Combating metastasis of breast cancer cells with a carboplatin analogue containing an all-trans retinoic acid ligand

Article abstract of DOI:10.1039/d0dt00507j

Pt-ATRA, a carboplatin analogue containing an all-trans retinoic acid (ATRA) derivative ligand, was synthesized via a click reaction. Upon cellular internalization, Pt-ATRA exhibits a dual function, releasing an active Pt(ii) moiety to induce cell apoptosis and ATRA to inhibit tumor metastasis.

Full text of DOI:10.1039/d0dt00507j

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Combating metastasis of breast cancer cells with a
carboplatin analogue containing an all-trans
retinoic acid ligand†
Cite this: DOI: 10.1039/d0dt00507j
Received 11th February 2020,
Accepted 20th March 2020
a,b
c
a
a
d
Yi Dai,‡ Hai Huang, ‡ Yang Zhu, Junjie Cheng, Ai-Zong Shen* and
a
DOI: 10.1039/d0dt00507j
Yangzhong Liu
*
rsc.li/dalton
1
4
Pt-ATRA, a carboplatin analogue containing an all-trans retinoic differentiation and arrest the proliferation of cancer cells;
acid (ATRA) derivative ligand, was synthesized via a click reaction. this activity could also render cancer cells more susceptible to
1
5–17
Upon cellular internalization, Pt-ATRA exhibits a dual function, other cytotoxic therapies.
For instance, ATRA strongly
releasing an active Pt(II) moiety to induce cell apoptosis and ATRA enhances the apoptosis induced by arsenic trioxide or cisplatin
1
8,19
to inhibit tumor metastasis.
in various human cancer cells.
Indeed, ATRA was found to
modulate the plasticity and inhibit the motility of breast
cancer cells, implying an anti-metastasis application.
Based on these finding, we hypothesized that the combination
of ATRA could enhance the anti-metastasis activity of platinum
drugs. Therefore, we designed a carboplatin analogue (Pt-
ATRA), in which the ligand cyclobutane-1,1-dicarboxylic acid
Breast cancer is the most common malignancy in women and
20
21,22
1
–3
has a high mortality rate.
Notably, among various causes,
4
metastasis is responsible for 90% of deaths of breast cancer.
Therefore, combating cancer metastasis is an urgent need for
the treatment of breast cancer in addition to the inhibition of
the proliferation of cancer cells. Although many advanced
methods, such as immunotherapy and targeted therapy, have
been developed for cancer treatments, the most widely used
agents for metastatic breast cancer are chemotherapeutics. For
instance, paclitaxel and platinum based anticancer drugs are
approved by the FDA for the treatment of metastatic breast
(
CDBCA) of carboplatin was replaced by an ATRA-bis(carboxy-
lato) derivative. Upon internalization, Pt-ATRA is hydrolyzed in
cells to release the activated platinum moiety and ATRA
(
Scheme 1). The inhibition of the proliferation and metastasis
of breast cancer cells has been analyzed, and the anti-meta-
stasis mechanism has been investigated.
5
–8
cancer.
The application of platinum drugs and paclitaxel
Pt-ATRA is prepared via a click reaction between the propy-
nyl ester of retinoic acid and a carboplatin analogue contain-
ing azide. The preparation of Pt-ATRA was designed using mild
experimental conditions since ATRA is sensitive to light and
heat. Therefore, the Cu-catalytic azide–alkyne click (CuAAC)
reaction was employed. In brief, diethyl 2-(6-bromohexyl)malo-
nate was synthesized by the reaction of diethyl malonate with
demonstrates an effective antitumor efficacy; however, it has
been reported that the chemotherapy could even increase the
9
,10
motility and invasion of cancer cells.
While reducing the
number cancer cells, the increased proportion of cancer stem
cells (CSCs) during chemotherapy is associated with cancer
1
1,12
recurrence and metastasis.
Therefore, improving the anti-
metastasis activity of platinum drugs is highly desired.
All-trans retinoic acid (ATRA) is a clinically used anti-cancer
1
3
agent with limited toxicity. ATRA has been found to induce
a
CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University
of Science and Technology of China, Hefei, Anhui, 230026, China.
E-mail: liuyz@ustc.edu.cn
b
College of Pharmaceutical Sciences, Anhui Xinhua University, Hefei, Anhui, 230038,
China
c
West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
d
The First Affiliated Hospital of University of Science and Technology of China, Hefei
230001, China
†
Electronic supplementary information (ESI) available: Experimental section;
NMR and ESI-MS spectra; images of migration, invasion, and nodules on the
lungs; etc. See DOI: 10.1039/d0dt00507j
Scheme 1 A schematic illustration of the anti-tumor and anti-meta-
‡
These authors contributed equally to this work.
stasis actions of Pt-ATRA.
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,6-dibromohexane under the conditions of potassium carbon-
ate and benzyl triethyl-ammonium bromide (TEBA), and
then reacted with sodium azide to replace the bromine atoms
to obtain diethyl 2-(6-azidohexyl)malonate (Scheme 2).
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1
2
3
After hydrolysis of diethyl 2-(6-azidohexyl)malonate with
NaOH, the resulting compound
NH (NO ] to form intermediate compound 2. Another
branch of synthesis is esterification of ATRA with propargyl
bromide under the conditions of Ce CO to form an inter-
1
reacted with [Pt
(
3
)
2
3 2
)
2
3
2
4
mediate compound 3. After the preparation of compounds 2
and 3, Pt-ATRA was generated via a click reaction catalyzed by
sodium ascorbate and copper sulfate. In addition, we syn-
thesized compound 5 as a control using the same synthetic
method as that for Pt-ATRA. All the reactions were conducted
under ambient conditions in order to reduce the destruction
of ATRA at high temperatures. The products were verified by
Fig. 1 In vitro and in vivo anti-tumor growth efficacy. (A) 4T1 cell viabi-
lity under different concentrations of the compounds from MTT assay.
NMR and ESI-MS (Fig. S1–S6, ESI†). Pt-ATRA is a light yellow (B) Tumor growth curves show the effects of the different treatments on
1
tumor volume. (C) The tumor weight in each group at the end of the
experiments. (D) The body weights of mice during the treatments. Balb/
solid and characterized as follow: H NMR (300 MHz, DMSO-
6
d ) δ 1.00 (s, 6H), 1.23 (brs, 6H), 1.42 (m, 2H), 1.56 (m, 2H),
c mice bearing 4T1 tumors were injected through the tail vein with PBS,
carboplatin, compound 5, ATRA and Pt-ATRA. The Pt(II) dose per injec-
1
=
2
(
(
.67 (s, 2H), 1.79 (brs, 4H), 1.98 (m, 6H), 2.30 (s, 3H), 3.50 (t, J
6.9 Hz, 1H), 4.14 (m, 6H), 4.33 (t, J = 7.0 Hz, 2H), 5.14 (s,
H), 5.84 (s, 1H), 6.19 (m, 3H), 6.41 (d, J = 15.3 Hz, 1H), 7.08 injection was equivalent to 5.4 mg kg− body weight (n = 5). PBS was
dd, J = 15.1, 11.4 Hz, 1H), 8.16 (s, 1H). ESI-MS: m/z = 794.8
calculated 794.3).
−1
tion was equivalent to 1.5 mg kg body weight and the ATRA dose per
1
used as a control. Mice were treated 6 times, every other day. Error bars
denote standard deviations.
The in vitro cytotoxicity of Pt-ATRA was evaluated on 4T1
breast cancer cells. MTT assay showed that the IC50 value of Pt-
ATRA (68.07 ± 3.92 μM) was comparable to those of carboplatin higher antitumor activity than that of compound 5 and ATRA,
50.62 ± 4.99 μM) and compound 5 (60.24 ± 6.35 μM) (Fig. 1A). and was almost equivalent to that of carboplatin (Fig. 1B and
(
This result suggests that these Pt(II) chelation complexes have C). More importantly, the body weights of the mice clearly
a similar potency in the inhibition of cell proliferation. The decreased during the treatment with carboplatin or ATRA,
cellular drug internalization analysis showed that the treat- while little affected by Pt-ATRA, implying the low toxicity of Pt-
ment of Pt-ATRA resulted in more platinum accumulation in ATRA at the dosage used in this measurement (Fig. 1D). These
cells in comparison to that with carboplatin, while a slightly observations suggest that Pt-ATRA possesses the same anti-
lower DNA platination level was detected by the treatment of cancer activity but with a lower toxicity relative to carboplatin
Pt-ATRA (Fig. S7†). The DNA platination levels are consistent at the same dosage.
with the results of the cytotoxicity assay.
3 2
Pt-ATRA is formed by the coordination of the Pt(NH )
The in vivo antitumor effect of these compounds was moiety with a chelation leaving group. This leaving group of
assessed on the 4T1 breast tumor bearing mice model. These the ATRA derivative contains a bis(carboxylato) ligand, and
compounds were administered through intravenous injection therefore the coordination structure of Pt-ATRA is similar to
every other day 6 times. The average tumor size and body that of carboplatin. It has been well-investigated that the
weight were monitored to evaluate the drug efficacy and tox- equation of carboplatin results in the release of the CDBCA
icity effects. The results showed that Pt-ATRA has a slightly ligand and generates the active Pt(II) moiety for DNA
2
5,26
binding.
On the other hand, the leaving group of Pt-ATRA
is formed through an ester bond between ATRA and the
hydroxyl group of a malonic acid derivative. Ester bonds are
typically designed in prodrugs since they can be broken by an
2
7–29
esterase catalyzed hydrolysis in the cellular environment.
Therefore, Pt-ATRA can produce both an active Pt(II) moiety
and ATRA in cells. Since ATRA is able to inhibit the motility of
breast cancer cells, the anti-metastasis effect of Pt-ATRA was
evaluated with a scratch assay on monolayer cells. The control
experiment showed that the scraped cells completely recovered
after 24 hours. The treatment of ATRA reduced the wound
healing rate to 13.53%, showing the inhibition of cell
migration by ATRA. The complex Pt-ATRA also reduced the
ability for migration and inhibited the wound healing rate to
Scheme 2 Synthetic route to Pt-ATRA and compound 5.
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Fig. 3 In vivo anti-tumor metastasis effects. (A) The number of meta-
static tumor nodules on the lungs of mice at the end of treatment. Error
bars denote standard deviations. Asterisks indicate p-values relative to
the control group (*: p < 0.05; **: p < 0.01; ***: p < 0.001). (B)
Representative histopathologic examinations of the lungs. The meta-
static tumors in the lungs are marked with black dotted circles.
Fig. 2 The effects of compounds on the migration and invasion of
cancer cells measured using a scratch test and transwell assay. 4T1 cells
were pre-incubated for 24 h with drugs at a dose of 50 μM. Analyses of
the lateral migratory cells (A) were obtained by measuring the wound
closure rate. Quantitative analyses of migratory cells (B) and invasive
cells (C) were obtained by measuring the UV absorbance at 550 nm of
crystal violet dye that was extracted from the migration or invasion cells
on the lower surface of the membrane. Error bars denote the standard
deviations of three independent experiments and asterisks indicate
p-values relative to the control group (*: p < 0.05; **: p < 0.01; ***: p <
0
.001, T test).
1
5.62%, which is comparable to that of ATRA (Fig. 2A and
Fig. S8†). By comparison, carboplatin and compound 5
demonstrated a much lower inhibition effect on the cell
migration, confirming the function of ATRA derivative as a
ligand on the platinum complex. Cell migration and invasion
Fig. 4 The expression of stemness-associated genes measured via
qRT-PCR on an mRNA level. Three genes (Sox2, Oct4 and Nanog) were
were also measured using a transwell experiment to further analyzed on 4T1 cells that were treated with equivalent doses of Pt(II)
1
−1
(
2
0
0.5 mg mL− ) and/or ATRA (1.9 mg mL ) in different formulations for
validate the anti-metastasis effect of Pt-ATRA. In agreement
with the scratch assay results, ATRA and Pt-ATRA effectively
inhibited the cell migration and invasion, while carboplatin
and compound 5 showed much less activity (Fig. 2B, C and
Fig. S9†).
4 h. Asterisks indicate p-values relative to the carboplatin group (*: p <
.05; **: p < 0.01; ***: p < 0.001).
As cancer stem cells (CSCs) are believed to be involved in
tumor metastasis,
was measured in order to understand the cause of the anti-
metastasis effect of Pt-ATRA. Three CSC-associated genes,
including Sox2, Oct4 and Nanog, were analyzed since they are
The anti-metastasis effect of Pt-ATRA was also assessed
in vivo on the 4T1 breast tumor bearing mice model. The
tumor nodules were clearly observed on the surface of the
lungs in the control group, showing a strong pulmonary meta-
stasis of 4T1 cells. Consistent with the in vitro assay, ATRA
inhibited the anti-metastasis and reduced the number of
tumor nodules from 16 to 4 (Fig. 3A and Fig. S10†).
Carboplatin and compound 5 only reduced the number of
tumor nodules to 13, indicating the marginal anti-metastasis
activity of these Pt(II) complexes. The coordination of the
ligand of the ATRA derivative significantly enhanced the anti-
metastasis activity of the Pt(II) complex, Pt-ATRA reduced the
tumor nodules to 5, which is similar to the treatment of free
ATRA. Furthermore, the tumor burden inside the lung tissue
was analyzed using H&E staining. The results clearly showed
that the treatment of ATRA and Pt-ATRA prevented tumor
growth inside the lung tissue more efficiently than compound
3
0–34
the expression of CSC-associated genes
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5–40
highly expressed in breast CSCs.
The RT-qPCR measure-
ment showed that the treatments of carboplatin and com-
pound 5 can slightly increase the gene expression level,
whereas the treatments of ATRA and Pt-ATRA suppressed the
expression of the correlated genes (Fig. 4). This result
suggested that ATRA and Pt-ATRA can reduce the stemness of
cancer cells, which is probably associated with the anti-meta-
stasis activity of Pt-ATRA.
Conclusions
5
and carboplatin. These results confirmed that the ligand of In summary, a carboplatin analogue (Pt-ATRA) was synthesized
the ATRA derivative designed in this work results in the in this work. Pt-ATRA is constructed by the coordination of Pt
remarkable anti-metastasis effect of ATRA.
(II) to a chelation ligand containing an all-trans retinoic acid
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ATRA) derivative. This design endows Pt-ATRA with both anti-
proliferation and anti-metastasis functions toward tumor cells.
Upon cellular internalization, Pt-ATRA is hydrolyzed to release
the activated platinum moiety and ATRA, exerting simul-
taneously anti-proliferation and anti-metastasis. In vitro cyto-
toxicity assays showed that Pt-ATRA has a similar potency to
Dalton Transactions
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Acknowledgements
This work was supported by the National Key R&D Program of
China (2017YFA0505400), the National Science Foundation of
China (21877103), and the Major Program of Development
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