Synthesis and biological evaluation of N-biphenyl-nicotinic based moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin Lymphoma

Article abstract of DOI:10.1016/j.canlet.2018.12.013

We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas, they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis. PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted an antiangiogenic effect by suppressing microvascular formation.

Full text of DOI:10.1016/j.canlet.2018.12.013

Accepted Manuscript
Synthesis and biological evaluation OF N-biphenyl-nicotinic based Moiety
compounds: A new class of antimitotic agents for the treatment of Hodgkin lymphoma
L. Porcelli, D. Stolfa, A. Stefanachi, R. Di Fonte, M. Garofoli, R.M. Iacobazzi, N.
Silvestris, A. Guarini, S. Cellamare, A. Azzariti
PII:
S0304-3835(18)30728-6
https://doi.org/10.1016/j.canlet.2018.12.013
CAN 14175
DOI:
Reference:
To appear in: Cancer Letters
Received Date: 17 July 2018
Revised Date: 5 December 2018
Accepted Date: 11 December 2018
Please cite this article as: L. Porcelli, D. Stolfa, A. Stefanachi, R. Di Fonte, M. Garofoli, R.M. Iacobazzi,
N. Silvestris, A. Guarini, S. Cellamare, A. Azzariti, Synthesis and biological evaluation OF N-biphenyl-
nicotinic based Moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin
lymphoma, Cancer Letters, https://doi.org/10.1016/j.canlet.2018.12.013.
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ACCEPTED MANUSCRIPT
Abstract
We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition
in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for
experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide
normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas,
they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest
in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such
cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis
which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of
microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis.
PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as
vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the
G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation
of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted
an antiangiogenic effect by suppressing microvascular formation.

ACCEPTED MANUSCRIPT
SYNTHESIS AND BIOLOGICAL EVALUATION OF N-BIPHENYL-NICOTINIC BASED
MOIETY COMPOUNDS: A NEW CLASS OF ANTIMITOTIC AGENTS FOR THE
TREATMENT OF HODGKIN LYMPHOMA
1
1,2
2
1
1
1
3
L. Porcelli , D. Stolfa , A. Stefanachi , R. Di Fonte , M. Garofoli , R.M. Iacobazzi , N. Silvestris , A.
4
2
1*
Guarini , S. Cellamare and A. Azzariti
1
3
4
Experimental Pharmacology Laboratory, Medical Oncology Unit, Haematology Unit, IRCCS Istituto
Tumori Giovanni Paolo II, Bari, Italy
2
Dipartimento di Farmacia-Scienza del Farmaco, Università di Bari, Bari, Italy
Keywords: novel microtubule-targeting agents, N-biphenylanilides, Hodgkin Lymphoma cell lines,
capillary morphogenesis
*
Correspondence:
Experimental Pharmacology Laboratory
IRCCS Istituto Tumori Giovanni Paolo II
Viale O. Flacco, 65
7
0124 Bari, Italy
List of abbreviations: List of abbreviations: classical Hodgkin Lymphoma (cHL), Microtubules
Targeting Agents (MTAs), Human Microvascular Endothelial Cells (HMVEC), Thin Layer
Chromatography (TLC), Normal Human Dermal Fibroblasts-Adult (NHDF-Ad), Hodgkin-Reed
Stemberg (HRS).

ACCEPTED MANUSCRIPT
Abstract
We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition
in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for
experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide
normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas,
they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest
in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such
cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis
which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of
microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis.
PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as
vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the
G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation
of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted
an antiangiogenic effect by suppressing microvascular formation.
2

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1
. Introduction
The clinical and basic research in cancer treatment frequently faces high cytotoxicity to non-
tumorigenic cells and multiple cancer resistance, developed in response to highly validated
chemotherapeutics. Among antimitotic drugs, the microtubules targeting agents (MTAs) such as
taxanes and vinca alkaloids are used in several hematological malignancies and solid tumors [1]. They
are categorized in destabilizers that bind to tubulin and avoid microtubule polymerization such as vinca
alkaloids and in stabilizers which in contrast are agents that bind to tubulin and stabilize microtubule
polymer, such as taxanes. Both classes of drugs, by suppressing the spindle-microtubule dynamics,
block mitosis at the metaphase–anaphase transition and induce apoptotic cell death through the
activation of the CDK1/cyclin B complex and the rapid phosphorylation/inactivation of the anti-
apoptotic protein Bcl-2, which is regarded as a hallmark of MTAs functionality because of its role as
“guardian of microtubule integrity” [2]. However, despite their clinical success, MTAs are highly toxic
to normal cells, therefore there is an urgent need for the discovery of new, more effective and less toxic
molecules, which once validated become novel therapeutics, principally for refractory or relapsed
patients.. Some years ago, some of us started a medium screening program of in-house chemical
libraries for identification of new antiproliferative substances, selected from a set of newly synthesized
P-gp modulators [3]. Most of the substances showed intrinsic cytotoxicity towards MCF7-ADR cell
line resistant to doxorubicin [4]. This evidence encouraged us to expand the series including non-
trimethoxy anilides members such as some nicotinamide derivatives. In 2017 we published a new
series of nitrobiphenyl nicotinamides that displayed a noteworthy antiproliferative activity against
MCF-7 and MDA-MB-231 breast tumor cell lines [5] and interestingly some of them induced a dose-
dependent accumulation of G2/M-phase cell population in MCF7 like vinblastine. This prompted us to
investigate if they blocked mitosis by suppressing the spindle-microtubule dynamics. Among such
compounds we choose PTA34, PTA73 and RS35 and here, in order to report the identification and
pharmacologic characterization of such N-biphenylanilides as a novel, highly potent and selective class
of MTAs we evaluated their effect on tubulin polymerization and their antitumor activity in a panel of
tumor cells and normal cells. The focus was the molecular and morphological features of mitotic arrest
and apoptosis induction in Hodgkin Lymphoma cell lines. In addition, because some MTAs have
recently received considerable interest as potential anti-angiogenic and vascular-disrupting agents, we
3

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tested the potential antiangiogenic effect, by assessing the ability to disrupt capillary morphogenesis of
human microvascular endothelial cells (HMVEC).
2
. MATERIALS & METHODS
2
.1 Experimental section
High analytical grade chemicals and solvents were purchased from commercial suppliers. When
necessary, solvents were dried by standard techniques and distilled. After extraction from aqueous
layers, the organic solvents were dried over anhydrous sodium sulfate. Thin layer chromatography
(
TLC) was performed on aluminum sheets precoated with silica gel 60 F254 (0.2 mm) (E. Merck).
Chromatographic spots were visualized by UV light. Purification of crude compounds was carried out
by flash column chromatography on silica gel 60 (Kieselgel 0.040−0.063 mm, E. Merck) or by
1
preparative TLC on silica gel 60 F254 plates or crystallization. H NMR spectra were recorded in
6 3
DMSO-d or CDCl at 300 MHz on a Varian Mercury 300 instrument. Chemical shifts (δ scale) are
reported in parts per million (ppm) relative to the central peak of the solvent. Coupling constant (J
values) are given in hertz (Hz). Spin multiplicities are given as s (singlet), br s (broad singlet), d
(
doublet), t (triplet), dd (double doublet), dt (double triplet), or m (multiplet). LRMS (ESI) was
performed with an electrospray interface ion trap mass spectrometer (1100 series LC/MSD trap system
Agilent, Palo Alto, CA). In all cases, spectroscopic data are in agreement with assigned structures.
Combustion analyses were performed by Eurovector Euro EA 3000 analyzer (Milan, Italy) and gave
satisfactory results (C, H, N within 0.4% of calculated values). The synthesis of compound PTA34 was
already described [5].
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i) Procedure for the Suzuki-Miyaura cross-coupling reaction: preparation of compounds 3-nitro-
[
1,1'-biphenyl]-4-amine a (Scheme 1).
A suspension of the 4-bromo-2-nitroaniline (1, 04g, 5.0 mmol), benzene boronic acid (0,92g, 7.5
mmol), and Pd(PPh3)4 (0.58 g, 0.50 mmol) in a 2 M aqueous solution of K2CO3 (7.5 mL) and 1,4-
dioxane (30 mL) was heated at 100 °C for 3 h under stirring. After cooling, the solvent was evaporated
under vacuum to dryness and the obtained residue was treated with dichloromethane. The suspension
was filtered on celite and the resulting residue was purified by silica gel column chromatography.
Yield: 50%. Eluent: Hexane 8/ AcOEt 2
1
HNMR (CDCl ) δ:8.38 (1H, d, J=2.1 Hz), 7.65 (1H, dd, J=2.1 Hz, J=8.7 Hz), 7.58-7.54 (2H, m), 7.44
3
(
2H,), 7.38-7.30 (1H, m), 6.90 (1H, d, J=8.7 Hz), 6.11 (2H, br s). LRMS (ESI) m/z 213.1 [M-H]-.
Synthesis of N-(3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (PTA 73).
The intermediate aniline a (0, 21g, 1 mmol) was dissolved in 10 ml of dry dioxane and then nicotinoyl
chloride hydrochloride (0,53g, 3 mmol), DMAP (0, 12g, 1 mmol) and triethylamine (2 mmol, 0.278
mL) were added. The mixture was refluxed for 18 hours. After cooling the obtained precipitate was
removed by filtration. The filtrate was evaporated and the residue was purified by silica gel column
chromatography.
Yield: 32 %. m.p.: 175-177 °C. Eluent: Hexane 7/ AcOEt 3
1
HNMR (CDCl ) δ: 11.40 (1H, s), 9.28 (1H, d, J=2.1Hz), 9.05 (1H, d, J=8.8 Hz), 8.85 (1H, dd, J=1.6
3
Hz, J=4.6 Hz), 8.53 (1H, d, J=2.1Hz), 8.30 (1H, dt, J=1.6 Hz, J=6.6 Hz), 7.98 (1H, dd, J=2.1 Hz, J=8.8
-.
Hz), 7.66-7.60 (2H, m), 7.54-7.47 (3H, m), 7.47-7.39 (1H, m). LRMS (ESI) m/z 318.0 [M-H]
Synthesis of N-(3-nitro-[1,1'-biphenyl]-4-yl)pyrimidine-5-carboxamide (RS 35).
The starting pirimydin carboxilic acid (1.5 mmol) was suspended under argon in thionyl chloride (2.0
mL) and stirred for 3h at room temperature. The unreacted excess of thionyl chloride was removed
under nitrogen flow to afford the corresponding benzoyl chloride. The solid thus obtained was used
without further purification in the condition described above to react with aniline. Yield: 10%. m.p.
2
18-220 °C: . Eluent: Hexane 8/ AcOEt 2
5

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1
HNMR (CDCl3) δ: 11.44 (1H, br s), 9.45 (1H, s), 9.35 (2H, s), 9.02 (1H, d, J=8.8 Hz), 8.54 (1H, d,
J=2.1 Hz), 8.0 (1H, dd, J=2.1 Hz, J=8.8 Hz), 7.65-7.62 (2H, m), 7.54-7.43 (3H, m).
-
LRMS (ESI) m/z 319.0 [M-H]
2
.2 In vitro tubulin polymerization assay
PTA34, PTA73 and RS35 were tested at 3 µm concentration in a tubulin polymerization assay, by
using the kit (Cytoskeleton Inc., Denver, CO, USA) Tubulin polymerization assay, Cytoskeleton, Inc.:
(
http://www.cytoskeleton.com/bk011p). Lyophilized tubulin (Cytoskeleton) was used to evaluate the
tubulin polymerization inhibiting activity. Vinblastine and paclitaxel at 3.0 µm were used as positive
controls for tubulin polymerization inhibition and stabilization, respectively. All compounds were
dissolved in DMSO and further diluted with sterile water to obtain a maximum DMSO concentration of
0
.1 %, which was used as solvent control. Polymerization was monitored by fluorescence enhancement
due to the incorporation of a fluorescent reporter into microtubules as polymerization occurred. In a 96
well plate, to a 0.5 mL of a 100V stock solution of each compound, 49.5 mL of supplemented tubulin
supernatant were added. Incubation was done in a temperature-controlled Multi-label Microplate
Fluorimeter, equipped with filters for excitation at 340–360 nm and emission at 420–460 nm (Victor 3
Model 1420-01296, PerkinElmer, Inc. MA, USA) at 378C, and fluorescence was measured at 460 nm
every minute for 60 min according to the recommended procedures.
2
.3 Drugs and chemicals
PTA34, PTA73 and RS35 were dissolved in DMSO. Further dilutions were made in medium
supplemented with 10% foetal bovine serum, 2 mM glutamine, 50,000 UL-1 penicillin and 80 µM
streptomycin.
2
.4 Cell lines
The classical Hodgkin lymphoma cell lines KM-H2, HDLM-2, L540, L428 and L1236 [6] were
generously provided by Prof. Rosaria De Filippi (University of Naples Federico II, Italy) and
authenticated through STR profile at the beginning of experiments. They were routinely cultured in
IMDM (Iscove's modified Dulbecco's MEM) supplemented with 10% fetal bovine serum, 100 U/ml
penicillin, 100 µg/ml streptomycin. The panel of solid tumor cells encompasses breast cancer cell lines
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MCF7, HCC1937 and MDA-MB-468; colon cancer cell lines Colo205, LoVo and KM-12; pancreatic
cancer cell lines Panc-1, AsPC-1 and MIA PaCa-2 and ovarian cancer cell lines SK-OV-3 and Caov-3.
All cells were purchased from ATCC. Breast cancer cells MCF7 were cultured in Eagle's Minimum
Essential Medium (EMEM) supplemented with 0.01 mg/ml human recombinant insulin and with 10%
fetal bovine serum; MDA-MB468 cells were cultured in Leibovitz’s L15 medium supplemented with
1
0% fetal bovine serum and HCC1937 were cultured in RPMI-1640 medium supplemented with fetal
bovine serum to a final concentration of 10%. Colon cancer cells Colo205 were cultured in RPMI-1640
medium with 10% fetal bovine serum; LoVo were cultured with F-12K with 10% fetal bovine serum
and Km 12 were cultured in RPMI-1640 medium supplemented with fetal bovine serum to a final
concentration of 10%Pancreatic cancer cells Panc-1 were culture in Dulbecco's Modified Eagle's
medium supplemented with fetal bovine serum to a final concentration of 10%; AsPC-1 were cultured
in RPMI-1640 medium with fetal bovine serum to a final concentration of 10% and MIAPaCa-2 were
cultured in Dulbecco's Modified Eagle's Medium with the addition of fetal bovine serum to a final
concentration of 10% and horse serum to a final concentration of 2.5%. Ovarian cancer cells Caov-3
were cultured with Dulbecco's Modified Eagle's Medium, supplemented with fetal bovine serum to a
final concentration of 10% and SK-OV-3 with McCoy's 5a medium with fetal bovine serum to a final
concentration of 10%. Human Dermal Microvascular Endothelial Cells (HMVEC) were purchased
from Lonza and cultured in EGM-2 Endothelial Growth Medium, EGM™-2MV BulletKit™ from
Lonza. NHDF-Ad Human Dermal Fibroblasts, Adult were purchased from Lonza and cultured in
FGM2 (Fibroblast Growth Medium-2 BulletKit™ from Lonza). cHL cell lines, solid tumor cell lines
HMVEC and NHDF-Ad were cultured in a humidified incubator at 37°C with an atmosphere
containing 5% CO2. MDA-MB-468 were cultured in a free gas exchange incubator at 37°C with
atmospheric air.
2
.5 Cell proliferation assay
Determination of cell growth inhibition was performed using the Cell Counting Kit-8 (Sigma-Aldrich),
after exposing cells to increasing concentrations of compounds for 24 and 72h in HRS cells and the
MTT assay for adherent cells. The IC50 was defined as the drug concentration yielding a fraction of
affected (no surviving) cells = 0.5, compared with untreated controls and was calculated utilizing
CalcuSyn ver.1.1.4 software (Biosoft, UK).
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2
.6 Cell cycle analysis
After two wash steps in ice-cold PBS (pH 7.4), cells were fixed in 4.5 ml of 70% ethanol and stored at -
2
5
0°C. For the analysis, the pellet was resuspended in PBS containing 1 mg/ml RNase, 0.01% NP40 and
0 µg/ml propidium iodide (PI) (Sigma). After an incubation time of 1 hour in ice, cell cycle
determinations were performed using a FACScan flow cytometer (Becton Dickinson), and data were
interpreted using the CellQuest software, provided by the manufacturer.
2
.7 Immunofluorescence on cells in suspension
Cell suspension was transferred to microcentrifuge for spinning. After two wash step in PBS 1X, cells
were spiked in 3.7% PFA and incubated at room temperature for 15 min. After two wash steps in PBS
1
X, cells were permeabilized with 0.3% Triton X-100 in PBS 1X for 5 minutes. Non-specific binding
sites were blocked for 30 min at room temperature with PBS containing 5% BSA and then cells were
incubated with a mouse anti-α-tubulin monoclonal antibody (Sigma-aldrich) diluted in PBS containing
4
% BSA for 60 min at room temperature. The staining of α-tubulin was followed by incubation with
FITC-conjugated anti-mouse antibody. After two wash steps in PBS 1X, the remaining liquid was
carefully aspirated and the cells pellet was resuspended in Vectashield with Dapi (Vector Laboratories),
dropped on slides and covered with coverslip for microscope examination (Leica).
2
.8 Cell apoptosis assays
Apoptosis detection was investigated by the Cell Death ELISAPLUS kit (Roche Molecular
Biochemicals, Milan, Italy) and the AnnexinV/PI assay (Becton Dickinson) followed by flow
cytometry (FACScan). The first is based on the detection of mono- and oligonucleosomes in the
cytoplasmic fraction of cell lysates by biotinylated anti histone-coupled antibodies, and their
enrichment in the cytoplasm is calculated as the absorbance of sample cells/absorbance of control cells.
The enrichment factor was used as a parameter of apoptosis and shown on the Y-axis as mean ± SE.
Experiments were performed according to manufacturer’s instructions. The second is a double-staining
method based on the analysis of phosphatidylserine exposure on the cell membrane by an Annexin-V
FITC/propidium iodide staining and allows detection of early apoptosis as Annexin V positive cells and
late apoptosis as Annexin V/PI positive cells.
2
.9 Western blot analysis
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Protein extracts were obtained by homogenization in RIPA buffer (0.5 M NaCl, 1% Triton X100, 0.5%
NP40, 1% deoxycolic acid, 3.5 mM SDS, 8.3 mM Tris HCl pH 7.4, 1.6 mM Tris base) added with
protease inhibitor cocktail (Sigma-Aldrich). Total proteins were measured and analyzed as described in
[
7]. Briefly 50 µg of total lysates were electrophoretically separated on 10% acrylamide gel (SDS–
PAGE by Laemli). Signal was detected by chemoluminescence assay (ECL-Plus, Amersham Life
Science, UK). All monoclonal antibodies utilized were provided by Cell Signalling-USA and Sigma-
Aldrich, St. Louis, MO-USA. A mouse-HRP and a rabbit-HRP (Amersham Pharmacia Biotech, Upsala
Sweden) were used as secondary antibody. β-actin expression was utilized as loading control.
2
.10 Detection of mitochondrial transmembrane potential using flow cytometry
Changes in mitochondrial membrane potential were detected by using 5,5′,6,6′-tetrachloro-1,1′,3,3′
tetraethyl benzimidazolylcarbocyanine iodide/chloride (JC-1), a cationic dye that exhibits potential-
dependent accumulation in mitochondria. Mitochondrial membrane depolarization was indicated by a
reduction of red fluorescence emission (JC-1 dimers monitored in FL2) and by an increase of green
fluorescence emission (JC-1 monomers monitored in FL1) with flow cytometry. Untreated and treated
cells were collected, washed with phosphate buffered saline (PBS), stained and analyzed through a
flow cytometer.
2
.11 In vitro capillary morphogenesis assay
Matrigel (0.5 ml) was pipetted into 96 well plate for tissue culture and left to polymerize as described
in [8]. Subsequently HMVEC were plated in complete MCDB medium, supplemented with 30% FCS,
and 20 mg/ml ECGS. Capillary morphogenesis was also performed in the presence of 1 µM PTA34.
The effects on the growth and morphogenesis of endothelial cells were recorded after 3 and 6 h with an
inverted microscope (Leica DMi8) equipped with CCD optics and a digital analysis system.
2
.12 Statistical analysis
All in vitro experiments were performed in triplicate, and results have been expressed as the mean ±
standard deviation (SD), unless otherwise indicated. Statistical differences of in vitro data were
assessed by the Student-Newman–Keuls test, while the Two tailed method was utilized for data
analysis of Tubulin polymerization assay. P-values lower than 0.05 were considered significant.
9

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Statistical analyses were performed using the GraphPad Prism software package version 5.0 (GraphPad
Software Inc., San Diego, CA, USA).
3
. RESULTS
3
.1 Cytotoxicity
The anti-proliferative activity of these novel N-biphenylanilides was tested on three breast cancer cell
lines, three colon cancer cell lines, three pancreatic cancer cell lines, two ovarian cancer cell lines, five
bona fide classical Hodgkin lymphoma cell lines and two bona fide normal cell lines. The cytotoxicity
of PTA34, PTA73 and RS35 was evaluated after one and three days of continuous exposure by Cell
Counting Kit-8 (CCK-8) proliferation assay in cHL cell lines and by MTT assay in solid tumors and
normal cells. All three compounds displayed strong antitumor efficacy in all tumor cell lines, whereas
PTA34 and PTA73 did not affect the proliferation of HMVEC and NHDF, thus showing to be safe to
the tested normal cells. RS35 slightly reduced cell proliferation (10% of untreated cells) of both normal
cell types (data not showed). The 24 hours exposure reduced the growth of tumor cells of about 50% at
1
µM in almost all cHL lines (data not shown); hence that concentration was utilized in the following
one day time experiments in cHL cell lines; while it was higher in solid tumors (data not shown). Three
days continuous exposure lowered the IC s in the nanomolar range in almost all cells, however
5
0
collectively the antitumor activity of N-biphenylanilides was stronger in cHL cell lines then in solid
tumors cells. The IC s, calculated after three days for all tumor cell lines, are reported in the Table I.
5
0
3
.2 Tested compounds induced G2/M cell cycle arrest
To verify whether the inhibition of cell proliferation was through the affection of cell cycle
progression, we detected the DNA content of cell nuclei by flow cytometry after staining with
propidium iodide. For this purpose we treated the cells with each compound at three concentrations
(
0.01 µM, 0.1 µM and 1µM) for 18h, 24h and 72h. All compounds induced a G2/M cell-cycle arrest
starting from 18h of exposure in all HL cell lines that further increased after 24h and interestingly the
extent of the arrest at each concentration of drug was stable until 72h, showing that no mitotic slippage
of cells occurred during the time of the arrest (data not showed). PTA34 induced the strongest G2/M
arrest from 50% to 70% of cells at 0.1 µM and 1 µM PTA34, respectively, in almost all cell lines
compared to the 50% and 60% induced by 1 µM PTA73 and RS35, respectively. PTA73 induced the
1
0

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highest sub-G0/G1 accumulation, namely 20% versus 8% and 11% induced by PTA34 and RS35,
respectively. A representative analysis of cell cycle of L1236 is reported in Fig. 1A, whereas the cell
cycle phases quantification from three different experiments performed in L1236 is reported in Fig. 1B.
Interestingly the strongest arrest at G2/M phase was found only in HRS cells and not in the other tumor
cells. In Fig.1C a representative analysis of cell cycle profile in some solid tumor cell lines, chosen
among the most sensitive to compounds, is reported. It shows that following treatment they did not go
into mitotic arrest comparable to that of HRS cells.. Therefore we decided to investigate the N-
biphenylanilides as MTAs only in HL cell lines.
3
.3 Tested compounds induce morphological features of prometaphase arrest in HRS cells
The morphological features of the nuclei of HRS cell lines were analyzed by fluorescent microscopy
after 48h of treatment with PTA34, PTA73, RS35 at 1µM after staining with DAPI (Fig. 2). The nuclei
of treated cells displayed distinct signs of arrest in the prometaphase stage of mitosis; namely the
nuclear membrane disappeared and the chromatin appeared condensed, spread and no chromosomal
alignment was detectable, as it happens during metaphase. These morphological features of nuclei
suggest that all compounds are mitotic inhibitors of HRS cells.
3
.4 Tested compounds cause the disruption of cellular microtubule network in HR cells
Because tested compounds markedly blocked the cell cycle in the M phase, we tested whether they
could directly affect the organization of the microtubule network. The microtubule network was
visualized by indirect immunofluorescence after staining with α-tubulin, whereas the nuclei were
revealed with DAPI staining. In control cells the microtubule network exhibited the normal
arrangement with microtubules traversing intricately throughout the cell while the nuclei were compact
and round shaped (Fig. 3). In the N-biphenylanilides treated cells (1µM PTA34, PTA73 and RS35) the
microtubules were destroyed, while the DAPI staining evidenced that the chromatin appeared
condensed; however it was spread and no sign of chromosomal alignment was found, and the most of
the cells showed pyknotic and fragmented nuclei indicating apoptotic cells (Fig.3).
3
.5 In vitro tubulin polymerization assay
In order to evaluate whether such compounds interfere with microtubules dynamics, we performed a
cell-free tubulin polymerization assay. The progression of tubulin polymerization was examined by
1
1

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monitoring the increase in fluorescence emission at 420 nm (excitation wavelength is 360 nm) for 1 h
at 37 °C. Vinblastine and paclitaxel, two known microtubule-targeting agents acting as disassembly
and assembly promoters, respectively, were used as controls [9]. The effects on tubulin polymerization
of the three substances are shown in Figure 4. As expected, paclitaxel was a stabilizer of tubulin
polymerization at 3 µM concentration, whereas vinblastine at the same concentration acted as an
effective inhibitor of tubulin polymerization. 3 µM PTA34 showed to perform an effective inhibition of
tubulin polymerization, since it achieved the same endpoint inhibition compared to vinblastine
(
p<0.0001 PTA34 vs control). RS35 was less effective than PTA34 in inhibiting tubulin polymerization
whereas PTA73 did not show any appreciable effect on the progression of tubulin polymerization
profile similar to control).
(
3
.6 Tested compounds induced apoptosis by causing mitochondrial δψ loss
To assess whether the mitotic arrest, was followed by induction of apoptosis we utilized the FITC
Annexin V staining to identify apoptosis at an earlier stage (24h) and a cell death Elisa assay after 72 h
of treatment to measure DNA fragmentation and histone release from the nucleus, occurring during late
stage of apoptosis. To carry on both apoptosis assays we treated cells with PTA34, PTA73 and RS35 at
0
.1µM and 1µM. We found that whilst all compounds induced in L540 a time and concentration
dependent (not showed) induction of early apoptosis starting from 24 h and of a significant late
apoptosis starting from 72 h of treatment; in other cells such as KMH2 and L1236 cells, the onset of
late apoptosis occurred already after 24h of treatment and increased with the concentration and time
exposure. A representative analysis of Annexin V/PI apoptosis performed by FACS in L540 cells is
reported In Fig.5A, whereas the fold change of Annexin V (early apoptosis) and AnnexinV/PI (late
apoptosis) positive cells in treated samples versus untreated ones is reported in Fig. 5B. The results are
representative of three distinct experiments performed in such cells. Figure 5C reports a time and
concentration dependent evaluation of late apoptosis induced by compounds showing that late
apoptosis was already induced at 0.1 µM by all compound in L540.
In order to assess the involvement of mitochondria in the apoptosis, we evaluated the change of
mitochondrial membrane potential (depolarization) after treatment with tested compounds.
Mitochondrial membrane depolarization was assayed by exposing the cells to JC-1 and visualized as a
reduction in the fluorescence signal in the FL2 channel (JC-1 dimers) and increase in FL1 channel (JC-
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1
monomers). It was assessed after 24 hours of treatment with increasing concentration of compounds
up to 1 µM in almost all HRS cells. Figure 6A reports a representative analysis of JC-1 performed by
FACS, whereas the quantification of membrane depolarization (JC-1 monomers increase) from
experiments performed in L1236, HDLM2 and L540 cells at 1 µM PTA34, PTA73 and RS35 is
reported in Fig. 6B. The results are representative of three distinct experiments performed in such cells.
3
.7 PTA34 affects Cyclin B1 expression and CDK1 activity
The Cdc2 (CDK1)/Cyclin B1 regulatory complex drives progression through G2 and the entrance into
and exit out of the M-phase of cell cycle; thus we determined the effects of compounds on CDK1. To
this purpose we evaluated the post-translational modifications of CDK1 crucial for its activation and
the expression level of Cyclin B1, which is the allosteric activator of CDK1, by evaluating the time
course of Cyclin B1 accumulation and destruction and CDK1 phosphorylation at Tyr-15 and Tyr-161
on L540 cell lysates on time 0 and after treatment with PTA34 (1µM), at 3h, 6h, 18h and 24h. Figure 7
reports the immunoblotting of CDK1, demonstrating that while the total CDK1 levels remained
relatively constant along the time course of CDK1 expression evaluation, the levels of CDK1
phosphorylated at Tyr-15 declined from 18h of treatment with PTA34 until 48h, while the p-
CDK1(Y161) was almost stable during the time course of treatment with PTA34, basically indicating
that CDK1 was activated during the mitotic arrest elicited by PTA34. Accordingly the level of Cyclin
B1 was up regulated starting from 6h of treatment until 24h, consistent with a sustained activation of
CDK1 along the G2/M blockage of cell cycle. The immunoblots representative of experiments
performed in L540 cells are reported in Fig.7.
3
.8 Apoptogenic effect of PTA34 resulted in S70 phosphorylation of Bcl-2, Mcl-1 and Bcl-2
downregulation and activating phosphorylation of pro-apoptotic protein BIM
Because of the pivotal role of Bcl-2 family of proteins in apoptosis, we evaluated the effects of PTA34
on the expression and activation of BH3-only pro-apoptotic protein BIM and on the expression of Bcl-
2
and Mcl-1, both playing a key role in the sensitivity to anti-tubulin chemotherapeutics. Furthermore
we evaluated the S70 phosphorylation of Bcl-2 which is induced by drugs affecting the integrity of
microtubules and is regarded as a post-translational modification which inactivates Bcl-2 anti-apoptotic
function [2,10,11]. As shown in Fig. 7, the time course of proteins expression performed on L540 cells
treated with PTA34 1µM, evidenced that Bcl-2 was reduced at 18h and 24h, while Mcl-1 started to be
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down-regulated soon after 3 hours of treatment and remained reduced until 24h. In agreement with the
potent induction of apoptosis and with the reduction of Mcl-1, we found a time dependent increase of
BIMEL expression starting from 3 hours until 48 h of treatment, while BIML and BIMS, which are
regarded as the two most apoptotic BIM isoforms [12], started to increase after 18h and remained
strongly expressed until 48h according with the occurrence of damage to microtubules and apoptosis
induction in such cells.
3
.9 PTA34 exerts anti-angiogenic effects in vitro
The matrigel in vitro assay was used to evaluate the anti-angiogenic properties of PTA34 on HMVEC
cells morfology. The cells were seeded on matrigel in presence of 1µM PTA34, then the vascular
structures formation was evaluated. Interestingly 1 µM of compound, which resulted in no anti-
proliferative effect on HMVEC cells after 24h, exerted a time dependent suppression of microvacular
formation starting from 4h (data not showed) compared to control and resulted in a complete inhibition
after 6h (Fig.8).
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4.
DISCUSSION
Targeting of microtubule assembly/disassembly with microtubule inhibitors represents an important
therapeutic strategy in both hematopoietic and solid tumors; though the effectiveness of such agents is
frequently limited from the onset of resistance and toxicity to normal cells [13]. The nitro-
biphenylanilides PTA34, PTA73 and RS35, that in the present work were tested against a panel of cell
lines from different solid tumor of origin and Hodgkin lymphoma cell lines, strongly inhibited cell
proliferation of almost all cells in the nanomolar range but they selectively caused cell-cycle arrest at
G2/M transition of HL cell lines, like the reference compounds vinblastine and paclitaxel. Therefore we
started with investigating whether the tested compounds selectively targeted the microtubule system
and the underlying mechanisms of antitumor effectiveness, basically in Hodgkin- Reed-Stemberg cells.
The results reported herein suggest that all compounds induced a promethaphase arrest of HRS cells,
however only PTA34 stops the tubulin polymerization comparable to vinblastine, and notwithstanding
that, while both PTA73 and RS35, did not affect tubulin polymerization, they resulted in defective
mitosis with similar features to PTA34 treated cells. Indeed the immunofluorescence showed that the
nuclear envelop was disassembled, the chromatin appeared condensed while no chromosomal
alignment was detectable and the microtubules appeared fragmented in compounds treated cells. Thus,
perhaps PTA73 and RS35 may have targeted mitotic machinery other than tubulin, as the key players
in the kinethocore-mitotic spindle interaction and this has caused a severe misalignment of the
chromosomes that affected microtubules spindle dynamics and prevented moving the poles and exiting
mitosis. According to the molecular effects elicited by microtubule-targeting drugs, such as paclitaxel
and vinblastine, PTA34 was found to induce many of the hallmarks associated with the activity of
antimicrotubule agents [14–16]. During mitotis the Cyclin B-Cdc2 kinase is activated to induce entry
into M-phase and when all chromosome’s kinetochores are attached to microtubules, the anaphase-
promoting complex/cyclosome (APC/C) inactivates Cyclin B1-CDK1 by promoting its degradation to
reform the nuclear envelope and for exiting the M-phase [17–19]. Accordingly, in PTA34-treated cells
the Cyclin B1-CDK1 is kept in an active state during the G2/M arrest and this evidently prevents APC
activation and the onset of anaphase. Characteristically during the G2/M phase arrest the microtubules
inhibitors induce apoptosis through the inactivation of prominent members of Bcl-2 family of proteins
which promote cell survival by blocking/sequestering cell death mediators such as BAX, BAK and
BIM [10]. BIM is a potent inducer of apoptosis. In the inactive form, BIM is bound to microtubules
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through the dynein light chain, while in the presence of microtubule damage, just like after treatment
with MTAs, it is released from microtubules to accumulate in mitochondria to trigger apoptosis [20].
PTA34 was found to reduce the expression of the anti-apoptotic proteins Bcl-2 and Mcl-1, both
notoriously involved in the sensitivity to MTAs, while it induced a strong increase of the most pro-
apoptotic isoforms of BIM, namely BIML and BIMS and consequent mitochondria membrane
depolarization Even more interesting is the sudden and lengthy period of phosphorylation of Bcl-2
.
induced by PTA34, followed by Bcl-2 dephosphorylation closely correlated with initiation of
apoptosis. The latter event is regarded as a switch for induction of apoptosis by vinblastine [21].
Therefore after damaging the microtubules of HRS cells, PTA34 determined the transition from the
mitotic block to the induction of apoptosis by eliciting
a
precise cycle of
phosphorylation/dephosphorylation of Bcl-2, the degradation of Mcl-1 and Bcl-2 and the activation of
BIM that connected microtubule damage to the induction of apoptosis mediated by mitochondria.
Speculating on the activity of mitosis-selective inhibitors, we suggest that N-biphenylanilides caused a
prolonged arrest in mitosis, culminating in mitotic cell death (MCD) in HRS cells and not in solid
tumor cells that we tested, perhaps because HRS cells notoriously harbor defective cell cycle regulation
[
22] and because of the pivotal role of anti-apoptotic proteins such as Bcl-2 in driving the survival of
such cells [23,24]. Indeed we found that N-biphenylanilides caused a strong and early inactivation of
Bcl-2 in HRS, instead this did not happen in solid tumors that we tested (unpublished data). Therefore
we suggest that the inactivation of Bcl-2 was the critical event that triggered the MCD in HRS, instead
different mechanistic pathways of cell death were induced by compounds in solid tumor cell lines.
Because tumor angiogenesis greatly influences tumorigenesis either of solid tumors and hematological
malignancies and that drugs belonging to the class of MTAs exert several antiangiogenic effects
[
25,26], we started the investigation of the antiangiogenic properties of N-biphenylanilides with
PTA34, in order to provide a broader evaluation of biological effect of such compound. We found that,
despite it did not inhibit the proliferation of either HMVEC and NHDF, thus suggesting a safety margin
for this compound in vivo, it was very effective in inhibiting the capillary morphogenesis of human
microvascular endothelial cells, perhaps because of its capability to inactivate Bcl-2, notoriously
connected to proangiogenic functions [27–30].
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5
. ACKNOLEDGEMENTS
The authors gratefully thank Prof. Rosaria De Filippi (University of Naples Federico II, , Italy) who
generously provided the Hodgkin lymphoma cell lines and Dr. Pinto Rosamaria and Dr. Petriella
Daniela for the technical support in the preparation of DNA extracts from HRS cells.
6
. FUNDINGS
The study was supported by the fund of Ricerca Corrente 2016 (Ministry of Health) of IRCCS Istituto
Tumori Giovanni Paolo II, Bari-Italy.
Conflict of interest
None
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Figures Legends
Figure 1
Effects on cell cycle of L1236 by 0.01-0.1-1µM PTA34, PTA73 and RS35. The modulation of cell
cycle phases was evaluated by FACS after staining cells with propidium iodide. In panel A, a
representative analysis of three independent experiments is reported. In panel B the bar graph shows
cell population % in each phase of cell cycle, demonstrating that all compounds induced a
concentration dependent increase of G2/M phase cell population. In panel C a representative analysis
of cell cycle profile shows that, following treatment with 1µM of all compounds, MDA-MB468, Caov-
3
and MIA PaCa-2 did not go into mitotic arrest comparable to that of HRS cells.
Figure 2
Immunofluorescent detection of nuclei in L540 cells after 1µM PTA34, PTA73 and RS35. Nuclei
were stained with dapi and the specimens were examined using a Leica (DMi8) immunofluorescence
microscope. Reported are images recorded with 100X magnification. The staining evidenced changes
of nuclei morphology indicating inhibition of promethaphase-methapahase transition in treated cells
compared to untreated ones.
Figure 3
Immunofluorescent detection of microbubules and nuclei in L540 cells after 1µM PTA34, PTA73
and RS35. Microtubules were revealed using the primary anti-α-tubulin followed by a FITC-
conjugated second antibody. Counterstain of nuclei was with dapi. The specimens were examined
using a Leica (DMi8) immunofluorescence microscope. Reported are images recorded with 40X
magnification, demonstrating the disruption of microtubules and the formation of pyknotic and
fragmented nuclei.
Figure 4
Effect of PTA34, PTA73 and RS35 on tubulin polymerization. Tubulin polymerization was
monitored by the increase of fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at
3
7°C. All compounds were tested at 3 µM. Paclitaxel and vinblastine were used as positive controls for
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tubulin polymerization inhibition and stabilization, respectively. The analysis evidenced that PTA34
inhibited tubulin polymerization like vinblastine.
Figure 5
Induction of apoptosis by PTA34, PTA73 and RS 35. Apoptosis was evaluated, by using Annexin
V/PI staining followed by FACS analysis and cell Death Elisa Kit. In Fig.5A a representative analysis
of Annexin V/PI apoptosis performed by FACS is reported, whereas in Fig. 5B the fold change of
Annexin V (early apoptosis) and AnnexinV/PI (late apoptosis) positive cells in treated samples versus
untreated ones is reported. In Fig. 5C a time and concentration dependent evaluation of late apoptosis
induced by compounds is reported showing that late apoptosis was already induced at 0.1 µM by all
compound in L540. The results are the mean ±SD of three independent experiments ( *p<0.05).
Figure 6
Effects of compounds on mitochondrial membrane depolarization by JC-1 analysis. Membrane
depolarization was visualized as a reduction in the fluorescence signal in the FL2 channel (JC-1
dimers) and as an increase in FL1 channel (JC-1 monomers). In Fig. 6A is reported a representative
analysis of JC-1 performed by FACS in HDML-2, L540 and L1236. In Fig. 6B the bar graph shows the
percentage of JC-1 monomer-positive cells. The results are the mean ± standard deviation of three
independent experiments. *P<0.05 versus the control.
Figure 7
Time-course of PTA34 effect on protein levels related to mitotic arrest and apoptosis in L540
cells. Protein expression was determined by Western blot analysis using a primary antibody followed
by a second HRP-conjugated antibody. For loading differences evaluation, the blots were stripped and
reacted with an antibody against β-actin.
Figure 8
Capillary morphogenesis after treatment with PTA34. Microvascular formation after 6h from
seeding on matrigel is reported in HMVEC untreated and 1 µM PTA34 treated specimens. Pictures
shows how PTA34 completely disrupted the formation of microvascular formation.
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Table I: Inhibitory effect of N-biphenylanilides against human cancer cells
IC50 (µM±SD)
Cell Line
PTA34
PTA73
RS35
-
3
-3
-3
L540
L1236
59x10 ±0.002
548.6x10 ±0.004
5.0x10 ±0.0004
-3
-3
-3
1.8x10 ±0.0004
0.3x10 ±0.00002
3.8x10 ±0.005
-3
-3
-3
L428
30.4x10 ±0.002
2.3x10 ±0.0004
1.0x10 ±0.0003
-
3
-3
-3
HDLM-2
KM-H2
268.4x10 ±0.004
13.4x10 ±0.0006
1.1x10 ±0.0005
-
3
-3
-3
63.9x10 ±0.002
1.15±0.5
0.63±0.2
0.88±0.5
1.5±0.9
0.2x10 ±0.00006
0.2x10 ±0.0001
Colo205
LoVo
0.24±0.12
0.1±0.02
0.74±0.6
2.611±0.4
0.26±0.05
3.6±0.5
0.1±0.04
0.17±0.02
0.19±0.05
5.34±1.1
5.47±0.8
6.3±0.6
Km 12
MCF-7
MDA-MB-468
HCC1937
Panc-1
0.88±0.05
6±0.1
1.9±0.2
0.9±0.3
1.02±0.05
1.5±0.1
AsPC-1
MIA PaCa-2
SK-OV-3
Caov-3
1.8±0.3
1.6±0.2
0.3±0.1
0.006±0.001
2.6±0.2
1.7±0.3
21.1±1.2
0.15±0.05
33.2±1.1
0.07±0.01
0.06±0.004

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