Invest Clin 65(2): 230 - 252, 2024 https://doi.org/10.54817/IC.v65n2a09

Corresponding Author: Diego Fernández-Lázaro. Department of Cellular Biology, Genetics, Histology

and Pharmacology, Faculty of Health Sciences, University of Valladolid, Campus of Soria, Soria, Spain.

E-mail: diego.fernadez.lazaro@uva.es

Mechanisms of programmed cell death:

structural and functional pathways.

A narrative review.

Diego Fernández-Lázaro

1,2,3

Begoña Sanz

4,5

and Jesús Seco-Calvo

3,4,6

Department of Cellular Biology, Genetics, Histology and Pharmacology, Faculty

of Health Sciences, University of Valladolid, Campus of Soria, Soria, Spain.

Neurobiology Research Group, Faculty of Medicine, University of Valladolid, Valladolid,

Spain.

SARCELLOMICS® Research Group, León, Spain.

Department of Physiology, University of the Basque Country, Leioa, Spain.

Biocruces Bizkaia Health Research Institute, Barakaldo Spain.

Institute of Biomedicine, University of León, León, Spain.

Keywords: apoptosis; caspases; mitochondrial/intrinsic pathway; extrinsic pathway;

necroptosis; autophagy.

Abstract. Apoptosis, necroptosis, and autophagy are cellular mechanisms

by which cells are programmed to die under various physiological and devel-

opmental stimuli. A multitude of protein mediators of programmed cell death

have been identified, and apoptosis, necroptosis, and autophagy signals have

been found to utilize common pathways that elucidate the proteins involved.

This narrative review focuses on caspase-dependent and caspase-independent

programmed cell death systems. Including studies of caspase-dependent pro-

grammed cell death, extrinsic pathway apoptotic mechanisms, phosphatidyl-

serine (PS), FAS (APO-1/CD95), tumor necrosis factor (TNF) receptor type

1 (TNF-R1) and TNF-related apoptosis-inducing ligand (TRAIL), and intrinsic

or mitochondrial pathway such as cytochrome C, the Bcl-2 family of proteins

and Smac/Diablo. The Bcl-2 family has apoptotic mediators Bcl-2-associated X

protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), Bcl-2-interacting

protein BIM (Bim), Bcl-2 agonist of cell death (Bad), Bid, Bcl-2 adenovirus E1B

19kDa-interacting protein 1 NIP3 (Bnip3), BMF, HRK, Noxa and PUMA and an-

tiapoptotic proteins such as Bcl-2 itself, Mcl-1, Bcl-w, A1, and Bcl-XL. Moreover,

caspase-independent programmed cell death pathways include the mitochon-

drial pathway with the protein mediators apoptosis inducing factor (AIF) and

endonuclease G, and the pathways necroptosis, and autophagy. Understanding

programmed cell death from those reported in this review could shed substan-

Mechanisms of programmed cell death. A narrative review 231

Vol. 65(2): 230 - 252, 2024

Mecanismos de muerte celular programada: vías estructurales

y funcionales. Una revisión narrativa.

Invest Clin 2024; 65 (2): 230 – 252

Palabras clave: apoptosis; caspasas; vía mitocondrial/intrínseca; vía extrínseca;

necroptosis; autofagia.

Resumen. La apoptosis, la necroptosis y la autofagia son mecanismos celu-

lares mediante los cuales las células se programan para morir bajo una amplia

gama de estímulos fisiológicos. Esta revisión describe en los sistemas de muerte

celular programada dependientes e independientes de la caspasa. Los estudios

incluidos sobre la muerte celular programada dependiente de la caspasa inclu-

yen mecanismos apoptóticos de la vía extrínseca que incluyen fosfatidilserina

(PS), FAS (APO-1/CD95), receptor del factor de necrosis tumoral (FNT) tipo

1 (FNT-R1) e inductor de la apoptosis relacionada con ligando FNT (TRAIL)

y vía intrínseca o mitocondrial como el citocromo C, la familia de proteínas

Bcl-2 y Smac/Diablo. La familia Bcl-2 tiene mediadores apoptóticos, proteína

X asociada a Bcl-2 (Bax) y antagonista/asesino homólogo de Bcl-2 (Bak), pro-

teína BIM que interactúa con Bcl-2 (Bim), agonista de la muerte celular de

Bcl-2 (Bad), Bid, proteína 1 que interactúa con el adenovirus E1B 19kDa de

Bcl-2, NIP3 (Bnip3), BMF, HRK, Noxa y PUMA y proteínas antiapoptóticas como

la propia Bcl-2, Mcl-1, Bcl-w, A1 y Bcl-XL. Además, las vías de muerte celular

programada independientes de la caspasa incluyen la vía mitocondrial con los

mediadores proteicos factor inductor de apoptosis (FIA) y endonucleasa G, las

vías necroptosis y autofagia. Comprender la muerte celular programada a partir

de los contenidos descritos en esta revisión podría arrojar luz sustancial sobre

los procesos de la homeostasis biológica y podría proporcionar la capacidad de

modular la respuesta de muerte celular programada y conducir a nuevas inter-

venciones terapéuticas en una amplia gama de enfermedades.

Received: 19-10-2023 Accepted: 18-11-2023

INTRODUCTION

Every hour, billions of cells die in us,

and our tissues do not shrink because of a

natural regulation whereby cell death is

balanced by cell division. It is necessary to

control both death and cell division in dif-

ferentiated cells to balance the different cell

populations, avoiding affecting the adjacent

cells

The process in which cells eliminate

themselves in a controlled manner is called

tial light on the processes of biological homeostasis. In addition, identifying

specific proteins involved in these processes is mandatory to identify molecular

biomarkers and therapeutic targets. Furthermore, it could provide the ability to

modulate the programmed cell death response and could lead to new therapeu-

tic interventions in a disease.

232 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

programmed cell death. Programmed cell

death plays an important role during em-

bryonic development, maintaining tissue

homeostasis in the body and eliminating

damaged cells

. In contrast, excessive or

defective cell death contributes to a broad

spectrum of human pathologies. Low-rate

cell death can result in the formation of

cancer and autoimmune diseases

, whereas

high-rate cell death can result in neurode-

generative disease, immunodeficiency, or

muscle atrophy

Knowledge of specific/differential pro-

teomic expression in each cell death is es-

sential for the early detection, diagnosis, and

prognosis of cell-death-related diseases. This

knowledge is also crucial for the use of more

precise and personalized pharmacological

treatments

. Cell death can be divided into

three groups: programmed, regulated, and

accidental

. Programmed cell death is pres-

ent in embryonic development and tissue

homeostasis, such as apoptosis and necrop-

tosis. Regulated cell death is that which, pro-

grammed or not, can be induced or inhibited

by a specific molecular mechanism through

pharmacology or genetic interventions, for

example, the release of neutrophil extracel-

lular traps (NETs), a regulated form of neu-

trophil cell death known as NETosis, modu-

lates neutrophil toxic effects. Accidental cell

death is triggered by external physical condi-

tions, such as ischemia, freeze-thaw cycles,

or high concentrations of pro-oxidants, an

example of this type of death are oncosis and

necrosis

. Two mechanisms of programmed

cell death are distinguished: apoptotic cell

death, dependent on caspases such as extrin-

sic and intrinsic apoptosis, and non-apoptot-

ic cell death, independent of caspases, such

as autophagy and necroptosis

MATERIALS AND METHODS

Search strategy

The present study is a narrative lit-

erature review comprising scientific stud-

ies conducted between May and July 2023

that sought to group and describe caspase-

dependent and caspase-independent pro-

grammed cell death, describing the molecu-

lar mechanisms of apoptosis, necroptosis,

and autophagy. The bibliographic search was

carried out in the following electronic data-

bases: Medline (PubMed), Sci-ELO, Scopus,

Science Direct, Cinahl, EMBASE - Excerpta

Medica Data Base, LILACS, Google Scholar,

Dialnet, and Cochrane Library Plus. The key-

words used for the search were: programmed

cell death, apoptosis, caspases, caspase in-

hibitory proteins, mitochondrial / intrinsic

pathway, extrinsic pathway, necroptosis, au-

tophagy, phosphatidylserine, FAS (APO-1/

CD95), tumor necrosis factor (TNF) recep-

tor type 1 (TNF-R1) and TNF-related apopto-

sis-inducing ligand (TRAIL) and cytochrome

C, linked by the Boolean operators “AND”

and “OR”. Additional records were gleaned

by conducting a ‘snowball’ search, checking

the reference lists of publications eligible

for full-text review, and using ResearchGate

to identify potential articles not included in

the databases used in the study.

Inclusion and exclusion criteria

The following inclusion criteria were

applied to select the articles: (1) Access to

the full text; (2) be a review, clinical trial,

observational study, or case report/study;

(3) identify caspase-dependent and cas-

pase-independent programmed cell death;

(4) describe the molecular mechanisms of

apoptosis, necroptosis, and autophagy; (6)

studies whose publication date is from the

beginning of the databases until July 2023;

(6) languages were restricted to English,

German, French, Italian, Spanish, and Por-

tuguese. The exclusion criteria applied were:

(2) Publications not related to programmed

cell death and/or describe its molecular

mechanisms; (2) duplicate documents.

Data extraction

After searching the databases for stud-

ies, the search titles were checked to iden-

tify duplicates and possible publications to

Mechanisms of programmed cell death. A narrative review 233

Vol. 65(2): 230 - 252, 2024

add. After reading the abstract, a full-text re-

view of the selected studies was performed.

Two reviewers (D.F.-L. and J.S.-C.) scruti-

nized and synthesized data from all selected

studies into a comprehensive table using a

standardized data extraction. A third review-

er (B.S.) resolved all disagreements between

them.

RESULTS AND DISCUSSION

Programmed Cell death

Apoptotic cell death functions individu-

ally and selectively and is executed through

a highly stereotyped series of biochemical

events that ensure rapid non-inflammatory

cell elimination. For this reason, the apop-

totic physiological process is produced and

characterized by decreased cell size, vesicle

formation, and condensation of the nucleus.

This series of transformations regulates the

control of morphogenesis and organogenesis

during embryonic development, in addition

to tissue homeostasis in adult organisms

Non-apoptotic cell death is usually de-

scribed as a secondary mechanism for defi-

cient apoptotic conditions. However, it is also

possible that non-apoptotic programmed

cell death mechanisms may function in first-

order lines; for example, autophagic cell

death is carried out during metamorphosis

in insects. This autophagic cell death elimi-

nates a tissue in its entirety

In 1972, Kerr et al.

coined the term

apoptosis to differentiate it from the death

of natural origin, necrosis. The word comes

from Greek, which refers to the leaves that

fall from the trees or the petals that fall from

the flowers. The prefix apo means “distance,

outside or part” and ptosis “fall,” which lit-

erally means “fall from”. Apoptosis is associ-

ated with caspases, a family of cysteine pro-

teases that control not only apoptosis but

also proliferation, differentiation, cell shape,

and cell migration.

Apoptosis is a form of programmed cell

death

, which occurs because of tissue and

cell aging or in response to different external

agents such as ionizing radiation and chemo-

therapeutic agents

. It can be considered a

process that facilitates the elimination of de-

fective cells, so the alteration in the regula-

tion of genes involved in cell death by apop-

tosis can cause and be associated with the

development of different neoplasms, autoim-

mune diseases, viral infections, and neurode-

generative diseases

. Apoptosis is an active

process where the cells react and execute

their death, programmed, by themselves

Apoptotic death is considered when a cell

has lost its individuality or reached a “point

of no return” at which the cell definitively

loses its function. Apoptotic cell death trig-

gers two stages. In the first stage, biochemi-

cal mediators attempt to repair a damaged

cell. If they fail, the cell enters the second

stage or execution phase, where structural

changes occur that lead the cell to death

. These structural changes affect the cell

membrane, intracytosolic organelles, and

the nucleus

. The cytoskeleton collapses,

the nuclear envelope disassembles, redis-

tributing the nuclear pores, the nuclear pro-

tein is altered, and the nuclear chromatin

condenses and fragments, becoming dense

clumps, with an electrophoretic “ladder”

pattern, which migrate towards the nuclear

membrane that shapes. DNA and RNA cleav-

age, due to the activation of Ca

+ and Mg

dependent endonucleases that cleave ge-

nomic DNA through the internucleosomal

spaces

. Also in the mitochondria, DNA de-

grades, and the endoplasmic reticulum los-

es its envelope; its cisterns widen and fuse

. The phospholipids of the cell membrane

change orientation and the phosphatidyl-

serine residues are exposed to the external

environment; the fragmentation of the phos-

pholipids is induced because of these disrup-

tions, the integrity of the plasma membrane

is lost, and the mitochondrial membrane

potential decreases. On the surface of the

plasma membrane, fragments of membrane-

enclosed cytoplasm called apoptotic bodies

protrude and are shed, which are cytoplas-

mic remnants surrounded by a membrane

234 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

such that they are rapidly engulfed by an

adjacent cell or macrophage when released

into the external environment, without caus-

ing an inflammatory response

12,14,15

Caspases

Cysteine aspartate-specific proteases

(Caspases, EC 3.4.22.-) are synthesized as

inactive 30-50 kDa precursors (zymogens)

that have three domains: an amino-terminal

domain (prodomain), a domain that will re-

sult in a large subunit (p20) containing the

active site, and another domain that will end

in a smaller subunit catalyst (p10) C-ter-

minus

(Fig. 1). In the presence of appro-

priate stimuli, a proteolysis process occurs

between the domains, generating the active

fragments. There are two types of caspases:

initiator caspases (Caspase-2, 8, 9, and 10)

that are activated in response to signs of

stress or cell damage and that protect and

activate effector caspases (Caspase-3, 6, and

7), these will oversee the direct proteolysis

of different substrates that will lead to the

death of the cell. One of their first identified

substrates was Poly ADP-ribose polymerase

(PARP)

17,18

Initiator caspases present in their N-

terminal region one or two essential adapter

domains for their function. In contrast, ef-

fector caspases do not have these domains.

There are two fundamental ways caspases can

be activated (intrinsic and extrinsic path-

ways of caspase-dependent apoptosis), but

although both pathways converge on effec-

tor caspases, they require different caspases

to initiate the process. Thus, the activation

of the extrinsic pathway mainly causes the

recruitment of Caspase-8, and the intrinsic

pathway principally causes the recruitment

of Caspase-9

18,19

During the process of apoptosis, there

is a massive activation of caspases, which

specifically cut proteins in cysteine residues

located near aspartic acid. Caspases initi-

ate a cascade of events that converge into

a common effector caspase pathway that

leads to the execution of apoptosis

12,20

. The

apoptotic machinery of the cytoskeleton has

inactive precursors or initiating procaspases

8-10

that are activated by proteolytic cleavage

and are catalyzed by other already active cas-

pases; here, the process remains reversible.

When activated, the initiator procaspases

and cell-specific target proteins cleave and

activate the executor procaspases (3, 6, and

7). From Caspase-3, the process is irrevers-

ible

17,19

Apoptosis constitutes a complex series

of positive and negative events with mul-

tiple positive and negative regulators and

is integrated into other critical intracellu-

lar pathways such as cell cycle progression,

phosphorylation-mediated signals, and DNA

Fig. 1. Structure of Caspases.

CARD: caspase recruitment and activation domain; DED: Death Effector Domain; p20: large subunit (p20);

p10: small subunit (p10).

Caspases contain three domains: an N-terminal prodomain, a large subunit (p20) containing the active center

with cysteine within a conserved QACXG motif, and a small subunit (p10) at the C-terminus.

Mechanisms of programmed cell death. A narrative review 235

Vol. 65(2): 230 - 252, 2024

damage repair

. Apoptosis is carried out

mainly by two alternative pathways of apop-

tosis induction divided into i) apoptosis me-

diated by death receptors expressed on the

cell surface or extrinsic pathway; ii) apopto-

sis mediated by the mitochondrial or intrin-

sic pathway

. Signaling by both pathways

induces the activation of caspases, and each

pathway uses its own initiator caspases and

activation complex

12,20

Inhibitors of Apoptosis Proteins (IAPs)

Inhibitors of apoptosis proteins (IAPs)

could inhibit apoptosis by selectively bind-

ing and inhibiting Caspase-3 and Caspase-7,

but not Caspase-8. IAPs block the caspase

cascade and inhibit cell death in response to

proapoptotic stimuli

. There are currently

eight protein members of this family, but

two of them, survivin and X-linked inhibitor

of protein apoptosis (XIAP), are particularly

interesting. In this sense, survivin is the only

IAP associated with the mitotic spindle

. Its

expression depends on the cell cycle

. It has

a double function since it inhibits apoptosis

through direct and indirect interaction with

caspases and regulates the cell cycle

. Sur-

vivin is expressed in embryonic tissue and

is overexpressed in tumor cells, associated

with resistance to chemotherapy, but not in

normal adult tissues

24,25

. XIAP is possibly the

best-studied IAP both at the structural level

and at the level of its mechanism of action

Furthermore, XIAP is the only member

of this family that can inhibit both effector

and initiator caspases. XIAP is frequently el-

evated in tumor cells, leading to resistance

to chemotherapy

. Therefore, XIAP is an

optimal therapeutic target based on its func-

tions; moreover, the inhibition of XIAP re-

stores cellular chemosensitivity

27,28

Caspase-dependent pathways of apoptosis

Extrinsic pathway: recipients of death

The extrinsic pathway is activated by

ligands of the family of Tumor necrosis fac-

tor (TNF) proteins. Some ligands can induce

apoptosis; when they bind to their receptors,

they trigger caspase activation and initiate

apoptosis

. Death receptors are character-

ized by having cysteine-rich extracellular

domains. They all have in common a death

domain (DD) domain in the cytoplasmic re-

gion. In general, the binding of ligands to

death receptors induces their trimerization,

and subsequently, the DD domains recruit

adapter molecules that will activate Cas-

pase-8 and, when activated, activate Cas-

pase-3

. Extrinsic apoptosis is related to

death receptors on the plasma membrane,

such as phosphatidylserine (PS), FAS (APO-

1/CD95), TNF receptor type 1 (TNF-R1),

and TNF-related apoptosis-inducing ligand

(TRAIL)

Phosphatidylserine (PS)

In cells, the negatively charged PS is

only localized to the cytosolic side of the

lipid bilayer of the plasma membrane, but

when it is translocated to the outer mono-

layer of the cell, it acts as an “eat me” signal,

so it is considered as a marker of extrinsic

apoptosis. In addition to expressing signals

on the cell surface that stimulate apoptosis,

PS also blocks inflammation in the phago-

cytic cell by inhibiting the production of cy-

tokines proinflammatory signaling proteins

. It must be considered that apoptotic cells

must not only activate the signals that in-

duce cell death but also inactivate or lose

the death signals

Fas (APO-1/CD95)

Fas (APO-1 / CD95) is ubiquitously ex-

pressed on the cell’s surface as a membrane

protein of 40 kDa, which is highly expressed

in T lymphocytes and activated natural kill-

er (NK) cells

. The activation of Fas at the

cell surface is done by binding the Fas ligand

(FasL) to the surface of a cytotoxic lympho-

cyte. The death domains of the cytosolic tails

of Fas death receptors recruit adapter pro-

teins, which in turn recruit procaspase ini-

tiators such as procaspase-8, procaspase-10,

or both, forming the death-inducing signal-

236 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

ing complex (DISC)

. In practice, once ac-

tivated in the DISC, the initiator caspases

activate the next executor procaspases in

the cascade, inducing apoptosis. This path-

way begins with the formation of the DISC

in which an adapter molecule called Fas as-

sociated death domain (FADD) and procas-

pase-8 intervene. FADD binds to Fas through

their respective DD domains and to pro-

caspase-8 through a death effector domain

(DED). The oligomerization of procaspase-8

in the DISC complex results in the activation

of Caspase-8 and subsequent activation of

other caspases. Depending on the cell type,

Caspase-8 can directly activate Caspase-3

or proteolyze the carboxy-terminus of BH3

interacting domain death agonist (Bid), a

proapoptotic protein from the Bcl-2 family.

Translocation of the truncated form of Bid

into the mitochondria will activate the mito-

chondrial pathway

35,36

(Fig. 2).

The Fas / Fas ligand (FasL) system par-

ticipates in the elimination of T and B lym-

phocytes, viruses-infected cells, and cancer

cells. Doxorubicin and methotrexate (cyto-

toxic agents) or immunomodulatory drugs

(IMiDs) activate this pathway to achieve cell

death in malignant cells in cancer disease

Furthermore, FasL can also interact

with the DcR receptor, a soluble secreted

receptor of the TNF superfamily. Some cells

produce “decoy” receptors on the cell sur-

face with a ligand-binding domain but no

death domain; therefore, they can bind to

a death ligand but do not trigger apoptosis.

When FasL binds to DcR3, it inhibits FasL/

Fas apoptotic activity, thus acting as a “de-

coy.” Cells can also produce intracellular

blocking proteins such as FADD-like IL-1β-

converting enzyme (FLICE)-inhibitory pro-

tein (FLIP), which resembles procaspase but

lacks a proteolytic domain; FLIP competes

Fig. 2. Extrinsic apoptotic pathway by FAS cell death receptors.

FAS is a cell surface receptor that, when binding to its ligand, causes apoptosis. (APO-1/CD95); FASL: Fas

ligand; FADD: Fas Associated Death Domain; DD: Death Domain; DED: Death Effector Domain; DISC:

Death-Inducing Signaling Complex; Bid: BH3 Interacting Domain Death Agonist Protein.

Diagram of apoptotic signals of the extrinsic pathway: mediated by FAS receptors of cell death. Caspase-8 can

directly activate Caspase-3 or proteolyze the carboxy-terminal end of Bid, a proapoptotic protein of

the Bcl-2 family. The translocation of the truncated form of Bid to the mitochondria will activate the

mitochondrial pathway.

Mechanisms of programmed cell death. A narrative review 237

Vol. 65(2): 230 - 252, 2024

with procaspases-8 and 10 for binding sites

on DISC and thus inhibits the activation of

these initiating procaspases

38,39

Tumor necrosis factor (TNF) receptor type

Tumor necrosis factor-α (TNF-α) is a

type II transmembrane protein that medi-

ates the inflammatory response, regulation

of immune cells, and cytotoxicity. TNF-α

binds to tumor necrosis factor receptor 1

(TNF-R1), also known as death receptor 1

(DR1), and tumor necrosis factor receptor

2 (TNF-R2). TNFR1 and TFNR2 are involved

in pro-survival signaling and proliferation by

activating the nuclear factor kappa B (NF-

kB) pathway

31,40

TNF-R1 can be found in all cell types,

and TNF-R2 is mainly expressed in immune

and endothelial cells

. TNF-R1 is ubiqui-

tously expressed, like Fas, whereas its ligand

TNF-α is only expressed on activated macro-

phages and lymphocytes in response to in-

fections

. TNF-R1 has a death domain that

can trigger apoptosis by activating the cas-

pase cascade. Upon activation, TNF binds

to its receptor via trimerization of TNFR1.

Subsequently, a TNFR-associated death do-

main protein (TRADD) adapter molecule is

added that induces association with FADD

and activation of Caspase-8. In addition to

the apoptotic pathway, TNF induces other

signal transduction pathways from TRADD

that trigger the activation of NF-κB and c-

Jun Kinase (JNK)/Ap-1

41,42

(Fig. 3).

TNF-Related Apoptosis-Inducing Ligand

(TRAIL)

TRAIL is a type II homotrimeric trans-

membrane protein expressed on the surface

of T cells, macrophages, and NK cells, mod-

ulating the immune response

. Zinc bind-

ing is essential for recognizing the receptor

and the subsequent induction of apoptosis

Fig. 3. Extrinsic apoptotic pathway by Tumor Necrosis Factor Receptor type 1 cell death receptors.

TNF: tumor necrosis factor; TNFR1: Tumor Necrosis Factor Receptor; TRADD: Tumor Necrosis Factor Recep-

tor (TNFR)-Associated Death Domain protein; RIPs: Receptor Interacting Proteins; FADD: Fas Associa-

ted Death Domain; Bid: BH3 Interacting Domain Death Agonist Protein.

Signaling pathway of the TNF receptor 1. TRADD adapter molecule is added, which induces the association

with FADD and the activation of Caspase-8.

238 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

by stabilizing the trimeric conformation in

TRAIL residue Cys230, which is essential

When TRAIL binds to DRs, it induces recep-

tor trimerization, which triggers the extrin-

sic apoptotic pathway in transformed cells

without affecting non-transformed cells

TRAIL binds to two death receptors

(DR), DR 4 and DR5, and “three decoys

receptors” (DcR) (DcR1, DcR2, and osteo-

protegerin (OPG)). TRAIL-R1 or DR4 and

TRAIL-R2 or DR5, with 60% homology, can

trigger apoptosis and determine whether

a cell is resistant or sensitive to TRAIL

DcR1, or TRAIL-R3, is a GPI-anchored pro-

tein lacking the intracellular and transmem-

brane domains, while DcR2 or TRAIL-R4 has

an intracellular portion containing a trun-

cated DD; both receptors are unable to in-

duce apoptosis after binding of TRAIL

45,46

OPG is a soluble receptor that can be re-

leased from the cardiovascular system, gas-

trointestinal tract, lung, kidney, bone, and

immune cells

. OPG binds TRAIL and many

ligands, including another member of the

TNF family, the receptor activating nuclear

factor kB ligand (RANKL)

Intrinsic pathway: mitochondrial death

pathway

Intrinsic apoptosis is activated from

within the cell in response to injury or other

forms of stress, such as DNA damage, lack

of oxygen, UV radiation, nutrient or survival

signals, oxidizing agents, drugs, and growth

factors

(Fig. 4).

Although mitochondria were consid-

ered a passive element in apoptotic cell

death, which only reflected damage to

critical functions due to cell death

, this

apoptotic pathway depends on the release

Fig. 4. Potential signals of the activation of the intrinsic apoptotic pathway.

PBK: Protein kinase B; AKT: serine-threonine kinase (also known as protein kinase B (PKB) phosphorylated by

PDK1 kinase.; BAD: Bcl-2 agonist of cell death; BIM: B-cell lymphoma 2 interacting mediator of cell

death; BIMF: proapoptotic protein BIM family; BID: BH3-interacting domain death agonist; BCL-XL:

B-cell lymphoma-extra-large; BCL-2: B-cell lymphoma 2; BAX: bcl-2-like protein 4; NOXA: (Latin for

damage) is a proapoptotic member of the Bcl-2 protein family; p53: Tumor protein P53; PUMA: p53

upregulated modulator of apoptosis.

In signaling activation of the mitochondrial pathway of apoptosis, the BH3 domain is essential for apoptotic

activity. Proteins that inhibit apoptosis and/or promote cell survival include Bcl-2, and Bcl-XL, loca-

ted in the outer mitochondrial membrane, and the proteins Bim, Bad, Bid, Bimf, and Bax are found

mainly in the cytosol and can be translocated to mitochondria in response to apoptotic stimuli.

Mechanisms of programmed cell death. A narrative review 239

Vol. 65(2): 230 - 252, 2024

into the cytosol of mitochondrial proteins

that normally reside in the intermembrane

space

Cytochrome-C

Cytochrome C is a protein that partici-

pates in the electron transport chain located

in the intermembrane space of the mito-

chondria, and that can be used as a biomark-

er of the apoptosis process

. Cytochrome C,

performs an entirely different function; after

being released into the cytosol, it binds to

a procaspase-activating protein called apop-

totic protease-activating factor-1 (Apaf1),

causing the oligomerization of Apaf-1 into a

heptameric wheel-like structure called apop-

tosome

. In the apoptosome, Apaf1 pro-

teins recruit initiator procaspase molecules

(procaspase-9); these are activated into Cas-

pase-9 and induce an apoptotic cascade acti-

vating one of the following chain executing

procaspases-3, -6, and -7 inducing apopto-

sis

(Fig. 5).

Bcl-2 family proteins

Bcl-2 family of proteins controls and reg-

ulates the entire process of intrinsic apopto-

sis. Bcl-2 family proteins actively participate

in this pathway, which, through interactions

between them, regulates the permeabili-

zation of mitochondria and the release of

apoptogenic proteins into the cytosol

. The

Bcl-2 family of proteins contains at least one

conserved domain, known as Bcl-2 homology

domains (BH [BH1, BH2, BH3 and BH4]).

The proteins of the Bcl-2 family are classi-

fied based on their function and structure:

i) Antiapoptotic proteins, which contain the

domains BH1 and BH2; ii) Proapoptotic pro-

Fig. 5. Intrinsic pathway by Cytochrome C-mediated apoptosis.

Apaf-1: Apoptotic protease-activating factor 1; IAP: Inhibitors of Apoptosis Protein; PARP: Poly (ADP-ribose)

polymerase; BID: BH3 Interacting Domain Death Agonist; BIM: B-cell lymphoma 2 interacting me-

diator of cell death; BAD: Bcl-2 agonist of cell death; BCL-2: B-cell lymphoma 2; MCL-1: Induced

myeloid leukemia cell differentiation protein; BCL-XL: B-cell lymphoma-extra-large; BAX: bcl-2-like

protein 4; BAK: Bcl-2 homologous antagonist/killer; Smac / DIABLO: Second Mitochondria-derived

Activator of Caspase.

Release to the cytosol binds to the Apaf-1 protein and procaspase-9, forming the apoptosome complex, indu-

cing caspase-9 and the caspase activation cascade.

240 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

teins containing the BH1, BH2, and BH3 do-

mains; iii) Proapoptotic proteins containing

only the BH3 domain

. Most of the antiapop-

totic members maintain sequence conserva-

tion in their four domains, while those with

proapoptotic activity have less conservation

of the first α-helix BH4 segment. Thus, the

proteins of the Bcl-2 family are proapoptotic,

and others are antiapoptotic. They can bind

to each other in various combinations and

form heterodimers in which the two proteins

mutually inhibit each other. However, when

a more significant proportion of these activi-

ties occur, the cell’s susceptibility to death

or survival is determined

55,56

Antiapoptotic proteins

Antiapoptotic Bcl-2 proteins, such as

Bcl-2 itself, Mcl-1, Bcl-w, A1, and Bcl-XL, are

found on the cytosolic surface of the outer

mitochondrial membrane, endoplasmic re-

ticulum, and nuclear envelope, where they

help maintain membrane integrity. This

group of proteins inhibits apoptosis and/

or promotes cell survival

. The three anti-

apoptotic proteins of the Bcl-2 family, Bcl-

2, Bcl-XL, and Mcl-1, prevent the activation

of the mitochondrial apoptosis pathway, as

demonstrated in multiple myeloma (MM)

In MM, the defect in the cell death pathways

is frequently due to an imbalance in the ex-

pression of the Bcl-2 family proteins. The

Bcl-2 gene has been implicated in resistance

to apoptosis induced by dexamethasone but

not melphalan in patients with MM. Bcl-XL

is expressed in most MM cell lines and cells

from patients; increased expression is fre-

quently detected in the relapsed patient and

correlates with resistance to chemotherapy.

Mcl-1 is expressed in virtually all MM lines

and fresh cells from patients. The induction

of apoptosis in MM cells has been related to

a decrease in the expression of Mcl-1

. Also,

for acute myeloid leukemia (AML), higher

expression of Bcl-2, Bcl-XL, and Mcl-1 and

lower expression of Bax increase resistance

to apoptosis in CD34+ populations than in

CD34- populations, mainly due to AML

Furthermore, increased expression of Bcl-

2 and Bcl-XL blocks doxorubicin-induced

apoptosis. Mcl-1 levels are increased in pa-

tients with recurrent AML

Pro-apoptotic Bcl2 proteins

Proapoptotic Bcl-2 proteins comprise

two subfamilies: the BH1-4 proteins that

share four different homology domains (Bcl-

2-associated X protein [Bax] and Bcl-2 ho-

mologous antagonist/killer [Bak] and the

proteins with restricted homology to BH3.

The BH3 domain is essential for apoptotic

function

. Among the members of the Bcl-2

family that induce apoptosis, with bounded

homology to the BH3 domain, the following

proteins are grouped: Bcl-2-interacting pro-

tein BIM (Bim), Bcl-2 agonist of cell death

(Bad), Bid, Bcl-2 adenovirus E1B 19kDa-

interacting protein 1 NIP3 (Bnip3), BMF,

HRK, Noxa and p53 upregulated modulator

of apoptosis (PUMA). These proteins are the

largest subclass of the Bcl-2 protein family

. Bak protein is tightly bound to the outer

membrane of the mitochondria even in the

absence of an apoptotic signal, whereas Bax

is localized primarily in the cytosol and only

translocates to the mitochondria if an apop-

totic signal is activated. Bax and Bak acti-

vation depend on activated “BH3 one” pro-

apoptotic proteins. Bax and Bak act on the

endoplasmic reticulum’s and nuclear mem-

branes’ surface; when activated in response

to endoplasmic reticulum stress, they re-

lease Ca

from the endoplasmic reticulum

into the cytosol. Bax and Bad are essential

gateways for cell death through mitochon-

dria

. Restricted homology to BH3 proteins

provides the crucial link between the apop-

totic stimulus and the intrinsic apoptosis

pathway; its BH3 domain binds to a long hy-

drophobic groove of the Bcl-2 antiapoptotic

proteins and neutralizes or inhibits their ac-

tivity. In some cells, the extrinsic apoptotic

pathway recruits the intrinsic pathway by

amplifying the caspase cascade that kills the

cell. In this way, Bid is the link between the

Mechanisms of programmed cell death. A narrative review 241

Vol. 65(2): 230 - 252, 2024

two pathways. Also, Bid, Bim, and PUMA can

inhibit all Bcl-2 antiapoptotic proteins

62,63

Klee et al.

have investigated mitochondrial

membrane permeabilization, which depends

not only on the canonical mitochondrial Bak

and Bax pathway to activate the death pro-

gram. Therefore, these investigators have

also found that the “only-BH3” molecules,

Bim and PUMA, can induce the release of

cytochrome c and apoptosis with the mere

presence of Bak in the endoplasmic reticu-

lum

. This pathway for transmitting apop-

totic signals from the endoplasmic reticu-

lum to mitochondria involves coordinated

communication mediated by the calcium

and ER1α/TRAF2 ER-stress surveillance ma-

chinery

61,63,65

Second Mitochondria-derived Activator

of Caspase (Smac / Diablo)

Second mitochondria-derived activa-

tor of caspase (Smac / Diablo) binds by its

N-terminal end to the mitochondria and, in

the intermembrane space, proteolyzes, leav-

ing free the domain that allows its union

with the IAPs

. The loss of mitochondrial

potential simultaneously triggers the re-

lease of cytochrome-C and Smac/Diablo.

Smac/Diablo, in the cytoplasm, is capable

of binding to IAPs (XIAP, c-IAP1, c-IAP2, and

survivin), inhibiting their function and en-

hancing caspase activation and triggering

the mitochondrial apoptosis pathway

. The

release of Smac / Diablo is inhibited by Bcl-

2 and Bcl-xL

. In MM cells, Smac plays a

functional role in mediating the activation

and apoptosis of Caspase-9 induced by dexa-

methasone treatment

Caspase-independent pathways

of apoptosis

Mitochondrial caspase - independent

pathway

The loss of mitochondrial potential in-

creases the permeability of the mitochondri-

al membrane, and the result is the release of

these proteins, such as AIF (apoptosis-induc-

ing factor) and Endonuclease G (Endo-G)

that activate a caspase-independent apopto-

sis pathway

AIF (Apoptosis Inducing Factor)

Apoptosis-inducing factor (AIF) is a

highly phylogenetically conserved protein,

essential for embryonic development, which

is synthesized in the form of an immature

precursor; it is translocated to the mito-

chondria, and in the intermembrane space it

is proteolyzed, the mature form has oxidase

activity

. In response to death signals, AIF

leaves the mitochondria and travels through

the cytosol to the nucleus, where it binds to

DNA, causing chromatin condensation and

DNA fragmentation into fragments of ap-

proximately 50 kb

(Fig. 6). AIF is neces-

sary to induce PARP-dependent death. The

processing and activation of PARP occur in

response to DNA damage. PARP initiates a

signal in the nucleus that induces the re-

lease of AIF from the mitochondria. AIF then

moves from the mitochondria to the nucleus

and induces chromatin condensation and

DNA fragmentation

69,70

Endonuclease G (Endo-G)

Endonuclease G (Endo-G) is an essen-

tial protein for mitochondrial DNA replica-

tion. It was isolated from the mitochondrial

fraction treated with the proapoptotic active

form of Bid: tBid. Once released to the cyto-

sol, it is transferred to the nucleus, where it

fragments DNA, even in the presence of cas-

pase inhibitors (Fig, 6). Endo-G cooperates

with exonuclease and DNase, facilitating

DNA processing

71,72

. Apoptotic endonucle-

ase acts cooperatively to fragment DNA and

ensure the irreversibility of apoptosis. How-

ever, very little is known about the potential

regulatory linkages between endonucleases.

Therefore, deoxyribonuclease deactivation is

caused by cutting. Also, alternative splicing

of DNase I pre-mRNA skipping exon 4 occurs

in response to overexpression of Endo-G in

242 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

cells

16,72

. Likewise, a strong correlation was

identified between the expression levels of

Endo-G and DNase I splice variants in human

lymphocytes. In fact, T cells downregulate

the mRNA levels of the active full-length DN-

ase I variant. They also upregulate the levels

of the inactive spliced variant, which acts in

a dominant-negative fashion

70,71

Necroptosis

Necroptosis is a form of programmed

cell death since it is genetically regulated;

it is characterized by cell inflammation, mi-

tochondria dysfunction, plasma membrane

permeabilization, and the release of cyto-

plasmic content into the extracellular space,

causing inflammatory reactions in the cells

of the surrounding tissue. This form of cell

death is also associated with mitochondrial

reactive oxygen species (ROS) and, unlike

apoptosis, does not involve DNA fragmen-

tation

. Necroptosis has been reported to

occur in a wide range of human diseases, in-

cluding retinal ischemia-reperfusion injury,

acute pancreatitis, brain trauma, retinal

detachment, and Huntington’s disease

73,74

In addition, necroptosis has been linked to

models of inflammation, including intestinal

inflammation and systemic inflammatory re-

sponse syndrome (SIRS)

75,76

. Perhaps the de-

tailed knowledge of this cell death pathway

can be used to develop drugs that temporar-

ily prevent or block this process to delay the

death of some types of cells or, conversely, it

could also serve to eliminate selectively, for

example, tumor cells

Activation Pathways of Necroptosis

Necroptosis is triggered as a form of

immunity against pathogens, under poor

conditions to trigger apoptosis. In necrop-

tosis, as in apoptosis, TNF activates TNFR1,

which induces the activation of a serine/

threonine kinase interaction protein (RIP1),

making integrating a joint inflammatory

and necroptotic response possible

. RIP3

is activated upon phosphorylation by the

serine/threonine kinase RIP1

. Necropto-

sis is RIP3-dependent as RIPK3 protein ki-

nase activity determines whether cells die

by apoptosis or necroptosis. Perhaps necrop-

tosis is fundamentally characterized by the

Fig. 6. Proapoptotic factors as Cytochrome-C, Smac/Diablo, Endonuclease, and Apoptosis Inducing Factor

are released from the mitochondria.

RIP3: Receptor-interacting serine/threonine-protein kinase 3; RIP1 Receptor-interacting serine/threonine-

protein kinase 1; FLIPs: FLICE-inhibitory proteins; FAAD: Fas-associated death domain protein.

In response to death signals, AIF leaves the mitochondria and moves through the cytosol to the nucleus,

where it binds to the DNA, causing chromatin condensation and DNA fragmentation.

Mechanisms of programmed cell death. A narrative review 243

Vol. 65(2): 230 - 252, 2024

activation of RIP1 or RIPK3, while the cas-

pase cascade is inhibited

. Giampietri et al.

have proposed a model that differentiates

the production of cell death by apoptosis

and by necroptosis; the dimerization of Cas-

pase-8 produces apoptosis while in necrop-

tosis it does not occur. The dimerization of

C-Flip S and caspase-8 produces a reduction

in caspase-8 activity, and it may not produce

either apoptosis or necroptosis, finally, the

heterodimerization of C-FLIPs and Caspase-8

produces inhibition of Caspase-8 and leads

to the production of necroptosis (Fig. 7).

Regulation Pathways of Necroptosis

Liu et al.

have shown that the Akt

and mTOR pathways regulate necroptosis by

inducing RIPK1 activation in neuronal cell

death. Just as it has also been verified that

necrostatin -1 is an inhibitor of all the bio-

chemical events carried out in this type of

cell death. In addition, other investigators

have reported that necroptosis is paired

with a mixed lineage kinase domain-like pro-

tein (MLKL) gene, an important substrate

of RIP3, and the plasma surface pores are

constituted by said protein (Fig. 8). These

pores cause the absorption of too much wa-

ter, so the cells ultimately burst. The block-

ade of MLKL activity leads to the inhibition

of necroptosis

. In this sense, Dondelinger

et al.

have proposed that a domain of four

activated MLKL molecules is required to

induce its oligomerization and trigger cell

death.

On the other hand, it has been found

that phosphatidylinositol (PIP) recruits the

MLKL protein to the plasma membrane.

Of note, recombinant MLKL lacks positive

charges and induces leakage of liposomes

containing both PIP and BAX, supporting a

model in which MLKL induces necroptosis

by directly permeabilizing the plasma mem-

brane. Consequently, inhibition of PIP for-

mation specifically inhibits TNF mediated by

necroptosis but not apoptosis

Autophagy

The term autophagy was introduced in

1996 by De Duve and Wattiaux, who defined

the vacuolization process for transporting

intracellular material to lysosomes for deg-

radation

. Autophagy is derived from the

Greek auto and phagos; it literally means

“self-feeding.” Its function mainly regulates

intracellular homeostasis since cytoplasmic

materials (long-lived proteins and damaged

organelles) are degraded in lysosomes and

recycled to produce new building blocks and

maintain energy metabolism

. From a mor-

phological point of view, autophagy has been

classified as a form of programmed cell death

associated with the massive accumulation of

autophagosomes in the cytoplasm, which

frequently, but not always, seems to be ac-

companied by increased blood flow; massive

autophagy triggers caspase-independent,

necrosis-like death

. It has been shown that

autophagy participates in natural processes

such as growth, embryonic development,

or aging. Also, it participates in the death

that occurs in mammary cells after lactation

and the death of some cancer cells that lack

apoptotic modulators, such as Bax and Bak

or caspases

85,86

. Dysfunction of this process

has been linked to cardiovascular and respi-

Fig. 7. Pathway of specific activation of necroptosis.

RIP3: Receptor-interacting serine/threonine-protein

kinase 3; RIP1 Receptor-interacting serine/

threonine-protein kinase 1; FLIPs: FLICE-in

hibitory proteins; FAAD: Fas-associated death

domain protein.

Necroptosis signaling pathways mainly comprise hete

rodimerization to c-FLIPs that reorganize the

catalytic site procaspase-8, producing caspa

se-8 inhibition, which induces necroptosis.

244 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

ratory diseases, neurodegenerative diseases,

metabolic diseases, and cancer

86,87

Autophagy is a type of programmed cell

death since more than 30 genes have been

identified in yeast that regulate autophagy,

and it is seen as a survival mechanism to com-

bat environmental stress factors

. Autopha-

gy could be induced in response to oxidative

or metabolic stress and can also be induced

through starvation, which is very commonly

used in research

. On the other hand, to

demonstrate that cell death in an in vivo or

in vitro model is caused by autophagy, it is

necessary to demonstrate that said death is

inhibited by agents interrupting the autopha-

gic pathway

. These agents can be chemi-

cal (agents directed against VPS34), genetic

(siRNA 3-methyl-adenine), or modulators of

autophagy (AMBRA 1, ATG 5, beclin)

Types of autophagy

Three types of autophagy are identified: i)

macroautophagy; ii) microautophagy; iii) chap-

erone-mediated autophagy

. The macroau-

tophagy process, as described by Kwanten et al.

begins with the formation of a phagophore,

a double membrane structure (also known as

an isolation membrane) that sequesters cyto-

plasmic material (long-lived proteins and or-

ganelles), which subsequently elongates to cre-

ate an autophagosome. The autophagosome

fuses with a lysosome to form an autolysosome,

where its contents will be degraded by lyso-

somal proteases (e.g., cathepsins) and other

hydrolytic enzymes

90,91

. According to Kwanten

et al.

88,

phagophore formation is regulated by

the ULK1 complex (initiation), which is under

the control of the mammalian target of ram-

pamycin (mTOR) complex and the beclin-1/

VSP34 interaction complex (nucleation). Two

large, conjugated ubiquitin-like complexes

are responsible for double membrane elonga-

tion: light chain 3 (LC3)-II and ATG5-Atg12-

ATG16L1. ATG7 is one of the proteins required

to form both elongation complexes. Autopha-

gosomes are generated on or in the vicinity of

the endoplasmic reticulum. However, it is not

clear whether the ER membrane is used di-

rectly for autophagosome formation. Recent

studies suggest that additional membranes

derived from the Golgi complex, mitochondria,

and the plasma membrane also contribute

to autophagosome formation

90,91

. Therefore,

autophagosome formation involves multiple

and complex processes

. Macroautophagy is

considered to play the most critical role in au-

tophagy

(Fig. 9).

Fig. 8. Recruitment of Mixed Lineage Domain-Like Protein Kinase by Phosphatidylinositol. Pathway to the

plasma membrane.

MLKL: Mixed Lineage Domain-Like Protein Kinase

The MLKL protein has become a specific and crucial protein. The 4-Helical Bundle Domain (4HBD) in the

N-terminal region of MLKL is required to induce its oligomerization. and trigger cell death.

Mechanisms of programmed cell death. A narrative review 245

Vol. 65(2): 230 - 252, 2024

Microautophagy process is considered

when a small portion of the cytoplasm is di-

rectly involved by the lysosome / late endo-

some (Fig. 10). Autolysosome formation is

mediated by the accumulation of small mem-

brane structures that envelop portions of

the cytoplasm. Phagophores are not formed,

and membrane structures invaginate direct-

ly into lysosomes, where degradation occurs

by direct absorption of cytoplasmic cargo.

In Chaperone-Mediated Autophagy

(CMA), the proteins to be degraded are

delivered selectively to lysosomes; they are

recognized by heat shock-like proteins 70

(HSC70) and by co-chaperones; proteins

in the degradation phase are internalized

through lysosomal membrane-associated

protein 2A (LAMP2A)

91,92

(Fig. 11).

Autophagy: a selective process

Degradation in the autophagy process

was believed to be non-selective; however, it

has been determined that there are selective

pathways to digest specific components, such

as “mitophagy” or selective autophagy of mi-

tochondria

, “peroxyphagy” (peroxisomes),

“ribophagy” (ribosomes) or “xenophagy”

(invading microbes), this phenomenon is

called selective autophagy, and in the case

of mitochondria, it serves to maintain their

homeostasis

70,84

. Thus, macroautophagy

can be non-selective (random uptake of in-

tracellular material) and selective (specific

load capture). The morphological and bio-

chemical characteristics of autophagy and

apoptosis are different. In this regard, cells

undergoing autophagy show an increase in

autophagic vesicles (autophagosomes and

autophagolysosomes). While chromatin

condensation is partial in autophagic cells,

DNA fragmentation does not occur. The two

processes, autophagy and apoptosis, are not

always mutually exclusive and can occur si-

multaneously in the same type of cells

In summary, homeostasis is maintained

between cells produced by mitosis and cell

death in the human body. In this sense, this

study is a narrative review that reports scien-

tific research in which an attempt has been

made to group programmed cell deaths,

explaining the molecular mechanisms that

involve structural and functional proteomic

pathways that intervene by inducing and

inhibiting each one of the proteomic path-

ways. In our study, caspase-dependent pro-

Fig. 9. Macroautophagy process from isolation membrane to autolysosome.

In the autolysosome, the inner membrane and luminal content of the autophagic vesicle are degraded by

lysosomal enzymes that act optimally within this acidic compartment.

Fig. 10. Endosomes result from the microautopha-

gy process.

Microautophagy is conserved from yeast to mam-

mals and contributes to the degradation of

organelles (e.g., peroxisomes, ER, nucleus),

protein complexes such as the proteasome,

and single proteins.

246 Fernández-Lázaro et al.

Investigación Clínica 65(2): 2024

grammed cell deaths and caspase-indepen-

dent programmed cell deaths are described.

Although classifying and describing pro-

grammed cell death processes is somewhat

complex, depending on which aspects are

analyzed, their grouping and knowledge of

the factors that trigger cell death vary great-

ly. This study could offer the basis for the de-

sign of new pharmacological treatments and

discover new potential molecular biomarkers

for early diagnosis that serve to cure or mod-

ulate the course of some diseases. For this,

it is necessary to understand the proteomic

signaling mechanisms of programmed cell

death since their alteration contributes to a

wide variety of diseases, one of which is can-

cer, which constitutes a global public health

problem due to its high mortality.

ACKNOWLEDGMENTS

The authors want to thank the SARCEL-

LOMICS® Research Group (Spain), for their

collaboration on infrastructure computer

support and for presenting their collabora-

tion in advising on the methodological de-

sign and interpretation of the results.

Funding

No funding was received.

Competing interests

The authors declare that they have no

competing interests.

ORCID numbers of authors

• Diego Fernández-Lázaro (DFL):

0000-0002-6522-8896

•

Begoña Sanz (BS):

0000-0002-6354-6401

• Jesús Seco-Calvo (JSC):

0000-0002-7818-9777

Authors’ contributions

DFL: searched and selected the litera-

ture and wrote the original draft. JSC and

BS conceived the study and reviewed and

edited the manuscript. DFL designed and

created the figures. Data authentication is

not applicable. All authors have read and ap-

proved the final manuscript.

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