QU Science Disease Message and Therapeutic Target Potentials Worksheet

Bioscience Reports (2019) 39 BSR20180992
https://doi.org/10.1042/BSR20180992
Review Article
Apoptosis and apoptotic body: disease message and
therapeutic target potentials
Xuebo Xu1,* , Yueyang Lai1,* and Zi-Chun Hua1,2,3
1 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China; 2 Changzhou High-Tech Research Institute of Nanjing
University and Jiangsu TargetPharma Laboratories Inc., Changzhou 213164, China; 3 Shenzhen Research Institute of Nanjing University, Shenzhen 518057, China
Correspondence: Zi-Chun Hua (zchua@nju.edu.cn)
Apoptosis is widely known as programmed cell death eliciting no inflammatory responses.
The intricacy of apoptosis has been a focus of an array of researches, accumulating a wealth
of knowledge which led to not only a better understanding of the fundamental process,
but also potent therapies of diseases. The classic intrinsic and extrinsic signaling pathways of apoptosis, along with regulatory factors have been well delineated. Drugs and
therapeutic measures designed based on current understanding of apoptosis have long
been employed. Small-molecule apoptosis inducers have been clinically used for eliminating morbid cells and therefore treating diseases, such as cancer. Biologics with improved
apoptotic efficacy and selectivity, such as recombinant proteins and antibodies, are being
extensively researched and some have been approved by the FDA. Apoptosis also produces membrane-bound vesicles derived from disassembly of apoptotic cells, now known
as apoptotic bodies (ApoBDs). These little sealed sacs containing information as well as
substances from dying cells were previously regarded as garbage bags until they were discovered to be capable of delivering useful materials to healthy recipient cells (e.g., autoantigens). In this review, current understandings and knowledge of apoptosis were summarized
and discussed with a focus on apoptosis-related therapeutic applications and ApoBDs.
Introduction
* These authors contributed
equally to this work.
Received: 21 June 2018
Revised: 30 November 2018
Accepted: 07 December 2018
Accepted Manuscript Online:
07 December 2018
Version of Record published:
18 January 2019
Apoptosis is a highly regulated process of cell death. Unlike necrosis which is a traumatic version of cell
death, apoptosis is a rational and active decision made to sacrifice specific cells for the greater benefits
of the organism. It is a normal physiologic process routinely carried out in multicellular organisms [1].
While many enigmas still remain in relevant research areas, it has been well-documented today that apoptosis confers advantages to multicellular organisms in a coordinated manner whereby organisms maintain
homeostasis and fine-tune life cycle [2]. Its importance, both to multicellular organisms and to researchers,
can be inferred from various biological responses and changes pertaining to apoptosis, e.g., embryonic development, cell renewal and turnover, and externally induced cell death (chemicals, radiations, etc.). One
widely exploited function of apoptosis in the biomedical field is obliteration of cancer cells in response
to extrinsically applied apoptosis-inducing stimuli such as small-molecule drugs. With apoptosis in the
research spotlight, the profound therapeutic potential of apoptosis has enabled researchers to develop
promising therapeutic solutions focusing on voluntary death of aberrant cells. A spectrum of drugs and
therapies exploiting apoptosis has been proven effective against diseases. Funds and research efforts are
being extensively invested in apoptosis-based research and clinical trials.
One notable feature of apoptosis is it exerts its effects mainly through action of a type of serine proteases
known as caspases. A death signals are relayed through signaling pathways which ultimately lead to activation of caspases responsible for the execution of cell destruction [3]. Both external and internal stimuli,
coupled to extrinsic and intrinsic apoptosis pathways, can initiate apoptosis. Despite many dissimilarities
in different pathways, they converge to form apoptotic bodies (ApoBDs) which are eventually engulfed
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by phagocytes in a process known as efferocytosis [4]. Traditional views regard efferocytosis as the end point of
apoptosis and therefore it biochemically concludes the life of morbid cells, whereas accumulating evidence converge
to implicate on transfer, recycling and even reuse of materials packaged in ApoBDs [5]. A substantial part of current interest in apoptosis-related researches lies in apoptotic body formation. Apoptotic cells can also be effectively
phagocytosed as a whole. In this review, current knowledge about apoptosis is discussed with a focus on therapeutic
applications and ApoBD.
Apoptosis and its involvements in vivo
Many forms of cell death exist, most of which can be triggered by various stimuli and physiological conditions. Among
them, apoptosis and necrosis have been a well-known duo frequently being compared. In 1965, John Foxton Ross Kerr
at University of Queensland made the first distinction between apoptosis and necrosis. Further refinements aiming
to better interpret apoptosis followed. But it was not until 1972 when the term ‘apoptosis’ was first proposed by Kerr
et al. [6]. Apoptosis has attracted much interest due to its intricate nature and diverse roles in maintaining a healthy
and self-sustainable biological entity. Necrosis, on the other hand, is a form of cell injury in response to acute external
damages and trauma, resulting in passive cell death which elicits inflammatory response [7]. Comparisons between
apoptosis and necrosis reflect the unique features of each other and help researchers to elucidate their respective
biological significance.
In cell cycle, apoptosis acts as a fail-safe measure that ensures fidelity and quality of proliferation. While certain
degrees of genetic variation are allowed and favored for evolution, reproduced cells with extensive genetic errors and
cellular damages are subjected to apoptosis. A key player in the cell cycle machinery, p53, initiates apoptosis in certain
cell types. p53 is an extensively studied tumor suppressor. Overwhelming evidence points to its exceeding importance
in prevention of cancer development. The p53 tumor suppressor gene is most frequently mutated (mutated in over
50% of all human cancers) in cancer cells [8], rendering the restrictive mechanism ineffective. Tumorigenesis is likely
to commence when the p53-based preventive system malfunctions or loses its function completely.
Stimuli such as DNA damage, hypoxia, and expression of certain oncoproteins (e.g., Myc, Ras) activates the
p53-dependent apoptotic pathway [4]. Once committed, p53 paves way for apoptosis by activating pro-apoptotic
factors (e.g., Bax) while suppressing antiapoptotic factors (e.g., Bcl-2) [9]. As a well-known tumor suppressor, p53
has been recognized for its critical function to initiate apoptosis in cell cycle, along with the ability to induce cell
arrest and DNA repair in recoverable cells. There are many other cell cycle regulators besides p53 that can influence
apoptosis (e.g., pRb, p21). Nevertheless the mechanistic details of apoptosis in cell cycle are beyond the scope of this
review.
Many researches highlighted the importance of apoptosis in the self-defense mechanism, or in other words, the
immune system. The immune system is in charge of defensing the host against an array of external pathogens. Apoptosis is an integral part of the immune system where it facilitates to maintain a homeostasis of the immune system.
For example, apoptosis is burdened with the responsibility to regulate immune responses, i.e., to induce death of T
and B cells at certain time point to limit an immune response because a prolonged response would otherwise be
deleterious to self. Second, the immune system depends upon apoptosis to eliminate unneeded T and B cells to be
functionally mature [10]. For example, immune cells targeting self-antigens must be killed by apoptosis to prevent an
attack on self. Or B cells that fail to generate antibodies of higher affinity for antigens are subjected to apoptosis as
well. Lastly, cytotoxicity of certain types of cells (i.e., cytotoxic T lymphocyte and natural killer cells) is conferred by
apoptosis. The well-coordinated killing protocol allows these cells to destroy target cells with themselves remaining
intact. Cytotoxic T lymphocyte (CTL) can induce death in target cells through two pathways, one of which involves
perforin and granzymes. Perforin and granzymes are contained within the granules excytosed from the CTLs in a
directed manner. T cell receptors on CTL help to recognize a target cell (e.g., a cell infected by virus) and unload the
granules on the surface of the target cell. Perforin, which is a protein capable of forming pores on the surface of cells,
is released in a degranulation process and aid the entry of granzyme into the cell by punching holes on the cell surface
[11]. Granzyme, which is also a serine protease, is key to DNA degradation associated with apoptosis in the target
cells [12]. Distinct from the extrinsic and intrinsic pathways of apoptosis, perforin–granzyme-mediated apoptosis is
exclusively employed in cytotoxic killing mediated by T cells.
Apoptosis plays an indispensable and irreplaceable role both under physiological and pathological conditions.
Anomalies in apoptosis have become a major field of interest to researchers and are associated with a broad spectrum
of pathological conditions, e.g., developmental defects, autoimmune diseases, cancer, etc. Some diseases pertain to
deficiency of apoptosis while others pertain to its redundancy. For example, one of the hallmarks of cancer is evasion of apoptosis, meaning insufficient apoptosis overwhelmed by the limitless replicative potential of cells [13]. On
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the other hand, too much apoptosis is linked to certain pathological conditions such as acquired immune deficiency
syndrome (AIDS). AIDS is a type of autoimmune disease caused by human immunodeficiency virus (HIV) infection
[14]. HIV infects it host through binding to CD4 receptors on T cells, followed by subsequent internalization into T
cells. Once inside the T cells, HIV increases the expression of Fas receptor which in turn incurs excessive apoptosis
of T cells [15].
Morphology and biology of apoptosis
Certain morphological changes exhibited by apoptotic cells have been well identified and documented. These changes
include cell blebbing and shrinkage, nuclear fragmentation, condensation and fragmentation of genetic materials
(chromatin and nucleosomal DNA), and formation of small vesicles known as ApoBDs. Cells undergo a characteristic shrinkage during the early stage of apoptosis. Cell size becomes smaller while the contents within the cells
experience a denser packing. Another phenomenon known as pyknosis (irreversible condensation of chromatin)
also occurs concomitantly at the early stage of apoptosis. These changes can be observed by light microscopy while
electron microscopy can better visualize subcellular changes like pyknosis. Following the early stage of apoptosis,
another phenomenon known as karyorrhexis, which refers to fragmentation of the nucleus, ensued. Karyorrhexis
is accompanied by further blebbing of the plasma membrane that tears apoptotic cells into small ApoBDs. Current
knowledge states that ApoBDs contain remnants of apoptotic cells (i.e., cytoplasm, organelles, and nuclear contents)
where the contents of apoptotic cells are randomly distributed to each apoptotic body. Therefore, a specific organelle
or a nuclear content may be or may not be present in a particular apoptotic body. These ApoBDs are then engulfed by
phagocytes, e.g., neutrophils, macrophages, and dendritic cells (DCs), for final degradation. Phagocytosis marks the
terminal step of an apoptotic cycle and it is believed that the essence of this step is to prevent spillage of hazardous
materials packed within the apoptotic cells into the surroundings. The reason for forming ApoBDs has remained
elusive. Details regarding ApoBDs will be discussed in the next section.
The biological mechanism of apoptosis is exceedingly sophisticated. A complete implementation of apoptosis involves interplay of a wide array of proteins and signal transducers as well as cascades of signaling pathways. Two
major apoptosis pathways exist: extrinsic and intrinsic. The extrinsic pathway refers to receptor-mediated initiation
of apoptosis. The death receptors in the extrinsic pathway are all anchored to the cell membrane by their transmembrane regions. Upon interaction with an extracellular ligand, membrane receptors relay death signals into intracellular
space via their cytoplasmic death domains. Membrane receptors involved in apoptosis belong to the tumor necrosis
factor (TNF) receptor superfamily, whose activation depends upon two major ligands: TNF and Fas. TNF and its receptors, namely TNFR-1 and TNFR-2, are responsible for initiating a major apoptosis pathway: the TNF pathway. The
interaction between TNF and its receptors has been shown to relay death signal through recruitment of two adaptor
proteins: TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD), and
effect programmed cell death through the action of caspases. TNF ligands form homotrimers which bind to TNF receptors on the membrane. Upon binding, adaptor proteins are recruited (TRADD, FADD, and RIP) to TNF receptors
on the cytoplasmic side where death domains on the adaptor proteins interact with its counterparts on the receptors.
FADD recruits its downstream interactor, procaspase-8 in this case, via homotypic interaction of the death effector
domains (DED). Procaspase-8 then undergoes auto-cleavage to yield active caspase-8 which initiates the execution
phase of cell death. Caspase-8 cleaves procaspase-3 via proteolytic cleavage to produce active caspase-3 responsible
for the final execution of proteolytic degradation of a variety of intracellular proteins. Fas-mediated pathway undergoes a much similar signaling process. FasL (Fas ligand) triggers the pathway by binding to Fas receptors (also
known as CD95). FADD associates with the Fas receptors and recruits caspase-8. Fas receptors, along with FADD
and procaspase-8, form the death-inducing signaling complex (DISC).
A third extrinsic apoptotic pathway has been shown to be therapeutically exploitable, the TNF-related apoptosis
inducing ligand (TRAIL) pathway. TRAIL, alternatively known as Apo2L, was first identified by its sequence homology to FasL [16]. It is a type II transmembrane protein containing an extracellular region which, upon proteolytic
cleavage by protease, releases a soluble portion that acts as a ligand. Trimeric TRAIL binds to receptors on the membrane, namely DR4 and DR5, which then trigger intracellular signaling cascade similar to the Fas pathway [17,18].
The TRAIL-DR4/DR5 pathway is proposed to function in a wide range of physiological processes such as T cell activation and tumorigenesis. TRAIL is recognized as a tumor suppressor for its ability to exclusively induce apoptosis
in malignant cells and xenografts, rendering TRAIL an ideal antitumor agent [16,19]. The TRAIL pathway also functions independently of p53 which is frequently mutated in cancer cells, yet endowing TRAIL with another crucial
therapeutic advantage [20]. However, a considerable number of cancers are resistant to TRAIL treatment [21]. Research on TRAIL has led to emergence of different variants of TRAIL possessing enhanced therapeutic efficacy than
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Table 1 Caspases in mammalian cells
Protease
Other names
Recognition sequence
Functions in apoptosis
Casp-1
ICE
YVAD
Cleavage of pro-interleukin, involved in
death receptor-mediated apoptosis
Casp-2
ICE-1
Casp-3
CPP32, Yama, apopain
Initiator or effector
Casp-4
ICErel-II, TX, TCH-2
Casp-5
ICErel-III, TY
Casp-6
Mch2
Casp-7
Mch3, ICE-LAP3, CMH-1
Effector
Casp-8
FLICE, MACH, Mch5
Initiator of death receptor-mediated
apoptosis
DEVD
Effector
Cleavage of pro-interleukin
Cleavage of pro-interleukin
VEID
Effector
Casp-9
ICE-LAP6, Mch6
Initiator
Casp-10
Mch4
Initiator of death receptor-mediated
apoptosis
Casp-11
ICH3
Cleavage of pro-interleukin; initiator of
death receptor-mediated apoptosis
Casp-12
Initiator in ER-mediated apoptosis
Casp-13
Also known as ERICE, functions unknown
[33,34]
Casp-14
Filaggrin proteolysis and UV protection [35]
Casp-15
Unknown
its precursor. Yao et al. first reported that RGD-TRAIL exhibited enhanced antitumor effect than wild type TRAIL
in multiple tumor cell lines [22]. Receptor-specific TRAIL variants (i.e., specific to either DR4 or DR5) have also
been shown to be more efficacious [23]. Another research group led by Dr. Hua recently engineered a novel variant
of TRAIL constructed by linking Annexin V and TRAIL [24]. This variant, designated as TP8, showed surprisingly
higher antitumor potency than RGD-TRAIL towards A549 cells.
The intrinsic pathway of apoptosis is characterized by non-receptor-mediated initiation and mitochondrial regulation. In the intrinsic pathway, stimuli directly generate intracellular signals which lead to biochemical changes within
the cell. When a stimulus is present, it elicits disruption of the mitochondrial transmembrane that dissipates the membrane potential, rendering it more permeable. The stimulus also results in formation of mitochondrial permeability
transition pore (MPT) on the outermembrane that channels pro-apoptotic factors into the cytosol [25]. Apoptosome
cleaves procaspase-9 to yield active caspase-9 which in turn activates the effector caspase (i.e., caspase-3). Another
group of proteins known as SMACs (small mitochondria-derived activator of caspases) bind to IAP (inhibitor of
apoptosis proteins) following their release into the cytosol [26]. SMACs deactivate IAP, allowing apoptosis to proceed. Other inhibitors of IAP have been reported such as DIABLO and Omi [27,28]. These proteins engage in the
caspase-dependent pathway of apoptosis. A second group of proteins known as apoptosis-inducing factors (AIFs)
function in the caspase-independent pathway of apoptosis [29]. AIFs are anchored to the inner membrane of the
mitochondria. Upon cleavage by calpain, which is a calcium-dependent protease, AIFs can translocate to the nucleus
by nuclear localization signal (NLS) to induce DNA fragmentation and chromatin condensation [30].
Furthermore, research has shown that endoplasmic reticulum (ER) is also involved in apoptosis. Redundant accumulation of proteins and disturbance of calcium homeostasis within ER can trigger ER stress and therefore apoptosis.
Mouse bearing caspase-12 knock-out are resistant to apoptosis, indicative of caspase-12’s involvement in ER-mediated
apoptosis pathways [31]. Caspase-12, which is located on the membrane of ER, is essential to ER-mediated apoptosis. ER response elicits caspase-12 expression while translocating cytoplasmic caspase-7 to ER membrane where
caspase-12 is activated and apoptosis ensues [32].
Caspases are pivotal components of apoptosis (Table 1). To date, two types of caspases have been defined, the
initiator caspase and effector/executioner caspase. As previously mentioned, caspase-8 and -9 are initiator caspases
while caspase 3 is effector caspase. Furthermore, other caspases such as caspase-2, -10, and -11 belong to the initiator
category while caspase-6 and -7 belong to the effector category [36,37].
Members of the Bcl-2 protein family are responsible for regulation of apoptosis [38]. Two subgroups of this family
perform opposite functions regarding apoptosis: they can be categorized as either pro-apoptotic or antiapoptotic
(pro-survival). Table 2 illustrates the detailed classification and description of each member in the Bcl-2 family. In
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Table 2 Bcl-2 family members
Protein encoded
Functions
Bcl-2
Apoptosis inhibitor, binds Bax and Bak
Bcl-x
L form inhibits, S form promotes apoptosis, binds Bax and Bak
Bcl-w
Apoptosis inhibitor
Bax
Pro-apoptotic, binds Bcl-2, Bcl-x1 , and EIB1-9K
Bak
Pro-apoptotic or Apoptosis inhibitor, binds Bcl-2, Bcl-x1 , and EIB19K
MCL-1
Apoptosis inhibitor
Bad
Pro-apoptotic, binds Bcl-2 and Bcl-x
Ced-9
Apoptosis inhibitor in nematode, homolog of Bcl-2
EIB19K
Apoptosis inhibitor in adenovirus, binds Bax and Bak
Figure 1. Defective clearance of apoptotic cells leads to inflammatory response and autoimmunity
addition, p53 has been shown to regulate members of the Bcl-2 protein family [39]. For example, p53 can directly
induce transcription of Bax which is a pro-apoptotic protein [40].
Role of apoptosis in diseases
As previously described, apoptosis is a highly controlled physiological process in multicellular organisms. Research
on molecular mechanism of apoptosis reveals that anomalies in apoptosis lead to many diseases. Therefore, factors
involved in apoptosis regulation are of tremendous diagnostic and intervention value in curing diseases [41].
Defective apoptosis is associated with many types of illness including autoimmune diseases, neurodegenerative
diseases bacterial and viral diseases, heart diseases, and cancer [42,43].
Several reports have linked autoimmune diseases directly to dysregulated apoptosis and impaired clearance of
apoptotic cells [44–49]. Rapid clearance of apoptotic cells is crucial to induce immunological tolerance and prevent
inflammatory responses (Figure 1). Under physiological conditions, free apoptotic cells are rare in normal tissues
[50–52]. However, inefficient ingestion of dying cells by phagocytes or increased rate of apoptosis can lead to secondary necrosis of free apoptotic cells, which induces secretion of pro-inflammatory cytokines. Autoantigens released
from dying cells causes activation of T cell and expression of B cells, which generates autoantibodies and therefore
autoimmunity (Figure 1). Many autoimmune diseases are consequences of impaired clearance of apoptotic cells such
as systemic lupus erythematosus (SLE), connective tissue disease, and rheumatoid arthritis (RA) [53,54]. Defective
apoptosis of immune cells can also cause autoimmune diseases such as multiple sclerosis and autoimmune lymphoproliferative syndromes [55,56]. Typically, the malfunctioning Fas/FasL apoptotic pathway interferes with normal
apoptosis of lymphocytes leading to lymphocyte redundancy which in turn beget autoimmunity [57]. Furthermore,
experimental results have shown that mouse lacking Fas on T lymphocytes showed lupus-like symptoms and lymphocyte hyperplasia [58].
While dwindled apoptosis of immune cells facilitates autoimmunity and fosters autoimmune diseases, too much
apoptosis is as well a pathological condition [59]. Neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease are all characterized by excessive apoptosis of neurons. For example, Alzheimer’s
disease is caused by accumulation of b-amyloids at lesion sites where b-amyloids induce abnormal apoptosis of neurons. Infections by HIV causes accelerated apoptosis CD4+ T cell, leading to onset of AIDS [60]. Myocardial infarction
is caused by acute apoptosis of myocytes [61].
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To summarize, impaired clearance of apoptotic cells, and too little or too much apoptosis are both disease-prone.
Thus tuning apoptosis to a balanced state can serve to treat diseases [62]. If autoimmune lymphocytes cannot be
cleared by apoptosis, these cells will attack autoantigens, triggering autoimmune diseases [63]. Research has shown
that repeated administration of autoantigens to individuals with autoimmune diseases can cure them by inducing
apoptosis and therefore clearance of lymphocytes that specifically attack these autoantigens. Members of the Bcl-2
protein family have become important drug targets because their abnormal expression causes many diseases [64].
Bcl-2, first member of its protein family and inhibitor of apoptosis, is pivotal to the occurrence and progression of
cardiovascular diseases. A typical example is cardiac infarction which is caused by the lack of Bcl-2 expression and
therefore excessive death of cardiomyocytes. Allowing overexpression of Bcl-2 in cardiomyocytes prevents apoptosis
and has become an early stage therapeutic measure for cardiac infarction [65]. Evasion of apoptosis has become a
prerequisite for occurrence of tumor. Some tumors, like follicular lymphoma, are related to elevated expression of
Bcl-2 which can be counteracted by RNA interference and treatment by antisense oligonucleotides. Topoisomerase
inhibitors, antimetabolic agents and alkylating agents belong to the class of cytotoxic drugs whose efficacy is realized
through apoptosis induction of tumor cells [59]. For tumor cells expressing Fas, anti-Fas can specifically target these
cells and induce rapid cell death. However, Fas is expressed on normal cells including peripheral while blood cells,
T cells, and hepatocytes, therefore restricting the application of anti-Fas agonists. For apoptosis induced by cerebral
ischemia, such as suffocation of newborns and cerebral ischemia in elderlies, along with Alzheimer’s disease, retinal
degeneration, transplant rejection, and neurodegenerative diseases are all caused by excessive apoptosis. Therefore,
inhibiting caspases is an effective therapeutic plan [66]. Cells undergo complex signaling pathways from apoptosis
initiation to execution, involving a large network of proteins whereby blocking of any grid in the network can achieve
therapeutic purpose [67]. For example, nerve growth factors inhibit apoptosis and apparently suit therapeutic needs
to diseases of extensive apoptosis; or increasing Bcl-2 expression which lowers neuron’s sensitivity to apoptotic stimuli
can inhibit pathological apoptosis of neurons in response to neurotoxic factors. Besides, small-molecule inhibitors of
caspases, e.g., Z-VAD-FMK, has been proven useful in treating ALS in animals [68]. For AIDS treatment, antiretrovirus drugs are major players in the market despite their low efficacy and severe side effects. If the therapeutic strategy
shifts its focus to shielding T cells from apoptosis and combines T cell protection with retrovirus destruction, the outcome will become much more satisfactory. Moreover, chronic myocardial ischemia and hypoxia cause apoptosis of
cardiomyocytes and cardiac fibroblasts, which is preventable and treatable by blocking apoptosis [69]. In a word, designing treatment strategy based on the role of apoptosis in diseases associated with apoptosis anomalies holds great
promises in restoring health and therefore is of critical research and application values (Table 3 ).
ApoBD and the formation of ApoBD
Clearance of apoptotic cells has been a major field of study in apoptosis-related researches. Efficient and timely clearance of apoptotic cells is crucial to avoid immune response to autoantigens and prevent perturbation to ambient cells
and tissues [86]. Majority of cells undergoing apoptosis are eliminated by phagocytes in the form of small vesicles
known as ApoBDs. ApoBDs are relatively large vesicles compared with exosomes and microvesicles. ApoBDs have a
diameter of 800–5000 nm while exosomes and microvesicles have a diameter of 30–100 and 50–1000 nm, respectively
[87,88]. Furthermore, ApoBDs contain DNA, RNA, and proteins, similar to exosomes. However, the only marker of
ApoBDs discovered so far is phosphatidylserine (PS) [89]. Although definitive answer to the purpose of ApoBDs is
currently absent, it is proposed that breakage into ApoBDs contribute to more efficient clearance of apoptotic cells
and are important in controlling immune responses [90].
Breakage of apoptotic cells into ApoBDs was previously believed to be a random process until a recent discovery
revealed that a well-coordinated process is responsible for the formation of ApoBDs. As shown in Figure 2, one of
the earliest and most identifiable morphological changes is deformation of cells that appears as membrane blebbing.
Formation of blebs is a result of increased hydrostatic pressure within the cell following contraction mediated by
actomyosin [91]. Notably, apoptotic blebs are distinct from necrotic blebs, which are generally larger, independent of
actomyosin contraction, and are generated after membrane permeabilization [92]. Repeated process of blebbing and
retraction of apoptotic cells gives rise to formation of ApoBDs packed with organelles and other cellular contents such
as chromatin [93]. Another phenomenon known as apoptotic volume decrease (AVD) is also required for apoptotic
body formation [94]. Cytoskeletal disruption which inhibits AVD halts the formation of ApoBDs [95]. AVD is an early
event occurring concomitantly with membrane blebbing, leading to shrinkage of apoptotic cells. It is noteworthy that
apoptotic blebs are different from necrotic blebs in many ways [96].
It is now assumed that membrane blebbing is a prerequisite for apoptotic body formation [97]. However, different
cell types exhibit different forms of membrane deformation such as membrane protrusions including microtubule
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Table 3 Apoptosis-based drugs and therapies
Molecular target
Reagent
Principle
Experimental effects
Clinical trial/status
Company/reference
TRAIL receptors
HGS-ETR2
Agonistic TRAIL-R2 mAb
Apoptosis induction in
tumor cell lines
Phase 1, up to 10 mg/kg
every 2 weeks with
minimal toxicity
[70]
TRA-8
Agonistic TRAIL-R2 mAb
Apoptosis induction in
tumor cell lines
Preclinical
Sankyo Co., Ltd.
Dulanermin
Recombinant forms of
TRAIL
Dulanermin plus
bevacizumab was well
tolerated with no
occurrence of DLTs and
demonstrated antitumor
activity in NSCLC
Phase 2, 8 mg/kg/d for 5
days and 20 mg/kg/d for 2
days every 3 weeks in
combination with PCB
[71]
Mapatumamab
Agonistic TRAIL-R1 mAb
Safe and has promising
clinical activity in patients
with follicular lymphoma
Phase 1b/2
[72]
CD95/Fas
APG101
Target CD95 ligands
Restore immune function
while effectively inhibiting
tumor cell growth
Phase 2
ApoGenix
TNF
HUMIRA (Adalimumab)
Recombinant human
IgG1k mAb against TNF-α
Inhibition of TNF-α
FDA approved for RA,
psoriasis, ankylosing
spondylitis (AS), Crohn’s
disease
CAT/Abbott
Recombinant TNF-αs
Combination of TNF and
chemotherapy
Apoptosis induction in
tumor-associated blood
vessels
Approved for isolated limb
perfusion therapy in
melanoma
[73,74]
Infiximab (Remicade)
Mouse/human TNF-α
antibody
Anti-inflammatory, induces FDA approved for RA and
also apoptosis in
Crohn’s disease
macrophages
Enbrel (Etanercept)
Recombinant TNF-R2/IgG Anti-inflammatory in RA,
fusion protein
Crohn’s disease and other
inflammations
Approved for US, some
patients with severe side
effects (infections,
neurologic and
hematologic disorders)
Peptidomimetic irreversible
caspase inhibitor
Phase 2 started for chronic
HCV infection, phase 2
opened for HBV infection
and ischemia/reperfusion
injury of liver transplants
Centocor/ScheringPlough
Amgen/Wyeth
For RA and AS
Pan-caspase
IDN-6556
Antiapoptotic,
anti-inflammatory, and
antifibrotic in models of
liver damage
Idun
Phase 2 in compensatory
and decompensated
cirrhosis patients
MX1013
Dipeptide pan-caspase
inhibitor
Prevents apoptosis in
animal models of
myocardial infarct, stroke,
and acute liver failure
Preclinical, developed for
myocardial infarct, stroke,
acute liver failure
Maxim
RGD peptides
Caspase activators
Apoptosis induction in
tumor cell lines
Preclinical
Merck-Frosst, Maxim
Caspase-1
VX-740 (Pralnacasan)
ICE inhibitor
Anti-inflammatory in
rheumatoid and
osteoarthritis models
Phase 2, RA patients
showed anti-inflammatory
effects
Vertex/Aventis
Caspase-3
Immunocasp-3
Cell-permeable HER2 mAb
fused to caspase-3
Growth inhibition in nude
mice xenograft models
containing
PSMA-overexpressing
LNCaP cells
Preclinical
[75]
Caspase-6
Immunocasp-6
Cell-permeable HER2 mAb
Growth inhibition of
fused to caspase-6
HER2-positive tumors in a
mouse xenograft model
Preclinical
[76]
Caspase-9
FKBP12/caspase-9 fusion
protein
Chemically inducible
dimerization of caspase-9
Antiangiogenic in mouse
models upon induction of
caspase-9 dimerization
Preclinical
[77]
IAPs and SMAC
Embelin
Herbal cell-permeable
XIAP inhibitor
Binds to XIAP BIR3,
activates caspase-9,
induces apoptosis in
XIAP-overexpressing cells
Preclinical
[78]
Continued over
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Table 3 Apoptosis-based drugs and therapies (Continued)
Molecular target
Reagent
Principle
Experimental effects
Clinical trial/status
Company/reference
AEG35156/GEMR640
XIAP antisense
oligonucleotide
Exhibits antitumor activity
alone or in combination
with chemotherapeutics in
cancer xenograft models
Phase 1, phase 1/2 study
of patients with
relapsed/refractory AM
Aegera/Hybridon [79]
SM-406
Antagonists of IAP proteins Induces rapid degradation
of cellular cIAP1 protein
and inhibits cancer cell
growth in various human
cancer cell lines
Phase 1
[80]
GDC-0917
Cell-permeable inhibitor of
XIAP, cIAP-1 and cAIP-2
Potentiates apoptosis in
combination with TRAIL
and TNF, lead structure for
development of IAP
antagonists
Phase 1
Genentech
LCL161
Antagonists of IAP proteins
Oral administration of
Phase 2, neoadjuvant
LCL161 inhibits tumor
clinical trial of this weekly
growth in a mouse model
combination in triple
of multiple
negative breast cancer is in
myeloma142 \ 143
progress
APG-1387
Small-molecule IAP
inhibitors
Promote the process of
apoptosis by blocking the
activity of IAPs
Advanced solid tumors,
hematologic malignancies
in clinical trials
Ascentage Pharma
Survivin
LY2181308
Survivin antisense
construct
Preclinical studies show
antitumor activity in a
broad range of cancers
Phase 1
ISIS, Eli Lilly
Antiapoptotic Bcl-2
members
ApoG2
Targeting Mcl-1
Induced apoptosis and
displayed promising results
in different types of
lymphomas
Preclinical
Ascentage Pharma
ABT-263
Inhibit Bcl-2, Bcl-XL, and
Bcl-w
Orally efficacious in an
established xenograft
model of human small cell
lung cancer, inducing
complete tumor
regressions in all animals
Phase 1 in small cell lung
cancer and hematological
malignancies
[81]
AT-101
Against B-cell lymphomas
A potent inhibitor of the
antiapoptotic Bcl-2 family and displayed a synergistic
effect when sequentially
members, Bcl-2, Bcl-XL,
combined with 4-HC in
and Mcl-1
diffuse large B-cell
lymphoma
Phase 2
[82]
Obatoclax
Inhibit Mcl-1 and
antagonize
Mcl-1-mediated resistance
Phase 3 trial
(NCT01563601) for
Obatoclax in combination
with Carboplatin and
Etoposide compared with
chemotherapy arm alone is
underway in naı̈ve patients
with advanced-stage small
cell lung cancer
Gemin X
Genasense
Bcl-2 18-mer-antisense
oligonucleotide
Kills drug-resistant chronic Phase 3: FDA fast-track
lymphocytic leukemia
status for melanoma,
(CLL) cells, delays
multiple myeloma, CLL.
Phase 3 for non-small cell
development of fatal
lymphoma in mice,
lung cancer, phase 2 for
increases dacarbazine
hormone-refractory
effectiveness in melanoma
prostate cancer
models
Aventis/Genta Inc.
Venetoclax
BCL-2 inhibitor
Treatment of patients with April 11, 2016 approved by
CLL who have the 17p
the US FDA
deletion mutation (del 17p)
and who have previously
received at least one
therapy
AbbVie
Induce Bax-mediated
apoptosis in
cholangiocarcinoma
Novartis
Continued over
8
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Table 3 Apoptosis-based drugs and therapies (Continued)
Molecular target
Reagent
Principle
Experimental effects
Clinical trial/status
Company/reference
APG-1252
Bcl-2 / Bcl-XL inhibitors
Selectively inhibit the Bcl-2
protein family members
Bcl-2 and Bcl-XL to restore
tumor cell apoptosis,
thereby killing the tumor, is
intended for the treatment
of small cell lung cancer
and other solid tumors
Phase 1
Ascentage Pharma
Pro-apoptotic Bcl-2
members
Pazopanib
Inducing PUMA expression
Effective antineoplastic
agents that show
promising clinical activity in
a variety of carcinoma
Preclinical
[83]
p53
INGN201
p53-expressing adenovirus
Apoptosis induction in
tumor cell lines and
xenograft models
Phase 3 for head and neck
cancer, clinical trials for
other advanced solid
tumors
Invitrogen Therapeutics
SCH58500
p53-expressing adenovirus
Apoptosis induction in
tumor cell lines and
xenograft models
Phase 3 for advanced
ovarian cancer
Schering-Plough
ONYX-015
p53 delivery with mutant
adenovirus
Virus demonstrates
Phase 2/3 for combination
significantly greater
therapy of advanced
antitumor activity against
squamous cell cancer;
mutant p53 tumors in vivo phase 1/2 trials for several
other cancers
Onyx
C-terminal p53 peptides
Stabilization of wt and
reactivation of mutant p53
Restore transactivation
and growth-suppressing
function of mutant p53
Preclinical
Aventis
Preclinical lead
compounds
Roche
Nutlins
Drugs bind to the p53
Imidazoline derivatives that
pocket of Mdm2 and
antagonize p53/Mdm2
inhibit protein interaction in
interaction
vitro and in vivo
Early clinical trials for blood
cancers
Small-molecule antagonist
of p53/Mdm2 interaction
Compounds with
presumably insufficient
specificity
Preclinical
[84]
Small peptide compounds Small-molecule antagonist
of p53/Mdm2 interaction
High-affinity peptide that
stimulates the p53
pathway
Preclinical
[85]
Chalcones
APG-115
Small-molecule inhibitors
that target the MDM2-p53
protein interaction
MDM2-p53 inhibitor,
potently activates
p53-regulated apoptosis.
For sarcoma, primary liver
cancer, primary gastric
cancer and other tumors
can form a highly effective
inhibition, animal test
administration allows
tumor tissue completely
disappear
Phase 1
Ascentage Pharma
RG7388
p53-MDM2 inhibitors
Selectively binds to the
p53 site on the MDM2
surface, isolating p53 from
MDM2, resulting in
activation of the apoptotic
program following P53
stabilization, thereby killing
cancer cells
Phase 3 of acute myeloid
leukemia
Roche
Companies: Abbott Laboratories (www.abbott.com), Abbvie Inc. (www.abbvie.com), Aegera Therapeutics Inc. (www.aegera.com), Ascentage Pharma (www.ascentagepharma.com), Amgen (www.amgen.com), ApoGenix (www.apogenix.de), Aventis (www.aventis.com), CAT (Cambridge Antibody Technology, www.cambridgeantibody.com), Centocor (www.centocor.com), Eli Lilly and company (www.lilly.com), Gemin
X Biotechnologies (www.geminx.com), Genentech (www.gene.com), Genta Incorporated (www.genta.com), Hybridon (www.hybridon.com),
Idun Pharmaceuticals, Inc. (www.idun.com), Invitrogen therapeutics (www.invitrogen.com), ISIS Pharmaceuticals (www.isispharm.com), Maxim
Pharmaceuticals (www.maxim.com), Merck-Frosst Canada & Co. (www.merckfrosst.ca), Novartis (www.novartis.com), Onyx Pharmaceuticals
(www.onyx-pharm.com), Roche (www.roche.com), Sankyo Co., Ltd. (www.sankyo.co.jp), Schering-Plough (www.sch-plough.com), Vertex Pharmaceuticals, Inc. (www.vpharm.com), Wyeth (www.wyeth.com).
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Figure 2. Different ways of cell disassembly into ApoBDs
Patterns of apoptotic breakage into ApoBDs are illustrated in this figure. Majority of cells employ membrane blebbing, while some
cells exhibit other types of membrane protrusions such as microtubule spikes, apoptopodia, and beaded apoptopodia.
spikes, apoptopodia, and beaded apoptopodia [89]. For example, neurons and some epithelial cells use microtubule
spikes, instead of membrane blebbing, to form ApoBDs. Human jurkat T cells, primary mouse thymocyte, and fibroblasts forms apoptopodia following membrane blebbing, while apoptotic THP-1 cells and primary human neutrophils
displayed beaded apoptopodia structure. It is notable that beaded apoptopodia appears to be the most efficient way
of producing ApoBDs, generating approximately 10–20 ApoBDs at the same time [89]. Beaded apoptopodia represents a unique mechanism of rapid formation of ApoBD. Interestingly, beaded apoptopodia and formation of ApoBDs
were observed in neutrophils genetically engineered to be incapable of membrane blebbing, providing direct evidence
that membrane blebbing is not required for formation of ApoBDs. Instead, other processes alone, or together with
membrane blebbing, can promote disassembly of apoptotic cells and formation of ApoBDs. However, the detailed
mechanism that ultimately rips cells into single ApoBD is still unclear. Researchers believe that a concerted effort
of both intracellular and extracellular factors is required to shatter apoptotic cells into smaller vesicles and some
unidentified force is there to separate membrane protrusions from the main cell body.
The role of ApoBDs in cell clearance
Clearance of ApoBDs has been a focus in apoptosis-related researches. Phagocytes recognize ‘find-me’ signals released
by apoptotic cells and ‘eat-me’ signals on the ApoBDs to engulf them [98]. Ingestion of cell corpses by phagocytes is
termed efferocytosis as previously mentioned in the Introduction section. Efferocytosis is a four stage process that
begins with locating the target cells [99]. ‘Find-me’ signals, which are soluble mediators released by the apoptotic
cells, mediate attraction of phagocytes to the vicinity of apoptotic cells. Release of soluble signals starts at the very
beginning of apoptosis where a gradient is set up by these mediators [86]. Signals are then recognized by receptors
on the phagocytes, and triggers directional migration of phagocytes along the gradient towards ApoBDs. The second
stage features ‘Eat-me’ signal recognition on ApoBDs. The ‘eat-me’ signals are represented by PS which becomes
10
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Figure 3. ApoBDs are implicated in intercellular communications
DNA, RNA, proteins, and cytokines are contained within ApoBDs. Transfer of macromolecules are mediated by ApoBDs upon
engulfment of ApoBDs by recipient cells, in which contents of ApoBDs are released. Appropriate molecules released by ApoBDs
commence downstream biological response or signaling pathways in recipient cells, therefore achieving the purpose of intercellular
communication.
externalized and serves as signals for recognition and anchorage of phagocytes, allowing them to initiate the engulfing
process [100]. The third stage is engulfment. Cytoskeletal rearrangement and modification of the phagocytes occur
to enable ingestion of ApoBDs [101]. The last stage is digestion of cellular remnants through lysosomal degradation.
The ‘eat-me’ signal PS can interact with a calcium phospholipid binding protein, Annexin V. Annexin V is a widely
expressed protein belonging to the annexin superfamily. It is largely involved in blood coagulation due to its ability
to bind PS [102]. Therefore, Annexin V derivative has been developed to identify and image apoptosis [103–105].
Although ApoBDs can readily be engulfed by phagocytes, intact apoptotic cells can be engulfed just as effectively
[96]. In fact certain cell types do not fragment and form ApoBDs. However, in vitro studies showed that suppression of apoptotic blebbing, therefore formation of ApoBDs, impaired clearance of apoptotic cells by monocytes and
macrophages [106,107].
ApoBDs are biological entities carrying functional
biomolecules
Extracellular vesicles (e.g., exosomes and microvesicles) play a critical role in intercellular communications by transporting intracellular signaling molecules [108] Likewise, ApoBDs are vesicles that encapsulate residual ingredients
of dying cells. Traditional views believed that apoptotic cells are phagocytosed to prevent deleterious impacts on the
surroundings. A specific apoptotic body may contain a wide variety of cellular components, i.e., cytosol, degraded
proteins, DNA fragments or even an intact organelle (Figure 3). An interesting question to ask is whether these cellular
contents, are randomly distributed to each apoptotic body.
Recent studies have unraveled involvement of ApoBD in progression, metastasis, and formation of microenvironment of tumor. The discovery of ApoBD has offered new insights and explanations for physiological and pathological conditions. During the period of apoptosis, membrane blebbing promotes distribution of nuclear material into
ApoBDs [109]. Horizontal transfer of DNA can occur between adjacent whereas different types of cells. For example,
DNA packaged into lymphoma-derived ApoBDs was engulfed by surrounding fibroblasts, resulting in the integration
of lymphoma-derived DNA into the fibroblast genome [110]. Transfer of oncogenes (h-ras and c-myc) by ApoBDs to
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recipient cells lacking p53 facilitates tumor formation [111]. Apart from DNA, ApoBDs of endothelial cells mediate
transfer of miRNA (i.e., miRNA-126) to healthy endothelial cells, and induce expression of a chemokine CXCL12 in
recipient cells. In mouse models bearing atherosclerosis, repeated administration of ApoBDs containing miRNA-126
ameliorate the condition, at which this therapeutic effect may be mediated through induction of CXCL12 expression in luminal cells of aortic root plaques and recruitment of endothelial progenitor cells to repair blood vessels
[112]. Interestingly, DNA and RNA are packaged into ApoBDs of HL-60 cells, a type of granulocyte [113]. Although
the molecular mechanism is to be elucidated, an accumulation of reports has shown that functional molecules (i.e.,
DNA, RNA, and proteins) can indeed be packaged into ApoBDs, and different type of molecules follow different
signaling pathways and therefore produce varied biological outcomes.
Apart from nucleotides, proteins can be transferred by ApoBDs to phagocytes, such as macrophages and DC
cells, for immunoregulation. ApoBDs of thymocytes from BALB/c mouse are enriched with autoantigens and
pro-inflammatory factors, along with 142 different protein including histones, hsp90, and many immunoproteins
[114]. Similarly, ApoBDs from lymphoblasts contain histones 1, 2A, 2B, 3, and 4, as well as autoantigen La/SSB 974.
Shotgun proteomics identified 11 different proteins unique to ApoBDs of biliary epithelial cells as compared with
their healthy counterparts [115]. These proteins include Annexin A6, hsp6, LDL receptor-related proteins. These
proteins are implicated in many immune pathways, e.g., activation of NF-κB, ERK, and Notch pathways, and signaling pathways mediated by IL8 and CXCR2. Recent studies also reported a drastic difference in 1028 proteins [89].
Many research teams reported distribution of autoantigens into membrane vesicles and ApoBDs from a variety of
cells (e.g., epithelial cells, T cells, cardiomyocytes) [116]. It is noteworthy that human primary monocyte-derived
macrophage can engulf ApoBDs containing autoantigens, suggesting a possible route through which autoantigens
can be transferred to professional phagocytes [117]. Furthermore, endothelial cells-derived ApoBDs contain precursor and processed pro-inflammatory factor IL-1α. ApoBDs containing IL-1α promote expression of chemokine IL-8
from healthy endothelial cells, thereby facilitating invasion of neutrophils into mouse peritoneum [118].
Contents of within the ApoBDs may be used for a number of purposes such as activation of immune system,
recruitment of dying cells and regeneration of damaged tissues. Rubartelli et al. first reported that DC can selectively
engulf ApoBDs [119]. DC is a type of phagocytes capable of engulfing ApoBDs rich in auto-antigens. It is first reported
in 1998 by Albert et al. that DCs acquire antigens from ApoBDs and induce class I-restricted CTLs [120]. The same
group later reported a continuation of this study that ApoBDs contain processed antigens that can be directly used by
DCs for cross-presentation [121]. In bone marrows, dying osteocytes release ApoBDs containing receptor activator
of nuclear factor kappa-B ligand (RANKL) to recruit replacement osteoclasts [122].
Schiller and co-workers observed that immunogenic molecules enter ApoBDs in the early stage of apoptosis before
DNA fragmentation [123]. JAK1/STAT3 in hepatic stellate cells (HSC) pathway was activated through engulfment of
ApoBDs derived from HepG2 cells, which also activated PI3K/Akt/NF-kB survival pathway to a lesser degree. Antiapoptotic proteins Mcl-1 and A1 were upregulated, leading to survival of HSC and spread of liver fibrosis [124].
Marin-Gallen et al. recently revealed therapeutic capacity of ApoBDs [125]. They reconstituted peripheral tolerance
of type I diabetes by forced intake of in ApoBDs derived from in vitro cells by apoptosis-resistant DC cells. Therefore,
these DC cells expressed lowered level of CD40 and CD86, and pro-inflammatory cytokines. As a result, autoimmunity against cells is reduced. These results suggest that ApoBDs can act as regulators at the cellular level and therefore
possess conspicuous biological significance.
Furthermore, ApoBDs are carriers of contagious materials of pathogens. For example, ApoBDs of HIV-infected T
cells can transfer proteins and genomic contents of HIV to adjacent epithelial cells where transcription and expression of viral proteins are furthered and assembly of HIV takes place [126]. Besides, ApoBDs of prion-infected neurons
must be effectively eliminated to prevent spreading of prions and onset of prion diseases [127].In mouse models bearing tumor xenografts, ApoBDs were detected in the blood [128]. Moreover, murine fibroblasts lost contact inhibition
and became tumorigenic after engulfing ApoBDs derived from cells transfected with oncogenes [129]. These results
suggest that genetic information can also be transferred by uptake of ApoBDs. Tumor cells release ApoBDs to environment upon apoptosis induced by treatments, which promotes tumor invasion and metastasis [127,130]. Hence,
ApoBDs can function as a ‘Trojan horse’ for infectious agents [131].
To conclude, ApoBDs are messengers containing legacy of its progenitors, whose destiny lies far beyond being
‘garbage bags’ as we previously assumed. They can be recycled to initiate an array of biological responses. Further
understanding of ApoBDs is essential to complete delineation of apoptosis.
12
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Concluding remarks
The more efforts invested, the more we realize how complex the process of apoptosis is. It is a highly coordinated process of both morphological and biochemical change. Depending on the cell type and disassembly pattern, ApoBDs of
different size and amount can be generated. Therefore, an obvious question can be asked: why the universal process
of apoptosis can present itself in various ways? Why different cell types employ different ways of breakage? What
controls the release, size and contents of ABs? By solving these mysteries, we become one step closer to complete understanding of programmed cell death under both physiological and pathological conditions. Lastly, it is encouraging
that drugs that block or promote ApoBD formation have been advanced to the stage of clinical trials [132]. Therefore,
therapies and drugs designed based on regulation apoptosis and ApoBD formation hold great promises in curing
intractable diseases [133].
Funding
This work was supported by the Chinese National Natural Sciences Foundation [grant numbers 81630092, 81773099, 81570790,
81573338]; the National Key R&D Research Program by Ministry of Science and Technology [grant numbers 2017YFA0506002,
2017YFA0104301]; the Shenzhen Science and Technology Innovation Committee [grant number JCYJ20160331152141936]; and
Shenzhen Peacock Plan [grant number KQTD20140630165057031].
Competing Interests
The authors declare that there are no competing interests associated with the manuscript.
Abbreviations
AIDS, acquired immune deficiency syndrome; AIF, apoptosis-inducing factors; ApoBDs, apoptotic bodies; AVD, apoptotic
volume decrease; CLL, chronic lymphocytic leukemia; CTL, cytotoxic T lymphocyte; DC, dendritic cell; DED, death effector domains; ER, endoplasmic reticulum; FADD, fas-associated death domain protein; HIV, human immunodeficiency virus;
HSC, hepatic stellate cells; PS, phosphatidylserine; RA, rheumatoid arthritis; TNF, tumor necrosis factor; TRADD, TNF
receptor-associated death domain.
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