Potent Anticancer Efficacy of First-In-Class CuII and AuIII Metaled Phosphorus Dendrons with Distinct Cell Death Pathways

Article abstract of DOI:10.1002/chem.202001014

First-in-class Cu^II and Au^III metaled phosphorus dendrons were synthesized and showed significant antiproliferative activity against several aggressive breast cancer cell lines. The data suggest that the cytotoxicity increases with reducing length of the alkyl chains, whereas the replacement of Cu^II with Au^III considerably increases the antiproliferative activity of metaled phosphorus dendrons. Very interestingly, we found that the cell death pathway is related to the nature of the metal complexed by the plain dendrons. Cu^II metaled dendrons showed a potent caspase-independent cell death pathway, whereas Au^III metaled dendrons displayed a caspase-dependent apoptotic pathway. The complexation of plain dendrons with Au^III increased the cellular lethality versus dendrons with Cu^II and promoted the translocation of Bax into the mitochondria and the release of Cytochrome C (Cyto C).

Full text of DOI:10.1002/chem.202001014

A Journal of
Accepted Article
Title: Potent anticancer efficacy of first-in-class Cu(II) and Au(III)
metaled phosphorus dendrons with distinct cell death pathways
Authors: Jean Pierre Majoral, Liang Chen, Yu Fan, Jieru Qiu, Régis
Laurent, Jin Li, Jérôme Bignon, Serge Mignani, Anne-Marie
Caminade, and Xiangyang Shi
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To be cited as: Chem. Eur. J. 10.1002/chem.202001014
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Potent anticancer efficacy of first-in-class Cu(II) and Au(III)
metaled phosphorus dendrons with distinct cell death pathways
Liang Chen [a, b, c], Yu Fan [a], Jieru Qiu [b, c], Régis Laurent [b, c], Jin Li [a], Jérôme Bignon [d], Serge
Mignani [a, e, f],* Anne-Marie Caminade [b, c], Xiangyang Shi [a, f]* and Jean-Pierre Majoral [b, c]
*
induced carcinogenesis effects.[4] The main limitation of metallo-
drugs is their potential instability in the blood and their oxidation
profile.[5]
Abstract: First-in-class Cu(II) and Au(III) metaled phosphorus
dendrons were synthesized and showed significant antiproliferative
activity against several aggressive breast cancer cell lines. The data
suggest that the cytotoxicity increases with reducing the length of the
alkyl chains, whereas the replacement of Cu(II) by Au(III)
considerably increases the antiproliferative activity of metaled
phosphorus dendrons. Very interestingly, we found that the cell
death pathway is related to the nature of the metal complexed by the
plain dendrons. Cu(II) metaled dendrons showed a potent caspase-
independent cell death pathway; whereas Au(III) metaled dendrons
Nanotechnology represents a huge multidisciplinary field
encompassing chemistry, biology, physics, engineering, etc.,
and it has appeared to be a benefit to humanity in a wide variety
of multidisciplinary domains including therapeutic domains
(termed nanomedicine) such as in the fields of cancer,
cardiovascular disease, and central nervous system disorders.[6]
Thus, the rational design of nanoparticles encapsulating or
complexing anticancer drugs (such as doxorubicin liposomes
(Doxil®) and paclitaxel albumin (Abraxane®), which have been
U.S. Food and Drug Administration (FDA) approved in 1995 and
2005, respectively) plays a pivotal role to treat cancer.[7]
displayed
a
caspase-dependent apoptotic pathway. The
complexation of plain dendrons with Au(III) increased the cellular
lethality versus dendrons with Cu(II) and promoted the translocation
of Bax into the mitochondria and the release of Cytochrome C (Cyto
C).
Introduction
The discovery of cisplatin (cis-diaminodichloroplatinum(II)) in
1965 by Rosenberg, through a serendipitous approach, played a
pivotal role in the discovery of many metallo-drugs used today in
the treatment of cancer.[1] Cisplatin is the first approved
anticancer drug based on an inorganic complex, and it is now
used to treat a wide range of cancers.[2] This built the foundation
of the modern era for the rapid development of anticancer
metallo-drugs using several types of metals such as palladium,
ruthenium, osmium, iron, gold, copper, and rhodium.[3] Note that
other metals such as nickel, cadmium, chromium, and arsenic
[a]
L. Chen, Y. Fan, J. Qiu, J. Li, Prof. Dr. S. Mignani, Prof. Dr. X. Shi
State Key Laboratory for Modification of Chemical Fibers and Polymer
Materials, College of Chemistry, Chemical Engineering and
Biotechnology,
Figure 1. Schematic 2D chemical structures of dendrons and corresponding
dendrimers.
Donghua University
Shanghai 201620, People’s Republic of China
E-mail: xshi@dhu.edu.cn
[b]
[c]
L. Chen, J. Qiu, Dr. R. Laurent, Prof. Dr. A. M. Caminade, Prof. Dr. J.
P. Majoral
Laboratoire de Chimie de Coordination, CNRS,
205 Route de Narbonne, 31077 Toulouse CEDEX 4, France
E-mail: majoral@lcc-toulouse.fr
L. Chen, J. Qiu, Dr. R. Laurent, Prof. Dr. A. M. Caminade, Prof. Dr. J.
P. Majoral
Université de Toulouse, INPT,
INPT, 31077 Toulouse CEDEX 4,France
Prof. Dr. J. Bignon
Institut de Chimie des Substances Naturelles du CNRS
91198 avenue de la Terrasse, Paris Gif-sur-Yvette Cedex, France
Prof. Dr. S. Mignani
Universitꢀ Paris Descartes, PRES Sorbonne Paris Citꢀ, CNRS UMR
860, Laboratoire de Chimie et de Biochimie Pharmacologiques et
Toxicologique
45, rue des Saints Pꢁres, 75006 Paris, France
E-mail: serge.mignani@parisdescartes.fr
Prof. Dr. X. Shi, Prof. Dr. S. Mignani
Within the nanomedicine field, the remarkably unique and
tunable properties of monodisperse dendrimers and dendrons
have offered the opportunity to open new avenues in the domain
of oncology. They are mainly used as nanocarriers of drugs,
small interfering RNA (siRNA), and anti-sense therapy (gene
delivery), as well as in diagnostics.[8] Dendrimers are
hyperbranched globular macromolecules, they are repetitively
branched molecules with a branched mode of growth and are
prepared by iterative reactions. Dendrons are also
monodisperse, owning wedge-shaped dendrimer sections with
multiple terminal groups. They generally have a single distinct
chemically addressable group at the focal point.[9]
[d]
[e]
The 2D schematic difference between dendrons and
dendrimers (generations 1, 2 and 3) is illustrated in Figure 1.
Due to their high degree of molecular uniformity, and perfect
[f]
CQM - Centro de Química da Madeira, Universidade da Madeira,
Campus da Penteada, 9020-105 Funchal, Portugal
E-mail: serge.mignani@staff.uma.pt
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Figure 2. Synthetic pathways of 9a, 9b, and metaled phosphorus dendrons 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III), and 1GC17-Au(III).
control of their shape, size, and surface groups, dendrimers and
dendrons offer a variety of drug-design options and important
suitable alternatives for the delivery of active biomolecules in
oncology applications.[9-10] Interestingly, several examples of
dendrimer- conjugated metallo-drugs have been pointed out
such as poly (alkylidenimine) dendrimers functionalized with Ru
metal,[11] and multivalent Cu(II)-conjugated phosphorus
dendrimers developed by several of us (S. M., A-M. C.; and J-P.
M.).[12] Thus, in 2013, novel Cu(II)-phosphorus dendrimers -
generations 1 (12 Cu(II)), 2 (24 Cu(II)), and 3 (48 Cu(II)) - were
prepared in good overall yield and displayed moderate to good
antiproliferative properties against KB (epidermal carcinoma)
and leukaemia HL60 cell lines (promyelocyte). The most potent
phosphorus dendrimer came from generation 3 (termed 1G3-
Cu(II), Figure S1), with 48 Cu(II) on the surface. 1G3-Cu(II) also
showed sustainable antiproliferative activities against a panel of
tumor cells such as HCT116 (human colon cancer), MCF7
(hormone-responsive breast cancer), OVCAR8 (ovarian
carcinoma), and U87-MG (human glioblastoma-astrocytoma,
epithelial-like) cancer cell lines. Outstandingly, 1G3-Cu(II)
demonstrated a good safety ratio based on its IC50 non-cancer
cells/IC50 cancer cells ratio. MRC5 (proliferative human lung
fibroblasts) and the quiescent EPC (endothelial progenitor cells,
Cyprinus carpio) have been selected as non-cancer cell lines.
The IC50s against cancer cell lines ranged between ~200 and
~800 nM, and against non-tumor cell lines between 800 and
1400 nM. Recently, Del Olmo, N. S et al. introduced the same
bidentate chelator with Cu(II) in the carbosilane dendrimer series
affording similar in vitro antiproliferative activities.[13]
Thereafter, in line with our previous work (vide supra), S.
Mignani and J-P. Majoral et al. conjugated the most potent G3
phosphorus dendrimer with Au(III) in place of Cu(II) giving 1G3-
14]
Au(III).[12,
The complexation of the dendrimer with Au(III)
strongly increased the antiproliferative activity against both KB
and leukemia HL-60 cancer cell lines versus the corresponding
1G3-Cu(II), showing IC50s in the low nanomolar range while
maintaining a good safety ratio.
Taking inspiration from the potent antiproliferative activity of
the phosphorus dendrimers 1G3-Cu(II) and 1G3-Au(III), in this
manuscript we describe the synthesis and strong antitumor
properties of four first-in-class multivalent Cu(II) and Au(III)
metaled phosphorus dendrons (generation 1, 10 terminal
groups) bearing two different types of linear alkyl chains (termed
1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III) and 1GC17-Au(III)).
Outstandingly, Cu(II)-phosphorus dendrons and Au(III)-
phosphorus dendrons displayed a distinct cell death pathway.
Results and Discussion
As shown in Figure 2, we prepared four different dendrons
bearing two distant linear alkyl chains (C11H23 and C17H35), and
bearing ten N-(pyridin-2-ylmethylene) ethanamine groups to
complex Cu(II) and Au(III) to afford 1GC11-Cu(II), 1GC17-Cu(II),
1GC11-Au(III) and 1GC17-Au(III). These dendrons were
prepared with good overall yields. The regioselective
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Figure 3. Hydrodynamic size of the metaled dendrons in normal saline (A) and medium (B) at different time periods; Cell uptake in 4T1 cells after incubation with
Au(III) metaled dendrons (C) or Cu(II) metaled dendrons (D) at the dendrons concentration of 10 μM for different time periods; Thioredoxin reductase activity of
4T1 cells incubation with 1GC17-Au(III) and 1-GC11-Au(III) at the dendrons concentration of 10 μM (E).
condensation of 4-hydroxybenzaldehyde (2) with the
hexachlorocyclotriphosphazene (1) (THF, room temperature)
afforded the AB5 monomer (3) in 76% yield. Then, 3 was
treated with the amino-phenol-derivatives 4a and 4b (cesium
carbonate, THF, room temperature) to give the dendrons 5a
and 5b in 85% yield. Thereafter, the resulting dendrons were
treated with (1- methylhydrazinyl)phosphonothioic dichloride (6)
(dichloromethane, room temperature) to afford 7a and 7b (90%
Au(III) and Cu(II) metaled dendrons exhibited approximately
similar hydrodynamic sizes at different time periods in both
media, indicating their good colloidal stability.
The primary objective of this study was to investigate the
antiproliferative activity of the four metallo-dendrons prepared.
Two different breast cancer cell lines were chosen for
antiproliferative activity: 1) 4T1, mouse breast adenocarcinoma
cells which are highly tumorigenic and invasive unlike most
tumor models used; and 2) MCF-7, human breast
adenocarcinoma cells which are widely studied as epithelial
cancer cells, as well the normal fibroblast NIH-3T3 cells and the
human fetal lung fibroblast cells MRC5 for safety purposes.
yield),
which
were
treated
with
4-(2-((pyridin-2-
ylmethylene)amino)ethyl)phenol (8) (cesium carbonate, THF,
room temperature), to give the dendrons 9a and 9b in 85%
yield. Finally, the complexation of 9a and 9b with CuCl2 and
HAuCl4 (dimethylformamide, room temperature) gave the
corresponding metaled-dendrons 1GC11-Cu(II), 1GC17-Cu(II),
1GC11-Au(III) and 1GC17-Au(III) in 78–81% yield. 1GC11-
Cu(II) and 1GC17-Cu(II) contain 10 CuCl2 on their surface,
whereas 1GC11-Au(III) and 1GC17-Au(III) incorporate 10
[AuCl2]+/[AuCl4]- on their surface. Monitoring the progress of the
reaction was performed using 31P NMR, 1H NMR, and 13C NMR
spectral analysis and inductively coupled plasma-optical
emission spectrometry (ICP-OES) analysis, which corroborate
the structure of the metaled phosphorus dendrons (Supporting
Information).
Table 1. Anti-proliferative activities of dendrons 9a, 9b, 1GC11-Cu(II),
1GC17-Cu(II), 1GC11-Au(III)) and 1GC17-Au(III).
IC50 (µM)[a]
4T1*
MCF-7*
NIH-3T3*
MRC5*
9a
>100
>100
>100
NT
9b
>100
>100
>100
NT
1GC11-Cu(II)
1GC17-Cu(II)
1GC11-Au(III))
1GC17-Au(III)
0.585±0.11
1.025±0.09
0.164±0.04
0.339±0.06
1.489±0.27
2.755±0.25
0.286±0.07
0.802±0.15
1.491 ± 0.22
4.075 ± 0.65
0.468 ± 0.12
0.132 ± 0.06
2.46 ±1.20
2.66 ± 0.64
2.71 ± 0.71
3.18 ± 0.27
The hydrodynamic sizes of the four metaled dendrons
were analyzed via dynamic light scattering (DLS). The results
are shown in Table S1. Interestingly, the hydrodynamic sizes of
the metaled dendrons with shorter alkyl chain length (C11)
(~255 nm) are smaller than those with the longer alkyl chain
length (C17) for the same metal (~ 298 nm). Meanwhile, the
presence of different metal on the surface plays a role in
hydrodynamic size of the metaled dendrons.
[a] Cells were incubated dendrons at eight concentrations (0.01 to 50 μM) for 72
h and the data were represented as “mean ± SD” (n = 3).
Firstly, we compared the anti-proliferative properties of two
phosphorus dendrons bearing Cu(II) as the metal moiety and
with two different linear alkyl chains, C11 (1GC11-Cu(II)) and
C17 (1GC17-Cu(II)) against 4T1, MCF-7, NIH-3T3, and MRC5
cells. As shown in Table 1, in the absence of metal ions, the
dendrons 9a and 9b did not display any inhibitory effect on cell
proliferation (IC50s >100µM) against the two cell lines (MCF-7
and NIH-3T3), whereas the introduction of Cu(II) increased the
To explore the stability of the metaled dendrons, the
hydrodynamic sizes of the metaled dendrons dispersed in both
o
normal saline and complete cell culture medium at 37 C were
monitored at different time periods. As shown in Figure 3A-B,
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antiproliferative activity against 4T1 and MCF-7 cells with IC50s
between 0.6 and 2.8 µM. The dendron 1GC11-Cu(II) showed
safety ratios of ~2.5 (IC50 NIH-3T3/IC50 4T1), ~4 (IC50
MRC5/IC50 4T1), 1 (IC50 NIH-3T3/IC50 MCF-7), and ~1.7 (IC50
MRC5/IC50 MCF-7), whereas, the dendron 1GC17-Cu(II)
showed safety ratios of ~4 (IC50 NIH-3T3/IC50 4T1), ~3 (IC50
MRC5/IC50 4T1), ~1.5 (IC50 NIH-3T3/IC50 MCF-7), and 1 (IC50
MRC5/IC50 MCF-7). Clearly, these data demonstrated that the
complexation of phosphorus dendrons with Cu(II) boosts the
antiproliferative activity versus nonmetallic dendrons, and the
safety ratio was related to the metal considered, to the nature of
the alkyl chain and to the nature of normal and tumoral cells
used.
previously mentioned, the safety ratio for the considered metal
is related to the nature of the alkyl chain and to the nature of
normal cell lines used.
Then, we evaluated the cellular uptake of the metaled
dendrons in 4T1 cells after the cells were treated with the
compounds at different time periods up to 24 h (Figure 3C-D).
Clearly, all compound increased the Au or Cu uptake with the
extension of incubation time. Interestingly, the metaled
dendrons with shorter alkyl chain length (C11) exhibit more
uptake than those with the longer alkyl chain length (C17) for the
same metal. Hence, for the same metal, the metaled dendrons
with C11 chain display a better therapeutic efficacy than those
with C17 chain (see Table 1).
Thereafter, our attention was directed to the replacement
of Cu(II) by Au(III). Previously, we showed that the replacement
of Cu(II) in the phosphorus dendrimer generation 3 (1G3-Cu(II))
by Au(III) (1G3-Au(III)) strongly boosted the antiproliferative
activities against the KB cancer cell lines and HL60
(promyelocytic) cell lines (vide supra) while keeping good a
safety ratio. In this direction, we prepared the metallo-dendrons
Taken together, these data fully confirm the strong
anticancer profile of phosphorus dendrons bearing linear alkyl
chains (C11H23 and C17H35) and complexed with Au(III). As in
the copper series, the nature of the alkyl chain plays a major
role in the safety ratio profile.
In order to extend the panel of cancer cell line profile, we tested
the antiproliferative activities of 1GC11-Cu(II), 1GC17- Cu(II),
1GC11-Au(III), and 1GC17-Au(III) against leukemia HL-60,
human colon cancer HCT116, and the chronic myeloid
leukemia cell line K562. As shown in Table 2, moderate
antiproliferative activities were observed regardless of the metal
complexed and the length of the alkyl chains, and IC50s were
between ~1.3 and ~8 μM.
1GC11-Au(III)
and
1GC17-Au(III)
(Figure
2).
The
antiproliferative activities of 1GC11-Au(III) and 1GC17-Au(III)
are presented in Table 1.
As observed in the phosphorus dendrimer series (vide
supra), the replacement of Cu(II) by Au(III) strongly improved
the antiproliferative activity. 1GC11-Au(III) and 1GC17-Au(III)
showed IC50s of ~0.16–0.8 μM against 4T1 and MCF-7 cells.
The improvement was between 3- and 5-times in favor of Au(III)
versus Cu(II). Interestingly, the safety ratios for 1GC11-Au(III)
were ~3 and ~1.7, for 4T1 and MCF-7 versus NIH-3T3 cells,
and ~17 and ~9, for 4T1 and MCF-7 versus MCR5 cells,
respectively. The safety ratio for 1GC17-Au(III) were ~0.3 and
~0.1, for 4T1 and MCF-7 versus NIH-3T3 cells, and ~11 and
~4, for 4T1 and MCF-7 versus MCR5 cells, respectively. As
The next study is the comparison of the antiproliferative
activities of the generation 1 metaled phosphorus dendrimers
bearing 12 metal groups and the dendrons of generation 1
bearing 10 metal groups (Figure 4). Table 2 presents the
antiproliferative activities of the dendrimers 1G1-Cu(II), 1G1-
Au(III) and, the dendrons 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-
Au(III), and 1GC17-Au(III) against HL-60 and HCT116. The
Figure 4. Schematic representation of 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III), 1GC17-Au(III), 1G1-Cu(II) and 1G1-Au(III).
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dendrimers 1G1-Cu(II) and 1G1-Au(III) are ~2 times more
potent (IC50s: 0.65 and 1 μM) than the corresponding dendrons
1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III)), and 1GC17-
Au(III) against HL-60, and the same potencies have been
observed against HCT116. This suggests that the scaffold of
dendrimers and dendrons may play a role in regulating the
different anticancer activities of the metal complexes.
probably due to the fact that C11 dendrons could be more
significantly taken up by cells than the C17 dendrons.
For the mechanisms of interaction of gold compounds with
this TrxR enzyme, related researches were assumed that the
“soft”, catalytically relevant, selenate group should constitute
the common anchoring site for gold-based inhibitors; this
assumption was grounded on the concept that “soft” gold ions,
are able to form strong bonds with “soft” Lewis donors.[15]
Therefore， a reasonable mechanisms of the Au(III) metaled
Table 2. Anti-proliferative activities of 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-
Au(III) and 1GC17-Au(III).
phosphorus dendrons could be that these compounds act as
gold(III) carriers. The “soft” gold(III) could be released possiibly
through a intracellular reductive environment when these
metaled compounds engulfed by cells. Then, The “soft” gold(III)
will reduce the activity of TrxR.
IC50 (µM)[a]
HL-60*
HCT116*
3.37±0.87
3.03±0.254
2.09±0.23
4.44±0.87
3.26±0.71
2.90±0.87
K562*
1GC11-Cu(II)
1GC17-Cu(II)
1GC11-Au(III)
1GC17-Au(III)
1G1-Cu(II)
1.93±1.37
2.65±0.65
1.33±0.25
1.34±0.30
1.00±0.20
0.65±0.04
2.36±0.20
7.8 ± 1.28
1.46 ± 0.04
2.31 ± 0.20
4T1 cells were selected due to the potent anti-proliferative
activities of 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III) and
1GC17-Au(III) (Table 1). With the purpose of evaluating the
apoptotic features of 4T1 cells by the action of phosphorus
dendrons with and without metals (9a, 9b, 1GC11-Cu(II),
Dendrons
Dendrimers
1G1-Au(III)
[a]
Cells were incubated dendrons and dendrimes at eight concentrations
1GC17-Cu(II),
1GC11-Au(III)
and
1GC17-Au(III)),
(0.01 to 50 μM) for 72 h and the data were represented as “mean ± SD” (n =
morphological change were analyzed using the fluorescence
microscopic imaging technique after 24 h at two concentrations
of phosphorus dendrons, 10 µM (Figure 5A) and 20 μM (Figure
5B). As shown in Figure 5, significant morphological changes
including granular apoptotic bodies and the appearance of
membrane blebbing were observed (Figure S11 and S12,
experimental section). As shown in Figure 5A, the four metaled
phosphorus dendrons enhanced the frequency of apoptotic
nuclei in 4T1 cells (marked with an arrow). Clearly, these
morphological observations suggested extensive DNA cleavage
to produce high molecular weight fragments (HMW) associated
with weak chromatin condensation.[16]
3).
In order to evaluate the cell death pathway of 1GC11-
Cu(II), 1GC17-Cu(II), 1GC11-Au(III) and 1GC17-Au(III),
several studies were performed including the thioredoxin
reductase activity, fluorescence imaging studies, flow
cytometric analysis and Western blot assays.
Recent studies have revealed that several cytotoxic Au(III)
complexes are potent thioredoxin reductase (TrxR) inhibitors,
which is beneficial for the improved generation of reactive
oxygen species.[1] To clarify if the intracellular TrxR can be
inhibited by the Au(III) dendrons, the TrxR activity in 4T1 cells
was evaluated. As shown in Figure 3E, after incubation of the
Au(III) dendrons (10 µM) with cells for different time periods, the
activity of TrxR decreased gradually with the incubation time.
Obviously, gold metaled dendrons are effective inhibitors of
TrxR. In addition, the Au(III) dendrons with C11 chain inhibited
the TrxR activity more significantly than those with C17 chain,
The rate of the generation and the abundance of HMW
fragments significantly depended on the following: 1) apoptotic
agent used, 2) the duration of the stimulus, and 3) the cell type
considered.[16] In this assay, 4T1 cells were treated with the four
different metaled phosphorus dendrons at different
Figure 5. Fluorescence microscopic images of 4T1 cells treated with 9a, 9b, 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III)) and 1GC17-Au(III) at 10 μM for 24 h
(A) and at 20 μM for 24 h (B). Scale bar in each panel represents 100 μm.
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Figure 6. (A) Cell cycle analysis of the 4T1 cells after incubation with 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III)) and 1GC17-Au(III) at the dendrons
concentration of 10 μM for 24h; (B) The percentages of the cellular distribution in different cell cycle phases of the 4T1 cells; (C) Western Blot assay of the
expression of protein related to S phase and apoptosis in 4T1 cells after incubation with different dendrons ([dendron] = 10 μM) for 24 h. The GAPDH protein was
used as a reference; (D) Relative protein expression levels in 4T1 cells after incubation with the different dendrons ([dendron] = 10 μM) for 24 h.
concentrations for 24 h. At high concentrations (20 µM),
metaled phosphorus dendrons stimulated the autophagosome
process and the formation of HMW fragments (Figure 5B).
These data suggest that the apoptosis is a consequence of
direct damage produced on nuclear DNA and autophagosome,
highlighting the strong proapoptotic potential of these novel
metaled dendrons. In addition, when 4T1 cells were treated
with 9a and 9b, no morphological changes of cells were
observed when compared to control cells treated with PBS
(Figure S11 and S12).
G0/G1 phase after a 24 h exposure. Particularly, phosphorus
dendrons complexed with Au(III) (1GC11-Au(III) and 1GC17-
Au(III)) were more potent promoters of cell cycle arrest than
Cu-complexed dendrons in 4T1 cells (phase G0/G1, ~ 45%;
phase G2/M, ~ 10%; and phase S, ~ 45%).
In order to elucidate the molecular mechanism of the cell
cycle arrest and apoptosis induced by the metaled phosphorus
dendrons using 4T1 cells, the cell cycle- and apoptosis-related
proteins were inspected using Western blotting (Figure 6C).
The cyclin E protein is regularly synthesized during the cell
cycle, reaching a peak in late G1 and early S phase.[17] CyclinE-
CDK2 is essential for the progression through the G1-phase of
the cell cycle and initiation of DNA replication (G1- and S-
phase transitions).[18] As shown in Figure 6D, for the treatment
with 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III) and 1GC17-
Au(III) during 24 h, up-regulation of Cyclin E and weak up-
regulation of CDK2 were observed. However, for 1GC17-Cu(II),
weak up-regulation of Cyclin E and CDK2 were observed,
indicating that the cellular percentage of phase G0/G1 is similar
for 1GC17-Cu(II) and the control group (~45 %). Additionally,
the expression of tumor suppressor P53 were also detected.
P53 regulates the cell cycle restriction point which is related to
the DNA damage checkpoint. In response to DNA damage, P53
is activated and turns on transcription of one of its downstream
genes, then the downstream genes activate cyclin-Cdk
complexes to stop DNA replication.[19] After treatment of the
cells with metaled phosphorus dendrons, the cells displayed a
high level of P53 versus the control group. Previous
experiments showed that up-regulation of P53 induces G2/M
cell cycle arrest.[20] Consequently, the metalated dendrons
It is commonly accepted that the cytotoxicity of
antiproliferative agents is primarily associated with cell cycle
arrest in G1, S or G2/M phases which trigger the final apoptosis
pathway and cell death. Since metaled phosphorus dendrons
displayed the ability to promote autophagosome and apoptotic
effects against 4T1 cells, we addressed the question about the
perturbation effect of the metaled phosphorus dendrons on cell-
cycle progression (Figure 6). To evaluate this effect, the impact
of the metaled dendrons 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-
Au(III)) and 1GC17-Au(III) at 10 μM was investigated in 4T1
cells treated for 24 h. The flow cytometric analysis of the cell
cycle distribution is presented in Figure 6A, whereas the
percentages of the cellular distribution in different cell cycle
phases (G0/G1, S, and G2/M) are given in Figure 6B.In the
control group, cells contained 2n chromosomes (phase G0/G1,
~45 %) and few cells were in an active DNA synthesis stage
(phase S, ~30 %) or already engaged in the mitosis process
(phase G2/M, ~ 22 %).However, for Cu-metaled phosphorus
dendrons (1GC11-Cu(II) and 1GC17-Cu(II)), cells are in the
mitosis phase (S) (~45%) without a substantial increase of the
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10.1002/chem.202001014
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[Au(III)Cl2]+ on their surface, and with C11H23 and C17H35 linear
alkyl chains were synthesized, and showed significant
antiproliferative activity against cancer cell lines such as 4T1
and MCF-7 (breast cancer). The complexation of phosphorus
dendrons with Cu(II) or Au(III) boosts its antiproliferative activity
compared to nonmetallic dendrons. The safety ratio depends
on the metal, the nature of the alkyl chain, and the nature of the
normal cells used. The antiproliferative activities against 4T1
and MCF-7 cells showed IC50s between 0.6 and 2.8 µM. Similar
to the phosphorus dendrimers, the replacement of Cu(II) by
Au(III) strongly improved the anti-proliferative activities. 1GC11-
Au(III) and 1GC17-Au(III) dendrons showed IC50s of ~0.16–0.8
μM against 4T1 and MCF-7 cells. In addition, short alkyl chain
length of the dendrons renders Au(III) complexes with more
significant antiproliferative activity to kill cancer cells. Cell death
pathway analysis reveals that the metaled dendrons could alter
the cell cycle- and apoptosis-related protein status of cells,
resulting in cell cycle S-phase arrest and apoptosis. In
particular, Au(III)-complexes induced the caspase-dependent
cellular lethality by promoting the translocation of Bax to the
mitochondria and the release of Cyto C, whereas the Cu(II)-
complexes are weak activators of caspase-3, in line with their
moderate antiproliferative activity in cancer cells.
influenced the expressions of cell cycle regulatory proteins
which altered the cell cycle distribution. In addition, the
expression of cytochrome C (Cyto C), Caspases-3, and Bax
were also detected. Caspases-3 are known to play a pivotal
role in both initiation and execution of apoptosis.[21] It has been
reported that caspase-dependent apoptosis requires the
release of proteins sequestered in the mitochondria of cancer
cells but the high inner membrane potential prevents the
opening of mitochondrial pores.[21] One of the key controls to
open the mitochondrial membrane pores is the regulation of the
level of Bax.[22] During the 24 h treatment with 1GC11-Au(III))
and 1GC17-Au(III), the cells displayed up-regulation of Cyto C,
Caspase-3, and Bax, while 4T1 cells treated with the Cu(II)-
dendrons did not display significative changes in the regulation
of Cyto C, Caspase-3 and Bax when compared to the control
cells treated with PBS. In a nutshell, the 4T1 cells treated with
Au(III)-dendrons exhibited higher levels of caspase-3 and Bax
than the group of Cu(II)-dendrons.
Taken together, these studies demonstrated that the
dendrons complexed with Au(III) increased the cellular lethality
(4T1 cells) and the cell death pathway is related to caspase-
dependent process, promoting the translocation of Bax to the
mitochondria and then the release of Cyto C, inducing the
activation of the apoptosis process.
Taken together, these studies showed that these first-in-
class metaled phosphorus dendrons represent a novel class of
anticancer nano-drugs, and their development will open new
avenue to tackle cancer. We thus ought to see the emergence
of novel dendron-based particles to be applied against difficult
diseases such as cancers and distant metastases.
To sum up, Au(III)-complexed dendrons (1GC11-Au(III)
and 1GC17-Au(III)) clearly demonstrated their ability to
promote cell death in a caspase-dependent pathway by
facilitating the translocation of Bax to the mitochondria and the
release of Cyto C, whereas Cu(II)-complexed dendrons
(1GC11-Cu(II) and 1GC17-Cu(II)) are weak activators of
caspase-3. Also, copper-based dendrons showed weak
antiproliferative activities in contrast with Au(III)-complexed
ones. Table 3 summarizes the mechanism of actions of the
phosphorus dendrons 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-
Au(III) and 1GC17-Au(III) versus phosphorus dendrimers 1G3
and 1G3-Cu.
Acknowledgements
This research is financially supported by the National
Natural Science Foundation of China (21911530230, 21773026,
and 81761148028) and Sino-French Cai Yuanpei Programme.
S. Mignani and X. Shi acknowledges the support of FCT –
Fundacao para a Ciencia e a Tecnologia with Portuguese
Government funds through the CQM Strategic Project PEst-OE/
QUI/UI0674/2013, and ARDITI-Agencia Regional parao
Desenvolvimento da Investigacao Tecnologia through the
project M1420-01-0145-FEDER-000005 – Centro de Quımica
da Madeira – CQM+ (Madeira 14-20 Program). J-P. Majoral, A-
M. Caminade and R. Laurent thank the CNRS (France) for
financial support.
Table 3. General view of cell death pathway of phosphorus dendrons
prepared in this study in comparison with the corresponding dendrimers.
Nanoparticles
Translocation of
Bax
Caspase-3
activation
Phosphorus dendrons
Strong effect (4T1):
1GC11-Au(III)) and
1GC17-Au(III)
Strong effect (KB
and HL60):
Weak effect (4T1):
1GC11-Cu(II),
1GC17-Cu(II)
Strong effect (KB
and HL60): 1G3
Phosphorus
dendrimers[12, 14]
1G3-Cu(II)
Conflict of interest
There are no conflicts to declare.
Conclusion
Keywords: anti-proliferative activities • metaled phosphorus
dendrons • fluorescence imaging • flow cytometric analysis
In summary, first-in-class Cu (II) and Au (III) metaled
phosphorus dendrons (generation 1) bearing 10 Cu(II)Cl2 or
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Entry for the Table of Contents
FULL PAPER
First-in-class
metaled phosphorus dendrons were
developed and displayed potent
Cu(II)
and
Au(III)
Liang Chen, Yu Fan, Jieru Qiu, Régis
Laurent, Jin Li, Jérôme Bignon, Serge
Mignani,*
Xiangyang
Majoral*
Anne-Marie
Shi* and
Caminade,
Jean-Pierre
anticancer activity stemming from
different cell death pathways. Cu(II)
metaled dendrons showed
caspase-independent cell
a
potent
death
Page No. – Page No.
pathway; whereas Au(III) metaled
dendrons displayed caspase-
dependent apoptotic pathway. These
metaled phosphorus dendrons
Potent antitumoral efficacy of first-in-
class Cu(II) and Au(III) metaled
phosphorus dendrons with distinct
cell death pathways
a
represent a novel class of anticancer
nano-drugs, and their development
will open new avenue to tackle
cancer.
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