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Citation: Tolos, A.M.; Moisa, C.;
Dochia, M.; Popa, C.; Copolovici, L.;
Copolovici, D.M. Anticancer Potential
of Antimicrobial Peptides: Focus on
Buforins. Polymers 2024,16, 728.
https://doi.org/10.3390/
polym16060728
Academic Editors: Ariana Hudita,
Bianca Gˇ
alˇ
a¸teanu and Huacheng
Zhang
Received: 6 December 2023
Revised: 4 March 2024
Accepted: 5 March 2024
Published: 7 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
polymers
Review
Anticancer Potential of Antimicrobial Peptides: Focus
on Buforins
Ana Maria Tolos (Vasii) 1, 2, † , Cristian Moisa 1, † , Mihaela Dochia 1, Carmen Popa 1,2, Lucian Copolovici 1,3
and Dana Maria Copolovici 1, 3, *
1Institute for Research, Development and Innovation in Technical and Natural Sciences, Aurel Vlaicu
University, Elena Dragoi St., Nr. 2, 310330 Arad, Romania; anamaria.tolos@yahoo.com (A.M.T.);
cristian.moisa@uav.ro (C.M.); dochiamihaela@yahoo.com (M.D.); camy_popa2007@yahoo.com (C.P.);
lucian.copolovici@uav.ro (L.C.)
2Biomedical Sciences Doctoral School, University of Oradea, University St., Nr. 1, 410087 Oradea, Romania
3Faculty of Food Engineering, Tourism and Environmental Protection, Aurel Vlaicu University,
Elena Dragoi St., Nr. 2, 310330 Arad, Romania
*Correspondence: dana.copolovici@uav.ro
†These authors contributed equally to this work.
Abstract: In seeking alternative cancer treatments, antimicrobial peptides (AMPs), sourced from
various life forms, emerge as promising contenders. These endogenous peptides, also known as
host defense peptides (HDPs), play crucial roles in immune defenses against infections and exhibit
potential in combating cancers. With their diverse defensive functions, plant-derived AMPs, such as
thionins and defensins, offer a rich repertoire of antimicrobial properties. Insects, amphibians, and
animals contribute unique AMPs like cecropins, temporins, and cathelicidins, showcasing broad-
spectrum activities against bacteria, fungi, and viruses. Understanding these natural peptides holds
significant potential for developing effective and targeted therapies against cancer and infectious
diseases. Antimicrobial peptides (AMPs) exhibit diverse structural characteristics, including
α
-
helical,
β
-sheet, extended, and loop peptides. Environmental conditions influence their structure,
connecting to changes in cell membrane hydrophobicity. AMPs’ actions involve direct killing and
immune regulation, with additional activities like membrane depolarization. In this review, we
focus on antimicrobial peptides that act as anticancer agents and AMPs that exhibit mechanisms
akin to antimicrobial activity. Buforin AMPs, particularly Buforin I and II, derived from histone
H2A, demonstrate antibacterial and anticancer potential. Buforin IIb and its analogs show promise,
with selectivity for cancer cells. Despite the challenges, AMPs offer a unique approach to combat
microbial resistance and potential cancer treatment. In various cancer types, including HeLa, breast,
lung, ovarian, prostate, and liver cancers, buforins demonstrate inhibitory effects and apoptosis
induction. To address limitations like stability and bioavailability, researchers explore buforin-
containing bioconjugates, covalently linked with nanoparticles or liposomes. Bioconjugation enhances
specificity-controlled release and combats drug resistance, presenting a promising avenue for targeted
cancer treatment. Clinical translation awaits further evaluation through
in vivo
studies and future
clinical trials.
Keywords: antimicrobial peptides; anticancer; buforins
1. Introduction
Non-communicable illnesses are becoming a widespread global public health issue.
Cancer is the second leading cause of morbidity and mortality worldwide [
1
]. It con-
tinues to be a major health burden, estimated to have killed 10 million people in 2018
and up to 12 million in 2020, despite considerable research and decades of therapeutic
approaches [2,3].
Most antitumor medications used in cancer treatment cannot distinguish
between cancer cells and normal cells (they are not selective), causing side effects such as
Polymers 2024,16, 728. https://doi.org/10.3390/polym16060728 https://www.mdpi.com/journal/polymers
Polymers 2024,16, 728 2 of 14
hair loss, diarrhea, and a weakened immune system. Acquired drug resistance, which,
according to the literature, accounts for up to 90% of cancer patients’ mortality, is one of
the main challenges of chemotherapy [
4
]. Consequently, scientists in the pharmaceutical
industry have been attempting to create chemotherapy medications that are less hazardous
to healthy cells and have more efficacy [
5
]. The primary goal of the researcher is to de-
velop new medicines that target cancers while also extending survival times, reducing
unfavorable side effects, and improving patient quality of life [6].
Several approaches have been studied to reduce the negative effects of chemotherapy.
Two of these have received particular notice: encapsulation in nanocarriers and conjugation
of drug molecules to water-soluble polymers [
7
]. There is a significant possibility of
improving various approaches to treating cancer thanks to the swift development of
nanotechnology and the discovery of nanomedical agents [8].
Creating antibody–drug conjugates (ADCs), which combine the cytotoxic effects of
chemotherapy with monoclonal antibodies, has demonstrated promising efficacy with
minimal side effects compared to conventional treatment [
9
]. Recently, interest has grown
in nanotheranostics, a theory that integrates many capabilities into a single nanotechnology-
based, multifunctional system for drug delivery and cancer imaging in one package [
10
].
Often made from peptides, antibodies, integrin receptor ligands, aptamers, and other molec-
ular components, these medications consist of recognition domains (detect appropriate
markers on endothelial cells) and effector domains (contribute to the curative effect). Due
to their small size, precise chemical structure, and stability, peptides utilized as ligands offer
significant benefits in cellular targeting. The large-scale, high-purity production of peptides
is simple and economical. The peptides are highly selective to target cells and tissues,
biocompatible, and can simultaneously have many ligands coupled to the delivery vehicle
to maximize binding to the target [
7
]. Because of their target particularity to corresponding
endogenous receptors, a variety of natural peptide analogs, including hormone peptides, so-
matostatin, cholecystokinin/gastrin, bombesin, and Arg-Gly-Asp (RGD-receptor-mediated
peptide analogs), receptor-targeting peptides, dual-receptor-targeting peptides, head-to-tail
cyclic peptides, peptide vaccines, theranostic agents, and self-assembled peptide-based
nanoprobes have been proposed for diagnosis and treatment. Their applications as thera-
peutic agents are constrained by proteolytic degradation. There are several ways to avoid
this inconvenience, including cyclization, hybridization, the addition of D-amino acids,
and alteration of the peptide’s C- and N-termini by therapeutic or targeted compounds [
6
].
A new possible anticancer treatment is using selenium-conjugated peptides (STPs).
Selenium compounds can potentially slow cancer cell development and trigger apopto-
sis in cancer cell culture models [
11
]. Selenobevacizumab and Selenotrastuzumab were
developed by Khandelwal et al. by combining the humanized IgG1 antibodies with re-
dox selenium and assessed their efficacy against the normal human mammary epithelial
cell line and the triple-negative breast cancer (TNBC) cell lines [
12
]. Also, the antitumor
action of Seleno-
β
- lactoglobulin was analyzed by Yu et al. on human gastric cancer
cells [
13
]. Zeng et al. combined the RGD peptide with the selenadiazole derivative (RGD-
SeD) for better selectivity and specificity. RGD-SeD showed antitumor effectiveness in
α
V
β
3 integrin-overexpressing HepG2 liver cancer cells [
14
]. According to Yang et al., the
selenium-conjugated selenadiazole peptide could enter cells by receptor-mediated endo-
cytosis via clathrin-mediated and nystatin-dependent lipid-raft-mediated pathways after
being attached to nanoparticles. Ranjitha et al. observed a comparable impact on colorectal
cancer cells [15].
Other promising anticancer therapeutic compounds are marine peptides. These have
gained attention for a variety of reasons. Unlike proteins or antibodies, they are smaller,
easier to manufacture, more easily changed, capable of traversing cell membranes, less
likely to interact with other drugs, and more versatile in chemical and biological processes.
Another benefit is fewer adverse side effects from liver or renal buildup [16].
For the treatment of diabetes, cardiovascular diseases, and several cancers, including
head and neck, brain, breast, prostate, cervical, and ovarian cancers, peptide drugs have
Polymers 2024,16, 728 3 of 14
been created as prospective targeted treatments. Clinical trials for more than 60 therapeutic
peptide medications are currently being conducted. The U.S. Food and Drug Administra-
tion (FDA) has approved 15 peptides in the last few years [
17
]. These peptide drugs serve
as radionuclide carriers (b, g-emitters) and inhibitors of angiogenesis, cytotoxic medicines,
and fluorescence probes (visible, Near-IR, Far-IR). The peptide analogs can also be utilized
as hormones and vaccines [6].
This review aimed to present the identification and applications of AMPs with anti-
cancer potential, with a main candidate being buforin and its derivatives.
2. Antimicrobial Peptides (AMPs) with Anticancer Potential
The use of traditional chemotherapies for cancer treatment, which are based on hor-
mone agonists/antagonists, antimetabolites, and alkylating drugs, is restricted due to the
development of multi-drug resistance by cancer cells and significant side effects [
18
]. A
number of bodily functions are also impacted by these medications, which might result
in unfavorable side effects include anemia, exhaustion, constipation, diarrhea, chest dis-
comfort, mucositis, rash, and vomiting [
19
]. A suitable and focused treatment plan may be
suggested in light of the difficulties in cancer therapy, with the potential to enhance quality
of life and results.
As an alternative to existing chemotherapeutic treatments, antimicrobial peptides
(AMPs) with cancer-selective cytotoxicity, have drawn attention recently. Compared to
the anticancer therapies currently in use, these peptides have a number of advantages,
including reduced intrinsic cytotoxicity, a lower chance of resistance developing, and
additive effects in combination therapy [18,20].
AMPs offer many benefits, such as rapid onset of action, low toxicity, low risk of
resistance, and broad-spectrum activity, which have made them attractive candidates for
clinical treatments. The clinical application of AMPs as antineoplastic drugs is hampered
by a number of factors, including their high cost of manufacture, short half-life, severe
immune reaction, and collateral damage on normal mammalian cells, despite the fact that
there is promise for their translation into clinical practice [21].
A presentation of the list of several AMPs with anticancer activities, like lactoferricin,
magainin II, NRC-3, NRC-7, BR2, pardaxin, cecropin B, gomesin, LL-37, buforin IIb, CopA3,
gaegurins, citropin 1.1, tachyplesin I-III, their mechanism of actions, and the sequence-
based computational methods that are free and suitable to discover new peptide sequences
with potential anticancer properties was published in a review by Kordi et al. [22].
Lactoferricin-derived peptides both from bovine and human milk were shown to
present
in vitro
anticancer activity toward leukemia and breast cancer cells when the
antimicrobial core sequence is maintained [23].
Cecropins are found in the skin of various frog species, including the African clawed
frog and the leopard frog Rana pipiens. They have broad-spectrum activity against bacteria,
viruses, and fungi [24], and also against ovarian and endometrial cancer cells [25].
Several temporins with antimicrobial activities against Gram-positive bacteria (e.g.,
S. aureus,B. megaterium, and Streptococcus pyogenes), Gram-negative bacteria (e.g., E. coli,
P. aeruginosa,Aeromonas hydrophyla), and fungi (C. candida) exhibited specific anticancer
properties against cell lines like MCF-7 (human breast cancer), HeLa, and NCI-H460.
The AMP human cathelicidin LL-37 (hCAP18) that is derived from epithelial cells and
leukocytes has two different mechanisms of action, depending on the type of cancer and
also on the tissue origin of the tumor [
26
,
27
]. LL-37 can stimulate the growth, migration,
and tumor formation in breast, prostate, and lung cancers, while in T-cell leukemia, colon,
and gastric cancer, it inhibits metastasis [22,28].
Human intestinal defensin 5 (HD-5) has shown
in vivo
colon carcinogenesis activ-
ity without affecting the normal cells [
29
]. Another defensin, Laterosporulin 10 (LS10),
displayed cytotoxicity against cancer cells (MCF-7, HEK293T, HT1080, HeLa, and H1299)
without affecting the tested prostate epithelium cells (RWPE-1) [30].
Polymers 2024,16, 728 4 of 14
The magainin II peptide demonstrated cytotoxic and antiproliferative effects against
lung adenocarcinoma cells (A549) [
31
], bladder cancer cells (RT4, 647V, and 486P) [
32
],
breast cancers cells (MDA-MB-231), and human mesothelioma (M14K) [
33
] while showing
no impact on normal human fibroblasts (3T3) [32].
2.1. Buforin Peptides Identification
Buforins are members of the non-lytic AMP family with the specific N-terminal section
of histone H2A shared by all its members [
34
–
36
] (Figure 1). This portion of the protein is
responsible for specifying the direct interaction with nucleic acids [
35
,
37
,
38
], inhibiting the
cellular processes of the bacteria by binding to DNA and RNA [
39
]. They have significant
antibacterial potential and promote inflammatory reactions, even though histone-derived
peptides do not play a part in the replication process [34,35].
Polymers 2024, 13, x FOR PEER REVIEW 4 of 14
displayed cytotoxicity against cancer cells (MCF-7, HEK293T, HT1080, HeLa, and H1299)
without affecting the tested prostate epithelium cells (RWPE-1) [30].
The magainin II peptide demonstrated cytotoxic and antiproliferative effects against
lung adenocarcinoma cells (A549) [31], bladder cancer cells (RT4, 647V, and 486P) [32],
breast cancers cells (MDA-MB-231), and human mesothelioma (M14K) [33] while showing
no impact on normal human fibroblasts (3T3) [32].
2.1. Buforin Peptides Identification
Buforins are members of the non-lytic AMP family with the specific N-terminal sec-
tion of histone H2A shared by all its members [34–36] (Figure 1). This portion of the pro-
tein is responsible for specifying the direct interaction with nucleic acids [35,37,38], inhib-
iting the cellular processes of the bacteria by binding to DNA and RNA [39]. They have
significant antibacterial potential and promote inflammatory reactions, even though his-
tone-derived peptides do not play a part in the replication process [34,35].
Figure 1. The amino acid sequence of buforins and their analogs.
Kim et al. (2000) reported that the histone variant H2A was the precursor for a 39-
amino acid sequence [40,41] named buforin I that was sampled and isolated for the first
time from the gastric tissue of Bufo bufo gargarizans, and presented promising antibacterial
effects against a broad spectrum of pathogens, both G+ bacteria (B. subtilis, S. aureus) and
G- bacteria (E. coli, S. typhimurium) as well as fungi (C. albicans, S. cerevisiae) [35,40,42–44].
When buforin I was compared with other amphibian antimicrobial peptides (AMPs) like
magainin 2, it demonstrated significantly stronger antimicrobial activities against micro-
organisms in vitro [40]. Magainin 2 is known to inhibit the growth of certain bacteria by
preventing them from reproducing. This feature is achieved by binding to the bacterial
membrane and disrupting its integrity. Magainin 2 also can inhibit the growth of certain
fungi by preventing them from reproducing, similar to its activity on bacteria [45]. Buforin
I is known to have bactericidal activity but also antiviral activity, which is achieved by
binding to viral envelope proteins and disrupting the viral replication cycle [46].
Buforin II is a helix-hinge-helix residual 21 amino acid antimicrobial peptide derived
from histones present in the gastric tissue of the Asian toad [34,38,40,43,47–50] or obtained
with the help of Lys-C endoproteinase from buforin I, and usually contain some residues
Thr
16
to Lys
36
, presenting a higher antimicrobial activity than its parent peptide [40].
A derivative analog of buforin II named buforin IIb presents more substantial anti-
bacterial and anticancer potentials [51–54] and includes at the C-terminus an α-helical se-
quence that exhibits in microorganisms a higher cytolytic activity than buforin II
[38,50,55–57].
Figure 1. The amino acid sequence of buforins and their analogs.
Kim et al. (2000) reported that the histone variant H2A was the precursor for a 39-
amino acid sequence [
40
,
41
] named buforin I that was sampled and isolated for the first
time from the gastric tissue of Bufo bufo gargarizans, and presented promising antibacterial
effects against a broad spectrum of pathogens, both G+ bacteria (B. subtilis,S. aureus) and
G- bacteria (E. coli,S. typhimurium) as well as fungi (C. albicans,
S. cerevisiae) [35,40,42–44].
When buforin I was compared with other amphibian antimicrobial peptides (AMPs) like
magainin 2, it demonstrated significantly stronger antimicrobial activities against microor-
ganisms
in vitro
[
40
]. Magainin 2 is known to inhibit the growth of certain bacteria by
preventing them from reproducing. This feature is achieved by binding to the bacterial
membrane and disrupting its integrity. Magainin 2 also can inhibit the growth of certain
fungi by preventing them from reproducing, similar to its activity on bacteria [
45
]. Buforin
I is known to have bactericidal activity but also antiviral activity, which is achieved by
binding to viral envelope proteins and disrupting the viral replication cycle [46].
Buforin II is a helix-hinge-helix residual 21 amino acid antimicrobial peptide derived
from histones present in the gastric tissue of the Asian toad [
34
,
38
,
40
,
43
,
47
–
50
] or obtained
with the help of Lys-C endoproteinase from buforin I, and usually contain some residues
Thr16 to Lys36, presenting a higher antimicrobial activity than its parent peptide [40].
A derivative analog of buforin II named buforin IIb presents more substantial antibac-
terial and anticancer potentials [
51
–
54
] and includes at the C-terminus an
α
-helical sequence
that exhibits in microorganisms a higher cytolytic activity than buforin II [38,50,55–57].
Derived from buforin II, Jang et al. designed several analogs named buforin III a-d.
These derivatives maintained the crucial structural features and the general hydropho-
bicity necessary for exerting antimicrobial effects [
58
]. In obtaining buforin IIIa, the cell-
penetrating motif was changed while keeping the rest of the structure identical to buforin
IIb. For the following peptides, namely buforins III b, c, and d, the cell-penetrating motif
Polymers 2024,16, 728 5 of 14
from buforin IIIa was maintained, while the
α
-helical structure and C-terminal sequences
were changed [58].
Furthermore, to obtain targeted medication delivery systems with reduced normal cell
cytotoxicity and higher cancer cell affinity [
59
], several designers created cell-penetrating
peptides like BR 1-3 which were synthesized and studied from the buforin IIb
model [50,56,60].
2.2. Buforins’ Preparation and Characterization
Usually, AMPs have a low molecular weight and are composed of less than 50 amino
acid bases, generally of a positive charge (+2 to +9) at pH 7 due to high lysine and arginine
amounts [61]. More than 30% of their amino acids are hydrophobic [39,44,53,58].
Immunohistochemical (IHC) and biochemical analyses were used in determining
that the mechanism of producing buforin I takes place within the gastric mucosal cells
of the Asian toad after excessive production of histone H2A that surpasses the amount
needed for DNA packaging and is further deposited in secretory granules [
40
,
42
,
43
]. Pepsin
processes the extra histone H2A within the gastric lumen, resulting in the solid antimicrobial
peptide buforin I, providing a protective layer to the stomach mucosal surface to which
it adheres [
40
]. Within the stomach, the secreted HCl interacts with inactive pepsinogen,
which is converted to active pepsin [40].
2.3. Buforins’ Mechanisms of Action
The molecular process of membrane penetration and the disruption of AMPs depends
on various variables, such as the sequence of amino acids in the membrane, the amount
of peptides, and the lipids in the membrane [
39
]. Almost all AMPs tend to be classified
into two mechanistic pathways depending on their cell membrane interaction [
61
–
64
].
The first category focuses on cell wall disruption peptides that can distinguish between
bacterial and human or host cells [
43
,
65
–
67
], and their cell wall penetrative properties
include the following models: carpet, staves of a barrel, micellar aggregate, and toroidal
pores (e.g., magainin-2, protegrin-1) [
39
,
67
]. Therefore, with the rise of AMPs in the volume
outside the bacterial cells, different reactions eventually lead to the induction of apoptosis
(inflammation or electrolyte disequilibrium) [
62
,
68
]. The second category of AMPs focuses
on non-disruptive cell wall interactions (intracellular targets—altering protein synthesis,
enzyme activity, binding, and interfering in DNA and RNA replication) [
39
,
63
,
67
]. Buforins
are included in the latest category.
Even though the processes through which AMPs and members of the buforin family
exhibit their action have not yet been fully explored and understood, models that have
revealed intracellular and extracellular interference are generally recognized [
61
], and there
is evidence that buforins can kill microorganisms by entering their cells and binding to
nucleic acids [
34
,
49
,
69
,
70
]. Furthermore, members of this family can penetrate lipid bilayers
thanks to the transient formation of peptide–lipid supramolecular complex pores [35].
Although there are similarities in the structure of buforin II and other AMPs (amphi-
pathic
α
-helical peptides), their mechanism of action is different. Buforin II acts without
bacterial cell lysis
in vitro
. It presents strong DNA and RNA affinity [
34
,
38
,
40
,
69
,
71
,
72
] and
produces microbial cell aggregation [
70
]. Buforin II presents bactericidal effects for various
G+ and G−microorganisms and fungi [73].
Several studies [
49
,
74
,
75
] compare the peptide–lipid interaction of buforin II to that
of magainin 2 without producing significant lipid flip-flop or membrane permeabiliza-
tion [
40
]. Other studies have observed that buforin II amide mimics the structure and
composition of the Gram-negative bacterial cell membrane of Escherichia coli, adhering and
generating peptide–DNA condensates inducing structural alterations within the bacterial
cell [34,69,72,76,77], without disrupting the plasma membrane [40,43,53,58,70,78].
In rat models, it was tested individually or in association with antimicrobial drugs
and had a high potential for treating Acinetobacter baumannii sepsis [34,70,78].
Buforin IIb is a histone-H2A-derived synthetic analog of buforin II [
38
,
51
] that contains
an
α
-helical sequence (3xRLLR) [
38
] and is altered to improve its selectivity for cancer
Polymers 2024,16, 728 6 of 14
cells leading to cell death [52,61] without disrupting the normal cells [42,71]. Like buforin
II, it interacts with the gangliosides in cancer cells, enters the cell without rupturing its
membrane, and triggers mitochondrial apoptosis by activating caspase-9 [
2
,
61
] through
mitochondria-dependent pathways [
52
]. Nonetheless, emerging evidence underscores the
collaboration between the endoplasmic reticulum (ER) and mitochondria in signaling cell
death. This investigation delves into the mechanism behind buforin-IIb-induced apoptosis
in human cervical carcinoma HeLa cells, with a focus on ER stress-mediated mitochondrial
membrane permeabilization [52].
Negatively charged elements present on cancer cells’ surfaces, like gangliosides, phos-
phatidylserine (PS), and heparan sulphate (HS) can serve as target molecules for interactions
with peptides. The study published by Lee at. al in 2008 indicates that gangliosides on
cancer cell surfaces act as particular binding sites for buforin IIb, enabling it to differentiate
between cancer cells and normal cells [79].
Given the therapeutic potential and action mechanisms of buforin IIb against a broad
spectrum of microorganisms, the possibility of developing resistance through target modi-
fication is unlikely [
80
]. However, even if buforin IIb has several remarkable characteristics
of being a new antimicrobial or cancer treatment, the earlier analysis revealed that at higher
amounts, it also attacked human cells [58].
To improve the therapeutic potential of the AMPs, analogs of buforin III, designated
Buf III a-d, were synthesized from buforin IIb. These analogs were designed to retain key
attributes such as hydrophobicity and certain structural elements crucial for maintaining
antimicrobial efficacy. This strategic preservation ensures that the modified peptides exhibit
potent antimicrobial activity while potentially offering an enhanced therapeutic index [
58
].
These analogs acted like their parent AMPs, easily penetrated bacterial cell membranes,
and effectively were bound to DNA in vitro [58].
3. Anticancer Activity of Buforins
Cancer is a complex medical condition involving chronic low-level inflammation [
81
],
and anticancer peptides (ACPs) are a category of AMPs capable of targeting cancer cell
membranes. Although the inhibition or cancer-killing mechanisms are uncertain, there
are similarities to those of antimicrobial activity, presenting both cell wall disruptive and
non-disruptive mechanisms [
62
]. However, unlike healthy normal cell membranes, the
surfaces of cancer cells have significant amounts of phosphatidylserine, which represents
an important factor in cell selectivity and tumor specificity and delivers apoptosis [62,71].
Alongside cancer cell specificity, some ACPs like Dermaseptins B2 and B3 can re-
strain endothelial cell development and inhibit cancer cell proliferation [
62
]. Other ACPs
lead to cancer cell apoptosis through DNA fragmentation. Both buforin II and buforin
IIb present high anticancer activities and specificity through the cell surface ganglio-
sides
interaction [53,61]
toward 60 cancer cell lines from the National Cancer Institute
(USA) [50,52,70,79,82]
like leukemia, CNS tumors, lung and renal cancers, prostate, melanoma,
breast, and colon cancers [
38
,
83
], and display cytotoxic activity by disrupting mitochondrial
functions causing apoptosis without affecting the cellular membrane [84].
3.1. Buforins Used for HeLa Cells
Recent studies explored the anticancer activity of buforins against HeLa cells, suggest-
ing that these peptides have the potency to be delivery vectors and effective therapeutic
agents [
40
,
43
,
52
]. Buforin I led to the growth inhibition and apoptosis of HeLa cells, pre-
senting cytotoxic effects [
35
,
42
], and similar results were observed for buforin II [
50
,
55
,
85
].
Buforin IIb was determined to be highly selective toward HeLa cells by LC-MS/MS analysis,
which confirmed its capability to enter cancer cells [
86
]. There are unknown mechanisms
within the anticancer activity of buforins on HeLa cells, all demonstrating that buforin pep-
tides accumulate at the cancer cell membranes without disrupting them [
87
] and inducing
apoptosis through buforins’ intracellular accumulation [
35
,
42
]. Buforins also initiate cell
death by binding to HeLa cell surface receptors, inhibiting cell proliferation and angio-
Polymers 2024,16, 728 7 of 14
genesis to slow tumor growth [
50
,
55
].
In vivo
animal studies using models of HeLa cell
xenografts have given important insights regarding buforins’ anticancer effects [59,79].
3.2. Buforins Used for Breast Cancer
Buforin IIb exhibited efficacy against breast cancer cells MX-1, MCF-7, and
T47-D [79,82,88]
with little or no effects on normal cells [
71
], where it hinders tumor growth in a mouse
xenograft model by two mechanisms (anti-vasculogenic and anti-angiogenic). The glyco-
sylation process in breast cancer cells facilitates their interaction with this antimicrobial
peptide (AMP), exerting its anticancer effects [
51
,
88
]. A combination of doxorubicin and
nisin had an anticancer effect against the MCF-7 cell line [89].
3.3. Buforins Used for Lung Cancer
In their study, Lee et al. (2008) [
79
] employed an NCI-H460 lung cancer cell line
utilizing a transplantation technique to generate tumor xenografts. Their findings indi-
cated that the administration of buforin IIb could effectively inhibit the progression of
cancer xenografts
in vivo
(>5 mg/kg), suggesting that buforin IIb holds promise as a new
therapeutic intervention for the management of cancer [38,61,79].
3.4. Buforins for Ovarian Cancer
For ovarian cancer cell lines (A2780 and A2780cisR) and normal non-cancerous human
dermal fibroblasts (NHDF),
in vitro
cytotoxic analysis for testing cell viability and prolif-
eration was performed using the microculture MTS assay. Buforin IIb presented similar
cytotoxic results to cis-[Pt(NH
3
)
2
(malBuf
–2H
], a new Pt-buforin IIb conjugate and better
than cis-Pt[(NH
3
)
2
(malonate)] [
82
], suggesting that peptides could have the potential of
being a new therapeutic alternative while overcoming resistance [90].
3.5. Buforins for Prostate Cancer
Prostate cancer is one of the most frequent types of cancer present in male individuals.
While the action mechanism of buforin IIb for this type of cancer is still not fully understood,
it has been determined that it inhibits cell proliferation and induces apoptosis for PC-3 and
Du-145 cancer cells in a dose-dependent manner (IC50 less than 8 µM) [51].
3.6. Buforins for Liver Cancer
Purified buforin IIb was tested on liver cancer cells HepG2 using different time frames
and concentrations, analyzing cell viability and inhibitory effects. A concentration of 1.0
µ
M
buforin IIb for 24 h presented the best results compared to control samples regarding cell
proliferation and apoptosis.
In vitro
, almost 50% cell migration was observed, while
in vivo
experiments on HepG2 xenografts were performed on inoculated male mice. Tumor size
(volume and weight) was inhibited, and cell apoptosis was observed [38].
4. Anticancer Activity of Buforin-Containing Bioconjugates
The search for effective and targeted anticancer therapies has led to exploring innova-
tive strategies that leverage the unique properties of antimicrobial peptides. Bioconjugates
incorporating antimicrobial peptides have emerged as promising novel and effective anti-
cancer therapies [91].
As presented earlier, buforins, a family of small cationic peptides derived from hi-
stones, have demonstrated promising anticancer properties. Their ability to selectively
target cancer cells while sparing normal cells makes them attractive candidates [
66
]. How-
ever, challenges such as low stability, limited bioavailability, and potential off-target effects
have hindered their clinical translation. Researchers have explored the development of
buforin-containing bioconjugates as innovative therapeutic strategies for cancer treatment
to enhance their efficacy and specificity further. Harnessing the potential of buforins
through bioconjugation offers a unique strategy to improve their specificity, bioavailability,
and therapeutic efficacy in cancer treatment [55,82,92].
Polymers 2024,16, 728 8 of 14
Bioconjugates are hybrid molecules that result from the covalent linkage of two or
more distinct moieties, such as peptides, antibodies, or drugs, to create multifunctional en-
tities [
93
]. This approach combines different therapeutic agents, enhancing their individual
properties and enabling targeted drug delivery [
94
,
95
]. For buforins, several bioconjugation
strategies have been explored, including peptide conjugation with nanoparticles, liposomes,
and polymeric carriers. The carrier choice depends on the bioconjugate’s desired proper-
ties, including stability, release profile, and targeting ability. These approaches improve
buforin stability and bioavailability, enabling controlled release and targeted delivery to
cancer cells.
The bioconjugation of buforins with various entities, such as nanoparticles, antibodies,
or targeting ligands, aims to improve their pharmacokinetics, biodistribution, cell-specific
delivery, and targeting of cancer cells [
96
]. Strategies for buforin-bioconjugate design
include chemical conjugation, genetic fusion, and supramolecular structures [
2
]. These
approaches can modulate their physicochemical properties and tailor their anticancer
activity [
87
]. Surface modification of nanoparticles or liposomes with targeting ligands,
such as antibodies or peptides, enhances their specificity for cancer cell receptors, leading
to increased cellular uptake [97,98].
Nanoparticle-based buforin bioconjugates, for example, can enable the slow and con-
trolled release of the peptide, prolonging its presence within the tumor microenvironment
and enhancing therapeutic efficacy. This targeted delivery minimizes off-target effects
and improves the therapeutic index of buforin-containing bioconjugates. By incorporating
buforins into carriers, researchers have achieved the controlled and sustained release of the
peptides, leading to prolonged exposure to cancer cells. This sustained exposure amplifies
the cytotoxic effects, resulting in improved efficacy against cancer cells.
The synthesis of a Pt(II)-peptide conjugate was reported by Parker et al. (2016) [
82
]
using buforin IIb as a cell-penetrating guidance peptide and Pt(II) as a cancer cell cytotoxic
load. The authors synthesized the D-stereoisomeric form of buforin IIb that was reacted
with a malonate-type linker to obtain an O,O’-bidentate linker that was further complexed
with cis-[Pt(NH
3
)
2
(H
2
O)
2
]
2+
to produce cis-[Pt(NH
3
)
2
(malBuf
–2H
)]. The amino acid se-
quence side chains from buforin IIb are compatible and non-reactive with Pt, resulting
in a Pt-buforin IIb conjugate more cytotoxic and selective to the ovarian cancer cell line
(A2780cisR) than the parental AMP buforin IIb. Additionally, combining buforins with
other anticancer agents in bioconjugates exhibited synergistic effects, leading to greater
effectiveness in inhibiting tumor growth and inducing apoptosis.
Libardo et al. (2015) [
99
] obtained an sh-buforin (RAGLQFPVGRVHRLLRK-NH
2
)
conjugate containing an amino-terminal Cu
2+
and Ni
2+
binding unit (ATCUN): ATCUN-
AMP. The ATCUN motif influenced the nuclease activity and made these peptide conjugates
more active and stable than the parental AMP [
91
]. The antibacterial potency was influenced
by the stereochemistry of the derivatives containing L- and D-amino acids in the AMP
sequence; it was higher for ATCUN-D-sh-buforin than ATCUN-L-sh-buforin.
In their study, Cuellar et al. (2018) [
100
] immobilized buforin II on magnetite nanopar-
ticles. The results indicate that membrane translocation succeeded without promoting
disruption and affecting cell viability in the mammalian and bacterial cells investigated.
However, antimicrobial activity was affected; buforin-II–magnetite conjugates presented
no antimicrobial activity compared to buforin II.
Another study on magnetite nanoparticles and buforin II conjugates was performed
by Perez et al. (2019) [
92
]. For better molecular flexibility, buforin II was immobilized
on polyetheramine-modified magnetite to increase the penetration properties in diverse
cell-lines, with high biocompatibility but with a decrease in antimicrobial effects compared
to the parent peptide.
A specific anticancer effect of a buforin IIb and anionic peptide (a derived maga-
inin sequence) conjugate, bonded via a matrix metalloproteinase (MMP) cleavable linker,
was obtained and investigated by Jang et al. (2011) [
55
]. The resulting buforin conju-
Polymers 2024,16, 728 9 of 14
gate presented better anticancer effects than buforin alone in the pathological-producing
MMP cells.
One of the major challenges in cancer treatment is the development of drug resistance
in cancer cells. Bioconjugates offer a potential solution by bypassing drug efflux pumps
and other resistance mechanisms [94,95].
Combination therapies involving buforin-containing bioconjugates with chemother-
apeutic drugs or other AMPs have shown promising results in preclinical studies. The
synergistic effects of these combinations result in enhanced cytotoxicity and reduced drug
resistance in cancer cells [
91
]. Bioconjugates can facilitate the co-delivery of multiple
therapeutic agents, maximizing their combined anticancer potential. While promising,
translating buforin-containing bioconjugates from preclinical studies to clinical applications
requires rigorous evaluation.
In vivo
studies have provided valuable insights into their
pharmacokinetics, toxicity profiles, and antitumor effects [
82
]. Future clinical trials will be
crucial in assessing the safety and efficacy in human subjects. Further research is needed to
optimize bioconjugation strategies, assess long-term safety, and conduct clinical trials to
evaluate their effectiveness and tolerability in cancer patients.
Buforin-containing bioconjugates represent a novel and promising approach in can-
cer therapy, combining the unique anticancer properties of buforins with the benefits
of bioconjugation strategies. Bioconjugation enhances the anticancer potential by im-
proving specificity, cellular uptake, and therapeutic efficacy while mitigating off-target
effects and drug resistance. The improved targeting, sustained activity, and synergistic
effects with other therapeutic agents make these bioconjugates attractive candidates for
personalized and targeted cancer treatment. As research progresses, the development of
buforin-containing bioconjugates holds great potential in improving cancer patient out-
comes and advancing the field of anticancer therapeutics. This strategy holds promise for
overcoming the limitations of free buforins and offers a novel and exciting direction in this
field. Continued research and development in this area may eventually lead to the clinical
use of buforin bioconjugates as a potent and selective therapy against cancer.
5. Conclusions and Perspectives
Non-communicable illnesses, especially cancer, continue to pose a significant global
public health challenge. Despite decades of research and therapeutic approaches, cancer
remains a major cause of morbidity and mortality worldwide. Chemotherapy, a commonly
used treatment, has several drawbacks, including non-selective targeting and drug resis-
tance. To address these issues, scientists in the pharmaceutical industry are exploring
various approaches, including nanocarriers and antibody–drug conjugates, to improve
cancer treatment efficacy and reduce side effects.
Among the potential solutions, using selenium-conjugated peptides (STPs) and marine
peptides show promise as alternative cancer treatments. The search for novel antimicrobial
medications has become increasingly urgent due to the growing problem of microbial
resistance to conventional antibiotics. AMPs from various sources, such as plants, insects,
amphibians, and animals, have demonstrated strong antimicrobial activity against bacteria,
fungi, viruses, and parasites. These AMPs offer potential as novel therapeutics to combat
infectious diseases and cancer.
The discovery and development of targeted therapies, nanotechnology-based ap-
proaches, and natural peptides like AMPs provide hope for advancing cancer treatment
and antimicrobial strategies. As research in these areas continues, the medical community
can look forward to improved treatments that enhance patient outcomes and quality of life.
However, further research and clinical trials are needed to fully understand and harness
the potential of these novel therapeutic strategies.
Antimicrobial peptides (AMPs), such as the buforin family, have emerged as a promis-
ing source of new antibiotics. Buforins exhibit potent antimicrobial and anticancer proper-
ties, by disrupting bacterial cell membranes or interfering with intracellular processes. They
Polymers 2024,16, 728 10 of 14
offer potential advantages over conventional antibiotics, including fast action, reduced
resistance buildup, and environmental friendliness.
The practical application of buforins as therapeutic agents faces challenges such as
high production costs, limited bioavailability, and potential toxicity to mammalian cells. To
address these limitations and enhance the therapeutic potential of buforins, researchers
have explored the development of buforin-containing bioconjugates. Bioconjugation strate-
gies involving the covalent linkage of buforins with nanoparticles, liposomes, or targeting
ligands aim to improve their stability, bioavailability, and specificity for cancer cells. These
bioconjugates offer controlled release and targeted delivery, minimizing off-target effects
and potentially overcoming drug resistance in cancer cells.
While promising, translating buforin-containing bioconjugates from preclinical studies
to clinical applications requires further evaluation and optimization.
In vivo
studies using
animal models have provided valuable insights, and future clinical trials will be essential
to assess their safety and efficacy in human subjects.
Developing buforin-containing bioconjugates represents a significant step forward
in the quest for effective and targeted antimicrobial and anticancer therapies. Continued
research and clinical investigations will determine their potential as valuable additions to
the arsenal of modern medicine in the fight against infectious diseases and cancer.
Author Contributions: Conceptualization, D.M.C.; data curation D.M.C.; software, C.M. and D.M.C.;
writing—original draft preparation, C.M., A.M.T., M.D., C.P., L.C. and D.M.C.; writing—review
and editing, C.M., A.M.T., M.D., C.P., L.C. and D.M.C.; supervision, D.M.C.; project administration,
D.M.C.; funding acquisition, D.M.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by a grant of the Ministry of Research, Innovation and Digitiza-
tion, CNCS-UEFISCDI, project number PN-III-P4-PCE-2021-0639, within PNCDI III.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: No data was used for the research described in the article.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the
study’s design; in the collection, analyses, or interpretation of data; in the writing of the manuscript;
or in the decision to publish the results.
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