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Mechanisms of Tumor Cell Necrosis

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56 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 death accompanied by a rapid efflux of cell constituents in extra-

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 bio-
chemical 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 mito-
chondrial 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: gabai@bu.edu
cellular 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

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Mechanisms of Tumor Cell Necrosis Current Pharmaceutical Design, 2010, Vol. 16, No. 1 57

Table 1. 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 C-
terminal 22-aa peptide (aa 361-382) linked to truncated 17-aa
peptide from Drosophila antennapedia homeobox domain (to
58 Current Pharmaceutical Design, 2010, Vol. 16, No. 1 Gabai et al.

facilitate cellular uptake) can induce selective necrosis in p53-

mutant 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 RIP1-
induced 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 nicotina-
mide) 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).
Mechanisms of Tumor Cell Necrosis
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 com-
ponent 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]. Accor-
dingly, 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 anti-
apoptotic 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 mecha-
nism under chronic ischemic conditions. As a result, apoptosis-
resistant 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 pathophysiolo-
gical 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 mito-
chondrial 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.
Current Pharmaceutical Design, 2010, Vol. 16, No. 1 59
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. Pro-
necrotic effect of peroxinitrite in U937 is mediated by hydrogen
perioxide apparently generated by mitochondria, since mito-
chondrial 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 anti-
oxidative 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 (death-
promoting) factors, mitochondria initiate or amplify the caspase-
dependent 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, 1-
methyl-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, 3-
nitropropionic 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 fibro-
sarcoma 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
60 Current Pharmaceutical Design, 2010, Vol. 16, No. 1
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 depen-
dent 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 hypergly-
cemia consists in activation of Bcl-2 expression and phos-
phorylation of the proapoptotic protein Bad [89]. Increased expres-
sion of Bax in glioblastoma stimulated apoptosis, but coexpression
of Bcl-XL, surprisingly, switched cell death to necrosis [90]. Phos-
phorylation 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 cis-
platinum, VP-16, and daunorubicin [91].
A recent work from Susin’s lab unraveled a role of mito-
chondria 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 Bcl-
2/Bcl-xL proteins is believed to consist in preservation of mito-
chondrial 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
B-
crystallin, can also protect cardiomyocytes from ischemia-induced
necrosis in vitro and in vivo [108,109]. The protective effect of
Mechanisms of Tumor Cell Necrosis
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 mecha-
nism 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 caspase-
expressing 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
Current Pharmaceutical Design, 2010, Vol. 16, No. 1 61
depletion in CD95-stimulated Jurkat cells was suppressed by the
pan-caspase inhibitor zVAD.fmk [127]. This inhibitor (but not z-
DEVD.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 bromo-
phenacyl bromide or AACOCF3) prevented neuronal death [143].
62 Current Pharmaceutical Design, 2010, Vol. 16, No. 1
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 TNF-
induced 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 protec-
tion 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 exclu-
sively 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
Gabai et al.
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, exter-
nalization 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]. Interes-
tingly, 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 macro-
phages 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 apop-
tosis, 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
Mechanisms of Tumor Cell Necrosis
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 bet-
ween three main modes of death: apoptosis, necrosis, and auto-
phagic cell death. For neoplastic cells of hematopoetic origin,
apoptosis is a prevalent form of cell death in vitro and in vivo,
Current Pharmaceutical Design, 2010, Vol. 16, No. 1 63
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 effi-
ciency 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
64 Current Pharmaceutical Design, 2010, Vol. 16, No. 1
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 relation-
ships 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 T-
lymphocytes 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
Gabai et al.
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