Lakesla Apoptosis

Biochimica et Biophysica Acta 1863 (2016) 2977–2992
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamcr
Review
Activation of apoptosis signalling pathways by reactive oxygen species

Maureen Redza-Dutordoir, Diana A. Averill-Bates ⁎
Département des Sciences Biologiques (TOXEN, BIOMED), Université du Québec à Montréal, Montréal, Québec, Canada
a r t i c l e i n f o a b s t r a c t
Article history: Reactive oxygen species (ROS) are short-lived and highly reactive molecules. The generation of ROS in cells exists
Received 9 June 2016 in equilibrium with a variety of antioxidant defences. At low to modest doses, ROS are considered to be essential
Received in revised form 12 September 2016 for regulation of normal physiological functions involved in development such as cell cycle progression and pro-
Accepted 15 September 2016
liferation, differentiation, migration and cell death. ROS also play an important role in the immune system, main-
Available online 17 September 2016
tenance of the redox balance and have been implicated in activation of various cellular signalling pathways.
Keywords:
Excess cellular levels of ROS cause damage to proteins, nucleic acids, lipids, membranes and organelles, which
Oxidative stress can lead to activation of cell death processes such as apoptosis. Apoptosis is a highly regulated process that is es-
Environmental stress sential for the development and survival of multicellular organisms. These organisms often need to discard cells
Apoptosis that are superfluous or potentially harmful, having accumulated mutations or become infected by pathogens. Ap-
Mitochondria optosis features a characteristic set of morphological and biochemical features whereby cells undergo a cascade
Death receptor of self-destruction. Thus, proper regulation of apoptosis is essential for maintaining normal cellular homeostasis.
Endoplasmic reticulum ROS play a central role in cell signalling as well as in regulation of the main pathways of apoptosis mediated by
mitochondria, death receptors and the endoplasmic reticulum (ER). This review focuses on current understand-
ing of the role of ROS in each of these three main pathways of apoptosis. The role of ROS in the complex interplay
and crosstalk between these different signalling pathways remains to be further unravelled during the coming
years.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction implicated in the activation of various cellular signalling pathways and

transcription factors including phosphoinositide 3-kinase (PI3K)/Akt,
1.1. Reactive oxygen species mitogen-activated protein kinases (MAPK), nuclear factor (erythroid-
derived 2)-like 2 (Nrf2)/Kelch like-ECH-associated protein 1 (Keap1),
Reactive oxygen species (ROS) are generally small molecules that nuclear factor-κB (NF-κB) and the tumour suppressor p53, which can
are short-lived and highly reactive [1,2]. They can be oxygen-derived activate cell survival and/or cell death processes such as autophagy
free radicals like superoxide anion (O•− 2 ) and the hydroxyl radical and apoptosis [3–6]. Redox-regulated signal transduction is often
(OH•), or non-radical molecules such as hydrogen peroxide (H2O2) through reversible oxidation of thiol proteins. Nevertheless, under-
(Fig. 1). The generation of ROS in cells exists in equilibrium with a standing the physiological importance and mechanisms of redox signal-
wide variety of antioxidant defences. These include enzymatic scaven- ling at the cellular level requires further clarification [2,4].
gers such as superoxide dismutases (SOD), catalase, glutathione perox- On the other hand, if the antioxidant detoxification systems fail to
idase and peroxiredoxins, as well as non-enzymatic scavengers such as maintain low, tolerated levels of ROS, then excess cellular levels of
vitamins C and E, glutathione (GSH), lipoic acid, carotenoids and iron ROS can be deleterious and trigger oxidative stress. Oxidative stress is
chelators [1]. defined as “a serious imbalance between the generation of ROS and an-
At low to modest doses, ROS are considered to be essential for the tioxidant defences in favour of ROS, causing excessive oxidative dam-
regulation of normal physiological functions involved in development age” [1]. Excess cellular levels of ROS can cause damage to proteins,
such as cell cycle progression and proliferation, differentiation, migra- nucleic acids, lipids, membranes and organelles such as mitochondria
tion and cell death [3]. ROS also play an important role in the immune [1]. Enhanced production of ROS has been associated with the develop-
system and in maintenance of the redox balance [4]. ROS have been ment of pathologies such as cancer, diabetes, atherosclerosis, stroke, ar-
throsis, amyotrophic lateral sclerosis (ALS) and neurodegenerative
disorders such as Parkinson's and Alzheimer's diseases [7–9]. However,
⁎ Corresponding author at: Département des Sciences Biologiques, Université du
clinical evidence supports an association rather than a causative role of
Québec à Montréal, CP 8888, Succursale Centre-Ville, Montréal, Québec H3C 3P8, Canada. ROS with diseases, and the molecular mechanisms involved are yet not
E-mail address: [email protected] (D.A. Averill-Bates). fully understood [8,9].
http://dx.doi.org/10.1016/j.bbamcr.2016.09.012
0167-4889/© 2016 Elsevier B.V. All rights reserved.
2978 M. Redza-Dutordoir, D.A. Averill-Bates / Biochimica et Biophysica Acta 1863 (2016) 2977–2992
Fig. 1. Environmental stress generates ROS that cause cellular damage and apoptosis. Environmental stress attributed to toxic compounds (xenobiotics) can lead to generation of
xenobiotic-derived free radicals through one-electron reduction catalysed by cytochrome P450 reductases. Xenobiotic-derived free radicals react rapidly with oxygen to produce
superoxide (O2•−), which either reacts with nitric oxide (NO•) to produce peroxynitrite (ONOO −), or undergoes dismutation to generate H2O2, catalysed by SOD. H2O2 can be either
detoxified by antioxidants such as catalase and GSH/glutathione peroxidase, or can generate OH• by the metal-catalysed Fenton reaction. OH• and ONOO − cause damage cellular
proteins, lipids and nucleic acids, which can lead to demise of the cell by apoptosis.
ROS can be generated in cells by both exogenous (see Section 1.2) xenobiotics including quinone compounds undergo redox cycling. This
and endogenous stimuli. Endogenous production of ROS, especially involves continuous generation of free radicals and ROS by one-
O•−
2 , arises mainly from leaks during mitochondrial electron transport electron transfer through successive oxidation and reduction reactions
chain activity [10]. The two major sites for superoxide production are [8]. The initial one-electron reduction of a toxic compound to generate
complexes I and III of the respiratory chain. Superoxide can also be a drug-derived free radical is catalysed by flavoenzymes such as
produced by NADPH oxidases (Nox) localised at the membrane, as NADPH cytochrome P450 reductases. The drug-derived free radical sub-
well as by xanthine oxidases and enzymatic activation of cytochrome sequently reacts rapidly with oxygen to generate the superoxide free
P450 reductases [5]. O•− •
2 can react with nitric oxide (NO ), a reactive ni- radical and other ROS such as H2O2 (Fig. 1). In the presence of metals,
trogen species (RNS), to generate the powerful oxidant peroxynitrite OH• can be produced from H2O2 by the Fenton reaction [11].
(ONOO−) [2,10] (Fig. 1). Otherwise, O•− 2 is rapidly dismutated into O2 At sufficient doses, powerful oxidants such as OH• and ONOO−, and
and H2O2 by SOD. H2O2 is used in inflammatory processes or can be likely H2O2, cause severe damage to macromolecules [1]. This can lead
reduced by metal ions such as iron or copper to form OH• through the to cell death by processes such as apoptosis and/or necrosis (Fig. 1)
Fenton reaction [8,11]. OH• is highly reactive and harmful to biological (see Section 4.2). Moreover, ROS such as O•− 2 and H2O2 can induce au-
macromolecules. To avoid the formation of OH•, H2O2 is detoxified by tophagy, which is mostly considered to be a cell survival response, al-
antioxidant enzymes such as catalase and glutathione peroxidases though it can lead to cell death (see review [16]) (see Section 4.1).
[12] (Fig. 1). Induction of autophagy would most likely occur during exposure to
modest doses of ROS.
1.2. Environmental factors and oxidative stress
2. Signalling pathways of apoptosis
Living organisms are constantly exposed to variations in their envi-
ronmental parameters, such as temperature, pH, oxygen pressure, me- 2.1. General features
tabolite concentrations, hormonal and immune signals. Moreover,
they are continually assaulted by toxic components found in their Apoptosis is a tightly regulated and highly conserved process of cell
environment [13,14], such as pesticides, aldehydes, chlorinated by- death during which a cell undergoes self-destruction [17]. It is an essen-
products, heavy metals, micro- or nano-particles, as well as UV and ion- tial process in multicellular organisms that eliminates undesired or su-
ising radiation [15]. When the balance between environmental con- perfluous cells during development or neutralises potentially harmful
straint and the capacity to sustain cell and tissue homeostasis is cells with DNA damage, thus preventing carcinogenesis [15,18]. The
disturbed, a stress situation arises. Environmental stressors can cause regulation of apoptosis is crucial for maintaining normal cellular ho-
more or less serious damage to cells and tissues, influence disease de- meostasis. However, the deregulation of apoptosis has been linked to
velopment, or even trigger death of cells, tissues or individual organisms numerous pathologies such as chronic inflammation, atherosclerosis,
[13]. Prolonged or acute exposure to environmental stressors can cause cancer, respiratory disorders, and neurodegenerative diseases such as
deleterious effects on health. Environmentally-related diseases include Alzheimer's and Parkinson's diseases [18].
cancer, diabetes, immunosuppression, chronic lung disease and neuro- Apoptosis is a well-characterised process presenting distinctive
degenerative disorders [14]. morphological and biochemical features [17]. It is distinguished by cell
Numerous exogenous factors including environmental pollutants shrinking, membrane blebbing, chromatin condensation and nuclear
are able to generate pro-oxidant compounds such as ROS [8,14,15] fragmentation, followed by the formation of apoptotic bodies that are
(Fig. 1). These factors include UV and ionising radiation, quinone com- digested by phagocytosis by neighbouring cells or macrophages. This re-
pounds, inflammatory cytokines, chemicals found in tobacco smoke, en- sults in the elimination of dying cells with minimal damage to sur-
vironmental toxins and various pharmaceutical agents. Many toxic rounding cells and tissues.
M. Redza-Dutordoir, D.A. Averill-Bates / Biochimica et Biophysica Acta 1863 (2016) 2977–2992 2979
Apoptosis can be triggered by a variety of extrinsic and intrinsic sig- Several newer caspases were identified although they are not well
nals. These include different stresses such as ROS, RNS, DNA-damaging characterised. Caspases-15, -17 and -18 are absent in mammals, with
agents (e.g. radiation), heat shock, serum deprivation, viral infection the exception of caspase-16 [20]. Caspase-14 is present only in terrestri-
and hypoxia [15,18]. The exposure to xenobiotics such as pesticides, en- al mammals and its expression is limited to epidermal tissue and
vironmental pollutants and chemotherapeutic drugs can also trigger ap- epithelia [20].
optosis, which is often mediated by ROS. Cell to cell variations are an Apoptosis can be triggered by three main signalling pathways, up-
important determinant of cell fate, as to whether cells will survive or stream of caspase activation. These include an extrinsic pathway involv-
die by apoptosis following a toxic insult [14]. These variations can result ing death receptors localised at the cell surface [30], or intrinsic
from differences in triggering signals and/or cellular state (e.g. genetic, pathways involving mitochondria or the ER [32,33].
phenotypic or stochastic fluctuations), cell cycle phase or cellular micro-
environment [19]. The main mechanisms that regulate apoptosis are 2.2.1. Mitochondrial pathway
discussed in the following sections. Mitochondria are essential organelles whose primary function is to
convert nutrients into energy (ATP), which is subsequently exported
and used throughout the cell [34]. This function is facilitated by distinct
2.2. Main mechanisms of regulation of apoptosis proprieties of the outer and inner mitochondrial membranes [35]. The
outer mitochondrial membrane (OMM) is permeable to molecules up
Most, but not all apoptotic pathways lead to the activation of to 5 kDa, which allows the exchange of respiratory chain substrates
cysteine-dependent aspartate-specific proteases (caspases) [20]. and products between mitochondria and the cytosol. The inner mito-
Procaspases exist as inactive zymogens that require cleavage to gener- chondrial membrane (IMM) is highly impermeable, an essential charac-
ate their active proteolytic forms. The latter are heterodimers composed teristic for generating the electrochemical potential necessary for
of two small (~ p10) and two large (~ p20) subunits that contain two oxidative phosphorylation and ATP production.
cysteine active sites. Certain initiator caspases can cleave other caspases, Mitochondria also have an important role in triggering and regulat-
which leads to their activation further downstream in the apoptotic ing apoptosis (see review, [36]). The regulation of IMM permeability ap-
cascade [20]. Apoptotic caspases consist of upstream initiators like pears to be critical in this process. Mitochondrial-dependent apoptosis
caspases-8, -10, -2 and -9 and downstream effectors such as caspases- appears to involve the mitochondrial permeability transition pore
3, -6 and -7. However, unlike the other initiator caspases, caspase-2 ap- (MPTP) complex, whose molecular nature remains to be elucidated
pears to play a role in both the initiation and execution phases of apo- [37,38]. The MPTP complex is thought to be composed of cyclophilin
ptosis, since it does not cleave other caspases [21,22]. Although they D, a molecular chaperone in the mitochondrial matrix bound to the
are classified as inflammatory caspases [23], caspases-4 and -12 are adenine–nucleotide–translocator (ANT) in the IMM, which interacts
involved in apoptosis through the endoplasmic reticulum (ER) [24,25]. with the voltage dependent-anion-channel (VDAC) in the OMM [39].
Caspases-11 and -13 are the murine and bovine orthologues of VDAC and ANT form points of connection between the IMM and
caspase-4 [20]. It should be noted that most caspases have subsequently OMM. Under conditions of stress, the inner membrane permeability in-
been found to also possess non-apoptotic roles [19,20]. creases, which allow free passage of molecules of less than 1.5 kDa, in-
The proteolytic activity of caspases involves cleavage of their sub- cluding protons, into the mitochondrial matrix, leading to disruption
strates at aspartate residues [26,27]. There are at least 1000 substrates of oxidative phosphorylation [38]. Moreover, this causes osmotic swell-
of effector caspases such as caspase-3 and -7, which are located in the ing of the mitochondrial matrix and compression of vesicles created by
cytosol, cytoskeleton and nucleus. They include structural proteins infolding of the intercristal space. Together with increased outer mito-
such as actin, lamin A, gelsolin and fodrin, proteins involved in DNA re- chondrial membrane permeabilisation (MOMP), this leads to release
pair like the poly(ADP-ribose)polymerase (PARP) and in cell cycle reg- into the cytosol of pro-apoptotic proteins such as cytochrome c, apopto-
ulation such as p21, the E3 ubiquitin-protein ligase (or mouse double sis inducing factor (AIF), endonuclease G (endoG), and second
minute 2 homolog (Mdm2)) and the inhibitor of caspase-activated mitochondria-derived activator of caspases/direct inhibitor of apoptosis
DNase (ICAD) [28]. During apoptosis, effector caspases can cleave the (IAP)-binding protein with low pI (Smac/Diablo) (Fig. 2) [36]. This leads
p75 subunit of complex I in the mitochondria, which disrupts the elec- ultimately to apoptotic cell death by both caspase-dependent and
tron transport chain and hence the critical function of ATP production -independent mechanisms [36]. Once in the cytosol, cytochrome c
[29]. Furthermore, caspases cleave p21-activated kinase 2 (PAK2) and forms a complex with apoptosis activating factor-1 (Apaf-1) and
Rho-associated kinase-1 (ROCK-1), which regulate actin polymerization procaspase-9, called the apoptosome, in the presence of dATP. This re-
and actin–myosin contractility. This leads to abnormal reorganization of sults in auto-activation of caspase-9, which in turn activates the execu-
the actin cytoskeleton and membrane blebbing [30]. The activation of tioner caspases-3, -6 and -7 (Fig. 2) [36]. Smac/Diablo can bind to
effector caspases leads to onset of the characteristic morphological fea- inhibitor of apoptosis proteins (IAP), which are caspase inhibitors,
tures of apoptosis and ultimate dismantlement of the cell. thus preventing them from exerting their anti-apoptotic proprieties
Caspases-1, -4, -5, -11 (in rodents) and -12 are involved in inflam- (Fig. 2). Furthermore, AIF and EndoG translocate into the nucleus and
matory processes. The functions of caspases-4 and -5 are not well take part in caspase-independent apoptosis. The role of EndoG in cell
known [20]. Inflammatory caspases such as caspase-1 and -5 are acti- death is still unclear, whereas AIF can bind directly to DNA, where its
vated in platforms known as the inflammasome [30,31]. The DNase activity causes chromatin condensation and DNA fragmentation
inflammasome generally responds to inducers of inflammation as well [36].
as infectious agents such as viruses, bacteria and fungi. Caspase-1 pro- The mitochondrial pathway of apoptosis, and particularly MOMP, is
motes the secretion of two inflammatory cytokines, interleukin (IL) 1β regulated by proteins belonging to the B-cell-lymphoma protein 2
and IL18. In addition, caspase-1 can cleave Bid and activate caspases-3 (Bcl-2) family (Fig. 2). There are more than 30 proteins and related
and -7 to stimulate apoptosis, although this type of cell death is members of this family. These proteins contain up to four conserved
generally referred to as pyroptosis. Caspase-12 appears to abrogate Bcl-2 Homology domains (BH1-4) that are essential for their functions
the inflammatory response by inhibiting caspase-1 [20]. The activation (see reviews, [40,41]). BH3 functions as a death domain. Bcl-2 family
of caspase-11 is not entirely clear although it appears to be an upstream proteins are divided into two groups: anti-apoptotic survival proteins
activator of caspase-1 [20]; in infected macrophages, pro-inflammatory and pro-apoptotic proteins. The six anti-apoptotic members of this fam-
caspase-11 produced caspase-1-dependent IL1β and IL18. On the con- ily include Bcl-2, Bcl-XL, Bcl-W, Bcl-B, A1 and Mcl-1, which each have
trary, caspase-11 induced cell death (lactate dehydrogenase (LDH) re- three or four BH domains. The pro-apoptotic proteins Bax, Bak and
lease) in a caspase-1-independent manner [31]. Bok possess two or three BH domains. Other pro-apoptotic members
Fig. 2. Activation of the mitochondrial (intrinsic) pathway of apoptosis by ROS. ROS generated exogenously or endogenously can activate p53 and/or c-Jun N-terminal kinase (JNK), which
activate pro-apoptotic Bcl-2 proteins (red circles) that can inhibit the functions of anti-apoptotic proteins (green oblongs). ROS cause oxidation of cardiolipin, which relinquishes
cytochrome c (Cyt c), allowing its release into the cytosol. Moreover, ROS cause mitochondrial membrane depolarisation and/or opening of Bax/Bak channels on the OMM, which
allows release of AIF, Endo G, cytochrome c and Smac/Diablo into the cytosol. Cyt c then forms the apoptosome complex in the cytosol together with Apaf-1 and procaspase-9, leading
to caspase-9 activation. Caspase-9 then activates effector caspases such as caspase-3, resulting in cleavage of cellular proteins and cell demise by apoptosis. AIF and Endo G translocate
to the nucleus and appear to be involved in DNA fragmentation. ROS can directly cause damage to nuclear and mitochondrial DNA.
of this family include the BH3-only proteins Bad, Bik, Bmf, Hrk, SOUL, Bak. Bax and Bak then transition from inactive monomers to high mo-
Bid, Bim, Puma and Noxa. Most cells contain a variety of pro- and anti- lecular weight homo-oligomers. This process facilitates MOMP by
apoptotic Bcl-2 family proteins. Regulation of the cellular balance be- forming channels large enough to allow leakage of pro-apoptotic factors
tween these pro- and anti-apoptotic Bcl-2 proteins is pivotal for the de- such as cytochrome c into the cytosol (Fig. 2) [48].
termination of cell fate, by either promoting survival or mitochondrial-
mediated apoptosis. Under normal conditions, most pro-apoptotic Bcl-2 2.2.1.1. Calcium and mitochondrial apoptosis. Another function of mito-
family proteins such as Bid, Bax and Bad are found in the cytosol. On the chondria is their pivotal role in calcium homeostasis. Under physiolog-
other hand, the anti-apoptotic Bcl-2 and Bcl-XL proteins are localised ical conditions, the mitochondrial Ca2+ pool is small, but it can rise
mainly at the OMM, where they form heterodimers with Bax, Bim, under pathological conditions. Mitochondria are located in proximity
Bak and Bad, thus inhibiting their pro-apoptotic proprieties. The func- to the ER and the local concentration of Ca2+ released by the ER can
tion of Bok is not clear [40,42]. reach high levels, which are taken up by mitochondria through a Ca2+
Under conditions of stress, relative expression of pro- and anti- uniporter. The Ca2+ concentration within the matrix plays a critical
apoptotic Bcl-2 proteins is modified (Fig. 2). BH3-only Bcl-2 proteins role in stimulating Ca2+-sensitive matrix dehydrogenases that provide
are activated either transcriptionally or post-transcriptionally for the NADH for oxidative phosphorylation and ATP production (see review
initiation of apoptosis [42]. Stress due to heat shock caused an increase [36]).
in cellular expression of pro-apoptotic relative to anti-apoptotic Bcl-2 Ca2+ plays a key role in the initiation and effectuation of cell death
proteins, creating a disequilibrium favouring apoptosis [43]. Stress con- by apoptosis [36]. Perturbation of intracellular Ca2+ homeostasis is
ditions such as DNA damage and growth factor withdrawal caused also associated with cell death by necrosis and some forms autophagic
targeting of the anti-apoptotic protein Mcl-1 for degradation by the cell death, which will not be discussed in detail in the present review
ubiquitin-proteasome system [30]. Most pro-apoptotic Bcl-2 family pro- (see review [36]) (see Sections 4.1 and 4.2).
teins are activated under stress stimuli: Bid is cleaved into t-Bid by mul- Mitochondrial Ca2+ accumulation can regulate opening of the MPTP
tiple proteases including caspases-2, -8, -10 and calpain; Bax and Bak complex in the IMM [36]. This is accompanied by osmotic swelling and
form homo-oligomers; Bad is released from its sequestering protein, triggers rupture of the mitochondrial membrane, which leads to release
14-3-3 [44]. Activated pro-apoptotic Bcl-2 proteins then translocate to of pro-apoptotic proteins such as cytochrome c into the cytosol. Cell
the OMM. Some activated BH3-only members, such as Bad, Bik, Bmf, death during Ca2+ overload appears to require binding of cyclophilin
Hrk and SOUL, only form heterodimers with anti-apoptotic proteins D to the c subunit of the F0 ATP synthase for MPTP opening [49].
like Bcl-2, Bcl-XL and Bcl-W, but not with A1 and Mcl-1 [45]. Other A link was suggested between calcium and Bcl-2 family proteins
BH3-only proteins such as Bim, Puma and t-Bid bind with high affinity during apoptosis [36]. Calpain activation caused a decrease in Bcl-2 pro-
and inhibit all of the anti-apoptotic Bcl-2 proteins [46]. Noxa teins and activation of mitochondrial apoptosis. Another role for Ca2+ in
antagonises only Mcl-1 and A1 [47]. The binding of these BH3-only pro- apoptosis is its ability to dephosphorylate Bad through Ca2+-mediated
teins to anti-apoptotic proteins liberates any complexed pro-apoptotic activation of the protein phosphatase calcineurin [50]. This triggers dis-
proteins so that they can activate the pro-apoptotic proteins Bax and sociation of Bad from 14‐3-3 in the cytosol and its translocation to
mitochondria where it can block the anti-apoptotic action of Bcl-XL. Fur- super-family [15,42,51]. TNF-Rs implicated in apoptosis are located at
thermore, cleavage of Bid by calpain triggered mitochondrial apoptosis the plasma membrane and contain an intracellular death domain
mediated by tBid [36]. (DD). These receptors include Death receptor 1 (DR1) (also known as
Moreover, Ca2+ overload in mitochondria can activate a calpain that TNF-R1, CD120a, p55), DR2 (Fas, CD95 or Apo-1), DR3 (Apo-3,
is localised in the intermembrane space [36]. AIF is anchored in the IMM TRAMP, LARD or TNFRSF25 (TNF receptor superfamily 25)), DR4 (TNF-
and undergoes cleavage by calpain, producing a fragment that is re- related apoptosis-inducing ligand (TRAIL)-R1 or Apo-2), DR5 (TRAIL-
leased from mitochondria. The AIF fragment translocates to the nucleus R2 or TRICK2) and DR6 (TNF receptor superfamily member 21
where it causes considerable DNA fragmentation and chromatin con- (TNFRSF21)) [52].
densation. AIF appears to play an important role in apoptotic cell DR2-mediated apoptosis is initiated by binding of the Fas ligand
death of neurons and certain cancer cells. (FasL) to its specific transmembrane receptor Fas, which triggers recep-
tor trimerisation (Fig. 3) [42]. This is a key step for recruitment of the
2.2.2. Death receptor pathway adaptor molecule Fas-associated death domain (FADD) to the intracel-
Apoptosis can be activated through an extrinsic pathway involving lular surface of the receptor, through interactions between their respec-
death receptors from the tumour necrosis factor receptor (TNF-R) tive DD domains, and formation of the death-inducing signalling
Fig. 3. Activation of the death receptor (extrinsic) pathway of apoptosis by ROS. Transmembrane death receptors such as Fas, TRAIL-R1/2 and TNF-R1 can be activated by ROS. Death
receptor-mediated apoptosis involves recruitment of adaptor protein FADD and procaspase-8 or -10 to the cytoplasmic surface of the receptor to form the DISC. This results in
activation of caspases-8/-10, which can directly activate caspases-3/-6/-7 and trigger apoptosis. Caspases-8/-10 can also cleave Bid to produce tBid, which activates a crosstalk pathway
between death receptors and mitochondria. tBid translocates to mitochondria where it blocks anti-apoptotic activity of Bcl-2 and Bcl-XL, and activates Bax and Bak. This leads to
release of cytochrome c and Smac/Diablo and activation of the mitochondrial pathway of apoptosis. Caspase-2 undergoes activation either by caspase-8 or at the PIDDosome complex
composed of procaspase-2, PIDD and RAIDD. Caspase-2 can activate the mitochondrial pathway through Bid cleavage. Daxx can interact with Fas, leading to activation of ASK-1 and
JNK, and mitochondrial apoptosis (see Fig. 2). In addition to promoting apoptosis through caspase-8, TNF-R1 can activate survival pathways mediated by adaptor protein TRADD, RIP
and Traf-2, leading to activation of survival genes by NF-κB.
complex (DISC). FADD then interacts with initiator procaspases-8 or -10 respective DD domains. The PIDDosome appears important for
through their respective death effector domains (DED), leading in turn caspase-2 processing and auto-activation in response to DNA damage
to their auto-activation (Fig. 3) [52]. This is followed by direct activation [20,62]. This pathway links caspase-2 to p53-mediated cell death.
of downstream effectors such as caspases-3 and -7, a process that occurs However, in certain experimental models, caspase-2 activation oc-
in type I cells. However, in type II cells, once activated, caspase-8 or -10 curred in the absence of RAIDD and PIDD, suggesting the existence of
can cleave BH3-only protein Bid to form truncated Bid (t-Bid). tBid then an alternative platform for its activation. During p53-mediated apopto-
undergoes translocation to the OMM, where it triggers translocation sis, caspase-2 was activated by recruitment to the Fas DISC and process-
and insertion of pro-apoptotic Bax into the OMM, activation of Bax/ ing by caspase-8 [66]. Caspase-2 is also activated by caspase-3 [67].
Bak and MOMP. Consequently, tBid contributes to amplification of mito- Furthermore, DNA damage activates the Ataxia telangiectasia mutat-
chondrial apoptosis through modification of the balance between pro- ed/Ataxia telangiectasia and Rad3 related (ATM/ATR) pathway, which
apoptotic and anti-apoptotic members of the Bcl-2 family [53]. Apopto- can induce caspase-2-dependent apoptosis when checkpoint kinase 1
sis mediated by the death receptors TRAIL-R1 and -R2 is very similar to (Chk1) is suppressed, thus circumventing the requirement for p53 [68].
Fas-mediated apoptosis, but instead implies TRAIL as the death ligand Compared to the effector caspases-3 and -7, there are few known
(Fig. 3) [54,55]. cleavage substrates of activated caspase-2 [21]. These include Mdm2,
The Fas and other death receptors are widely distributed on the sur- αll-spectrin, Bid, ROCK-2, protein kinase C δ (PKCδ), ICAD, PARP and
face of most cell types and are also important in control of the immune plakin [20,21]. Most of these proteins are also substrates of calpains
response [42]. The membrane-bound, rather than soluble form of FasL, and other caspases. The cytoskeletal-associated protein desmoplakin
appears to be a more potent inducer of apoptosis. Fas can also activate appears to be specifically cleaved by caspase-2 and not by other
receptor-interacting protein (RIP) kinase-mediated non-apoptotic cell caspases [21]. The cleavage of Bid by activated caspase-2 leads to
death by necroptosis when caspase-8 is inactivated or absent. More- MOMP and mitochondrial apoptosis [20], although this is much less ef-
over, Fas can also activate other non-death signalling processes that ficient than caspase-8-mediated MOMP and apoptosis. Mdm2 binds to
lead to proliferation or differentiation. p53, targeting it for ubiquitination and degradation by the 26S protea-
The signalling mechanisms through the TNF-R1 transmembrane re- some, when it is no longer needed [15]. Hence, caspase-2-mediated
ceptor are different from those for the Fas and TRAIL-R1/R2 receptors cleavage of Mdm2 increases the stability of p53 and enhances its func-
[52]. The binding of TNF-α to TNF-R1 causes trimerisation of TNF-R1, tions, such as growth arrest and induction of apoptosis [20,69]. Mdm2
which results in recruitment of the TNF-R-associated adaptor protein cleavage results in a positive feedback loop: p53 induces PIDD upregu-
with death domain (TRADD) to its cytoplasmic side (Fig. 3). This com- lation, while PIDD stabilizes p53 through activation of caspase-2.
plex can promote either cell survival or cell death, depending on the More recently, caspase 2 was reported to play a role in regulation of
other factors it associates with [52]. Indeed, recruitment of RIP and the stability of several cytoskeletal proteins during apoptosis [21]. These
TNF-associated factor-2 (Traf-2) to TRADD leads to activation of tran- proteins include stathmin-1, tropomyosin, myotrophin and profilin.
scription factors such as nuclear factor kappa B (NF-κB) [56]. This in They are all associated with actin or tubulin filaments, and their func-
turn can activate an inflammatory response, or factors that promote tion is to stabilise integrity of the cytoskeleton. They were degraded in
cell survival through up-regulation of antioxidants such as manganese a caspase-2–dependent manner during exposure to agents that cause
superoxide dismutase (MnSOD) and anti-apoptotic proteins such as DNA damage, ER stress or microtubule destabilisation [21]. These pro-
Bcl-XL, X-linked inhibitor of apoptosis (XIAP) and cellular inhibitors of teins were not directly cleaved by caspase-2 but were targeted for
apoptosis (cIAP) 1 and 2 (Fig. 3) [56,57]. On the other hand, the associ- ubiquitination and proteasomal degradation in cells exposed to apopto-
ation of TRADD with FADD and procaspase-8 leads to the activation of tic stimuli.
effector caspases-3, -6 and -7 and hence apoptosis, either directly, or Interestingly, the antioxidants SOD1 (CuZnSOD) and thioredoxin
through t-Bid-mediated amplification of the mitochondrial pathway were among the cytosolic proteins that were affected by active
(Fig. 3) [58]. caspase-2 and found to be less abundant [21]. This suggests that
The mechanisms regulating whether TNF-R1 binding will result in caspase-2 can inhibit antioxidants, which would promote ROS-
cell survival or cell death involve the FLICE inhibitory protein (FLIP), mediated apoptosis.
which is a transcriptional target of NF-κB [56,57,59,60]. The domain Besides its putative role in apoptosis, caspase-2 has been shown to
structure of FLIP is similar to that of caspase-8, but lacks a catalytic cys- take part in non-apoptotic functions such as cell cycle regulation as a
teine. Indeed, high concentrations of c-FLIP, a procaspase-8 competitive checkpoint protein, tumour suppression and cancer prevention, and
inhibitor that prevents it from associating with FADD, promote cell sur- DNA repair [20,62,63].
vival, whereas cleavage of RIP by caspase 8 promotes apoptosis [53].
Low concentrations of c-FLIP, however, promote caspase-8 recruitment 2.2.3. Endoplasmic reticulum pathway
and activation.
2.2.3.1. UPR and ER stress. The ER is a vital organelle in the secretory
2.2.2.1. Role of caspase-2. Pioneering work about caspase-2 has been car- pathway and is also involved in lipid biosynthesis and regulation of
ried out by the group of Jürg Tschopp [61]. Caspase-2, the most con- Ca2+ flux (see reviews, [70,71]). It is involved in synthesis, folding, traf-
served caspase identified, is thought to have an important role in ficking and post-translational modifications of proteins. This includes
regulation of apoptosis [22,61]. However, its function is not entirely proteins that reside in the ER as well as those destined for other organ-
clear and is controversial, compared to the other caspases [20]. As well elles such as the Golgi and lysosomes, the plasma membrane and the
as being located in the cytosol, caspase-2 contains a nuclear localisation extracellular space [70]. Indeed, control mechanisms exist in the ER to
signal (NLS) sequence and is transported into the nucleus, although its ensure high quality protein folding, which is essential for cell survival,
function in the nucleus is not presently known [62]. function and homeostasis. Exposure to adverse environmental stimuli
Caspase-2 activation occurs in response to various stress stimuli in- can disturb ER homeostasis, which leads to accumulation of misfolded
cluding DNA damage, metabolic imbalance, cytoskeletal disruption, ER and unfolded proteins in the ER lumen by a phenomenon known as
stress, heat shock [63,64] and H2O2 [65]. Mechanisms responsible for ER stress [70]. Conditions that cause ER stress include exposure to envi-
caspase-2 activation are not entirely clear, although this caspase can as- ronmental toxins, energy or nutrient deprivation, disturbance of calci-
sociate with the p53-inducible death domain-containing protein (PIDD) um or redox homeostasis, hypoxia, inflammation, increased protein
and the adaptor molecule RIP-associated ICH-1 homologous protein synthesis, impaired protein degradation and defective autophagy
with a death domain (RAIDD), forming the PIDDosome complex [20, [15,18,70]. The induction of ER stress activates a survival response
61] (Fig. 3). PIDD and RAIDD interact in the PIDDosome via their known as the Unfolded Protein Response (UPR), which helps cells to
adapt to ER stress and restore homeostasis [18,70]. UPR activation can ER kinase (PERK), inositol-requiring protein-1α (IRE-1α) and activating
promote cell survival or cell death, depending on the severity of ER transcription factor-6α (ATF-6α) [72,73]. Under normal conditions,
stress. these three proteins are sequestrated in the ER lumen by interactions
The UPR involves three signalling mechanisms: i) transient inhibi- with the chaperone binding immunoglobulin protein (BiP)/glucose-
tion of translation, thereby decreasing influx of new proteins into the regulated protein 78 (GRP78), which keeps them inactive (Fig. 4A)
ER; ii) up-regulation of genes encoding for chaperones and antioxi- [15,70]. However, during ER stress, BiP binds preferentially to unfolded
dants, in order to increase folding, and for proteins involved in ER- proteins that accumulate in the ER. This process releases the three ER
associated protein degradation (ERAD); and iii) activation of ERAD, stress sensors, which then become activated [15,70]. BiP also directs
which allows translocation of unfolded or aggregated proteins from misfolded proteins for degradation by the ERAD system.
the ER to the cytosol where they are ubiquinated and degraded by the To transiently attenuate global protein synthesis, activated PERK
proteasome [15,18,70,71]. These mechanisms are controlled mainly by phosphorylates the eukaryotic initiation factor 2α (eIF2α), which
three sensors of the ER stress response: protein kinase RNA (PKR)-like takes part in general inhibition of the initiation of mRNA translation
Fig. 4. (A) ROS activate the ER stress response and (B) cause apoptosis through the ER. A. Low dose stress stimulates activation of three ER stress sensors, PERK, ATF-6α and IRE1α that are
involved in the unfolded protein response (UPR). Bip is released from these sensors and binds to unfolded proteins in the ER lumen. Together, the three ER stress sensors inhibit protein
translation, increase chaperone expression and enhance ER-associated protein degradative (ERAD) pathways. Activation of PERK phosphorylates eIF2α, which inhibits general protein
translation. This allows selective translation of ATF-4, which activates transcription of chaperones such as BiP, and upregulates genes for the UPR and ERAD. ATF-6α undergoes
proteolytic cleavage in the Golgi apparatus. IRE1α activation causes alternative splicing of XBP1 mRNA, leading to expression of the active XBP1 transcription factor. IRE-1α and ATF-
6α both activate transcription pathways that increase the UPR and ERAD. B. Higher dose ER stress triggers apoptosis through several pathways. Caspases-9 and -3 can be activated by
mitochondrial-dependent and independent pathways. Activated IRE1α recruits TRAF2 and ASK1, causing activation of JNK and mitochondrial apoptosis. ER caspase-4/-12 is activated
by calpain, and possibly caspase-7, leading to activation of caspase-9 and caspase-3. Cleavage of BAP31 at the ER membrane by caspase-8 generates a p20 fragment, which leads to
activation of mitochondrial apoptosis. Ca2 + released from the ER is taken up by mitochondria, leading to depolarization of the inner mitochondrial membrane (IMM). Activation of
PERK and ATF-6α induces CHOP, which appears to activate apoptosis by up-regulating expression of pro-apoptotic genes such as Bax and Bim and/or by inhibiting expression of the
Bcl-2 gene.
Fig. 4 (continued).
[15,70]. However, phosphorylated eIF2α specifically upregulates trans- targeted to the nucleus where it activates genes that encode for proteins
lation of the ATF-4 mRNA. The transcription factor ATF-4 then translo- involved in the UPR. These proteins include chaperones such as BiP, ER
cates to the nucleus where it activates UPR genes that encode for protein 57 (ERp57), GRP94 and proteins involved in ERAD [70].
proteins such as the chaperone BiP, as well as those involved in amino The UPR thus constitutes an adaptive, anti-apoptotic survival re-
acid biosynthesis and transport, and the antioxidant response sponse during exposure to ER stress [70]. However, when the UPR
(Fig. 4A) [70]. ATF-4 also activates the transcription factor CCAAT- fails to restore homeostasis in the ER under conditions of severe or
enhancer-binding protein homologous protein (CHOP) (or growth ar- prolonged ER stress, cells undergo ER stress-mediated apoptosis
rest and DNA damage-inducible gene 153 (GADD153)). [15,18,70,74].
IRE-1α and ATF-6α both activate transcription pathways that in-
crease protein folding, transport and degradation (see review [70]). 2.2.3.2. ER stress and apoptosis. There are several different mechanisms
Once activated, IRE-1α catalyses the alternative splicing of the X-Box by which cells can undergo apoptosis following unresolved ER stress
binding protein (XBP-1) mRNA, which leads to expression of a function- [18,71,72,74]. These mechanisms involve several of the factors involved
al transcription factor XBP-1s (Fig. 4A). XBP-1s translocates to the in the UPR, and can be dependent on, or independent of mitochondria.
nucleus where it activates transcription of genes encoding for chaper- In addition to its role in selective splicing of XBP-1 mRNA during the
ones located in the ER as well as for degradation pathways, thus increas- ER stress response, IRE-1α appears to play a central role in the switch
ing the capacity of the ER to deal with damaged and unfolded proteins. from a survival response to cell death [18,70,72]. During prolonged con-
Furthermore, IRE-1α activation can promote mRNA decay to reduce the ditions of ER stress, IRE-1α can directly bind to unfolded proteins [75].
protein folding load in the ER. This process is known as regulated IRE- IRE-1α can also interact with Traf-2, which then recruits and activates
dependent decay (RIDD) [70]. Finally, when ATF-6α is released from apoptosis signalling kinase-1 (ASK-1) [76]. This causes phosphorylation
BiP, it translocates to the Golgi where it undergoes cleavage and activa- and activation of c-Jun N-terminal kinase (JNK) and p38 MAPK, which
tion (Fig. 4A). The cytosolic cleavage fragment of ATF-6α is then can promote apoptosis (Fig. 4B) [70,71,76]. Indeed, activated JNK
translocates to the mitochondrial membrane, where it blocks Bcl-2 and ER stress induces the formation of a complex between procaspase-8
allows activation of pro-apoptotic members of the Bcl-2 family that are and Bap31, an integral membrane protein of the ER [71,76]. This results
critical for cytochrome c release from mitochondria and induction of ap- in cleavage of the cytosolic tail of Bap31 by caspase-8, leading to produc-
optosis [15]. tion of a pro-apoptotic p20Bap31 cleavage fragment (Fig. 4B). The p20
Although Bcl-2 family proteins are best characterised at the mito- fragment can then direct pro-apoptotic signals between the ER and mi-
chondria level, they are also localised at the ER and nuclear membranes tochondria [76]. p20 triggers Ca2+ efflux from the ER where it is rapidly
[77,78]. IRE-1α-mediated JNK activation causes phosphorylation and taken up mitochondria, and mitochondrial recruitment of dynamin-
inactivation of Bcl-2 located at the ER membrane [76]. Phosphorylation related protein 1 (Drp1). Once Ca2+ levels in the mitochondrial matrix
by JNK inhibits anti-apoptotic Bcl-2 [71], which leads to its proteasomal reach a critical threshold, the MPTP becomes activated, resulting in in-
degradation [79]. ER-localised Bcl-2 has been shown to inhibit apoptosis creased MOMP. This culminates in pro-apoptotic cytochrome c release
and to regulate ER Ca2+ homeostasis, whereas pro-apoptotic members from mitochondria and cytochrome c-dependent cell death [76].
of the Bcl-2 family such as Bax and Bak are able to promote Ca2+ release ER-localized Bap31 can interact with the outer membrane mito-
from the ER, in a similar manner to their action at the mitochondrial chondrial fission protein Fission 1 homolog (Fis1) [87]. Fis1 is the recep-
membrane [71,76,80]. Consequently, when inactivated by JNK, Bcl-2 is tor for cytosolic Drp1 and appears to be involved in its recruitment to
no longer able to inhibit pro-apoptotic members of the Bcl-2 family mitochondria [88]. Drp1 mediates scission of the OMM, which leads to
and unable to regulate Ca2+ efflux from the ER [81]. This leads to dramatic fragmentation and scission of the mitochondrial network.
massive and/or prolonged influx of Ca2+ into mitochondria, which can Concurrent with cytochrome c release, the mitochondrial network un-
activate the MPTP complex and trigger mitochondrial swelling and dis- dergoes fission.
ruption of the OMM, resulting in release of pro-apoptotic factors into The Fis1–Bap31 complex at the ER-mitochondria interface serves as
the cytosol and apoptosis [36]. Activated JNK can also phosphorylate a platform, known as the ARCosome, which recruits and activates
and activate the ER pro-apoptotic Bcl-2 protein Bim [76]. Under physio- procaspase-8, thereby linking two major organelles that are involved
logical conditions, pro-apoptotic activity of Bim is inhibited by binding in intrinsic apoptotic signalling [39]. It was proposed that Ca2+ release
to dynein motor complexes [82]. Phosphorylation by JNK releases Bim from the ER could be a mechanism that allows the cell to achieve signif-
from these complexes and allows activation of Bax-mediated apoptosis icant amplification by engaging mitochondria in apoptosis.
[83].
Under conditions of severe ER stress, the PERK-eIF2α-ATF-4 path- 3. ROS and Apoptosis
way also activates the transcription factor CHOP, which in turn
upregulates expression of pro-apoptotic proteins such as DR5, Bim At lower doses, ROS such as H2O2 have been linked to induction of
and PUMA, and downregulates expression of Bcl-2 (see reviews cell survival responses, whereas higher doses activate death processes
[71,76]). Among other things, CHOP downregulates Bcl-2 by repressing such as apoptosis [6]. At lower doses, ROS activate the tumour suppres-
its promoter, and promotes activation and translocation of Bax to mito- sor protein p53 [6]. p53 plays a key role in the control of cellular stress
chondria (Fig. 4B) [76]. Another manner in which CHOP can cause apo- responses, inducing either cell cycle arrest in order to promote DNA re-
ptosis is through activation of growth arrest and DNA damage-inducible pair and survival, or cell death by apoptosis, depending on the context
34 (GADD34), which stimulates dephosphorylation of elF2α and re- [89]. Under normal conditions, p53 has a short life-time and is main-
verses the translational block [76]. This increases protein synthesis tained at low levels through ubiquitination by Mdm2, leading to its
and contributes to additional accumulation of unfolded proteins in the proteasomal degradation. When the cell faces stress conditions or
ER, ATP depletion, oxidative stress and apoptosis. In addition, this allows DNA damage, p53 is released from Mdm2 and stabilised by post-
the translation of mRNAs encoding for pro-apoptotic proteins. In addi- translational modifications, thus avoiding proteasomal degradation, be-
tion, CHOP can be regulated post-translationally by p38 MAPK phos- fore associating with DNA [89]. If the stress is low, p53 induces cell cycle
phorylation, which increases its transcriptional activity [76]. p38 is a arrest, DNA repair and senescence. However if damage is too severe, p53
substrate of ASK-1, which is recruited to the IRE1α–TRAF2 complex can regulate apoptosis transcriptionally by down-regulating pro-
during ER stress [76]. survival proteins such as Bcl-2, Bcl-XL, IAPs and survivin, and upregulat-
Another mechanism of ER-mediated apoptosis involves the caspase- ing pro-apoptotic members [89]. p53 activates the transcription of pro-
12/caspase-4 pathway [15,18]. Caspase-12/-4 can be activated by sever- apoptotic genes that are crucial for inducing the intrinsic pathway of
al different mechanisms. Procaspase-12 in mice is bound to the cytosolic apoptosis, such as Bax, Bid, Puma, Noxa and Apaf-1, but also extrinsic
face of the ER membrane and can be activated by cleavage involving the pro-apoptotic factors such as Fas, FasL, DR-4 and DR-5 [89]. Moreover,
Ca2+-dependent cysteine protease m-calpain [18,24,84]. Cytosolic cytosolic p53 can translocate to mitochondria where it can interact di-
caspase-7 translocates to the ER membrane and can also cleave and ac- rectly with anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 and
tivate caspase-12 in response to excess ER stress [15,24]. Under normal the pro-apoptotic proteins Bax and Bak, which allows MOMP, release
conditions, Traf-2 forms a complex with procaspase-12. However, dur- of pro-apoptotic factors and apoptosis [45]. In addition, cytosolic p53
ing excess ER stress, Traf-2 also takes part in caspase-12 processing by can activate Bax directly by causing its structural rearrangement [45].
releasing it, thus allowing its activation. This appears to provide a link p53 thus enhances mitochondrial membrane permeabilisation and sub-
between the IRE-1α-Traf-2 complex and ER stress–induced apoptosis sequent release of pro-apoptotic factors from mitochondria [90].
[18]. During ER stress, pro-apoptotic Bcl-2 family member Bim is Apoptosis induced by H2O2 has been associated with increased pro-
recruited to the ER membrane, where it can intervene in caspase-12 tein expression of p53, Puma, Noxa and Bax, and p53 phosphorylation at
processing [85]. On the other hand, anti-apoptotic proteins such as Ser15 and Ser46, in several cell types including rat neural AF5, glioma,
Bcl-XL can dimerise with Bim, inhibiting its translocation to the ER colon cancer and human cervical carcinoma HeLa cells [91–94]. During
membrane [85]. Once activated, caspase-12 can activate caspase-9 H2O2-induced apoptosis in HeLa cells, p53 was an upstream factor for
that in turn processes caspase-3, in a cytochrome c-independent path- the upregulation of Puma and translocation of Bax to mitochondria [92].
way [15,18]. However, the role of caspase-12 as an initiating caspase The redox regulation of caspase activity appears to involve post-
in ER stress-induced apoptosis is unclear. In humans, functional translational modifications of their catalytic site cysteine residues [56,
caspase-12 is lacking in most cells because the caspase-12 gene contains 95]. The catalytic site cysteines of most caspases are susceptible to oxi-
several inactivating mutations [86]. Instead, caspase-4 appears to carry dation, whereas procaspase-9, procaspase-3 and caspase-3 are suscepti-
out the equivalent function in ER stress-induced apoptosis [25]. ble to S-glutathiolation [56,95]. While caspases can be activated by
Caspase-2 has also been implicated in initiating ER stress-mediated oxidants such as H2O2 [65,92], oxidants can also irreversibly inactivate
apoptosis [71]. their enzymatic activity [95]. This is the case for caspases-3, -8 and -9.
Moreover, cellular GSH can also regulate caspase-3 activity, mediated by play a central role in protecting cells against activation of apoptosis by
S-glutathiolation, which decreases accessibility for proteolytic cleavage, different stimuli. This topic is discussed in detail in the following review
resulting in resistance to apoptosis. However, the overall control of this [56].
process and its consequences for apoptosis induction are still not eluci-
dated [56]. For example, are there other apoptotic components up- 3.2. ROS and the death receptor pathway
stream of caspase-3 that are targets for S-glutathiolation and which
specific cysteines are susceptible? Links between ROS and extrinsic-induced apoptosis also exist. The
cytokine TNF-α is an important regulator of the complex signalling net-
3.1. ROS and the Mitochondrial Pathway works that promote either cell survival, or cell death by apoptosis [52].
TNF-α can also cause caspase-independent cell death by necrosis
The mitochondrial pathway of apoptosis is activated in response to a (necroptosis), which involves ROS generated from either mitochondrial
variety of cellular stresses, including mitochondrial DNA (mtDNA) dam- or non-mitochondrial sources [99].
age, growth factor deprivation, heat shock, hypoxia, ER stress and devel- ROS and TNF-α influence each other in a positive feedback loop [57].
opmental cues [15,36,56,95]. ROS have been tightly linked to activation At low doses, ROS play an important role in regulating the TNF-R1 sig-
of the mitochondrial pathway [36]. Indeed, mitochondria are the loca- nalling pathway, whereas at higher doses, this leads to DNA damage
tion where most intracellular ROS are produced, as a result of leakage and cell death. There is considerable debate as to whether mitochondri-
from the respiratory electron transport chain [10]. Mitochondria- al ROS can activate or suppress the transcription factor NF-κB. It is clear
derived ROS are then able to target nearby structures such as mtDNA, that ROS can mediate activation of NF-κB through TNF-R1 signalling,
which is susceptible to oxidative damage [36]. mtDNA damage would which leads to upregulation of antioxidants such as catalase and
impair mtRNA transcription of proteins involved in the electron trans- MnSOD [57]. These antioxidants can offset TNF-induced apoptosis by
port chain, causing disruption of respiratory chain function, further in- neutralising mitochondrial-derived ROS. In addition, NF-κB stimulates
creases in ROS generation, leading to loss of mitochondrial membrane the transcription of anti-apoptotic genes such as IAP and Bcl-XL, which
potential and impaired ATP synthesis [36]. These events culminate in can prevent induction of TNF-R1-mediated apoptosis by blocking
apoptosis through the mitochondrial pathway. caspase-8 activation [12]. However, it is generally accepted that TNF-
ROS such as H2O2 and superoxide can cause cytochrome c release derived ROS can inhibit NF-κB activation, decreasing NF-κB-mediated
from mitochondria and induction of apoptosis through the mitochon- survival signalling, thus promoting apoptosis [100]. On the other hand,
drial pathway [56,65,96,97]. Protein components of the MPTP such as mitochondria-derived ROS appear to promote, rather than inhibit,
VDAC, ANT and cyclophilin D are targets of ROS and undergo oxidative TNF-mediated NF-κB activation [100]. It appears that mitochondrial
modifications, which will stimulate MPTP opening [56]. H2O2 caused ROS inactivate the phosphatases that regulate activity of the kinases
initial mitochondrial membrane hyperpolarisation that led to collapse controlling NF-κB signalling. Consequently, ROS-mediated phosphatase
of mitochondrial membrane potential (ΔΨm), mitochondrial transloca- inhibition would cause enhanced phosphorylation of the inhibitor IκB,
tion of Bax and Bad, and cytochrome c release [95]. Significant loss of cy- triggering its degradation and allowing NF-κB activation.
tochrome c from mitochondria will further increase ROS generation due The mechanisms by which TNF stimulation leads to increased mito-
to disruption of the electron transport chain. chondrial ROS generation in cells are not understood. Once activated by
In HeLa cells, H2O2-induced apoptosis through the mitochondrial TNF-R1, caspase-8 can bind to ROS modulator-1 (ROMO-1) that is locat-
pathway was mediated by p53 as an upstream factor [92]. H2O2 caused ed in the OMM [57]. ROMO1 then sequesters Bcl-XL, which triggers loss
both caspase-dependent and caspase-independent apoptosis. Caspase- of mitochondrial membrane potential and increases generation of ROS.
dependent apoptosis involved loss of mitochondrial membrane perme- ROS in turn trigger activation of ASK-1. Under normal conditions, ASK-1
ability, cytochrome c release into the cytosol, and caspase-9-mediated remains inactive by binding to reduced thioredoxin [95]. However, ROS
activation of caspase-3. Caspase-independent apoptosis involved re- oxidise thioredoxin, which releases ASK-1, allowing it to activate its
lease of AIF from mitochondria and its translocation to the nucleus downstream targets JNK and p38 MAPK, leading to TNF-induced apo-
[65]. Furthermore, JNK activation by oxidative stress could phosphory- ptosis. ASK-1 can also be inactivated by protein phosphatase-5, which
late and inactivate anti-apoptotic factors such as Bcl-2 and Bcl-XL, is regulated by ROS [57]. ROS can also inactivate JNK-inactivating phos-
while phosphorylating and activating pro-apoptotic members of this phatases, which leads to JNK activation, followed by cytochrome c re-
family [12]. lease, caspase-3 activation and apoptosis.
ROS can oxidise other targets such as the phospholipid cardiolipin, Fas-mediated apoptosis can also be activated by ROS such as H2O2
which binds cytochrome c to the outer leaflet of the IMM [95]. Under [65]. In HeLa cells, H2O2 caused up-regulation of FasL, FADD transloca-
normal conditions, cytochrome c shuttles electrons between complexes tion to the plasma membrane and caspase-8 activation. Once activated,
III and IV of the mitochondrial electron transport chain. Cardiolipin ox- caspase-8 processed caspase-2. Activated caspase-8 and caspase-2 both
idation by ROS decreases cytochrome c binding and increases the level caused cleavage of Bid to t-Bid, thus enhancing mitochondrial-induced
of free cytochrome c potentially released through the OMM in the cyto- apoptosis [92]. Furthermore, p53 was an upstream initiating factor in
sol, where it initiates the apoptotic cascade [95]. the activation of Fas-mediated apoptosis by oxidative stress [65]. H2O2
Oxidative stress and mitochondrial Ca2+ accumulation can both trig- also induced death receptor-mediated apoptosis in other cell types.
ger opening of the pore in the IMM, by a process known as mitochondri- H2O2 increased mRNA levels for FasL and Fas in murine intestinal epi-
al permeability transition (MPT) [36]. Although Ca2+ is a key factor in thelial cells [101] and up-regulation of Fas in HUVECs [102]. In HL-60
pore opening, other factors such as ATP depletion, oxidative stress, cells, H2O2 caused activation of caspase-8 and caspase-3 [103].
low pH and high inorganic phosphate can also facilitate pore opening During stress conditions, death-associated protein 6 (Daxx) can in-
[98]. This leads to osmotic swelling and OMM rupture, followed by re- teract with Fas through its DD, leading to activation of ASK-1, which in
lease of mitochondrial proteins such as cytochrome c. The consequence turn activates JNK [104] (Fig. 3). JNK triggers Bcl-2 phosphorylation,
of pore opening appears to depend on the level of Ca2+; higher concen- thus favouring apoptosis through the mitochondrial pathway.
trations favour necrosis while lower levels promote apoptosis. Further- Members of the Nox family are an important source of ROS (O•2−)
more, ROS generation can significantly increase Ca2+-induced cell generation in cells [105]. Noxs are transmembrane proteins that are
deterioration [36]. In fact, ROS decrease the level of Ca2+ required to specifically located in subcellular compartments such as lipid rafts, ca-
trigger MPT and subsequent cell damage. veolae, endosomes and the nucleus [106]. For Fas and TNF-α, death
Disruption of cellular GSH redox status due to GSH oxidation or GSH ligand-receptor binding caused lipid raft formation, recruitment and ac-
efflux can contribute to oxidant-mediated apoptosis. GSH appears to tivation of Nox, and ROS generation. These processes constituted lipid
raft-derived redox signalling platforms, which promoted death receptor also interfere with protein folding by inactivating the PDI/ERO1 thiol-
activation and induction of apoptosis [107]. The physiological signifi- disulfide exchange and/or by causing incorrect disulfide bond
cance of ROS-dependent receptor-mediated apoptosis compared to formation.
the classical ligand/receptor-induced activation of apoptotic signalling Activation of the UPR by mild oxidative stress appears to be an adap-
is not completely understood [95]. tive response to maintain cell survival and function, whereas more se-
ROS have been implicated in the regulation of TRAIL-mediated apo- vere or prolonged oxidative stress and protein misfolding can initiate
ptosis [106], although the mechanisms involved are not clear. In partic- apoptotic signalling pathways [76]. During persistent ER stress, activa-
ular, ROS can upregulate gene expression for DRs such as TRAIL, which is tion of IRE1α leads to ASK and p38 MAPK activation [18], which further
abrogated by antioxidants such as catalase and N-acetylcysteine. In ad- activate CHOP and contribute to generation of ROS [109]. Among other
dition, ROS-mediated upregulation of TRAIL appears to be mediated by things, CHOP activates ERO1 transcription [76], which could lead to
factors such as CHOP and p53. Furthermore, substances that increase acute ROS production [109], favouring an over-oxidizing environment.
ROS generation appear to sensitize cells to TRAIL-mediated apoptosis This would exacerbate PDI activity, causing extra disulfide bond forma-
[106]. tion and re-oxidation of PDI in a vicious loop, as well as accumulation of
misfolded proteins [74]. ERO1 can also activate the inositol triphosphate
3.3. ROS and the ER Pathway receptor (IP3R), which stimulates massive Ca2+ transport from the ER to
mitochondria, thereby triggering mitochondrial-mediated cell death
In the ER lumen, the redox status is very important because protein [76]. Furthermore, ER-stress-mediated apoptosis is under the control
folding and disulfide bond formation require an oxidant environment. of JNK, which is regulated by ASK-1, whose function is directly con-
The oxidizing environment of the ER lumen has a high ratio of oxidized trolled by oxidative status. Indeed, thioredoxin-mediated ASK-1 oxida-
to reduced glutathione (GSSG/GSH) compared to the reducing environ- tion is necessary for ASK-1 conjugation with Traf-2 and subsequent
ment of the cytosol [108]. Disulfide bond formation is under the control activation of JNK [95].
of chaperone proteins such as the protein disulfide isomerase (PDI) In addition to eIF2α, PERK can phosphorylate nuclear erythroid 2
[109]. PDI is the most abundant chaperone in the ER. During formation p45-related factor 2 (Nrf2) [113]. This leads to dissociation of the
of disulfide bonds in nascent proteins, PDI catalyses the oxidation of two Nrf2-Keap1 complex and promotes the expression of genes containing
cysteine residues between polypeptide substrates, while itself undergo- antioxidant response elements (ARE), thereby preventing oxidative
ing two electron reduction at its active site. The ER membrane- stress by induction of antioxidant genes such as heme oxygenase 1
associated protein, endoplasmic reticulum oxidoreductin-1 (ERO1), is (HO-1) [113]. Nrf2 also increases expression of glutamate cysteine li-
capable of restoring PDI to its oxidized state, but this leads to ROS gen- gase (GCL), the main enzyme involved in synthesis of the antioxidant
eration [109]. Electrons are transferred from protein thiol groups of PDI GSH.
to O2 and ERO1, through a flavin adenine dinucleotide (FAD)-depen- During ER stress, ROS can stimulate Ca2+ release from the ER lumen
dent reaction. FAD acts as an electron acceptor and its reduced form [109]. There are tight interactions between the ER and mitochondria
(FADH2) is reoxidised by a reaction with O2, which results in generation through specialised ER compartments such as mitochondria-
of H2O2. It is not clear whether the ER-derived H2O2 produced during associated membranes (MAM), which control the transport of small
oxidative folding is toxic or acts as a signalling molecule [110]. Further- molecules and ions between these organelles [39]. Due to proximity of
more, increased levels of GSH in the ER can lead to excessive generation mitochondria to the ER, high levels of Ca2+ released from the ER are
of H2O2 through ERO1. Glutathione (GSH) is a substrate for PDI- then taken up by mitochondria [36]. The increased levels of Ca2+ in mi-
catalysed oxidation, which leads to increased levels of GSSG when tochondria stimulate the Ca2+-sensitive matrix dehydrogenases, which
ERO1 is active. The increased levels of H2O2 and GSSG cause a distur- increase metabolic activities by providing NADH for mitochondrial res-
bance in the redox equilibrium of the ER lumen and lead to ER stress. piration and ATP production [36]. Increased mitochondrial respiration
Moreover, the activities of both ERO1 and PDI are redox regulated [110]. results in an increase in mitochondrial ROS generation through com-
The impairment of ER proteostasis is another condition that would plexes I and III. Furthermore, mitochondrial Ca2+ overload stimulates
increase the need for ERO1-derived disulfides [110]. Misfolded proteins opening of the MPTP, leading to release of ATP, GSH and cytochrome c
in the ER are thought to be subjected to fruitless cycles of aberrant disul- to further increase ROS levels [110].
fide bond formation and reduction. This would increase production of Interestingly, ROS can induce expression of the pro-apoptotic BH3-
ERO1-derived H2O2, which would in turn cause ER stress and activate only Bcl-2 protein Bim [114]. In addition, JNK phosphorylates Bim,
UPR signalling. However, the source and nature of ER stress-derived which releases it from the inhibitory dynein complexes, thus allowing
ROS, and whether they are ERO1-derived, is not entirely clear. activation of Bax-Bak [83]. Ca2+ release from the ER through Bax-Bak
Furthermore, ROS levels may increase when GSH levels are depleted channels allows signal transmission from the ER to mitochondria and
due to reduction of improperly-paired disulfide bonds in misfolded pro- Bax-dependent apoptosis.
teins. It was estimated that about 25% of cellular ROS may be generated There appears to be a functional connection between ER stress and
from the formation of disulfide bonds in the ER during oxidative protein mitochondrial ROS production. It should be noted that ERO1 is a tran-
folding [111]. Proteins with many disulfide bonds would play a larger scriptional target of the ATF-4/CHOP pro-apoptotic pathway, which
role in ROS generation in the ER compared to those with fewer disulfide stimulates mitochondrial ROS production [70,110]. Mitochondrial ROS
bonds. The generation of ROS as by-products of protein oxidation in the production was dependent on levels of ERO1, which is concentrated in
ER could then cause ER stress-mediated cell death. Other antioxidants MAMs. ERO1 also promotes IP3R-mediated Ca2+ efflux from the ER
found in the ER include glutathione peroxidases 7 and 8, and that is taken up by mitochondria through the MAM [76]. Therefore, ER
peroxiredoxin 4, which all detoxify H2O2 [110]. The ER-antioxidant sys- stress induces oxidative stress and impairs mitochondrial function,
tem is thus essential for maintaining ER homeostasis because changes in which leads to cell death in a CHOP-dependent manner.
the redox status result in oxidative modification and/or misfolding of The pro-oxidant H2O2 activated the UPR in several different cell
newly-synthesised proteins, which triggers ER stress. types. A relatively short exposure (15 min) of HeLa cells to H2O2
ROS generation could increase during protein misfolding through (15–50 μM) activated the UPR: the expression of the ER stress sensors
changes in oxidative phosphorylation, which would arise from Ca2+ re- p-PERK, p-eIF2α and p-IRE1α increased, while cleavage of ATF6 oc-
lease from the ER or energy depletion. However, excess ROS are able to curred [24]. H2O2 (50 to 500 μM, 24 h) caused induction of GRP78/Bip,
trigger protein misfolding, leading to UPR activation and induction of p-PERK and p-eIF2α in human oral keratinocytes and oral cancer cells
CHOP, which can be prevented by the antioxidant butylated [115]. In mesenchymal stem cells, H2O2 (120 μM, 6 to 24 h) increased
hydroxyanisol (BHA) [112]. Furthermore, excess ROS production could the expression of Bip [116]. On the other hand, longer exposure times
(1−3 h) to H2O2 induced ER-mediated apoptosis. In HeLa cells, expres- such as cancer [118]. More research is required to clearly delineate
sion of CHOP increased along with cleavage of the calpain inhibitor how autophagy triggers cell death.
calpastatin, leading to increased enzymatic activity of the ER proteases
calpain, caspase-4, caspase-12 and caspase-7 [24]. The activation of ER 4.2. Necroptosis
caspase-4 and caspase-12 was decreased by the calcium chelator
BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the Necrosis was originally classified as an accidental form of cell death,
roles of calcium, calpain and caspase-7 in the activation of ER- where there was explosive release of cellular contents following rupture
mediated apoptosis by H2O2. Further downstream in the signalling cas- of the cytoplasmic membrane [14]. Necroptosis eventually emerged as a
cade, caspase-4 and caspase-12 caused activation of caspase-9 and highly regulated process that can be activated by TNF-α and a pan-
caspase-3, followed by ER-dependent DNA fragmentation and apoptosis caspase inhibitor (z-VAD-FMK) [119]. Similar to necrosis, morphologi-
[24]. Caspase-4 cleavage and up-regulation of CHOP were induced by cal features of necroptosis include cellular rounding, swelling, cytoplas-
peroxide (200 to 500 μM, 24 h) in human oral keratinocytes and oral mic granulation and plasma membrane rupture. This leads to release of
cancer cells [115]. The exposure of mesenchymal stem cells to H2O2 cellular contents into the surrounding tissue, thus provoking an inflam-
(120 μM) for times ranging from 6 to 24 h caused cleavage of matory response [51]. Necroptosis is a regulated pathway of cell death
procaspase-12 [116]. In rat hepatocytes, the pro-oxidant tert- that contributes to certain diseases, notably those associated with in-
butylhydroperoxide (tBOOH) caused calpain-mediated cleavage of flammation and cancer (see reviews [51,120]). It is thought to be a
procaspase-12 at the ER and its translocation to the nucleus, where in- form of cell death that compensates for the inhibition of apoptosis due
creased caspase-12 activity was found [117]. Therefore, the adaptive to certain cellular contexts or caused by viruses, cancer, etc.
survival response (UPR) dominated during milder conditions of pro- Necroptosis is activated in response to death receptors (e.g. TNF-R1,
oxidant stress, whereas ER-mediated apoptosis occurred under more Fas), Toll-receptors, viruses and DNA damage [51,120]. In the presence
severe conditions [24]. of TNF-α, complex 1 consisting of Rip1, TRADD, cIAP1/2 and Traf
forms on the cytoplasmic side of TNF-R1 [121]. Rip1 is involved in this
signalling pathway and its polyubiquitination by cIAP1/2 leads to acti-
4. Other Regulated Cell Death Pathways
vation of the transcription factor NFκB. When caspase-8 is inactivated,
deubiquitination of Rip1 leads to its association with Rip3. When Rip1
Since the discovery of apoptosis, several other regulated pathways of
and Rip3 become phosphorylated, they interact to form the necrosome
cell death have been recognised [105]. These include autophagy,
(complex 2). In the absence of Rip3, Rip1 can mediate apoptosis. Rip3
necroptosis and anoikis. These modalities will not be described in detail
appears to facilitate a molecular switch from apoptosis to necroptosis.
in this review and the reader is referred to recent reviews on these
Mixed lineage kinase domain-like (MLKL) protein and PGAM5L, a
subjects.
mitochondrial protein, both interact with Rip3 and are involved in the
necroptosis signalling cascade [121]. MLKL is phosphorylated by Rip3
4.1. Autophagy and acts downstream of Rip3 as an adaptor protein that is essential for
necroptosis induction. PGAM5L is a protein phosphatase that is involved
Autophagy (or macroautophagy) is a regulated cellular catabolic in the Rip1 and Rip3 containing necrosome. PGAM5L recruits Drp1 to
process that eliminates damaged or superfluous cytoplasmic contents mitochondria, which expedites mitochondrial fission and necroptotic
and organelles (e.g. mitochondria, ER) (see review, [118]). It is a normal cell death. Interestingly, PGAM5L is a substrate for Keap1, which is an
process that is involved in important functions such as cell growth, de- inhibitor of Nrf2. The transcription factor Nrf2 activates the antioxidant
velopment, aging and immunity [15]. Deregulation of autophagy has response and thus contributes to detoxification of intracellular ROS
been implicated in several human diseases such as cancer, cardiac dis- [113]. This provides a molecular link between ROS and the necroptotic
ease, and neurodegenerative disorders such as Alzheimer's, Parkinson's signalling pathway. In addition, ROS have been implicated in the induc-
and Huntington's diseases [15,105]. Different conditions of stress (e.g. tion and execution of cell death by necroptosis [122].
hypoxia, nutrient starvation, hormonal imbalance, oxidative stress, pro- The understanding of mechanisms of necroptosis is not very ad-
tein aggregates) can enhance autophagy. Autophagy is generally a stress vanced and future mechanistic studies are required. These mechanisms
adaptation pathway that promotes cell survival during starvation or are likely to be dependent on cell type and tissue. Once these mecha-
acts as a defence mechanism against different environmental stresses. nisms are more clearly delineated, necroptosis is likely to play an impor-
During autophagy, the cytoplasmic contents of a cell are sequestered tant role in the development of novel cancer treatment strategies. In
within double membrane vacuoles (autophagosomes) [118]. The addition, improved understanding of necroptosis should advance treat-
outer membrane of the autophagosome fuses with the membrane of ly- ments for multiple inflammation-based diseases. There are other
sosomes to form an autolysosome, in which cytoplasmic contents are emerging forms of regulated necrosis such as pyroptosis and ferroptosis
degraded by lysosomal enzymes such as cathepsins B, D and L [15]. [120], whose mechanisms will be surely uncovered during the coming
Under conditions of nutrient deprivation, degradation of cytoplasmic years.
material during autophagy generates amino acids and energy to main-
tain cell survival [118]. Autophagy is regulated by a full set of 4.3. Anoikis
autophagy-related genes (Atg) and by the mammalian target of
rapamycin (mTOR) kinase signalling pathway [15,105,118]. mTOR ki- Anoikis was first characterised by Frisch and Francis in 1994 [123]. It
nase is the major inhibitory signal that turns off autophagy when suffi- is a cell death program where a cell dies when it becomes detached from
cient quantities of nutrients and growth factors are available. the extracellular matrix (ECM) (see reviews [121,124]). For certain cell
Although autophagy is generally recognised as a cell survival pro- types such as epithelial cells, attachment to the ECM is essential for their
cess, it can also act as a caspase-independent cell death pathway. In ability to carry out vital cellular functions such as metabolism, prolifer-
terms of morphology, autophagic cell death is characterised by dying ation and survival. Detachment from the ECM can trigger a wide variety
cells containing abundant autophagosomes and the lack of phagocyte of cellular changes. A major role of anoikis is to limit the progression of
participation in cell death [118]. Beyond the morphology, the process cancer. For cancer cells to undergo metastasis, they need to inhibit
by which autophagic cells subsequently die is not clear and has been anoikis [121]. Hence, resistance to anoikis facilitates the progression of
the subject of controversy. Is this cell death by autophagy or cell death tumour cells and enables metastasis.
with autophagy? Much less is known about the mechanisms that Anoikis is initiated through adhesion molecules that are involved in
regulate autophagic cell death and how this may relate to diseases cell-cell contacts such as E-cadherin and integrins that are involved in
cell-ECM [124]. E-cadherin is a transmembrane protein whose cytoplas- Abbreviations

mic extremity is bound to β-catenin, which is in turn bound to α- AIF Apoptosis inducing factor
catenin. This complex anchors E-cadherin to the actin cytoskeleton, ALS Amyotrophic lateral sclerosis
allowing it to regulate cell adhesion. E-cadherin appears to regulate AMPK AMP-activated protein kinase
anoikis through the RAF/ERK and PI3K/Akt pathways. The ligated con- Apaf-1 Apoptotic peptidase activating factor 1
formation of integrins can activate signalling pathways that promote ASK-1 Apoptosis signalling kinase 1
cell survival such as the PI3K/Akt pathway, whereas their unligated con- ATF-6α Activating transcription factor-6α
formation can stimulate apoptosis mediated by caspase-8. Atg Autophagy-related gene
ECM-detached cells have several metabolic defects including in- ATM Ataxia-telangiectasia mutated
creased levels of ATP and ROS, which can lead to non-apoptotic cell ATR Ataxia telangiectasia and Rad3 related
death (see review [121]). These regulated death pathways include Baf Bafilomycin A1
entosis, autophagy and necroptosis. Entotic cells are internalised by Bcl-2 B-cell-lymphoma protein 2
host cells through activation of Rho and ROCK, before undergoing BH Bcl-2 homology
non-autophagic degradation involving lysosomes. Some entotic cells BiP Binding immunoglobulin protein
can exit the host and return to their normal functions. ECM-detached Ca2+ Calcium (II)
cells can undergo autophagy to prolong cell survival until nutrient res- Chk1 Checkpoint kinase 1
cue mechanisms are activated. On the other hand, autophagy can be ac- CHOP CCAAT-enhancer-binding protein homologous protein
tivated as a death mechanism in cells under duress due to ECM- Daxx Death-associated protein 6
detachment. The loss of β1 integrin engagement can promote autopha- DD Death domain
gy. Necroptosis can be linked to ECM-detachment through its regulation DED Death-effector domain
of ROS, mediated by Rip3. Anoikis can also lead to cell death mediated DISC Death inducing signalling complex
by both intrinsic and extrinsic pathways of apoptosis [124]. The intrinsic DR Death receptor
pathway is mediated by MOMP and caspase-9 activation whereas the DRP Dynamin-related protein 1
extrinsic pathway involves the TNF-R death receptor and caspase-8 ac- ECM Extracellular matrix
tivation. Further advances are required to fully understand the mecha- eIF2α Eukaryotic initiation factor 2α
nisms involved in the molecular regulation of anoikis as well as the Endo G Endonuclease G
different modes by which these cells undergo cell death. ER Endoplasmic reticulum
ERAD ER-associated protein degradation
5. Concluding Remarks ERK Extracellular regulated kinase
ERO1 Endoplasmic reticulum oxidoreductin-1
Three different types of programmed cell death have been identi- ERp57 ER protein 57
fied: apoptosis, autophagy and necroptosis. An important concept is FAD Flavin adenine dinucleotide
that these cell death pathways each act as backup in the event that FADD Fas-associated death domain
the other pathways are inhibited. These cell death pathways modulate FasL Fas ligand
each other by mutual inhibitory processes and are controlled by multi- Fis1 Fission 1 homolog
ple feedback loops [51]. During recent years, our understanding of the FLIP FLICE inhibitory protein
mechanisms involved in regulation of the signalling pathways of apo- GADD153 Growth arrest and DNA damage-inducible gene 153
ptosis has evolved significantly. However, many important questions GCL Glutamate cysteine ligase
remain unanswered. These include further clarification of the molecular GRP94 Glucose-regulated protein 94
links that trigger the transition between cell survival and cell death for GSH Glutathione
each of the death receptor and ER signalling pathways. In addition, ap- H2O2 Hydrogen peroxide
optosis through the ER pathway appears to involve several distinct sig- OH• Hydroxyl radical
nalling mechanisms whose respective roles require clarification. HSP Heat shock protein
Moreover, the complex interplay between apoptosis and other signal- IAP Inhibitor of apoptosis protein
ling pathways involved in cell survival and cell death such as autophagy ICAD Inhibitor of caspase activated DNase
and necroptosis requires further mechanistic insights. Autophagy has IL Interleukin
been mainly characterised as a cell survival phenomenon. However, it IMM Inner mitochondrial membrane
appears to play a role in cell death by mechanisms that have not been IP3R Inositol triphosphate receptor
entirely clarified. Further confounding factors are the influence of differ- IRE-1α Serine/threonine-protein kinase/endoribonuclease-1α
ent cell types and the responses to different environmental factors. The JNK c-Jun N-terminal kinase
recent interest in the involvement of miRNAs in cell survival and death Keap1 Kelch like-ECH-associated protein 1
signalling pathways is likely to open up a multitude of new avenues of LDH Lactate dehydrogenase
research in this field. 3-MA 3-Methyladenine
ROS play a central role in cell signalling and in the regulation of the MAM Mitochondria-associated membranes
main pathways of apoptosis mediated by mitochondria, death receptors MAPK Mitogen-activated protein kinase
and the ER. ROS are also involved in other regulated pathways of cell Mdm2 Mouse double minute 2 homolog
survival and cell death such as autophagy and necroptosis. In particular, MLKL Mixed lineage kinase domain-like
the role of ROS in the complex interplay and crosstalk between these MOMP Mitochondrial outer membrane permeability
different signalling pathways remains to be further unravelled during MPT Mitochondrial permeability transition
the coming years. The deregulation of these different cell survival and MPTP Membrane permeability transition pore
cell death pathways is likely to have important pathological conse- mtDNA Mitochondrial DNA
quences for oxidative stress-associated diseases such as cancer, neuro- mTOR Mammalian target of rapamycin
degenerative diseases, ischemia-reperfusion injury and diabetes. mTORC1 Mammalian target of rapamycin complex 1
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Lakesla Apoptosis

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Biochimica et Biophysica Acta 1863 (2016) 2977–2992

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbamcr

Activation of apoptosis signalling pathways by reactive oxygen species

1. Introduction implicated in the activation of various cellular signalling pathways and

cell-ECM [124]. E-cadherin is a transmembrane protein whose cytoplas- Abbreviations

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