Utilizing genome-wide mrna profiling to identify the cytotoxic chemotherapeutic mechanism of triazoloacridone C-1305 as direct microtubule stabilization

Article abstract of DOI:10.3390/cancers12040864

Rational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often difficult due to limited information regarding the complete identification of a molecule’s biological activities. The increasing affordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound’s diverse effects on gene regulation. Here, we used an unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for delineating the antitumor mechanisms of other putative anticancer drug candidates.

Full text of DOI:10.3390/cancers12040864

cancers
Article
Utilizing Genome-Wide mRNA Profiling to Identify
the Cytotoxic Chemotherapeutic Mechanism of
Triazoloacridone C-1305 as Direct
Microtubule Stabilization
Jarosław Króliczewski 1 , Sylwia Bartoszewska 2 , Magdalena Dudkowska 3,†
,
Dorota Janiszewska 3,†, Agnieszka Biernatowska 4,†, David K. Crossman 5 , Karol Krzymin´ski 6,
Małgorzata Wysocka 6 , Anna Romanowska 6 , Maciej Baginski 7 , Michal Markuszewski 8
Renata J. Ochocka 1 , James F. Collawn 9, Aleksander F. Sikorski 10, Ewa Sikora 3 and
,
Rafal Bartoszewski 1,
*
1
Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, 80-416 Gdansk, Poland;
jakrol@windowslive.com (J.K.); renata@gumed.edu.pl (R.J.O.)
Department of Inorganic Chemistry, Medical University of Gdansk, 80-416 Gdansk, Poland;
sylwiabart@gumed.edu.pl
Laboratory of the Molecular Bases of Ageing, Nencki Institute of Experimental Biology of the Polish
Academy of Sciences, 02-093 Warsaw, Poland; m.dudkowska@nencki.pl (M.D.);
d.janiszewska@nencki.edu.pl (D.J.); e.sikora@nencki.edu.pl (E.S.)
Department of Cytobiochemistry, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw Poland;
agi.biernatowska@gmail.com
Department of Genetics, UAB Genomics Core Facility, University of Alabama at Birmingham, Birmingham,
AL 35233, USA; dkcrossm@uab.edu
Faculty of Chemistry, University of Gdansk, 80-308 Gdansk, Poland; karol.krzyminski@ug.edu.pl (K.K.);
malgorzata.wysocka@ug.edu.pl (M.W.); anna.romanowska@phdstud.ug.edu.pl (A.R.)
Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of
Technology, 80-233 Gdansk, Poland; maciej.baginski@pg.edu.pl
Department of Biopharmacy and Pharmacodynamics, Medical University of Gdansk, 80-416 Gdansk,
Poland; michal.markuszewski@gumed.edu.pl
Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham,
Birmingham, AL 35294, USA; jcollawn@uab.edu
2
3
4
5
6
7
8
9
10
Research and Development Centre, Regional Specialist Hospital, 51-154, Wroclaw, Poland;
afsikorski@gmail.com
*
†
Correspondence: rafalbar@gumed.edu.pl; Tel.: +48-58-349-32-14; Fax: +48-58-349-32-11
These authors contributed equally to this paper.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 February 2020; Accepted: 31 March 2020; Published: 2 April 2020
Abstract: Rational drug design and in vitro pharmacology profiling constitute the gold standard in
drug development pipelines. Problems arise, however, because this process is often difficult due
to limited information regarding the complete identification of a molecule’s biological activities.
The increasing affordability of genome-wide next-generation technologies now provides an excellent
opportunity to understand a compound’s diverse effects on gene regulation. Here, we used an
unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of
C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results
demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of
action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by
modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent
that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for
delineating the antitumor mechanisms of other putative anticancer drug candidates.
Cancers 2020, 12, 864; doi:10.3390/cancers12040864
www.mdpi.com/journal/cancers

Cancers 2020, 12, 864
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Keywords: microtubule; RNASeq; tubulin; cell cycle arrest
1. Introduction
The development of cytotoxic chemotherapy into molecularly targeted cancer drug discovery
provided an opportunity to deliver successful therapies that could change the lives of a large number
of cancer patients. However, reducing the safety concerns of any drug therapy requires a thorough
evaluation of the molecular mechanisms of the candidate molecules and, importantly, the prediction of
their off-target effects [1,2]. Nevertheless, despite their obvious benefits, early in-vitro pharmacological
profiling approaches that address the molecular mechanisms that define molecular anticancer effects
are rarely utilized [1].
Studies that focus on presenting the molecular mechanisms governing cytotoxicity, which are
currently fueling the anticancer development pipeline, often focus on single-pathway models that are
difficult to translate into the standard drug development process [1,2]. However, recent developments
and the decreasing costs of high-throughput genotyping technologies like deep sequencing [
allow investigators to address these research limitations.
3,4] now
Several lines of evidence have indicated that the triazoloacridinone derivative, C-1305 (Figure 1)
altered gene and protein expression and promoted apoptosis [ ]. Furthermore, the antitumor activity
of C-1305 [ ] was connected with its ability to intercalate to DNA and/or inhibit topoisomerase II
activity in in-vitro assays [ ]. The genome-wide impact of C-1305, however, and its exact mechanism
of action, remain unknown.
5
6,7
8
Figure 1. Triazoloacridinone derivative, C-1305. (
amino)-8-hydroxy-6H-[1,2,3]triazolo[4,5,1-de]acridin-6-one (C18H19N5O2)—C-1305; (
A
) Chemical formula of 5-((3-(dimethylamino)propyl)
) Predicted
B
3-dimensional (3D) structure of C-1305. The 3D structure was generated as described under materials
and methods section (red-oxygen, blue-nitrogen, black-carbon, grey-hydrogen).
In this study, we used an unbiased approach in lung cancer and colon cell lines (A549 and HTC
116, respectively) to identify universal early transcriptomic signatures of C-1305 cytotoxicity and to
highlight the novel pathways responsible for its biological activity. The cancer cell lines chosen for our
studies were previously reported as very sensitive to the potent anticancer activity of C-1305, [7–9].
Our results identified C-1305-mediated direct microtubule stabilization as the central mediator
that leads to apoptosis. Furthermore, we demonstrated that C-1305 promotes G2 cell cycle arrest
by modulating gene expression. The results indicate that C-1305 is the first identified microtubule
stabilizing agent coupled with topoisomerase II inhibitory activity. Our work provides a framework to
understand the effects of C-1305 on cancer cells and serves as a resource for delineating the antitumor
mechanisms of other putative anticancer drug candidates.

Cancers 2020, 12, 864
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2. Results
2.1. Evaluation of C-1305 Cytotoxicity Profile
The synthesis of compound C-1305 was performed by adopting and optimizing literature
descriptions [ 10]. C-1305 compound identity and purity were verified as described in the
6
,
methods section. Next, to determine when the C-1305 began to induce apoptosis, we performed
a time-course study and monitored A549 and HTC 116 viability using impedance-based real-time
analysis (Figure 2A–E).
Figure 2. Evaluation of C-1305 cytotoxicity profile. (
survival of A549 cells. The cell conductances (expressed as normalized cell index) of A549 cells were
accessed every 15 min following 48-hour treatment with C-1305 at different concentration 25 M, 10 M,
2.5 M, 1 M, 0.5 M, 125 nM, and 12.5 nM. The conductances were normalized to the last value prior
to the experiment start. ( ) IC50 curve for compound C-1305 as determined by real-time cell analysis.
A) Real-time cell analysis of C-1305’s effects on
µ
µ
µ
µ
µ
B

Cancers 2020, 12, 864
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IC50 was calculated after 48 h, as mean and standard deviation (SD), from three independent
measurements (n = 3). ) Real-time cell analysis of C-1305’s effects on HTC 116 cell survival.
The cell conductances (expressed as normalized cell index) of HTC 116 cells were accessed every
15 min following 48-hour treatment with C-1305 at different concentrations: 25 M, 10 M, 2.5 M,
M, 0.5 M, 250 nM, and 125 nM. The conductances were normalized to the last value prior to
the experiment start. ( ) IC50 curve for compound C-1305 as determined by real-time cell analysis.
IC50 was calculated after 48 h as a mean and standard deviation (SD) from 3 independent measurements
(n = 3). ( ) Real-time cell analysis of the effects of increasing concentrations of C-1305 on 16HBE14o
cell survival. The cell conductances (expressed as normalized cell index) of 16HBE14o cells were
M,
(C
µ
µ
µ
1
µ
µ
D
E
−
−
accessed every 15 min following 24-hour treatment with C-1305 at different concentrations: 25
2.5 µM, 5 µM. The conductances were normalized to the last value prior to the experiment start.
µ
The applied real-time monitoring system allows the evaluation of C-1305 dose-dependent changes
in a cellular index that represent cells’ viability, attachment, and morphology [11 14]. The cancer cell
–
lines chosen for our studies were previously used in studies testing the potent anticancer activity of
C-1305 [7–9].
C-1305 at concentrations below 10
until approximately 24 h, when the cell index numbers decreased. Furthermore, the C-1305 IC50 value
calculated based on this real-time assay after 48 h treatment was about 3 M in A549 (Figure 2A,B).
Similar, real-time survival profiles were obtained for C-1305 treatment in HTC 116 cells. C-1305 at
concentrations below 10 M had no significant effect on HTC 116 cell growth and survival until
µM had no significant effect on A549 cell growth and survival
µ
µ
approximately 18 h, when the cell index numbers decreased in a concentration-dependent manner.
Furthermore, the C-1305 IC50 value, calculated based on this real-time assay after 48 h treatment,
was about 10
real-time system corresponded well to the values obtained by the independent MTT assays: 3.08
for A549 (after 24 h, p = 0.0024) and 9.27 M for HTC 116 (after 24 h, p = 0.0019). Finally, C-1305 effects
on cell survival were also tested in immortalized normal human lung epithelial cell line (16HBE14o-).
The IC50 was 23.66 M (after 24 h the p = 0.0012), and as shown in Figure 2E, the C-1305 concentrations
µM in HTC 116 cells (Figure 2C,D). Notably, the C-1305 IC50 values obtained with the
µM
µ
µ
below 5 µM had no significant impact on cell growth.
The data obtained with real-time analysis was used to select appropriate doses for subsequent
RNAseq and biochemical analysis. Furthermore, the RNA samples prior to RNA-seq analysis were
pre-verified for transcriptomic activation of apoptosis related pathways via qPCR (Figure 3). We focus
on p53 and DNA damage signaling, since these pathways were previously proposed to be involved in
C-1305 cytotoxicity [8,15]. Since activation of apoptosis transcriptomic signaling should precede its
phenotypical effects, we focused on the 24 h exposure time points (base on our real-time assays results)
when the cytotoxic effects of C-1305 just started to be significant.
The 24 h exposure of the A549 and HTC 116 to C-1305 at their IC50 concentrations significantly
induced tumor protein p53 (TP53) expression and the downstream gene mRNA expression of Bcl-2
binding component 3 (BBC3). Meanwhile, there were no significant changes in these gene transcripts’
levels in 16HBE14o- cells exposed to a non-toxic (3 µM) concentration of C-1305 (Figure 3A,B). Similar
elevated patterns were also observed for the mRNAs of DNA damage inducible transcript 3 (DDIT3) and
growth arrest and DNA damage inducible alpha (GADD45A) (Figure 3C,D). However, these transcripts
were also slightly induced in 16HBE14o- cells.
Furthermore, C-1305 treatment resulted in increased expression of mitochondrial apoptosis
markers Bcl-2-associated X apoptosis regulator (BAX) and Bcl-2-associated agonist of cell death (BAD)
in cancer cells, when compared to controls and 16HBE14o- cells (Figure 3E,F). The mRNA levels,
however, of the other mitochondrial apoptosis marker, BH3-interacting domain death agonist (BID),
and the extrinsic apoptosis marker, Fas associated via death domain (FADD), remained unaffected in all
models (Figure 3G,H). Taken together, these data confirmed the activation of C3105-related apoptosis
transcriptomic pathways in the cancer cell model (A549 and HTC 116) and limited or no activation of
apoptotic signaling in the control cell model (16HBE14o−).

Cancers 2020, 12, 864
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Figure 3. Exposure of A549 and HTC 116 cells to C-1305 activates transcriptional apoptotic signaling.
A549 and 16HBE14o- cells were exposed to 3 M C-1305 for 24 h, whereas HTC 116 cells were exposed
to 10 M C-1305 for 24h and levels of ( ) TP53, ( ) BBC3, ( ) DDIT3, ( ) GADD45A ( ) BAX, ( ) BAD,
) BID, and ( ) FADD were measured with qPCR. The results from three independent experiments
µ
µ
A
B
C
D
E
F
(
G
H
(n = 6) are plotted normalized to TBP mRNA levels and expressed as a fold-change over the DMSO
vehicle controls. Error bars represent standard deviations. Significant changes (p < 0.05) are marked
with an asterisk.

Cancers 2020, 12, 864
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2.2. RNAseq Global Transcriptome Analysis
Since our real-time analysis of cell growth indicated that 24 h exposure to C-1305 (at IC50
concentrations) was sufficient to significantly alter A549 and HTC 116 cell growth and to activate
apoptosis-related transcriptional signals, we determined the genome-wide transcriptomic alterations
in A549 and HTC 116 upon C-1305 treatment. In brief, the A549 and HTC 116 cells were exposed to
3
µ
M or 10
indicated that a 24 h exposure of noncancer cells, epithelial lung cells (16HBE14o
resulted in minimal cellular damage and loss of viability (Figures 2E and 3), we included the treatment
of 16HBE14o as a negative control in our analysis. Finally, since genomic instabilities were reported
µ
M of C-1305, respectively, for 24 h one day after plating. Furthermore, since our data
−
), to 3 M C-1305
µ
−
for A549 cells [16], DMSO vehicle-treated cells (controls) were obtained after 8 and 24 h of cell culture.
Total RNA was extracted from the cells after 24 h exposure to C-1305 and 24 h and/or 8 h exposure to
vehicle and subjected to RNA sequencing.
As shown in Figure 4A (Data Set S1), after 24 h exposure to C-1305, 3776 and 1769 genes were
dysregulated (by at least 2-fold) in A549 cells. Notably, culturing A549 cells for 16 h in the presence of
DMSO vehicle affected the expression of 562 genes. Furthermore, despite no significant cytotoxicity in
16HBE14o- cells, the exposure to C-1305 dysregulated the mRNAs of 2405 genes. Hence, in order to
focus on universal transcriptome changes related to C-1305 cytotoxicity, we performed a step-by-step
signal selection. As shown in Figure 4, we first corrected the A549-C-1305-treated gene set with the
A549-vehicle-treated gene set. Next, the A549-C-1305-treated gene set was corrected with the non-toxic
16HBE14o—C-1305-treated gene set and the analysis was narrowed to the most significant expression
changes (p
≤
0.05). This approach resulted in the identification of 2040 and 353 genes dysregulated
upon C-1305 treatments in A549 and HTC 116 cells, respectively. In the final selection stage, we selected
153 common genes (26 upregulated and 127 downregulated) for which expression was significantly
changed in both of these cancer cell lines upon C-1305 exposure (Data Set S1).
Next, for the selected genes, we performed gene ontology analysis using three different software
approaches (Gene Analytics [17], Enricher [18] and WebGestalt [19]). As shown in Figure 4B and
Table 1, microtubule cytoskeleton organization and tubulin binding were the most significant gene
ontology (GO) molecular clusters among the dysregulated genes. This highlighted an important role
for the genes involved in chromosome segregation, mitotic spindle formation and cell cycle regulation
(Table 1).
Within this cluster of gene transcripts, the following genes’ mRNA levels were downregulated
upon C-1305 exposure: Aurora kinase B (AURKB), Polo-like kinase 1 (PLK1), cyclin-dependent kinase
1 (CDK1), kinesin family members 1C, 11, 14, 15, 18A, 18B, 20A, 22, 23 and 4A (KIFC1); cell division
cycle-associated 8 (CDCA8), baculoviral IAP-repeat-containing 5 (BIRC5); cyclins B1 and B2 (CCNB1
and CCNB2), cell-division cycle protein 20 (CDC20), Lamin B1 (LMNB1), centromere proteins F, J, F, K,
N, and Q (CENPF, CENPJ, CENPF, CENPK, CENPN and CENPQ); family with sequence similarity 83
member D (FAM83D), growth arrest-specific 2-like 3 (GAS2L3), nucleolar and spindle-associated protein
1 (NUSAP1), protein regulator of cytokinesis 1 (PRC1), spindle and kinetochore-associated complex
subunit 1 (SKA1) stathmin 1 (STMN1) (Figure 4B). Notably, the upregulated gene set were tumor
protein P53-inducible protein 3 (TP53I3), tumor Protein P53-inducible nuclear protein 1 (TP53INP1)
and Snail family transcriptional repressor 2 (SNAI2) and these had no significant assignment to the
cellular signaling pathways.

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Figure 4. Schematic of analysis and data filtration and analysis performed to identify gene ontology (GO)
terms of significantly dysregulated genes. ( ) Schematic of analysis and data filtration Following the
A
flirtation of gene transcript potentially related to A549 growth and no cytotoxic effects in 16HBE14o-cells.
The common gene transcripts dysregulated in both A549 and HTC 116 were selected. Well-established
gene transcripts (no predicted gene transcripts, gene names starting with “LOC”) with greater than
10 RPKMs per sample and with significance (p
between C-1305-treated and control groups were used in pathway analyses. (
≤
0.05) greater or equal to a 2-fold change in expression
) The GO clusters are
B
depicted for selected genes; clusters are listed followed by the enrichment score calculated by Enricher,
which is used to determine the percentage of the pie chart. The darker the color, the more enriched
the cluster.

Cancers 2020, 12, 864
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Table 1. Function of genes being significantly changed upon C-1305 treatment (p < 0.05) as assigned
with the Gene Ontology databases and verified with the literature.
Gene
BBC3
Function
Reference
[20]
Mediator of p53/TP53-dependent/independent apoptosis
Multifunctional transcription factor in the response to cell stresses; induces cell
cycle arrest and apoptosis in response to endoplasmic reticulum (ER) stress
Regulator of the mitochondrial apoptotic process
Promotes cell death
DDIT3
[21,22]
BAX
BAD
GADD45A
BID
[23]
[23]
[24]
[25]
ER stress dependent/apoptotic
Pro-apoptotic member of the B-cell lymphoma-2 (Bcl-2) protein family
Apoptotic adaptor molecule that recruits caspases to the activated Fas or
TNFR-1 receptors.
Tumor suppressor in many tumor types; induces growth arrest or apoptosis
Regulator of mitosis involved in the bipolar attachment of spindle
microtubules to kinetochores
FADD
TP53
[26]
[27]
[28]
AURKB
PLK1
CDK1
Kinetochore assembly
[29]
[30]
Regulate mitotic entry, centrosome separation and spindle assembly
Regulate chromosome congression and alignment and bipolar spindle
formation
KIFC1
[31]
KIF11
KIF14
KIF15
KIF18A
KIF18B
KIF20A
KIF22
Establishing a bipolar spindle during mitosis
Regulate chromosome congression and alignment
Involved in mitotic spindle assembly
Regulate chromosome congression and alignment
Microtubule movement and depolymerization
Cytokinesis
[32]
[31]
[33]
[34]
[35]
[31]
[36]
[31]
[31]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
Spindle formation and the movements of chromosomes during mitosis
Cytokinesis
KIF23
KIF4A
CDCA8
BIRC5
CCNB1
CCNB2
CDC20
LMNB1
CENPF
CENPJ
Anaphase spindle dynamics and cytokinesis
Mitosis regulation, microtubule stabilization and spindle assembly
Chromosome alignment and segregation during mitosis and cytokinesis
Control of the cell cycle at the G2/M (mitosis) transition
Control of the cell cycle at the G2/M (mitosis) transition
Mitotic regulation of the human anaphase
Interact with chromatin and promote senescence
Regulate kinetochore function and chromosome segregation in mitosis
Inhibits cell proliferation and induces apoptosis after G2/M arrest
Assembly of kinetochore proteins, mitotic progression and chromosome
segregation
CENPK
CENPN
CENPQ
[45]
[46]
[47]
Assembly of kinetochore proteins, mitotic progression and chromosome
segregation
Assembly of kinetochore proteins, mitotic progression and chromosome
segregation
FAM83D
GAS2L3
NUSAP1
PRC1
Cell proliferation, growth, and migration
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
Cytokinesis/Stabilize the formation of the actin and microtubule network
Promote the organization of mitotic spindle microtubules
Cytokinesis and microtubule assembly
Chromosome segregation and microtubule depolymerization
Regulation of the microtubule filament system by destabilizing microtubules
Generation of reactive oxygen species (ROS)/Apoptosis
Proapoptotic protein and regulator of transcription and autophagy
Transcriptional repressor
SKA1
STMN1
TP53I3
TP53INP1
SNAI2
PARP
Mediates poly-ADP-ribosylation of proteins/DNA repair
2.3. C-1305 Affects Microtubule Cytoskeleton Through the Induction of Tubulin Polymerization
Based on the results of the results of the RNAseq-based approach, we speculated that C-1305
cytotoxicity may result from the direct disorganization of the microtubule skeleton. This hypothesis
was further supported by the good correlation of C-1305’s transcriptomic profile with the transcriptomic
changes reported for other tubulin-directed anticancer drugs, including nocodazole and paclitaxel

Cancers 2020, 12, 864
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(Data Set S1). C-1305-related interruptions of the microtubule network could consequently cause
defects in mitotic spindle assembly and chromosome segregation leading to the deregulation of cell
cycle and eventually cell death. To test this hypothesis, we performed molecular docking of C-1305 to
the
for paclitaxel (tubulin polymerization stabilizing agent [58]) using the same parameters (Figure S7).
As shown in Figure 5, C-1305 could bind at multiple locations to both tubulin dimer and subunits,
with the free energy comparable to that calculated for paclitaxel ( 7.1466 kcal/mol). Notably, the
α/β tubulin dimer crystal structure (Figure 5). As a control, the molecular docking was calculated
α
β
−
predicted C105 binding sites, despite being located at the luminal side of the tubulin dimer, do not
overlap with the paclitaxel binding niche or the colchicine binding site [59,60].
Figure 5. Molecular docking identifies potential C-1305 binding to the α/β tubulin dimer. (A) Predicted
interactions between tubulin dimer and a C-1305 molecule. The favorable binding sites were identified
by judging from the largest number of conformations in the same position with the lowest binding
energy. The analysis showed minor differences among the top results of each binding site for C-1305,
supporting the robustness of the calculations. Additionally, the superposition of the structure of the

Cancers 2020, 12, 864
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paclitaxel co-crystallized with the tubulin (PDB accession code 1TUB) onto that obtained from the
docking of paclitaxel yielded a good fitting of both ligands. ( ). A close-up of the representative
snapshots of the simulations depicting the interactions of C-1305 at the luminal side of tubulin.
) Classification of results obtained from the docking of C-1305 into tubulin by SwissDock. The Full
Fitness and G values (kcal/mol) for the most favorably ranked complex (cluster Rank = 0) are shown.
B
(
C
∆
Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for
Biocomputing, Visualization, and Informatics at the University of California, San Francisco [61]. Protein
(1TUB) is shown as ribbon and ligands are shown as sticks, colored by element. The control structure
of tubulin, with colchicine and molecular docking results for paclitaxel, are provided in Figures S6 and
S7 respectively. In Figure S7, we will add the three-dimensional structure of the
(PDB: 1TUB) [62] used for molecular docking, showing that paclitaxel binds to a unique pocket on
-tubulin near GDP binding site, which controls molecular docking of the paclitaxel molecule and
α/β-tubulin dimer
β
tubulin protein and tubulin dimer crystal structure (1TUB) superimposed with the computed tubulin
dimmer allosteric structure. The ZINC96006020 (Paclitaxel) entry obtained from the ZINC database
(http://zinc.docking.org) was used for ligand structure generation [63].
The molecular docking results strongly supported the hypothesis that C-1305 could directly
affect tubulin polymerization. To further examine this possibility, we performed an in-vitro tubulin
polymerization assay.
As shown in Figure 6A, C-1305 strongly stabilized the tubulin polymerization process in a
dose-dependent manner. Paclitaxel and vinblastine were used as a control for stabilizing and
destabilizing the polymerization of tubulin, respectively. The calculated effective concentration
of C-1305 on tubulin polymerization (EC50) was 2.74 µM (Figure 6B). Notably, although C-1305,
like paclitaxel, promoted tubulin polymerization and possibly stabilized microtubules, these two
compounds affected the polymerization process with different kinetics (Figure 6C). The most significant
difference was observed in the time required to achieve exponential microtubule polymerization
(Lag), which was 1 minute for paclitaxel and 4–8 min for C-1305. Furthermore, both C-1305 and
paclitaxel significantly and comparably increased tubulin polymerization rate as well as higher levels
of polymerized tubulin at the equilibrium phase (Figure 6D). Taken together, the data indicate that
C-1305 acts as an efficient tubulin polymerization stabilizing agent in vitro.
Notably, the molecular docking studies predicted that the C-1305 does not compete for tubulin’s
binding site with paclitaxel, and the kinetics of these two compounds’ impact on tubulin polymerization
differed as well. This observation suggests that C-1305 effects on tubulin polymerization could result
from different mechanisms of interaction than for paclitaxel. Hence, to further examine this hypothesis,
we analyzed the combined effects of C-1305 and paclitaxel co-treatment on tubulin polymerization
in vitro. As shown in Figure 6E, these compounds complemented each other’s effects on tubulin
polymerization, significantly improving the polymerized tubulin level at equilibrium phase and
decreasing the lag time during the co-treatment (Figure 6F). The complementary effects were also
well reflected in the maximal velocity of tubulin polymerization, as shown in Figure S8, which was
significantly higher than for any of these compounds alone (Figure 6G).
We then tested whether C-1305 had similar effects on tubulin polymerization in A549 cells exposed
to this compound for 6 h and 24 h, given that these cells were more sensitive to the compound
treatments as shown by lower IC50 values when compared to HTC 116. As shown in Figure 7A and
Figure S9, C-1305 treatment (equal to or above 1 and 3
µM concentrations for 24 h and 6 h, respectively)
dramatically reduced the soluble tubulin fraction and led to a significant accumulation of the polymeric
tubulin fraction. Notably, the effects of 5 µM C-1305 were comparable to the 10 µM paclitaxel treatment
(Figure 7A).

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Figure 6. C-1305 acts as an efficient tubulin polymerization stabilizing agent in vitro and complements
paclitaxel effects on tubulin polymerization ( ) The kinetics of the polymerization of 3.0 mg/mL purified
A

Cancers 2020, 12, 864
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tubulin with microtubule stabilizers was monitored turbidimetrically at a 340 nm wavelength (OD340 nm).
The increasing concentration of tubulin polymers as a function of time displays the characteristic
sigmoidal curve for tubulin filament formation. The curve is typically divided into the lag phase
(nucleation), the growth phase (polymerization), and the plateau phase [64]. The square data points in
a line in each graph indicate microtubule polymerization alone (control). Vinblastine, a destabilizer
of tubulin polymerization, was used as a control. C-1305 stabilized the polymerization of tubulin
in a cell-free system in a dose-dependent manner. (
of the OD340 nm change obtained with C-1305 treatment to the OD340 nm change of the control [65].
Due to observed tubulin oscillation at high doses of C-1305, to calculate EC50 for 10 M C-1305
concentration, we used a mean value from three independent experiments. ( ) The quantization
B) The EC50 rate was calculated as the ratio
µ
C
of tubulin polymerization parameters was calculated as described in [66]. Lag indicates the time
required to achieve exponential microtubule polymerization. The maximum relative rate and total
polymerization have been normalized to non-drug-treated tubulin (control). The graph is representative
of three independent experiments. (D) The velocity of tubulin polymerization in the growth phase
after C-1305 treatment was calculated as described previously [67]. The polymerization velocity was
calculated based on the ratio of the OD340 nm change to the reaction time. The velocity in the absence of
stabilizers was taken as 100%. * p < 0.05 versus control. Dose-dependent effect of C-1305 or paclitaxel on
in-vitro microtubule formation. The level of polymerization was measured by an increase in turbidity
(OD) at 340 nm. (
tubulin polymerization. (
E
) The synergic/additive effects of concomitant C-1305 and paclitaxel treatment on
) The quantization of tubulin polymerization parameters was calculated
F
as described in [66]. Data presented in the Figure S8 graph was used for calculation. Lag indicates
the time required to achieve exponential microtubule polymerization. The maximum relative rate
and total polymerization have been normalized to non-drug-treated tubulin (control). The graph is
representative of three independent experiments. (G) The velocity of tubulin polymerization in the
growth phase after C-1305, paclitaxel or concomitant C-1305 and paclitaxel treatment. The velocity in
the absence of stabilizers was taken as 100%. * p < 0.05 versus control.
We also analyzed C-1305’s effects on the microtubule network organization in A549 cells exposed
for 24 h to C-1305. Paclitaxel and vinblastine were used as polymerization stabilizing and destabilizing
reference controls. As shown in Figure 7B, the C-1305 treatments strongly interrupted the microtubule
network. Similar effects of the C-1305 treatments were also observed in the HTC 116 cells (Figure S10).
These were both detected with antibodies against the
α and β subunits of the tubulin dimer and led to
similar results in the paclitaxel treated cells by the thickening of microtubule fibers.
Collectively, the biochemical and immunochemical data confirmed the C-1305-specific impact
on tubulin polymerization in A549 cells. To test for the impact of C-1305 treatment on cell cycle
progression, the C-1305-treated A549 cells were arrested in the G2 phase of the cell cycle (Figure 8).
To distinguish cells arrested in G2 compared to in M phase, we analyzed the mitotic index using
MPM-2 assay. Cells were stained with MPM-2 antibody, which recognized a set of phospho-proteins
expressed specifically during mitosis, and then they were measured by flow cytometry. As shown in
Figure 8A,B in the A549 cell line treated with C-1305, there was a decrease of MPM-2-positive cells
in a time-dependent manner. Meanwhile, after paclitaxel, an accumulation of cells in mitosis was
observed. This would suggest that C-1305 caused cell cycle blockade rather in G2 then during mitosis.
This observation is in agreement with previous studies reporting C-1305-induced cell arrest at the
G2/M checkpoint [15
,68]. Notably similar effects on cell cycle arrest were reported for the microtubule
network interrupting agents [69–72].

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Figure 7. C-1305 stabilizes polymeric tubulin and changes the microtubule network in A549 cells.
) SDS-PAGE (bottom panel) and Western blot analysis (top panel) of soluble and polymeric
tubulin fractions in A549 cells after C-1305 treatment. Western blot analysis of soluble (S) and
polymeric (P) tubulin fractions using rabbit primary antibody against -tubulin (ab18207, Abcam) and
HRP-conjugated secondary antibodies (Bio-Rad) and detected using ECL (Amresco). The molecular
weight markers are indicated by the arrows on the left (Precision Plus Protein Kaleidoscope
(
A
β
™
™
Prestained Protein Standards BioRad, #1610375). Densitometry was performed using Image Lab
software v. 4.1 (Bio-Rad) and Gel-Pro Analyzer v. 4.0 (Media Cybernetics L.P); the amount in each band
was calculated in relation to the corresponding band of the control lane. The bottom panel total protein
loading controls SDS-PAGE of soluble (S) and polymeric (P) tubulin fraction isolated from A549 cells

Cancers 2020, 12, 864
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after 24h of treatment. The results of the soluble and polymeric tubulin fractions in A549 cells treated
with C-1305 for 6 h are shown in Figure S9. Raw Western blot files are shown in Figure S12. ( ) The
B
C-1305 effects on the microtubule network were observed by immunofluorescence using a Confocal
Scanning Laser Microscope (LSM) and Transmitted Light (TL). A549 cells were incubated in the absence
and the presence of C-1305 (3 or 10
Cells were fixed and processed for immunostaining with antibodies against
-tubulin (right panel) for 2 h, then reacted with Alexa fluoro-conjugated secondary antibody. The scale
bar is 10 µm. Paclitaxel and vinblastine were used as the reference compound.
µM), paclitaxel (3
µM) and vinblastine (3 or 10
µM) for 24 h.
α
-tubulin (left panel) and
β
Figure 8. C-1305 induces G2 cell arrest in A549 cells. Cytometric analysis of MPM-2-positive cells
after C-1305 or paclitaxel treatment ( ). Representative dot blot of mitotic, MPM-2-positive cells in
A549 cell line treated with C-1305 or paclitaxel for 6 or 24 h, respectively. MPM-2-positive cells were
gated, quantified, and shown as a percentage next to the gate. ( ). Percentage of MPM-2-positive cells
SD from three independent experiments. * p < 0.05; ** p <0.001; *** p < 0.0001
A
B
presented as means
versus control cells.
±

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3. Discussion
Rational drug design, together with in-vitro pharmacology profiling, constitute the gold standard
of drug development pipelines that allow for the cost-effective reduction of safety-related drug attrition
at an early stage during development. However, precise chemical targeting is difficult due to potential
adverse side effects and thus requires an understanding of the molecular mechanisms underlying the
molecule’s biological activity. Hence, although a large number of potential compounds have been in the
drug pipeline, relatively few of them have reached clinical trials or contributed to therapy development.
Consequently, many potent promising compounds were left behind or forgotten due to an inability
to explain their biological activities in numerous complex gene pathways and cell types. However,
the recent development and increasing affordability of genome-wide next-generation technologies
provides an opportunity for non-biased determination of the molecular changes brought about by a
compound’s multiple activities, and thus revalidation and testing of these forgotten drug candidates is
now possible.
In our approach, we exploited label-free, real-time monitoring of cell survival and genome-wide
RNA-Seq to revalidate molecular mechanisms underlying the cytotoxicity of triazoloacridone C-1305
in two cancer cell lines. The compound was first synthesized at Gdansk University of Technology in
late 1980s [
6], during the development of synthetic DNA-interacting agents, and displayed robust
cytotoxic and cytostatic effects towards both cancer cell lines and solid tumors in vitro
[
7]. Despite
non-cellular studies that confirmed the ability of C-1305 to covalently bind DNA (preferably in
guanidine-rich regions) [73], this phenomenon was not cytotoxicity correlated [74]. In the early 2000s,
subsequent research focused on the impact of C-1305 on DNA topoisomerase II activity [8], a crucial
regulator of cellular DNA topology [75]. These studies indicated that C-1305 had very mild and
“unusual” topoisomerase II poison activity in vitro, but these results did not clarify the observed robust
cytotoxic effects [
8]. Hence, despite many other, often contradictory, reports [76–78], the mechanism of
C-1305-induced apoptosis remained undefined.
Since the vast majority of previous attempts to explain C-1305 biological activity have been based
on steady-state cytotoxicity assays (often after 72 h exposure), we utilized real-time monitoring of
cell survival to determine the time course of C-1305 activity. The results indicate that a significant
cell growth inhibition begins at about 24 h after C-1305 administration, which confirms previous
studies [8,15]. This was demonstrated in both cancer cell lines and indicated a significant induction of
the apoptotic markers’ mRNA levels. Furthermore, comparing the mRNA expression of these markers
in cancer cell lines with the mRNA levels in non-cancer cells, we find that their expression correlates
well with the observed cytotoxic effects of the C-1305 in the cancer cell lines.
Taking advantage of these differences, we selected appropriate conditions to follow the
C-1305-induced changes in the transcriptome with next-generation sequence profiling. Since these
RNA-seq results show dramatic differences in C-1305 transcriptomic profiles depending on the cell line
tested, we focused on changes in gene expression on mRNAs that were independent of cancer cell line
type or vehicle, and thus potentially related to the apoptotic activity of C-1305. This approach resulted
in the selection of 153 transcripts that could potentially determine the mechanism of C-1305 activity.
The selected mRNAs were mainly related to the microtubule cytoskeleton organization and tubulin
binding and critical for the regulation of chromosome segregation, mitotic spindle formation, and cell
cycle regulation. Taken together, these data suggest that C-1305-induced apoptosis results from the
direct disorganization of its microtubule cytoskeleton and related deregulation of its cell cycle.
The most straightforward explanation of such effects of C-1305 treatment would depend on
this compound’s direct interaction with tubulin. The hypothesis that C-1305 could directly affect
tubulin polymerization was strongly supported by the molecular-docking-based identification of this
compound’s binding locations within α/β tubulin dimers. The in vitro tubulin polymerization assays
demonstrated that C-1305 efficiently stabilized the tubulin polymerization process in a dose-dependent
manner. Notably, the C-1305-related increase in tubulin polymerization rate, polymerized tubulin level
at equilibrium phase, and velocity were comparable to those observed for paclitaxel, a classical tubulin

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polymerization stabilizing agent. Furthermore, our analysis indicated that C-1305 and paclitaxel
do not compete for the same tubulin binding sites, and that these two compounds are additive on
tubulin polymerization. This suggests that C-1305 could potentially support paclitaxel-based therapies;
however, this hypothesis will require further studies. Finally, we confirmed the proposed tubulin
polymerization-related mechanism of C-1305 biological activity using biochemical and immunochemical
analyses. The C-1305 treatment resulted in polymeric tubulin accumulation in cells, and strongly
interrupted microtubule networks, in a similar manner to paclitaxel, and was accompanied by cell
cycle arrest in the G2 phase. Upon C-1305 treatment, there was no accumulation of mitotic marker
MPM-2, which was observed in paclitaxel-treated cells. However, it cannot be excluded that the
differences between C-1305 and paclitaxel cell effect could be due to the different concentrations of
drugs. The observed G2 arrest was not only previously reported for C-1305 [15], but also for many other
tubulin destabilizing agents [69–72]. Support for our results also comes from previous reports that cells
depleted of poly(ADP-Ribose) polymerase 1 (PARP1) or functional p53 are more sensitive to C-1305
and cell cycle arrest [15]. Notably, both p53 and PARP-1 are crucial regulators of the centrosomes,
which play an important role in microtubule function and cell division [79
shown that loss of p53 involves extensive alterations in microtubule composition and dynamics [83],
and confers sensitization to paclitaxel [69], similar to the PARP inhibitors [84 85]. Finally, numerous
promising anti-cancer compounds were shown to be both topoisomerase poisons and anti-microtubule
agents, having a similar pleiotropic activity to C-1305 [86 88]. Notably, however, since all these are
–82]. Other studies have
,
–
topoisomerase inhibitors that destabilize microtubules, C-1305 is, to the best of our knowledge, the first
known molecule that shares both topoisomerase inhibition and microtubule-stabilizing properties.
Nevertheless, further studies are necessary to establish if such an unusual activity of anti-cancer agent
can have beneficial effects [88].
4. Materials and Methods
4.1. Cell lines and Culture Conditions
Human bronchial epithelial 16HBE14o cells were obtained as previously described (Sigma-
−
Aldrich, catalog no. #SCC150, Poznan´, Poland [89]. Human colorectal carcinoma HTC 116 (ATCC
CCL-247) cells and human small lung carcinoma A549 cells (ATCC CCL-185) were obtained from
ATCC. 16HBE14o cells were cultured in Minimum Essential Modified Eagle’s Medium (Invitrogen,
−
Warsaw, Poland) with 10% fetal bovine serum (FBS), while HTC 116 cells were cultured in McCoy’s 5a
Medium (Invitrogen) with 10% FBS. A549 cells were cultured in RPMI 1640 media supplemented with
10% FBS in a humidified incubator at 37 ◦C in 5% CO2. All experiments were conducted at a final
confluence of 70–80%.
4.2. Chemicals
5-{[3-(Dimethylamino)propyl]amino}-8-hydroxy-6H-[1,2,3]triazolo[4,5,1-de]acridin-6-one
(C18H19N5O2, MW = 337.38 g/mol) (compound C-1305) was synthesized in several steps by adopting
an optimization of literature descriptions [6,10]. In the first step 6-chloro-3-nitro-2-{[4-(benzyloxy)-
phenyl]amino}benzoic acid was obtained by Ullman condensation of 2,6-dichloro-3- nitrobenzoic
acid and 4-(benzyloxy)aniline hydrochloride in the presence of triethylamine in anhydrous ethanol
at reflux. The next step was cyclization of the above anthranilic acid derivative by the action
of phosphorus oxychloride at reflux. Subsequent stages of the synthesis were removal of the
benzyl group from the 8-OH group, reduction of 4-nitroacridin-9(10H)-one derivative to the
respective 4-aminoacridin-9(10H)-9-one, followed by diazotization of the 4-amino group. The last
step of the C-1305 synthesis route was the substitution of the halogen atom (5-Cl) in the latter
compound by 3-(dimethylamino)propan-1-amine. Purification of C-1305 was performed by applying
gravitational liquid chromatography, using silica gel as a stationary phase and gradient type of
elution (chloroform/methanol/ammonia mixtures). Detailed information concerning the synthesis of

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compound C-1305 and its isolation in chemically pure form is provided in Electronic Supplementary
Material (ESI) (Figure S1).
The compound purity and identity were verified with RP-HPLC, elemental analysis, 1H NMR
spectroscopy and HR MS spectrometry. Representative chromatograms and spectra together with
experimental settings are presented in Figures S2–S5 (ESI). Summary of C-1305 analyses: RP-HPLC:
98.2% (average area under the main signal, n = 3); elemental analysis (% found/calculated):
1
C = 63.76/64.08, H = 5.64/5.68, N = 20.20/20.76; H NMR (500 MHz, CDCl3, room temperature):
δ
1.80–1.85 ppm (quintet, 2H, J = 6.8 Hz); 2.18 ppm (s, 6H); 2.36 ppm (t, 2H, J = 6.9 Hz); 3.57–3.61
ppm (q, 2H, J = 6.4 Hz); 7.16 ppm (d, 1H, J = 9.3 Hz); 7.42 ppm (dd, 1H, J1 = 8.8 Hz, J2 = 3 Hz); 7.74
ppm (d, 1H, J = 2.4 Hz); 8.29 ppm (d, 1H, J = 9.3 Hz); 8.35 ppm (t, 1H, J = 9.3 Hz); 9.46 ppm (t, 1H,
J = 6.4 Hz, NH); 10.3 ppm (s, 1H, OH). ESI-QTOF MS: m/z = 338.23 (M+1)+, 310.22 (M+1 28)+. UV-Vis
−
(H2O/DMSO = 9/1 v/v): 258.2 nm (
(ε = 43253 M−1 cm−1
, 422.2 nm (
are provided in Figure 1.
ε ε
= 55060 M−1 cm−1), 298.5 nm ( = 36580 M−1 cm−1), 405.1 nm
)
ε
= 44570 M−1 cm−1). The C-1305 chemical formula and 3D structure
◦
The two independent batches of C-1305 were tested. C-1305 was stored in the dark at 4 C.
Prior to the experiments, the compound was dissolved in DMSO at 3 mM stock solution. Paclitaxel was
purchased from Cytoskeleton (BK006P, Cytoskeleton Inc., Denver, CO, USA) and vinblastine (catalog
no. V1377) and DMSO (catalog no. S-002-D) were purchased from Sigma-Aldrich. Paclitaxel was
dissolved in DMSO at 2 mM stock solution and stored at µM
70 ◦C. Vinblastine was stored as 100
−
stock in water at −20 ◦C.
4.3. Isolation of RNA and Small ncRNA
Total RNA containing the small ncRNA fraction was isolated using miRNeasy kit (217004, Qiagen,
Wroclaw, Poland ). RNA concentrations were calculated based on the absorbance at 260 nm. RNA
samples were stored at −70 ◦C until use.
4.4. Next Generation RNA Sequencing Analyses
Cells treated with C-1305 or DMSO vehicle (as specified in the Results section) were used
for the RNA isolation and analyses. Following total RNA isolation, samples were validated with
quantitative real-time PCR for apoptosis activation prior to further analysis. Following rRNA depletion,
the remaining RNA fraction was used for library construction and subjected to 75-bp single-end
sequencing on an Illumina HiSeq 2000 instrument (San Diego, CA, USA). Sequencing reads were
aligned to the Gencode human reference genome assembly (GRCh38 p7 Release 25) using STAR
version 2.5.3b [90]. Transcript assembly and estimation of the relative abundance and tests for
differential expression were carried out with HTSeq-count version 0.9.1 and DESeq2 for those samples
with biological replicates, and with Cufflinks and Cuffdiff for those samples without biological
replicates [91–93]. The resulting data were validated with quantitative real-time PCR.
The GeneAnalytics
™ web server (geneanalytics.genecards.org) [17] was used to place the Next
Generation Sequencing (NGS) results into a physiological context. Furthermore, the analyses were
limited to experimentally verified interactions and no extended gene enrichment set analyses were
performed. The results of GeneAnalytics analysis were further verified with Enricher [18] and
WebGestalt [19]. Furthermore, the transcript changes crucial for GO assignment were independently
verified for these gene level changes upon 8 h and 24 h exposure of A549 cells to C-1305, with qPCR.
4.5. Measurement of mRNA Levels Using Quantitative Real-Time PCR (qRT-PCR)
We used TaqManOne-Step RT-PCR Master MixReagents (Applied Biosystems, Carlsbad, CA,
USA as described previously using the manufacturer’s protocol [11]. The relative expressions were
calculated using the comparative relative standard curve method [94]. We used TATA-binding protein
(TBP) mRNA, TFRC and 18S as the relative controls for our studies. TaqMan probes ids used were:
DDIT3—Hs00358796_g1; GADD45A—Hs00169255_m1; BBC3—Hs00248075_m1; TBP—Hs4332659_m1;

Cancers 2020, 12, 864
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18 S—Hs99999901_s1; FADD Hs04187499_m1; BID Hs00609632_m1; TP3 Hs01034249_m1; BAX
Hs00180269_m1; BAD Hs0018930_m1; TFRC Hs00951083_m1.
4.6. Cell Viability Assays
For real-time monitoring of cell viability, we used the Roche xCeligence system as we described
previously [95]. Briefly, 16HBE14o cells (2,000 cells per well), A549 cells (7,500 cells per well) or HTC
−
116 cells (15,000 cells per well) were seeded in 16-well PC plates 12 h prior to the treatment. Control
cells were cultured in the presence of DMSO vehicles. Treated cells were incubated with different
concentrations of C-1305 for the next 48 h, and every 15 min the cell conductances (cell index) were
recorded. All experiments were performed in triplicate with two independent repeats. Control cells
were cultured in the presence of DMSO vehicles. RTCA software v. 1.2.1 (ACEA, Biosciences Inc.,
San Diego, CA, USA) was used to calculate the normalized cell index and the cells’ growth curve slopes.
MTT assays of cell viability were performed in 96-well plates. One day after plating, 16HBE14o
−
cells (15,000 cells per well), A549 cells (7,500 cells per well) or HTC 116 cells (2,000 cells per well)
were cultured with different concentrations of C-1305. After a 24-hour incubation with the specified
compounds, medium containing 1 mg/mL MTT was added to cells for a final concentration of 0.5 mg/mL
and incubated at 37 ◦C for 4 h. The medium was aspirated, and the formazan product was solubilized
with DMSO. The absorbance at 630 nm (background absorbance) was subtracted from absorbance
at 570 nm for each well. There were six replicates for each tested concentration. DMSO was used as
control and the concentration in the medium did not exceed 0.1%. The IC50 values were calculated
from dose–response curves using the four-parameter logistic function.
The permeability of C-1305 to cross the cell membrane was reported previously [
confirmed with confocal microscopy (Figure S11)
9] and also was
4.7. Molecular Docking
In the absence of crystal structures that would indicate an exact location for the C-1305 ligand,
a blind docking was performed for C-1305 with the use of the 1TUB PDB file as a receptor [62].
The crystal structure contained one paclitaxel with 420 amino acid residues and GTP in
another with 427 amino acid residues and GDP in -tubulin. The paclitaxel was removed from the
complex before docking. Blind docking was performed with the use of the SwissDock web server [96
α-tubulin and
β
]
based on the docking software EADock DSS [97], whose algorithm consists of the following steps:
many binding modes are generated either in a box (local docking) or in the vicinity of all target cavities
(blind docking). Simultaneously, their CHARMM energies are estimated on a grid and the binding
modes with the most favorable energies are evaluated with fast analytical continuum treatment of
solvation (FACTS) and clustered. The mol2 file of C-1305 and colchicine molecules with all hydrogens
and 3D coordinates was generated using UCSF Chimera and MarvinSketch. The program Avogadro,
version 1.2.0, was used to perform geometric optimization of unbound C-1305 with 500 steps of steepest
descent using the MMFF94 force field [98]. The data for tubulin dimers with complexed colchicine
molecules was obtained by engineering the crystal structure of colchicine-bound tubulin that was
downloaded from the protein data bank (4O2B, PDB)[99], (Figure S6).
4.8. In Vitro Tubulin Polymerization Assays
The direct effect of C-1305 on tubulin polymerization was determined by turbidity using a
microtubule polymerization assay kit (BK006P, Cytoskeleton Inc., Denver, CO, USA) following the
manufacturer’s protocol [100]. Briefly, bovine brain tubulin (3.0 mg/mL) in polymerization assay buffer
(80 mM Pipes, pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, 10% glycerol) was added to pre-warmed
96-well microtiter plates at 37 ◦C containing compound at various concentrations in DMSO (2% final).
After mixing, the rate of polymerization at 37 ◦C was followed by absorption at 340 nm for 45 min at
1 min intervals, using a thermostated Tecan Infinite M200Pro microplate spectrophotometer (Tecan
Group Ltd., Männedorf, Switzerland). For this assay, paclitaxel was used as the positive control and

Cancers 2020, 12, 864
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vinblastine (Sigma-Aldrich) was used as a reference inhibitor. All experiments were repeated three
times and the measurements and calculations were conducted with Tecan Magellan V7.0 software,
Statistica 13.1 (Dell Inc., 2016, Palo Alto, CA, USA). The half-maximal effective concentration (EC50)
was defined as the concentration of the drug that gives half-maximal response between the baseline
and maximum after a specified exposure time.
4.9. Determination of the Monomeric and Polymeric Tubulin with Western Blot
Levels of soluble (monomeric, cytosolic) and polymeric (insoluble, cytoskeletal) tubulin were
determined in A549 cells as was described previously [101], with some modification. Briefly, A549 cells
at 70% confluency were incubated with or without different concentrations of C-1305 in 6-well plates
for 24 h. After 24 h, the cells were washed twice in PBS and then incubated for 4 min at 37 ◦C in 150
µL
of microtubule stabilization buffer (25 mM Hepes, pH 6.9, containing 2 mM MgCl2, 2 mM EGTA,
0.5% Triton X-100, 20 L protease inhibitor mixture (Halt Protease Inhibitor Cocktail (100 ), cat. 78430,
Thermo Scientific), and 25% glycerol to permeabilize the cells and release the soluble tubulin. The cell
lysates were vortexed briefly, and the soluble fractions were separated by centrifugation (15,000 g for
10 min at room temperature). The supernatant (200 L) containing the soluble tubulin was transferred
to a new tube. Next, the remaining cytoskeletal fractions (polymeric tubulin) were lysed with ~200
µ
×
×
µ
µL
of 1 RIPA buffer and protease inhibitor. Soluble and polymerized tubulin samples were stored at
×
−80 ◦C for future analyses.
The effect of C-1305 on the polymerized amount of tubulin in A549 cells was analyzed by
SDS-PAGE (Mini-PROTEAN TGX Stain-Free, Bio-Rad, Warsaw, Poland) and western blot analysis.
The protein concentrations for both the fractions of tubulin were measured by DC protein assay kits
(Bio-Rad). Protein (20 µg) from both the soluble and polymeric fraction was taken and subjected to
electrophoresis. The protein band from gels were transferred onto the poly(vinylidenedifluoride)
(PVDF) membrane via the semidry system. After blocking with 5% bovine serum albumin (BSA) in
TBST (TBS containing 0.1% Tween 20) for 60 min, the membrane was immunoblotted overnight at 4◦C
with primary antibodies against β-tubulin (ab18207, Abcam, Cambridge, UK). The membranes were
then probed with (H + L) HRP-conjugated secondary antibodies (Cat# 170-6515, Bio-Rad) for 60 min at
room temperature. The intensity of the bands was analyzed using ChemiDock system and Image Lab
software v. 4.1 (Bio-Rad) and Gel-Pro Analyzer v 4.0 (Media Cybernetics, L.P., Silver Spring, MD, USA).
4.10. Immunofluorescence Microscopy
Briefly, A549 cells were grown in 6-well plates (8
before treatment with 3 or 10
×
104 cells/well) and serum-starved for 16 h
M C-1305/paclitaxel/vinblastine respectively for 24 h in a humidified
µ
cell culture chamber (37 ◦C, 5% CO2). After incubation, cells were washed twice with PBS and fixed
in 2% formaldehyde for 15 min, washed and permeabilized in 0.1% Triton X-100 for 15 min at 4 ◦C,
washed again then blocked in FBS for 30 min. Immunostaining was carried out with appropriate
primary antibodies against α-tubulin (catalog no. ab52866, Abcam) or β-tubulin (catalog no. ab18207,
Abcam), followed by staining with secondary Alexa-633 antibodies (catalog no. A-21071 Thermofisher
Scientific, Warsaw, Poland). Cells were washed as above and incubated for 15 min with mounting
medium containing DAPI (ab104139, Abcam). Images were acquired on an LSM 510 META microscope
(Carl Zeiss, GmbH, Jena, Germany) using a PLAN-APOCHROMAT 63 /1.4 OIL DIC M27 objective.
×
Image acquisition was performed using ZEN 2009 Light Edition software. Representative cells were
chosen to indicate significant differences between controls and treated cells.
4.11. Mitotic Index
Mitotic index was estimated by the MPM-2 assay. The anti-phosphorylated Ser/Thr-Pro MPM-2
antibody (Merck Millipore, Poznan´, Poland) recognizes a phosphorylated epitope in proteins that are
phosphorylated at the onset of mitosis. After treatment with C1305 (IC50 dose) or paclitaxel (500 nM)
for 6 or 24 h, the medium was discarded, and the cells were collected by trypsinization and fixed

Cancers 2020, 12, 864
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in 4% paraformaldehyde (Sigma Aldrich) for 15 min at room temperature and permeabilized with
ice-cold 70% ethanol, then kept until staining at
20 ◦C. For cell staining, the cells were treated with 3%
−
BSA in PBS with 0.1% Triton X-100, and incubated with the anti-MPM-2 antibody (1:1000) then with
the secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Thermofisher Scientific)
(1:1000). The expression of MPM-2 phospho-proteins was determined by flow cytometry using the
CellQuest Software; 10,000 events were counted for each sample (FACSCalibur, Becton Dickinson
Warsaw, Poland).
4.12. Statistical Analysis
Results were expressed as means
±
standard deviations (SD). Statistical significance among means
was determined using the Student’s t-test (two samples, paired and unpaired) after verification of
normal distribution of data by Shapiro–Wilk test or the Kruskal–Wallis One Way Analysis of Variance
on Ranks [102] and Dunn’s Method [103] (ANOVA on ranks).
4.13. Data Accessibility
Deep sequencing data were deposited in Gene Expression Omnibus (GEO) at accession number:
GSE143649 [104].
5. Conclusions
In summary, although further studies are necessary to test the details of the molecular relationship
between C-1305 and the microtubule cytoskeleton and how it can be translated into therapy,
the non-biased approach presented here resulted in the identification of a novel mechanism for
C-1305-induced apoptosis. The results demonstrate that this triazoloacridone, besides being an unusual
topoisomerase poison, binds to tubulin and stabilizes the microtubule network and simultaneously
induces cell cycle arrest. Finally, taking into account that in the pre-NGS era, numerous promising and
advanced molecules were abandoned due to problems with determining their mechanism of action as
well as potential side effects, we provide an example of a modern approach that could result in their
reinstatement in the drug development pipeline.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/4/864/s1,
Synthesis of C-1305, Figure S1: Scheme of synthesis of compound C-1305, Figure S2: Melting profiles of compound
C-1305, Figure S3: RP-HPLC chromatograms of purified compound C-1305 (neutral form), Figure S4: Mass spectra
(positive ionization mode) of compound C-1305, Figure S5: 1H NMR spectrum of compound C-1305, (CDCl3,
room temperature), Figure S6: The α-tubulin and β-tubulin heterodimer structure with colchicine, Figure S7:
Tubulin dimmer crystal structure (PDB, 1TUB) superimposed with the computed tubulin dimmer with paclitaxel
molecule, Figure S8: Dose-dependent effect of C-1305 or paclitaxel on in vitro microtubule formation. The level
of the polymerization was measured by an increase in turbidity (OD) at 340 nm, Figure S9: C-1305 stabilized
the polymeric tubulin and changed the microtubule network in A549 cells, Figure S10: The C-1305 effects on
the microtubule network were observed by immunofluorescence using a Confocal Scanning Laser Microscope
(LSM) and Transmitted Light (TL). HTC 116 cells were incubated in the absence and the presence of C-1305 (10 or
20
against
secondary antibody. The scale bar is 10
µ
M) and paclitaxel (0.5 or 3
µ
M) for 24 h. Cells were fixed and processed for immunostaining with antibodies
-tubulin (right panel) for 2 h, and then reacted with Alexa fluoro-conjugated
m. Paclitaxel was used as the reference compound, Figure S11: Cell
α
-tubulin (left panel) and
β
µ
morphology after C-1305 treatment. HCT 116 cells were treated with 10 mM C-1305 (IC50 10 mM) for the
indicated time, Figure S12: (A) Raw, not cropped supporting Western blot files of soluble and polymeric tubulin
fraction in A549 cells after C-1305 treatment. A blue box denotes the cropped area shown in Figure 7A (main
manuscript). To shown molecular weight marker (Precision Plus Protein™Kaleidoscope™ Prestained Protein
Standards, Bio-Rad). Western blot was scanned with white EPI illumination before Chemiluminescent analysis
and next merged. (B). Raw data used for relative mean band intensity calculation of different tubulin fraction after
C-1305 treatment with respect to untreated cells presented in Figure 7A.
Author Contributions: Conceived and designed the experiments: J.K., E.S., R.B. Performed the experiments: J.K.,
S.B., M.D., D.J., A.B., M.W., A.R., K.K., R.B. Analyzed the data: A.F.S., M.D., J.K., D.K.C., K.K., E.S. Contributed
reagents/materials/resources: K.K, A.F.S., E.S., M.B., M.M., R.J.O. Wrote the paper: R.B., J.F.C. All authors read the
final version of the manuscript.
Funding: This work was funded by National Center for Research and Development Program under contract
Strategmed3/306853/9/NCBR/2017 (TARGETTELO) to R.B., E.S., M.M.

Cancers 2020, 12, 864
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Acknowledgments: We would like to thank the Microbiology Department of Medical University of Gdansk for
providing a Tecan Infinite M200Pro microplate spectrophotometer.
Conflicts of Interest: The authors declare no potential conflicts of interest.
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