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Humanin: A mitochondrial-derived peptide in the treatment of

apoptosis-related diseases

Abu Hazafa, Ammara Batool, Saeed Ahmad, Muhammad Amjad,

Sundas Nasir Chaudhry, Jamal Asad, Hasham Feroz Ghuman,
Hafiza Madeeha Khan, Muhammad Naeem, Usman Ghani

PII: S0024-3205(20)31432-6
DOI: https://doi.org/10.1016/j.lfs.2020.118679
Reference: LFS 118679

To appear in: Life Sciences

Received date: 24 August 2020

Revised date: 19 October 2020
Accepted date: 25 October 2020

Please cite this article as: A. Hazafa, A. Batool, S. Ahmad, et al., Humanin: A
mitochondrial-derived peptide in the treatment of apoptosis-related diseases, Life Sciences
(2018), https://doi.org/10.1016/j.lfs.2020.118679

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as the addition of a cover page and metadata, and formatting for readability, but it is
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© 2018 Published by Elsevier.

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Humanin: A Mitochondrial-derived Peptide in the Treatment of

Apoptosis-related Diseases
Abu Hazafaa, Ammara Batoola, Saeed Ahmadb, Muhammad Amjadc, Sundas Nasir
Chaudhrya, Jamal Asadd, Hasham Feroz Ghumanc, Hafiza Madeeha Khane, Muhammad
Naeemf, Usman Ghania
Affiliations
a
Department of Biochemistry, Faculty of Sciences, University of Agriculture, Faisalabad,
38000, Pakistan
b
Centre of Biotechnology & Microbiology, University of Peshawar, Pakistan

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c
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture,

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Faisalabad, 38000, Pakistan -p
d
Department of Biochemistry, University of Health Sciences Lahore, Pakistan
e
School of biological sciences, University of the Punjab, Lahore, Pakistan
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f
College of Life Sciences, Hebei Normal University, China
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Running Head: Humanin as anti-death agent

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Total words: 4900 (excluding references, tables, and figure captions)

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Funding: None

Figures: 3
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Tables: 2

Correspondence author:

(Abu.9093270@talmeez.pk) A. Hazafa

Phone: +92-305-6517149

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Abstract

Humanin (HN) is a small mitochondrial-derived cytoprotective polypeptide encoded by

mtDNA. Humanin exhibits protective effects in several cell types, including leukocytes, germ
cells, neurons, and tissues against cellular stress conditions and apoptosis through regulating
various signaling mechanisms, such as JAK/STAT pathway and interaction of BCL-2 family
of protein. The HN is an essential cytoprotective peptide in the human body that regulates the
mitochondrial functions under stress conditions. The present review aims to evaluate HN
peptide’s antiapoptotic activities as a potential therapeutic target in the treatment of cancer,
diabetes mellitus, male infertility, bone-related diseases, cardiac diseases, and brain diseases.

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Based on in vitro and in vivo studies, HN significantly suppressed the apoptosis during the
treatment of bone osteoporosis, cardiovascular diseases, diabetes mellitus, and

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neurodegenerative diseases. According to accumulated data, it is concluded that HN exerts
the proapoptotic activity of TNF-α in cancer, which makes HN as a novel therapeutic agent in
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the treatment of cancer and suggested that along with HN, the development of another
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mitochondrial-derived peptide could be a viable therapeutic option against different oxidative
stress and apoptosis-related diseases.
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Keywords: Humanin; Cell survival; Cytoprotection; Apoptosis; Infertility; Mitochondria-

derived peptide; Diabetes mellitus; Cancer; Oxidative stress; BCL-2.
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1. Introduction
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Humanin (HN) is a natural short mitochondrial polypeptide with antiapoptotic and

neuroprotective effects [1]. HN was initially discovered in 2001 by Hashimoto using a cDNA
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library made from tissues of a healthy portion of Alzheimer’s patient brain [2-4]. HN was the
first mitochondrial-derived peptide (MDP) [5] that encoded by 75-bp in an open reading
frame (ORF) portion within mitochondrial 16S ribosomal RNA (rRNA) [6, 7]. The relatively
small HN structure allowed researchers to identify each amino acid within the polypeptide
chain and its role through a systematic single amino acid substitution technique [8]. Under
physiological conditions, HN is produced by tissues in several organs, including kidney,
skeletal muscles [9, 10], brain, heart, and liver [11, 12]. After endogenous production of HN
by cells, it secreted into the blood circulation and transported to several target cells [11].
Recently published data suggested that endogenous peptide (HN) protects cells against
various diseases, including oxidative stress and cytotoxicity induced by various stimuli [13]
with broad-spectrum activity on pancreas, brain, testis, and heart [4, 14]. Besides

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cytoprotection, HN administration also has several metabolic benefits in promoting and

improving insulin sensitivity and cardiac functions [15, 16]. HN considers a fundamental
molecule in cell metabolism, and its role as a source of endocrine/paracrine/autocrine
protective stress response factors has been extensively demonstrated in a recent couple of
years [17]. Based on the observations, HN becomes an attractive candidate in recent years
due to many biological progressions, including anti-apoptosis, cell stress response
inflammation, and substrate metabolism [18-20].

HN is a mitochondrial-derived peptide that is produced in response to cellular stress [21]. In

human, HN is produced from small gene peptide such as Humanin that found within 16S
ribosomal RNA gene (MT-RNR2) in the mitochondria genome [22]. HN peptide contains 21

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amino acids when its mRNA is translated within the mitochondria and 24-amino acids when

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its peptide moves to the cytoplasm. However, the number of amino acids in HN depends on
translational site [21, 23]. The study revealed that both forms of HN are functional and
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significantly inhibit apoptosis in many diseases including cancer, but it is still unclear which
is more common. This support HN's multiple roles as a signaling molecule and suggests
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unappreciated complexity [11]. The emerging evidence suggested that the circulation level of
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HN varies with age that could act as a critical biomarker for many pathological conditions.
The HN exerts its functions by binding through the trimeric receptors. Therefore, HN
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transported outside the cell immediately after its translation to show its cytoprotective action
[17].
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HN itself appears to be significant in protecting cell death in many tissues [22]. HN secreted
from cells that bind to cellular membranes and also found in plasma [24]. HN binds to
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trimeric receptors composed of the cytokine receptor WSX-1, heterotrimeric transmembrane

gp130, and ciliary neurotrophic factor receptor α (CNTFR). Moreover, an endogenous
peptide, HN is an agonist peptide of the formyl peptide receptor 2 (FPRL-2) [25-27]. HN also
interacts with those receptors, which involve in WSX-1/gp130/CNTFR and induce
heterodimerization of intracellular domains of WSX-1 and gp130, and mediates the HN
signals mainly via JAK2, MAPKs, JNK, and STAT3 pathways. The dimerized STAT3 moves
to nucleus where it regulates gene expression to exert cytoprotective action. The evidence
reported that HN showed its cytoprotective function by regulating the intrinsic or
mitochondrial apoptotic pathway via mediating intracellular localization of protein family of
BCL-2. HN upregulates the expression of BCL-2 and suppresses the BAX expression to
inhibit apoptosis [17, 28]. Moreover, HN could also be made interaction with other BCL-2

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protein family members like BID and BAX, and regulates their translocation to suppress the
production of apoptosome and promote the expression of caspase-3 in mitochondria [29].

Apoptosis is the most important cause of death in many cell lines such as neurons, male germ
cells, and lymphocytes [30, 31]. Many diseases, including neurodegenerative, autoimmune,
cardiovascular, cancer, and infectious diseases that are established as a leading cause of
disability and death worldwide, are caused due to insufficient or excessive apoptosis [32].
The accumulated data reported that HN significantly suppressed the chemotherapy-induced
apoptosis in normal blood cells while sensitized tumor cells to chemotherapy [33]. Age-
related diseases occur due to the dysfunction of mitochondrial peptides and oxidative stress.

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Recently, many studies revealed that HN plays a vital role in reducing apoptosis in age-
related diseases and myocardial fibrosis by targeting oxidative stress [34]. Similarly, Qin and

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his co-workers observed that HN treatment effectively reduced the age-related diseases,
including apoptosis and myocardial fibrosis in rat models, and suggesting that HN could be a
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viable option in reducing age-related diseases [35].
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The present review aims to investigate the events associated with apoptosis condition and to
evaluate the antiapoptotic effect of humanin (HN) under various stress conditions as a
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therapeutic target in the treatment of cancer, diabetes mellitus, male infertility, bone-related
diseases, cardiac diseases, and brain diseases. Moreover, the present review discussed the
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structure and mechanism of HN for a better understanding.

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2. Structure of HN peptide
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According to emerging evidence, HN is a first member of a new class of signaling molecule

that is initially identified from the mitochondrial genome (see Fig. 1 (a). Initially, HN was
revealed as a neuroprotective molecule, but later it was noticed that HN regulates many
pathways, including apoptosis in several diseases like growth hormone, cancer, and brain-
related diseases [36]. The accumulated data revealed that HN is composed of 24 or 21-
different amino acids including Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-
Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, which are encoded by a 75-bp open reading
frame (ORF), and each amino acid have a distinct function as presented in Fig. 1 [8]. HN is
transcribed by a polycistronic mitochondrial gene, namely MT-RNR2, which encodes the
16S (large ribosomal subunit) of mitochondrial RNA (mtRNA) [17].

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According to a recent study, number of amino acids (24 or 21) in HN depends on

translational sites in the cytoplasm or mitochondria. However, the exact site of translation is
not fully understood yet. A few recent studies on rats revealed that HN translation was carried
out in mitochondria, which conclusively showed antiapoptotic activities [37]. HN bears three
regions, including negatively charges C-terminal (PVKRRA), positively charged N-terminal
(MAPR), and central hydrophobic region (GFSCLLLLTSEIDL). Maftei et al. [38] reported
that the residues from 3 to 19 are fundamental domain or core domain for HN
neuroprotective function. They also observed that this region directly bonded with Amyloid β
protein (Aβ) and prevent the self-aggregation. The aggregation of Aβ (39-43 amino acids)
oligomer is a crucial stage in the pathogenic process of neuron associated diseases like

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Alzheimer’s disease (AD), that promotes the neuronal cell death and subsequent memory loss

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[39]. However, HN and its derivatives like Gly14-humanin (HNG) significantly showed a
neuroprotective effect in spatial memory protection and secured the STAT3 pathway from Aβ
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disruption by activating the tyrosine kinases [40].
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A study demonstrated that Cys at eight positions of HN-polypeptide chain is a critical amino
acid in antiapoptotic action [17]. Similarly, Bodzioch and his co-workers revealed that Pro at
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19-position could effectively facilitate the secretion of HN and Pro-Thr-Ser at 3, 13, and 14-
positions respectively helped in receptor binding (see Fig. 1 (b) [41]. Furthermore, a study
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stated that the secondary structure of HN also depends on specific amino acid sequences. For
instance, Ser at 7th and Leu at 9th position successfully prevents self-aggregation in dimer or
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oligomer of HN-polypeptide chain during neuroprotective function [17]. The circular

dichroism and NMR studies of synthetic HN in 2, 2, 2-trifluoroethanol (TFE; 30%) and
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aqueous solution revealed that HN exists in helical like structure with Gly5 to Leu18
sequence order spanning in 30% TFE solution and adopts unstructured confirmation in
equilibrium with a turn-like structure in an aqueous solution that provides nascent helix with
Gly5-leu10 and Glu15-Leu18 sequence order spanning [42]. Another NMR study of HN in
aqueous solution reported that Leu9 and Leu10 are proximal to Phe6 aromatic ring which
helped in forming a hydrophobic binding and broken by substitution of Leu10 either by
acidic or basic residue [43]. However, Ala-scanning analysis of Pro residues from 3 to 19 of
HN observed that Cys8, Leu12, Pro3, Leu9, Pro19, Ser14, and Thr13 are listed as crucial
residues for neuroprotective effects of HN. The findings also stated that substitution of these
residues by Ala (non-polar amino acid) did not affect the configuration of HN. However,
substitution of Ser14 by Gly significantly increased the neuroprotective activity of HN that

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might be due to the conformational flexibility of HN [42, 44]. The data reported that HN
exerts its mechanism either bind with membrane trimeric receptors or intracellular receptors,
including FPRL1 and CNTFR/WSX-1/gp130, and insulin-like growth factors to regulates the
cell survival ability in several diseases by inducing antiapoptotic activities, inhibiting
oxidative stress, cell proliferation, and promoting insulin sensitivity [45]. Similarly, Jia and
his research group revealed that few important analogs of HN, including HN-S7A, HN-C8P,
and HN-LI2A (antagonist) play a significant role in suppressing cyclophosphamide-induced
germ cell apoptosis [46].

3. HN and its mechanism of action

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The intrinsic or mitochondrial pathway of apoptosis is primarily regulated by members of the
B-cell lymphoma 2 (BCL-2) protein family [47]. A BCL-2 family of proteins includes both

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proapoptotic and antiapoptotic members. The members of BCL-2 family are usually arranged
based on their participation in apoptosis and the presence of different BCL-2 homology (BH)
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domains. BH enables the interactions of family members to facilitate antiapoptotic and
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proapoptotic functions [48]. Generally, BCL-2 protein family is classified into three major
subfamilies, including proapoptotic (BID, BIM, BAD, and BMF), antiapoptotic (BCL-XL,
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BCL-W, BCL-B, MCL-1, and MCL-1L), and pore-forming executioner proteins (BAX,
BAK, and BOK). Only proapoptotic and antiapoptotic subgroups are usually involved in the
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apoptosis regulation, and executioner proteins only participate in the activation of pore-
formation [48, 49]. Careful regulation determines the fate of a cell between life and death [32,
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50]. Pathologies of BCL-2 are of great prominence for disease states where apoptosis is mis-
regulated [32, 51]. To induce the intrinsic apoptotic pathway of cell death, proapoptotic BCL-
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2 homology domain (BHD) proteins interact with BH3-only domain (BOD) proteins to
trigger mitochondrial outer membrane permeabilization (MOMP) that resulted in apoptosis
(see Fig. 2) [32, 52, 53].

HN exerts antiapoptotic action through an intrinsic mitochondrial mechanism by modulating

expression and intracellular translocation of BCL-2 family of protein. HN analogs can
interact with proapoptotic protein, namely BAX, to upregulate BCL-2 expression [29, 54].
HN can effectively bind to BAX and tBID in the cytoplasm and prevent translocation across
the mitochondrial membrane that precludes pore formation facilitated by proapoptotic BCL-2
proteins. Although HN cannot move these proteins out of membrane, HN interaction with
membrane-bound BAX or tBID impairs the additional recruitment of cytosolic BAX. BCL-2-
associated X apoptosis regulator (BAX) is a critical factor to initiates the intrinsic apoptosis

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pathway. A study revealed that HN blocked the translocation of BAX in outer membrane of
mitochondria to prevents apoptosis, but the molecular mechanism is still unknown [55].
Recently, an in vitro study demonstrated that BAX and HN undergo secondary and tertiary
structural rearrangements and form fibers. A study also revealed that fibrillation process
undergoes the changes in antiapoptotic properties of HN, which prevents BAX to initiate
mitochondrial outer membrane permeabilization (MOMP) and subjecting the cells to
apoptosis [56]. HN also prevents BAX oligomerization in a membrane, induced by tBID,
which promotes the release of cytochrome c and other caspase activating factors into the
cytoplasm regulated by mitochondrial permeability transition pore (mPTP) [55, 57-59]. The
release of cytochrome c and other caspase factors formed a complex, namely the

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“apoptosome” in the cytoplasm, and activates a new caspase such as caspase 9. This caspase

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9 further activates the effector caspases that are committed to apoptosis [60].

Similar to the intrinsic apoptotic pathway, HN can also exert an antiapoptotic action through
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extracellular receptors (gp130, WSX-1, CNTFR-α, and FPLR-1), which subsequently
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modulates the expression of proteins of BCL-2 family. HN exerts its cytoprotective effect
from extracellular space through binding receptors such as formylpeptide receptor-like-1
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(FPRL1) and trimeric complex receptor on the cell surface. HN binds with FPRL1, which
activates the downstream signaling cascade through ERK1/2 signaling pathway and leads to
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an antiapoptotic effect, which subsequently improves cell survival [8]. A trimeric receptor is
composed of different subunits, including receptor for ciliary neurotrophic factor α (CNTFR-
α), WSX-1, and glycoprotein 130 kDa (gp130) subunits [11, 17, 61, 62]. Trimerization of
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receptors induce activation of Janus kinases (JAK1 and JAK2), which subsequently activate
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signal transducers and activators of transcription 3 (STAT3). The dimerized STATs

translocate to the nucleus where they help in regulating the transcription during cell survival
[29].

4. HN as a therapeutic target
The discovery of humanin (HN) attracted several researchers due to its small molecular size
(21 or 24 amino acids) and high target specificity. HN was the first and best-characterized
peptide among all mitochondrial-derived peptides (MDPs). Initially, the circulating level of
HN was associated with an increased lifespan in aged rat models. Later on, HN’s longevity
expends to a set of biological activities ranging from anti-apoptosis and oxidative stress in
several organs, including liver, kidney, and heart to control metabolic events [63]. The

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emerging evidence suggested that HN and its analogs (HN-S14G) are being investigated as
potential therapeutic targets in the treatment of various diseases, including Alzheimer's
disease, age-related disease, cancer, diabetes mellitus, and Creutzfeldt-Jakob disease [64, 65].
Herein, we discussed some diseases in which HN could be an innovative therapeutic option
soon.

4.1. HN and male infertility

Cancer chemotherapies have increased the life expectancy and survival of cancer patients, but
these therapies also exerted severe adverse effects (germ cell apoptosis). One of the long-term
adverse effects of chemotherapy is infertility in male cancer survivors. Many
chemotherapeutic agents, including Doxorubicin, caused apoptosis in male germ cells, but

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they also exert side effects [46, 66, 67]. The mechanism of action of HN and its analogs

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significantly enhanced the chemotherapy-induced tumor-suppressive effects with protective
outcomes on healthy tissues and cells by regulating the divergent signaling pathways [68,
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69]. The localization of HN in normal and abnormal morphology of spermatozoa showed
different protective effects [70]. The emerging evidence suggested that HN is a novel
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component in regulating testicular homeostasis. Testis is the only part of the body that
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produced an extensive quantity of HN. In rat testis, HN is articulated in peritubular, Leydig,

and endothelial cells but not expressed in Sertoli cells [67]. While in human testis, HN is
extensively expressed in mitochondria of ejaculated sperms and spermatocytes [71]. A study
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reported that HN did not play any role in spermatogenesis under physiological conditions.
However, it showed the protective character during the injury of testis caused by testosterone
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impoverishment on spermatocytes and by a drug like dexamethasone on Leydig cells [67,

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72]. Leu et al. [73] reported that intratesticular injection of HN into mice model significantly
reduced spermatocytes apoptosis expressed by testosterone impoverishment on spermatocytes
in a GnRH antagonist hormone-independent model. Another study also revealed that
cytoprotective effect of HN in spermatocytes was due to the binding of HN with trimeric
receptors like CNTFR/WSX1/gp130 that trigger the STAT3 pathway [74].

Experiments proved the antiapoptotic effect of HN and its analogs both in vivo and in vitro
studies using different chemotherapeutic agents, as represented in Table 1. HN has been
recognized in many species, including rats, nematodes, and mice (see Fig. 3) [75]. Another
study reported that germ cell apoptosis induced by testicular hormonal deprivation mediates
through HN by IGFBP3 (Insulin-like growth factor-binding protein-3) action [76-78]. Studies

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suggested that interactions between HN (antiapoptotic) and IGFBP3 (proapoptotic) may play
an essential role in the regulation of testicular diseases and spermatogenesis [66, 79-81].

4.2. Antiapoptotic activity of HN in bone-related diseases

Osteoporosis is represented as one of the most common bone-related diseases characterized
by impairment of bone loss with a higher risk of bone fracture. Due to high mortality and
morbidity rate, osteoporosis is presented as a significant threat to postmenopausal women and
older individuals [82]. Although apoptosis is a part of a normal physiological process in
maintaining bone homeostasis, if this process is deregulated, it can cause many apoptosis-
related bone diseases (osteoporosis) [83]. Osteoporosis is a common metabolic bone disorder
in thigh bone of the hip, vertebrae of the spine, and the wrist that mainly resulted from an

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imbalance in the birth and death of those (apoptotic) cells triggered by BCL-2 family [84].

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Glucocorticoids (GCs) are widely used as an immunosuppressive and anti-inflammatory drug
in children and adults suffering from chronic disease. Nevertheless, recent findings suggested
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that GC drug showed side effects on the growth of plate chondrocytes [85]. Dexa (a GC drug)
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induced apoptosis by increasing the expression of proapoptotic protein, BAX, which
resultingly activates the caspase-8 in proliferative chondrocytes [54]. A recent study on HN
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suggested that HN peptide effectively prevents glucocorticoids-induced bone growth

impairment. However, the mechanism of action of HN in preventing osteoporosis is not clear.
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HN did not disturb the anti-inflammatory action of GC drug; instead, it prevents apoptosis.
HN also carried anti-inflammatory activity [54]. A study reported that Gly14-humanin (HNG;
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HN derivative) blocked the activation of proapoptotic protein, namely BAX, and prevent the
formation of a proapoptotic truncated BID (tBID) protein [54]. A similar study [82] also
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described that HN analog, namely HNGF6A, has a protective role in the osteoblast apoptosis
and osteoblast phenotype against H2O2.

4.3. Cardioprotective effect of HN

Apoptosis plays a major role in various cardiovascular diseases (CVD), including cardiac
ischemia/reperfusion (I/R) injury. I/R injury induces myocardial damage and causes
significant production of oxidative stress [11]. Oxidative stress induces cardiac apoptosis, one
of the most important causes of cardiomyocyte loss during ischemia-reperfusion (I/R) [86-
88]. The reactive oxidative species (O2-) led toward oxidative stress when it reached a
threshold level and triggered the opening of inner membrane anion channels (IMAC) or
mitochondrial permeability transition pores (mPTP) and resultingly release the O2- anion
species across the membrane. However, the involvement of the mPTP in this context is rather

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related to a necrotic response, particularly in response to ROS. The prolonged opening of

mitochondrial permeability transition pores (mPTP) by ROS causes a rupture in cardiac cells
[89-94]. In the past few decades, many efforts were made to reduce oxidative stress and
apoptosis during cardiac injury [11]. HN and its analogs seem to reduce apoptosis (caused by
oxidative stress) by binding with several receptors such as trimeric receptor [90].

Thummasorn et al. [90] experimented on ischemia/reperfusion (I/R) injured rats and observed
that there was no difference in the endogenous HN levels before I/R injury, but the level of
HN was tremendously decreased after I/R injury. They also revealed that HN analogs
significantly decreased the infarct size and arrhythmia prevalence to improve cardiac
dysfunction and cardiac mitochondrial function. Similarly, another study demonstrated that

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the level of plasma endogenous HN was interestingly decreased in patients with acute

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coronary disease compared to healthy volunteers. HN’s reduction might be due to the defense
mechanism against I/R damage during the early stages, in which HN excessively circulated to
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the myocardial injury side [95]. Patel and his research group experimented on 19 NYHA II-
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IV heart failure (HF) patients and showed that level of endogenous HN was interestingly low
in HF patients after six months of treatment accessed by Wilcox Rank-Sum test (p=0.039).
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They also found very little correlation between serum HN (P=0.0162) and NT-proBNP,
which suggested that HN could be an innovative biomarker in risk stratification of patients
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with heart failure with reduced ejection fraction (HFrEF) [96].

4.4. Neuroprotective effect of HN in brain

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The accumulated data suggested that necrosis is one of the major modes of cell death caused
by an accidental or uncontrolled form of cell death. Necrosis is considered to be associated
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with many clinical conditions such as brain stroke, Alzheimer’s diseases, myocardial
infarction, and many other pathological conditions [2, 97]. The most common
neurodegenerative Alzheimer’s disease is characterized by profound neuronal cell loss,
amyloid plaques, and vascular damage from plaque deposition and tau neurofibrillary tangles
[98, 99]. Amyloid β-peptide (Aβ) is a crucial molecule in triggering the neurodegenerative
cascades of Alzheimer’s disease (AD) [100], which increased considerably in AD patients
[101] Zhao et al. [102] reported that HN effectively prevents brain neurons, well-maintained
cell viability, and protein phosphatase 2A (PP2A) activity. They also revealed that HN
successfully protected the cortical neurons against Calyculin A (CA)-induced neurotoxicity
by blocking the phosphorylation at Thr231, Ser396, and Ser199/202 sites.

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HN peptide reduced the oxidative stress and induced apoptosis by Aβ-peptide [103, 104]. HN
peptide works as an inhibitor that interacts with toxic Aβ-oligomer by interfering with Aβ-
peptide formation and its biological properties [105]. A study by Cohen et al. [106] suggested
that a novel HN derivative, namely AGA(C8R)-HNG17, protects neuronal cell necrosis both
in vivo and in vitro experiments. In vitro findings showed that AGA(C8R)-HNG17
impressively exerted the necrosis protective action in neuronal cell lines of NSC-34 and PC-
12. They also revealed that AGA(C8R)-HNG17 testified the necrosis protective effects in
traumatic brain injury associated mice model by targeting mitochondria and mediates ATP
synthase levels, and suggested that AGA(C8R)-HNG17 could be a viable therapeutic option
in the reduction of necrosis associated diseases like myocardial infarction, stroke, and

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traumatic brain injury. Other studies also found a new generation of HN peptide, Colivelin

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(CL), that exerts beneficial effects in neurodegenerative disease through binding to cell
surface receptors and subsequent activation of JAK/STAT3 signaling as a major pathway
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[107, 108]. CL protects against cell death induced by AD-causative genes and Aβ peptide,
even at very low concentrations (100 fM-10 pM) [107, 109].
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4.5. HN action in diabetes mellitus
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Diabetes mellitus (DM) is one of the most menacing diseases characterized by high blood
glucose level [110]. Pancreatic beta-cell (β-cells) dysfunction is one of the key contributors to
DM pathophysiology. High glucose (HG) level induces cell apoptosis and insulin secretion
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impairments [111]. Apoptosis of β-cells is the primary mode of cell death, vital for
pathogenesis and treatment of Type 1 diabetes [112]. HN is considered a novel cytoprotective
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hormone in DM, and represents a protective role in islet cell apoptosis and improves glucose
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tolerance in vitro and in vivo studies [10, 64, 113]. Recent studies demonstrated that isolated
islets express the BAX proapoptotic protein at a higher level than antiapoptotic protein BCL-
2 in diabetes [114]. HN and its analogs are useful for preventing and treating diabetes and
promoting β-cell survival, as presented in Table 2 [114]. A study conducted by Kwon et al.
[10] showed that systemic administration of HN inhibited β-cell apoptosis and islet
inflammation and delayed pathological progression of diabetes in non-obese diabetic mice.
Hoang et al. [112] observed that HN effectively controlled glucose tolerance, delayed onset
of diabetes in nonobese diabetic mice (NOD) over 20 weeks of treatment, and inhibited
apoptosis in β-cells in vitro induced by cytokine. Wang and his coworkers [115] reported that
HN impressively prevents high-glucose-induced affection of monocyte THP-1 cells to human
umbilical vein endothelial cells (HUVECs) to inhibits the hyperglycemia. They also stated

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that HN successfully blocked high glucose-induced secretion of interleukin-1β (IL-1β) and

tumor necrosis factor-α (TNF-α) to treat the hyperglycemia related endothelium dysfunction.
Muzumdar et al. [116] reported that intracerebroventricular administration of HN
significantly promotes insulin sensitivity in different body organs, including liver, which
results in the reduction of hepatic glucose production by inducing fatty acid metabolism and
Akt signaling pathway. The protective role of HN and its analogs offers the treatment of type
2 diabetes as well. HNGF6A has potent effects on insulin secretion with well-known effects
on insulin action [64]. Evaluation of the mechanism of action reveals that hypothalamic
STAT-3 activation is required for insulin-sensitizing action of HN. STAT-3 signaling and
activation in the hypothalamus are well known to play central role in treating diabetes [20,

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112, 116].

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4.6. HN and cancer
Cancer is a diverse group of diseases with uncontrolled growth of cells. Impaired apoptosis is
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recognized as a hallmark in cancer development [117]. Cells induce apoptosis due to the
accumulation of DNA mutations or oncogene activations (E1A, EGFR, and HER2/neu) and
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exposure to cellular stress [118]. Inadequately controlled apoptosis leads to the development
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of metastasis, malignancy, and chemotherapy resistance [119]. Apoptosis initiates either an

intrinsic (mitochondrial or BCL-2 -regulated) pathway or extrinsic (death receptor-mediated)
pathway [120]. The upregulation of BCL-2 family of proteins (intrinsic pathway) gained
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interest in the development of cancer, including diffuse large B-cell lymphoma (DLBCL) and
chronic lymphocytic leukemia (CLL) [51, 121]. Tumor necrosis factor α (TNF-α) ligand is
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required for the initiation of extrinsic apoptosis pathway [119, 122]. TNF-α ligand binds to a
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receptor and brings a conformational change in the intracellular region that promotes adapter
proteins and death-causing signaling complex formation [123]. TNF-α is an essential
pleiotropic cytokine expressed in several biological processes, including apoptosis,
proliferation, differentiation, and inflammation. TNF-α triggers two TNF-family receptors
such as TNF-R1 and TNF-R2 during signal transduction. TNF-R1 contains the death domain
in its C-terminal domain responsible for apoptosis induction in carcinoma [124].

Cell death modulation could possess a possible significant impact on the treatment of many
diseases, especially cancer. It is also possible that apoptosis inhibition may provide a
prolonged opportunity for cells to repair their damaged DNA or rebuild homeostasis state and
returns to a healthy state [32]. A study conducted on HN regarding cancer led to concerns
that it might be tumor-promoting in triple-negative breast cancer (TNBC) because HN

12
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antagonizes the proapoptotic BAX initially [33]. However, the comforting data revealed that
it inhibits tumor that must be further investigated [69]. A most recent study revealed that the
inhibition of HN peptide by circNOL10 (circular RNA) significantly promotes the reduction
of lung cancer both in vivo and in vitro studies by regulating the expression of transcription
factors, including sex comb on midleg-like 1 (SCML1) [125]. Gottardo et al. [29]
experimented to observe the effect of HN in TNF-α in both tumor pituitary and normal cells
of rats and reported that HN significantly inhibited TNF-α-induced apoptosis effects in GH3
cells (somatolactotrope cell line). They also revealed that STAT3 pathway suppressed the
inhibitory effect of HN on TNF-α-induced apoptosis in both anterior pituitary and normal
cells and suggested that HN involved in pituitary tumorigenesis. Similarly, HN (5 µM)

of
successfully inhibited the proapoptotic effect of TNF-α in lactotropes, anterior pituitary,

ro
somatotropes, and pituitary tumor cells in both male and female rats [126].

A study by Mottaghi-Dastjerdi et al. [127] found that the upregulation of HN in

-p
tumorigenesis is an important molecular event. Investigation of HN in the tumor of pituitary
cells in rat models reported that apoptosis induced by TNF-α of anterior pituitary cells. HN
re
peptide is released from cells that modulate proapoptotic effect induced by TNF-α in anterior
lP

pituitary cells [128]. A study on male mice model with pulmonary melanoma metastases
shows potent HN analog HNG (S14G-humanin). HNG protects healthy male germ cells and
na

leukocytes against cyclophosphamide (CP) that induce apoptosis and sensitize tumor cells to
CP-induce tumor suppression in male mice [68]. Unlike direct antitumor properties, HNG has
ur

a protective effect on normal cells but not tumor cells [129]. However, based on evidence, it
is suggested that more study is needed on HN against different cancer cells.
Jo

Summary of the mechanism of action of HN in signaling cascades involved in different

diseases is schematically represented in Fig. 4.

5. Concluding remarks
In summary, humanin (HN) and its derivatives showed protective effects in many diseases,
including cardiovascular diseases and age-related diseases. Since its discovery in 2001, HN’s
role has been demonstrated in many biological processes, including oxidative stress and anti-
apoptosis. Apoptosis (programmed cell death) is one of a vital process to affirms cell
homeostasis, which maintains the delegate balance between cell death and cell survival. The
uncontrolled apoptotic condition led to the loss of vital cells and resulted in serious health
diseases like age-related and bone-related diseases. Over the past few years, HN became an
13
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impressive therapeutic agent due to its cytoprotective, oxidative stress, and antiapoptotic
effects in various cell types, including neuronal, male germ cells, and cardiac cells. HN
mediates its action through multiple mechanisms by binding to various specific cellular
trimeric receptors (FPLR1/2, CNTFR-α, and gp-130). HN effectively exerted several
cytoprotective effects in multiple diseases, including the protection of bones from
osteoporosis condition, damage of cardiac cells, and beta cells from oxidative stress in
cardiac and diabetic conditions respectively, and male germ cells by inducing anti-apoptotic
activities. HN and its analogs could be used as good therapeutic targets against
neuroprotective and neurodegenerative diseases. It is suggested that more study is required to
understand the mechanism and recognize the translation site of HN for a better

of
understanding. Moreover, it is recommended that more mitochondria-derived peptides

ro
(MDPs) developed shortly, which could be proven as potential therapeutic targets along with
HN for the treatment of many lethal diseases including cancer.
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6. Outstanding questions
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1. Could the use of humanin be an innovative therapeutic option in treating different cancers
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like prostate, head and neck, liver, and lung cancers?

2. Could the use of HN with other drugs be a safe option in treating cardiovascular and
neuroprotective diseases?
na

3. What will happen in the case of overexpression of HN in the body? Would it lead to a few
lethal diseases or act as a productive agent?
ur

4. Could the use of natural products be a potential medication to activate the HN, which
Jo

would target the apoptotic cycle in lethal diseases like cancer and germ cell infertility?
5. Could the use of HN-analogs, including HN‐ GF6A and S14G-HN, be an essential
therapeutic medicine in the treatment of age-related diseases such as reperfusion, stroke,
Alzheimer’s disease, and amyotrophic lateral sclerosis?
6. Could HN and its analog (S14G-HN) replace the current diabetes mellitus treatment
strategies soon?
7. Could the development of new mitochondrial-derived peptide be a temporary or a
permanent therapeutic option against oxidative stress and apoptosis-related diseases?

Acknowledgement

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Hereby, we extend our gratitude to A.Q. Research Group, Pakistan for reviewing the article
and providing helpful comments.

CRediT authorship contribution statement

A. Hazafa, A. Batool, and U. Ghani: Conceived the presented data, Writing - original draft,
Software, & Supervision. S. Ahmad, M. Amjad, J. Asad, and S. N. Chaudhry: Developed
the theory, Formal analysis, & Investigation. H. M. Khan, M. Naeem, and H. F. Ghuman:
Software.
Compliance with ethical standards
Conflict of interest

of
The authors declared no conflict of interest.
Funding

ro
None.
Informed consent
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For this type of study informed consent is not required.
re
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List of Tables
Table 1. Antiapoptotic effects of HN and its analogous in the treatment of male infertility and chemotherapy side effects with
different chemotherapeutic drugs.

Drug Study type Dose Mechanism of HN/analogue HN Reference

action of a drug

o f characteristic

Cyclophosphamide In vivo 5 mg/Kg (BW) Circulate to

p
cancer cells and ro
HN-S14G
analogue
Reduce CP-
induced germ
[46]
(CP)
damages DNA

e - cell apoptosis

P r
and leading to
apoptosis

l
Doxorubicin ex vivo 10 µM Intercalating HN Reduce DOX- [46]

a
(DOX) DNA to suppress induced germ

n
proliferation and cell apoptosis

Ethane In vivo
u r
80 mg/kg (BW)
induce apoptosis
Induce apoptosis
Rat HN protein
Reduce germ [67]

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dimethanesulfonate at the middle cell apoptosis at
(Rattin)
stages on early and late
(EDS)
seminiferous stages
epithelium cell
middle stages
Temozolomide In vivo 50 mg/kg (BW) Use in pediatric HNG (where the Reduce TMZ- [66]
(TMZ) brain cancer serine in position induced germ
14 of HN cell apoptosis
substituted by
glycine)

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Cyclophosphamide In vivo 200 mg/kg (BW) CP decreases HN-S14G Saved CP- [68]
(CP) peripheral analogue induced
leukocytes and destruction of
use in cancer sperm counts,
treatment to spermatogonia
suppresses male germ cell
metastatic lung
melanomas
o f and total WBC

BW: body weight; HN: humanin.

r o
- p
r e
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n a
u r
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Table 2. The antiapoptotic effects of HN and its analogous in the treatment of diabetes mellitus (DM).

Humanin/ Study model HN Treatment time Function Reference

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analogue dose/concentration
HN‐ GF6A Sprague-Dawley rat 0.07 mg/kg/h (BW) 2-30 min Improve insulin [64]
sensitivity and help
in decreasing blood
glucose level

HN Sprague−Dawley rat 0.375 mg/kg/h (BW) 360 min

o fDecrease blood
glucose in
[116]

r o Sprague−Dawley
rats by STAT-3

- p phosphorylation

HNGF6A Zucker diabetic fatty

rat
0.05 mg/kg/h (BW)
re 90-240 min Decrease blood
glucose in Zucker
[116]

l P diabetic fatty rats

HN‐ GF6A In vitro

n a
50 ng/mL 15-120 min Promote glucose- [64]

u r stimulated insulin
secretion in βTC3

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cells

HN In vitro 1-10000 nmol/L 24 h Reduce cytokine- [112]

induced apoptosis in
β-cells by activating
the transcription 3

HN Nonobese diabetic 0.7 mg/kg/daily 6 weeks Protect the β-cells [112]

(NOD) mice (BW) and treat diabetes by
inhibiting apoptosis

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via promoting
serum starvation

HN 24 volunteers with 28-1800 pg/mL --- Decrease hepatic [130]

T2D (HbA1c < 7.0 glucose production
%), and 93 with T2D and increased

f
(HbA1c > 7 %) glucose disposal
into peripheral

oo tissue

HNG (S14G-HN) In vitro 1 μM 3h

p r Protect against high [131]

e - glucose-induced
apoptosis
BW: body weight; HN: humanin; T2D: type-2 diabetes.

P r
a l
r n
u
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List of Figure

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Fig. 1. The general representative structures of (a) human mitochondrial DNA and (b) humanin
peptide with specifications. (a) The mitochondrial DNA (mtDNA) is a complex genome in
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humans, which is encoded by 16,569 bp. The transcription in mtDNA initiated from two types of
promoters including (HSP1 and HSP2) in the central region. The mtDNA genome contains 2
rRNAs (12S and 16S RNA) (green and red), cytochrome b (blue), 1-6 ubiquinone
oxidoreductase subunits (sky color), 2 ATPases (ATPase 6 and 8) (dark green), and 1-3
cytochrome c oxidase subunits (orange). (b) This part is representing the structure of humanin
with the function of each amino acid in humans. The functions of each amino acid are marked
with a specific key in figure [36].

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Fig. 2. The mechanism of apoptosis by BCL-2 antagonists in response to stress. In response to

apoptotic stimuli including ROS or radiations, the process of apoptosis begins. The pro-apoptotic

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group of proteins (green box), including BOD [BCL-2-interacting mediator of cell death (BIM),
BH3-interacting domain death agonist (BID), and BID active truncated form (tBID)] are
upregulated and interact with anti-apoptotic proteins (yellow box) such as BCL-2. However, in
the absence of anti-apoptotic proteins, these (BOD) activate BHD proteins (orange box) [Bcl-2-
associated X protein (Bax) and BCL-2 antagonist/killer (BAK)] and resulted in the pore-
formation in a mitochondrial membrane (blue). The pro-caspase-9 (pink) bind with different
APAF-1 (dark green) to form the apoptosome (like a flower) which resultingly helped in the
formation of three different caspases (pink) including caspase 3, 6, and 7. Finally, these caspases
induced programmed cell death (apoptosis) [32, 132].

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Fig. 3. Alignment of humanin peptide sequence in different species.

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Fig. 4. The general mechanism of actions of anti-apoptosis, anti-cell death, and cell survival of
HN against different diseases. Humanin (HN) peptide encoded by mitochondrial DNA (mtDNA)
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which exerts its cytoprotective effect by regulating the intracellular and extracellular signaling
cascades. Inside the cell, HN (pink circle) binds to intracellular receptors (blue box), including
Bim, Bid, Bax, and prevent apoptosis (Step 1). In step 2, HN bind to trimeric membrane
receptors including WSX-1/gp-130/CNTFR-a to triggers the downstream signaling pathway to
induce cytoprotective property (red box), by the activation of JAK/STAT signaling pathway
(green box). HN also has the ability to bind with formyl peptide receptor-like 1 (FPRL-1) (green
and blue lines) which subsequently activates the extracellular kinase (ERK 1/2) (dark green box)
and provides cytoprotection and help in cell survival (purple box) (Step 3). HN can also reduce
ROS production, which subsequently opened the mitochondrial permeability transition pore
(mPTP) and caused anti-cell death activity (Step 4) [11, 34].

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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Graphical Abstract

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Highlights

 Humanin is a mitochondrial-derived peptide, comprises of 24 amino acids, and encoded by a

75-bp ORF.

 HN protects cells against various diseases, including diabetes mellitus, cardiovascular, and

neurodegenerative diseases.

 HN exerts pro-apoptotic activity of TNF-α in cancer, which makes HN as a novel therapeutic

agent.

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 HN exhibits anti-apoptotic activity by binding through extracellular CNTFR-α/gp130/WSX-

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1 trimeric receptors.

 -p
HN peptide could be a viable therapeutic option against oxidative stress and apoptosis-related
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diseases.
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