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Original Article
Topological properties and in vitro identification of
essential nodes of the Paclitaxel and Vincristine
interactomes in PC-3 cells
Claudia Delgado-Carre~
no
a,b
, Gina M
endez-Callejas
a,*
a
Group of Biomedical Research and Applied Human Genetics, Laboratory of Cellular and Molecular Biology, School of
Medicine, University of Applied and Environmental Sciences, U.D.C.A, Bogota, Colombia
b
Department of Chemistry, Faculty of Science, Javeriana University, Bogota, Colombia
article info
Article history:
Received 6 June 2018
Accepted 12 April 2019
Available online 31 October 2019
Keywords:
Prostate cancer
Interactome
Essential nodes
Microtubule-targeting agents
(MTAs)
Apoptotic proteins
abstract
Background: Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby
inducing apoptosis via mitochondrial pathway activation through the modulation in the
expression of the Bcl-2 family.
Methods: To describe topological features of the MTAs networks associated to intrinsic
apoptosis induction in p53-null prostate cancer cells, we predicted and compared the
interactomes and topological properties of Paclitaxel and Vincristine, and thus, the
essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics
were subjected to experimental analysis in PC-3 cell line.
Results: The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in
both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX,
and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis
demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells
exposed to both treatments. Immunoprecipitation analysis showed that treatments
induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the
role of BimEL as a de-repressor from sequestering complexes, in addition, new protein
complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing
data to the Vincristine network for p53-null cells in response to MTAs.
Conclusion: The differences in sensitivities, protein profiles, and protein complex kinetics
observed between the drugs confirmed that the selectivity and stimulation of the apoptotic
system vary depending on the cell's genotype, the drug used and its exposure period.
*Corresponding author. School of Medicine, University of Applied and Environmental Sciences, U.D.C.A, 222 street # 55-37, 111166,
Bogota, Colombia.
E-mail address: gmendez@udca.edu.co (G. M
endez-Callejas).
Peer review under responsibility of Chang Gung University.
Available online at www.sciencedirect.com
ScienceDirect
Biomedical Journal
journal homepage: www.elsevier.com/locate/bj
biomedical journal 42 (2019) 307e316
https://doi.org/10.1016/j.bj.2019.04.003
2319-4170/©2019 Chang Gung University. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
At a glance commentary
Scientific background on the subject
Cancer is a significant cause of morbidity and mortality
of the world population, being the first cause of non-vi-
olent death. Prostatic cancer has become the most
common malignancy among men. Network biology with
an experimental approach can be used to described
protein interaction, identify biomarkers and define the
topological features of cytostatic drugs among other
uses.
What this study adds to the field
Advance and variant forms of prostatic cancer are not
susceptible to common treatment which leads to the
pursuit of different classes of drugs, such as MTAs, in
which the resistance rate and protein interaction be-
tween drugs vary. Some protein interactions were pre-
viously described by network biology and then verified in
this paper, also, new interactions were found.
Network biology has been extensively used to describe how
proteins interact and coordinate cellular responses [1].
Proteineprotein network approaches have been applied to
gain a better understanding of cancer mechanisms [2],to
identify cancer subnetworks [3], to discover cancer-related
biomarkers [4], and to define the topological features of the
interactome of microtubule-targeting agents (MTAs) [5].
High cancer death rates and resistance or over-sensitivity
to conventional cancer therapies remain major challenges
for researchers [6,7]. Prostate cancer has become the most
common malignancy among men and is the second leading
cause of cancer death worldwide [8,9]. Prostate cancer is very
heterogeneous, often characterized by the presence of AR-
dependent and AR-independent cellular clones in the same
patient [10], its biological, hormonal, and molecular charac-
teristics are extremely complex [6].
Most prostatic cancers (PCs) are adenocarcinomas char-
acterized by a glandular formation, lack of basal cells, un-
controlled proliferation of malignant tumor cells and the
expression of androgen receptor (AR) and prostate-specific
antigen (PSA). These adenocarcinomas are indolent and
androgen-dependent (AD) [6], although localized PC may be
successfully treated with radical prostatectomy and external
beam radiation, many patients subsequently develop meta-
static disease. Since its growth is driven by androgens, they
are primarily treated with hormonal therapy, the androgen
ablation therapy (ADT), that aims to block or decrease the
expression of the androgen receptor, the hormonal therapy is
the standard treatment of hormone-naı
¨ve metastatic disease
[11e13]. Nearly all patients with metastatic prostate cancer
(PC) become resistant to the initial approach with ADT,
developing the state known as metastatic castration-resistant
prostate cancer (mCRPC) [10].
There are variant forms of prostatic epithelial malig-
nancies, such the Small-Cell Neuroendocrine Carcinoma
(SCNC), which are rare tumors that account for no more than
1% of all carcinomas of the prostate, they lack AR and PSA
expression. Although they may arise de novo, they are often
seen as recurrent tumors in patients who have received hor-
monal therapy with conventional prostatic adenocarcinoma
[6]. PC-3 is a cell line characteristic of SCNC since PC-3 cells
share a lot of traits with prostatic SCNCs, including the lack of
expression of the basal markers AR and PSA. PC-3 cells are
often referred to as aggressive, androgen-independent (AI),
and castration-resistant [6,14]. Nevertheless, the PC-3 line
presents a base deletion at codon 138 (GCC/GC) in the TP53
gene, resulting in a frame-shift mutation [15,16] that in-
fluences negatively p53 dependent-apoptosis through the
mitochondrial pathway, where it directly interact with
different Bcl2 family members, acting as a direct activator of
the Bax/Bak effectors, or as a sensitizer/de-repressor of Bcl-x/2
and Mcl-1 [17]. In human cancers, TP53 gene is frequently
mutated what not only leads to loss of its tumor suppressive
function but also acquires dominantenegative activities and
gains new oncogenic properties that increase drug resistance
[14,18,19].
Advanced and variants forms of these PCs are only tem-
porary or not susceptible at all to the androgen ablation
therapy, leading to the pursuit of different classes of drugs,
such as MTAs, which inhibit microtubule dynamics and
induce cell death via the mitochondrial intrinsic pathway
[12,20].
Microtubule-targeting agents are usually obtained from
natural sources, such as Paclitaxel, obtained from the Pacific
yew tree (Taxus brevifolia)[8], and Vincristine, obtained from
Madagascar periwinkle (Catharanthus roseus)[21]. These
agents disrupt microtubules dynamics, thereby inducing
apoptosis via mitochondrial pathway activation [19]. Prostate
cancer exhibits high levels of anti-apoptotic Bcl-2 and Bcl-xL
proteins in refractory and advanced disease, contributing to
the defective apoptosis associated with poor prognosis,
treatment resistance and disease progression [8,12,22e24].
The mitotic arrest is induced by two kinds of well-known
MTAs, including the microtubule-stabilizing agent Paclitaxel
[25] and microtubule-destabilizing agents, such as Vincristine
and Vinblastine [26], which are associated with the activation
of Raf-1 protein kinase and the phosphorylation of the anti-
apoptotic protein Bcl-2 [25]. This phosphorylation increases
the resistance to many anticancer drugs and may inactivate
the Bcl-2 protein, ensuring cell death after microtubule
rupture and DNA fragmentation in PC-3 cells [25,26]; addi-
tionally, it inhibits the ability of Bcl-2 to interfere with pro-
apoptotic proteins, such as Bax, leading to apoptosis [22].
Paclitaxel also induces the accumulation of the pro-apoptotic
protein Bim [12], causing a G2/M cell cycle block, mitochondria
damage, and p53-independent apoptosis [27].
Activation of the caspase cascade has been correlated with
the outset of apoptosis [28]. Caspase inhibition attenuates
apoptosis in prostate cancer cells in response to diverse
apoptotic stimuli, including androgen ablation. Therefore,
reduced caspase expression, which is a frequent event in
biomedical journal 42 (2019) 307e316308
prostatic cancer, has been linked with poor prognosis and/or
resistance to therapy in several human tumors. Caspases-1,
-3, and -9 are the three key caspases implicated in the
execution of apoptosis in prostate cancer cells and malignant
prostatic tissue, and there is dramatically reduced caspase-1
and -3 immunoreactivity among the tumor cells [29].
The secondary targets of the signaling pathways induced
by Paclitaxel and Vincristine in PC-3 cancer cells remain
poorly understood. Here, we described the topological prop-
erties and identified the essential nodes of Paclitaxel and
Vincristine interactomes against the human proteome. Then,
experimental validation of those essential nodes, corre-
sponding to pro- and anti-apoptotic proteins and their ki-
netics in p53-null PC-3 cancer cells in regard to the intrinsic
apoptosis induced by Paclitaxel and Vincristine was
conducted.
Materials and methods
Paclitaxel and Vincristine network prediction
The chemical information of Paclitaxel and Vincristine was
extracted from the PubChem database. After, the interaction
between proteins and the MTAs were integrated into a
network by using of the Search Tool for Interacting Chemicals
(STICH) 5.0 database [30] (http://stitch.embl.de/), with the
following criteria: a confidence score of 0.500, with a
maximum of 500 interactions. The prediction methods in the
program were active solely against the human proteome.
Next, each network was imported into Cytoscape 3.4.0 [31],
and the following topological parameters were obtained: De-
gree (k), which indicates the connection density of each node;
Betweenness Centrality (BC), which indicates the number of
times a node is visited; Closeness Centrality (CC), which in-
dicates what nodes are closer to the center of the network; and
Stress, which indicates how many times a particular node is
part of different shortest paths using the Network Analyzer
plugin included by default in Cytoscape.
To determine the functional richness of the network, a
hypergeometric distribution was used and the false dis-
covery rate (FDR) correction was included, using the Bio-
logical Network Gene Ontology (BiNGO) plugin. The FDR
correction, with a significance level of 0.05, is shown for two
descriptive categories of the clusters. The first represents
the biological function, and the second represents node
mapping in the Kyoto Encyclopedia of Genes and Genomes
(KEGG) [32].
Compounds, cell lines, and culture conditions
The Paclitaxel and Vincristine sulfate (Cayman Chemical) was
dissolved in dimethyl sulfoxide (DMSO) and for the experi-
ments, dilutions were made in supplemented Eagle's Mini-
mum Essential Medium (EMEM) with 10% (w/v) Fetal Bovine
Serum (Biowest), 2 mM L-glutamine, 5000 UI/ml penicillin and
5 mg/ml streptomycin. The prostate adenocarcinoma PC-
3 cells (ATCC
®
CRL1435™) were grown in supplemented EMEM
at 37 C in a humidified atmosphere containing 5% CO
2.
The 3-(4,5-methyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) cell viability assay
PC-3 cells were cultured to 80% confluence, then, seven
thousand cells approximately were seeded per well in a
96-well plate and were grown at 37 C, 5% CO
2
for 24 h.
Then, MTAs treatments were administered at different
concentrations, between 0,01 and 0.15 mg/mL for Vincris-
tine and between 0.005, and 0,0012 mg/mL for Paclitaxel.
Incubation was carried out for 48 h. Following the incu-
bation time, 100 mlof0.5mg/mLoftheMTTsolution,
dissolved in a medium without phenol red, were added
and incubated for 4 h. Formazan crystals were dissolved
with 100 ml of DMSO. The results were determined by the
optical density (OD) determined by the absorbance at
570 nm [33]. Estimation of the MTAs concentrations
required to reduce the 50% of cell viability (IC
50
)wasdone
using nonlinear regression from plotting cell survival (%)
versus drug concentration [mg/mL].
Immunofluorescence analysis
Seven thousand PC-3 cells approximately were seeded per
well in a 96-well plate and were grown for 24 h before
treatment. The cells were treated with Paclitaxel and
Vincristine at the IC
50
and incubated for 24 h. Later the cells
were fixed in methanol (20 C), and then with acetone at
20 C for 20 s. The microtubules were stained with anti-a-
tubulin monoclonal antibody DM1A (SigmaeAldrich) and
goat anti-mouse Alexa Fluor 488 (Molecular Probes), both
diluted in 5% BSA/TTBS (w/v) blocking solution. DNA
staining was done with 1.0 mg/mL of DAPI (Invitrogen)
[22,33]. Fluorescence was monitored using an epifluor-
escence microscope (Motic AE31), and the images were
captured with the MoticCamPro 282A and analyzed with
Motic Image plus 2.0 software.
Electrophoresis &western blotting
Protein extraction was carried out from approximately 6
million cells treated for 6, 16, 24 and 48 h with the MTAs at the
corresponding IC
50
using a lysis buffer (Tris HCl pH 8.0 20 mM,
NaCl 137 mM, Glycerol 10%, Np40 1%, EDTA 10 mM) [34], and
the protein concentration was determined by Bicinchoninic
Acid (BCA) Protein Assay Kit (Pierce). Fifteen mg of protein were
subjected to an 11% SDS-PAGE gel electrophoresis using a
mini-gel system (Protean II; Bio-Rad Laboratories). Proteins
were blotted using polyvinylidene fluoride (PVDF) membranes
[35,36]. The transferred membranes were blocked in 5% (w/v)
BSA/TTBS followed by overnight incubation with the
following primary antibodies: anti-a-Tubulin (Sigma-
eAldrich); anti-Bim; anti-Bax; anti-Bcl-2 (Gene-Tex); anti-
procaspase-3 (Novus) or anti-caspase-3 (Thermo-Fisher Sci-
entific). Next, the membranes were incubated with the
correspondent secondary antibody, either anti-mouse IgG
(SigmaeAldrich) or anti-rabbit IgG (Cell Signaling), for 1 h at
room temperature. The bands were visualized using a
Chemiluminescent Detection System (Thermo-Fisher Scien-
tific) [22]. Image J software was used to quantify and compare
the density of bands [37].
biomedical journal 42 (2019) 307e316 309
Co-immunoprecipitation
One-hundred and fifty mg of protein per treatment were mixed
with 2ug of primary antibody, in a dilution buffer eNaCl (Tris
HCl pH 7.4, EDTA 10 nM, NP40 1%, NaF 50 mM, PMSF 1 mM, and
protease inhibitors cocktail (Pierce) (Thermo Fisher Scientific)
1:100), and the samples were incubated at 4 C for 4 h. Later,
the protein mix was incubated with the washed resin (Protein
G) (Thermo-Fisher Scientific) at 4 C overnight. The next day,
the samples were washed with a þNaCl wash buffer (Tris HCl
pH 7.4 1 M, NaCl, EDTA 10 mM, NP40 1%, NaF 50 mM, PMSF
1 mM and protease inhibitors cocktail (Pierce) (Thermo Fisher
Scientific) 1:100) and were reduced with 35 ml of a 1x loading
buffer and boiling for 5 min. Subsequently, the immunopre-
cipitated complexes were subjected to electrophoresis and
western blotting with primary antibodies against the corre-
sponding essential nodes from intrinsic apoptosis pathway,
the detection of the proteins in the complex was conducted as
previously described and densitometry analyses were per-
formed using ImageJ software [37].
Results
Topological nodes that regulate the Paclitaxel and
Vincristine interactomes
The Paclitaxel and Vincristine interactomes showed several
secondary targets [Fig. 1]. The Paclitaxel network presented
166 nodes and 1497 edges. The evaluated topological features
showed positive correlations, indicative of highly connected
nodes, as follows: nodes/k (R: 0.857, R
2
: 0.657); neighbors/BC (R:
0.972, R
2
: 0.871) and neighbors/CC (R: 0.767, R
2
: 0.592) [Fig. 1A].
Then, to choose the essential nodes, the k values were reor-
dered in a decreasing fashion, and the top 30 nodes with the
highest values are showed [Table 1]. To determine the
importance of the essential nodes during the process the in-
formation flow in the network, a linear correlation analysis
between the BC and stress parameters was conducted. The
essential nodes TP53, AKT1, VEGFA, JUN, and CASP3 seemed
to control the information processing in the paclitaxel
Fig. 1 Topological nodes that regulate the interactome information process derived from Paclitaxel and Vincristine. (A, C) Stress
and BC values were used to identify the nodes that connect the most significant shortest in the interactomes and thus, define
how the information flow is distributed. (B, D) The essential interactomes were built based of the K parameter and shows the
most connected nodes in both interactomes.
biomedical journal 42 (2019) 307e316310
network [Fig. 1B], but also in the top the BCL 2, BAX and BCL2L1
nodes were identified confirming the mitochondrial-mediated
pathway apoptosis induction.
The Vincristine network presented 19 nodes and 42 edges.
This network also reveals essential nodes and highly signifi-
cant correlations were detected among their basic topological
parameters as follows: nodes/k (R: 0.913, R
2
: 0.770); neighbors/
BC (R: 0.999, R
2
: 0.926) and neighbors/CC (R: 0.807, R
2
: 0.695)
[Fig. 1C], and only two essential nodes, TP53 and CASP3,
associated to the intrinsic apoptosis, seems to be involved in
the information processing of the network [Fig. 1D].
Paclitaxel and Vincristine cause irreversible morphologic
damage in PC-3 cells
Vincristine and Paclitaxel inhibitory effect on PC-3 cells pro-
liferation was confirmed by MTT assay. However, the IC
50
values demonstrated that the cells were significantly more
sensitive to Paclitaxel [2.81 nM] than to Vincristine [44,8 nM]
[Fig. 2]. Those IC
50
data were used for all others in vitro
analyses.
Morphological analysis was performed to examine the
cellular damage that occurred after exposing PC-3 cells to
MTAs using immunofluorescence microscopy [Fig. 3A]. There
were variations in the cells on the morphological scale, such
as microtubule and nucleus integrity and cell shape and size
variations. Paclitaxel mainly affects the microtubules, causing
microtubule destabilization, and increase in cytosol size, the
appearance of some apoptotic bodies (marked with a red
arrow), and the emergence of different nuclear morphologies
[Fig. 3A]. On the other hand, the Vincristine effect on cell
proliferation was more evident than paclitaxel and the cells
had dense and compact damaged microtubules around the
nuclei, and, thus some apoptotic cells (marked with a red
arrowhead). The nuclei of PC-3 cells exposed to Vincristine
had irregular and small nuclei compared to the control but not
as quite as paclitaxel. Further, Paclitaxel-induced the
condensation and fragmentation of the nuclear material (in
blue) as well as a reduction of its perimeter [Fig. 3B].
Role of Paclitaxel and Vincristine treatments in the
expression of essential node proteins in PC-3 cells
The content of the essential nodes, the Bcl-2, Bim, Bax,
procaspase-3 and cleaved-caspase-3 proteins, was evalu-
ated by western blot at 0, 6, 16, 24 and 48 h of treatments
with Paclitaxel and Vincristine. Both drugs stimulated an
increase in the expression of Bim protein [Fig. 4A], and
caused a significant decrease in the level of anti-apoptotic
protein Bcl-2, leading the way for apoptosis. But the ef-
fects on Bax, procaspase-3 and cleaved caspase-3 proteins,
Table 1 Essential topological nodes from Paclitaxel and
Vincristine networks based on the k parameter.
Node k BC CC
Paclitaxel 165 0.621 1.000
TP53 74 0.022 0.645
AKT1 71 0.024 0.637
VEGFA 68 0.021 0.630
JUN 66 0.013 0.625
CASP3 60 0.010 0.611
EGFR 58 0.015 0.607
PTGS2 55 0.010 0.600
MAPK1 52 0.007 0.594
STAT3 50 0.008 0.589
CASP8 49 0.004 0.587
ERBB2 49 0.007 0.587
CDKN1A 47 0.003 0.583
CYCS 46 0.004 0.581
BIRC5 45 0.004 0.579
MAPK8 45 0.006 0.579
PARP1 45 0.005 0.579
PCNA 44 0.009 0.577
FASLG 44 0.005 0.577
BCL2 43 0.003 0.575
PIK3CA 43 0.006 0.575
PTEN 42 0.004 0.573
BAX 41 0.002 0.571
CDH1 40 0.008 0.569
MAPK14 39 0.004 0.567
MDM2 39 0.003 0.567
SOD2 37 0.005 0.563
BCL2L1 37 0.003 0.563
IL8 36 0.005 0.561
Vincristine 18 0.735 1.000
TP53 8 0.030 0.643
PTGS2 7 0.011 0.621
ABCB1 7 0.015 0.621
CASP3 6 0.009 0.600
CYP3A4 6 0.014 0.600
MAPK8 5 0.004 0.581
MAPT 5 0.016 0.581
CYP3A5 5 0.007 0.581
ABCG2 3 0.000 0.545
CYP3A7 3 0.001 0.545
MAPK10 2 0.000 0.529
TUBB2C 2 0.000 0.529
TUBB2A 2 0.000 0.529
BUB1B 1 0.000 0.514
ASRGL1 1 0.000 0.514
ACP1 1 0.000 0.514
MIXL1 1 0.000 0.514
TWF1 1 0.000 0.514
Fig. 2 Percentage of cell viability of PC-3 cells exposed to
different concentrations of Paclitaxel and Vincristine. The
inhibitory concentration of cell viability (IC
50
) was calculated
by non-linear regression using Graph Pad 6.0 software.
biomedical journal 42 (2019) 307e316 311
were divergent between the two drugs. Paclitaxel-induced a
slight decrease in the levels of Bax protein, which was
dependent on time, while Vincristine caused an increase
through a distinct period of the drug incubation. Paclitaxel
did not provoke considerable changes in the levels of the
procaspase-3form,buttheactiveformwasslightly
augmented. Furthermore, in the cells treated with Vincris-
tine, there was a decrease in the levels of procaspase-3 at
48 h, which was related with the decrease in the expression
of the activated form that was observed at the same time
[Fig. 4B].
In vitro proteineprotein interactions in PC-3 cells after
Paclitaxel and Vincristine treatment
Immunoprecipitation analyses showed that Bim protein
interacted with the essential nodes Bcl-2, Bax and cleaved-
caspase-3. However, the intensity of the bands indicated
that the function of Bim was dependent on the type of drug
[Fig. 5A]. The Bim/Bcl-2 interaction was similar in both
treatments, decreasing slightly at 48 h. Bim/Bax complex
levels increased significantly at 16 h only in the cells
exposed to Paclitaxel. Interestingly, new interactions have
been identified in cells that were exposed to vincristine,
sincetherewasanaugmentationintheinteractionbe-
tween Bim/cleaved-caspase-3 in a time-dependent manner
[Fig. 5B].
The Bcl-2/Bax interaction was significantly decreased at
48 h treatment in cells exposed to Paclitaxel [Fig. 5AandB].
In contrast, in cells exposed to Vincristine, the Bax protein
has not been completely released at 48 h from the anti-
apoptotic complex, elucidating the PC-3 mechanism to
Vincristine resistance. Another new proteineprotein inter-
action was revealed between cleaved-caspase-3 and Bcl-2,
Fig. 3 Morphological changes in PC-3. (A) Immunofluorescence micrograph showing the nuclear (in blue) and microtubular (in
green) effects in PC-3 cancer cells after 24 h of incubation with two microtubule-targeting agents, Vincristine and Paclitaxel. (B)
The nuclear perimeter of PC-3 cells before and after being treated with the MTAs Paclitaxel and Vincristine. Motic 2.0 software
was used to measure the nuclei.
Fig. 4 Comparison between the pro-apoptotic and anti-apoptotic protein levels in PC-3 cells treated for up to 48 h with two
MTAs, Paclitaxel, and Vincristine. (A) The readouts, defined as the protein complex levels, were normalized to the readouts of a-
tubulin, which are shown below each band. (B) The immunoblot images were quantitated by ImageJ and were then graphed
using GraphPad Prism 6. The readouts, defined as the arbitrary light units of the pro-apoptotic and antiapoptotic proteins, are
the relative densitometric units of each band.
biomedical journal 42 (2019) 307e316312
where the levels of this complex decreased in the cells
treated with Paclitaxel and increased after Vincristine
treatment, much like the levels of the Bim/cleaved-caspase-
3complex[Fig. 5B].
Discussion
Paclitaxel and Vincristine bind preferentially and reversibly to
the b-tubulin microtubule subunit, promoting the polymeri-
zation or depolymerization of microtubules, respectively;
both drugs disrupt microtubule dynamics [38]. However, the
networks here predicted show several secondary targets,
indicating their lack of specificity. The Paclitaxel interactome
was more highly expressed than the Vincristine interactome
and presented more pro- and anti-apoptotic proteins, sug-
gesting that this compound has pleiotropic moieties possibly
related to unspecific cellular activation and the regulation of
cell death and toxicity. The Vincristine network was more
specific towards activated proteins that regulate the induction
of cell death. According to preliminary computational results,
we observed that Paclitaxel has significant druggability to-
wards Bax, Bcl-2, and cleaved-caspase-3, indicating that this
molecule can induce non-regulated biochemical signaling,
and stimulate an apoptotic catastrophe [39] and even induce
paraptosis [Fig. 1][40].
The prostate cancer cell line PC-3 isan appropriate model to
study the possible causes of the ineffectiveness of the common
Fig. 5 Comparison between immunocomplex interactions in PC-3 cells treated for up to 48 h with two MTAs, Paclitaxel, and
Vincristine. (A) The readouts, defined as the protein complex levels, were normalized to the readouts of a-tubulin, which are
shown below each band. (B) The immunoblot images were quantitated by ImageJ and were then graphed using GraphPad Prism
6. The readouts, defined as the levels of the complexes of the pro-apoptotic and antiapoptotic proteins, were the relative
densitometric units of each band.
biomedical journal 42 (2019) 307e316 313
drugs used in the treatment of apoptosis-resistant phenotypes
cells with p53-null status, which alter the normal function of
the network determinants for each drug [Fig. 1]. Previously, it
was showed that PC-3 cells exposed to Paclitaxel had an
abnormal network of microtubules around the nucleus at 4e8h
after treatment, which induced the formation of multipolar
spindles. Nevertheless, those cells entered G2/M to form a
defective spindleechromosome complex that then caused
cycle arrest [22]. Cell proliferation assays indicated that a lower
concentration of Paclitaxel was sufficient to induce cell death in
comparison to Vincristine [Fig. 2], perhaps, because Paclitaxel
did target a higher number of nodes. Fluorescent images from
PC-3 cells treated with paclitaxel [Fig. 3A] showed enlarged cells
with damaged microtubules and irregular and multiple nuclei
suggesting mitotic catastrophe [39]. On the other hand,
Vincristine effect on cells was more evident, it resulted in
enlarged cells with enlarged and irregular nuclei, and aggre-
gation of compact and denser microtubules around a large
nucleus [Fig. 3A and B]. In addition, there was the appearance of
some apoptotic cells marked with a red arrowhead [Fig. 3A];
these results correspond with previous findings in several
resistant cells lines, which can be ascribed to drug efflux
pumps, mutations in tubulin that abrogates drug binding,
ineffective interaction with the target, deficient induction of
apoptosis and overexpression of prosurvival Bcl-2 family pro-
teins [39,41].
During apoptosis, it is expected the decrease of the anti-
apoptotic proteins Bcl-2 like [8,23,24]. However, the over-
expression of Bcl-2 and Bcl-xL in PC-3 cells might contribute to
the apoptosis-resistant phenotype [42,43]. In this respect, our
data indicate that the anti-apoptotic protein Bcl-2 decrease
occurred after both treatments, but a portion of the Bcl-2 pro-
tein remains in the cells, even after 48 h, contributing to the
resistance [Fig. 4A and B]. MTAs initiate the translocation of
Bim from microtubules to the mitochondria, allowing the Bax/
Bak activation and then inducing apoptosis [12,43].Nonethe-
less, the increasing of the pro-apoptotic Bim protein found after
both drug treatments, corresponded with several studies in
which Bim is reported as a tumor suppressor and is essential
for apoptosis induction [26,27,37,44]. Literature reports that Bim
level increases dramatically after paclitaxel treatment [12],ac-
cording to our findings, the level of Bim was higher in vincris-
tine treatment than the level induced by paclitaxel.
The forced expression of Bim increases Paclitaxel-
mediated killing of cells expressing an undetectable level of
Bim. Conversely, the knockdown of Bim, but not Bcl-2
expression, decreases the susceptibility of tumor cells to
Paclitaxel-mediated killing [27]. Similar observations were
made using a panel of breast and prostate cancer cell lines
[27,43]. Furthermore, it was proven that the depletion of Bim
levels prevented paclitaxel-induced Bax and Bak activation
[39]. Paclitaxel impairs microtubule function, causes G2/M cell
cycle blockade, mitochondria damage, and p53-independent
apoptosis. These results established Bim as a critical molec-
ular link between the microtubule toxic agent, Paclitaxel, and
apoptosis [27].
Prior to any antineoplastic treatment, Bim is bound by Bcl-
2, and there has been proven to be a displacement of Bim from
apoptotic proteins consistent with the Paclitaxel treatment at
48 h in four breast cancer cell lines (MCF-7, T47D, MDA-MB-
468 and BT20), 3 p53 mutant [43], similar to our findings,
where the Bim/Bcl-2 complex level started diminishing at 48 h
for both treatments, releasing Bim to initiate apoptosis via
Bax/Bak activation [Fig. 5B]. Likewise, the Bim/Bax complex
level decreased, indicating that Bim might act as a tumor
suppressor that dissociates the anti-apoptotic interaction and
promotes Bax release, replacing the p53 function absent in PC-
3 cells [37].
Additionally, the levels of the procaspase and the activated
form, cleaved-caspase-3 variated significantly indicating that
caspase-3 activation during cell death induction in PC-3 cells
depends on the drug mechanism. It was shown that Paclitaxel
and Vincristine drive the activation of caspase-3 in two breast
cells lines, MCF-7 and MDA-MB231, and as it's suggested that
those microtubules disrupting agents provoke a signaling
cascade and a cellular response to DNA damage [40,45]. The
activation of caspase-3 has been considered a remarkable
end-point of apoptosis independently of the activated
pathway, therefore, the activation of caspase-3 for both
treatments is concluded as the induction of apoptosis [Fig. 4B]
[46].
Furthermore, new interactions between the cleaved
caspase-3 and Bcl-2 or Bim were found, in which, Vincristine
interactions seems to increase in a time-dependent manner in
both treatments. Contrary to Paclitaxel interactions which
decrease also in a time-dependent manner [Fig. 5].
Conclusions
In this study, we reported secondary targets predicted to
microtubule-targeting agents, Paclitaxel and Vincristine and
we show differences in their effects on PC-3 androgen-in-
dependent prostate cancer cells. These are anticancer drugs
with a high clinical value that demonstrated noticeable
differences in their sensitivities, protein profiles and the
protein complex kinetics, confirming that selectivity in the
stimulation of the apoptotic system varies depending on the
cell's genotype, and the drug exposure period. Thus, they
might differ in the formation and quantification of new
protein immunocomplexes, such as the one between
cleaved-caspase-3 and the pro-apoptotic proteins Bim and
Bcl-2.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgements
This research was supported by Colciencias and Universidad
de Ciencias Aplicadas y Ambientales. Grant number 599-2014.
We thank Andr
es Gutierrez for the bioinformatics contribu-
tions during his stay at the U.D.C.A.
biomedical journal 42 (2019) 307e316314
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bj.2019.04.003.
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