In vitro and in vivo studies of gold(I) azolate/phosphane complexes for the treatment of basal like breast cancer

Article abstract of DOI:10.1016/j.ejmech.2018.06.002

Basal like breast cancer (BLBC) is a very aggressive subtype of breast cancer giving few chances of survival, against which cisplatin based therapy is a compromise among the anticancer activity, the resistance development and the severe side effects. With the aim of finding new anticancer agents alternative to cisplatin, seven gold(I) azolate/phosphane compounds were evaluated in vitro by MTT tests in human MDA-MB-231, human mammary epithelial HMLE cells overexpressing FoxQ1, and murine A17 cells as models of BLBC. Two compounds, (4,5-dichloro-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 1 and (4,5-dicyano-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 2 were found very active and chosen for an in vivo study in A17 tumors transplanted in syngeneic mice. The compounds resulted to be more active than cisplatin, less nephrotoxic and generally more tolerated by the mice. This study also provides evidence that both gold(I) complexes inhibited the 19 S proteasome-associated deubiquitinase USP14 and induced apoptosis, while compound 1's mechanism of action depends also on its ability to down-regulate key molecules governing cancer growth and progression, such as STAT3 and Cox-2.

Full text of DOI:10.1016/j.ejmech.2018.06.002

Accepted Manuscript
In vitro and in vivo studies of gold(I) azolate/phosphane complexes for the treatment
of basal like breast cancer
Valentina Gambini, Martina Tilio, Eunice Wairimu Maina, Cristina Andreani, Caterina
Bartolacci, Junbiao Wang, Manuela Iezzi, Stefano Ferraro, Anna Teresa Ramadori,
Oumarou Camille Simon, Stefania Pucciarelli, Guojun Wu, Q. Ping Dou, Cristina
Marchini, Rossana Galassi, Augusto Amici
PII:
S0223-5234(18)30492-6
10.1016/j.ejmech.2018.06.002
EJMECH 10472
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 May 2018
Revised Date: 31 May 2018
Accepted Date: 1 June 2018
Please cite this article as: V. Gambini, M. Tilio, E.W. Maina, C. Andreani, C. Bartolacci, J. Wang, M.
Iezzi, S. Ferraro, A.T. Ramadori, O.C. Simon, S. Pucciarelli, G. Wu, Q.P. Dou, C. Marchini, R. Galassi,
A. Amici, In vitro and in vivo studies of gold(I) azolate/phosphane complexes for the treatment of basal
like breast cancer, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.002.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
In vitro and in vivo studies of gold(I) azolate/phosphane complexes for the
treatment of Basal Like Breast Cancer
1,†
1,†
1
1,
1,
Valentina Gambini , Martina Tilio , Eunice Wairimu Maina , Cristina Andreani , Caterina Bartolacci ,
1
2
3
3
3
Junbiao Wang , Manuela Iezzi , Stefano Ferraro , Anna Teresa Ramadori , Oumarou Camille Simon , Stefania
1
4
4
1, *
3,†, *
1,†
Pucciarelli , Guojun Wu , Q. Ping Dou , Cristina Marchini , Rossana Galassi , Augusto Amici
1
School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, 62032, Italy.
2
Aging Research Centre, G. d’Annunzio University, Chieti, 66100, Italy.
3
School of Science and Technology, Chemistry Division, University of Camerino, Camerino, I-62032, Italy.
Barbara Ann Karmanos Cancer Institute, Department of Oncology, School of Medicine, Wayne State
4
University, Detroit, MI 48201, USA.
*
To whom correspondence should be addressed: Rossana Galassi, School of Science and Technology,
Chemistry Division, Via Sant’Agostino, 1 I-62032, Camerino, Italy Tel:+39 0737 402243; Email:
rossana.galassi@unicam.it; Cristina Marchini, School of Biosciences and Veterinary Medicine, via Gentile III
Da Varano, University of Camerino, Camerino, 62032, Italy. Tel: +39 0737 403275; Email:
cristina.marchini@unicam.it.
†
These authors contributed equally to this work.
Keywords: gold compounds, phosphane, breast cancer, anticancer agents, apoptosis, proteasome inhibitors
1

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Abstract
Basal like breast cancer (BLBC) is a very aggressive subtype of breast cancer giving few chances of survival,
against which cisplatin based therapy is a compromise among the anticancer activity, the resistance development
and the severe side effects. With the aim of finding new anticancer agents alternative to cisplatin, seven gold(I)
azolate/phosphane compounds were evaluated in vitro by MTT tests in human MDA-MB-231, human mammary
epithelial HMLE cells overexpressing FoxQ1, and murine A17 cells as models of BLBC. Two compounds, (4,5-
dichloro-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I)
1
and
(4,5-dicyano-1H-imidazolate-1-yl)-
(
triphenylphosphane)-gold(I) 2 were found very active and chosen for an in vivo study in A17 tumors
transplanted in syngeneic mice. The compounds resulted to be more active than cisplatin, less nephrotoxic and
generally more tolerated by the mice. This study also provides evidence that both gold(I) complexes inhibited
the 19S proteasome-associated deubiquitinase USP14 and induced apoptosis, while compound 1’s mechanism of
action depends also on its ability to down-regulate key molecules governing cancer growth and progression, such
as STAT3 and Cox-2.
2

ACCEPTED MANUSCRIPT
Introduction
Basal-like Breast Cancer (BLBC) is one of the most aggressive subtypes of breast cancer and it is associated
with the worst prognosis. BLBCs lack the expression of steroid hormone receptors (estrogen receptor and
1
progesterone receptor) and human epidermal growth factor receptor 2, limiting targeted therapeutic options.
BLBC treatment usually consists of conventional cytotoxic chemotherapy: although patients are quite sensitive
2,3
in the preoperative setting, they are nevertheless associated with short relapse-free and overall survival. Hence,
the development of new therapies with improved therapeutic indices is of paramount importance. In the search
of anticancer drugs, metal-based drugs gained great importance starting from the accidental discovery of
4
cisplatin, one of the leading agents in clinical use. Preclinical and clinical data reveal encouraging activity of
5
platinum-based chemotherapy drugs in BLBC, however, their continued use is greatly limited by severe dose
6
limiting side effects and intrinsic or acquired drug resistance. Among all the drugs containing metals other than
7
,8
platinum, gold compounds have turned out to be a cutting-edge class of anticancer compounds. Among all the
8,9
classes of gold compounds, the gold(I) phosphane complexes are potential good candidate for the anticancer
1
0,11
therapy.
and the
triethylphosphine]gold), has been recently reconsidered for the treatment of P388 leukemia,
In fact, Auranofin (ridaura), an old and reliable anti-rheumatic drug, made of a tetracetylthioglucose
gold(I)triethylphosphane fragment, [(2,3,4,6-tetra-O-acetyl-1-thio- -D-glucopyranosato-S-
β
12,13
[
lung’s non
14,15
16
small cell tumors,
as well as, in combination with other drugs, for the treatment of breast cancer. A strong
encouragement to continue the investigation in the anticancer activities of phosphane gold(I) complexes comes
1
7-19
from the upgrade in the understanding of the mechanism of action of Auranofin and its derivatives.
Seleno-
proteins such as Thioredoxine Reductase, were invested to possess a key role in a complicate network of
20,21
biological processes
involving redox metabolism and mithocondria and, even if they have been identified as
22
the major targets of gold(I) phosphane complexes contextually the concept that gold compounds are multitarget
3

ACCEPTED MANUSCRIPT
19,23,24
drugs grew up too.
The nature of the ligand, its slight modification as well as the gold coordination
19,25-27
environment lead to the activation of preferential cellular paths.
However, Auranofin appears to have
predominant antimitochondrial activity due to the inhibition or uncoupling of oxidative phosphorylation by
gold(I) phosphine complexes; comparing its activity to those of cationic ([Au(dppe) ]Cl) and ([Au(dppy) ]Cl)
2
2
(
dppe = bis(diphenylphosphino)ethane, dppy = bis(dipyridylphosphino)ethane) complexes, showing a similar
2
8
mechanism, the fine tuning of the hydro/lipophilic balancing gained attention in the gold drug design. Azolate
gold(I) phosphane complexes were found to possess a pronounced cytotoxic activity on many human cancer
29,30
cells, some of which were endowed with cisplatin or multidrug resistance.
These compounds were made with
pyrazolate or imidazolate and gold(I) triphenylphosphane or gold(I)TPA (TPA=1,3,5-triazaphosphaadamantane):
for these latter a lack of activity was observed. Thus, assuming the hydro/lipophilicity might be the critical
aspect on the design of effective anticancer gold(I) phosphane complexes with low systemic toxicity, planning
N-Au-P or P-Au-Cl skeletal structure, different polar functional groups have been introduced either in the
phosphane or/and in the azole ligand. Hence, a series of gold(I) complexes containing, in the azolate and/or in
the phosphane ligand, polar groups such as COOH, CH OH, Cl, CN, were synthesized and labelled as follow:
2
(
4,5-dichloro-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I)
triphenylphosphane)-gold(I) 2, (2-benzoic acid-diphenylphosphane)-gold(I)chloride 3, (4,5-dicyano-imidazolyl-
yl)-(4-benzoic acid-diphenylphosphane)-gold(I)) 4, (4-Benzoic acid-diphenylphosphane)-gold(I)chloride) 5, (4-
hydroximethyl-imidazolyl-1yl)-(2-benzoic acid-diphenylphosphanegold(I) and [tris(4-benzoic-
1,
((4,5-dicyano-1H-imidazolate-1yl)-
(
1
6
diphenylphosphane-acid)-gold(I)chloride] 7. Then, our goals have been: (i) to define the Structure Activity
Relationship (SAR) of the above mentioned complexes; (ii) to evaluate their anticancer efficacy in vitro; (iii) to
confirm their antitumor potential in vivo against a murine BLBC transplanted in a syngeneic host; and (iv) to
investigate the mechanisms underlying their anticancer activity. Among all the complexes, we identified
compounds 1 and 2 as the most powerful antitumor agents behaving as multi target anticancer agents.
Material and Methods
Synthesis of gold(I) phosphane complexes
4

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For the synthesis, 2-benzoic acid of diphenylphosphine, 4-benzoic acid of diphenylphosphine and other
chemicals were purchased and used without further purification. The complex Ph PAuCl was synthesized from
3
31
tetrachloride gold (III) acid and a double molar amount of PPh in ethyl alcohol as previously reported. Solid
3
Me SAuCl was synthesized by reducing HAuCl with an excess of Me S and by washing with methanol the
2
4
2
white solid obtained. Melting Points were not determined because most of the compounds decompose at low
temperature and the meltings were not clearly observed. (4,5-dichloro-1H-imidazolate-1-yl)-
(
triphenylphosphane)-gold(I) 1, (4,5-dicyano-1H-imidazolate-1yl)-(triphenylphosphane)-gold(I) 2, (2-benzoic
acid-diphenylphosphane)-gold(I)chloride and (4,5-dicyano-imidazolyl-1yl)-(4-benzoic acid-
diphenylphosphane)-gold(I)) 4, (4-Benzoic acid-diphenylphosphane)-gold(I)chloride) 5 were synthesized as
3
29,30
described in literature.
Samples for microanalysis were dried under vacuum (20°C, 0.1 torr) till constant
weight. C, H and N elemental analyses were performed by the CARLO-ERBA ELEMENTAL ANALYSIS mod.
–
1
1
106 mycro analyser at University of Camerino. Infrared spectra (4000 − 100 cm ) were recorded with
1
PERKIN-ELMER SYSTEM 2000 FT-IR spectrophotometer. H NMR spectra were recorded on an Oxford-400
1
Varian spectrometer. Chemical shifts, in ppm, for H NMR spectra are relative to internal Me
4
Si. Melting points
were determined with an instrument ELECTROTHERMAL ENGINEERING LTD Mod.9100. Mass spectra
ESI-MS) were obtained for negative ions through HP Series 1100 MSD spectrometer. Solutions (ca. 0.1 mM)
(
were prepared using MeOH as solvent. The experimental conditions were as following: the organic phase flow
was 300 µL/min, the drying gas flow (N ) was 10 L/min, the nebulization pressure was 30 psig, the temperature
2
of the drying gas was 350 °C, the value for the fragmentor was fixed to 30, the acquisition of the data was
performed by scanning in ranges from 500 to 2000 amu.
Synthesis of 4-hydromethyl)imidazolyl-1H-gold(I)-(2-benzoic-diphenylphosphane acid (compound 6)
5
3
0 mgs of solid 4-hydromethyl-imidazole (0.5 mmol) were dissolved in 5 mL of CH OH. To this solution 0.5
mL of a 1 M methanolic solution of KOH (0.5 mmol) were added. After magnetic stirring of a half of hour at
room temperature, 12 mL of a methanolic solution containing 275 mgs of 2-benzoic-diphenylphosphaneacid-
gold(I)-chloride were added (0.5 mmol). The suspension was stirred for two hours and then filtered off over a
5

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celite bed. The clear solution was concentrated to 10 mL and let to evaporate till a microcrystalline solid was
obtained Yield 63 %. M. p. 195-197°C.
1
H-NMR (CD
3
OD, δ): 8.11 (s, br 1H); 7.65-7.46 (m, 13H), 7.41 (t, 1H), 7.00 (s, 1H), 6.86 (t, 1H), 4.56 (s, 1H)
3
1
-1
P-NMR (CD
3
OD, δ): 33.21 (s). MIR (cm ): 3123 (s, br), 3055 (m, br), 2855 (m, br), 1702.9 (vs), 1596 (s),
1
1
579 (s), 1557 (s), 1479 (m), 1435 (s), 1371 (s), 1280 (m), 1256 (m), 1241 (m), 1206 (w), 1157 (w), 1101 (s),
063 (w), 1019 (m), 982 (m), 937 (m), 879 (m-w), 830 (s), 747 (s), 710 (s), 692 (vs). FIR (cm-1): 555 (m), 519
(
(
m), 543.6 (m-s), 500.8 (w), 433.8 (m), 393 (m), 285.7 (m), 227 (w), 175.6 (m), 153.4 (w), 135 (w). ESI (-)
+
CH OH, m/z) : 98 (45), 392 (30), 536.9 (100). ESI (+) (CH OH, m/z): 809 (100) [(2-COOH-Ph P) Au] .
3
3
2
2
Elemental analysis for C H AuN O P, calcd %: C 46.01, H 3.36, N 4.67. Found C 46.56, H 4.01, N 4.99.
2
3
20
2
3
Synthesis of tris(4-benzoic-diphenylphosphane-acid)-gold(I)chloride (compound 7)
The solution of 4-(diphenylphosphine)benzoic acid (208.3mg; 0.4 mmol) in CHCl was added dropwise to the
3
3
solution of dimethylsulfidegold(I)chloride (30 mg; 0.1 mmol) in CHCl (3 mL) at 0˚C The mixture was stirred at
r. t. overnight. A pale yellow precipitate was obtained. The suspension was filtered off through a gooch. The
solid was washed with few mL of CHCl and dried by vacuum. The crude product was crystallized dissolving in
3
3
few mL of CHCl and adding cyclohexane.
Yield. 86%. M. p. 139-140˚C
1
31
-1
H-NMR (CD
484 (s, br), 3054 (m, br), 1702.9 (vs), 1597.9 (s), 1560.2 (s), 1481 (m), 1434 (s), 1393 (s), 1310.9 (m), 1275 (m,
sh), 1225 (vs), 1183 (s),1122.6 b(m), 1091.8 (s), 1025.9 (m), 1016.8 (s), 998.9 (m), 855 (m-w), 791.9 (m), 764
3 3
OD, δ): 7.81 (d, 6H); 7.50 (t, 6H), 7.26 (m, 30H). P-NMR (CD OD, δ): 42.09(s). MIR (cm ):
3
-1
(
s, sh), 742.8 (s), 689.9 (vs).FIR (cm ): 534 (w), 513.5 (m), 367.8 (w), 295.68 (w), 176.95 (m), 139 (m), 125.7
(
m), 106 (w), 58.43 (m), 39.8 (m). ESI (-) (CH OH, m/z) : 305.2 (100), 536.9 (40). ESI (+) (CH OH, m/z): 808.9
3
3
+
(
100) [(2-COOH-Ph P) Au] . Elemental analysis for C H AuClO P , calcd %: C 56.80 , H 4.26. Found C
2 2 57 51 9 3
5
6.86, H 4.13.
Cell cultures
6

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A17 cells were established from spontaneous lobular carcinomas that arose in a FVB/neuT mice transgenic for
3
2,33
the activated form of rat HER-2/neu oncogene (FVB/neuT233), as previously described
.
A17 cells were
cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 20%
fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1% penicillin-streptomycin (P/S, Invitrogen, Carlsbad,
CA). MDA-MB-231 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured
in DMEM supplemented with 10% FBS and 1% P/S. MDA-MB-468 and HMLE/FoxQ1 cells were cultured in
DMEM supplemented with 10% FBS, 1% P/S or DMEM/HAM F12 (1:1) supplemented with 10% FBS, 1% P/S,
2
mM L-glutamine, 10 ng/mL human EGF, 0.5 µg/mL hydrocortisone and 10µg/mL insulin, respectively. To
3
4
maintain the Foxq1 gene in HMLE/FoxQ1 cells, 2.5-5 µg/mL Blasticidin was added to culture media. , 30
Cells were grown in a humidified atmosphere with 5% CO at 37°C.
2
Cell viability assay
The effects of gold(I) compounds (1-7), respect to cisplatin (cis-Diammineplatinum(II) dichloride, Sigma-
3
3
Aldrich, St. Louis, MO), on cell viability were evaluated by seeding 2.5*10 A17, 7*10 MDA-MB-231,
3
3
7
.5*10 MDA-M-468 and 3*10 HMLE/FoxQ1 cells/well in 96 wells plates in their respective complete
medium. The day after, fresh medium containing appropriate concentrations of compounds 1, 2, 3, 4, 5, 6, 7 and
cisplatin were added. After 24h and 48h, cell viability was determined using an MTT (Sigma Aldrich, St. Louis,
35
MO) assay, as previously described
.
The cytotoxicity of the compounds was reported as percentage of viable
cells relative to control cells. All the experiments were repeated three times.
Immunoblotting analysis
Lysates were separated by 4-20% gradient precast SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene
difluoride (PVDF) membranes (Immobilion P, Millipore). For DUB labeling assay, cell lysates were incubated
with Ub-VS for 30 minutes at 37°C, followed by boiling in reducing sample buffer and resolving by SDS-
PAGE. For immunoblotting analysis on HMLE/Fox Q1 cell extracts, primary antibodies against NMNAT3 (D-
1
0) (Santa Cruz Biotechnology, Inc.), Purified Mouse Anti-Human PARP (BD Pharmingen™), and USP14
(
Abcam) were used, respectively. After secondary antibody incubation, the immunoreactive bands were captured
7

ACCEPTED MANUSCRIPT
with ECL chemiluminescent substrates (Pierce Rockford) and detected by exposing to X-ray films (Kodak,
Rochester, NY, USA). For immunoblotting analysis on tumor lysates, primary antibodies to β-actin, STAT3,
phospho-STAT3, Cox-2, Slug and Snail were from Cell Signaling Technology. Secondary antibodies conjugated
with peroxidase were from Sigma-Aldrich. The immunoreactive bands were detected by using LiteAblot PLUS
(
Euroclone) reagents and images were acquired with ChemiDoc Imaging System (Bio-Rad).
Animals
Female FVB mice, line 233, 6 weeks old, were obtained from Charles River S.r.l. (Lecco, Italy), and housed
under controlled conditions. Mice were treated according to the European Community guidelines. The Animal
Research Committee of the University of Camerino authorized the experimental protocol.
Treatments and tumor growth
5
A suspension of 1*10 A17 viable cells in 200
µ
l of PBS was inoculated into mammary fat pad of 8-week-old
FVB females. 10 days after tumor challenge, mice were randomly divided in 4 groups (5 mice per group) and
treated with compound 1, compound 2, cisplatin or isotonic solution by intraperitoneal injection (via i.p.) in
36
accordance with a treatment schedule (protocol q3x4) previously reported. Briefly, 3 mg/kg/day of each
compound (12 mg/kg final amount) were administered four times, once every three days. Body weight of mice
and tumor growth was checked constantly until the end of the experiment, progressively growing masses >1 mm
2
mean diameters were considered as tumors. Tumor volume was calculated as 0.5 x d
1
x d
2
, where d
1
and d
2
are
the smaller and larger diameters, respectively. Tumors were surgically removed on day 24.
Histology and immunohistochemistry
Tumors and kidneys were harvested at the end of the experimental period, fixed in formalin and embedded in
paraffin or fixed in PFA 4% and frozen in cryo-embedding medium (OCT Bioptica, Milan, Italy). To detect
possible organ toxicity, kidney slides were stained with hematoxylin and eosin. Histopathological evidences of
37
acute tissue damage were semi-quantified according to the methodology described elsewhere
.
Two slides were
analyzed in a blind fashion evaluating the following parameters: atrophic glomerulus, dilated proximal
convoluted tubule, degenerated tubules, inflammatory cells infiltrate, desquamation of epithelial cells and cast
8

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formation. The severity and incidence of each parameter was scored as follows: - absent , -/+ scarcely; +/-
moderately; + frequently. For immunohistochemistry, tumor slides were incubated with the following primary
antibodies: anti-CD31 (550274) and anti-CD105 (550546) (BD Pharmingen, Milan, Italy). After washing, slides
were overlaid with appropriate secondary antibodies. Immunostaining was developed with DAB Chromogen
System (Dako, Milan, Italy) or Vulcan Fast Red (Biocare, Milan, Italy) alkaline phosphatase method. The
number of CD31/105 positive vessels was evaluated on digital images of controls, cisplatin, compound 1 and
compound 2 treated tumors (5 per group, 5 x 400 microscopic fields per tumor).
Metal trace examination
Gold and platinum content in explanted kidneys from mice treated with compound 1, compound 2, cisplatin or
isotonic solution was evaluated by ICP-MS. All solutions were prepared using ultrapure water obtained from a
Millipore Milli-Q system (resistivity 18.2 M
Ω cm). Optimized digestion of samples was carried out in a
microwave digestor (Berghof Speedwave four, Berghof, Eningen, Germany) with 5 ml of HNO (65%). Fifty
3
microliters of iridium solution (20 mg) was added as recovery standard. Digested solutions were diluted with
ultrapure water to obtain a solution with the correct acid concentration in order to perform analysis. The
concentrations of metals in the processed samples were measured by ICP-MS (7500cx series) with the operating
38
conditions described elsewhere
.
Calibration curves were obtained using aqueous standard solutions (1,5%
nitric acid) with appropriate stock standards dilutions (Fluka Analytical, Aldrich, Milan, Italy).
Table 3) Optimized digestion protocol for explanted kidneys.
Proteasomal chymotrypsin-like activity in HMLE/FoxQ1 breast cancer cells
9

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The HMLE/FoxQ1 cells were treated with compound 1 and compound 2 for different periods of time (0, 30, 60,
9
0, 120, 150, 180 minutes), lysed, and the protein concentrations were measured using a Bio-Rad protein assay
Bio-Rad). Whole-cell lysates (10 g) were incubated for 2 h at 37 C in 100 l assay buffer (20 mM Tris-HCl,
M fluorogenic peptide substrate Suc-LLVY-AMC (AnaSpec, Fremont, CA, USA).
(
µ
̊
µ
pH 7.5) with 20
µ
Proteasomal CT-like activity was measured using the Wallac Victor3 multi-label counter with an excitation lter
39
of 365 nm and an emission filter of 460 nm, as previously described.
Statistical analysis
Quantitative data are presented as means ± SEM from three independent experiments. The significance of
differences was evaluated with two-tailed Students t test, or one way ANOVA followed by Bonferroni or
Tukey’s Multiple Comparison post test. Statistical analysis was executed with GraphPad Prism Software (San
Diego, CA, USA), using p<0.05 as the critical level of significance (*p
≤ 0.05; **p ≤ 0.01; *** p≤0.001).
Results
Structure of the compounds
A set of gold(I) compounds having the N-Au-P or P-Au-Cl backbones were synthesized according to a general
scheme where the azolate salt was treated with the selected phosphane gold(I) chloride or tetrafluoborate (Figure
1
). It is known that compounds having a N-Au-P environment are more active than those with a C-Au-P
chemical structure. Thus, the gold(I) compounds 1, 2, 3 and 6 have the N-Au-P chemical backbone but,
compounds 1 and 2 contain polar groups such as chloride and cyano groups in the imidazole ring and they have
PPh as coligand, compound 3 and 6 possess one COOH group in the phosphane ligand for the former and a
3
2
CH OH group in the imidazole for the latter. Compounds 4 and 5 exhibit a P-Au-Cl backbone and they differ for
the position of the COOH group in the phenyl group of the phosphane ligand (compound 4, ortho position, and
compound 5, para position); lastly, the compound 7 contains the P-Au-Cl environment with the gold coordinated
to three 4-benzoic diphenylphosphane ligands. The white microcrystalline solids of compounds 1-7 were
prepared and used to prepare fresh stock solutions for the biological tests.
1
0

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N
N
Cl
Cl
N
N
N
N
N
N
N
N
Au
Au
Au
P
P
P
HO
O
Compound
1
Compound
2
Compound
3
OH
N
OH
O
Cl
N
O
Cl
Au
Au
Au
P
P
P
O
OH
OH
Compound
4
Compound
5
P
7
Compound
6
OH
Cl
O
Au
3
Compound
Figure 1. Schematic representation of the gold(I) compounds tested in this work.
Compounds 1 and 2 inhibited BLBC growth in vitro
The effect of the new gold(I) compounds (1-7), compared with that of cisplatin, on cancer cell viability was
estimated by MTT assay at 24h treatment using the human MDA-MB-231 cells and the murine A17 cells as
40
models of BLBC. Among the tested drugs, only compounds 1 and 2 exhibited a remarkable in vitro anticancer
efficacy against both murine and human cell lines, being able to decrease cell viability in a dose-dependent
manner with IC values at low µM concentrations, whereas compounds 3-7 failed in reducing significantly cell
50
1
1

ACCEPTED MANUSCRIPT
viability even at the highest tested dose (Table 1; supporting information Figure 1, 2). Interestingly, compounds
1
2
1
and 2 displayed a stronger antineoplastic activity respect to cisplatin. In particular, the response of MDA-MB-
31 cells to compounds 1 and 2 resulted to be stronger and even faster than that to cisplatin. In fact, compounds
and 2 showed IC50 values of 19.28 µM and 14.83 µM, respectively, after 24h treatment, whereas cisplatin
displayed an IC50 value of 50.49 µM only after a 48h treatment (Table 1). In addition, in vitro screening was
completed evaluating the cytotoxicity of the separate moieties of compounds 1 and 2, corresponding to free
azoles (ImH(Cl) and ImH(CN) for compounds 1 and 2, respectively), and triphenylphosphane moieties
2
2
(
Ph
3
PAuCl and the bare Ph
3
P) (supporting information Figure 4). Of note, only Ph
3
PAuCl moiety was able to
M and 18.29 M,
respectively. On the contrary, ImH(Cl) , ImH(CN) and Ph P displayed negligible cytotoxicity in both BLBC
decrease viability in both A17 and MDA-MB-231 cells at 24h, with an IC value of 22.27
µ
µ
50
2
2
3
cells highlighting the role of the metal centre in the anticancer activity of our gold compounds 1 and 2
supporting information Figure 3). The anticancer efficacy of compounds 1 and 2 was also confirmed on two
(
4
1
other in vitro models of breast cancer: the human BLBC MDA-MB-468 cells and human mammary epithelial
,
34 42
HMLE cells overexpressing FoxQ1, characterized by stemness traits and chemoresistance.
HMLE/FoxQ1
M and
.27 µM at 24h, respectively. Of note, cisplatin was less effective than compounds 1 and 2 also in HMLE/FoxQ1
line revealed to be the most responsive cells to both compound 1 and 2, displaying IC values of 7.41
µ
50
9
and MDA-MB-468 cells, inducing a significant decrease in cellular viability only after 48h treatment with an
IC50 value of 34.12 µM in HMLE/FoxQ1, and after 24h treatment with an IC50 value of 32.50 µM in MDA-MB-
4
68 cells (Table 1; supporting information Figure 3).
Table 1- IC50 Values of Compounds 1−2, cisplatin on A17, MDA-MB-231, MDA-MB-468 and HMLE/FOXQ1
breast cancer cells.
IC50
Compound
Cell Line
Time
a
Mean± SD [µM]
1
2

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A17
24h
11.38 ± 1.05
19.28 ± 1.06
13.62 ± 1.05
7.41 ± 1.06
MDA-MB-231
MDA-MB-468
HMLE/ FoxQ1
24h
24h
24h
Compound 1
A17
24h
24h
24h
24h
11.95 ± 1.04
14.83 ± 1.05
11.25 ± 1.14
9.27 ± 1.06
MDA-MB-231
MDA-MB-468
HMLE/FoxQ1
Compound 2
A17
24h
48h
24h
48h
15.86 ± 1.17
50.49 ± 2
MDA-MB-231
MDA-MB-468
HMLE/FoxQ1
Cisplatin
32.50 ± 1.12
34.12 ± 2.17
a
SD: standard deviation.
Table 1. Half Maximal Inhibitory Concentration (IC ) values of compounds 1,2 and cisplatin for A17, MDA-
50
MB-231, MDA-MB-468 and HMLE/FoxQ1 cells after 24h or 48h of treatment with increasing concentration of
each gold(I) compound. IC values are expressed as Mean ± SD ( M).
µ
50
Compounds 1 and 2 inhibited BLBC growth in vivo
We next investigated the in vivo antineoplastic effect of compounds 1 and 2, which showed the strongest
anticancer activity in vitro, against A17 cells, able to give rise to aggressive mesenchymal tumors when injected
3
2,33
into syngeneic mice.
BLBC, including expression of vimentin, cytokeratin 14, N-cadherin and Cox-2.
of compounds 1 and 2 was compared with that of cisplatin, known to be effective against A17 transplanted
As previously described, A17 transplanted tumors share molecular signature with
40,43
The antineoplastic activity
1
3

ACCEPTED MANUSCRIPT
36
tumors. 10 days after tumor challenge when tumors became palpable, 3 mg/kg/day of each compound were
3
6
administered 4 times, once every 3 days, in accordance with a previously reported protocol (q3X4). Here we
show that compounds 1 and 2 were able to reduce significantly A17 tumor growth. Of note, control tumors
displayed a rapid growth rate, completely counteracted by both compounds 1, 2 and cisplatin for all the duration
of treatment (from day 10 to 19) (Figure 2A). In addition, both gold(I) compounds were able to significantly
delay the growth of A17 tumors and to maintain them smaller than control ones even after the last drug
administration (day 19) until the end of the experiment, although their effect was not long lasting as the cisplatin
one (Figure 2A,B). Since A17 cells are able to give rise to highly vascularized aggressive tumors, we carried out
histological and immunohistochemical analyses to evaluate the effect of compounds 1 and 2 on A17 tumor
angiogenesis. In fact, several data support a central role for angiogenesis in breast cancer growth and
44
metastasis. Interestingly, both compound 1 and 2, similarly to cisplatin, were able to provoke a reduction in the
number of vessels (CD31/ 105 positive cells) in explanted tumors (supporting information Figure 5). Of note,
body weight did not significantly differ in mice treated with compounds 1 and 2 respect to the control ones,
suggesting that they have very low drug toxicity at the selected doses. On the contrary, cisplatin treated mice
showed a continuous significant reduction in body weight during the treatment, reaching the maximum weight
loss at day 19 (Figure 2C).
1
4

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Figure 2. Compounds 1 and 2 suppressed BLBC growth in vivo. FVB female mice were injected with syngeneic
A17 cells and treated with cisplatin, compound 1 or 2 or isotonic solution. Treatments schedule (q3x4) started
ten days after cell injection. A) Tumor growth curves in mice receiving the treatment schedule q3X4. Values are
mean ± SEM, n = 5/group; B) representative images of tumors explanted from control and treated mice at
sacrifice (upper panel) and relative tumor weight (lower panel) 24 days after tumor challenge, values are mean ±
SEM; C) effect of treatments on mean body weight of mice. Values are mean ± SEM. The significance was
determined by the One way ANOVA test followed by Tukey’s Multiple Comparison Post Test; each group was
compared to control. (*p
≤ 0.05; **p ≤ 0.01; *** p≤0.001).
Compounds 1 and 2 exhibited reduced nephrotoxicity respect to cisplatin
To better evaluate the toxicity of compounds 1 and 2 we carried out an extensive histopathological analysis of
explanted kidneys, common target organs of chemotherapy drugs. Importantly, hematoxylin and eosin (H&E)
staining (Figure 3) and relative semiquantitive analysis (Table 2) revealed a significant reduced nephrotoxicity,
similar for compound 1 and compound 2 treated mice, respect to cisplatin. In detail, histological changes in
kidneys after cisplatin treatment revealed a severe atrophy of glomeruli, which was apparently due to the
1
5

ACCEPTED MANUSCRIPT
reduction in its size, and moderate dilatation of proximal convoluted tubules with slogging of almost entire
tubular epithelium due to its desquamation. Cisplatin treatment also provoked marked degeneration of tubules
and moderate interstitial inflammatory cells infiltration (Figure 3 A,B,C) (Table 2). On the contrary, both
compounds 1 and 2 administrations caused just a moderate atrophy of glomeruli respect to cisplatin, whereas the
dilation of proximal convoluted tubule and the degeneration of tubules is comparable to controls. Interestingly,
we revealed no signs of inflammation neither after compound 1 or compound 2 treatment (Figure 3 A,D,E,F)
(
(
Table 2). Furthermore, analysis of gold and platinum traces by Inductively Coupled Plasma-Mass Spectrometry
ICP-MS) analysis in the kidneys of treated mice clearly demonstrated a good excretion of both gold(I)
phosphane compounds, significantly higher respect to cisplatin, that led to an accumulation of platinum in
kidneys (Figure 4).
Figure 3. Histopathological effects of compound 1, 2 and cisplatin in mouse kidney using hematoxylin and eosin
(
H&E) staining. A) Histology of normal control mouse kidney (isotonic solution); B,C) renal tissue of cisplatin-
treated mice; D) renal tissue of compound 2-treated mice; E,F): renal tissue of the compound 1-treated mice. (N)
normal glomeruli; (G) atrophic glomerulus; (PCT) dilated proximal convoluted tubule; (T) degenerated tubules;
(
I) inflammatory cells infiltrate; (D) desquamation of epithelial cells. (Magnification x 200).
1
6

ACCEPTED MANUSCRIPT
Isotonic solution Cisplatin Compound 1 Compound 2
score
-/+
score
+
Score
+/-
score
+/-
-/+
-/+
-
Atrophic glomerulus (G)
Dilated proximal convoluted tubule (PCT)
Degenerated tubules (T)
-/+
+/-
+
-/+
-/+
-/+
Inflammatory cells infiltrate (I)
Desquamation of epithelial cells (D)
-/+
+/-
+/-
-/+
-/+
+/-
+/-
Table 2. Semiquantitative analysis of histopathological features in kidneys after treatment with cisplatin,
compound 1 and compound 2: - absent , -/+ scarcely; +/- moderately; + frequently.
Figure 4. Gold and platinum content in kidneys of mice treated with compound 1, compound 2 or cisplatin
evaluated by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The data are presented as micrograms
of metal (Au or Pt) per gram of tissue. Values are mean ± SEM, n= 5/group. The significance was determined by
the One way ANOVA test followed by Tukey’s Multiple Comparison test (*** p≤0.001).
Compound 1 decreased Cox-2 expression in BLBC A17 transplanted tumors
A17 cells, beside the expression of mesenchymal markers, display constitutively active STAT3 transcription
1
7

ACCEPTED MANUSCRIPT
32,45
43,46
factor
and overexpression of Cox-2, that is one of its downstream target genes.
Cox-2 is frequently
40
associated with basal-like transcription pattern in human breast tumors, where it correlates with their
mesenchymal signature and their malignant phenotype regulating neoangiogenesis, invasion and anti-apoptotic
33,40,43,45,47
mechanisms.
Thus, the molecular mechanisms underlying the anticancer effects of compound 1 and 2
were analysed by western blot firstly evaluating Cox-2 expression level in A17 tumors explanted from treated or
untreated control mice. Our results show that both compound 1 and cisplatin treatments were able to induce a
marked downregulation of Cox-2 expression, which was correlated with a significant decreased level of
phospho-STAT3 (Figure 5). Furthermore, both compound 1 and cisplatin induced a significant reduction of both
Snail and the closely related gene Slug (Figure 5), which mediate Epithelial Mesenchymal Transition (EMT) in
43,47-50
invasive carcinoma cells and retain the mesenchymal aggressive phenotype.
On the contrary, compound 2
treatment did not affect neither Cox-2 expression nor STAT3 activation and it did not induce a decrease of Snail
and Slug targets, suggesting a mechanism of action different from the one exerted by compound 1.
1
8

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Figure 5. Effect of compound 1, compound 2 and cisplatin treatment on STAT3 activation and Cox-2, Snail and
Slug expression in A17 transplanted tumors. Representative image of western blot analysis (right panel) for the
expression levels of STAT3, p-STAT3, Cox-2, Snail, Slug and
FVB mice receiving isotonic solution (control), compound 1, compound 2 or cisplatin; densitometric
quantification of p-STAT3, Cox-2, Snail and Slug expression normalized on -actin (left panel) from three
independent experiments, performed with ImageJ Software. Values are mean ± SEM. The significance was
determined by the One way ANOVA test followed by Tukey’s Multiple Comparison test (*p 0.05; **p 0.01;
** p 0.001).
β-actin (loading control) in A17 tumors from
β
≤
≤
*
≤
Compounds 1 and 2 were able to induce apoptosis in HMLE/FoxQ1 breast cancer cells
To gain further insights into the mechanism underlying anticancer activity of both compounds 1 and 2, we
investigated their ability to induce apoptosis in HMLE/FoxQ1 cells, which revealed the higher sensibility to both
complexes in vitro, as previously described. Hence, HMLE/FoxQ1 cells were treated with compound 1 or
compound 2 for 8h or 24h and cleavage of poly (ADP-ribose) polymerase-1 (PARP), a hallmark of apoptosis,
was detected by western blot. Interestingly, both complexes were able to induce PARP cleavage after 8 to 24h
treatment, suggesting their strong ability to trigger apoptosis in HMLE/FoxQ1 cells (Figure 6).
Figure 6. Compounds 1 and 2 induce apoptosis. Representative image of western blot analysis of PARP
cleavage (89 kDa fragment) detected in HMLE/FoxQ1 cells upon treatment with compound 1 (7.5 µM) or
compound 2 (9.15 µM), β-actin was used as loading control.
1
9

ACCEPTED MANUSCRIPT
Compounds 1 and 2 induced accumulation of polyubiquitinated NMNAT3 (Nicotinamide mononucleotide
adenylyltransferase 3) in HMLE/FoxQ1 breast cancer cells
Dou lab has found that NMNAT3 protein can be degraded by ubiquitin-proteasome system (unpublished
results). NMNAT3 is the key enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis pathway in
mitochondria and cancer cells generally display aberrant NAD metabolism, hence targeting NMNAT3 could be
a strategy for treating different cancers. Thus, we investigated whether both compounds 1 and 2 had effect on the
level of ubiquitinated NMAT3 in HMLE/FoxQ1 cells. Figure 7 showed that both gold(I) compounds were able
to induce a time-dependent accumulation of polyubiquitinated NMNAT3 in treated HMLE/FoxQ1 cells,
suggesting a possible inhibition of 26S proteasome either at 20S (proteolytic core) or 19S (regulatory part) via
proteasome-associated deubiquitinases (DUBs) or both, leading to a block of degradation of ubiquitinated
proteins and eventually to apoptosis. However, we excluded that our gold complexes could suppress tumor
growth via direct inhibition of the 20S proteasome activity since they failed to inhibit 20S proteasomal
enzymatic activity, measured by a proteasomal chymotrypsin-like (CT-like) assay in HMLE/FoxQ1 cells (data
not shown).
2
0

ACCEPTED MANUSCRIPT
Figure 7. Effect of compounds 1 and 2 on ubiquitinated NMNAT3 in HMLE/FoxQ1 cells. Representative image
of western blot analysis for the expression of ubiquitinated NMNAT3 was shown with
control.
β-actin as a loading
Compounds 1 and 2 inhibited 19S-associated Ubiquitin specific peptidase 14 (USP14) in HMLE/FoxQ1
breast cancer cells
Auranofin, a gold(I)-containing complex and a clinically used antirheumatic agent, has been found to be a
13
proteasomal deubiquitinase inhibitor. Since accumulation of polyubiquitinated NMNAT3 in HMLE/FoxQ1
cells treated with compounds 1 and 2 did not correlate with inhibition of 20S proteasomal enzymatic activity, we
51
investigated their ability to inhibit the 19S proteasome-associated deubiquitinases (DUBs) by UB-VS
(
Ubiquitin vinyl sulfone, HA-tag) assay. Interestingly, compounds 1 or 2 were able to partially inhibit 19S DUB
USP14 after 1h and 3h treatment, as shown by the increased levels of unmodified USP14 protein (56 KDa) that
indicates a competitive binding (Figure 8). However, after 5h treatment both gold(I) compounds treated cells
2
1

ACCEPTED MANUSCRIPT
showed only the 66 kDa band (Ub-VS-bound form) which can be explained as decrease of inhibition of USP14
DUB.
Figure 8. UB-VS (Ubiquitin vinyl sulfone, HA-tag) assay for USP14 in HMLE/FoxQ1 cells: ability of
compound 1 and compound 2 to inhibit USP14. C–UB-VS= Control cells without UB-VS, C+UB-VS = Control
cells with UB-VS, DMSO–UB-VS= DMSO without UB-VS, DMSO+UB-VS = DMSO with UB-VS,
was used as loading control.
β-actin
2
2

ACCEPTED MANUSCRIPT
Discussion and conclusions
To date, BLBCs remain the most hard-to-treat breast tumor due to the lack of effective targeted therapies and the
52
occurrence of resistance phenomena, which correlates with a short relapse-free and overall survival.
Experimental research on the promising cell growth inhibiting effects of gold(I) complex Auranofin, definitively
triggered an immense interest in the research on gold complexes for cancer chemotherapy as innovative
8
alternative metal based drug. Hence in this work, seven new gold(I) complexes with different hydro/lipophilic
properties have been screened for their anticancer activity against in vitro models of BLBC. Consistently, we
found that micromolar concentration of compound 1 and compound 2 exhibited a unique in vitro dose-dependent
cytotoxic activity, higher than cisplatin, against murine A17 cells and human MDA-MB-231 cells. Interestingly,
the antitumor efficacy was confirmed also on both human MDA-MB-468 and HMLE/FoxQ1 BLBC cells, which
resulted to be the most sensible cells to both gold compounds treatment. The in vitro cytotoxic activity of free
azoles and phosphane moieties of compounds 1 and 2 was also assessed. Of note, just Ph PAuCl moiety
3
+
possessed the ability to hamper cell growth in vitro, suggesting that the lipophilic fragment Ph PAu is likely
3
responsible for the antiproliferative activity. A structure–activity relationship study on 63 Auranofin analogues
had already demonstrated the importance of the phosphine ligand as derivatives lacking this moiety were
5
3
significantly less active. These early evidences remark without any doubt that for this class of gold(I)
compounds the introduction of hydrophilic groups such as the alcoholic or the carboxylic functions both in the
azole and/or in the phosphane group hampers the expected anticancer activity, even though in literature is
54
reported that hydrophilic phosphane gold(I) complex were found active against several panels of tumor cells.
Furthermore, the presence of polar groups (COOH) in only one of the aryl substituents of the phosphine cation,
although it lowers the lipophilia and likely the side toxicity, negatively affects the anticancer activity, then the
idea to lower the potential toxicity of the active PPh
of large and lipophilic ligand such as the phenyl group on the phosphorous guarantees a good cellular uptake.
In the present study, Ph PAuCl resulted to be less effective than compound 1 and compound 2 on both A17 and
MDA-MB-231 cells at 24h treatment, suggesting that the introduction of azole ligands with polar functional
3
moiety has to be forsaken. On the other hand, the presence
25
3
2
3

ACCEPTED MANUSCRIPT
groups could be critical in determining the fate of anticancer properties of this new class of gold based drugs.
+
Moreover, previous study on both Auranofin and the Et PAuCl, aimed to disclose the role of the Et PAu ,
3
3
5
0,55
showed that for the latter the DNA interaction was preferred
highlighting that the easier chloride exchange
leads to alternative cellular path. So far, there have not been studies on the effects of compounds 1 and 2 in in
vivo experimental tumor models. Hence, in this study we investigated the antineoplastic activity of both
complexes against A17 transplanted tumors, representing a BLBC preclinical model. Of note, A17 cells are a
highly tumorigenic and invasive cell line, established from an FVB/neuT transgenic mammary tumor, strongly
33,40,45,47
related to human mesenchymal cancer stem cells and basal-like breast cancer.
Herein reported results
indicate that both compound 1 and 2 administration was able to hamper BLBC tumor growth in vivo. Of note,
the anticancer efficacy of both compounds 1 and 2 correlated with a reduced nephrotoxicity respect to cisplatin
due to the increased hydrophilicity of the azolate gold(I) phosphane complexes which allowed a good excretion
of gold from kidneys. Interestingly, the study performed on the molecular mechanisms underlying their
anticancer effects suggests a different mechanism of action between compound 1 and compound 2. Compound 1
induces a significant inhibition of STAT3 activation associated with a significant downregulation of Cox-2
expression. In fact, Cox-2 expression has been reported to be controlled by STAT3 transcription factor in A17
45
56
tumors. This is a relevant result considering Cox-2 role in tumor growth, invasiveness and angiogenesis and
that a preferential activation of STAT3 has been reported in BLBC, where it seems to be specifically required for
57,58
the maintenance of stem cell-like cancer cells.
Consistently, we found that phospho-STAT3 dowregulation
induced by compound 1 correlates also with decreased levels of both Snail and Slug transcription factors, known
to be involved in EMT. Thus, our data indicate that one of the main targets of compound 1 is STAT3
transcription factor, that represents a really good target for anticancer drugs conceived against BLBC, since its
down-regulation leads to a decreased expression of key molecules governing cancer growth and progression,
such as Cox-2 and the EMT inducers Slug and Snail.
On the contrary, probably due to a different structure of the whole molecule, the anticancer activity of compound
2
does not strictly depend on the same molecular targets affected by compound 1 suggesting a completely
different mechanism of action which remains to be further examined. Furthermore, preliminary in vitro studies
2
4

ACCEPTED MANUSCRIPT
on HMLE/FoxQ1 cells, allowed us to highlight alternative important mechanisms through which both
compounds 1 and 2 could exert their anticancer activity in vitro. First, both gold(I) complexes induced apoptosis
in HMLE/FoxQ1 cells as demonstrated by cleavage of PARP. The normal function of PARP is the routine repair
of DNA damage, but it is also a preferred substrate for several ‘suicidal’ proteases. In fact, cleavage of PARP by
caspases is considered to be a hallmark of apoptosis. Then, preliminary data suggest that the anticancer potential
of these new gold(I) complexes is based on their ability to inhibit 19S associated DUBs, although a more in
depth analysis of their mechanisms of action is necessary. In fact, compounds 1 and 2 targeted the
deubiquitylating function of USP14, one of the DUBs associated with the proteasome 19S, leading to an
accumulation of polyubiquitylated NMNAT3 in HMLE/FoxQ1 cells. Consistently, recent studies showed that
some metal complexes, including gold(I) Auranofin, acting as inhibitors of 19S-associated DUBs UCHL5 and
5
1
USP14, have promising antitumor effects
.
In conclusion, the present study demonstrates that compounds 1 and 2, targeting key molecules associated with
the malignant phenotype, provide an effective therapeutic strategy against BLBC associated with minimal side
effects.
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