS (in particular, superoxide O2-), and ROS is essential mediators of TNF-induced necrotic cell death since anti-oxidant BHA almost completely prevented cell death. Another important player in RIP1/Nox1 – dependent necrosis is MAP kinase JNK. This kinase is also involved in apoptotic signalling under many stressful conditions, and, in some cases, its transient activation may play role in mitogenic response and cell survival. However, in case of TNF-induced necrosis, sustained activation of JNK appears to be essential for cell death, since JNK inhibition, similar to inhibition of ROS production, protected cell from necrosis [29] (Fig. 1). It is quite possible that besides ROS, activation of JNK in TNF-induced necrosis is also mediated by ceramide [28], a known JNK stimulator. Unexpected link between genotoxic stress, poly (ADP-ribose) polymerase (PARP-1) and RIP1-JNK necrotic cascade has been found by Xu et al. [30]. DNA alkylating agents such as MNNG in high concentrations is able to induce necrosis via PARP-1 stimulation [10,31]. PARP-1 is a nuclear enzyme containing a Zn-

Fig. (1). Key role of RIP1 kinase in necrotic signaling. Diverse stimuli activate signaling pathways intercepting at the level of RIP1 kinase. See text for further explanation.

binding domain; upon activation by DNA breaks it attaches oligomers of ADP-ribose to itself and some other nuclear proteins. Excessive activation of PARP, for example, as a result of profound induction of DNA breaks, was believed to be a cause of cell necrosis due to ATP depletion [32,33] which is resulted from use of ATP for synthesis of the PARP substrate NAD+(see also section 2.3 below). PARP inhibition (e.g., by 3-aminobenzamide and nicotinamide) suppressed cell necrosis [32] or switched it to apoptosis, which was associated a marked increase in caspase activity [34,35]. During apoptosis, PARP, similar to RIP1, is normally inactivated by caspase-specific cleavage, forming an 89-kDa fragment, a biochemical hallmark of apoptosis. If this mechanism of PARP inactivation is not operational, for example, in a PARP mutant resistant to caspase cleavage, cells become more sensitive to necrosis induced by UV radiation or TNF [36,37]. In these cells expressing mutant PARP, as well as in their wild-type counterpart, inhibition of PARP activity reduced necrosis and increased apoptosis [37]. Hence, proteolytic or pharmacological inactivation of PARP is one of the ways to prevent cell elimination through necrotic pathway. Xu et al found that MNNG-induced necrosis, which depends on PARP activity, was significantly suppressed in RIP1 and JNK1 knockout cells [30]. Since poly-ADP ribosylation was not inhibited in these knockout cells, it places RIP1 and JNK downstream of PARP-1 (Fig. 1). Surprisingly, even necrosis induced by hydrogen peroxide and reactive nitrogen species (RNS) also depends on activation of TRAF2-RIP1-JNK signaling cascade [38,39]. Therefore, besides receptor-activated necrosis, RIP1-JNK signaling appears to be important for necrosis induced by some genotoxic agents, ROS and RNS (Fig. 1).


JNK and another related stress kinase, p38 are also involved in necrotic death of cells following transient energy deprivation in vitro, or ischemia/reperfusion of different organs in vivo [40]. Ischemia/reperfusion-induced necrosis was also inhibited by expression of dominant-negative form of Rac, an upstream component of stress-signaling cascade, although this protective effect may be also related to inhibition of ROS production [41], similar to effect of dominant-negative RAC on TNF-induced necrosis [29]. On the other hand, activation of MAP kinase ERK and AKT, which protect cells from stress-induced apoptosis, can protect against necrotic death as well. For example, AKT overexpression reduced necrotic zone formation in ischemic myocardium [42]. Accordingly, ischemia/reperfusion-induced ERK activation seems to be protective against necrosis, since its inhibition aggravated necrosis of myogenic cells in vitro [43] and myocardial infarction in vivo [44]. Therefore, it seems that proapoptotic (JNK, p38) and antiapoptotic kinases (AKT, ERK) play a similar role in necrosis. Of note, tumor cells often have higher activity of AKT and ERK signaling cascades which can make them potentially more resistant not only to apoptosis, but necrosis as well. On the other hand, Akt activity blocks autophagy, an important tumor cell survival mechanism under chronic ischemic conditions. As a result, apoptosisresistant cells with overexpression of active Akt are less resistant to ischemia than cells without such overexpression [22] (see also section 4). 2.2. Reactive Oxygen and Nitrogene Species Necrotic cell death is almost always accompanied by generation of ROS, and various anti-oxidants can prevent necrosis or switch it to apoptosis. Increased production of ROS and RNS can be caused by macrophages during immunological response, by mitochondria, and by some other mechanisms. Hydrogen peroxide, a component of ROS, is often used as a model reagent since it is produced as a factor of immune defense and during various stresses. It can cause both apoptosis and necrosis of cells [34, 45] which can be prevented by the antioxidants glutathione , N-acetylcystein (NAC), BHA and others. When applied together with some antitumor drugs (VP-16, doxorubicin, cisplatin, and AraC) subtoxic doses of H2O2 can switch cell suicide to necrosis [46,47]. This effect was probably associated with specific signaling role of H2O2 rather than with inhibition of caspases or PARP activation (see above) since much higher concentration of H2O2 was necessary for the latter effect [34, 46]. A decrease in cellular content of glutathione can also switch a form of cell death induced by ROS. For example, apoptosis of U937 tumor cells induced by Cd2+, cisplatin, and melphalan was switched to necrosis when glutathione synthesis was inhibited [48, 49]. Interestingly, ROS-induced necrosis can also be modulated by cell transformation. Transformation of 3T3 cells by SV40-T antigen promoted menadione-induced necrosis, which can be inhibited by blocking FAS receptors, although inhibitors of caspases were ineffective [50]. Along with ROS, another mediator of various pathophysiological processes is nitrogen oxide (NO). Because nitrosylation/ denitrosylation reaction is involved in regulation of caspase-3, a key apoptotic caspase, NO may inhibit apoptosis directly through caspase-3 nitrosylation [51], although other mechanisms of inhibition of apoptosis upstream caspase-3 may also exist [52,53]. The inhibition of apoptotic pathway may be the reason why NO can switch apoptosis to necrosis upon treatment with staurosporine, ceramide, FAS, and retinoids [52]. On the other hand, FAS-induced denitrosylation of caspase-3 by thioredoxin-2 is required for caspase-3 activation and promotion of apoptosis [54]. NO can also bind to iron of heme-containing complexes of respiratory chain and inactivate them, potentially leading to mitochondrial damage (see below). A deleterious effect of exogenous NO can be increased by Fe2+ ions and by blocking GSH synthesis while NAC and SH-group donors can protect against NO.


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Peroxinitrite is a highly active nitrogen compound that is formed in organisms and it is often used as a model simulating action of cytokines on effector cells. Peroxinitrite formation is caused by expression of inducible NO synthase (iNOS) and ROS generation as a result of reaction between NO and superoxide anione. Pronecrotic effect of peroxinitrite in U937 is mediated by hydrogen perioxide apparently generated by mitochondria, since mitochondrial inhibitor rotenone prevented necrosis [55]. Recently, necrosis of L929 tumor cells caused by prolonged incubation with IL-1 and IFN was shown to depend on expression of iNOS and NO production, which, in turn, was dependent on p38 MAP kinase and NF-kB (IKK) activities [56]. In most cases, antioxidants suppress both apoptotic and necrotic cell destruction. It seems that oxidative stress induces an apoptotic response when cells can maintain their reducing capacity against ROS, whereas necrosis is triggered when this reducing homeostasis is disturbed (e.g., by excess of ROS or damage of natural antioxidative systems). 2.3. ATP and Mitochondria Now it seems obvious that mitochondria play a crucial role in determination of cell fate under stresses. First, as a source of ATP, mitochondria chose between ATP-dependent or -independent programs. Second, they generate ROS that control form of cell suicide (see above), and finally, as a source of tanathogenic (deathpromoting) factors, mitochondria initiate or amplify the caspasedependent apoptotic program (mainly through efflux cytochrome c) or activate directly the execution phase (through efflux of apoptosis induction factor, AIF, and other factors). Apparently, maintenance of certain levels of ATP is required for execution of apoptotic programs. ATP or its derivate, dATP, is a cofactor of apoptosome [57], a high-molecular-weight complex consisting of APAF-1 and caspase-9 [58], which activates a major execution caspase, caspase-3. Besides apoptosome, ATP also seems necessary at other stages of the apoptotic program [59]. Generally, if the amount of ATP drops below some critical levels, this either can switch apoptotic cell death to necrotic (e.g., when cells exposed to genotoxic stress causing profound PARP activation, see section 2.1) or may cause necrosis by itself. For instance, in HeLa cells, inhibition of glycolysis and respiration leads to more than 97% decrease in ATP and, if ATP levels restored after 3 hr, cells die via apoptosis, but more prolonged energy deprivation evoked necrotic cell death [60]. Mitochondrial inhibitors alone can cause necrosis in some cells. Inhibitors of complex I of respiratory chain, such as rotenone, 1methyl-4-phenylpyridium, or 6-hydroxytriptamine, which simulate cell loss during Parkinson’s disease, caused necrosis of PC12 neuroblastoma cells [61]. Furthermore, inhibitors of complex II, 3nitropropionic acid, or complex III, antimycin A, also induced necrosis [62,63]. Mitochondrial inhibitors, however, usually do not affect viability of tumor cells with high level of glycolysis that are capable of maintaining ATP levels without any respiration (see, e.g., [64, 65]). Of note, drastic ATP depletion (below 3% to 5% of initial) for many hours resulted from hypoxia or starvation is not toxic for some cells (e.g., fibroblasts), whereas other cells (e.g., neuronal and cardiac cells) rapidly die via necrosis (see Ref. [65] for review). The reason for such different sensitivity of cells to ATP depletion is not clear, but may be associated with much more severe ionic imbalance (in particular, Ca2+ imbalance) in sensitive cells. On the other hand, necrosis can be induced in the cells with a normal amount of ATP (e.g., TNF-induced necrosis of L929 fibrosarcoma cells, see section 2.1), or peroxynitrite-induced necrosis of U937 cells [66]. This indicates that, although the ATP levels may control mode of cell death, there are other factors that contribute in final outcome. One such factor may be ROS produced by the mitochondrial respiratory chain, and this ROS generation may trigger a necrotic


program, as discussed above. It was hypothesized that when cellular anti-oxidative defense is limited, ROS caused oxidation of the key molecules and release of executor proteases, lipases, and nucleases from mitochondria [67]. The emergence of such dangerous mitochondria triggers the cell’s protective response in the form of autophagy with participation of caspases [67,68]. This hypothesis may explain why in some cells inhibition of caspases, while inhibiting TNF-induced apoptosis, may trigger necrotic cell death. Indeed, TNF may activate mitochondrial ROS generation, and such dangerous ROS-producing mitochondria are normally eliminated by caspase-dependent autophagy [67]. However, when caspases are inhibited, these mitochondria may trigger necrotic death of a whole cell. Interestingly, because some viruses encode caspase inhibitors to avoid apoptosis of infected cells, the ability to trigger necrosis when caspases are inhibited may be an important part of the cellular antiviral defence [69]. Being the source of apoptogenic factors (cytochrome c, Smac/Diablo, AIF), in addition to ROS, mitochondria can be the source of pro-necrotic factors as well. Under some conditions (e.g., high Ca2+, oxidative stress) mitochondria undergo drastic changes accompanied by deenergization of the inner membrane, swelling, and permeabilization, a process called mitochondrial permeability transition (MPT). This is usually an irreversible process leading to mitochondrial “death” (mitochondrial apoptosis or “mitoptosis” [60]). It was suggested that MPT may be inductor of necrotic cell death through release of some mitochondrial factors (e.g., Ca2+, proteases, lipases) [70,71]. Indeed, inhibitors of MPT such as CsA, or bonkrekic acid may protect from necrosis caused by oxidative stress, hypoxia–reoxygenation in vitro [72], or ischemia– reperfusion in vivo [73], or some other stimuli (Table 1). Cyclophilin D (CypD) is a mitochondrial matrix protein and indispensable component of MPT; recent studies showed that knockout of CypD gene induce resistance to necrosis induced by ROS and Ca2+ overload in vitro, as well as ischemia-reperfusion of heart and brain in vivo [74, 75]. Accordingly, knockout of CypD and specific inhibition of cyclophilin D with compound Debio-025 also protected mice from necrosis caused by muscular dystrophy [76]. Interestingly, necrostatin-1, a small molecule inhibitor of necrosis (Table 1), but not apoptosis which was recently found by Yaun’s lab [77], also prevented MPT in mitochondria [78,79]. Specific cellular target of necrostatin-1, however, appears to be RIP1 kinase (see section 2.1) rather than mitochondria [80], and protection of mitochondria is obviously a secondary effect of RIP1 inhibition (see section 2.1). Despite its name, apoptosis-inducing factor (AIF), when released from mitochondria, may also play a crucial role in necrosis, which was shown for arsenic trioxide-induced necrosis of human cervical cancer cells [81], and MNNG-induced necrosis of fibroblasts [3,31]. Release of AIF was also blocked by necrostatin-1 [82]. Recent study from D. Green’s lab showed that mitochondria can preserve their integrity and sustain cell survival despite complete loss of cytochrome c provided caspases are blocked and autophagy is activated. Important role in such survival is played by GAPDH, which both generate ATP via glycolysis and stimulates transcription of Atg12, a protein involved in autophagy [83]. Therefore, mitochondria may be the source of three relatively independent signals that trigger or switch cell death pathways: ATP, ROS, and apoptogenic/necrogenic factors such as cytochrome c and AIF. The final outcome of cell suicide is apparently dependent on interplay between these factors. 2.4. Proteins of the Bcl-2 Family Proteins of the Bcl-2 family play a very significant role in the determination of cell sensitivity to lethal signals. Antiapoptotic members of this family (Bcl-2, Bcl-X L, etc.) can inhibit not only

Gabai et al.

apoptotic, but also necrotic death. They delay or prevent necrosis evoked, for instance, by chemical anoxia [84], myocardial ischemia [85], hypoxia [86], TNF [28], or arsenic trioxide [11]. However, not all necrotic programs are suppressed by proteins of the Bcl-2 family, for example, necrosis caused by peroxinitrite [87], or the mitochondrial uncoupler 3-acetylpyridine [88]. A balance between the necrotic and apoptotic cell responses may also depend on a balance between pro- and antiapoptotic members of the Bcl-2 family. For instance, the anti-necrotic effect of chronic hyperglycemia consists in activation of Bcl-2 expression and phosphorylation of the proapoptotic protein Bad [89]. Increased expression of Bax in glioblastoma stimulated apoptosis, but coexpression of Bcl-XL, surprisingly, switched cell death to necrosis [90]. Phosphorylation status of Bcl-2 also may play a role in cell necrosis. For instance, suppression of inhibitory phosphorylation of Bcl-2 by Twist-1 protected from necrosis caused by anti-cancer drugs cisplatinum, VP-16, and daunorubicin [91]. A recent work from Susin’s lab unraveled a role of mitochondria in MNNG-induced, PARP-1 dependent necrosis [31] (see section 2.1). It was found that Bax, but not Bak knockout completely prevented necrosis; furthermore, Bcl-2 overexpression also suppressed necrosis. Activation of BAX was mediated by cytosolic protease calpain (see section 2.6 below) which leads to AIF release and necrosis [31]. A protein from the Bcl-2 family, BNIP3, which causes mainly necrotic cell death, has been discovered [92]. Pro-necrotic functions of this protein in transfected cells are manifested by earlier plasma membrane permeabilization, cytoplasm vacuolization, and autophagy of mitochondria. These morphological changes were accompanied by mitochondrial depolarization and ROS generation and were blocked by inhibitors of MPT CsA and bonkrekic acid [92]. Interestingly, integration of BNIP3 into the mitochondrial membrane upon necrosis of neurons was prevented by necrostatin-1 [82], which also may explain inhibitory effect of necrostatin on MPT and ischemic necrosis in cardiac cells (see section 2.3 above). Indeed, expression of dominant-negative form of BNIP3 in myocardium reduced ischemic injury (e.g., release of creatine kinase) [93]. BNIP3 accumulation is activated by hypoxia via HIF1 and it is highly expressed in some tumors, including those of breast, lung, and cervix, although in colorectal and pancreatic cancer it is often epigenetically silenced (see [94] for review). Apparently, at least in some tumors, suppression of BNIP3 provide them with growth advantage under conditions of hypoxia/ischemia that occurs during tumor development [95]. Thus, the main anti-apoptotic and anti-necrotic effect of Bcl2/Bcl-xL proteins is believed to consist in preservation of mitochondrial integrity (i.e., prevention of MPT, efflux of cytochrome c, and other proapoptotic/pronecrotic factors). 2.5. Heat Shock Proteins Heat shock proteins (Hsps) are other important regulators of cell death, and, along with other prosurvival proteins such as Bcl-2, they are often overexpressed in tumor cells [96-98]. The most studied of them are Hsp70 and Hsp27, and they were first described as inhibitors of apoptosis caused by diverse stimuli (see [97-99] for review). However, later it was established that Hsps can protect cells not only from apoptosis, but also from autophagic cell death [100], senescence [101, 102] and, apparently, mitotic catastrophe [103]. Their overexpression also protect cells from necrosis caused by heat shock [104], oxidative stress [105], nitric oxide [106], and ischemia/reperfusion [65]. For instance, after myocardial ischemia/ reperfusion, transgenic mice overexpressing Hsp70 in the heart had a smaller infarct zone, a lower level of creatine kinase (indicator of necrosis) in blood plasma, and better recovery of mechanical function [65,107]. Small Hsps, Hsp27 and its homolog Bcrystallin, can also protect cardiomyocytes from ischemia-induced necrosis in vitro and in vivo [108,109]. The protective effect of


Hsp70 in myocardial ischemia was not associated with preservation of ATP level during ischemia, but ATP recovery in the myocardium of Hsp70-expressing animals was faster and higher than in control [110]. These data may indicate that Hsp70 preserve mitochondrial functions during ischemia/reperfusion and/or accelerate the recovery of these functions. Furthermore, the protective effect of Hsp70 against NO-induced necrosis in human -cells was not associated with suppression of lipid peroxidation, but also with rescue of mitochondrial functions (tetrazolium reduction) [111]. However, since neither Hsp70 nor Hsp27 are localized to mitochondria, it seems unlikely that their protective action is associated with direct effect on mitochondrial structure. Probably, these chaperones suppress signal transduction pathways leading to mitochondrial damage and cell death. These pathways may include the stress kinases JNK and p38 (see section 2.1.). Indeed, activity of these kinases was markedly elevated during ischemia/reperfusion, and their inhibition suppressed necrosis in vitro and in vivo [112]. Because activation of JNK and p38 under in vitro “ischemia” of myogenic cells was reduced in Hsp70-expressing cells [40], these kinases may be the targets of anti-necrotic effect of Hsp70 in the myocardium. Interestingly, protection of the kidney from ischemia/ reperfusion by ischemic preconditioning was also associated with stress kinase suppression, although in this case it was Hsp27 rather than Hsp70 that was accumulated in the preconditioned kidney [113]. Although role of Hsps in protection of tumor cells from ischemia-induced necrosis occurring during tumor development has not been assessed directly, it seems quite probable since higher levels of Hsp27 or Hsp70 promote growth of tumors, while downregulation of these proteins suppresses it [114-118]. Therefore, the molecular chaperones Hsp70 and Hsp27, along with proteins of the Bcl-2 family, are powerful inhibitors of necrosis. It seems that, although they have quite different mechanism of action, the main targets of their protective effect are mitochondria, either directly (in case of Bcl-2/Bcl-xL) or indirectly (in case of Hsp70/Hsp27). 2.6. Proteases, Nucleases, and Phospholipases Cysteine proteases of the caspase family perform crucial functions in suicide elimination of cells: transduction of lethal signal via cascade of caspases and a final destruction of various protein targets (PARP, lamins, cytoskeletal proteins) [119]. In many models it is the only way of execution of apoptosis, because in the presence of endogenous or exogenous caspase inhibitors, or in the absence of caspase expression, suicidal programs either completely blocked or, more often, switched to a necrotic pathway. As we discussed in section 2.1, inhibition of caspases does not prevent TNF-induced death but rather switches it from apoptosis to necrosis. Accordingly, caspase inhibitors switched to necrosis cell death caused by many anticancer treatments such as irradiation, camphotechine, etoposide, or dexametasone, or inducers of MPT [120-122]. LCC human carcinoma cells deficient in caspases died via necrosis in the presence of a zinc chelator, while caspaseexpressing cells died via apoptosis [123]. Furthermore, in mice without Apaf-1 or caspase-3/caspase-9, apoptotic cell death during development switched to necrotic [124]. There are some models where caspase inhibition not only prevented apoptosis but also severely aggravated necrosis. In L929 fibrosarcoma cells, inhibition of caspases increased the cell’s sensitivity to TNF-induced necrosis by a factor of 1000 [125]. These data indicate that caspases may also play an antinecrotic role consisting of elimination of “harmful” mitochondria that produce high level of ROS, and if such mitochondrial killing fails, necrosis is triggered (see section 2.3). Additionally, caspase inactivation promote autophagy-mediated degradation of catalase which causes ROS generation and cell death [126]. However, in some circumstances the execution of the necrotic program requires caspase activation. Necrotic death caused by ATP


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depletion in CD95-stimulated Jurkat cells was suppressed by the pan-caspase inhibitor zVAD.fmk [127]. This inhibitor (but not zDEVD.fmk, an inhibitor of caspase-3) also reduced TNF-induced necrosis of fibrosarcoma cells [128], as well as TRAIL-induced necrosis of human colon cancer cells at low pH [26]. Of note, however, that at least in some cells (e.g., L929), zVAD.fmk is able to suppress cathepsine B activity [129]. During the past years, a number of data emerged demonstrating wide occurrence of caspase-independent programmed cell death, both apoptotic and necrotic [130, 131]. For instance, TNF-induced cell death of hepatocytes and some tumor cells apparently requires lysosomal cysteine protease cathepsine B [132]. However, in L929 cells, zVAD.fmk-induced necrosis was aggravated by cathepsin inhibitors [129]. Interestingly, cathepsine B expression appears to be higher in immortalized and transformed cells making them more sensitive to TNF-induced cell death [133]. Another cysteine protease, Ca2+-dependent calpain, may participate in ischemia-induced cell death of hepatocytes and neurons after ischemia/reperfusion [134,135]. Mitochondrial calpain I was shown to be able to cleave AIF [136], which may be critical for release of AIF from mitochondria and neuronal cell death [135]. Calpain also mediates activation of BAX and release of AIF from mitochondria in MNNG-induced necrosis [31], whereas in arsenic trioxide-induced necrosis calpain mediates cleavage of nuclear Bax [137]. Finally, some as yet unidentified serine proteases can participate in TNF-induced necrosis of L929 cells [138], necrosis of kidney cells induced by “chemical hypoxia” [139], CD47-induced necrosis of lymphoid cells [9], or Gleevec-induced necrosis of BCR-ABL-positive leukemic cells [18]. One of such serine proteases may be mitochondrial Omi/HtrA2, which is released from mitochondria upon Gleevec treatment [18]. Therefore, the role of caspases, key executor caspases in apoptosis, is more diverse in necrosis. Their inhibition may either suppress or activate necrosis depending on cell line and stimuli. It is probable that switching from apoptosis to necrosis in the presence of caspase inhibitors, at least in some cases, may be associated with ATP depletion due to PARP1 activation (see section 2.1). If caspase inhibition prevents caspase-dependent PARP inactivation, it may cause ATP depletion, blockade of ATP-dependent apoptosis, and triggering of necrosis. At present, however, little is known how caspases participates in necrosis. In most cases, however, caspases are dispensable for necrosis, and other proteases such as cathepsine, calpain, and serine proteases are involved in execution of this form of cell death. Along with proteolysis, necrosis is also accompanied by degradation of DNA. Degradation of DNA during necrosis usually occurs randomly, forming a “smear” pattern on agarose gels, while apoptotic DNA fragmentation occurs to oligonucleosome fragments forming a remarkable “ladder” pattern on the gels. The main apoptotic nuclease is CAD (caspase-activated DNase), whereas caspase-independent DNase I and II are probably implicated in necrosis. For instance, an increase in DNase I-like endonuclease activity was observed in the kidney cortex after ischemia/ reperfusion [140], and activation of DNase II was found in the necrotic hippocampus after global ischemia [141]. However, the mechanisms of activation of these nucleases are presently not known. Obviously, DNA damage during necrosis is also mediated by AIF. Activation of some phospholipases during necrosis, especially cytosolic Ca2+-dependent phospholipase A2 (cPLA2), has been also demonstrated (see [142] for review). Activity of cPLA2 was increased in hyppocampal slices immediately following exposure to ischemic conditions, and this enhancement lasted for at least 24 h; furthermore, pharmacological blockade of cPLA2 (by bromophenacyl bromide or AACOCF3) prevented neuronal death [143].


Likewise, TNF-induced necrosis of MCF7 cells was suppressed by cPLA2 inhibitors [144]. Knockdown of one of isoforms of cPLA2, Ca2+- independent cPLA2 also delayed hypoxia-induced necrosis of neuronal cells [145]. However, in peroxynitrite-induced necrosis of U937 cells, cPLA2 apparently plays a protective role [55]. In contrast to necrosis, cPLA2 activity was dispensable for TNFinduced apoptosis of HeLa cells; moreover, during apoptosis cPLA2 underwent caspase-dependent cleavage and inactivation [146]. Such inactivation of cPLA2 during apoptosis may represent a mechanism to avoid the inflammatory response against apoptotic cells that may be evoked by products of phospholipid hydrolysis. 2.7. Molecular Scenario of Necrotic Cell Death The data described in the previous sections suggest a possible molecular scenario of tumor cell necrosis. There are several receptors implicated in triggering necrosis; among them are TNF receptors and other receptors of this family (FAS, TRAIL), and TLR. Another important sensor is DNA: its damage may be induced either directly (e.g., by radiation or anticancer drugs) or indirectly through oxidative stress (e.g., upon ischemia/reperfusion or other ROS generating treatments). Massive DNA breaks may cause activation of PARP, depleting its substrate NAD+ and, subsequently, ATP, which may lead to necrosis due to energy deficiency. Stimulation of the receptors, oxidative stress, and DNA damage are powerful activators of RIP1 and JNK, which leads to mitochondrial damage. Indeed, mitochondria, besides their role in ATP generation along with glycolysis, obviously play the key role in determination of a pathway of cell suicide. Mitochondria are powerful sources of tanathogenic factors such as cytochrome c, AIF, and ROS, and they are the main targets of cell survival system (e.g., proteins of the Bcl-2 family) The amount of ATP may be the essential factor that determines the choice of the cell suicide pathway, but there are obviously other important (but yet unknown) factors. Finally, the last stage of necrotic destruction is the activation of proteases. In several models of necrosis, this destruction is executed by caspases, but in most cases inhibition of caspases during stresses may trigger necrosis rather than suppress it. This indicates that caspase activity is sometimes necessary, paradoxically, for protection of cells from stresses, possibly through caspase-mediated elimination of ROS-generating mitochondria. Among proteases probably involved in necrotic digestion are calpains, cathepsins, and serine proteases, but their cellular targets in necrotic cell destruction are yet to be elucidated. There are many questions, however, remain to be answered. Among them: are there any physiological stimuli activating exclusively necrosis in mammalian cells; what determines choice of suicidal pathway by mitochondria; how mitochondrial collapse is translated to collapse of plasma membrane. 3. NECROTIC DEATH AS A MODULATOR OF IMMUNE RESPONSE Histological data show necrosis as a phenomenon that involves large population of cells, contrary to apoptosis which involves individual cells. This type of cell destruction may be determined by combination of several causes: toxicity of some factors released by necrotic cells (e.g., ROS, NOS) and destroying adjacent cells (“bystander effect”), suppression of phagocytosis by ROS, and poor mobilization of macrophages. In contrast to apoptotic cells with their content being isolated before phagocytosis, necrotic cells present a powerful inflammatory and immunogenic stimuli. Cellular thanatogenic mechanisms usually trigger the apoptotic form of cell destruction to avoid inflammatory and autoimmune reactions that are potentially dangerous for an organism. However, in some cases the necrotic pathway is activated, and triggering necrosis instead of apoptosis is not just a cell’s failure, but may have a positive effect when a strong inflammatory response is

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necessary. Indeed, there are indications that choice of program of autodestruction occurs before initiation of the irreversible phase of cell response to a lethal signal. The hallmark of apoptosis, externalization of phosphatidylserine (PS), that designates a cell with an “eat me” message, is the earliest feature of apoptosis triggering [147]. However, in necrotic cells this feature is usually registered after plasma membrane destruction; therefore, necrotizing cells are not recognized by phagocytes and cannot be digested until their intracellular contents are spilled into the extracellular space [148]. In some cases, however, appearance of PS on necrotic cells is an early and sufficient event for phagocytosis, which occurs even before perforation of plasma membrane. Effective absorption of necrotic cells with inhibited caspases and not exposing PS suggest that, in addition to PS, other ligands of the “eat me” type should exist, among them are vitronectin receptors [149, 150]. Interestingly, in artificially mixed population of apoptotic and necrotic cells, macrophages preferred the necrotic cells [150]. One of the reasons for this preference may be abundant histidine-rich plasma protein (HRG) which selectively recognizes necrotic, but not apoptotic cells, and enhance their phagocytosis [151]. Additionally, apoptotic and necrotic cells induce different cell signaling events in bone marrow macrophages: apoptotic cells suppress ERK1/2, but activate JNK and p38 kinases, while necrotic cells activate ERK1/2, but have no effects on JNK and p38 [152]. Recent scanning electron microscopy study by Krysko et al. [153] also revealed difference in internalization mechanisms of apoptotic and necrotic cells by macrophages: apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, whereas necrotic cells were internalized by macro-pinocytotic mechanism involving formation of multiple ruffles directed toward necrotic debris. Depending on molecular signals from necrotic cells (which are alien to surrounding cells), diverse type of these cells (neutrophils, macrophages, and others) become involved in the immune response. It was found that necrotic cells are more efficient than apoptotic cells in their capacity to stimulate the antigen-presenting cells (APC) and T-cell response [154]. On the other hand, apoptotic cells induced in APC the secretion of cytokines that inhibit Th1 response [155]. Necrotizing tumor cells also potentiate maturation of dendritic cells and optimal presentation of tumor antigens [154]. These data indicate that a much more robust immune response is evoked during necrosis than from apoptosis. This may be physiologically important upon some dangerous situations such as viral or bacterial infection, trauma, or abnormal (transformed) cells, when strong stimuli produced by necrosis are required for mobilization of all cell defense forces (dendritic cells, monocytes, and neutrophils). Accordingly, activation of necrosis of colon tumor in vivo by depletion of cytochrome c, a critical component of apoptosis, markedly decreased their tumorigenic ability [156]. Importantly, this decreased tumorigenicity was dependent on host immune system, since in immunodeficient animals the cytochrome c – depleted cells grew as fast as control cells. What signals does the immune system receive from necrotic cells? Some of the signals are already known; among them are high mobility group box 1 (HMGB1), Hsp70, calreticuline, and uric acid (see [157] for review). When delivered in the extracellular space, these substances activate APC, including dendritic cells [157]. HMGB1 protein is a powerful pro-inflammatory factor; it is passively released by necrotic cells whereas in apoptotic cells it tightly bound to chromatin [158]. HMGB1 can either bind to TLR4 or RAGE (receptor for advanced glycation end products) (Fig. 2). Furthermore, HMGB1 along with Hsps and uric acid released by necrotic cells is a potent adjuvant in vivo. Important role in inflammation is also attributed to Hsp70 [159]. Its elevated levels in necrotic neoplastic cells markedly increased their immunogenecity by promoting the Th1 response and APC maturation [154]. Thus, Hsp70 is not only a marker of necrosis, but also a specific signal for the immune system. Hsp70 has high immunogenicity by itself and



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Fig. (2). Necrosis as a modulator of immune response. Necrotic cells release a variety of factors stimulating macrophages (M) via their specific receptors, which leads to generation of inflammatory cytokines by macrophages

increases immunogenicity of some other macromolecular antigens [160]. There are several receptors activated by Hsp70, among them CD91, CD40, CD14, and TLR receptors [98, 161] (Fig. 2). This activation of macrophages and dendritic cells by necrotic cells is accompanied by inhibition of secretion of anti-inflammatory cytokines (IL-10, TGF-), and by release of pro-inflammatory mediators (TNF-, L-1, IL-6, MIP-2, IL-8) and chemokines (Fig. 2). Exposed to the factors of necrotic cells, dendritic cells enter the mature state that is characterized by appearance of specific markers (CD40, CD80, CD86), costimulating molecules (B7.1, B7.2), stimulation of T-cell proliferation etc. Immunization of animals with dendritic cells loaded with necrotic tumor cells markedly suppressed the growth of appropriate tumors (see ref [162] for review). On other hand, most cytokines that promote inflammation (e.g., TNF- or IL-6) can activate NF-kB signaling pathway in tumor cells that protects them from apoptosis and promote tumor cell proliferation (see [163] for review). 4. NECROTIC DEATH IN ANTI-CANCER THERAPY AND TUMOR DEVELOPMENT Extensive studies of tumor cell death during the last decade allow to come to several conclusions regarding connections between three main modes of death: apoptosis, necrosis, and autophagic cell death. For neoplastic cells of hematopoetic origin, apoptosis is a prevalent form of cell death in vitro and in vivo,

which, apparently, coincides with high apoptotic sensitivity of corresponding normal cells such as thymocytes, lymphocytes etc. Radiation and anti-neoplastic drugs at clinically relevant doses kills these cells mostly via apoptosis, and necrosis of these cells can be observed only when apoptosis is blocked, e.g., by caspase inhibitors or overexpression of anti-apoptotic proteins of Bcl-2 family. For this type of neoplasia, inhibitors of proteins of Bcl-2 family may be a promising way to increase their apoptotic sensitivity and efficiency of anti-cancer therapy. For most widespread tumors of epithelial origin such as breast and prostate cancers, apoptosis can be rarely found in vivo during tumor development or after anti-cancer therapy, and in vitro it can be caused only by relatively high (unattainable in clinic) doses of radiation and drug. Accordingly, most normal epithelial cells are also relatively resistant to apoptosis. Moreover, there is no dependence between apoptotic sensitivity of epithelial tumors and their response to anti-cancer therapy. For instance, high expression of anti-apoptotic Bcl-2 protein in solid tumors paradoxically correlate with better prognosis [164, 165] and there was no apparent correlation between sensitivity of tumor to therapy and expression of other pro- and anti-apoptotic members of BH3-family. At the same time, necrosis of solid tumors in vivo can be observed quite often after anti-cancer treatment or during tumor development. Since conventional anti-cancer drugs or radiation can


not directly induce necrosis at the clinically relevant doses, the observed tumor necrosis in vivo apparently is secondary, as a consequence of mitotic catastrophe (i.e., death after division of cells with irreparable damage of DNA). The most common cause of necrosis during tumor development is obviously inadequate oxygen and nutrient supply (metabolic stress) of fast-growing tumor cells. Recent elegant studies from E. White lab demonstrated relationships between apoptosis, autophagy and necrosis during tumor development [166, 167]. It was found that transformed epithelial cells with disabled apoptosis can survive energy starvation in vitro and in vivo by activating authophagy to provide essential nutrients. However, if autophagy of apoptosis-resistant cells is also disabled, which is often happens in tumors (e.g., by activation of Akt-mTOR signaling pathway), these cells die via necrosis [22]. Therefore, autophagy plays an important role in preventing necrosis of tumor during development. Paradoxically, however, despite high level of necrosis, cancer cells with disabled apoptosis and autophagy forms faster growing and more aggressive tumors. One of the reason for this is that autophagy was found to mitigate DNA damage and genomic instability which is generated during metabolic stress in vitro and in vivo [168]. Such genomic instability (e.g., aneuploidy) is a common feature of most aggressive tumors, and it may accelerate tumor progression by generating mutations promoting invasion and metastasis. Another possible reason why necrosis in tumor is associated with cancer progression may be chronic inflammation caused by necrotic cells (see section 3). Inflammatory forms of cancer (e.g., breast cancer) are the most aggressive forms, and it was suggested that immune cells infiltrating tumor, besides trying to kill cancer cells, also provide essential cytokines to surviving cells (see [169] for review). Among these cytokines are TNF , IL-6, TGF and some others, and their stimulatory role on tumor growth has been shown [163]. On the other hand, recent studies from Zitvogel lab clearly demonstrated that outcome of anticancer chemotherapy and radiotherapy depends not only on ability of these agents to kill cells directly, but also on their capacity to stimulate immune response (see ref [157, 170, 171] for review). In particular, regression of tumors after treatment with doxorubicin or gamma-radiation was dependent on T-cell mediated immunity since it was severely compromised in immunodeficient (nude) mice. It was found that release of HMGB1 by dying tumor cells and its interaction with TLR4 receptor on APC is critical for cross-priming of anti-tumor Tlymphocytes in vivo [157, 170, 172] (see also section 3 above). Since release of HMGB1 is a specific marker of necrosis (see section 3 above and ref [173]), this mode of death rather than apoptosis is apparently necessary for tumor regression. Therefore, current data suggest that tumor cell necrosis may play dual role: if it occurs after anti-cancer therapy, it may provide favorable patient’s response due to cell elimination and activation of immune response by dying tumor cell; on the other hand, necrosis occurring during tumor development may leads to more aggressive and faster growing tumors due to stimulatory role of inflammation on tumor growth and genomic instability of tumor cells. ABBREVIATIONS MPT = Mitochondrial permeability transition PCD = Programmed cell death PARP = PolyADPribose polymerase RIP1 = Receptor-interacting protein 1 ROS = Reactive oxygen species RNS = Reactive nitrogen species TNF = Tumor necrosis factor

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Received: June 18, 2009

Accepted: June 30, 2009

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