J. Braz. Chem. Soc., Vol. 00, No. 00, 1-8, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
#These authors contributed equally to this work.
Morpho/Proteomic Comparative between High Grade Pleomorphic Sarcoma and
Metastasis Diagnosed in an Old Captive Common Hippo
Adriana R. Silva,a,# José T. J. G de Lacerda,b,# Bernadete Faria,b Isabela W. da Cunha,c
Vitor P. de Andradec and Nilson A. Assunção*,a,b
aInstituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo,
04039-032 Diadema-SP, Brazil
bEscola Paulista de Medicina, Universidade Federal de São Paulo, 04023-062 São Paulo-SP, Brazil
cDepartamento de Patologia, A.C. Camargo Cancer Center, 01509-010 São Paulo-SP, Brazil
Old age is a risk factor for cancer development in humans and animals, and studies have
shown that tumors in animals are acceptable models for studying human cancers, considering the
similarities between their factors. This work was conducted in a 53-year-old captive female common
hippo (Hippopotamus amphibious) with a left leg tumor and metastatic mass. Histopathological and
immunohistochemical analyses were carried out with a ﬁnal diagnosis of a high grade pleomorphic
sarcoma. A proteomic study using mass spectrometry was added in order to identify further aspects
of the primary tumor and metastasis which could improve our understanding, and each tissue showed
a proteomic proﬁle indicative of its pathologic state with signiﬁcant differences between healthy
tissue, primary and metastatic tumors. Low levels of β-actin in primary tumors were identiﬁed,
and this may be associated with a possible consequence of cytoskeleton dynamic modiﬁcation. In
metastatic tissue, these dynamics may be affected by the presence of HSP chaperone 60.
Keywords: Hippopotamus amphibious, high grade pleomorphic sarcoma, cytoskeleton,
β-actin, mass spectrometry
Oncogenic phenomena in animals include a wide
variety of benign and malignant tumors that develop during
the life of the animal and have several health consequences.1
Species with large bodies tend to have a lower cancer rate,
which seems to indicate the presence of complex tumor
detection systems (Peto’s paradox).2 To illustrate this
paradox, in contrast with humans, who have only two copies
of the TP53 tumor suppressor gene, elephants, the largest
of the land mammmals, have 20 copies of the TP53 gene.
This allows for extra efﬁciency in their systems, enabling
the correction of DNA damage or inducing apoptosis in
mutated cells. As a result, only 3% of elephants develop
Despite some comparative cancer and lifespan analyses
between wild and zoo animals showing that captive animals
were susceptible to cancer and decreased longevity.5,6
Tidière et al.7 revealed that mammals from zoo populations
generally lived longer than their wild counterparts (84% of
species). Of particular interest, the common hippopotamus
(Hippopotamus amphibius), a robust mammal with an
estimated 40-year lifespan in nature and with slow pace of
life, has increased longevity in captivity.7-9 The relationship
between life expectancy and environment may be associated
with the low caloric diet and reduced metabolism present
in captive animals, which slows ageing and the appearance
of cancer cases. This is also true for humans.10,11
Paradoxically, the exceptional longevity these captive
animals exhibit can be associated with cancer due to several
factors such as telomere shortening, increased cellular
senescence, accumulation mutation of DNA in stem and
progenitor cells required for active replenishment during
the lifespan of the organism, mutations in tumor suppressor
genes and metabolic changes.12-14
Based on available database information and proteomics
approach, it is possible to differentiate proteomes in healthy
and pathological conditions to obtain an understanding of
cancer at the molecular level for the purpose of describing
Morpho/Proteomic Comparative between High Grade Pleomorphic Sarcoma and Metastasis Diagnosed J. Braz. Chem. Soc.
molecular processes and identiﬁcation of biomarkers.15,16
Thus, the objective of this study was to differentiate
the proteomes of healthy skeletal muscle, high grade
pleomorphic sarcoma and heart metastasis diagnosed in
a common hippo, located at a zoo, with high longevity,
and to identify possible biomarkers responsible for the
development and progression of these conditions.
Animal aspects and samples collection
The animal under examination was a female common
hippo (Hippopotamus amphibious) aged 53 years
presenting with prolonged anorexia and weight loss.
Clinical and radiological examinations of the posterior and
upper limbs revealed severe bone disease and osteoarthritis
of both metacarpophalangeal joints. During autopsy,
a 27 cm pink to gray main tumor mass attached to the
left tibial crest near the cranial epiphysis was detected.
There were also several white to gray nodules in the lungs
(2 to 5 cm), heart (0.5 to 1.0 cm), mesenteric lymph nodes
(1.5 cm) and liver (12 cm). Tissue samples were collected
from a primary tumor present in the paw, healthy skeletal
muscle around the tumor, and metastatic tissue from heart.
All the samples remained at −80 °C until the time of the test.
Histopathological and immunohistochemical analysis
Samples were ﬁxed in 10% neutral buffered formalin,
embedded in parafﬁn, sectioned at 4 µm, and stained with
hematoxylin and eosin (H&E). Immunohistochemistry was
also performed on tumor sections according to a previously
described protocol.17 Based on histological ﬁndings, primary
antibodies were used to detect antigens from mesenchymal
tissues with Vimentin (V9), muscle with Desmin (D33) and
α-SMA (1A4), histiocytes with CD 34 (QBEnd 10), CD45
(2B11-PD7/26) (DakoCytomation, Glostrup, Denmark)
and Lysozyme (polyclonal), neuroendocrine tissues with
S100 (polyclonal) and epithelial tissues with cytokeratin
cocktail (AE1AE3), and EMA (E29). All antibodies used
were from DakoCytomation, Denmark. Normal tissues
from the hippopotamus were used as negative and positive
controls for the reactions.
Sample preparation for proteomic analysis
Samples (500 mg) were ground in liquid nitrogen,
homogenized and solubilized in 500 µL of lysis buffer
(25 mmol L−1 Tris base pH 8.5, 1% Triton X, 4% CHAPS,
1 mmol L−1 phenylmethylsulfonyl ﬂuoride) with 750 µL
of protease inhibitor (Roche, Basel, Switzerland). The
homogenate was centrifuged at 5000 rpm at 4 °C for 10 min
with 125 µL of trichloroacetic acid containing 80 mmol L−1
dithiothreitol (DTT). The supernatant was discarded, and
the precipitate was washed with 500 µL of ice-cold acetone
containing 20 mmol L−1 DTT and centrifuged at 13,000 rpm
at 4 °C for 10 min; the washing and centrifugation steps
were repeated 3 times. The supernatant was discarded and
the precipitate was dried by evaporation and resuspended in
extraction buffer (40 mmol L−1 Tris base pH 8.5 containing
7 mol L−1 urea, 2 mol L−1 thiourea, 4% CHAPS and
65 mmol L−1 DTT), and solubilized at 300 rpm overnight
at 4 °C. The sample was centrifuged at 13,000 rpm at 4 °C
for 10 min, and detergent was removed from the supernatant
with Pierce Detergent Removal Spin Columns (Thermo
Fisher Scientiﬁc Inc., USA) according to the manufacturer’s
recommendations. Total protein was determined using the
Proteins were reduced with 5 mmol L−1 DTT for 30 min
at 56 °C, then alkylated with 14 mmol L−1 iodoacetamide
for 30 min in the dark. Proteins were further reduced with
5 mmol L−1 DTT for 30 min at 56 °C, with the addition of
50 mmol L−1 NH4HCO3 plus CaCl2 1 mmol L−1 (100 µL) for
pH adjustment and reduction of self-enzymatic proteolysis.
The chemicals used were purchased from Sigma-Aldrich,
USA. The samples were subjected to enzymatic digestion
overnight at 37 °C with trypsin 1:50 (m/m) (sequencing-
grade modiﬁed trypsin, Promega, USA), and the reaction
was quenched with the addition of 0.1% triﬂuoroacetic
Nano high performance liquid chromatography tandem
mass spectrometry (HPLC-MS/MS) analysis
The solutions containing the eluted peptides were
concentrated in a SpeedVac at 20 °C and resuspended in 2%
acetonitrile + 0.1% formic acid. Concentrates were injected
into ultra-high performance liquid chromatography systems
(UHPLC) Nano-Advance (Bruker Daltonics, Germany)
in a reverse phase C18 column 150 × 0.1 mm at a ﬂow of
0.1 mL min−1 and injection volume of 5 µL. Formic acid
gradients consisted of 0.1% formic acid in water (solvent
A) and 0.1% formic acid in acetonitrile 95% (solvent B),
with an initial concentration of 2% solvent B and a gradual
increase to 10-90% solvent B over 120 min. The MS data
was acquired with a micrOTOF QII mass spectrometer
(Bruker Daltonics, Germany). The acquisitions were made
in the range of m/z 200-1500, with the following parameters:
quadrupole ion energy 6.0 eV; collision energy 12 eV s−1;
radio frequency (RF) collision 600 Vpp; 140 µs time transfer;
and pre-pulse 14 µs storage. The time-of-ﬂight repetition
Silva et al. 3Vol. 00, No. 00, 2017
rate was 5.0 kHz frequency 2 GHz with ﬂight tube 8600 V,
reﬂector 1700 V, 1700 V detector source, TOF 2140 V
detector and collision energy 8 keV. The MS/MS spectra
were obtained by collision-induced dissociation (CID).
The MS/MS spectra were analyzed using Peaks Studio
7.0 software (Bioinformatics Solutions, Waterloo, Canada)
for de novo analysis and multi-round database search.19
Based on the low number of proteins available in the
hippopotamus database, the de novo sequenced peptides
with average local conﬁdence (ALC) scores ≥ 50% were
selected for database searches against Mammalian databases
from UniProt20 in the ﬁrst round and NCBI21 in the second
round. The MS/MS spectra were carried out with precursor
mass tolerance of 10 ppm, fragment mass tolerance of
0.5 Da, trypsin as speciﬁc-enzyme, with up to 02 missed
cleavages and 01 non-specific, carbamidomethylation
on cysteine (+57.02) as ﬁxed modiﬁcation, oxidation on
methionine (+15.99 Da) as the variable modiﬁcation and
false discovered rate (FDR) < 5%.
Peptides with length of amino acids ≥ 6 and ALC ≥ 65%
obtained by de novo analysis not matching any sequence in
the database searches were submitted to homology search
in BLAST analysis22 using the sequence presets against the
UniprotKB (Mammalian) database and hits E-values < 2
was used as the limit parameter.
PANTHER analysis was used to classify protein
function23 in healthy, tumor and metastasis tissues based
on Gene Ontology (GO) terms. The percentage values were
therefore normalized to a total value of 100%.
Results and Discussion
High grade pleomorphic sarcoma diagnosed by the morpho-
The tumor was composed of plump spindle cells
forming fascicular and storiform patterns, with admixed
bizarre pleomorphic and multinucleated giant cells on a
background of mononuclear inﬂammatory cells. There were
an average of three mitotic ﬁgures per 10 high power ﬁelds
and bizarre mitoses were common. The cystic, gritty areas
seen grossly proved to be foci of myxoid degeneration,
necrosis and mineralization. The tumor was high positive
for vimentin, had patchy SMA staining and was negative for
all other markers, consistent with a high grade pleomorphic
sarcoma, not otherwise speciﬁed (Figure 1).
Many of these tumors showed focal immunoreactivity for
smooth muscle actin, but stains for desmin and h-caldesmon
are typically negative.24 The high grade undifferentiated
pleomorphic sarcoma (UPS) manifests in a broad range
of histologic appearances, although the most common
form consists of a mixture of storiform and pleomorphic
areas, and it is diagnosed when no identiﬁable speciﬁc
line of differentiation exists, excluding dedifferentiated
types of pleomorphic sarcomas (diagnosis of exclusion).
Overall, the morphologic and immunohistochemical results
described are consistent with a conclusive diagnosis of this
Protein proﬁling describes differences in pathological status
Currently, little information about the proteome of
common hippos exists, but MS/MS analysis enables the
observation of the proteomic changes in the different tissues,
which can be compared to differentiate the physiopathological
status among them. Of the proteins identiﬁed, 25 were found
in healthy skeletal muscle, 27 in primary tumors, and 15 in
metastatic tumors (Supplementary Information); uncommon
characteristics were shown between normal and tumor
tissues based on protein class annotated in GO terms. These
differences were observed mainly by the number of proteins
associated to the cytoskeleton, in healthy tissue (36.7%),
which decreased in the pathological tissues. Meanwhile,
proteins identiﬁed in primary and metastasis tumors revealed
enrichment for nucleic acid binding, which is absent in
normal tissue (Figure 2).
The nucleic acid binding PANTHER protein class
includes RNA and DNA binding proteins, helicases and
nucleases. The abundance of these proteins differentially
encoded in malignant samples may suggest altered
transcriptional activity in tumor samples.26 Evidence
suggests that activated nucleic acid binding proteins might
play a vital role in cell growth, abnormal cell proliferation,
and metastasis, and may form a network center during
Thus, when comparing diseased and control tissues, it
is relevant to consider the amount associated with the GO
category as well as the number and function of the proteins.
The normal muscle sample was characterized by structural
proteins related to contractile muscle function, the formation
of microtubules, and the composition/maintenance of the
cytoskeleton, with effects on cell division. These proteins
included actin, myosin, tropomyosin, titin, ﬁlamin, dynein,
receiver skeletal protein tyrosine kinase, protein kinase
PAK 3, microtubule-associated protein ASPM (abnormal
spindle microtubule) and various isoforms.28-31 The presence
of these proteins seems to indicate there was no stress at
the cellular level.
Morpho/Proteomic Comparative between High Grade Pleomorphic Sarcoma and Metastasis Diagnosed J. Braz. Chem. Soc.
The primary tumor had proteins in common with healthy
tissue, but with a peculiar proteome. Proteins associated
with DNA damage were detected along with a possible
mutation in a protein commonly found in healthy tissue,
which may elucidate the cellular mechanism of the protein.
Excess soluble histone proteins is an indicative of damage
to the nucleosome, which impacts DNA replication and
may negatively affect the repair of the DNA molecule.32,33
An increase in protein synthesis by the methionyl-tRNA
synthetase enzyme is also related to oncogenesis,34 as is
an increase in the transcription factor that binds the EFL 1
promoter, responsible for increasing transcription in tumors
and during angiogenesis.35,36
Some proteins that act to restore homeostasis were also
detected, including the DNA repair protein PMS237 and
CTCF tumor suppressor,38 as well as proteins involved in
stress response, including the small chaperone crystalline
alpha39 and the chaperone HSP 60.40 The presence of
vimentin was detected; this protein was associated with the
cytoskeleton in tumor cells and is responsible for increasing
adhesion and metastasis, but it is poorly expressed during
myogenesis in normal cells.41,42 The different intracellular
dynamics and tissue organization in tumors could also be
observed in changes in the levels of TMPRSS matriptase,
which increases collagen degradation and the effects of
Figure 1. Comparative photomicroscopy of the histological differences between tissues show progression and differentiation of tumor from skeletal muscle
and aspects of heart metastasis. (a) Skeletal muscle tissue; (b) transition of skeletal muscle to tumor tissue; (c, d) tumor tissue; (e, f) heart metastatic tissue.
Hematoxylin and eosin (H & E).
Silva et al. 5Vol. 00, No. 00, 2017
of tumors which do not require anchoring.46 The initiator of
transcription factor 2 alpha (GCN2) is a protein expressed
in the absence of nutrients. It is related to various pathways
of tumor progression and angiogenesis47,48 and increases
ﬁbroblast growth factor.49
The efrin A2 receptor is associated with oncogenesis
and with the progression of metastatic growth factors,
such as ﬁbroblast growth factor.50 Proteins involved in
cell proliferation were also found, including GSG2, a
kinase responsible for mitotic histone production51 and
guanine nucleotide exchange factor RASGRF1, which is
responsible for cell proliferation and migration related to
metastatic in tumorogenesis.52 However, this tissue also
showed the presence of tumor suppressor CHD1.53
Decreased level β-actin in primary tumor in response to
possible cytoskeleton dynamic modiﬁcation
De novo sequenced peptides used to identify proteins
had MS/MS spectra manually verified. Based in the
retention time (RT), m/z ratio of precursor ion, and sequence
coverage, we identiﬁed peptides of β-actin protein that
were present in all tissues. Based on the extension to
the spectral counting technique by the average of the
total ion current (TIC) of MS/MS spectra this peptides,
it was estimated their relative abundance of the protein,
similarly to a label-free quantiﬁcation.54
Despite similarities among malignant samples, the
β-actin present in primary tumors showed minor abundance
compared with the other tissues, but there was no signiﬁcant
difference between healthy and metastatic tissues. Some
differences in sequence coverage may be considered
tolerable error-fragmentation, i.e., AG → K55 (Figure 3).
Based on both literature reports and the proteomic
proﬁle of the primary tumor generated, we suggest that
the reduction of β-actin (actin cytoplasmic 1) is the result
of the progressive increase of the cytoskeleton through a
dynamic process involving changes in its dynamics, and
for the presence of protein connections that bind to this
structure with subsequent alteration of gene expression in
the tumor. The presence of vimentin and crystalline alpha
proteins increases stability of the cytoskeleton by reducing
The results of decreased depolymerization is an
increase of the cytoskeleton with membrane protrusion,
tumor progression and metastasis, as well as a change
of polarization during mitosis leading to cellular
pleomorphism.58,59 The presence of HSP 60 in the primary
tumor indicates the refurbishment of misfolding proteins
and affects the stability of the cytoskeleton, thus enhancing
the progression of carcinogenesis.60
Figure 2. Pie diagram of the distribution of protein functions in the
(a) healthy skeletal muscle, (b) primary tumor, and (c) metastatic tumor,
according to their molecular functions as determined using PANTHER.
The metastatic tumor proteome had a profile that
differed from the other tissues, suggesting that this
type of tumor has a specialized molecular mechanism.
The genetic constitution of the primary tumor and its
metastasizing cells may be quite different.45 Stromal
epithelial protein 1 (EPSTI1) was observed; this protein
induces stromal ﬁbroblasts in tumors, it is characteristic of
invasive metastasis, and has been described as a signature
Morpho/Proteomic Comparative between High Grade Pleomorphic Sarcoma and Metastasis Diagnosed J. Braz. Chem. Soc.
β-Actin is present only in non-muscle cells, and it
has the capability to alternate from a globular (G-actin)
to polymeric (F-actin). The G-actin isoform is converted
into F-actin by nucleation and subsequently polymerizes
and depolymerizes through a recycling process
(treadmilling).61-63 Decreased levels of β-actin can be a
consequence of cytoskeletal organization changes causing
reduction of organization during cell division and increased
protrusions, cell motility and chromatin remodeling.64,65
Signiﬁcant levels of β-actin in metastatic tissue suggest
a heterogeneous mechanism for formation of the primary
tumor. Based on the metastatic tissue proteome, HSP 60
may be associated with senescent cells output ﬁbroblast,
i.e., ﬁbroblasts reached by a state in which cell division is
limited and cannot be induced.66 Thus, active ﬁbroblasts
were stimulated by growth factors detected and responsible
for distinctive features within the tissue with collagen
presence. This explains the levels of β-actin, which is
not typically modiﬁed in the ﬁbroblasts.65 Therefore, this
protein measurement may be indicative of the activation of
response mechanisms on the development and progression
Figure 3. Differences of the β-actin levels evaluated for MS/MS spectra of two speciﬁc peptides present in all tissues. The peptide 1 in healthy tissue
exhibited an m/z 488.73, z +2, RT 14.98 min and 4.07E4 TIC; in the primary tumor it exhibited an m/z 488.74, z +2, RT 15.08 min and TIC 2.15E4; and
on metastatic tissue it exhibited an m/z 488.72, z +2, RT 15.4 min and 3.73E4 TIC. The peptide 2 in healthy tissue had an m/z 627.95, z +3, RT 19.63 min
and 2.66E5 TIC; in primary tumor had an m/z 627.95, z +3, RT 19.1 min and 9.48E4 TIC; and metastatic tumor had an m/z 627.93, z +3, RT 19.59 min
and 2.58E5 TIC. Amino acids with ALC% > 90% are colored in red.
Silva et al. 7Vol. 00, No. 00, 2017
We performed an in depth proteomic analysis of a high
grade pleomorphic sarcoma of a common hippo and offered
new opportunities of research based on the hypothesis that
pleomorphic sarcoma tumor samples may be differentiated
by an increase in nucleic acid binding protein class and
for the low levels of β-actin and key cytoskeleton proteins
as possible candidates for cell organization and dynamics
in carcinogenesis. We also recognize that high-grade
pleomorphic sarcomas are complex neoplasia and that
the proteomic ﬁndings seen in this case require further
characterization in other animals and in human tumor
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF ﬁle.
The authors thank to FAPESP (São Paulo Research
Foundation) by grant (2012/02514-9) as well as CAPES
(Coordination for the Improvement of Higher Education
Personnel) and FINEP (Brazilian Innovation Agency) for
ﬁnancial support. The authors specially thank the São Paulo
Zoological Park Foundation (SPZPF) for all support.
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Submitted: June 3, 2017
Published online: August 17, 2017