Critical Reviews in Oncology/Hematology 82 (2012) 249–258

Necroptosis: An emerging form of programmed cell death

Wei Wu, Peng Liu ∗ , Jianyong Li ∗
Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, PR China
Accepted 10 August 2011

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
2. Signaling pathway of necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
2.1. Initiators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
2.2. Formation of the complex I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
2.3. The formation of complex II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
2.4. The formation of necrosome complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
3. Execution of necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
3.1. Reactive oxygen species (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
3.2. ANT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
3.3. RNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
3.4. PLA2 and LOXs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
4. Necroptosis and other types of programmed cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
4.1. Necroptosis and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
4.2. Necroptosis and autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
4.3. Necroptosis and PARP-induced programmed necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
5. Necroptosis in physiological and pathological processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
6. Necroptosis and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Abstract
Necrosis plays an important role in multiple physiological and pathological processes. Recently, a relatively new form of necrosis has been
characterized as “necroptosis”. Morphologically, necroptosis exhibits the features of necrosis; however, necroptosis exhibits a unique signaling
pathway that requires the involvement of receptor interaction protein kinase 1 and 3 (RIP1 and RIP3) and can be specifically inhibited by
necrostatins. Necroptosis has been found to contribute to the regulation of immune system, cancer development as well as cellular responses
to multiple stresses. In this review, we will summarize the signaling pathway, biological effects and pathological significance of this specific
form of programmed cell death.
© 2011 Elsevier Ireland Ltd. All rights reserved.

Keywords: Necroptosis; Apoptosis; Cell death; Cancer

∗ Corresponding authors. Tel.: +86 25 83781120; fax: +86 25 83781120.

E-mail addresses: liupeng8888@yahoo.com.cn (P. Liu), lijianyonglm@medmail.com.cn (J. Li).

1040-8428/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2011.08.004
250 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
1. Introduction
Cell survival has been the subject of active research for
several decades, and over time research studies have evolved
to include a further investigation of cell death. There are
two principal types of cell death according to morphological
appearance: apoptosis and necrosis [1]. Apoptosis is charac-
terized by the rounding up of cells, pseudopod retraction,
pyknosis, chromatin condensation, nuclear fragmentation,
and the appearance of apoptotic bodies [2]. Apoptosis is
executed in intrinsic or extrinsic pathways in response to var-
ious cell death stimuli and is regulated by a specific class
of compounds, such as the caspase cascade [3]. Necrosis is
morphologically identified by the swelling of organelles, an
increased cell volume, disruption of the plasma membrane,
and loss of intracellular contents. It has been previously
demonstrated that necrosis can be triggered not only by death
domain receptors, but also through AIF-mediated chromati-
nolysis or alkylation-induced DNA damage [4–6]. In contrast
to apoptosis, which has been demonstrated to involve pro-
grammed cell death, necrosis has been commonly viewed as
an accidental and unregulated event. However, an increasing
body of evidence indicates that necrosis can also be exe-
cuted by regulated mechanisms [7]. The recently coined term
“necroptosis” refers to one particular form of programmed
necrosis induced by stimulating death receptors with ago-
nists such as TNF␣, FasL, and TRAIL. Necroptosis has its
own unique signaling pathway which requires the involve-
ment of receptor interaction protein kinase 1 and 3 (RIP1
and RIP3), and can be specifically inhibited by necrostatins
[4,8,9]. Compared to apoptosis, of which signaling path-
ways that have been studied for many years, the underlying
mechanisms of necroptosis remain poorly understood. In
this review, we will provide a detailed description of the
mechanisms of necroptosis and briefly discuss the potential
impact of necroptosis on physiological processes and tumor
pathogenesis.
2. Signaling pathway of necroptosis
2.1. Initiators
Necroptosis is induced by a class of death receptors that
include TNFR1, TNFR2, and Fas. Upon binding with their
agonists, these death receptors induce cells toward either sur-
vival or death depending on the circumstances. Until recently,
death receptors were considered to induce apoptosis only.
However, it has been recently demonstrated that death recep-
tors potentially leads to necroptosis with the involvement of
RIP1 when the apoptotic pathway is blocked [4,10]. It has
also been established that the agonists of the Toll-like receptor
(TLR) induce caspase-independent necrosis [11,12]. Consis-
tently, multiple genes involved in the TLR pathway are found
in necroptotic signaling, indicating that the TLR pathway
may be involved in necroptosis.
Since there is more than one kind of necroptotic initiator, it
is not clear whether or not there is a common downstream sig-
naling pathway. Among initiators, the TNF-␣/TNFR-induced
pathway has received the most intensive scrutiny. The molec-
ular mechanisms of TNFR ligation induced necroptotic
signaling are described below.
2.2. Formation of the complex I
TNF-␣ is produced by activated macrophages [13] and is a
homotrimer protein with each subunit containing 157 amino
acids. Although currently TNF-␣ is commonly considered an
apoptotic inducer, it was first identified as an agent capable of
causing tumor cell necrosis [14,15]. New evidence demon-
strating that TNF-␣ is an inducer of programmed necrosis
has recently emerged.
TNFR1 or TNFR2, located on the cell surface, are specific
receptors of TNF-␣. As TNFR2 does not have a death domain,
TNFR1 plays the principal role in TNF-␣-mediated activation
and triggers a series of intracellular events.
Initially, TNF-␣ binds to the extracellular portion of
TNFR1, creating allosteric changes in the intracellular por-
tion of TNFR1 [16]. There are four cysteine-rich domains
(CRDs) in TNFR1. The first CRD, known as pre-ligand
assembly domain (PLAD), is essential for the formation
of pre-assembled receptors which can bind to TNF-␣ with
higher affinity [17]. After binding to TNF-␣, the silencer
of death domains (SODD) is released from the intracellu-
lar domain of TNFR1 with the help of various proteins and
enzymes. TNFR1 and TNFR2 trigger the downstream signal-
ing by forming complex I with proteins containing a death
domain, such as TNF-␣ receptor-associated death domain
(TRADD), RIP1, Fas-associated death domain (FADD), and
several E3 ubiquitin ligases, such as TNF-␣ receptor asso-
ciated factor 2/5 (TRAF2/5), inhibitor of apoptosis proteins
(IAPs) cIAP1 and cIAP2. Ubiquitination of these proteins is
important for the regulation of the activity of complex I and
impacts the outcome of the cell [18].
RIP1 is a member of the RIP family exhibiting a homol-
ogous N-terminal kinase domain. The ubiquitination state
of RIP1 determines whether it functions as a pro-survival
molecule or a kinase promoting cell death. RIP1 is initially
recruited to complex I by TNFR1, and is polyubiquitinated
by TRAF2/5, cIAP1, and cIAP2 at the position of lysine 63
[19,20]. The ubiquitination of RIP1 initiates the recruitment
and activation of the IKK complex and NEMO and promotes
the activation of the NF-␬B pathway, inducing a pro-survival
cellular mode. Deubiquitination of RIP1 could inhibit NF-
␬B pathway and this leads to an inclination of cell death
pathways. Two proteins have been shown to exhibit the activ-
ity of RIP1 deubiquitination in the regulation of the NF-␬B
pathway. Cylindromatosis (CYLD) is encoded by a tumor-
suppressor gene Cyld. It blocks the activation of NF-␬B by
cleaving Lys63 -linked polyubiquitin chains from several pro-
teins [21]. Tumor cells carrying inactive CYLD exhibit an
elevated proliferation activity and a decreased apoptosis rate
 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
[22,23]. A20 removes Lys63 -linked ubiquitin by trigging the
proteasome-dependent degradation of E3 ligases, such as
TRAF2 and cIAPs, and negatively regulates NF-␬B pathway
[24,25]. It has been previously demonstrated that ubiquitina-
tion of RIP1 is necessary for activation of the NF-␬B pathway
[26]. However, the kinase activity of RIP1 is not a requirement
for this process [27,28]. Thus, the key element in the regula-
tion of the TNF-induced NF-␬B pathway is the ubiquitination
status of RIP1, regardless of whether the RIP1 kinase is acti-
vated. Complex I is situated at the crossroads of cell survival
and death, switching between various signaling pathways in
response to an array of stimuli and microenvironments.
2.3. The formation of complex II
Following deubiquitination, RIP1 is released from com-
plex I to the cytoplasm, and subsequently recruited to
complex II. TRADD is also released from complex I after
the internalization of ligand-bound TNFR1. Internalization
of ligand-bound TNFR1 is essential for the formation of
complex II, as it has been previously demonstrated that
the inhibition of TNFR1 internalization leads to a resis-
tance to apoptosis in cells [29]. Complex II, also known as
the death-inducing signaling complex (DISC), is composed
of TRADD, FADD, RIP1, and caspase-8. Knock-down of
CYLD inhibits TNF-induced necroptosis [12], which indi-
cates that the deubiquitination of RIP1 is an important step
in TNF-induced necroptosis. However, there is no other evi-
dence regarding whether other deubiquitinating enzymes,
such as A20, are as essential as CYLD to necroptosis. Inhibi-
tion of cIAPs accelerates complex II formation by decreasing
the ubiquitination of RIP1 [30]. TRAF2, another E3 ligase,
has been demonstrated to be essential for TNF-␣ induced
necrosis because TRAF2−/− cells show resistance to TNF-
␣ induced necrosis [31]. This effect is potentially due to
the fact that TRAF2 is required for the formation of com-
plex I. FADD is one of the domains recruited to complex
II and its effect on necroptosis varies depending upon cell
type. FADD is essential for necroptosis induced by TNF-
␣ in mouse embryonic fibroblasts (MEFs) [18], but it is not
required for necroptotic process in leukemic Jurkat cells [32].
For T cells in a proliferative phase, FADD acts as a negative
regulator in necroptosis [33]. The mechanism underlying the
various roles of FADD remains unclear. It has been reported
that TRADD is generally necessary for necroptosis with the
exception of necroptosis induced by Smac mimetics [34,35].
Thus, whether TRADD is required for necroptosis poten-
tially depends upon the type of stimulus involved. Complex
II may activate two downstream signaling pathways: apopto-
sis and necroptosis. When capase-8 is activated, it deprives
RIP1 of its activity by cleaving it, driving complex II into a
pro-apoptosis mode. Cleavage of RIP1 by caspase-8 not only
abrogates the stimulatory role of RIP1 in the activation of
NF-␬B pathway [36], but also exhibits a negative effect on
necroptosis because the kinase activity of RIP1 is crucial in
251
Fig. 1. Formation of necrosome After binding to TNF-␣, the intracel-
lular domain of TNFR1 summons a series of proteins, including TNF
receptor-associated death domain (TRADD), receptor-associated death pro-
tein1 (RIP1), and Fas-associated death domain (FADD). Meanwhile, several
E3 ligases, such as TNF receptor associated factor 2/5 (TRAF2/5), inhibitor
of apoptosis proteins (IAPs) cIAP1 and cIAP2 are also recruited to TNFR1.
Those proteins mentioned above form “complex I”. RIP1 is ubiquitilatized
in complex I, and has the ability to activate of the I␬B kinase (IKK) complex
and NF-␬B essential modulator (NEMO), leading to the activation of pro-
survival NF-␬B pathway in consequence. When RIP1 is deubiquitilitized by
cylindromatosis (CYLD), it is released to the cytoplams, and forms complex
II with TRADD, FADD, RIP3 and caspase-8. While caspase-8 is activated, it
would cleave RIP3 and induce apoptosis through caspase cascade. However,
when caspase-8 is blocked, phosphorylated RIP1 and RIP3 form necrosome
and initiate necroptosis.
necroptosis [35]. Additionally, RIP3 is cleaved at Asp328 by
caspase-8 under apoptotic stimuli, suppressing the ability of
RIP3 to induce caspase-independent cell death [37]. When
the apoptotic pathway is blocked, necroptosis prevails.
2.4. The formation of necrosome complex
When the apoptosis pathway is inhibited, a complex
named “necrosome” is formed (Fig. 1). Necrosome is primar-
ily composed of RIP1 and RIP3, and it distinctly enhances
necroptosis [38]. RIP3 accelerates recruitment of RIP1 to the
necrosome, and the kinase activity of both proteins is required
in this process [39]. The RIP1 kinase inhibitor Nec-1 inhibits
complex II formation, and RIP1 recruitment to complex II
is crucial for the induction of pronecrotic complex II kinase
activity. However, RIP1 kinase activity is not required for the
formation of complex I. It has been previously reported that
RIP3 is required in RIP1 phosphorylation in TNF-␣ induced
necroptosis. However, the phosphorylation mediated by RIP3
is very weak and is similar to the level of RIP1 autophos-
phorylation [40]. Additionally, only ubiquitinated RIP1 is
252 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
detectable in necroptosis-resistant cells that exhibit a low
RIP3 expression level, indicating that RIP3 may facilitate
the deubiquitination of RIP1.
Similar to other RIPs, RIP3 has an N-terminal kinase
domain. However, its C-terminus lacks a death domain or
CARD motif. The biological function of RIP3 remains con-
troversial. It has been reported that RIP3 is able to inhibit
RIP1 from activating the NK-␬B pathway [40]. Alternatively,
RIP3 could activate the NK-␬B pathway when it is over-
expressed [41]. However, the absence of RIP3 does not inhibit
NK-␬B pathway activation [42]. Recent evidence demon-
strates that RIP3 is critical in necroptosis induced by various
stimuli [39,43]. It has been reported that RIP3 knockdown
leads to a notable inhibition of necroptosis in HT-29 cells
[43]. Studies have demonstrated a low RIP3 expression level
in cells resistant to necroptosis, and transfection of these cells
with the RIP3 gene enables them to undergo necroptosis when
the apoptotic pathway is blocked [44]. Phosphorylation of
RIP3 is essential in necroptosis, but the exact mechanism
remains unclear [39]. A recent report identified RIP3 as an
essential mediator in TNF-␣ induced necroptosis using a
screening technique involving RNA interference [44]. The
interaction between RIP1 and RIP3 is based upon RIP homo-
typic interaction motif (RHIM). Mutations of RHIMs in RIP1
or RIP3 may block the formation of necrosomes and protect
cells from necroptosis [45]. Moreover, RIP3 kinase activity
is also required for the interaction between RIP1 and RIP3
[39].
Although RIP1 and RIP3 have been reported to be
necessary for necroptosis in most experimental models,
there are some contradictory reports. The necroptosis
induced by TCR in FADD−/− T cells has been demon-
strated to be RIP1-dependent only [36]. Studies on murine
cytomegalovirus infection have identified a RIP3-dependent,
RIP1-independent necroptosis induced in infected cells [46].
Briefly, kinase activity of RIP1, RIP3, and the interaction
of RIP1 and RIP3 is required to guarantee the activation
of necroptotic pathway. Aside from the molecules discussed
above, other necroptotic regulatory factors exist.
As mention above, apoptosis suppression may drive cells
to use necroptosis as an alternative cell death mechanism.
Some caspase inhibitors, such as zVAD.fmk and BocD.fmk,
could induce necroptosis through autocrine production of
TNF-␣ [47]. However, treatment with Smac mimetic, which
mimics the function of Smac protein, could only result in
apoptotic cell death, even though it can also induce autocrine
of TNF-␣ [48]. Most apoptotic inhibitors require the pres-
ence of exogenous TNF-␣ to induce necroptosis. It has been
demonstrated that only certain cell types are able to execute
necroptosis in response to TNF-␣ stimulus when the apopto-
sis pathway is inhibited or inactive. These cells include mouse
fibrosarcoma L929 cells, human T cell leukemia Jurkat cells,
human monocytic leukemia U937 cells, MEFs, and human
colon cancer HT-29 cells. Among these cell lines, L929 cells
are most extensively utilized in experiments related to necrop-
tosis, as TNF-␣ induces necrosis prominently to apoptosis in
L929 cells, and L929 cells could commit necroptosis treated
with TNF-␣ or z-VAD alone, or both simultaneously [49]. An
experiment performed in Harvard Cell Biology Department
[12] showed that necroptosis might be controlled at tran-
scriptional level, indicating a possible genetically explanation
about the cellular choice of necroptosis.
Necrostatin-1 (Nec-1) was initially defined as an inhibitor
of necroptosis because of the specific inhibitory effect of
RIP1 [9,50]. It provides neuron-protection regarding brain
trauma, hypoxia, and ischemia/reperfusion injuries [51,52].
Further studies have revealed the underlying mechanism with
greater detail: the inactivation of RIP1 blocks the recruitment
and interaction of RIP3 and RIP1, thus inhibits the formation
of necrosome, which is essential for necroptosis [40]. It is
well established that RIP1 is the only active site of Nec-
1 [50]. A recent report indicated that Nec-1 protected cells
from necrosis by blocking the reduction of mitochondrial
membrane potential (MMP) and activating the NF-␬B path-
way, supporting mitochondrion as a potential active site of
Nec-1 [53]. Further investigations are needed to confirm the
new characteristics of Nec-1, as those characteristics may
only emerge in certain cell types or under certain stimulus.
Aside from Nec-1, other members of the Necrostatin family,
such as Nec5 [54] and Nec7 [55] have also been found to be
capable of inhibiting necroptosis too, although their targets
remain to be elucidated.
3. Execution of necroptosis
Execution necroptosis is equally important, but much
less clarified compared to the upstream signaling pathway.
It is unlikely that necrosomes could lead to cell death by
destroying cellular organelles directly, as no obvious co-
localization of necrosome or RIP3 has been found in any
cellular organelles [43]. Thus, necrosome may function
as an upstream signal and potentially triggers the execu-
tion of cell death through various mechanisms. It has been
demonstrated that necroptosis shares identical sub-cellular
events with necrosis, such as oxidative burst, mitochondrial
membrane hyperpolarization, lysosomal membrane perme-
abilization and plasma membrane permeabilization, but the
mechanisms underlying those processes might be different
from necrosis [56]. We will discuss these subcellular events
in detail in the following sections.
3.1. Reactive oxygen species (ROS)
ROS potentially leads to cell death by directly oxidizing or
triggering various downstream pathways [57]. It has been pre-
viously demonstrated that TNF-␣ induced necrosis requires
the participation of ROS [58], although the exact mechanism
that triggers ROS production remains poorly understood.
Mitochondria are the principal producers of ROS in cells
[56,59,60]. RIP3 accelerates mitochondrial ROS production
and mitochondrial metabolism through the activation of a
 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
series of metabolism-related enzymes. In T293 cells, RIP3
enhances the activity of glycogen phosphorylase (PYLG),
glutamate-ammonia ligase (GLUL), and glutamate dehydro-
genase 1 (GLUD1) during TNF-␣ induced necroptosis [61].
All of these enzymes are essential for ROS production. PYLG
is the rate-limiting enzyme in glycogen degradation and the
glucose-1-phosphate released by PYLG is important in gly-
colysis. GLUL and GLUD1 are key enzymes in the process
of oxidative phosphorylation. Moreover, by enhancing these
metobolism-related enzymes, RIP3 may also participate in
the decision regarding the choice of cell death because varia-
tion in energy metabolism status potentially leads to differing
mechanisms of cell-death [62].
NADPH oxidases are a family of enzymes specifically
important in ROS production [63]. Several of the oxidases,
such as Nox1, Nox2, Nox3, Nox4, and p47phox, have been
shown to be upregulated in the presence of TNF-␣ [64–67].
Nox1 is activated by TNF-␣ and thus generates superoxides
in MEFs [66]. In this process, Nox1 forms a complex with
TRADD, RIP1, and a small GTPase called Rac1 [64]. Hence,
for necrosis induced by TNF-␣, RIP1 is required in ROS pro-
duction [31]. However, in HT-29 cells, ROS is not required for
necrosis induced by TNF-␣, Smac mimetic, and zVAD.fmk
[43].
The c-Jun NH2-terminal kinase (JNK) plays a duel role
in TNF-␣-induced necrosis. On one hand, JNK plays a pro-
survival role and suppresses apoptosis induced by TNF-␣;
on the other hand, JNK is a pro-necrotic signal and initi-
ates TNF-␣-induced necrotic cell death in fibroblasts [68].
JNK is activated downstream of RIP1 and TRAF2 in necro-
sis [69,70], and TRAF2 is essential for JNK activation [71].
JNK leads to necrosis by producing ROS, potentially by
impacting mitochondrial function [72], whereas intracellu-
lar production of ROS activates the pro-necrotic effect of
JNK [57]. Therefore, JNK and ROS form a positive feedback
loop and enhance necrosis. Recently, it has been reported that
JNK potentially promotes autocrine production of TNF-␣ by
mediating activating protein-1 (AP-1) activation in L929 cells
treated with zVAD.fmk, consequently accelerating necropto-
sis [47].
3.2. ANT
Beyond the production of ROS, mitochondria also con-
tribute to necrotic cell death through an ADP/ATP-related
pathway. Synthesis of ATP in mitochondria requires the
normal activity of adenine nucleotide translocase (ANT),
an ADP/ATP carrier located in the inner mitochondrial
membrane. The activity of ANT is potentially affected by
interaction with VDAC and cyclophilin D (CYPD). CYPD is
an important regulator of mitochondrial permeability transi-
tion pore (MPTP) [73]. RIP1-dependent inhibition of ANT
has been reported in the programmed necrosis induced by
TNF-␣ and zVAD.fmk in U937 cells [74]; zVAD.fmk poten-
tially blocks the ability of ANT to transport cytoplasmic ADP,
thereby inducing a massive ATP depletion in mitochondria.
253
RIP1 and TNF-␣ have both been demonstrated to be essential
for the binding of zVAD.fmk to ANT. This study also reported
that CYPD exhibits a protective role against cell death by sup-
pressing zVAD.fmk binding to ANT. It has been shown that
CYPD is consistently up-regulated in several human tumor
tissues including breast, ovary, and uterus [75]. However,
another study reported that CYPD was required in oxidative
damage-induced cell death [76].
3.3. RNS
Nitric oxide (NO) is produced in endothelial cells by
endothelial nitric oxide synthase (eNOS). It affects many
physical and pathological processes, such as vascular relax-
ation, inflammation, cell proliferation, and cell death [77–80].
NO interacts with mitochondria [81], and influences cellular
bioenergetics as well as oxygen consumption [82,83]. NO can
lead to programmed cell death in endothelial cells in a manner
similar to the necroptosis induced by TNF-␣ [84], and RIP1,
RIP3, and ROS are all involved in the process. However, no
receptor is required here. Since necrotic cell death is inhib-
ited by Nec-1 and is dependant upon RIP3, and possibly RIP1
in addition, it may represent a type of necroptosis. However,
its mechanism is very different from the necroptosis induced
by TNF-␣. Further investigation is warranted to clarify the
underlying mechanisms in NO-induced necroptosis.
3.4. PLA2 and LOXs
PLA2 is a family of enzymes that liberate and cleave
free fatty acids and lysophospholipids at the sn-2 posi-
tion of glycerophospholipids. cPLA2 (calcium-dependent
cytosolic type) is a member of PLA2 primarily responsi-
ble for initiating arachidonic acid (AA) metabolism [85].
The transmission and activation of cPLA2 requires calcium
and phosphorylation [86,87]. cPLA2 plays an important role
in TNF-␣-induced necrotic cell death in L929 cells and
MEFs, as well as in chemical or oxidant-induced necrosis in
renal epithelial cells [88–90]. Lipoxygenase (LOXs) acts as a
downstream effector of PLA2 and is activated by an increased
concentration of calcium resulting from the generation of
free fatty acids. LOXs contributes to lipid hydroperoxidation
and leads to the disruption of organelle and plasma mem-
branes [91]. It has been reported that LOXs is involved in
both apoptosis and necrosis induced by TNF-␣ [92,93].
4. Necroptosis and other types of programmed cell
death
4.1. Necroptosis and apoptosis
Necroptosis and apoptosis induced by death receptor liga-
tion share the same pathway from initiation to the formation
of complex II. Complex II is composed of FADD, RIP1,
RIP3, and caspase-8. When caspase-8 is inhibited, both RIP1
254 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
and RIP3 are phosphorylated and the “necrosome” is acti-
vated, inducing necroptosis in consequence. However, when
caspase-8 remains activated, it attenuates the activity of RIP3
through cleavage and forms the complex IIa with FADD and
RIP1, leading to apoptosis. Among the factors that affect the
switch between apoptosis and necroptosis are: the nature of
the stimulus, type of cell, and activity of caspases. As previ-
ously mentioned, only certain types of stimulus are capable
of inducing necroptosis, and not all cell types are able to
die through necroptosis. In concordance with this finding, a
recent study demonstrated that shikonin, previously known as
an apoptosis inducer [94], was able to induce necroptosis in
MCF-7 and HEK293 cells, but only apoptosis in HL60 cells
[95]. However, necroptosis in HL-60 cells can be induced
by DT-GMCSF (diphtheria toxin granulocyte-macrophage
colony-stimulating factor), a novel fusion toxin treatment
for AML [96]. These results underscore the importance
of proper stimuli to trigger necroptosis. The exact mecha-
nism of cell type-dependence and stimulus-dependence of
necroptosis remains unclear. The cells capable of exhibiting
necroptosis appear to have nothing in common. Conversion
between necroptosis and apoptosis has been identified in the
same cell line, in accordance to differing stimuli and cir-
cumstance [97]. The alternation of mitochondrial membrane
potential may partially contribute to the conversion. In cer-
tain circumstances, necroptosis and apoptosis could occur
simultaneously in cells [3].
4.2. Necroptosis and autophagy
Autophagic cell death is characterized by massive
autophagosomes, a two-membrane vesicle containing cyto-
plasmic organelles or degenerating cytosol. Autophagy has
proven to be protective to cells, as knock-down of autophagy-
related genes appears to promote cell death [98]. However,
as some investigations have recently discovered, autophagy
may also contribute to necroptotic cell death [50,52,99,100]
even though it is not indispensable for necroptosis [101].
The mechanism underlying autophagy activation remains
controversial. It has been reported that a signaling pathway
involving caspase-8, RIP1, JNK, and c-JUN may contribute
to autophagy activation in L929 cells [102]. c-Src was
recently reported to be associated with caspase-8 in L929
cells at resting state. zVAD.fmk Treatment releases the acti-
vated c-Src, and triggered c-Src-dependent JNK and ERK
signaling pathways [103]. In proliferative T cells, FADD
and caspase-8 work in concert to limit autophagy and pro-
tect T cells from necroptosis [99]. Recently, it has also
been reported that necroptosis promotes autophagy, as Nec-
1 reduced LC3 levels in FADD−/− cells and rescued the
cycling and proliferation of FADD−/− T cells [99] simul-
taneously. This phenomenon indicates the possibility that
autophagy and necroptosis may potentially form a posi-
tive feedback loop and reinforce the process of cell death.
Autophagy might accelerate cell necrosis in two ways. Firstly,
autophagy induces an increased level of ROS released primar-
ily form mitochondria [103]. Catalase is a scavenger enzyme
of ROS. It has been reported that catalase is discomposed
by autophagy, leading to an imbalance of intracellular ROS
[104]. Secondly, PARP1 is over-activated by autophagy and
leads to necrotic cell death. However, whether PARP1 acti-
vation occurs upstream or downstream of ROS production
remains controversial [105]. In contrast to the results men-
tioned above, it has been reported that autophagy protects
L929 cells from necroptosis induced by zVAD.fmk [98].
Rapamycin, a molecule that increases the flux of autophagy,
was demonstrated to inhibit zVAD-induced cell necrosis.
Moreover, suppression of autophagy enhanced the necropto-
sis induced by zVAD.fmk. Given that zVAD.fmk are capable
of inhibiting the activity of cathepsins and calpains and
that the two proteases proved to be essential for autophagy,
it is possible that zVAD.fmk suppresses autophagy by
blocking cathepsins and calpains directly, and that the sup-
pression of pro-survival autophagy leads to necroptosis in
L929 cells.
4.3. Necroptosis and PARP-induced programmed
necrosis
Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear
enzyme activated by DNA-strand breaks, and plays a key
role in repairing DNA damage. Over-activation of PARP-1
potentially leads to NAD + reduction, ATP depletion, cellu-
lar energy failure, and release of apoptosis-inducing factor
(AIF) from mitochondria to nucleus, which result in mas-
sive DNA damage and necrotic cell death. PARP-1-mediated
necrosis contributes to several pathological processes, such
as ischemia-reperfusion, inflammation, ROS-induced injury,
and glutamate excitotoxicity. Dysfunction of mitochondria
is a key step in PARP-1-induced necrosis. The mecha-
nism of PARP-1 induced mitochondria dysfunction remains
unknown. It is potentially associated with the modification
of Bcl-2 families, essential regulators of membrane perme-
ability in mitochondria. BNip3 is a member of the BH3-only
Bcl-2 family. A recent study reported that BNip3 mediated
TNF-␣-induced necroptosis in alveolar lung cells [106]. In
PARP-1 induced necrosis, calpains are required for translo-
cation of AIF from mitochondrial membrane to nucleus
[107]. Calpain is a protease regulated by intracellular calcium
level. The released AIF binds to DNA, leading to chromatin
condensation and DNA fragmentation, and finally induces
necrosis [108]. Inhibition of PARP fails to protect Jurkat cells
from necroptosis, indicating that PARP activation is poten-
tially not be involved in necroptosis [50]. However, it has
been previously reported that Nec-1 blocks the transloca-
tion of AIF from mitochondria to nucleus and suppresses the
activation of PARP-1 [109,110]. These findings suggest the
possibility that PARP-1 induced necrosis acts downstream
of necroptosis. Moreover, PARP-2, the closest homologue to
PARP-1, has been identified as one of the essential regulators
of necroptosis by a genome-wide siRNA screen study [12].
 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
5. Necroptosis in physiological and pathological
processes
Necroptosis may play a regulatory role in the development
of the immune system. It is related to the regulation of T
cell proliferation [33,99] and macrophage survival [12]. An
increased expression of necroptosis related genes have been
found in mouse immune cells [12]. RIP3 expression is found
to be elevated in immunologic organs, such as thymus and
spleen in mice [43].
Programmed necrosis plays an important role in the
response to viral infection by serving as a backup mechanism
of cell death other than apoptosis. Virus-infected cells, which
are resistant to apoptosis, are found to be highly sensitive to
necroptosis [111]. Knock-down of RIP3 in vaccinia virus
(VV) infected cells apparently protects the cells from cell
death induced by activated T cells or TNF-␣. Additionally,
TNF-␣ potentially induces necroptosis without the presence
of zVAD.fmk in VV infected cells [39], indicating that viral
infection may serve to sensitize infected cells to necroptosis.
Regarding the innate immune response following viral infec-
tion, it has been demonstrated that the inflammation caused
by VV infection is evidently reduced in RIP3−/− mice [112],
as well as TNFR1−/− and TNFR2−/− cells [32,112], sug-
gesting the necroptotic pathway plays a key role in triggering
inflammation.
Recently, a viral inhibitor of RIP3-dependent necroptosis
has been reported [46]. The findings indicated that murine
cytomegalovirus (MCMV) M45, which encodes the viral
inhibitor of RIP activation (vIRA), potentially inhibits RIP
induced necrotic cell death and accelerates viral replication.
It has been demonstrated that M45 inhibits TNFR/FasL/TNF-
␣-induced necroptosis by either inhibiting RIP1, RIP1–RIP3
complex formation, or RIP3 alone [113,114].
Necroptosis is also involved in several types of tissue
damage. Enhanced expression of RIP3 was detected in pan-
creatitis tissue induced by cerulein, and deletion of RIP3
was shown to prevent the tissue damage [39,43]. Moreover,
necroptosis participates in hypoxia injury, ischemia reperfu-
sion damage of brain and retina, cortical impact, myocardial
infarction, and excitotoxicity in neurons [51,52,115–117].
6. Necroptosis and cancer
Necroptosis serves as a “backup” pathway of cell death
differing from apoptosis. Defects in necroptosis or variations
of necroptosis-related genes may contribute to the patho-
logical process of human malignancies. Our research group
has discovered necroptotic defects in chronic lymphocytic
leukemia (CLL) cells (unpublished results). RIP3 SNPs have
been identified in non-Hodgkin lymphoma patients. Genetic
variation of TNF and TLR signaling pathways have also been
detected [118]. Inactive mutation of CYLD was found to
accelerate proliferation and migration in epidermal cancer
cells [22].
255
Necroptosis may accelerate cancer cell death or enhance
the sensitivity of tumor cells to anti-cancer treatment
[96,100,119,120]. A study on pediatric acute lymphoblastic
leukemia (ALL) revealed the important role of necroptosis
in overcoming the glucocorticoid resistance of ALL cells
[100]. Additionally, research has demonstrated that necropto-
sis participates in acute myeloid leukemia (AML) cell death
induced by a novel fusion toxin treatment [96]. Other research
has demonstrated that necroptosis is able to overcome resis-
tance to cancer drugs mediated by P-glycoprotein, Bcl-2,
and Bcl-xL in cancer cell lines [95]. All these results indi-
cate necroptosis may be a potent therapeutic target for the
treatment of cancer.
7. Conclusions
As a newly identified form of cell death, necroptosis plays
an important role in immune system regulation, tissue injury,
and cancer development. Necroptosis is might be triggered by
TNF-␣ and caspase inhibitors. RIP1 and RIP3 are critical reg-
ulators of necroptosis, and necrostatin-1, an inhibitor of RIP1,
specifically blocks necroptosis. Necroptosis is a potential
therapeutic target for many diseases. Inhibition of necrop-
tosis potentially protects cells from severe injury induced
by trauma or inflammation. Hence, the necrostatin family
may serve as a potential therapeutic tool for diseases such
as ischemic/reperfusion of brain and myocardiac infarction.
Acceleration of necroptosis may provide a backup route to
cell death for tumor cells, particularly for those resistant to
apoptosis-inducing cancer therapy.
Reviewers
Junying Yuan, Ph.D., Harvard Medical School, Depart-
ment of Cell Biology, 250 Longwood Avenue, Boston, MA
02115, United States.
Tao Cheng, M.D., University of Pittsburgh School of
Medicine, Department of Radiation Oncology, Pittsburgh,
PA, United States.
Conflict of interest
No conflict of interest is declared.
Acknowledgements
This work was supported by National Natural Science
Foundation of China (30500603, 30871104, 30971295,
81170486, 81170485), International Cooperation Program
of Jiangsu Province (BZ2010041), the Program for Devel-
opment of Innovative Research Team in the First Affiliated
Hospital of NJMU, and A Project Funded by the Priority Aca-
256 W. Wu et al. / Critical Reviews in Oncology/Hematology 82 (2012) 249–258
demic Program Development of Jiangsu Higher Education
Institutions.
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Biographies
Dr. Peng Liu is a professor of Department of Hematology
at the First Affiliated Hospital of Nanjing Medical University.
He specializes in clinical hematology with a research focus in
molecular biology of leukemia. He also has a strong interest in
developing novel strategies to improve the efficacy of cancer
chemotherapy.
Dr. Jianyong Li is a professor and Director of Department
of Hematology at the First Affiliated Hospital of Nanjing
Medical University. He has a strong interest in clinical and
basic study of lymphoid malignacies.