Ribonucleases. possible new approach in cancer therapy
In the review, the use of the ribonucleases for cancer therapy is discussed. Using of epigenetic mechanisms of regulation — blocking protein synthesis without affecting the DNA structure — is a promising direction in the therapy. The ribonucleases isolated from different sources, despite of simila...
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irk-123456789-1361332018-06-16T03:11:28Z Ribonucleases. possible new approach in cancer therapy Shlyakhovenko, V.O. Reviews In the review, the use of the ribonucleases for cancer therapy is discussed. Using of epigenetic mechanisms of regulation — blocking protein synthesis without affecting the DNA structure — is a promising direction in the therapy. The ribonucleases isolated from different sources, despite of similar mechanism of enzymatic reactions, have different biological effects. The use of enzymes isolated from new sources, particularly from plants and fungi, shows promising results. In this article we discuss the new approach for the use of enzymes resistant to inhibitors and ribozymes, that is aimed at the destruction of the oncogene specific mRNA and the induction of apoptosis. 2016 Article Ribonucleases. possible new approach in cancer therapy / V.O. Shlyakhovenko // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 2–8. — Бібліогр.: 47 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/136133 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Reviews Reviews Shlyakhovenko, V.O. Ribonucleases. possible new approach in cancer therapy Experimental Oncology |
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In the review, the use of the ribonucleases for cancer therapy is discussed. Using of epigenetic mechanisms of regulation — blocking
protein synthesis without affecting the DNA structure — is a promising direction in the therapy. The ribonucleases isolated from different
sources, despite of similar mechanism of enzymatic reactions, have different biological effects. The use of enzymes isolated from new
sources, particularly from plants and fungi, shows promising results. In this article we discuss the new approach for the use of enzymes
resistant to inhibitors and ribozymes, that is aimed at the destruction of the oncogene specific mRNA and the induction of apoptosis. |
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Article |
| author |
Shlyakhovenko, V.O. |
| author_facet |
Shlyakhovenko, V.O. |
| author_sort |
Shlyakhovenko, V.O. |
| title |
Ribonucleases. possible new approach in cancer therapy |
| title_short |
Ribonucleases. possible new approach in cancer therapy |
| title_full |
Ribonucleases. possible new approach in cancer therapy |
| title_fullStr |
Ribonucleases. possible new approach in cancer therapy |
| title_full_unstemmed |
Ribonucleases. possible new approach in cancer therapy |
| title_sort |
ribonucleases. possible new approach in cancer therapy |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2016 |
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Reviews |
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http://dspace.nbuv.gov.ua/handle/123456789/136133 |
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Ribonucleases. possible new approach in cancer therapy / V.O. Shlyakhovenko // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 2–8. — Бібліогр.: 47 назв. — англ. |
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Experimental Oncology |
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AT shlyakhovenkovo ribonucleasespossiblenewapproachincancertherapy |
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2025-07-10T00:42:02Z |
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2025-07-10T00:42:02Z |
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2 Experimental Oncology 38, 2–8, 2016 (March)
RIBONUCLEASES. POSSIBLE NEW APPROACH IN CANCER THERAPY
V.O. Shlyakhovenko
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
In the review, the use of the ribonucleases for cancer therapy is discussed. Using of epigenetic mechanisms of regulation — blocking
protein synthesis without affecting the DNA structure — is a promising direction in the therapy. The ribonucleases isolated from different
sources, despite of similar mechanism of enzymatic reactions, have different biological effects. The use of enzymes isolated from new
sources, particularly from plants and fungi, shows promising results. In this article we discuss the new approach for the use of enzymes
resistant to inhibitors and ribozymes, that is aimed at the destruction of the oncogene specific mRNA and the induction of apoptosis.
Key Words: RNases, RNA cleavage, cytotoxicity, apoptosis, cancer therapy.
Ribonucleases (RNases) are a large group of hy-
drolytic enzymes that degrade ribonucleic acid (RNA)
molecules. They are nucleases that catalyze the break-
down of RNA into smaller components. RNases are
a superfamily of enzymes which catalyzing the degra-
dation of RNA operate at the levels of transcription and
translation. They can be cytotoxic because the cleavage
of RNA renders illegible its information. Initially it was
assumed that the function of these enzymes is a simple
destruction of polyribonucleotides already fulfilled their
physiological role. Later, however, it became clear that
the RNases present in all living cells and biological flu-
ids, like pro- and eukaryotes, perform many important
vital functions. Some members of this family possess
a high molecular heterogeneity, with some isoforms that
exhibit extremely high specificity for cleavage of specific
loci in the polyribonucleotide chains.
RNases are ubiquitous, with a very short lifespan
in an unprotected environment. The cytotoxic ef-
fects of RNases include the RNA cleavage leading
to the inhibition of the protein synthesis and inducing
apoptosis [1]. The cytotoxic effect can be produced
by applying RNase on the outside surface of the cell
but it was found that cytotoxicity increases about
by 1000 times when enzyme is artificially introduced
into the cytosol, indicating internalization into the cell
as the rate limiting step for toxicity [2]. Mi et al. [3]
showed evidence that RNASEL Arg462Gln and Asp-
541Glu polymorphisms are associated with prostate
cancer risk and could be low-penetrance prostate
cancer susceptibility biomarkers.
There are two types of RNases, endoribonucle-
ases and exoribonucleases. Endoribonucleases are
RNase A, RNase P, RNase H, RNase III, RNase T1,
RNase T2, RNase U2, RNase V1, RNase I, RNase
PhyM and RNase V. Exoribonucleases includes
polynucleotide phosphorylase (PNPase), RNase PH,
RNase II, RNase R, RNase D, RNase T, oligoribonucle-
ase, exoribonuclease I and exoribonuclease II. These
enzymes differently cleave various RNA species.
Recently, researchers have paid attention to RNases
as possible agents for the cancer treatment. The idea
of using RNases for the treatment of cancer is to find
an enzyme selectively damaging cancer cells without
affecting the surrounding normal [4].
Today, several RNases allocated as possible candi-
dates for anticancer agents [5–9]. The interferon (IFN)
antiviral investigations and prostate cancer genetics
have merged on a single-strand specific, regulated
endoribonuclease. The 2´,5´-oligoadenylate (2´-5´A)
system is an IFN-regulated RNA decay pathway that
provides innate immunity against viral infections.
The biologic action of the 2´-5´A system is mediated
by RNase L, an endoribonuclease that becomes enzy-
matically active after binding to 2´-5´A. RNase L is also
implicated in mediating apoptosis in response to both
viral and nonviral inducers. To study the cellular effects
of RNase L activation, 2´-5´A was transfected into
the human ovarian cancer cell line Hey1B. Activation
of RNase L by 2´-5´A resulted in specific 18S rRNA
cleavage and induction of apoptosis, as measured
by TUNEL and annexin V binding assays. In contrast,
the dimeric form of 2´-5´A, ppA2´p5´A, neither acti-
vated RNase L nor caused apoptosis. Treatment with
IFN-beta prior to 2´-5´A transfection enhanced cellular
RNase L levels 2.2-fold and increased the proportion
of cells undergoing apoptosis (by 40%). However,
rRNA cleavages after 2´-5´A transfections were not
enhanced by IFN-beta pretreatments, indicating that
basal levels of RNase L were sufficient for this acti vity.
Apoptosis in response to RNase L activation was ac-
companied by cytochrome C release from mitochon-
dria. Induction of apoptosis by either 2´-5´A alone
or by the combination of 2´-5´A and IFN-beta was
effectively blocked with either the pancaspase inhibi-
tor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-
fmk. Therefore, activation of RNase L by 2´-5´A leads
to cytochrome C release into the cytoplasm and then
to caspase activation and apoptosis. These results
suggest potential uses for 2´-5´A in augmenting the
anticancer activities of IFN [10].
Submitted: September 30, 2015.
Correspondence: E-mail: AFDA@onconet.kiev.ua
Abbreviations used: 2´-5´A — 2´,5´-oligoadenylate; BS-RNase —
bovine seminal RNase; HPC1 — hereditary prostate cancer;
HPV — human papillomavirus; HVJ — hem-agglutinating virus
of Japan; IFN — interferon; mRNA — messenger RNA; PNPase —
polynucleotide phosphorylase; RI — ribonuclease inhibitor protein;
RIPs — ribosome-inactivating proteins; RNases — ribonucleases;
siRNA — small interfering RNA; Topo — topoisomerase.
Exp Oncol 2016
38, 1, 2–8
REVIEW
Experimental Oncology 38, 2–8, 2016 (March)38, 2–8, 2016 (March) (March) 3
The RNASEL gene, a strong candidate for the
hereditary prostate cancer 1 (HPC1) allele, encodes
a single-stranded specific endoribonuclease involved
in the antiviral actions of IFNs. Xiang et al. [11] have
shown that RNase L is activated enzymatically after
binding to unusual 5´-phosphorylated, 2´,5´-linked
oligoadenylates (2´-5´A). Biostable phosphorothio-
ate analogues of 2´-5´A were synthesized chemically
and used to study the effects of naturally occurring
mutations and polymorphisms in RNASEL. The 2´-
5´A analogues induced RNase L activity and caused
apoptosis in cultures of late-stage, metastatic hu-
man prostate cancer cell lines DU145, PC3, and
LNCaP. However, DU145 and PC3 cells were more
sensitive to 2´-5´A than LNCaP cells, which are
heterozygous for an inactivating deletion mutation
in RNase L. The RNase activities of missense variants
of human RNase L were compared after expression
in a mouse RNase L(-/-) cell line. Several variants
(G59S, I97L, I220V, G296V, S322F, Y529C, and D541E)
produced similar levels of RNase L activity as wild-type
enzyme. In contrast, the R462Q variant, previously
implicated in up to 13% of unselected prostate cancer
cases, bound 2´-5´A at wild-type levels but had a 3-fold
decrease in RNase activity. The deficiency in RNase
L activity (R462Q) was correlated with a reduction in its
ability to dimerize into a catalytically active connect.
Furthermore, RNase L (R462Q) was deficient in cau-
sing apoptosis in response to 2´-5´A consistent with its
possible role in prostate cancer development. Authors
support the notion that RNASEL mutations allow tumor
cells to escape a potent apoptotic pathway [11].
Genetics studies from several laboratories
in the U.S., Finland, and Israel, support the re-
cent identification of the RNase L gene, RNASEL,
as a strong reason for the HPC1. Results from these
studies suggest that mutations in RNASEL predispose
men to an increased incidence of prostate cancer,
which in some cases reflect more aggressive disease
and/or decreased age of onset compared with non-
RNASEL linked cases. RNase L is a uniquely regulated
endoribonuclease that requires 5´-triphosphorylated,
2´,5´-linked oligoadenylates (2´-5´A) for its activity.
The presence of both germline mutations in RNASEL
segregating with disease within HPC-affected families
and loss of heterozygosity in tumor tissues suggest
a novel role for the regulated endoribonuclease in the
pathogenesis of prostate cancer. The association
of mutations in RNASEL with prostate cancer cases
further suggests a relationship between innate im-
munity and tumor suppression. It is proposed that
RNase L functions in counteracting prostate cancer
by virtue of its ability to degrade RNA, thus initiating
a cellular stress response that leads to apoptosis [12,
13]. The HPC1 allele maps to the RNASEL gene en-
coding a protein (RNase L) implicated in the antiviral
activity of IFNs.
To investigate the possible role of RNase L in apop-
tosis of prostate cancer cells, Malathi et al. [14]
decreased levels of RNase L by several fold in the
DU145 human prostate cancer cell line through the
stable expression of a small interfering RNA (siRNA).
Control cells expressed siRNA with three mismatched
nucleotides to the RNase L sequence. Cells deficient
in RNase L, but not the control cells, were highly
resistant to apoptosis by the RNase L activator, 2´-
5´A. At the same time, the RNase L-deficient cells
were also highly resistant to apoptosis by combination
treatments with a topoisomerase (Topo) I inhibitor
(topotecan) and tumor necrosis factor-related apop-
tosis-inducing ligand TRAIL (Apo2L). In contrast, cells
expressing siRNA to the RNase L inhibitor RLI (HP68)
showed enhanced apoptosis in response to Topo I in-
hibitor alone or in combination with TRAIL. An inhibitor
of c-Jun NH(2)-terminal kinases reduced apoptosis
induced by treatment with either 2´-5´A or the com-
bination of camptothecin and TRAIL, thus implicating
c-Jun NH(2)-terminal kinase in the apoptotic signaling
pathway. Likewise, prostate cancer cells were sensitive
to apoptosis from the combination of 2´-5´A with either
TRAIL or Topo I inhibitor, whereas normal prostate
epithelial cells were partially resistant to apoptosis.
These findings indicate that RNase L integrates and
amplifies apoptotic signals generated during treatment
of prostate cancer cells with 2´-5´A, Topo I inhibitors,
and TRAIL [14].
Montia et al. [15] found that an extracellular RNase
is involved in the control of ovarian tumorigenesis. They
shown that the loss of function of RNase T2, an ancient
and phylogenetically conserved RNase, plays a crucial
role in ovarian tumorigenesis.
The above data were the basis for many attempts
to use RNases for cancer therapy. Currently, it is shown
that the RNases isolated from different sources, have
antitumour activity. In some cases, studies reported
to the clinical trials.
Bovine seminal RNase (BS-RNase). The RNase
found in bull semen, displays antitumor, antispermato-
genic, and immunosuppressive activities. Besides its
unique structure and enzymatic properties seminal
RNase belongs to an interesting group of RNases, the
RISBASES (RNases with Special, Biological Actions),
other members of which include angiogenin, selec-
tively neurotoxic RNases, a lectin and the incompa-
tibility factors from a flowering plant [16].
BS-RNases, a dimeric protein found to be homoge-
neous by several standard criteria of purity, is hetero-
geneous when analyzed by ion-exchange chromato-
graphy on carboxymethylcellulose. Three increasingly
cationic subforms can be separated. The heterogene-
ity is due to the presence of two types of subunits, al-
pha and beta, which make up three isoenzymic dimers:
alpha 2, beta 2, and alpha beta. Deamidation reactions
can convert the most cationic beta 2 subform into the
alpha beta subform, which in turn can be converted
into stable alpha 2 subform. These conversions involve
the hydrolysis of 2 mol of differentially labile amide
groups per mol of protein. The ratios alpha 2: alpha
beta: beta 2 are constant in all preparations of seminal
RNase tested; they are independent of the purification
4 Experimental Oncology 38, 2–8, 2016 (March)
procedure as well as of the biological source of the
enzyme (seminal plasma or seminal vesicles). These
results indicate that deamidations occur in vivo before
the protein is secreted from the seminal glands. They
also suggest that heterogeneity of seminal RNase
reflects a physiological need of distinct molecular
forms of enzyme or, alternatively, a process which
leads to the aging of the protein [17]. The cytotoxic
effect of BS-RNase on tumor cells is accompanied
by the induction of apoptosis [17].
Sinatra et al. [18] provide ultrastructural and flow
cytometry evidence of apoptotic death following BS-
RNase treatment, in normal cells and phytohemag-
glutinin-stimulated lymphocytes. Transmission and
scanning electron microscopy, which were fully sup-
ported by flow cytometry data, showed typical features
of apoptosis, such as decreased cell size, chromatin
condensation, fragmentation in micronuclei, and the
presence of apoptotic bodies. BS-RNase is a homo-
logue of bovine pancreatic ribonuclease (RNase A).
Unlike RNase A, BS-RNase has notable toxicity for
human tumor cells. Wild-type BS-RNase is a homodi-
mer linked by two intermolecular disulfide bonds. This
quaternary structure endows BS-RNase with resistance
to inhibition by the cytosolic ribonuclease inhibitor
protein (RI), which binds to RNase A and monomeric
BS-RNase. Authors report on the creation and analysis
of monomeric variants of BS-RNase that evade RI but
retain full enzymatic activity [19]. The cytotoxic activity
of these monomeric variants exceeds that of the wild-
type dimer by up to 30-fold, indicating that the dimeric
structure of BS-RNase is not required for cytotoxicity.
Dimers of these monomeric variants are more cytotoxic
than wild-type BS-RNase, suggesting that the cytoto-
xicity of the wild-type enzyme is limited by RI inhibition
following dissociation of the dimer in the reducing
environment of the cytosol. Finally, the cytotoxic acti-
vity of these dimers is less than that of the constituent
monomers, indicating that their quaternary structure
is a liability. These data provide new insight into struc-
ture — function relationships of BS-RNase. Moreover,
BS-RNase monomers described herein are more toxic
to human tumor cells than is any known variant or homo-
logue of RNase A including ranpirnase, an amphibian
homologue in phase III clinical trials for the treatment
of unresectable malignant mesothelioma [20].
Onconase (ranpirnase) is a first-in-class therapeu-
tic product based on Alfacell’s proprietary RNase tech-
nology. A natural protein isolated from the leopard frog
Rana pipiens, ranpirnase has been shown in the labo-
ratory and clinic to target cancer cells while sparing
normal cells. Ranpirnase triggers apoptosis, the natu-
ral death of cells, via multiple molecular mechanisms
of action [21]. Ranpirnase is a novel RNase which
preferentially degrades tRNA, thus leading to inhibition
of protein synthesis and, ultimately, to cytostasis and
cytotoxicity. Ranpirnase has demonstrated antitumor
activity both in vitro and in vivo in several tumor mod-
els [22, 23]. The maximum tolerated dose emerging
from phase I studies was 960 g/m2, with renal toxicity
as the main dose-limiting toxicity. A large phase II trial
showed that ranpirnase has disease-modifying activity
against malignant mesothelioma. Ranpirnase proved
to be general caspase inhibitors strongly suggest that
the signaling pathways triggered by the death stimuli
diverge into one pathway leading to caspase activa-
tion (PARP processing, DNA degradation, and annexin
V binding) and another leading to mRNA degradation.
In addition, data obtained show that extensive mRNA
degradation, although apparently a general early
apoptotic event, is not sufficient to induce cell death
per se. The molecular mechanisms implicated in apop-
tosis-induced mRNA degradation are not known.
The apoptotic stimuli leads to activation of an RNase
dependent on an upstream apical caspase (inhibited
by Z-VAD-fmk, but not p35, and unable to cleave
PARP) in a similar manner as CAD (caspase-activated
DNase) activation. Conversely, activation of the RNase
may be independent of caspase activation but still
inhibited by Z-VAD-fmk [24]. Ranpirnase has been
granted fast track status and orphan-drug designation
for the treatment of malignant mesothelioma by the
Food and Drug Administration. Additionally, ranpirnase
has been granted orphan-drug designation in the
Euro pean Union and Australia.
In April, 2006 Alfacell released interim data from
the company’s ongoing Phase IIIb randomized clinical
trial of ranpirnase and doxorubicin for the treatment
of malignant mesothelioma. The study reached the first
interim analysis at 105 events (patient deaths) of the
total 316 patients enrolled. Interim data demonstrate
that the overall median survival time favored the ran-
pirnase plus doxorubicin treatment group (12 months)
over the doxorubicin group (10 months). It has been
tested and found to be cytotoxic to cancer cells be-
cause of its enzymatic activity against RNA.
Ranpirnase is internalized by endocytosis and
released into the cytosol of the cancerous cell, where
it selectively degrades tRNA beyond repair. In doing so,
ranpirnase inhibits protein synthesis, stops cell cycle
proliferation, and induces apoptosis [25].
USA-based Tamir Biotechnology (formerly Alfacell)
says that scientists supported by the National Institute
of Allergy and Infectious Diseases (NIAID) reported
test results confirming two of its lead compounds
showed excellent in vitro antiviral activity and no cel-
lular toxicity at dose levels tested for human papillo-
mavirus (HPV). Testing was performed using the HPV
11 strain, which along with HPV type 6, is responsible
for 90% of genital or anal warts [26].
Ranpirnase is a cytotoxic RNase which targets
tumor cells in vivo and in vitro. To date, cellular tRNA
appeared to be the major target for ranpirnase medi-
ated cytotoxic activity. Saxena et al. [27] demonstrated
that ranpirnase can also cleave double-stranded
RNA (dsRNA). Incubation of ranpirnase at 37 °C with
GAPDH gene-dsRNA (~440 bp long) and dsRNA lad-
der showed degradation of dsRNA into a spectrum
of smaller dsRNA fragments. Moreover, incubation
of dsRNA substrates at 40 °C under similar conditions
Experimental Oncology 38, 2–8, 2016 (March)38, 2–8, 2016 (March) (March) 5
markedly potentiated further cleavage of dsRNAs. The
recently discovered double-stranded RNase activity
of ranpirnase suggests another mechanism for induc-
ing cell death/apoptosis in malignant phenotypes via
the RNA interference mechanism involving siRNA and
miRNA [27]. Another enzyme sialic acid-binding lectin
(SBL), isolated from oocytes of Rana catesbeiana,
is leczyme and has both lectin and RNase activities [8].
SBL agglutinates various kinds of tumor cells but not
normal cells. SBL agglutination activity is not affected
by mono- or oligosaccharides. However, SBL-induced
agglutination and antitumor effects are inhibited by si-
alomucin but not asialomucin. SBL causes cancer-
selective induction of apoptosis by multiple signaling
pathways, which target RNA [8].
RNase T1 is an endonuclease (EC 3.1.27.3), iso-
lated from fungi, which cuts the single-stranded RNA
molecules after guanine residues, i.e., at the 3´ end.
RNase T1 is the best known representative of a large
family of ribonucleolytic proteins secreted by fungi,
mostly Aspergillus and Penicillium species. Ribotoxins
stand out among them by their cytotoxic character.
They exert their toxic action by first entering the cells
and then cleaving a single phosphodiester bond lo-
cated within a universally conserved sequence of the
large rRNA gene, known as the sarcin — ricin loop.
This cleavage leads to inhibition of protein biosynthe-
sis, followed by cellular death by apoptosis. Although
no protein receptor has been found for ribotoxins,
they preferentially kill cells showing altered membrane
permeability, such as those that are infected with virus
or transformed.
The most-studied form of the enzyme RNase
T1 is the mold of Aspergillus oryzae and Penicillium
species. RNase T1 is a small protein α + β, consisting
of 104 amino acid residues. Protein structure contains
four antiparallel beta pleated sheets covered nearly
five turnovers long alpha-helix. RNase T1 has two di-
sulfide bonds Cys2-Cys10 and Cys6-Cys103, the latter
of which is involved in the installation [28] and the full
restoration of disulfide bonds leads to the unfolding
of the protein [29].
Due to the specificity guanine RNase T1 frequently
used for cleavage of RNA denatured prior to sequenc-
ing. Like other RNases, for example RNase A, RNase
T1 is a popular subject for study of protein folding [30].
So, ranpirnase and α-sarcin are known to be toxic
to tumor cells, and on the other hand, their structure
is related to that of RNase T1, latter is noncytotoxic
because of its inability to internalize into tumor cells.
In their study, Yuki et al. [31] internalized RNase T1 into
human tumor cells via a novel gene transfer reagent,
hem-agglutinating virus of Japan (HVJ) envelope vec-
tor, which resulted in cell death. This cytotoxicity was
drastically increased by pretreatment of HVJ envelope
vector with protamine sulfate, and was stronger than
that of ranpirnase, which is in phase III human clinical
trials as a nonmutagenic cancer chemotherapeutic
agent. Furthermore, internalized RNase T1 induced
apoptotic cell death programs. Because its cytotoxic-
ity is unfortunately not speci�c to tumor cells, it can-�ctotumorcells,itcan-c to tumor cells, it can-
not at present be developed as an anticancer drug.
However, authors believe that RNase T1 incorporated
in HVJ envelope vector will be a unique anticancer
drug if HVJ envelope vector can be targeted to tumor
cells [28, 32].
Specific complexes of protein and RNA carry out
many essential biological functions, including RNA
processing, RNA turnover, RNA folding, as well as the
translation of genetic information from mRNA into pro-
tein sequences. Messenger RNA (mRNA) decay is now
emerging as an important control point and a major
contributor to gene expression. Continuing identifica-
tion of the protein factors and cofactors, and mRNA
instability elements responsible for mRNA decay allow
researchers to build a picture of the special processes
involved in mRNA decay and its regulation [33].
Presently known data demonstrate that apoptosis-
induced mRNA degradation is an early event triggered
by different apoptotic signals, occurring not only in in vi-
tro models but also in vivo. It is not restricted to genes
with a specific function, since besides 28S rRNA, it af-
fects mRNA coding for proteins implicated in a variety
of functions, including cell type-specific functions
such as HLA-I, IAα, TCRβ, and CD69, structural func-
tions such as β-actin, or control of cell survival, such
as BAX. There is evidence that potassium channels,
the NF-κB signaling pathway, and various caspases
play a role in exogenous RNase-induced apoptosis [9].
Apoptosis-induced mRNA degradation is an active
process that induces a similar decay kinetics (t1/2 be-
tween 1.5 and 3 h) of mRNAs with very different intrinsic
half-lives (from 20 min to > 10 h). Kinetic experiments
using stimuli diverge into one pathway leading to cas-
pase activation (PARP processing, DNA degradation,
and annexin V binding) and another leading to mRNA
degradation were held. In addition, the data obtained
show that extensive mRNA degradation, although ap-
parently a general early apoptotic event, is not sufficient
to induce cell death per se. The molecular mechanisms
implicated in apoptosis-induced mRNA degradation
are not known. Nonetheless, it seems that the apop-
totic stimuli leads to activation of an RNase dependent
on an upstream apical caspase (inhibited by Z-VAD-fmk,
but not p35, and unable to cleave PARP) in a similar
manner as CAD activation. Conversely, activation of the
RNase may be independent of caspase activation but
still inhibited by Z-VAD-fmk [34].
Actibind and RNase T2. Actibind, a protein that
is produced by the black molds Aspergillus niger,
a well-known microorganism used in bio and food
industry [32]. In plants, actibind binds actin, a major
component of the intracellular structure in plants, in-
terfering with the plants’ pollen tubes and halting cell
growth. Actibind can also affect mammalian cancer
development [35]. RNase T2, was also subsequently
found to bind actin in human and animal migrating
cells, such as the cells that are responsible for new
blood vessel formation (angiogenesis) in tumors thus
blocking the blood supply to the tumors, actibind
6 Experimental Oncology 38, 2–8, 2016 (March)
halted the ability of malignant cells to move through
the blood stream to form new metastases [36].
A further plus is that actibind is not toxic to normal
cells, thereby significantly minimizing the risk of side
effects. The fungal actibind and the human RNase
T2 represent the basis for a new class of drugs that
could be used as a front-line therapy in the fight
against cancer.
α-Sarcin. α-Sarcin, mitogillin, and restrictocin
are small (approximately 17 kDa) basic ribosome-
inactivating proteins (RIPs) produced by the Aspergilli
that catalytically inactivate the large ribosomal subunits
of all organisms tested to date. These three fungal ribo-
toxins act as specific RNases by hydrolyzing one single
phosphodiester bond in the universally conserved
alpha-sarcin domain of 23–28S rRNAs and are among
the most potent inhibitors of protein synthesis known.
Previous molecular studies of ribotoxins indicated
that they belong to the superfamily of RNases and
analysis of the mitogillin gene employing PCR-mediated
site-specific mutagenesis suggests that certain do-
mains in ribotoxins, which share homologies with motifs
in ribosome-related proteins, may be responsible for the
targeting of ribotoxins to the ribosome. The applications
of the ribotoxins as tools in research and their using
as therapeutic and diagnostic agents are reviewed [37].
α-Sarcin is a potent polypeptide toxin (cyclising RNase)
of 150 residues secreted by the fungus Aspergillus gi-
ganteus MDH18894 that belongs to the type 1 (those
having only single polypeptide chain) group of the
ribosome-inactivating enzyme. It is the most significant
member of the family of fungal ribotoxins that display
a 3-dimensional structure.
His50, Glu96, His137 residues of α-sarcin in-
volved in the mechanism of catalysis. The hydrolysis
of 3´-5´phosphodiester bond of the substrate yielding
2´-3´ cyclic mononucleotide and then conversion of the
intermediate into the corresponding 3´-monophos-
phate derivative as the final product of the reaction.
Thus, α-sarcin acts by cleavage of the phosphodiester
bond of 28S rRNA, stops the protein synthesis. In addi-
tion to this enzymatic activity α-sarcin interacts with the
lipid bilayers promoting their fusion and leakage. Any
toxin that is able to produce more than 90% of inhibition
of protein synthesis may induce apoptosis [38, 39].
α-Sarcin is active against transformed or virus-
infected mammalian cells, in the absence of any other
permeabilizing agent [40, 41]. RNases distinct from
ubiquitin-like peptides and proteins were isolated
from several mushroom species. One of RNases was
isolated from Pleurotus sajor-caju; it exerts an anti-
proliferative action on hepatoma and leukemia cells,
and anti-mitogenic action on mouse spleen cells [42].
Recently was shown that antiproliferative activity can
be attributed to enzymes which inactivate ribosomes
by eliminating one or more adenosine residues from
rRNA. During the past decade, mushroom inactivating
proteins were isolated from several species including
Calvatia caelata, F. velutipes, H. marmoreus, Lyophyl-
lum shimeiji, and Pleurotus tuber-1 ribosome [43, 44].
RNase P. RNase P is unique from other RNases
in that it is the ribozyme, i.e. RNA that acts as a cata-
lyst in the same way that the protein based enzyme
would. Its function is to cleave off the extra or precur-
sor sequence of RNA on RNA molecules. In the current
cancer therapy, the main problem is to distinguish be-
tween the cancer cells and the normal cells. There are
certain chimeric molecules, which are specific to the
cancer cells, which can act as the specific targets and
thus solving the problem [45]. M1 RNA is the catalytic
subunit of RNase P and this subunit catalyses the hy-
drolytic removal of 5´-leader sequence of t-RNA. Stu-
dies of the substrate recognition by M1 RNA have led
to the development of the strategy of gene targeting
by M1 RNA [45]. M1 RNA can be targeted to the mRNA
simply by the addition of the so-called guide sequence
at the 3´-terminal. So this now becomes as M1-GS,
which has mRNA as its target. Cleavage of the mRNA
will not allow the formation of the fusion proteins, which
are specific for the cancer cells. The utility of the M1-
GS in the cancer was shown by its use against BCR-ABL
oncogene model [45]. This BCR-ABL oncogene was
created by the translocation of the sequences from
ABL gene on chromosome 9 to the BCR gene on the
chromosome 22 [46]. Two oncogenes were created
BCR-ABL p190 and BCR-ABL p210. Both of these
differ and have identical ABL derived sequences but
differ in the number of the BCR nucleotides. These
chimeric molecules so formed by the chromosomal
rearrangement would be specific to the cancer cells
and thus serve as the excellent targets. These BCR-
ABL oncogenes are responsible for the myelogenous
leukemia and acute lymphoblastic leukemia [47]. BCR-
ABL oncogenes inhibit the apoptosis by Bcl-2 pathway
as a part of their oncogenic phenotype. Inhibition of the
BCR-ABL expression would thus reverse this phenotype
and the cells die by apoptosis. It should be noted that
M1-GS should target only at the junction sequences
of the transcribed mRNA. If not so then the mRNA of the
normal cells would be cleaved and thus the resultant
damage to the normal cells would occur. This gene
therapy promises to be an effective strategy for the
future treatment of the cancer.
Using only this BCR-ABL system, model has created
the RNase P technology for the inhibition of the chimeric
gene products and the efficiency of the agents has not
been evaluated in the animal models to our knowledge
and thus the efficiency of the delivery process is still
a major problem to be investigated and solved. But
surely this advancement provides a new therapeutic tool
for the treatment of cancer and holds some promise for
more selective, non-toxic cancer therapy in the future.
These data show that the sources of bioactive
RNases may be of different origin. It is known that
a huge amount of unexplored RNases contain plants.
Vegetation, as well as the world of fungi, presents
a potentially inexhaustible source of these enzymes.
It can be expected that some of these will possess
anti-tumor activity. From the above it is clear that fur-
Experimental Oncology 38, 2–8, 2016 (March)38, 2–8, 2016 (March) (March) 7
ther study of antitumor action of RNases is possible
in several directions.
1. The search for new sources of the enzymes with
the new physiological properties. In this regard, we can
point to such a source as the plants and fungi. It is known
that the flora is characterized by an exceptionally large
variety of RNases. It can be expected that some of these
will possess anti-tumor activity. An important source
of the enzymes can also be mushrooms. Well-known
antitumor activity of many fungi at least partially may
be explained by their enzymatic activity.
2. The study must be focused on the search for new
inducers of RNases activity in the tissues, including
tumor tissue. The same way the latent RNase L is ac-
tivated by 2´-5´A which in turn is synthesized under
the influence of IFN.
3. Chemical modification of known RNases to en-
hance their resistance to intracellular inhibitors.
4. Creation of new ribozyme-type highly specific
RNases aimed to the precise destruction of the pro-
ducts of oncogenes.
5. Perspective can be also combined use of RNases
with known anticancer drugs. We believe that further
development of these studies will be a prerequisite for
the creation of new effective agents for the treatment
of cancer.
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