Traceable in-cell synthesis and cytoplasm-to-nucleus translocation of a zinc Schiff base complex as a simple and economical anticancer strategy

Article abstract of DOI:10.1039/c9cc03480c

We have proposed a facile and cheap cancer therapeutic strategy by in-cell synthesizing theranostic Zn Schiff base complexes with nuclear targeting and DNA damaging capability. The in-cell synthesis can be traced via a green-to-blue fluorescence shift, an

Full text of DOI:10.1039/c9cc03480c

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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Traceable
In-cell
Synthesis
and
Cytoplasm-to-Nucleus
Translocation of A Schiff base Zinc Complex as A Simple and
Economical Anticancer Strategy
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www. rsc.org/
†
†
Qian Cao [a] , Jing Yang [a] *, Hang Zhang [a], Liang Hao [a], Gang-Gang Yang [a], Liang-Nian Ji [a], and
Zong-Wan Mao [a]
*
We have proposed a facile and cheap cancer therapeutic strategy
On the other hand, luminescent zinc complexes have been
by in-cell synthesizing theranostic Zn Schiff base complexes with widely explored as cellular imaging agents, in which Zn(II) can
nuclear targeting and DNA damaging capability. The in-cell stabilize the structure of the organic ligand and improve the
synthesis can be traced via green-to-blue fluorescence shift, rigidity, thus exhibiting enhanced photo-stability and quantum
and the subsequent cytoplasm-to-nucleus translocation yield.12-18 This also includes Zn Schiff base complexes.
realizes cancer-specific therapy both in vitro and in vivo.
Moreover, some transition metal Schiff base complexes have
been reported possessing unique medicinal activities ca. anti-
bacteria, anti-tumor and anti-virus.19-20 Inspired by these,
rational design of theranostic nuclear-targeted Zn Schiff base
complexes is very promising, which can simultaneously induce
and monitor the therapeutic effects, thus giving insights into
the anticancer mechanisms. However, such zinc complexes
have not yet been reported.
In this work, we have proposed a facile and cheap cancer
therapeutic strategy, in which a dated Schiff base (L1) is turned
into a promising theranostic agent by simply using zinc salt as
an auxiliary reagent to generate a Zn-Schiff base complex
(ZnHL1) in living cells, exhibiting nuclear permeability, DNA
damage capability and anti-cancer activity. As shown in
Scheme 1, the in-cell synthesis of ZnHL1 can be traced in real-
time through the Zn2+ induced green-to-blue fluorescence
changes and cytoplasm-to-nucleus translocation. The disulfide
linked Schiff base L1, positively charged ZnHL1 and neutral ZnL1
complex were synthesized and characterized by ESI-MS, 1H
NMR, and elemental analysis (Fig. S1-S4, ESI†). ZnHL1 is found
insoluble in most solvents (water, methanol, ethanol, diethyl
ether) and only partly soluble in DMF and DMSO, which may
seriously restrict its biological application. The solubility of
neutral complex ZnL1 was even worse.
Initially, the selectivity of L1 towards a series of different
biologically relevant metal ions such as Na+, K+, Ag+, Ca2+, Mg2+,
Fe2+,Fe3+, Co2+, Ni2+, Cu2+, Mn2+, Cd2+, Al3+, Cr3+ was studied.
UV-Vis spectra show that introduction of Zn2+ into the solution
of L1 induces an intensive new absorption centered at 365 nm,
which is not prominent in other metal ion cases (Fig. S5A, ESI†).
Under 365 nm excitation, L1 exhibits weak green fluorescence
in a DMSO-H2O (1:1, v/v) solvent; upon introducing various
metal ions (3 equiv), Zn2+ is the only one that induces a green-
to-blue fluorescence change, while other metal ions only
induce negligible fluorescence enhancement (< 10%) or
Zinc(II) is the second most abundant transition metal in
human body, and zinc homeostasis alternations have been
linked to many disease.1 Nowadays, zinc complexes have
attracted a lot of interest in the field of cancer therapy based
on the fact that 1) zinc(II) is significantly non-toxic even at
higher doses compared with other metals (Fe, Cu, Hg, ect),
which is beneficial to biocompatibility;2-4 2) owing to the ability
in assisting Lewis activation, nucleophile generation, and rapid
ligand exchange, zinc complexes can be adept in the catalysis
of hydrolytic reactions, such as DNA hydrolysis and cleavage,
thus making the anti-cancer activity possible.5-6 Although many
Zn complexes have been developed in this purpose and some
of them exhibit excellent DNA (duplex or quadraplex) binding
affinities in vitro,7 their anti-cancer efficacy at cellular level are
very limited and the mechanisms of action are still not very
clear. This is because most of these complexes localize in the
cytoplasm rather than the nucleus, resulting in negligible
inhibitory activity towards DNA expression in living cells.8
There are also a few examples of nuclear penetrated zinc
complexes, such as a nitro-substituted ZnSalen reported
by Laura,9 a Zn-terpyridine complex reported by Tian,10 and a
Zn-naphthalimide complex reported by Guo,11 however, their
cytotoxicity is still very low. To the best of our knowledge,
nuclear-targeted anti-cancer zinc complexes are very rare and
the in vivo anticancer investigations are still lacking.
a. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry, Sun Yat-Sen University, Guangzhou 510275 (P. R. China). E-mail:
yangjing8@mail.sysu.edu.cn (J. Yang); cesmzw@mail.sysu.edu.cn (Z.-W. Mao).
†These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [characterization of ligand
and Zn complexes; metal ion selectivity experiment; ATP assay; changes in the
nuclear morphology of apoptotic cells; and so on]. See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C9CC03480C
ATP depletion
DNA damage
Zn2+
Zn
Zn
Cytoplasm-to-nuclear
translocation
Cancer cell
apoptosis
Zn
S
S
S
S
+
Zn2+
N
N
O
N
N
Zn
HO
OH
O
L1
H
ZnHL1
L2
GSH
S
Zn2+
N
SH
N
OH
air
OH
quench the emission (Fig. 1A and S5B, ESI † ). Competition
experiment was also conducted by adding more equivalents of
other metal ions, showing that most of them do not interfere
the blue fluorescence is gradually emerging and the final
spectrum is similar to that of ZnHL1. This cascade “on-off-on”
phenomenon suggests that even if GSH can cleave the S-S
Scheme 1 Zn2+ induced intracellular synthesis of Zn-Schiff base
complex, indicated by the green-to-blue fluorescence changes
followed by the cytoplasm-to-nucleus translocation, resulting
in ATP depletion, nuclear DNA damage and cancer cell
apoptosis.
Fig. 1 (A) Photographs of L1 in the presence of different metal
ions (3 equiv) under 365 nm UV lamp; (B) UV/Vis absorption
and (C) fluorescence changes of L1 (30 µM) with increasing
concentrations of Zn2+ ions (0-40 µM) in DMSO-H2O (1:1, v/v)
solutions; (D) Job-plot indicates the 1:1 binding stoichiometry
between L1 and Zn2+ by absorption changes at 405 nm.
with the fluorescence analysis of Zn2+ significantly (Fig. S5C,
ESI†). Although excess Al3+, Fe3+ and Cu2+ quench the
fluorescence, these free metal ions are trace in cells which will
not affect the cellular probing of abundant exogenous Zn2+.
These results suggest the specific fluorescence response of L1
to Zn2+, which is visible to naked eyes.
bond of L1 to generate non-emissive L2
,
oxidative
interconversion of L2 can take place in the presence of Zn2+ to
still give ZnHL1 as the final product. The oxidative
interconversion has also been proved by FTIR in previous
literature.21
Secondly, Zn2+ titration experiments were conducted in
DMSO-H2O (1:1, v/v) solvent. UV-Vis absorption spectrum of L1
shows a gradual decrease at 315 and 410 nm with the
concomitant formation of a new band at 365 nm upon Zn2+
addition (Fig. 1B). Simultaneously, weak fluorescence of L1 is
gradually enhanced and the emissive maxima is gradually blue-
shifted from 515 nm to 455 nm (Fig. 1C), in which the intensity
ratio of the blue and green fluorescence (I455/I565) displays a
good linear relationship with the zinc ion concentrations in the
0-20 µM range (Fig. S6A, ESI†).The complexation stoichiometry
and binding affinity of L1 with Zn2+ is found to be 1:1 by Job-
plot (Fig. 1D) and 0.93×104 M-1 by absorption methods using
Benesi-Hildebrand equation, respectively (Fig. S6B, ESI†).
Moreover, upon Zn2+ addition the molecular ion peak of L1
(m/z = 361.00) disappears with the concomitant formation of a
cluster peak centered at m/z = 442.60, which has been
assigned to [ZnHL1 + H2O]+ (Fig. S7A, ESI†), and the NMR signal
of phenolic OH of L1 (13.3 ppm) gradually decreases (Fig. S7B,
ESI†). These results validate the formation of ZnHL1 complex.
Thirdly, although L1 exhibits good stability at physiological
pH at least in 24 h (Fig. S8, ESI †), it may easily undergo S-S
All the above results provide sufficient evidence proving the
facile formation of ZnHL1 by simple mixing Zn2+ and L1 in
solvent, and the green-to-blue fluorescence change can be
used as an indicator for the intracellular formation of ZnHL1, no
matter Schiff base undergoes S-S bond cleavage or not.
Accordingly, in-cell synthesis of ZnHL1 is highly probable and
can be traced by confocal microscopy (ex= 405 nm). Initially,
living A549 cells was incubated with L1 (40 M) at 37oC for 6 h
to make sure the sufficient cellular uptake of the ligand. After
refreshing the culture medium, cells were further incubated
with Zn2+ (100 M) and cellular fluorescence at different time
period was analyzed with a dual-emission mode (em: green
channel 520±20 nm; blue channel 450±20 nm). As shown in Fig.
2A, L1 displays green fluorescence mainly localizing in
cytoplasm. Upon Zn2+ addition (0.5 h), a significant increase in
the blue fluorescence is observed accompanied with a
dramatic drop of the green fluorescence, indicating the in-cell
formation of ZnHL1, which mainly localizes in cytoplasm at
early stage. Co-localization experiment indicates that the blue
fluorescence from ZnHL1 is not colocalized with mitochondria-
or lysosome-specific dyes (Fig. S10, ESI † ). With prolonged
incubation time (up to 12 h), most of the blue fluorescence
migrates from cytoplasm into nucleus, finally exhibiting a high
degree of co-localization (pearson’s correlation coefficient
0.80) with the commercial DNA stain Syto59 (Fig. 2B). This
indicates the excellent nucleus penetrating ability of the in-cell
synthesized ZnHL1. Average Fblue/Fgreen ratios measured from
five cells in Fig. 2A shows a gradual increase with incubation
1
bond reduction in the presence of SH containing species. H
NMR spectra show that after reacting with ehyl thioglycolate
the signals of L1 are significantly reduced accompanied with
new signals rising, for example, the O-Ha and C-Hb peaks of L1
at 13.30 and 8.59 ppm disappear accompanied with new O-Ha’
and C-Hb’ peaks emerging at 9.92 and 5.65 ppm, indicating the
S-S reduction (Fig. S9A, ESI † ). This is also confirmed by
fluorescence spectroscopy (Fig. S9B, ESI † ), in which green
fluorescence from L1 is completely quenched after incubating
with GSH (5 mM) for 10 min. However, after addition of Zn2+
2 | J. Name., 2012, 00, 1-3
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time (Fig. S11, ESI†). According to ctDNA titration and EB λex = 405 nm; (B) Colocalization of intracellular synthesized
View Article Online
DOI: 10.1039/C9CC03480C
competition experiments (Fig. S12, ESI†), ZnHL1 may interact ZnHL1 (40 μM, 12 h) with nuclear dye Styo-red (λex/λem
with DNA after translocating to the nucleus, which is similar to 622/645 ± 20 nm) in A549 cells. Scale bar: 5 µm.
other ZnSalen complexes.7,13 The in-cell synthesis and
=
Table 1 IC50 (μM) values of tested compounds towards
distribution of ZnHL1 complex was also demonstrated by ICP-
MS, in which A549 cells were incubated with different
concentrations of L1 (0, 20 and 40 μM) for 12 h and then
treated with the same amount of Zn2+ (100 μM) for another 12
h. As shown in Fig. S13 (ESI†), Zn(II) contents in the whole cell
and nucleus both increase in a L1 dose-dependent manner,
indicating the intracellular generation of various
concentrations of ZnHL1, ca. 60% of which is localized in
nucleus. All these results demonstrate the efficient nuclear
internalization of in-cell synthesized ZnHL1, as indicated by the
green-to-blue fluorescence change and subsequent cytoplasm-
to-nucleus fluorescence translocation.
different cell lines.
Compound A549
MCF-7
>100
HeLa
>100
>100
HLF
L1
>100
>100
>100
>100
ZnCl2
>100
ZnHL1
ZnCl2 + L1
Cisplatin
50.3 ±4.5 41.2 ±2.7 87.4 ±6.7 97.5 ±3.4
21.5 ±2.1 19.3 ±2.5 16.3 ±1.9 95.5 ±7.8
22.3 ±2.1 24.2 ±1.7 15.5 ±1.2 27.5 ±2.3
Zn2+ (100 µM, 36 h) or L1 (100 µM, 36 h) alone to induce
apoptosis is negligible; however, dramatically dose-
a
dependent increase in the percentage of apoptotic cells is
observed from 24.5 ± 0.5 % (10 µM) to 61.4 ±0.82 % (40
µM) based on the in-cell synthesis of different amount of ZnHL1
(Fig. 3A). Moreover, owing to the nuclear penetrating and
staining ability, in-cell synthesized ZnHL1 can induce cell
apoptosis and track nuclear condensation and fragmentation
simultaneously (Fig. S15, ESI†).
Secondly, in-cell synthesized ZnHL1 induces a substantial
decrease in the intracellular ATP levels in dose-dependent
manner (25-100 µM), whereas free ligand L1 or zinc salt did not
affect the ATP levels even at a high concentration of 100 µM
Then the in vitro cytotoxicity against several different
cancerous (A549, HeLa, MCF-7) and normal (HLF) cell lines was
determined by 48 h MTT assay. As shown in Table 1, treatment
with either zinc salt or L1 alone does not affect the cell viability
even at 100 M. Pre-prepared ZnHL1 solution exhibits a
medium cytotoxicity against cancer cells with IC50 values of
40~90 M. Interestingly, when the cells are pre-treated with
Zn2+ (100 M,
2 h) and then incubated with various
concentrations of L1 for another 46 h incubation, significantly
enhanced cytotoxicity against cancer cell lines is observed with
IC50 values of 15-20 M, which are similar to that of cisplatin.
By contrast, its cytotoxicity against normal cell line HLF is much
lower with IC50 values of ca. 95~98 M, suggesting the
capability of ZnHL1 to selectively kill cancer cells. This may be
due to the relatively higher accumulation of ZnHL1 in cancer
cells (Fig. S14, ESI†).
Furthermore, the anticancer mechanism of in-cell
synthesized ZnHL1 was investigated (Fig. 3). Firstly, Annexin
V/PI double staining experiment shows that the capability of
(Fig. 16, ESI†).
Fig. 3(A) Annexin V/PI assay of A549 cells treated with ZnCl2, L1
,
Zn2++ L1 at the indicated concentrations for 36 h. (B) Gel
Fig. 2 (A) Confocal images of A549 cells incubated with L1 (40
μM, 6 h) followed with ZnCl2 treatment (100 μM, up to 12 h),
electrophoresis showing the cleavage of pUC19 DNA (0.5 lg); (C)
Immunofluorescence of γH2AX (DNA breaks foci) in A549 cells
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such Zn Schiff base complex can be tracked in real-time via the
View Article Online
Zn2+ induced green-to-blue fluorescDeOncI:e10.1s0h3i9f/tC9CanCd0348t0hCe
subsequent cytoplasm-to-nucleus translocation. Owing to the
nucleus penetrating capability and DNA hydrolysis activity, in-
cell synthesized Zn-Schiff base complex exhibits excellent anti-
cancer activity and cancer-specificity both in vitro and in vivo.
In the future work, the inorganic zinc salt can be replaced by
approved nutritious zinc supplements, which is more
conducive to clinical application.
This work was financially supported by the National
Natural Science Foundation of China [21837006, 21572282,
21401217], 973 Program [2015CB856301], Natural Science
Foundation of Guangdong Province [2017A030313041], China
treated with various compounds for a total incubation time of
36 h. ZnCl2: 100 µM, L1:100 µM; ZnHL1: 40 µM; In-cell
synthesized ZnHL1 (ZnCl2 + L1): 40 µM. DAPI: λex/λem =405/461
± 20 nm; γH2AX antibody: Daylight 549. Scale bar: 5 µm.
Postdoctoral
Science
Foundation
[2018M643290],
Fig. 4 In vivo anti-tumor efficacy of PBS (control), L1, ZnCl2, as-
prepared ZnHL1 and in-cell synthesized ZnHL1 (Zn2++ L1). (A)
Volume changes of MCF-7 tumors and (B) Photos of resected
tumors from mice.
Fundamental Research Funds for the Central Universities.
Notes and references
§
There are no conflicts to declare.
Thirdly, DNA cleavage experiment and immunofluorescence
assay were conducted, in which phosphorylated histone 2AX
(γH2AX) is an established marker forming foci at the site of
DNA breaks and activates DNA damage response.22 Fig. 3B-C
shows that ZnHL1 at 25 µM already causes substantial cleavage
of pUC19 DNA from its SC (supercoiled) to the NC (nicked
circular) form, while both the in-cell synthesized and pre-
prepared ZnHL1 (40 µM, 36 h) can induce a large amount of
DNA breaks foci in living cells. However, zinc salt or L1 alone
(100 µM) does not damage DNA at all. These results
demonstrate that in-cell formation of ZnHL1 can effectively
induce DNA damage response and trigger cancer cell apoptosis.
Finally, the anti-tumor efficacy was evaluated in the nude
mice bearing MCF-7 tumors. When the tumor grew to ca. 130
mm3, mice were intratumorally injected with PBS (control), L1
(2 mg/kg), ZnCl2 (2 mg/kg), pre-prepared ZnHL1 (1 mg/kg), and
ZnCl2 + L1 combination (1 mg/kg), where the mice was injected
with ZnCl2 (1 mg/kg) followed with another injection with L1 (1
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.
After 14 days treatment all mice were sacrificed for tumor
collection. As shown in Fig. 4, treatment with L1 alone does not
affect the tumor growth at all while ZnCl2 exhibits a small
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ZnHL1 group (Fig. S17, ESI †). Moreover, all the treated nude
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