Metallo-supramolecular Complexes Enantioselectively Eradicate Cancer Stem Cells in Vivo

Article abstract of DOI:10.1021/jacs.7b07490

Cancer stem cells (CSCs) are responsible for drug resistance, metastasis and recurrence of cancers. However, there is still no clinically approved drug that can effectively eradicate CSCs. Thus, it is crucial and important to develop specific CSC-targetin

Full text of DOI:10.1021/jacs.7b07490

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Article
Metallo-supramolecular complexes
enantioselectively eradicate cancer stem cells in vivo
Hongshuang Qin, Chuanqi Zhao, Yuhuan Sun, Jinsong Ren, and Xiaogang Qu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07490 • Publication Date (Web): 13 Oct 2017
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Metallo-supramolecular complexes enantioselectively eradicate
cancer stem cells in vivo
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Hongshuang Qin,a Chuanqi Zhao,a Yuhuan Sun,a,b Jinsong Ren,a and Xiaogang Qu*a
aLaboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Ap-
plied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China.
bUniversity of Chinese Academy of Sciences, Beijing 100039, China.
KEYWORDS :cancer stem cell • telomerase • G-quadruplex DNA • chiral recognition • supramolecular complex
ABSTRACT: Cancer stem cells (CSCs) are responsible for drug resistance, metastasis and recurrence of cancers. However, there
is still no clinically approved drug that can effectively eradicate CSCs. Thus, it is crucial and important to develop specific CSC-
targeting agents. Chiral molecular recognition of DNA plays an important role in rational drug design. Among them, polymorphic
telomeric G-quadruplex DNA has received much attention due to its significant roles in telomerase activity and chromosome stabil-
ity. Herein, we find that one enantiomer of zinc-finger-like chiral metallohelices, [Ni2L3]4+-P, a telomeric G-quadruplex-targeting
ligand, can preferentially reduce cell growth in breast CSCs compared to the bulk cancer cells. In contrast, its enantiomer,
[Ni2L3]4+-M, has little effect on both populations. Further studies indicate that [Ni2L3]4+-P can repress CSC properties and induce
apoptosis in breast CSCs. This is different to the bulk cancer cells. The inhibition of breast CSC traits is involved in the nuclear
translocation of hTERT. The apoptosis is associated with the induction of telomere uncapping, telomere DNA damage and the deg-
radation of 3′-overhang. Moreover, [Ni2L3]4+-P, but not [Ni2L3]4+-M, has the ability to reduce tumourigenesis of breast CSCs in vivo.
To our knowledge, this is the first report that chiral complexes show significant enantio-selectivity on eradicating CSCs.
INTRODUCTION
back to form a T-loop structure, in which the 3′-overhang in-
serts the double-stranded region to form a D-loop structure
which can stabilize the T-loop.11,12 A specialized protein com-
plex including TRF1, POT1, TRF2, RAP1, TIN2 and TPP1,
called shelterin, binds and protects the 3′-overhang and main-
tains telomere length and structure.13 Telomere deprotection
due to the progressive telomere shortening or telomere damage
results in cell growth arrest and apoptosis.14,15 The predomi-
nant mechanism of telomere maintenance depends on telomer-
ase, a reverse transcriptase which can add TTAGGG repeats to
the ends of telomeres.16 Telomerase is highly expressed in
CSCs and most types of cancer cells but not in normal somatic
cells.17,18 Intriguingly, it has been demonstrated that CSCs are
more sensitive to telomerase inhibitors than bulk cancer
cells.19,20 Therefore, targeting telomerase and telomere repre-
sents a promising anticancer therapeutic strategy that has the
ability to preferentially deplete CSCs with little effects on
normal cells.
Cancer recurrence is closely related to the presence of cancer
stem cells (CSCs), a rare cell subpopulation capable of self-
renewal and differentiation.1 Tumors which have larger pro-
portions of CSCs compared with well differentiated tumors are
linked to the lowest life expectancy.2 CSCs also play a key
role in the distant metastasis of cancer cells. Indeed, clinical
studies have shown that metastatic tumors possess much
greater proportions of CSCs than the primary tumors.3 Con-
ventional therapeutic approaches such as chemotherapy and
radiotherapy effectively remove the bulk of cancer cells, but
are unable to eliminate CSCs due to specific resistance mech-
anisms. Surviving CSCs differentiate and regenerate new tu-
mor cells, causing tumor recurrence and metastases.4 To im-
prove patient survival, treatments must be capable of eliminat-
ing the entire population of cancer cells, especially CSCs.
Although many potential anti-CSC agents aimed at various
targets, such as multiple kinases, certain organelles and vul-
nerable microenvironments, have been identified, they often
cause severe side effects.5-8 Therefore, there is an urgent need
to find more effective targets to discover novel anti-CSC com-
pounds with little effects on normal somatic cells.
Traditional telomerase inhibitors need a long lag period to
induce cell senescence and apoptosis, because telomere short-
en depends on cell proliferation and DNA replication.21,22 Fur-
thermore, alternative lengthening of telomeres (ALT) might be
activated when the telomerase activity is inhibited in cancer
cells, which is mediated by recombination and is one of the
major limitations for the clinical application of telomerase
inhibitors.23,24 Previous reports have shown that telomere DNA
cannot be elongated by telomerase when the 3′-overhang
forms G-quadruplex (G4) structure.25 Thus, the use of com-
pounds to induce and stabilize telomere G-quadruplex struc-
ture has become a promising strategy for anticancer agent
development.26,27 This strategy has been proved effective to
Telomeres have attracted much attention for anticancer
therapy, as the maintenance of telomeres is required for cancer
cell immortality. Telomeres are distinct nucleotide sequences
at chromosome ends that protect chromosome structural integ-
rity from degradation, illegitimate recombination and fusion.9
Human telomere DNA includes a duplex region (2–15 kb)
containing long arrays of tandem TTAGGG repeats and a sin-
gle-stranded overhang (50–400 nucleotides) at the 3′ end of
the G-rich strand.10 Telomeric DNA has been found to loop
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Figure 1. NiP, but not NiM, inhibits telomerase activity in breast CSCs. (A) G-quartet of guanine residues linked by Hoogsteen hydrogen
bonds. (B) Structures of the M-enantiomer and P-enantiomer of [Ni2L3]4+ cation. (C) Representative illustration of NiP selectively recog-
nizing of human telomere G4 DNA. (D, F) Inhibition of telomerase activity mediated by NiP in MDA-MB-231 and MCF-7 mam-
mospheres. Mammospheres were exposed to different concentrations of NiP and NiM for 7 days, then standard TRAP assay was per-
formed with equivalent amounts of protein. (E, G) Quantification of telomerase activity was performed as the percent of the control sample
without treatment of NiP and NiM. The results were shown as the means ± SD.
shorten telomeres and to perturb telomere function directly,
which would result in short-term DNA damage and apoptosis
in cancer cells.28,29 A large number of G4 ligands have been
designed and synthesized in recent years to perturb telomerase
and telomere function for cancer treatment.30-38 However, few
G4 ligands exhibit high selectivity towards telomere G4 DNA.
Chiral molecular recognition of DNA has attracted much at-
tention because of its important application in rational drug
design.39,40 Human telomere G4 DNA (Figure 1A) is polymor-
phic, and its structural transition is associated with many im-
portant life events.26,41,42 Chiral recognition of telomeric G4
can offer a novel strategy for development of G4 selective
ligands. Chirality is closely associated with the specificity and
binding activity of ligands to G4. Thus, targeting of telomeric
G4 DNA by chiral compounds can identify highly selective
agents which are more specific and effective to inhibit te-
lomerase activity and disturb telomere structure in CSCs. We
have recently reported that one enantiomer of chiral metallo-
helices, [Ni2L3]4+-P (NiP; see structure in Figure 1B) is able to
selectively recognize and stabilize telomeric G4 (Figure 1C)
and inhibit telomerase activity.43,44
apoptosis and has little effect on senescence, whereas NiP
promotes cell apoptosis and senescence in the bulk cancer
cells.45 Further studies reveal that telomere uncapping with the
delocalization of TRF2 and POT1 from telomeres, telomere
DNA damage and the degradation of 3′-overhang result in the
apoptosis of breast CSCs. The reduction of CSC properties is
associated with the nuclear translocation of hTERT. Moreover,
NiP has the ability to reduce tumourigenesis of breast CSCs in
vivo. Overall, our data indicates that the enantiomer NiP can
effectively deplete breast CSCs in vitro and in vivo. To the
best of our knowledge, this is the first example that chiral
complexes show contrasting enantio-selectivity on eradicating
CSCs. Our work will shed light on the application of chiral
agents in anti-CSC therapy.
RESULTS
For verifying the effects of chiral metallo-supramolecular
complexes (NiP and NiM) on CSCs, MDA-MB-231 and
MCF-7 cell lines containing inherent breast CSC subpopula-
tion were used. These two types of cells were cultured in an-
chorage-independent and serum-free culture condition to form
mammospheres, which grew robustly from single cell and
enriched with breast CSCs.46 The primary mammosphere for-
mation assay showed that MDA-MB-231 and MCF-7 cells
contained a CSC population of 0.39% and 1.04% respectively
Herein, we investigate the effects of chiral supramolecular
complexes (NiP and NiM) on breast CSCs, and find that NiP,
rather than NiM, preferentially inhibits cell growth in breast
CSCs compared to the bulk cancer cells. Intriguingly, NiP has
remarkable different roles in breast CSCs and bulk cancer
cells. NiP inhibits breast CSC properties, induces breast CSC
(Figure
S1A-B).
Using
CD44+/CD24−/low
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Figure 2. NiP, but not NiM, eliminates breast CSCs. (A) Reduction of breast CSC proportion induced by NiP in MDA-MB-231 and MCF-
7 monolayer cells. MDA-MB-231 and MCF-7 cells were treated with NiP or NiM (5.12 μM), or transfected with 2′-O-MeRNA and
TRF2ΔBΔM for 3 weeks, then 5000 cells were cultured in CSC medium for 7 d to form mammospheres. The mammosphere formation was
determined by microscopic examination. Scale bar = 100 μm. (B, C) The mammospheres were quantitated. (D, E) Inhibition of mam-
mosphere formation induced by NiP in serial passaging of breast CSCs. The secondary mammospheres of MDA-MB-231 and MCF-7 cells
were incubated with NiP or NiM and subcultured every 7 d for 3 weeks. The mammosphere formation was determined by microscopic
examination. Scale bar equals 100 μm. (F, G) MDA-MB-231 and MCF-7 cells were cultured in ultra-low attachment plates or adherent
monolayer culture, and treated with NiP or NiM (5.12 μM) for 21 d. The cell viability was determined at the indicated time. The results
were shown as the means ± SD of three separate experiments. **P < 0.01.
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as breast CSC surface markers,47-49 we observed that the per-
centage of breast CSC population increased to 96.7% and 98.3%
respectively in the secondary spheres (Figure S1C). Typical
microscopy images of the mammospheres at three different
passages showed that there was no apparent change in the
morphology of the mammospheres with serial passaging (Fig-
ure S1D), suggesting that our method was effective for breast
CSC enrichment.
[Ni2L5 ]4+ (NiP-3, NiM-3, NiP-5 and NiM-5),51,52 and tested
3
their effects on telomerase activity in MDA-MB-231 mam-
mospheres (Figure S3-4). The inhibitory effects of these de-
rivatives on telomerase were less than NiP (the IC50 value of
NiP-3 and NiP-5 on telomerase inhibition were 4.34 ± 0.24
and 1.21 ± 0.12 μM, respectively).
Telomerase inhibition associates with growth suppression,
so we assessed whether NiP could deplete CSC proportion in
MDA-MB-231 and MCF-7 monolayer cells. In this assay,
5.12 μM was used because this concentration of NiP induced
almost complete inhibition on telomerase activity but had no
acute toxicity on breast CSCs (Figure S5A-B). MDA-MB-231
and MCF-7 cells were exposed to NiP and NiM for 3 weeks,
and then the cells were cultured in CSC medium for 7 d to
form mammospheres. As shown in Figure 2A-C, NiP treat-
ment inhibited the formation of mammospheres in MDA-MB-
231 and MCF-7 cells, especially, no larger mammosphere (>
150 μm in MDA-MB-231 cells and > 80 μm in MCF-7 cells)
was observed in the cells treated with NiP, indicating that NiP
was able to deplete breast CSCs from bulk tumor populations
in these two cell lines. For clarifying the inhibition of mam-
mosphere formation mediated by NiP was dependent on te-
lomerase activity or telomere structure, MDA-MB-231 and
MCF-7 cells transfected with 2′-O-MeRNA (an inhibitor of
telomerase activity)53 or transfected with TRF2ΔBΔM mutation,
which would lead to dysfunctional telomere,54 were also sub-
jected to mammosphere formation assay. Transfection of 2′-O-
MeRNA did not inhibit mammosphere formation in both
MDA-MB-231 and MCF-7 cells, however, obvious inhibition
was observed in the cells transfected with TRF2ΔBΔM (Figure
2A-C), this was similar to the cells treated with NiP, indicating
that mammosphere formation inhibition caused by NiP was
dependent on telomere structure, but not telomerase activity.
The expression of TRF2ΔBΔM in MDA-MB-231 and MCF-7
cells was detected by western blot assay targeting Myc-tag
We first examined the effects of the two enantiomers on te-
lomerase activity. The telomerase was prepared from the sec-
ondary mammospheres and subjected to a modified TRAP-G4
assay as described previously.50 As shown in Figure S2A-B,
telomerase activity was inhibited by NiP in a concentration-
dependent manner and almost complete inhibition was ob-
served at the concentration of 320 nM. The IC50 value for NiP
on telomerase inhibition was 73.14 ± 2.23 nM. Furthermore,
these concentrations of NiP had no inhibitory effect on te-
lomerase substrate internal control. Moreover, slight inhibition
of telomerase activity was found in the group treated with
NiM, indicating that the two enantiomers had chiral selectivity
on inhibition of telomerase activity.
We further investigated whether there was a difference be-
tween the two enantiomers in telomerase inhibition in living
breast CSCs. MDA-MB-231 and MCF-7 mammospheres were
exposed to NiP and NiM at concentrations ranging from 0.16
to 5.12 μM for 7 days. Then telomerase activity was tested
with a conventional TRAP assay.43 As shown in Figure 1D-G,
treatment of NiP induced a concentration-dependent inhibition
of telomerase activity in breast CSCs. The IC50 values of NiP
on telomerase inhibition were 1.08 ± 0.10 and 2.03 ± 0.09 μM
in MDA-MB-231 and MCF-7 mammospheres, respectively. In
contrast, only slight inhibition of telomerase activity was ob-
served in the cells treated with NiM. Taken together, NiP, but
not NiM, had the ability to inhibit telomerase activity in breast
CSCs. We also synthesized the methyl-substituted (at 3’- and
5’-position) supramolecular compounds [Ni2L3 ]4+ and
3
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(Figure
S6A-C),
furthermore, transfection of It has been demonstrated that growth inhibition mediated by
telomere-targeting agents often involves in DNA damage.27
Thus, we tested whether NiP could induce DNA damage in
breast CSCs. As shown in Figure S7A-B, the phosphorylation
of H2AX (γ-H2AX), a common marker of DNA double-strand
break,9 was significantly increased after 2 weeks treatment
with NiP (5.12 μM). Immunofluorescence results showed that
53BP1, another DNA damage response factor,9 and γ-H2AX
formed foci during NiP treatment (Figure S7C-D), confirming
that NiP could induce DNA damage response. Quantitative
analysis results revealed that the percent of cells containing
53BP1 and γ-H2AX foci exceeded 80% after treatment with
NiP (Figure S7E-F). In contrast, the same concentration of
NiM had little effect on DNA damage. The up-regulation of γ-
H2AX and more 53BP1 and γ-H2AX foci were also observed
in the cells transfected with TRF2ΔBΔM, but not 2′-O-MeRNA.
Collectively, these results indicated that the growth inhibition
of breast CSCs mediated by NiP involved in the induction of
DNA damage.
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To determine whether DNA damage response induced by
NiP occurred at telomeres, double immunofluorescence exper-
iment was performed in breast CSCs. Confocal microscopy
showed that most of the 53BP1 and γ-H2AX foci induced by
NiP co-localized with TRF1 (Figure 3A), forming the telo-
mere dysfunction-induced foci (TIFs).9,55 Quantitative results
showed that the cells with more than four 53BP1/TRF1 or γ-
H2AX/TRF1 co-localizations was significantly increased after
NiP treatment (the percentage of TIFs-positive cells up to
nearly 70% upon treatment), with an average number of about
twenty TIFs per cells (Figure S8A-B). These results were fur-
ther confirmed by ChIP-qPCR,50,56 as shown in Figure S8C,
the amount of 53BP1 and γ-H2AX combined with telomeres
in the cells treated with NiP was obviously increased (P <
0.01). Telomere DNA damage induced by NiP was very po-
tent because the percentage of TIFs-positive cells was similar
to that tested in the cells transfected with TRF2ΔBΔM (Figure
S8A).55 However, slight telomere DNA damage response was
observed in the cells treated with NiM or transfected with 2′-
O-MeRNA.
Figure 3. NiP induces telomere DNA damage and apoptosis. (A)
Representative confocal images of merged 53BP1 (green)/TRF1
(red) or γ-H2AX (green)/TRF1 (red). MDA-MB-231 mam-
mospheres were treated with NiP and NiM (5.12 μM), or trans-
fected with 2′-O-MeRNA and TRF2ΔBΔM for 2 weeks, then the
cells were stained with the antibodies against 53BP1
(green)/TRF1 (red) or γ-H2AX (green)/TRF1 (red) and DAPI
(blue). Scale bar equals 5 μm. (B) Cell apoptosis was detected by
Annexin V-FITC/PI double staining in MCF-7 and MDA-MB-
231 mammospheres exposed to NiP or NiM (5.12 μM), or trans-
fected with 2′-O-MeRNA and TRF2ΔBΔM for 3 weeks.
It has been reported that the formation of quadruplex struc-
tures at telomeres induced by G-quadruplex-binding ligands
may lead to delocalization of telomere binding proteins, result-
ing in telomere uncapping and DNA damage.56-58 Therefore,
we examined the localization of TRF1, POT1 and TRF2,
which are telomere binding proteins and induce telomeric
DNA damage and telomere dysfunction when they dissociate
from telomeres9,54,55 in breast CSCs treated with NiP. ChIP-
qPCR assay showed that NiP had little effect on the binding of
TRF1 to telomeres (Figure S9A). However, significant reduc-
tion of the binding of POT1 and TRF2 was observed in the
cells treated with NiP (P < 0.01). Confocal microscopy results
also revealed that NiP markedly delocalized TRF2 and POT1
from the telomeres which were represented by TRF1 foci
(Figure S9B). The percentage of cells with more than four
POT1/TRF1 or TRF2/TRF1 co-localizations was reduced to
less than 20% in the cells treated with NiP, similar to that test-
ed in the cells transfected with TRF2ΔBΔM (Figure S9C-D).
Furthermore, the expression of TRF2 and POT1 did not
changed (Figure S9E), indicating that the reduction of TRF2
and POT1 at telomeres induced by NiP was not due to the
down-regulation of these proteins.
pcDNA3.1 (empty vector) did not affect mammosphere for-
mation in both MDA-MB-231 and MCF-7 cells (Figure S6D).
We further examined the effects of NiP and NiM on mam-
mosphere formation in serial passaging. The secondary mam-
mospheres of MDA-MB-231 and MCF-7 cells were incubated
with NiP and NiM and subcultured every 7 d for 3 weeks. As
shown in Figure 2D-E, mammosphere formation was signifi-
cantly inhibited after treatment with NiP for 2 weeks, followed
by almost complete inhibition after additional 1 week, sug-
gesting that NiP could eliminate breast CSCs in serial passag-
ing. In contrast, there was no evident effect on mammosphere
formation in the cells treated with NiM.
To test whether NiP could predominantly inhibit cell growth
in breast CSCs, the sensitivity of monolayer cells and mam-
mospheres to NiP was examined. As shown in Figure 2F-G, a
time-dependent inhibition of cell viability was observed in
both populations after treatment with NiP, however, mam-
mospheres were more sensitive to NiP than the cells in mono-
layer cultures (P < 0.01), indicating that NiP preferentially
inhibited cell growth in breast CSCs compared with the bulk
cancer cells.
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were also obtained by transfection of TRF2ΔBΔM, but not 2′-O-
MeRNA.
Next, we investigated whether NiP-mediated telomere dys-
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function could result in senescence and apoptosis in breast
CSCs. To detect cell senescence, the expression of p21 and
p16 (the major markers of senescence) was examined in breast
CSCs after exposure to NiP for 3 weeks. As shown in Figure
S12, NiP had little effect on the expression of p21 and p16,
indicating that NiP could not induce senescence. However,
NiP induced significant apoptosis (35.1% in MDA-MB-231
mammospheres and 29.4% in MCF-7 mammospheres) in
breast CSCs (Figure 3B). Similar results were obtained by
transfection of TRF2ΔBΔM, but not 2′-O-MeRNA. Overall,
these results indicated that NiP, but not NiM, had the ability to
induce telomere uncapping and activate telomere DNA dam-
age response, resulting in growth suppression and apoptosis in
breast CSCs.
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Growing evidence suggests that human telomerase reverse
transcriptase (hTERT), the catalytic subunit of telomerase, can
be phosphorylated at site tyrosine 707 and reversibly translo-
cate from the nucleus to cytoplasm in response to cell
stress.61,62 To investigate whether the localization of hTERT
was changed by NiP treatment, western blot and immunofluo-
rescence assay were applied. Western blot results showed that
the phosphorylation level of hTERT was significantly in-
creased on exposure to NiP (Figure S13). Immunofluorescence
results revealed that hTERT translocated to cytoplasm after
NiP treatment, as observed in the cells transfected with
TRF2ΔBΔM (Figure 4A). It has been reported that hTERT is a
key regulator of the stemness of CSCs, which can regulate the
transcription of stemness markers, such as Oct4, Klf4 and Myc,
to modulate CSC stemness.63-65 Therefore, we tested whether
the stemness of CSCs was affected by the nuclear transloca-
tion of hTERT. RT-PCR results showed that the expression of
stem cell markers (Aldh1, Oct4, Lin28a, Klf4, Nanog and
Bmi1) in MDA-MB-231 mammospheres treated with NiP for
2 weeks was distinctly reduced, whereas little effect was ob-
served in NiM-treated cells (Figure S14). These results were
further evidenced by qRT-PCR (Figure 4B). Flow cytometry
analysis also showed that the stem cell marker CD44 was dras-
tically reduced after treatment by NiP with the up-regulation
of CD24, which resulted in significant reduction of
CD44+/CD24−/low population (Figure 4C). Meanwhile, we ob-
served that the overall expression of hTERT was reduced after
NiP treatment, whereas this effect was slight in the cells treat-
ed with NiM (Figure 4D). Collectively, these results demon-
strated that NiP, but not NiM, had the ability to repressed
breast CSC traits.
Figure 4. NiP induces translocation of hTERT from nucleus to
cytoplasm and represses breast CSC properties. (A) The subcellu-
lar localization of hTERT in MDA-MB-231 mammospheres treat-
ed with NiP and NiM (5.12 μM), or transfected with 2′-O-
MeRNA and TRF2ΔBΔM was analyzed by staining with the anti-
body for hTERT. Representative images were presented. Scale
bar = 20 μm. (B) qRT-PCR analysis of the expression of CSC
makers (Aldh1, Oct4, Lin28a, Klf4, Nanog and Bmi1) in MDA-
MB-231 mammospheres treated with NiP and NiM for 2 weeks.
**P < 0.01. (C) The proportion of CD44+/CD24−/low cells in
MDA-MB-231 mammospheres incubated with NiP and NiM was
measured by flow cytometry. (D) Western blot assay of the ex-
pression of hTERT in MDA-MB-231 mammospheres treated with
NiP and NiM for 2 weeks. Equal protein loading was evaluated by
β-actin.
Previous studies have shown that when the telomere cap-
ping is altered, the unprotected telomeres can be accessed and
labeled by terminal deoxytransferase (TdT) which can add
Cy3-conjugated deoxyuridine to the naked telomere ends.59 So
we investigated TdT signals in the cells treated with NiP to
confirm telomere uncapping. As shown in Figure S10A-B,
most of the TdT signals (nearly 70%) co-localized with TRF1
in NiP-treated cells, indicative of robust telomere uncapping.
Similar results were observed in the cells overexpressing
TRF2ΔBΔM. However, little TdT-Cy3 was detectable in the
cells transfected with 2′-O-MeRNA.
To evaluate the effect of NiP on tumour-initiating capacity
of breast CSCs in vivo,66,67 MDA-MB-231 cells were subcuta-
neously injected into nude mice at decreasing dilutions (5×105,
1×105 and 1×104 cells) and treated with NiP, NiM or PBS
(control) for 4 weeks. As shown in Table 1 and Figure 5A-B,
the number and size of the tumours in NiP-treated group were
less than PBS- and NiM-treated groups for 5×105 cells injected.
Furthermore, there was no tumor formation in the mice seeded
with 1×105 cells after treatment with NiP. In contrast, signifi-
cant tumor formation was observed in control and NiM-treated
groups injected with the same number of cells, suggesting that
NiP, but not NiM, could decrease the tumorigenic potential of
breast CSCs in vivo. To further verify the effect of NiP on
CSCs in vivo, primary tumour cells
The loss of TRF2 and POT1 from telomeres can induce sin-
gle-stranded G-overhang degradation.57,60 Therefore, we
measured the telomeric G-overhang length by hybridization
protection assay (HPA) as described previously.57,60 As shown
in Figure S11A, the telomeric G-overhang length was signifi-
cantly reduced after treatment with NiP for 2 weeks (P < 0.01),
whereas little reduction was observed in the total length of
telomeres, suggesting the depletion is specific to single-
stranded G-tails. Furthermore, the degradation of G-overhang
was accompanied with the emergence of micronuclei, indica-
tive of severe genotoxicity (Figure S11B-C). These results
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Table 1. The effects of NiP and NiM on tumorigenesis in
vivo
have potential to remove both CSCs and bulk cancer cells, and
1
2
3
4
5
6
7
8
Treatment
Control
Cell number
Tumor formation
500 000
100 000
10 000
5/6
3/6
0/6
5/6
2/6
0/6
2/6
0/6
0/6
NiM
NiP
500 000
100 000
10 000
9
10
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12
13
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15
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22
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58
59
60
500 000
100 000
10 000
Different numbers of MDA-MB-231 cells were injected subcuta-
neously and mice were treated with NiP or NiM once a day. The
numbers represent ‘the number of mice with tumorigenesis/the
number of mice in every group’.
isolated from tumor tissues were subjected to mammosphere
formation assay. Single cells were seeded into ultralow adher-
ence plates, and cultured in CSC medium for 7 d, then mam-
mosphere formation was tested. As shown in Figure 5C-D, the
number of mammospheres generated from the cells derived
from NiP-treated tumours were obviously reduced compared
with control group, especially, there was no larger mam-
mosphere (> 150 μm) was observed in NiP-treated group. In
contrast, the number and size of mammospheres come from
the cells separated from NiM-treated tumours were similar to
that in control group, suggesting that NiP rather than NiM
could diminish breast CSC population in vivo. Moreover, no
body weight drop was observed in all groups (Figure S15A).
Importantly, the histologic appearance of major organs, such
as heart, liver, lung, kidney and spleen, was normal in NiP-
treated group (Figure S15B), indicative of the extremely low
toxicity of NiP.
Figure 6. Schematic illustration of telomerase activity inhibition,
telomere DNA damage, breast CSC property reduction, apoptosis
and tumourigenesis inhibition induced by NiP in breast CSCs.
Telomeric DNA is postulated to loop back to form a T-loop struc-
ture, in which 3′-overhang inserts double-stranded region to form
a D-loop structure which can stabilize the T-loop. During DNA
replication, the 3′-overhang can be accessed and elongated by
telomerase. However, in the presence of NiP, but not NiM, the G-
rich telomeric strand self-assembles into a G4 structure, leading to
telomere elongation block. The persistence of G-quadruplex re-
sults in telomere uncapping, telomeric DNA damage, 3′-overhang
degradation and apoptosis. Meanwhile, cell stress mediated by
NiP can induce hTERT translocation from nuclear to cytoplasm,
leading to the inhibition of breast CSC traits. The induction of
breast CSC trait loss and apoptosis caused by NiP result in the
abrogation of tumour initiation in vivo.
have little effect on normal cells. Moreover, it has been
demonstrated that CSCs are more sensitive to telomerase in-
hibitors than the bulk cancer cells.19,20 Therefore, targeting
telomerase represents a promising anticancer therapeutic strat-
egy with the ability to deplete CSCs predominantly. Telomeric
G4 ligands as one type of telomerase inhibitors have great
potential in the development of CSC-targeting drugs because
the agents not only can inhibit telomerase activity resulting in
telomere shortening in the long term but also can disturb telo-
mere structure in the short term.28,29 In this study, we reported
that chiral metallo-supramolecular complex NiP, which dis-
played preferential binding to telomere G4, had the ability to
induce telomerase inhibition and dissociation of telomere as-
sociated proteins from telomeres, leading to telomere DNA
damage and apoptosis in breast CSCs (Figure 6). Similar re-
sults were obtained by transfection of TRF2ΔBΔM (a mutant of
TRF2), but not 2′-O-MeRNA (a telomerase inhibitor), demon-
strating that NiP induced CSC apoptosis through destroying
telomere structure before the length of telomere is shortened.
Figure 5. NiP inhibits tumour-initiating capacity of breast CSCs in
vivo. (A) Photographs of the dissected tumors. MDA-MB-231
cells were injected subcutaneously and mice were treated with
NiP or NiM once a day for 4 weeks. (B) Tumor growth curves of
tumor-bearing mice after NiP or NiM treatment. (C) Mam-
mosphere formation of the cells separated from tumor tissues
treated with NiP or NiM. (D) The mammospheres were quantitat-
ed. **P < 0.01.
Drug development aims to discover novel therapeutic
agents targeting the key enzymes, protein, nucleic acids, and
the interactions of protein-nucleic acid, receptor-ligand or
protein-protein to mitigate or cure relevant diseases. Almost
all of the enzymes, nucleic acids and proteins are chiral, and
DISCUSSION
Telomerase is highly expressed in CSCs and most cancer cells
but not in normal somatic cells.17,18 Thus, telomerase inhibitors
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these biomolecules have stereoselectivity towards their bind- treated cells (Figure S9A), implying that telomeres could be
ing ligands.68 Indeed, it has been demonstrated that chirality
can determine drug efficacy and safety.69 Therefore, chiral
recognition of targets is crucial for design and development of
drugs. Chiral drugs have played an important role in human
disease treatments. Chiral compounds account for more than
50% of currently used drugs and this ratio exhibits an increas-
ing tendency.68 In some cases, one enantiomer is active, while
the other may show side effects, including toxicity. Thus, in-
vestigation of the effect of individual enantiomers is important
for new drug discovery.68 The structural transition of telomeric
G-quadruplex DNA is associated with many significant life
events. Chiral recognition of telomeric G-quadruplex in CSCs
can discover highly selective agents which can specifically
and effectively inhibit telomerase activity and perturb telo-
mere function in CSCs. Targeting of telomere G4 DNA via
chiral compounds may offer a novel strategy for development
chiral drugs that can eliminate CSCs. Our previous studies
have demonstrated that one enantiomer of chiral metalloheli-
ces, NiP, rather than NiM, has the ability to selectively stabi-
lize telomere G4 DNA.43,44,70 The steric configuration match-
ing of NiP, but not NiM, with the G-quadruplex DNA plays a
key role for their enantioselective interaction. When interact-
ing with G-quadruplex DNA, NiP stacks at the end G-quartet
of G-quadruplex by hydrophobic interaction. In addition, for
these metal complexes, central metal ions Ni2+ are coordina-
tively saturated in octahedron geometry and hence would not
coordinate directly to the DNA. Thus, the cationic charges of
Ni2+ further favor their electrostatic interactions with G-
quadruplex DNA (Figure 1C).43,44,70 Our study reported here
showed that enantiomer NiP could observably eliminate breast
CSCs via inducing telomere DNA damage, breast CSC prop-
erty loss and apoptosis. In contrast, enantiomer NiM had little
effect on breast CSCs. To our knowledge, this is the first re-
port that chiral compounds show enantio-selectivity in eradi-
cating CSCs.
represented by TRF1 foci to investigate the localization of
other telomere binding proteins, such as TRF2 and POT1.
However, confocal microscopy and ChIP-qPCR results both
revealed that the binding of POT1 and TRF2 to the telomeres
was significantly reduced after treatment with NiP (Figure
S9A-D). The different localization of these telomeric proteins
after treatment could be attributed to the difference of binding
sites. POT1 binds specifically to 3′ single-stranded G-
overhang.74 TRF2 binds the single-/double-stranded DNA
junction and the telomeric duplex DNA, and promotes the 3′-
overhang to invade the double-stranded region to form the T-
loop structure.9 In contrast, TRF1 is more prone to bind long
tracts of duplex DNA and maintains telomere length.73 Fur-
thermore, it has been reported that TRF1 possesses higher
affinity to telomere DNA than other shelterin subunits, for
example, the binding affinity of TRF1 to telomeric DNA is
approximately four times higher than TRF2.75 Therefore,
TRF2 and POT1 would be more prone to delocalize from te-
lomeres than TRF1 when telomere DNA forms the structure of
G-quadruplex.
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It has been confirmed that the anticancer effect of G4 agents
is strongly impacted by the length of telomeres, and short te-
lomere is more sensitive to G-quadruplex ligands than longer
telomere.30,57,76 Furthermore, although it is long enough to
form T-loop, short telomere is more prone to DNA damage
than longer telomere.73,77 Our results showed that NiP had
little effect on normal cells (Figure S16), however, NiP could
induce telomere uncapping and telomere DNA damage, lead-
ing to 3′-overhang degradation and apoptosis in breast CSCs.
These selective effects of NiP on breast CSCs may be due to
normal cells possess longer telomeres than cancer cells and
CSCs. Thus, targeting telomere G-quadruplex is a promising
strategy for eliminating CSCs.
In summary, our results show that one enantiomer of chiral
metallohelices, NiP, rather than NiM, has the ability to pre-
dominantly reduce cell viability in breast CSCs compared to
the bulk cancer cells, and has little effect on normal cells. Fur-
ther studies demonstrate that NiP is able to induce breast CSC
apoptosis at 3 weeks before the total length of telomeres is
reduced. The short-term effect of NiP is involved in the loss of
telomere associated protein (POT1 and TRF2) from telomeres,
which lead to series of telomere DNA damage, degradation of
G-overhang and apoptosis. Meanwhile, cell stress caused by
NiP induces the translocation of hTERT from nucleus to cyto-
plasm, accompanied by down-regulation of hTERT and CSC
markers (Aldh1, Oct4, Lin28a, Klf4, Nanog and Bmi1), indic-
ative of the reduction of breast CSC traits. Furthermore, NiP,
but not NiM, can reduce tumourigenesis of breast CSCs in
vivo. Our work suggests that chiral recognition of telomere
DNA is an efficient way for design of anticancer agents capa-
ble of eliminating CSCs.
In addition to telomere length-dependent function, hTERT,
the catalytic subunit of human telomerase, can also modulate
gene expression in CSCs.63 There is emerging evidence that
hTERT can regulate the transcription of pluripotency markers,
such as Oct4, Klf4 and Myc, to modulate CSC stemness.64,65
Our results showed that hTERT translocated from the nucleus
to cytoplasm after treatment with NiP, meanwhile, the expres-
sion of CSC markers (Aldh1, Oct4, Lin28a, Klf4, Nanog and
Bmi1) were reduced after NiP treatment (Figure 4B). We rea-
soned that the reduction of hTERT in nucleus induced by cell
stress could perturb the network of pluripotency transcription
factors, resulting in the inhibition of CSC properties, accom-
panied by down-regulation of CSC markers and hTERT whose
expression is closely correlated with stem cell-like proper-
ties.71,72 The perturbation of the pluripotency transcription
factor network may be one of the reasons that CSCs are more
sensitive to NiP than the bulk cancer cells.
MATERIALS AND METHODS
Telomeres are protected from DNA damage by the shelterin
complex, which includes TRF1, POT1, TRF2, RAP1, TIN2
and TPP1.9 Removal of individual shelterin subunits can cause
the perturbation of T-loop/D-loop structure, resulting in the
activation of specific DNA damage response pathways, such
as ATM and ATR pathways.73 TRF1, TRF2 and POT1 directly
bind with telomeric DNA, while TIN2, TPP1 and RAP1 per-
form their protective function through interaction with other
shelterin subunits.73 Our ChIP-qPCR results showed that there
was no distinct dissociation of TRF1 from telomeres in NiP-
Mammosphere culture. For mammosphere culture in vitro,
MDA-MB-231 and MCF-7 cells were seeded at 5000 cells/ml
in 6-well ultralow adherence plates (Corning Inc., USA) in
serum-free DMEM/F12 with 20 ng/ml recombinant epidermal
growth factor (Sigma, USA), 20 ng/ml basic fibroblast growth
factor (Gibco), 4 μg/ml heparin (Sigma), and 1% penicil-
lin/streptomycin (Gibco).78 Medium was changed every 48 h.
After 7 days, mammospheres were subcultured at 5000
cells/ml.
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Tumour tissues isolated from PBS, NiM or NiP-treated
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mice were mechanically dissociated followed by enzymatic
dissociation (300 U/ml of collagenase and hyaluronidase for 2
h at 37 °C).78 After filtration, the cell suspension was centri-
fuged for 10 min at 5000 rpm. Then the cells were resuspend-
ed and plated at 5000 cells/ml in ultralow adherence plates.
After 7 days, mammospheres were tested by microscopic ex-
amination.
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ASSOCIATED CONTENT
Supporting Information
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Materials and Methods in detail. Characterization of mam-
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TRF2ΔBΔM. NiP induces DNA damage response in breast CSCs.
The effect of NiP on telomere uncapping. NiP induces TdT-
Cy3 signal in breast CSCs. NiP induces the emergence of mi-
cronuclei in breast CSCs. The phosphorylation level of
hTERT induced by NiP. RT-PCR analysis of the expression of
CSC makers. The body weights of mice. H&E staining of five
organs. The effects of NiP and NiM on HEK293 cells. Primer
sequences for PCR experiments.
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AUTHOR INFORMATION
Corresponding Author
* xqu@ciac.ac.cn.
(27) Balasubramanian, S.; Neidle, S. Curr. Opin. Chem. Biol. 2009,
13, 345.
(28) Neidle, S. FEBS J. 2010, 277, 1118.
Funding Sources
973 Project and NSFC. No competing financial interests have
been declared.
(29) Riou, J.-F. Curr. Med. Chem. Anticancer Agents 2004, 4, 439.
(30) Phatak, P.; Cookson, J.; Dai, F.; Smith, V.; Gartenhaus, R.;
Stevens, M.; Burger, A. Br. J. Cancer 2007, 96, 1223.
(31) Kim, M.-Y.; Vankayalapati, H.; Shin-Ya, K.; Wierzba, K.;
Hurley, L. H. J. Am. Chem. Soc. 2002, 124, 2098.
ACKNOWLEDGMENT
This work was supported by 973 Project (2012CB720602), and
NSFC (21210002, 21431007, 21533008).
(32) Rodriguez, R.; Pantoş, G. D.; Gonçalves, D. P.; Sanders, J. K.;
Balasubramanian, S. Angew. Chem. 2007, 119, 5501.
(33) Haudecoeur, R.; Stefan, L.; Denat, F.; Monchaud, D. J. Am.
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