Tumor Biol.

(2016) 37:8471–8486
DOI 10.1007/s13277-016-5035-9

REVIEW

Major apoptotic mechanisms and genes involved in apoptosis

Yağmur Kiraz 1,2 & Aysun Adan 1 & Melis Kartal Yandim 2 & Yusuf Baran 1,2

Received: 7 December 2015 / Accepted: 28 March 2016 / Published online: 9 April 2016
# International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract As much as the cellular viability is important for the Introduction

living organisms, the elimination of unnecessary or damaged
cells has the opposite necessity for the maintenance of homeo- The main goal for a cell is to stay alive during the lifetime.
stasis in tissues, organs and the whole organism. Apoptosis, a Holding the key of proliferation events as much as death
type of cell death mechanism, is controlled by the interactions mechanism has vital importance for the cells to keep the bal-
between several molecules and responsible for the elimination ance between living and death cells in the body. As one of the
of unwanted cells from the body. Apoptosis can be triggered cellular death mechanisms, apoptosis, also known as pro-
by intrinsically or extrinsically through death signals from the grammed cell death, can be defined as the process of a proper
outside of the cell. Any abnormality in apoptosis process can death of any cell under certain or necessary conditions.
cause various types of diseases from cancer to auto-immune Apoptosis is a part of natural homeostatic mechanism to keep
diseases. Different gene families such as caspases, inhibitor of the number of the cells constant in an organism and helps the
apoptosis proteins, B cell lymphoma (Bcl)-2 family of genes, tissue to eliminate increasing number of unwanted/unneeded
tumor necrosis factor (TNF) receptor gene superfamily, or p53 cells that are damaged or no longer manageable during devel-
gene are involved and/or collaborate in the process of apopto- opment, growth or aging [1]. It also plays crucial roles in
sis. In this review, we discuss the basic features of apoptosis early development and differentiation of the embryo in order
and have focused on the gene families playing critical roles, to generate a full and decent organism. Although the term of
activation/inactivation mechanisms, upstream/downstream ef- Bapoptosis^ was firstly used by Kerr et al. in 1972 to identify a
fectors, and signaling pathways in apoptosis on the basis of distinguished type of cell death, the first description and un-
cancer studies. In addition, novel apoptotic players such as derstanding of the programmed mechanisms of apoptosis was
miRNAs and sphingolipid family members in various kind derived from the studies on the development of the nematode
of cancer are discussed. Caenorhabtidis elegans in 1999 [2, 3].
Apoptosis is a defense mechanism against damaged,
stressed, or stimulated cells by any agents to prevent accumu-
Keywords Intrinsic/extrinsic pathway . Bcl-2 . Caspase . lation of non-functional cells in the tissues. If apoptosis is not
TNF . TRAIL . p53 be mediated properly in unwanted cells, the mutations could
continue to accumulate in the cells that eventually could lead
to generation of cancer and other diseases such as auto-
immune diseases, AIDS, or some of neurodegenerative disor-
* Yusuf Baran ders [4]. However, apoptosis is the best defined and well-
ybaran@gmail.com
understood Bprogrammed^ type of cell death; there are many
different types of cellular death mechanisms such as
1
Department of Molecular Biology and Genetics, Faculty of Life and pyroptosis, necrosis, or autophagy, and some others might
Natural Sciences, Abdullah Gül University, 38080 Kayseri, Turkey not yet be discovered [5, 6]. Apoptosis regulatory pathways,
2
Department of Molecular Biology and Genetics, İzmir Institute of including a number of gene families, orchestrate the specific
Technology, İzmir 35430, Turkey morphological and biochemical changes in the cells during the
8472 Tumor Biol. (2016) 37:8471–8486

process. We touched on these significant chances briefly and time, the cytoplasmic scaffold proteins and cell junction pro-
focused on the genes involved in apoptosis with details. teins such as actin, β-catenin, spectrin, or Gas2 are deactivated
by cleavage and the cell loses its integrity by the function of
Morphological and biochemical processes of apoptosis caspases [14].
Mitochondria have complex and important roles by provid-
Many biochemical events and a series of morphological ing various pro-apoptotic signals, creating a downstream cas-
changes occur at the early stage and increasingly continue till cade of apoptosis activation. The balance between the pro-
the end of apoptosis process. Some of the changes such as cell apoptotic and anti-apoptotic molecules keeps the cellular ho-
shrinkage, chromatin condensation, or nuclear differences meostasis stable and determines the cell fate, which is either
could be observed by microscopic techniques [1, 7]. apoptosis or proliferation. Mitochondria have a leading role in
Morphological event cascade including cytoplasmic filament releasing a number of important apoptosis inducing molecules
aggregation, nuclear condensation, cellular fragmentation, including cytochrome c, SMAC, apoptosis-inducing factor, or
and plasma membrane blebbing finally results in the forma- endonuclease G as a result of permeabilization of mitochon-
tion of apoptotic bodies. All the morphological hallmarks of drial membrane. Permeabilization is triggered by pro-
apoptosis can be gathered under three headings; (i) the changes apoptotic B cell lymphoma (Bcl)-2 family proteins, while
occur in nucleus; (ii) cell membrane and cytosolic changes; (iii) the integrity of mitochondrial membrane is maintained by
those happen in mitochondria [1, 8]. Chromatin condensation, anti-apoptotic members of Bcl-2 family [10, 15, 16].
DNA fragmentation, and nuclear fragmentation are the nucle- Several biochemical changes such as protein modifica-
ar changes that could be observed with light and fluorescence tions/degradations, DNA and chromatin deteriorations, and
microscopy during apoptosis. The apoptotic cell loses its as- synthesis of cell surface markers form morphological process
sociation with other cells at initiation stage of the process by during apoptosis. Caspases are mainly responsible for these
different signals breaking the connection. This separation changes with their extensive capabilities to cleave certain mol-
followed by apoptotic body formation and resulted in ecules from one or more specific points, causing degradation
blocking the inflammatory reaction of the cells, since they and inactivation of target protein. Moreover, they can also
package their ingredient and do not release any contaminant inhibit the negative regulatory domains of specific proteins,
outside the cell. Then, the buddies are rapidly phagocytized by which leads to the activation of subjected molecule [17, 18].
other neighbor cells and these absorber cells do not produce They are also involved in DNA fragmentation process.
any signal that causes any inflammatory response. Also, mi-
tochondria play an important role by interacting with many Intrinsic and extrinsic apoptotic pathways
different apoptotic/anti-apoptotic proteins and releasing signal
molecules [9, 10]. Apoptosis can be stimulated by two different pathways: (i)
Chromatin condensation and nuclear fragmentation are the intrinsic pathway (or mitochondria) that mainly occurs via
major modifications observed in the nucleus, eventually release of cytochrome c from the mitochondria, which acti-
resulting in pyknosis (chromatin condensates irreversibly that vates different caspases as downstream signals, and (ii) extrin-
signs cell death) and followed by karyorrexis (nuclear frag- sic pathway when Fas death receptor is activated by a signal
mentation, the last event in nucleus during apoptosis) [11]. coming from the outside of the cell. After the activation of
The fragmentation of double-stranded DNA into 180–200 different intermediate molecules by signaling cascade, both of
bp sequences in length by the help of caspase proteins is also the pathways meet up at the final caspase activation step and
another essential hallmark of nuclear events during apoptosis. commonly lead to cleavage of different proteins [19] (Fig. 1).
Caspases are responsible for DNA repair during replication as
well as the termination stage of apoptosis. They are also in- Intrinsic pathway
volved in the fragmentation in apoptosis together with DNA
fragmentation factors (DFFs) and endonucleases. Most of the The intrinsic pathway of apoptosis is independent from a re-
nuclear changes of apoptosis observed by electron microsco- ceptor signaling, and mitochondria-associated stimuli create
py or even light microscopy make the apoptosis process to be an intracellular signaling. The inner activation of this pathway
determined easily [11, 12]. makes the cell undergo apoptosis in either a positive or nega-
As soon as the apoptosis is initiated in the cells, they lose tive manner. The positive stimuli (e.g., toxic materials, viral
the connection between the neighboring cells; membrane infections, and radiations) directly activate all the mediators
shrinks and the cell packs its cytosolic ingredients into apo- for apoptosis, whereas negative stimuli (loss of growth fac-
ptotic buddies. The apoptotic buddies will be eliminated by tors, different cytokines, or certain type of hormones) work in
phagocytotic cells that recognize phosphatidylserine, which is contrast to positive one and eliminate the factors that suppress
normally located in the inner side of the cell membrane and apoptosis in the cells and cause apoptotic activation [1, 20]. In
flips to the outer membrane during apoptosis [13]. At the same addition to different infections or cytokine-mediated intrinsic
Tumor Biol. (2016) 37:8471–8486 8473

Fig. 1 Two main apoptotic pathways; intrinsic and extrinsic pathways. In located on the membrane of the mitochondria such as Bcl-2 and Bcl-
the extrinsic pathway, interaction between the death receptors and their XL. Release cytochrome c, APAF-1 complex, and pro-caspase9 can be
ligands initiates the pathway, resulting in caspase 8 activation. This gathered in the cytosol, which is called apoptosome. The formation of this
activation can be inhibited by cFLIP. Caspase 8 can directly induce complex will result in the activation of caspase 9 followed by the
apoptosis or activates caspase 3 or Bid, which lead to apoptosis. On the transformation of pro-caspase-3 to caspase 3, which is the last step for
other hand, intrinsic pathway can be initiated by DNA damage. As a apoptosis. The cross talk between extrinsic and intrinsic pathways of
response, the cells can trigger apoptosis through mitochondrial pathway, apoptosis is regulated by Bid, a pro-apoptotic member of Bcl-2 family.
which starts with the activation of the pro-apoptotic member of the Bcl-2 The cleavage of Bid is mediated by caspase 8, which induces apoptosis by
family, Bax. Anti-apoptotic proteins inhibiting the action of Bax are releasing cytochrome c release from the mitochondria

apoptosis activation, DNA damage also majorly induces apo- nucleus will be fragmented together with the breaking of nu-
ptosis as a protection mechanism of the cells that do not let self clear membrane [23]. This stage is the initial event for extrin-
to continue proliferation with an imperfect DNA sequence. sic and intrinsic pathways of apoptosis, where caspase-3
DNA damage or any other type of apoptosis stimuli basically cleaves the different proteins such as kinases, DNA control
causes the changes in the trans-membrane potential of mito- proteins, cytoskeletal proteins, or inhibitor of endonucleases.
chondria, which result in the release of pro-apoptotic proteins DNA condensation, membrane blebbing, and all the morpho-
into the cytoplasm. logical changes are regulated by caspases as a common mech-
Cytochrome c, Smad, or high-temperature requirement anism for both intrinsic and extrinsic trigger [17].
protein A2 (HtrA2)/Omi are a group of pro-apoptotic mole- On the other hand, another group of molecules released by
cules released from mitochondria and cause the activation of mitochondria including endonuclease G or AIF are also pro-
caspase protein cascade [21, 22]. Cytochrome c interacts with apoptotic proteins but involved in the process at the later
Apaf-1, resulting in the formation of Bapoptosome^ complex, stages. These molecules are trans-located into the nucleus
which activates pro-caspase-9. After active caspase-9 acti- where they first cause an elementarily DNA fragmentation
vates caspase-3, the final cascade is become activated and and chromatin condensation which is defined as Bstage 1,^
8474 Tumor Biol. (2016) 37:8471–8486

and an advanced condensation and DNA fragmentation by the Ced-3 was reported to be essential for the cell death, which
help of caspase-3 at later stage is called as Bstage 2^ [24]. is mainly conducted by the caspase Ced-3, Ced-4 activating
All the intrinsic apoptosis events are primarily controlled by Ced-3, and Ced-9 inhibiting apoptosis [33, 34]. There are 14
Bcl-2 family of proteins and p53 tumor suppressor protein different caspases in mammals, and they are basically classi-
which is majorly involved in the activation of Bcl-2 family fied as the initiators including caspase-2, -8, -9, and -10; and
proteins. The members of Bcl-2 protein family can act as either the effectors including caspase-3, -6, -7, and -14; and also the
pro-apoptotic (Bax, Bak, Bid, Bim, Puma, Noxa, Bad, and cytokine activators including caspase-1, -4, -5, -11, -12, and -
Blk) or anti-apoptotic (Bcl-2, Bcl-XL, Bcl-X, and BAG) and 13 [35, 36]. Structurally, while initiator caspases have long N-
also determine the membrane integrity of mitochondria and are terminal pro-domain known as caspase recruitment domains
involved in the process of cytochrome c release [1, 25]. (CARDs) including more than 90 amino acids, effector
caspases have shorter sequences known as death effector do-
Extrinsic pathway main (DED) including 20–30 amino acids. Since caspases
firstly synthesized as zymogens, they are subsequently acti-
Apoptosis triggered by extrinsic pathway is primarily mediat- vated during the apoptotic process. While initiator caspases
ed by signaling through membrane-bound death receptors that are self-activated, effector caspases are activated by initiator
belong to tumor necrosis factor (TNF) gene superfamily. The caspases via internal cleavages [37]. Rather than apoptosis,
initial signal is provided by the interactions between the li- most of caspase family members are functional in cellular
gands and cell membrane death receptors such as Fas ligand/ proliferation, survival, and inflammation, whereas some of
FasR, TNF/TNF R1, Apo2L/DR4, or TNF-related apoptosis- them are essential for apoptosis [38, 39].
inducing ligand (TRAIL) R1, which is resulted in ligation of Caspase-1, the first identified caspase, is interleukin-1b
death domains of these receptors [26]. Binding of Fas ligand processing enzyme (ICE), and it is known as Ced-3 homo-
to its receptor induces the binding of adaptor protein, Fas- logue [40]. Caspase-1 is involved in cytokine activator group
associated death domain (FADD), while TNF/tumor necrosis of caspase family since inflammatory cytokines, pro-IL-1b
factor receptor (TNFR) interaction causes the binding of and pro-IL-18, are the main substrates for caspase-1 [39].
TNFR-associated death domain (TRADD), which is resulted While caspase-1 is not essential for apoptotic signaling, it is
in pro-caspase-8 activation. Pro-caspase-8 is activated auto- essential in inflammation process [41].
catalytically by the help of death-inducing signaling complex The second identified caspase, caspase-2, containing
(DISC). Active caspase-8 either induce Bid, thus intrinsic CARD, plays important roles in DNA damage-, metabolic
pathway also become involved and activated with an outside abnormality-, and ER stress-induced apoptosis [42].
signal, or caspase-3 and caspase-7 and the activation process Caspase-2 is known as a substrate for both caspase-3 and
of apoptosis is terminated with the same final pathway as caspase-8 [43, 44]. Caspase-2 activation comprises the forma-
intrinsic stimuli does [1, 19, 27, 28]. Bid is the pro-apoptotic tion of PIDDosome complex including RIP-associated ICH-1/
member of Bcl-2 family, exhibiting a common molecule be- ECD3 homologous protein with death domain (RAIDD) that
tween intrinsic and extrinsic pathways of apoptosis. Caspase- have CARD and death domain (DD) and p53-induced protein
8 causes the cleavage and myristoylation of cytoplasmic Bid with death domain (PIDD) [45]. Functional properties of
protein, leading to its movement through mitochondria. Then, caspase-2 have still not been clarified thoroughly.
apoptosome formation is induced by cytochrome release via Caspases-3, -6, and -7 are involved in the effector caspase
Bak and Bax molecules [29]. group, and they act in a similar manner in the apoptotic pro-
The extrinsic activation of apoptosis can also be inhibited cess [46]. Caspase-3 is activated via both extrinsic and intrin-
via two different ways. The one is binding of FLICE-like sic apoptotic pathways [47]. Despite there are limited infor-
inhibitory protein (cFLIP) to FADD and pro-capase-8 and mation about caspase-6 and -7 rather than caspase-3, it is
blocking their activity, and the other way is inhibition of known that while caspase-3 suppression results in the inhibi-
caspase-8 biogenesis by a protein named Toso which is firstly tion of apoptosis, suppression of caspase-6 and -7 do not sig-
described in T cells [30, 31]. nificantly affect the apoptotic process [48]. Furthermore,
In the next sessions, we will discuss all the gene families caspase-3 was reported to be crucial for PARP cleavage and
involved in intrinsic or extrinsic pathways of apoptosis with DNA fragmentation which are hallmarks of apoptosis [49].
their main player and their functions. However, some studies showed that under the conditions that
both caspase-3 and caspase-7 were knocked out, the cells
Caspase family members could undergo cell death in an alternative manner via necrosis.
Studies with caspase-3/caspase-7 double-knockout thymo-
Caspase family comprise conserved cysteine aspartic-specific cytes and mouse embryonic fibroblasts showed that thymo-
proteases, and members of caspase family are considerably cytes remain sensitive to Fas-mediated apoptosis, whereas fi-
crucial in the regulation of apoptosis [32]. In C. elegans, broblasts become resistant [49, 50].
Tumor Biol. (2016) 37:8471–8486 8475

Functionally well-known member of caspase family, cas- of aspartic acid into the caspase active region [67].
pase-8, is crucial factor for TNF-induced extrinsic apoptotic Additionally, XIAP can also inhibit caspase-9 activity via
pathway [51]. Pro-caspase-8 is recruited to DISC by FADD, binding the third BIR to the N-terminus of pro-caspase-9,
and dimerization or trimerization triggers pro-caspase-8 acti- resulting in the prevention of caspase-9 dimerization [68].
vation via reciprocal cleavage. Caspase-8, in turn, cleaves and Similarly, another mammalian IAPs, cIAP1/BIRC2, and
activates caspase-3, -7, Bid, and also NF-kB [52, 53]. cIAP2/BIRC3 have three BIR domains and a RING domain
Caspase-8 activation is regulated by cFLIP that is structurally in their structures [63]. Rather than inhibiting directly caspase
homologous to caspase-8 but does not have caspase activity activity, they inhibit apoptosis indirectly. They can bind IAP
[54]. FLIPS, the short isoform of cFLIP, controls the DISC inhibitor, second mitochondrion-derived activator of caspase/
formation in a negative manner. However, the long isoform direct inhibitor of apoptosis-binding protein with low pI
of cFLIP, FLIPL, has reciprocal effect on DISC formation and (SMAC/DIABLO), trigger NF-kB and MAPK activity, and
caspase-8 activity. While some studies reported FLIPL to be also they can trigger proteasomal degradation of caspases
inducer of DISC formation, some reports showed that it could [69].
be an inhibitor [55, 56]. Inhibitor and inducer effects of FLIPL Bruce/BIRC6, a member of IAP family, is mainly found in
on caspase-8 activity depend on FLIPL levels. At low concen- secretory organs, testis, lymphatic cells, and brain. Similar to
trations, generation of heterodimers between FLIPL and pro- survivin, Bruce located at the outer membrane of the trans-
caspase-8 or pro-caspase-10 induces their activity. However, Golgi network bears only one BIR domain. Bruce can inhibit
at higher levels of FLIPL, caspase-8 activation diminishes and caspase-3, -6, -7, -8, and -9, and also, it can trigger the
NF-kB activation increases [57, 58]. proteasomal degradation of SMAC/DIABLO. Many studies
Caspase-9, the initiator caspase, is an important factor for showed that Bruce is upregulated in ovarian and brain cancer
the generation of apoptosome complex in the mitochondrial cell lines, resulting in development of resistance against apo-
pathway. Once cytochrome c is released from the mitochon- ptotic agent [70].
dria, it binds to Apaf1 which is the receptor for cytochrome c IAP antagonists, SMAC/DIABLO, HtrA2/Omi, and
in the cytoplasm [59]. Cytochrome-c and Apaf-1 generate XIAP-associated factor 1 (XAF1) are known as potent inhib-
apoptosome, and then, pro-caspase-9 binds to Apaf-1. itors of IAPs. SMAC/DIABLO bears a mitochondrial
Afterward, pro-caspase-9 is activated via reciprocal cleavage, targeting signal (MTS) at N-teminus, and it become mature
and by this way, apoptosome complex also become activated. after the MTS cleavage [21]. Once apoptosis is triggered,
Then, caspase-3 is cleaved and activated via caspase-9 found SMAC/DIABLO is delivered into the cytosol, and
in the active apoptosome complex [60]. SMAC/DIABLO homodimers bind to IAPs via its Ala-Val-
Pro-Ile sequence in N-terminal domain [71]. Interaction of
Inhibitors of apoptosis proteins and inhibitors of apoptosis SMAC/DIABLO with XIAP via the second and third BIRs
protein antagonists results in caspase-3 and caspase-9 release [72]. It was reported
that SMAC is overexpressed in several types of solid tumors
Inhibitors of apoptosis proteins (IAPs) were firstly discovered such as colon, stomach, prostate, ovary, and lung cancers [73].
in Baculovirus as gene products. All IAPs have baculovirus Likewise, HtrA2/Omi delivery is triggered via apoptotic in-
IAP repeats (BIRs) that composed of one or more zinc finger duction and then, it binds to IAPs through its IAP-binding
motifs [61]. The first identified IAP, OpIAP, inhibits pro- motif (IBM) [74]. XAF1, the other potent IAP antagonist,
caspase cleavage and activation rather than direct inhibition binds to BIR domains of IAPs such as XIAP, cIAP1, and
of caspase activity [62]. NAIP, the first identified mammalian cIAP2 and by this way, promotes apoptosis [75]. A study
IAP, was reported to be related to the generation of immune suggesting XAF1 as a prognostic marker for colon cancer
response against bacterial infection. Hence, it is not directly showed that XAF1 is overexpressed in colon cancer cells as
correlated with caspase inhibition [63]. compared to adenoma cells which are benign [76].
Survivin/BIRC5, another identified mammalian IAP, was
firstly reported as caspase inhibitor, but now, it is known that Bcl-2 family members
survivin does not directly inhibit caspase activity. Mechanism
of action of survivin, bearing one BIR domain, is to assemble Bcl-2 family members, which play important roles in regulat-
with centromeres and p21Waf1 at the beginning of mitosis ing apoptotic signaling, are divided into three subfamilies in-
[64, 65]. cluding (i) pro-survival subfamily members (Bcl-2, BclXL,
XIAP/BIRC4, the mostly clarified mammalian IAP, has BclW, MCL1, and BFL1/A1), (ii) BH3-only subfamily mem-
three BIR domains and a RING domain; it is located on X bers (Bad, Bim, Noxa, and Puma9), and (iii) pro-apoptotic
chromosome; and also, it is quite effective in apoptosis inhi- mediator subfamily members (Bax and Bak) [77, 78].
bition via inhibiting caspase activity [66]. Mainly, caspase-3 Basically, all of the members of Bcl-2 family share typical
and caspase-7 can be inhibited by XIAP via inserting a residue characteristic functions; (i) they dimerize with other members
8476 Tumor Biol. (2016) 37:8471–8486

of Bcl-2 family, (ii) they contribute to the regulation of mito- characterized DRs of which ligands are CD95 ligand
chondrial homeostasis by binding proteins, and (iii) they con- (CD95L/FasL), TNFα, lymphotoxin-α (these two bind to
tribute to outer mitochondrial membrane pore formation [79]. TNFRI), and TRAIL (these two bind to TRAIL-R1 and
The members of BH3-only subfamily members become TRAIL-R2), respectively [94]. The genes encoding TNF su-
activated under stress conditions like growth factor depriva- perfamily receptors and ligands are the scope of this part to-
tion and DNA damage. Active BH3-only proteins, in turn, gether with their significant contribution to apoptosis
inactivate members of pro-survival subfamily via binding that specifically.
promotes the activation of the members of pro-apoptotic sub-
family members. Active pro-apoptotic subfamily members, Fas cell surface death receptor and Fas ligand
Bak and Bax, then provide cytochrome c release from the
mitochondria through permeabilizing the outer mitochondrial The proteins encoded by Fas cell surface death receptor (FAS)
membrane [80]. and Fas ligand (FASLG) genes are members of the TNF su-
In several types of human malignancies, the balance be- perfamily. Fas also known as CD95/APO-1/DR2 is one of the
tween the expression levels of Bcl-2 family genes is broken best studied DRs with molecular weight of 48 kDa. Fas gene
down, and the equilibrium changes to the pro-survival sub- occupies about 25 kb on human chromosome 10 with nine
family member direction. In this case, cancer cells can escape exons in which exon 6 codes for trans-membrane domain
from apoptotic signals and therefore develop resistance [95]. This receptor containing a DD plays a crucial role in
against therapeutic agents [81, 82]. Additionally, Bcl-2 family the regulation of programmed cell death and is involved in
members are also considered in cancer therapy due to their the pathogenesis of various malignancies such as cancer. On
therapeutic potentials [83]. In the clinical trials, the BH3-only the other hand, FasLG gene is located on human chromosome
mimetic agents targeting Bcl-2 are being investigated in order 1, which encodes a type II trans-membrane protein called
to find alternative potent therapeutic approaches in several FasL (CD95L) present at the surface of activated immune
types of malignancies [84–86]. cells such as T cells and natural killer cells [93]. Therefore,
Fas/FasL interaction results in the elimination of infected and
TNF gene superfamily transformed cells, which is generally used by immune cells to
avoid cancer development.
One of the most important ways of triggering apoptosis is The interaction of Fas with FasL results in changes in the
mediated through death receptors (DRs), which are classified conformation and aggregation of receptor on the plasma mem-
in TNF superfamily including also ligands such as TNF, brane and triggers an initial signaling event through protein-
TRAIL, and Fas ligand (FasL) [87]. The induction of apopto- protein interactions. The main structural changes take place in
sis by these ligands is initiated by binding to their specific DD of receptor, which recruits FADD through its DD. Then,
membrane receptors [88, 89]. TNF superfamily is known to FADD interacts with pro-caspase-8 and -10. After activation
comprise 19 ligands and 29 receptors that function in highly of these caspases via auto-cleavage, they are released in the
different processes in the body including inflammation, apo- cytosol as active caspases resulting in the apoptotic cell death.
ptosis, proliferation, and invasion [90]. Even though all mem- The complex CD95/FADD/caspase-8/-10 is called DISC
bers of TNFR superfamily are generally trimeric type I trans- stands for Bdeath-inducing signaling complex^ [96].
membrane proteins and possess cysteine-rich extracellular In the literature, there are various kinds of studies identify-
subdomains, they are actually different in their primary struc- ing the roles of Fas/FasL in order to induce apoptosis in sev-
ture, which make them unique to recognize their ligand in a eral malignancies especially in cancer. Escaping from apopto-
specific and exclusive manner [91]. These DRs also contain a tic stimuli is a very well known feature of cancer cells which
homologous cytoplasmic cysteine-rich BDD^ which is respon- might develop multiple strategies to inhibit apoptotis mediated
sible for transmission of apoptotic signals from cell surface to by CD95. In a majority of cancer types from different origins,
intracellular signaling pathways [92]. Because, adapter mole- somatic mutations in CD95 gene were found to be a common
cules such as FADD and TRADD have these death domains way to trigger the development of resistance toward apoptosis.
as well to interact with DRs. The ligands included in TNF For instance, 5′ region of CD95 gene was analyzed in terms of
superfamily share a common extracellular TNF homology somatic mutations in nodal diffuse large B cell lymphoma and
domain (THD), which is involved in the formation of defined mutations were shown to be included in progression
homotrimers via non-covalent bonding [93]. The extracellular of diseases due to inhibition of apoptosis [97]. Another com-
domain of most TNF ligands undergoes proteolytic cleavage mon way to become resistant to CD95-induced apoptosis is
to make a soluble ligand, even though they are synthesized as the regulation of surface expression of receptor [98]. In a study
type II trans-membrane proteins [93]. performed by Ivanow et al. (2006) [98], the treatment of mel-
CD95 (DR2/Fas/APO-1), TNF receptor 1 (DR1/TNFR1), anoma cells having increased surface expression of Fas recep-
TRAIL-R1 (DR4), and TRAIL-R2 (DR5) are the best tor with soluble FasL resulted in the induction of apoptosis.
Tumor Biol. (2016) 37:8471–8486 8477

CD95-mediated apoptosis can also be blocked by decreasing higher OPG expression and resistance of ovarian cancer cells
expression of FADD or caspase-8 [99, 100]. to TRAIL-induced apoptosis.
In a recent study, the effect of a toxic steroid on human Similar to apoptosis induction by Fas/FasL, binding of
bladder cancer cells was found to be related to increased ex- TRAIL to its DRs, DR4 and DR5 recruits the adaptor protein
pressions of Fas and FasL in vitro and in vivo at both messen- FADD and inactive versions of caspase-8 and -10 to form the
ger RNA (mRNA) and protein levels [101]. Zhong et al. DISC complex in which caspase-8 and -10 become activated
(2015) [102] showed that the mechanism of resistance to by auto-proteolytic cleavage and released in the cytosol to
Fas-mediated apoptosis in human hepatocellular carcinoma activate effector caspases [111].
cells (HCCs) and overexpression of oxysterol-binding pro- The size of human TRAIL gene is ~20 kb, containing five
tein-related protein 8 (ORP8) which is normally downregulat- exons and four introns. The first exon encodes for the trans-
ed in HCC was found to induce apoptosis by upregulating membrane domain and the cytoplasmic domain, whereas
FasL. exons 4 and 5 code for the extracellular domain responsible
for the interaction of TRAIL with its receptors. Exon 5 also
encodes for the C-terminal amino acids along with containing
TRAIL the 3′-untranslated region (3′-UTR) and poly-A tail [112].
There are various variants of TRAIL; however, only one spe-
TRAIL (also called as APO2 ligand) is a type II trans- cific isoform of TRAIL (TRAILα) is responsible for its
membrane protein and processed proteolytically at the cell cancer-selective apoptosis induction potential [113].
surface to form a soluble ligand. TRAIL is grouped into the TRAIL gene is subjected to strict regulation due to its crit-
TNF cytokine family, which was discovered based on its ex- ical role in the induction apoptosis. Altered expression of
tracellular domain sequence homology with CD95L and TNF TRAIL gene has been found in various kind of disease.
[103]. TRAIL-mediated apoptosis takes place after its binding Multiple studies have been performed to analyze single-
to its DD containing receptors, TRAIL receptor 1 (death re- nucleotide polymorphisms (SNPs) in various patient popula-
ceptor 4, DR4), and TRAIL receptor 2 (death receptor 5, DR5) tions. In a recent study, peripheral blood samples were ana-
[104]. There are also three other TRAIL receptors, which do lyzed in terms of SNPs in TRAIL promoter and substitution of
not possess apoptotic ability and function as decoys. Decoy C to T at position -723 was found to be significantly associated
receptors 1 (DcR1) and 2 (DcR2) are expressed on the cell with sporadic breast cancer and decreased TRAIL mRNA
surface similar to DR4 and DR5. Even though their extracel- levels due to transcriptional repression [114]. Bos et al.
lular and ligand-binding domains show significant homology (2009) found that TRAIL mRNA expression decreased
to DR4 and DR5 and are fully functional, they lack of func- in breast cancer patients with brain metastasis [115].
tional intracellular DD [105]. Therefore, increased expression TRAIL has been considered as a potential therapeutic tar-
of either DcR1 or DcR2 provides resistance against TRAIL- get due to its selective apoptosis inducing action in cancer
induced apoptosis [104]. In a recent study, it was shown that cells as compared normal cells [116]. There are several ongo-
coexpression of DcR1 and DcR2 with DR4/DR5 on the same ing and completed clinical phase studies targeting TRAIL
cell can block apoptosis. However, TRAIL was engineered in apoptotic pathway by using agents including monoclonal an-
order to escape from binding to DcRs, which were found to tibodies and recombinant human proteins, and these
still exert trans-cellular regulation originating from stromal studies are giving promising results with no significant toxic
cells and affect tumor cells. Therefore, it is important to target effects [117].
these decoy receptors selectively to gain maximum efficacy
[106]. Another recent study showed that DR4 and DR5 were
upregulated while DcR1 and DcR2 downregulated in colon TNF and TNF receptor 1–2 (TNFR1 AND TNFR2)
cancer cells after their treatment with a non-steroidal anti-in-
flammatory drug. Therefore, colon cancer cells became sensi- TNF is expressed as a trans-membrane 26-kDa protein and
tive to TRAIL-induced apoptosis [107]. The fifth TRAIL re- then undergoes proteolytic cleavage to form 17-kDa trimeric
ceptor is osteoprotegerin (OPG), which is a secreted receptor soluble cytokine. The resulting TNF functions by binding to
for TRAIL with low affinity. OPG has sequence homology to two different receptors, TNRF1 and TNFR2, which are also
TNF receptor superfamily, however but lacks of a trans- trimeric trans-membrane proteins [90]. TNF is produced by
membrane domain. OPG can bind to TRAIL and prevents immune cells including activated natural killer cells, T cells,
its interaction with death receptors, thus preventing Jurkat and activated monocytes/macrophages and a wide range of
cells from undergoing TRAIL-mediated apoptosis [108]. non-immune cells such as fibroblasts [118]. TNF/TNFR sig-
Therefore, OPG has an anti-apoptotic effect by preventing naling is well known to be involved in various cellular func-
interaction between TRAIL and the death receptors [109]. tions such as apoptosis, cell proliferation, and differentiation
Lane et al. (2012) [110] showed the relationship between [119].
8478 Tumor Biol. (2016) 37:8471–8486

TNF-mediated apoptosis is generally carried out by and encodes a type II trans-membrane protein. Similar to other
TNFR1 (DR1), since only TNFR1 is known to contain DD TNFR family members, DR3 has a DD in its cytoplasmic part
[87]. Binding of TNF to TNFR1 triggers the recruitment of and initiates apoptotic signaling [87].
TRADD protein through its DD. Then, TRADD interacts with Most of the studies have been related to DR3 involvement
FADD resulting in the recruitment of pro-caspase-8, which is in immune system modulation due to its frequent expression
proteolytically cleaved to active caspase-8. Caspase-8 then on lymphoid tissues such as the spleen, thymus, and periph-
activates caspase-3 responsible for apoptotic cell death eral blood lymphocytes [131]. The role of DR3 in the devel-
[120]. The activity of caspase-8 is strictly regulated by a neg- opment of some human malignancies has also been enlight-
ative inhibitor protein cFLIP that contains DED instead of ened. DR3 expression was shown to be increased in human
DD. cFLIP interacts with pro-caspase-8 to prevent its contin- colon cancer cells treated with cordycepin, a deoxy form of
uous recruitment to the TNFR1 DISC [121]. TNF-induced adenosine, resulting in apoptosis induction [132]. A specific
cell death occurs only under stress conditions such as altered phenolic compound triggered apoptosis in human non-small-
cell metabolism, inhibition of cell cycle progression, and pro- cell lung cancer (NSCLC) cells by increasing the expression
tein synthesis [120]. Therefore, it only induces apoptosis in of DR3 [133]. Silencing of DR3 via siRNA approach reversed
transformed cells (cancer cells), virus-infected cells, or its growth inhibitory effect.
stressed cells, not in normal healthy cells. Interestingly,
TNFR2 has been shown to function in cell proliferation unlike p53
TNFR1 [122].
In a recent study, IL32-α, a novel cytokine, was found to p53 is encoded by human TP53 gene localized on the short
inhibit colon cancer cell growth in an experimentally generat- arm of chromosome 17 with a molecular mass of 43.7 kDa
ed colon cancer model by increasing TNFR1-induced cell [134]. It occupies 19.200 bp including 11 exons. There are
death signaling, which was evidenced by increased ex- various p53 isoforms based on alternative splicing of TP53
pression of TNFR1. Yu et al. [123, 124] found a new way of gene; some of which play opposite roles as compared to p53,
apoptosis induction in adenosine-treated colon cancer cells, while others have similar functions like full-length p53 [135].
which included increased expression of TNFR1. TNFα secre- Human p53 protein is composed of the following three
tion increased in acute myeloid leukemia cells after treatment different domains with important functions: the DNA-
with SMAC mimetic and IFNα combination [125]. Inhibition binding domain, the N-terminal trans-activational domain,
of TNFα and TNFR1 by a pharmacological inhibitor and and the C-terminal oligomerization domain [136]. DNA-
genetic silencing, respectively, reduced SMAC mimetic/ binding domain binds to response elements of target genes,
IFNα-triggered apoptosis. Tao et al. suggested that survivin, whereas the N-terminal trans-activation domain forms binding
an inhibitor of apoptosis, inhibitor induced programmed cell sites for several negative or positive regulators. The C-
death in Wilms tumor cells by increasing expression of terminal domain undergoes alternative splicing and posttrans-
TNFR1 signaling [126]. lational modifications [137].
As well as TNFα, lymphotoxin α (LTα) which is secreted p53 acts as tetrameric transcription factor, which controls
as a homotrimer binds to TNRF1 [127]. Even though both the expression of a large set of genes involved in significant
TNFα and LTα bind signal via TNFR1, LTα was found to cellular processes including DNA damage detection, cell cy-
possess less ability to induce TNRF-mediated cell death in an cle arrest, apoptosis, DNA repair, and senescence [134]. It is
early study [128]. However, a recent research by Etemadi et al. both involved in intrinsic and extrinsic pathways of apoptosis
displayed that LTα has same potential to induce apoptosis via by inducing transcription of several proteins like PUMA, Bid,
TNFR1 signaling [129]. Bax, TRAILR2, and CD95, which is called transcription-
Genetic changes in the promoter region of TNFα have dependent apoptotic pathway of p53 taking place in the nu-
been well studied in several human diseases to find correlation cleus [138]. In this pathway of p53-dependent apoptosis, the
between apoptosis induction and TNFα expression. De trans-activation domain of p53 interacts with the players of
Oliveria et al. studied the effect of a polymorphism, TNF-α- basal transcription machinery such as the transcriptional coac-
857 C/T, on gastric cancer patients, found that this mutation tivator p300/CBP [139]. In normal healthy cells, p53 levels
decreased mRNA level of TNFα resulting in resistance to are very low due to its rapid turnover; however, if a damage is
apoptosis [130]. detected in the cells, p53 becomes stabilized resulting in-
creased p53 level [140]. p53 stability is controlled by mouse
Death receptor 3 double minute 2 (MDM2) gene, which is an E3 ubiquitin
ligase that negatively regulates p53 stability through
Death receptor 3, also known as APO-3, has four characteris- ubiquitination, thus proteosomal degradation. MDM2 also in-
tic cysteine-rich motifs with molecular weight of 53.5 kDa. hibits the interaction between the p53 trans-activation domain
Death receptor 3 (DR3) gene is localized on chromosome 1 and the components of transcription machinery [141]. MDM2
Tumor Biol. (2016) 37:8471–8486 8479

has been found to be overexpressed in many cancer cells, patients, missense mutations in DNA binding motif were cor-
which leads to neutralization of interaction between p53 and related with poor survival [154, 155].
transcription machinery components, thus impairing p53 has been considered as a significant player of apoptosis
transcription-dependent apoptosis [142, 143]. in many studies, and there is a growing accumulation of arti-
p53 has the ability to activate intrinsic pathway of apopto- cles revealing its role in many malignancies. In a recent study
sis by inducing the transcription of especially apoptotic Bcl-2 by Wang et al., acute pro-myelocytic leukemia cells were sub-
family genes such as PUMA [144]. p53 induces PUMA jected to apoptosis when treated with combination of
mRNA expression immediately in response to DNA damage tetraarsenictetrasulfide and arsenic trioxide through upregula-
by binding the two p53-responsive elements in the PUMA tion of p53 and its target gene Bax. Transcription-independent
promoter. p53 binding results in the acetylation of core his- role of p53 in apoptosis induction was identified in a study in
tones, H3 and H4, which is responsible for chromosome which p53 trans-located to mitochondria and induced mito-
decondensation and transcriptional activation [145]. chondrial membrane depolarization in HUVEC cells exposed
Similarly, TRAIL-R2 expression is induced by p53, which to heat stress [156, 157].
binds to a p53-responsive element in TRAIL-R2 promoter in There is an increasing attention to develop different strate-
order to induce extrinsic pathway of apoptosis [140]. As an gies that can modulate p53-dependent apoptotic pathways
alternative mechanism, p53 functions a transcriptional repres- such as inhibition of p53-MDM2 interaction using MDM2
sor of certain anti-apoptotic genes including survivin which inhibitors, restoring mutated p53 back to its wild-type form
promotes caspase activation [146]. p53 is also directly in- and p53 vaccines [158–160].
volved in apoptosome formation by activating transcription
of Apaf-1 gene including a p53 response element in its pro- MicroRNAs in extrinsic and intrinsic apoptotic pathways
moter [54]. In response to DNA damage, p53 is also displayed
to activate caspase-6 cleaving nuclear envelope protein lamin Changes in the apoptotic response in cancer can result in tu-
A and various transcription factors through a response element mor initiation, progression, and treatment resistance [3]. There
within the third intron of the gene [147]. are numerous studies including the roles of microRNAs in the
As well as transcription-dependent functions of p53, its control of apoptosis, and these microRNAs display their ef-
transcription-independent functions in terms of apoptosis have fects by directly targeting genes involved in both extrinsic and
been defined. p53 induces apoptosis by acting directly at mi- intrinsic pathways of apoptosis [161]. These microRNAs
tochondria. p53 trans-locates to the mitochondria in response (miRNAs) can be classified as oncogenic and tumor suppres-
to apoptotic signal, where it forms inhibitory complexes with sive miRNAs [162]. One of these miRNAs is miR-130a that
Bcl-XL and Bcl-2 causing the permeabilization of the mito- was found to reduce drug resistance in non-small-cell lung
chondrial membrane and cytochrome c release [148]. The cancer by targeting MET proto-oncogene and to sensitize this
interaction between p53 and Bcl-XL and Bcl-2 is mediated cancer cells to TRAIL-induced apoptosis by inhibiting miR-
by p53 trans-activation domain like p300/CBP binding, even 221 and miR-222, which are upregulated by MET and in-
though these two modes of p53-dependent apoptosis induc- volved in TRAIL resistance [163]. Therefore, miR-221 and
tion occur in different cellular compartments [149]. Moreover, miR-222 are oncogenic miRNAs that induce drug resistance
cytosolic p53 might induce the activation of pro-apoptotic and block apoptosis in several cancer types such as gliomas
Bax via direct protein-protein interactions [150]. Leu et al. [164]. Another oncogenic miRNA exerting its anti-apoptotic
displayed that p53 interacts with pro-apoptotic mitochondrial effects by inhibiting FasL directly is miR-21, which is found
membrane protein Bak, which makes Bak undergo oligomer- to be upregulated in advanced pancreatic cancer patients
ization and releases cytochrome c from mitochondria. Binding [165]. miR-24 regulates apoptosis by binding to coding se-
of p53 to Bak damages interaction between Bak and anti- quence of Fas-associated factor 1 (FAF1) mRNA and induced
apoptotic Mcl-1 [151]. apoptosis of several different types of cancer [166]. miR-21
Mutations in p53 gene have been considered most common also negatively regulates PTEN, a tumor suppressor gene,
genetic changes in cancer. Mutant p53 proteins can both lose involved in the apoptotic pathway through the formation of
their native tumor suppressor activity and provide active tu- DISC complex in many tumors such as breast and gastric
mor development [152]. Missense mutations form the major- cancers [167, 168]. In gastric cells, miR-21 upregulated and
ity of alterations in p53 gene, which commonly occur in the decreased PTEN expression, resulting in significant suppres-
DNA binding domain of p53, thus preventing p53 from pro- sion of trastuzumab-induced apoptosis [168]. miR-200c sen-
moting target gene expression [153]. Recently, Saleem et al. sitized cells to apoptosis by directly targeting Fas-associated
analyzed loss of function mutations in p53 gene in the patients phosphatase-1 (FAP-1), which is an inhibitor of Fas-induced
of oral squamous cell carcinoma and found that AGT to ACT apoptosis [169]. miRNA-886-5p inhibited apoptosis of hu-
missense mutation in DNA binding domain may result in man cervical cancer cells by downregulating Bax [170].
impaired p53 function. In chronic lymphocytic leukemia Zhou et al. (2010) [171] found that miR-125b was upregulated
8480 Tumor Biol. (2016) 37:8471–8486

in taxol-resistant breast cancer cells and suppressed apoptosis. NSCLC by the downregulation of miR-186, whose direct tar-
Bak1, pro-apoptotic Bcl-2 antagonist killer 1, was identified get is caspase-10 [185]. Based on all this evidence discussed
as the direct target of miR-125b. miR-25 and miR-32 could in this particular review, miRNAs play key regulatory roles in
directly target the pro-apoptotic function of Bim, therefore apoptosis and could be important therapeutic targets in cancer
suppressing apoptosis in ovarian and human myeloid leuke- (Fig. 2).
mia cells, respectively [172, 173]. In addition, miR-483-3p
might target PUMA, whose enforced expression protects cells Bioactive sphingolipids in apoptotic pathways
from apoptosis [174]. miR-34 decreases the expression of Bcl-
2, resulting in an increase in apoptosis [175]. In this study, Bioactive sphingolipids are a family of membrane lipids that
downregulation of miR-34 induced cell proliferation and in- have many regulatory roles in several cellular events such as
vasion in malignant mesothelioma. miR-15b/16 could down- cell proliferation, senescence, adhesion, migration, and also
regulate Bcl-2, thereby triggering apoptosis [176]. miR-153 apoptosis [186]. Ceramide, the central molecule of bioactive
induced apoptosis of glioblastoma cells by targeting 3′-UTR sphingolipid metabolism, was reported as a regulator of apo-
of Bcl-2 and Mcl-1 [177]. miR-491 could induce apoptosis in ptosis in many studies. Treatment of cancer cells with radia-
colorectal cancer cells by downregulating anti-apoptotic Bcl- tion and chemotherapeutics such as vincristine, daunorubicin,
XL [178]. There are several studies showing the roles of gemcitabine, and etoposide result in ceramide accumulation in
miRNAs in the regulation of caspase expression. miR-23a the cells as a secondary effect of these therapeutics [187].
and miR-24a blocked mitochondrial apoptosis by inhibiting Decreased ceramide levels result in the development of drug
the expression of caspase-9 [179, 180]. The downregulation of resistance in cancer cells [188]. In vitro studies indicated that
miR-23a increased the 5-FU-induced apoptosis in colon can- treatment of cancer cells with ceramide triggers the release of
cer cells [179]. Wu et al. (2014) [181] displayed that miR-421 cytochrome-c from the mitochondria [189]. Additionally, it
upregulated in human gastric cell lines and tissues. miR-421 was reported that anti-apoptotic Bcl-2 blocks ceramide chan-
downregulated the expression of caspase-3 and blocked the nels in an independent manner from Bak and Bax [190].
apoptosis of cancer cells. miR-106b-25 was shown to be up- Furthermore, ceramide also induces Bax-mediated apoptosis
regulated in human prostate cancer and functions by partly in several types of cancer including, breast, prostate, and colon
inhibiting of caspase-7 expression [182]. Floyd et al. (2014) cancers [191]. Ceramide was reported that it activates cathep-
[183] demonstrated that miR-582-5p and miR-363 inhibit ap- sin D, an inducer of apoptosis in a form of lysosomal aspartyl
optosis by directly targeting caspase-3 and caspase-9 in glio- protease, in response to gemcitabine treatment [192]. Unlike
blastoma. Caspase-3 has been shown to be a target of miR let- ceramide, the other member of sphingolipid family, sphingo-
7a in human squamous carcinoma cells and hepatocellular sine 1-P (S1P) is related to cell proliferation, survival, and also
carcinoma cells [184]. Curcumin induced apoptosis of inhibition of apoptosis [193]. S1P was reported to cause

Fig. 2 miRNAs involved in the

apoptotic pathways. Some of the
miRNAs can inhibit apoptosis by
targeting the death-receptor
pathway including miR-21,
miR-24, and miR-200c. In the
mitochondrial pathway, various
miRNAs could target Bcl-2
family proteins and caspases
including miRNA-886-5p,
miR-125b, miR-25, miR-32,
miR-483-3p, miR-34, miR-15b/
16, miR-153, miR-491, miR-23a
and miR-24a, miR-421,
miR-106b-25, miR-582-5p and
miR-363, miR let-7a, and
miR-186
Tumor Biol. (2016) 37:8471–8486 8481

angiogenesis by VEGF signaling, which then triggers RAS resistant and imatinib-sensitive K562 CML cell lines [209].
and MAPK signaling in cancer cells. By this way, S1P causes Additionally, when we treated K562 human CML cell lines
cytoskeleton reconstruction and apoptosis inhibition [194]. and HL60 human acute pro-myelocytic leukemia cell lines
While increased ceramide levels cause induction of apoptosis, with resveratrol in combination with ceramide analog,
increased S1P levels cause inhibition of apoptosis [195]. S1P GCS inhibitor, or SK1 inhibitor, apoptotic effects of res-
leads to drug resistance due to its anti-apoptotic function. veratrol increased synergistically in the cells treated with
Sphingosine kinase-1 (SK1), which catalyzes the conversion resveratrol and ceramide combination, while the cells treat-
of apoptotic ceramide to anti-apoptotic S1P, was also reported ed with resveratrol and GCS or SK1 combinations resisted
to decrease apoptotic effects of chemotherapeutic agents in to apoptosis as compared to the control group [210, 211].
prostate cancer [196]. Like S1P, glucosylceramide (GC), the Furthermore, our studies also showed that GCS and SK1
other important member of sphingolipid family, is also known inhibition in combination with nilotinib or dasatinib treat-
as a powerful anti-apoptotic molecule [193]. ment synergistically triggers apoptosis in K562 and Meg01
Glucosylceramide synthase (GCS), the enzyme catalyzing human CML cell lines [212, 213].
the conversion of apoptotic ceramide to anti-apoptotic GC, Briefly, while ceramide is a powerful apoptotic molecule,
was found to be increased in drug-resistant cancer cells glucosylceramide and S1P generated from ceramide by GCS
[193]. Treatment of pancreatic cancer cells with a ceramide and SK1 activities, respectively, are powerful anti-apoptotic
analogue causes the accumulation of ceramide in the mito- molecules, and therefore, alterations in intracellular levels of
chondria, and by this way, ceramide reduces drug resistance these sphingolipids could be a novel approach for cancer
and triggers apoptosis [197]. Additionally, treatment of pan- therapy.
creatic cancer cells with ceramide in combination with a GCS
inhibitor, PDMP, decreases tumor growth in vivo [198]. Summary and perspectives
Moreover, GC degradation increases ceramide generation
resulting in the trigger of apoptosis and also reduced tumor Apoptosis is highly regulated way of cell death, which is
growth in melanoma xenografts [199]. Reduced tumor growth crucial for all higher-level organisms to balance tissues
was also reported in mouse models with breast cancer treated homeostasis and control cell proliferation as well as re-
with C6:ceramide [200]. Furthermore, a type of sphingosine move damaged or unnecessary cells. Apoptosis has its
kinase inhibitor, SK2, was reported to trigger apoptosis, inhib- own morphological and biochemical properties where
it cell proliferation, and decrease tumor size in mouse models caspases play a central role at the end. Here, we have fo-
bearing hepatoma, mammary adenocarcinoma, and kidney cused on aspects of apoptosis in terms of critical genes and
carcinoma [201, 202]. Safingol, the first agent used in the their products together with their role in both intrinsic and
clinic due to its ability in sphingosine kinase inhibition, in- extrinsic pathways of apoptosis. In this specific vital pro-
creases apoptotic effects of chemotherapeutics used in cancer cess, diverse groups of molecules function compatibly for
therapy [203]. An FDA-approved drug that has inhibitory ef- strict regulation. Any alterations or abnormalities occur-
fects on SK1 and SK2 activities, FTY720, inhibits cell prolif- ring in apoptotic processes contribute to development of
eration and leads to apoptosis in mice bearing breast cancer or human diseases and malignancies especially cancer. Deep
melanoma [204]. In chronic myeloid leukemia (CML) cell understanding of apoptotic signaling mechanisms, individ-
lines, increase in serine palmitoyltransferase levels via BCR/ ual players, and genes involved in apoptosis have provided
ABL inhibition result in activation of apoptotic signals [205]. a great opportunity to develop novel agents that make ap-
GCS inhibition in CML cell lines bearing T315I mutation optosis deficient cells sensitive to apoptosis.
results in apoptosis via activating GSK-3 [206]. Another study
reported that ceramide triggers apoptosis via activating p38, Compliance with ethical standards
caspase-8, and c-Jun N-terminal kinase (JNK) in K562 CML
cells [207]. Many studies from our laboratory also showed Conflict of interest None
effects of bioactive sphingolipids on apoptosis. In K562
CML cell lines resistant to nilotinib, apoptotic ceramide
synthase-1 and Bax genes were found to be downregulated
while anti-apoptotic SK1 and GCS genes were overexpressed,
and inhibition of these overexpressed genes sensitized the References
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