Mediating K+/H+ Transport on Organelle Membranes to Selectively Eradicate Cancer Stem Cells with a Small Molecule

Article abstract of DOI:10.1021/jacs.0c02134

Molecules that are capable of disrupting cellular ion homeostasis offer unique opportunities to treat cancer. However, previously reported synthetic ion transporters showed limited value, as promiscuous ionic disruption caused toxicity to both healthy cells and cancer cells indiscriminately. Here we report a simple yet efficient synthetic K⁺ transporter that takes advantage of the endogenous subcellular pH gradient and membrane potential to site-selectively mediate K⁺/H⁺ transport on the mitochondrial and lysosomal membranes in living cells. Consequent mitochondrial and lysosomal damages enhanced cytotoxicity to chemo-resistant ovarian cancer stem cells (CSCs) via apoptosis induction and autophagy suppression with remarkable selectivity (up to 47-fold). The eradication of CSCs blunted tumor formation in mice. We believe this strategy can be exploited in the structural design and applications of next-generation synthetic cation transporters for the treatment of cancer and other diseases related to dysfunctional K⁺ channels.

Full text of DOI:10.1021/jacs.0c02134

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Mediating K/H Transport on Organelle Membranes to
Selectively Eradicate Cancer Stem Cells by a Small Molecule
Fang-Fang Shen, Sheng-Yao Dai, Nai-Kei Wong, Shan Deng, Alice Sze-Tsai Wong, and Dan Yang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c02134 • Publication Date (Web): 22 May 2020
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Mediating K+/H+ Transport on Organelle Membranes to Selectively
Eradicate Cancer Stem Cells by a Small Molecule
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Fang-Fang Shen, Sheng-Yao Dai, Nai-Kei Wong, Shan Deng, Alice Sze-Tsai Wong and Dan Yang*
ABSTRACT: Molecules that are capable of disrupting cellular ion homeostasis offer unique opportunities to treat cancer. However,
previously reported synthetic ion transporters showed limited value as promiscuous ionic disruption caused toxicity to both healthy
cells and cancer cells indiscriminately. Here we report a simple yet efficient synthetic K+ transporter that takes advantage of the
endogenous subcellular pH gradient and membrane potential to site-selectively mediate K+/H+ transport on the mitochondrial and
lysosomal membranes in living cells. Consequent mitochondrial and lysosomal damages enhanced cytotoxicity to chemo-resistant
ovarian cancer stem cells (CSCs) via apoptosis induction and autophagy suppression with remarkable selectivity (up to 47-fold). The
eradication of CSCs blunted tumor formation in mice. We believe this strategy can be exploited in the structural design and applica-
tions of next-generation synthetic cation transporters for the treatment of cancer and other diseases related to dysfunctional K+ chan-
nels.
to be drug-like18. In addition, their transport activity studies were
predominately restricted to in vitro characterization in liposomes19-
21. It is thus challenging but imperative to develop active small mol-
ecules that could promote cation transport inside living cells.
To develop new synthetic cation transporters, we utilize α-ami-
noxy acids22 as the main scaffold. Prior studies revealed that by in-
corporating two α-aminoxy acids into an isophthalamide unit, chlo-
ride channels23 and chloride dependent potassium channels24 can
be formed via self-assembly. Yet, their transport activities were still
unsatisfactory. Here we have performed structural modifications on
α-aminoxy acid monomer to obtain a molecule with much smaller
molecular weight but a drastically different mechanism and highly
improved K+ transport efficiency and selectivity.
INTRODUCTION
Ion transport proteins are located not only on the plasma mem-
brane but also on organelle membranes. They exhibit unique ion
and direction selectivity1. Major ions like K+, Na+, H+, Ca2+ and Cl−
are distinctively distributed in cellular compartments to maintain
electrochemical signals for physiological processes and support or-
ganelle functions. Small molecules that are capable of performing
transmembrane ion transport have shown effectiveness in restoring
ionic balance in disease models caused by defective ion transport
proteins2-5. Disrupting normal ion homeostasis with ion transport-
ers, on the other hand, was also demonstrated to be detrimental to
cancer cells6-8. However, previous examples were less therapeuti-
cally valuable as they were cytotoxic to many types of cells indis-
criminately6. Notably, different types of cells maintain subtly al-
tered bioelectric properties, such as membrane potential, to regulate
definite cell behaviors, including proliferation and differentiation9.
Identify and selectively disrupt the critical electrochemical gradi-
ents may serve as a potential strategy to develop new cell-specific
therapies.
RESULTS
Molecular design and ion transport mechanism. By
conjugating N- and C-termini with benzoyl group and aniline, re-
spectively, compound 1 was first synthesized from α-aminoxy O-
benzyl-serine (Fig. 1a). K+ transport activity of compound 1 was
evaluated in large unilamellar vesicles (LUVs) prepared from egg
yolk phosphatidylcholine (EYPC) encapsulating pH-sensitive dye
8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS)25. The results
shown in Figure 1b indicated poor K+ transport activity of this com-
pound. However, it was found that by modifying the N-terminal
group to 3,5-bis(trifluoromethyl)benzoyl group, compound 2 ex-
hibited significantly improved K+ transport ability at the concentra-
tion of 10 μM. The N-terminal replacement may exert two effects
that contribute to the improved K+ transport activity. First, with the
bis-CF3 groups, the estimated Clog P value was increased from 3.4
(compound 1) to 4.8 (compound 2)26, which corresponds to the im-
proved lipophilicity and thereby better transmembrane K+ transport
activity27. Second, the electron-withdrawing effect of the bis-CF3
substituents also increased the acidity of aminoxy amide NH. In
addition, to confirm whether this acidic proton contributes to the
K+ transport, compounds 3−6 were synthesized. As shown in Fig-
ure 1b, once the acidic aminoxy amide proton was replaced with a
methyl group (compound 3), the K+ transport activity vanished
K+ channels are the largest ion channel family that is expressed
in virtually all living organisms10. Ion flux across the subcellular
organelle membranes constitutes over 80% of total ion transport
processes. The major functions of organellar K+ transport are to
regulate osmotic homeostasis as well as to maintain membrane po-
tentials11. Many K+ transport processes inside cells are also coupled
with H+ or Ca2+ transport, which, in turn, regulate the pH levels
within each compartment and control organellar functions govern-
ing metabolism, enzyme activity and protein secretion12. Growing
evidence suggests that cancer cells, which show an abnormally
high propensity to proliferate, are associated with K+ channel
dysregulation13-14. For instance, several types of human cancer cells
including cancer stem cells (CSCs) have low expression of K+
channels and hyperpolarized mitochondrial membrane potentials
that contribute to apoptosis resistance15-17. Synthetic cation trans-
porters that can modulate such gradients may serve as selective an-
ticancer agents. Despite the fact that a considerable range of cation
transporters have been reported, the majority of them are too large
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To fully understand the transport mechanism, further studies were
performed. According to the previously reported method30, 39K
NMR technique was used to study the K+ binding stoichiometry
(Fig. S2). The calculated transport rate (see method) varied linearly
with the compound concentration in LUVs containing different
concentrations of K+ (75 mM, 100 mM and 125 mM) (Fig. S3).
This result strongly indicated the formation of a 1:1 complex be-
tween K+ and compound 2 during transmembrane transport.
Supported by the structure-activity relationship study, it is highly
likely that compound 2 transports K+ through a 1:1 carrier mecha-
nism. In the anionic form, it could bind to K+ through electrostatic
interaction as well as chelation, possibly by aminoxy oxygen atom
and the two carbonyl groups. In addition, the hydrophobic
sidechain OBn group and the N- and C-terminal substituents might
shield the hydrophilic K+ ion during transport. After entering into
liposomes, where the intravesicular pH value was around 6.8, com-
pound 2 can be re-protonated and release K+ ion. Compound 2 in
neutral form can freely diffuse through the membrane to complete
the carrier cycle (Fig. 1e).
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Fig. 1. Molecular design and transport mechanism studies. a, Chemical
structures of aminoxy acid monomers 1−6. b, K+ transport behavior of com-
pounds (10 µM) as evaluated by HPTS assay. c, X-ray crystallographic
structure of compound 2. All hydrogen atoms except N‒H are omitted for
clear presentation. Eight-membered ring hydrogen bond formation denotes
the adoption of N‒O turn conformation. d, CD spectrophotometer analysis
of compound 2 (0.5 mM) in CH3CN (black) and 50 mM DPC (red) at pH
7.4. e, Proposed transport mechanism of compound 2 in liposome assays.
Fig. 2. Ion transport activities of compound 2. a, K+/Na+ selectivity of syn-
thetic K+ transporter 2 at a concentration of 10 µM. b, Cl− transport activity
of compound 2 as evaluated by SPQ assay. c, Liposome membrane potential
as evaluated by safranin O assay. Val was used as a positive control. All the
molecules were used at the final concentration of 1 μM. d, Ion transport
activity of compound 2 (0.3 µM) in the presence or absence of the proton
ionophore FCCP (100 nM) or the potassium ionophore Val (25 nM).
completely. Similarly, without α-effect from the extra oxygen
atom, compound 4 that was built from amino acid serine exhibited
no K+ transport activity. These results indicated the critical roles of
free aminoxy amide NH for K+ transport. The K+ transport capacity
was also significantly reduced when the molecule was truncated
(compound 5) or when the side chain was completely removed
(compound 6). The X-ray crystallographic structure of compound
2, crystallized in ethyl acetate and hexane, confirmed the existence
of the anticipated N‒O turn in the solid state28-29 (Fig. 1c), which
was also supported by circular dichroism (CD) spectra obtained in
acetonitrile (Fig. 1d). However, the CD spectrum of compound 2
obtained in dodecylphosphocholine (DPC) buffered at pH 7.4
clearly showed the loss of its secondary structure. We hypothesized
that the bis-CF3 substituted aminoxy amide NH group is partially
deprotonated at physiological pH. Indeed, the pKa of aminoxy NH
group was found to decrease from around 8.3 to around 7.1 upon
the bis-CF3 substitution (Fig. S1). In our liposome assays, the ex-
travesicular pH value was maintained at around 8.0, at which the
predominant population of compound 2 would be the anionic form.
Selective K+ and H+ transport in liposomes. Next, the ion
selectivity of compound 2 was further evaluated in liposome assays
(see details in Methods). It was found that compound 2 exhibited
excellent K+/Na+ selectivity but did not transport Cl− (Figs. 2a and
2b) with EC50 values of 2.02 μM (4.04 mol%) for K+ and 47.26 μM
(94.52 mol%) for Na+, respectively (Figs. S4−S8). The EC50 values
of compound 2 for different ions and K+/Na+ selectivity were sum-
marized in Table 1. The selectivity of compound 2 towards other
alkali metal cations was also evaluated (Fig. S9). Compound 2 has
shown a selectivity trend of K+ > Cs+ > Li+ > Na+ in the presence
or absence of pH gradient, which implied that the transport ability
was not directly correlated with dehydration energy or sizes of
ions31. Compound 2 with remarkable K+ selectivity was able to es-
tablish stable membrane potential in safranin O assay32 at a con-
centration of 1 µM, which was comparable to that of natural potas-
sium ionophore valinomycin (Val) (Fig. 2c). At a concentration of
10 µM, no carboxyfluorescein release was detected, suggesting the
absence of pore formation in lipid bilayers (Fig. S10).
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Fig. 3. Selective K+/H+ transport on the inner mitochondrial membrane. a, Illustration of the transport behavior of compound 2 in the context of mitochondria.
b, Representative confocal images and quantitative fluorescence measurements representing kinetic changes in matrix pH of HeLa cells (n = 10−20 cells)
transfected with Mito-SypHer. Compound 2 was added at 180 s (mean ± s.e.m., n = 20−30 cells). c, Representative confocal images and quantitative fluores-
cence measurements of mitochondrial-targeting K+ probe KS6 representing kinetic changes in matrix K+ concentration in SKOV3 cells. Compound 2 was
added at 180 s (mean ± s.e.m., n = 20−30 cells). d, Quantification of measurements of mitochondrial membrane potential using probe JC-1 in HEYA8 cells
treated with compound 2 for 10 min (mean ± s.e.m., n = 20−30 cells, ***p < 0.001, one-way ANOVA) and kinetic changes in mitochondrial membrane
potential of HEYA8 cells. Compound 2 was added at 180 s (mean ± s.e.m., n = 20−30 cells). Scale bars denote to 20 μm.
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To identify relative K+/H+ transport of compound 2, the HPTS
assay was performed in the presence of Val and carbonyl cyanide-
4-(trifluoromethoxy)phenylhydrazone (FCCP) to counter-balance
the rate-limiting ion33. As shown in Fig. 2d, a significant increase
in HPTS fluorescence by compound 2 was observed in the presence
of Val but not FCCP. This suggested that compound 2 was an elec-
trogenic transporter with a H+ > K+ transport rate. This result fur-
ther confirmed our model that compound 2 can facilitate the move-
ment of both K+ and H+ down their electrochemical gradients inde-
pendently. In the presence of Val, the EC50 value of proton
transport activity was determined to be 0.15 μM (0.30 mol%) (Fig.
S11).
IMM. We speculated that the neutral form of compound 2 could
freely pass through IMM and enter the matrix, where it subse-
quently loses one proton and changes to the anionic form. Due to
the negative charges on IMM, this anionic form could be trapped
in the matrix, unless it forms a neutral complex with one K+ and
travels back to the intermembrane space. This cycle would lead to
H+ and K+ exchange in the matrix (Fig. 3a).
To confirm this hypothesis, matrix pH change was monitored by
real-time confocal imaging in HeLa cells transfected with the pH-
sensitive mitochondria-targeted fluorescent protein Mito-SypHer35
(Fig. 3b and S14a). Upon addition of compound 2, a spontaneous
decrease in the green to blue fluorescence ratio (I488/I405) was ob-
served, suggesting that matrix H+ influx was induced. This effect
was also confirmed in human ovarian cancer SKOV3 cells (Fig.
S14b). Matrix K+ concentration was also monitored by a mitochon-
dria-targeting K+ fluorescent probe KS636. An obvious drop in flu-
orescence intensity of KS6 occurred in SKOV3 cells shortly after
the addition of compound 2, which indicated K+ efflux (Figs. 3c
and S15). As ΔΨm was closely related to ionic balance, we next
monitored its change with a ratiometric fluorescent probe JC-1. A
decreased red to green fluorescence ratio of JC-1 was observed in
live-cell confocal imaging, in a dose-dependent manner, indicating
ΔΨm dissipation (Figs 3d and S16). Kinetics studies demonstrated
that an acute depolarization of ΔΨm occurred within 30 s upon
compound 2 addition, which suggested the faster rate of H+ influx
in the matrix than that of K+ efflux. This H+ influx and K+ efflux
processes on IMM agreed well with our hypothesis and the charac-
terized transport activity of compound 2 in liposomes.
Disruption of mitochondrial functions. Given that ion ho-
meostasis is coupled to mitochondrial functions37, we further eval-
uated the acute effects of compound 2 on mitochondrial ROS pro-
duction38, respiration and mitochondrial morphology in HEYA8
cells. Mitochondrial superoxide (O2•‒) production was monitored
with the chemoselective fluorescent probe HKSOX-2m39. A surge
in O2•‒ level occurred shortly after the challenge of cells with com-
pound 2 (Figs. 4a and S17). Disturbance to K+/H+ homeostasis in-
duced by cation transporter 2 also significantly dampened mito-
chondrial respiration. As illustrated in the oxygen consumption rate
(OCR) assay (Fig. 4b), following the addition of oligomycin (ATP
synthase inhibitor), compound 2 (10 µM) caused an immediate rise
Table 1. Summary of ion transport activities of compound 2.
EC50 (mol%)
Hill coefficient
EC50
+
RK+/RNa
16.0d
K+/Na+
K+
Na+
H+
K+
Na+
H+
4.04a 94.52b 0.30c
23.4
0.84a 3.66b 1.41c
a
EYPC vesicles encapsulating 75 mM K2SO4 were suspended in 75 mM
K2SO4 buffer, or b75 mM Na2SO4 buffer. cTested in the presence of 25 nM
Val in 75 mM K2SO4 buffer. dRatio of fractional transport activity (see Fig.
S8).
Selective K+/H+ transport on the inner mitochondrial
membrane. We thus asked whether compound 2 could promote
ion transport in living cells. We first investigated the changes of
cytosolic K+ concentration and the plasma membrane potential in
human ovarian cancer HEYA8 cells. Following the treatment with
compound 2, however, no significant ion transport across the
plasma membrane was detected (Figs. S12 and S13).
Mitochondria are double-membrane organelles, composed of the
inner membranes (IMM) and outer membranes (OMM). During ox-
idative phosphorylation (OXPHOS), the electron transport chain
complexes located on IMM pumped protons out from the matrix to
the intermembrane space (IMS), which generates the mitochondrial
membrane potential (ΔΨm) and pH gradient with higher pH level
in the matrix (pH ≈ 7.8) than that in the cytosol (pH ≈ 6.8)34. In
mitochondria, the pH gradient and ΔΨm would act as strong driv-
ing forces for compound 2 to facilitate K+ and H+ transport across
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in OCR of intact HEYA8 cells. This was due to the collapse of the
proton gradient, which allowed the oxygen consumption of com-
plex IV to reach the maximum. Subsequently, cells treated with
compound 2 showed no respiratory response to FCCP. These re-
sults corroborated the notion that our K+ transporter drastically im-
pairs cell respiration through the dissipation of pH gradient across
IMM40. Homeostasis of K+ and H+ is essential for maintaining the
matrix volume and structural integrity of mitochondria12, 37. There-
fore, we examined mitochondrial morphology in HEYA8 cells
treated with compound 2. Upon 1 h incubation, mitochondria un-
derwent a morphological change into a round shape, in contrast to
their characteristic tubular morphology in resting cells, suggesting
a stress induction (Fig. 4c). As a whole, these results indicated that
K+ and H+ transport across IMM mediated by compound 2 coordi-
nately resulted in the damage of mitochondrial functions.
Selective killing of salinomycin resistant ovarian
CSCs. CSCs, as a minor cell subpopulation in tumors, are resistant
to many current therapies and privileged with the capacity for self-
renewal, which leads to tumor relapse and poor prognosis41. Selec-
tive elimination of CSCs has been proposed as a promising direc-
tion to improve current anti-cancer treatments42-43. Accumulating
evidence suggests that many CSCs, including ovarian CSCs, are
enriched population of cells with CD133+ antigenic phenotype rep-
resenting ovarian CSCs50 was confirmed in spheres (Fig. 5b). Con-
sistent with literatures51-52, increased mitochondria mass (Fig. S18)
and hyperpolarized mitochondria membrane potential were de-
tected (Fig. S19) in CSCs. When the CSCs were treated with com-
pound 2, depolarization of mitochondria was clearly observed even
when the concentration was as low as 200 nM (Fig. S20). These
results were consistent with our previous studies in adherent cancer
cells and suggest that the ion transport activity of compound 2 was
conserved in CSCs.
9
It was found that compound 2 inhibited the growth of CSCs at a
concentration of 5 µM, which was otherwise non-toxic to adherent
cancer cells (Fig. 5c). Other non-transporting compounds 1, 3, 4, 5
and 6, on the other hand, did not show significant toxicity at the
same dose. Known resistance to paclitaxel (PTX) in these ovarian
CSCs was also observed. Interestingly, Val and FCCP, which
solely transport K+ and H+, respectively, showed lower toxicity to-
ward CSCs than cancer cells. Comparisons of IC50 values of K+
transporter 2, salinomycin and nigericin against ovarian CSCs and
cancer cells were shown in Figs. 5d and 5e. Compound 2 displayed
high selectivity towards ovarian CSCs and only moderate toxicity
towards adherent cancer cells and noncancerous cells (HEK293,
NIH3T3, and MDCK) (Fig. S21, Table 2). This selectivity was
found to be as high as 47-fold in the case of the IC50 of compound
2 towards SKOV3 CSCs and corresponding cancer cells. In con-
trast, the CSCs were resistant to both salinomycin and nigericin
(Figs. 5d,e) due to their abundant expression of ABC transporters
(ABCB1 and ABCG2)47 (Fig. S22). When HEYA8 cells were
treated with compound 2, the proportions of CD133+ subpopulation
declined (Fig. 6a). In contrast, PTX treatment increased this sub-
population. As an in vitro measurement of CSC activity, we tested
the ability of cells to form spheres when grown in suspension cul-
tures. In agreement with the above results, significantly fewer
spheres were observed in cells treated with compound 2 (Fig. 6b).
As a functional assessment of CSC inhibition, an in vivo tumor-
forming experiment was performed. Cells were pretreated ex vivo
with compounds for 2 days, then were allowed to recover for 10
days before injected subcutaneously into nude mice. This experi-
ment allows direct evaluation of the subsequent in vivo tumor-
forming ability of the treated cells. The recovery period is critical,
as it ensures tumor-forming ability is directly correlated to CSC
population among the cells but not due to the loss of viability. It
was found that cells pretreated with compound 2 showed signifi-
cantly decreased ability in tumor formation than the untreated cells
or those treated with PTX (Fig. 6c). Moreover, compound 2 itself
could also reduce the size of established tumor in nude mice model
(Fig. S23). Collectively, our results demonstrated that K+/H+
transport on the inner mitochondria membrane can lead to selective
eradication of ovarian CSCs.
Induction of apoptotic cell death and suppression of
autophagy. Mitochondrial oxidative stress and ΔΨm dissipation
are considered critical pro-apoptotic events to initiate mitochon-
dria-mediated (intrinsic) apoptotic cascades characterized by the
activation of caspases-9, caspase-353 and subsequently poly [ADP-
ribose] polymerase 1 (PARP-1)54. It was found that after 48 h treat-
ment with compound 2, a much higher proportion of apoptotic cells
were detected in CSCs than in cancer cells, even when CSCs were
treated with 10-fold lower concentrations (Fig. 7a). Immunoblot
showed that cleavage of procaspase-9, -3 and PARP-1 were de-
tected in CSCs treated with transporter 2, which indicated mito-
chondria-mediated apoptosis (Fig. 7b). Depolarization of ΔΨm
would initiate mitophagy through the PINK1/Parkin pathway55.
Following treatment with compound 2 for 2 h, merging of mito-
chondria (green) and lysosomes (red) was observed in HEYA8
cells. These observations serve as an early sign of mitophagy to
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Fig. 4. Synthetic cation transporters perturbed mitochondrial functions. a,
Representative confocal images and quantitative fluorescence measure-
ments of mitochondrial superoxide levels in HEYA8 cells stained with
HKSOX-2m (4 µM). Scale bars = 20 µm. Compounds at a final concentra-
tion of 5 μM were added at 180 s (mean ± s.e.m., n = 20−30 cells per group).
b, OCR as measured by the Seahorse instrument (mean ± s.e.m., n = 3). c,
Representative confocal images for analysis of mitochondrial morphology
of MitoTracker Green-stained HEYA8 cells treated with compound 2 (10
µM) for 1 h. Scale bars = 10 µm.
characterized by hyperpolarized ΔΨm, due to the oxidative phos-
phorylation (OXPHOS)16 dependence. The dissipation of ΔΨm
thus provides a viable strategy in selective eradication of CSCs44.
Salinomycin, a natural polyether K+ ionophore, was previously
identified as the leading anti-CSCs agent45-46. However, emerging
evidence suggested that CSCs overexpressing ATP-binding cas-
sette transporters (ABC transporters), as in the case of ovarian can-
cer47, are non-susceptible to salinomcin48. With our synthetic trans-
porter 2, we next tested the effect of K+/H+ transport on drug-re-
sistant ovarian CSCs. We utilized the CSC model of ovarian
SKOV3 and HEYA8 cells previously established by sphere-form-
ing in non-adherent, stem-cell-selective conditions (Fig. 5a)49. An
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Fig. 5. Synthetic cation transporters selectively killed ovarian CSCs. a, Microscopic images of HEYA8 cells cultured as spheres and adherent cells. b, HEYA8
cells cultured as spheres had higher proportions of CD133+ antigenic phenotype (mean ± s.e.m., n = 3, ****p <0.001). c, Viability of HEYA8 cancer cells and
CSCs following treatment of PTX (100 nM), Val (5 μM), FCCP (5 μM), compounds 1−6 (5 μM) for 48 h (mean ± s.e.m., n = 3, **p < 0.01, ***p < 0.001).
d, e, Representative viability curves of HEYA8 and SKOV3 cancer cells and CSCs following treatment of compound 2, salinomycin, or nigericin for 48 h
(mean ± s.e.m., n = 3).
Table 2. Summary of IC50 values (μM) of compound 2 on cancer cells, CSCs, and non-cancer cells.
HEYA8
Sphere Adherent
1.5 ± 1.3 23.4 ± 2.1
SKOV3
Sphere Adherent
0.9 ± 1.0 42.1 ± 5.2
HEK293
MDCK
NIH3T3
HEYA8
CSCs
selectivity
SKOV3
CSCs
selectivity
Non-cancer Cells
16-fold
47-fold
51.0 ± 1.0 25.1 ± 1.1 30.5 ± 1.1
Figure 6 Synthetic cation transporters reduces CSCs population in vitro and tumor-forming ability in vivo. a, In vitro effects of PTX (100 nM) or compounds
2 (15 μM) against CD133+ HEYA8 cell population following treatment for 72 h. Fluorescence for PE-conjugated anti-CD133 antibody was measured by flow
cytometry. b, Sphere-forming ability of HEYA8 cells following treatment as indicated for 48 h (mean ± s.e.m., n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
c, In vivo tumor forming ability of HEYA8 CSCs treated as indicated (mean ± s.e.m., n = 4 mice per group, **p < 0.01).
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indicate the fusion of autophagosomes with lysosomes56 (Fig. 7c).
ion transport across lysosomal membranes (Fig 7e). As shown in
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Next, protein levels of the autophagy components, i.e. microtu-
bule‑associated protein light chain 3 (LC3) and p62, were moni-
tored by immunoblotting. Upon treatment with compound 2 for 24
h, the conversion of LC3-I to LC3-II was detected in both HEYA8
adherent cancer cells and CSCs in a dose-dependent manner (Fig.
7d), suggesting the initiation of autophagy. However, the upregu-
lation of both LC3-II and p62 implies the suppression of subse-
quent proteolytic degradation. Again, the effects of compound 2
were more profound in CSCs.
Figure 7f, significantly decreased red fluorescence of lysosome pH
sensor acridine orange (AO) was observed in HEYA8 cells 1 h after
the addition of compound 2, which illustrated the pH increase of
lysosomes was caused by proton efflux from lysosomes. The lyso-
some alkalization induced by compound 2 was likely to inhibit fur-
ther proteolytic degradation process57. Altogether, we have demon-
strated that K+/H+ transport induced by compound 2 on mitochon-
drial and lysosomal membranes act coordinately to selectively in-
duce apoptotic cell death and suppress autophagy of ovarian CSCs.
9
We expected that the pH gradient existing between the cytosol
and lysosomal lumen (pH 4.5‒4.7) also provides a driving force for
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Fig. 7. Synthetic compound 2 induced apoptosis and suppressed autophagy in HEYA8 cancer cells and CSCs. a, Flow cytometry analysis of apoptosis in
HEYA8 ovarian cancer cells and CSCs following indicated treatment for 48 h. Data were acquired immediately after cells were stained with Annexin V-Alexa
488 and propidium iodide (PI). b, Immunoblots showing the levels of PARP-1, cleaved PARP-1, procaspase-9, procaspase-3, cleaved caspase-3 and β-tubulin
(internal loading control) in HEYA8 cells following indicated treatment for 48 h. c, Representative confocal images for analysis on co-localization of lyso-
somes (stained with LysoTracker Red) and mitochondria (stained with MitoTracker Green) in HEYA8 cells treated with compound 2 (10 µM) for 2 h. Scale
bar = 10 µm. d, Immunoblots showing the levels of p62, LC3-I, LC3-II and GAPDH (internal loading control) in HEYA8 cells following indicated treatment
for 24 h. e, Illustration of the transport behavior of compound 2 in the context of lysosomes. f, Representative confocal images of lysosomal pH probe acridine
orange in HEYA8 cells treated with compound 2 (10 µM for 1 h). Scale bars = 20 µm.
function on the mitochondrial and lysosomal membranes within
minutes upon addition. Our mechanistic studies have revealed that
compound 2 allows ion transport to take place in the above-men-
tioned sites by exploiting endogenous subcellular proton gradients
and membrane potential. The localized K+/H+ flux potently dis-
rupted mitochondrial and lysosomal functions, which are charac-
terized by the dissipation of mitochondrial membrane potential,
ROS production, uncoupling of respiration, mitochondrial morpho-
logical changes and pH alterations in lysosomes. All these effects
culminated in up to 47-fold selectivity in killing ovarian CSCs via
apoptosis induction and autophagy suppression. The selective de-
pletion of CSCs by compound 2 also blunted tumor formation in
mice. The exceptionally enhanced cytotoxicity of compound 2 to-
wards ovarian CSCs was ascribed to the higher dependence of
those CSCs on OXPHOS in mitochondria for energy production16.
DISCUSSION
Disrupting ion homeostasis with synthetic ion transporters has
been demonstrated to cause cell death. However, this approach has
shown limited clinical potential in cancer treatment as the resulting
ionic disruption would cause toxicity to cancer cells and normal
cells indiscriminately58. As different types of cells establish altered
ionic regulation to maintain their distinct physiology, we have suc-
cessfully demonstrated that targeting these critical electrochemical
gradients can serve as a promising strategy to attack cells selec-
tively.
In this study, we have developed a simple yet efficient synthetic
ion transporter, compound 2, which could selectively mediate
K+/H+ transport in living cells. It is found that compound 2, without
bearing any organelle-targeting moieties, could site-selectively
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In contrast, cancer cells favor the glycolysis pathway for metabo-
CONCLUSIONS
lism that is known as the “Warburg effect”. Our results are in line
with the recent findings that ovarian CSCs are more sensitive to-
wards extrinsic inducers of mitochondrial stress and damage in trig-
gering apoptosis59. It has also been reported that autophagy is es-
sential for CSCs' self-renewal capacity and pluriopotency60. The
suppressed autophagy processes as a result of lysosome alkaliza-
tion induced by compound 2 also contributed to its selective cyto-
toxicity towards ovarian CSCs.
In summary, a novel small-molecule K+/H+ transporter, com-
pound 2, was developed by using an α-aminoxy acid monomer. By
disturbing the critical mitochondrial and lysosomal ionic gradients
in cells, this molecule exhibits exceptional selectivity in killing
drug-resistant ovarian CSCs. We believe the structural and biolog-
ical insights from our study could guide the design, optimization
and application of small-molecule synthetic cation transporters as
well as other small molecules that selectively eradicate cancer stem
cells.
9
Previously, salinomycin was identified as the leading anti-CSCs
agent45. As a well-known potassium ionophore, salinomycin has
also been demonstrated to facilitate K+/H+ exchange across the in-
ner mitochondrial membrane40. It is possible that the K+/H+
transport behavior of salinomycin acts as the upstream effect that
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Methods
directs to the intricate cell responses for selective CSCs killing61-64
.
Base-pulsed HPTS assay. HPTS-loaded LUV suspension (100
μL) prepared as described above was added in isotonic HEPES
buffer (1.9 mL) and placed into a fluorometric cuvette. HPTS flu-
orescence was monitored with excitation at 403 and 460 nm, and
emission at 510 nm. At t = 100 s, DMSO solution of the test com-
pounds (20 μL) and 0.5 M NaOH/KOH aqueous solution (20 μL)
were added through an injection port. Addition of the base caused
about 1 pH unit increase in the extravesicular buffer. At t = 500 s,
40 μL of 5% Triton X-100 was added to lyse the liposomes. The
fluorescence ratio of F460/F430 of initial 100 s was set as 0% ion
transport and the final fluorescence ratio of F460/F430 induced by
Triton X-100 was set as 100% ion transport. DMSO or other sol-
vents as indicated was used as control.
Cancer stem cell cultures. Isolation and culture of spheres were
performed in serum-free stem-cell-selective condition as described
in literature49. Briefly, 1–2 weeks after seeding, non-adherent
spherical clusters of cells could be observed and were separated
from single cells by low-speed centrifugation. After 8th to 10th pas-
sages, the non-adherent spherical clusters of cells appeared as dis-
tinct spheres. Using this selection condition, HEYA8 spheres
(HEYA8 CSCs) could be enzymatically dissociated and reformed
into spheres within 3 days under stem-cell-selective condition. To
allow differentiation, dissociated sphere cells were plated on tissue
culture plates in medium (MCDB 105: M199 = 1:1) supplemented
with 10% FBS and 1% penicillin-streptomycin. The culturing
methods for other types of cells including MDCK, HEK293 and
NIH3T3 are provided in the Supplementary Information.
Measurement of adherent cell viability. Cells were plated in
triplicate in 0.1 mL full medium in 96-well plates for 24 h. After
that, the medium was changed to the freshly prepared medium with
various concentrations of the test compound. Cells were incubated
for another 48 h. Then the cell viability was measured by CellTiter-
Glo® Luminescent Cell Viability reagent according to the manu-
facture’s instruction. The luminescence at 550 nm was measured
using a microplate reader (DTX 880 Multimode Detector, Beck-
man Coulter). Data were analyzed by GraphPad software 7.00.
Measurement of CSCs viability. HEYA8 or SKOV3 CSCs (5 ×
104) were plated in triplicate in 10 mL serum-free MCDB105 me-
dium in a 100-mm Petri dish for 7 days to form spheres. Then test
compounds at different concentrations were added. The cells were
further incubated at 37oC for 48 h. After that cells were collected
by centrifugation and the medium was removed. Then 100 μL of
CellTiter-Glo® Luminescent Cell Viability reagent was added into
each tube, which was incubated for 10 min with shaking. After that,
the reagent was transferred into 96-well plates and cell viability
was measured using a microplate reader (DTX 880 Multimode De-
tector, Beckman Coulter).
Since emerging evidences have challenged the therapeutic poten-
tial of salinomycin due to drug extrusion by ABC transporters48,
the use of our newly developed molecule to induce K+/H+ transport
could guide us to achieve more potent, less toxic and resistance-
evasive therapies for CSCs elimination.
Our studies have also introduced a novel strategy for the devel-
opment of synthetic small-molecule K+ transporter. Building syn-
thetic molecules that could mimic the function of K+ channels has
attracted intensive interests. Despite the extended efforts, the ma-
jority of the prior examples were constructed with complex scaf-
folds, such as oligophenyl rods18, calix[4]arene65, pillar[n]arene66,
and cyclic peptides67-68 Recently, by using crown ether as cation
binding unit, several small-molecule K+ transporters have also been
reported32, 69. However, the ion transport activities of all those re-
ported molecules were constrained to in vitro characterization in
liposomes. The therapeutic values of synthetic cation transporters
have been underexplored. Here, by using α-aminoxy acids as a
novel K+ recognition scaffold, a simple K+ transporter with excel-
lent transport efficiency and K+/Na+ selectivity was constructed. It
was found that the increased acidity of aminoxy amide NH allowed
compound 2 to partially adopt anionic form in physiological pH
(7.4), which enabled its binding with K+ through electrostatic inter-
action and chelation. Other hydrophobic moieties of the molecule,
including the side chain, the N- and C-terminal carbonyl groups,
shielded the hydrophilic cation to lower the physical barriers during
the diffusion through membrane lipids. However, attempts to meas-
ure the K+ binding by using UV/vis spectroscopy, isothermal titra-
tion calorimetry and 1H NMR methods failed, as negligible change
was observed with KPF6 titration, in either acetonitrile or do-
decylphosphocholine (DPC) micelles (data not shown). These re-
sults indicated relatively weak cation binding affinity for com-
pound 2, suggesting that tight association with cation may not be
necessary for transport, instead, suitable initial cation recognition
and hydrophobicity should be more critical in efficient transmem-
brane movement. We anticipate that further studies could enable us
to define in more detail the structural underpinnings of K+/Na+ se-
lectivity of aminoxy acids. We believe this insight could further
guide the development of small yet more selective and efficient cat-
ion transporters into improved therapeutics.
More broadly speaking, our discoveries of small molecule-based
K+ transporter would also foster the development of new therapies
for other diseases related to dysregulation of K+ channels, such as
long QT syndrome, neuronal disorders, and Alzheimer's disease,
arise from potassium channel deficiency and cause erratic potas-
70
sium gradient build-up across compartment-specific membranes .
By understanding and harnessing the properties of such ion gradi-
ents, small cation transporters with the capacity to autonomously
perform K+ movement have the potentials to restore such K+ accu-
mulation and treat diseases.
Oxygen consumption rate assay. Cell respiration was measured
by using an XF24 Extracellular Flux Analyzer (Seahorse, Biosci-
ence), which measures the oxygen consumption rate (OCR). Ad-
herent HEYA8 cells were seeded at 50000 cells/well in 200 μL of
their culture medium and incubated for 24 h at 37oC in a humidified
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atmosphere with 5% CO2. The medium was then replaced with 670
Seahorse instrument. We thank The University of Hong Kong Li
Ka Shing Faculty of Medicine Faculty Core Facility for support in
confocal microscopy and flow cytometry.
µL/well of high-glucose DMEM without serum and supplemented
with 1 mM sodium pyruvate and 2 mM L-glutamine. The oxygen
consumption rate (OCR) was measured with an extracellular flux
analyzer (Seahorse) at preset time intervals upon the prepro-
grammed additions of the following compounds: oligomycin to 1
µM, FCCP to 500 nM, antimycin A and rotenone to 0.5 µM final
concentrations.
Measurement of the in vivo tumor-forming ability. All animal
experiments were approved by the Committee on the Use of Live
Animals in Teaching and Research (CULATR) at the University of
Hong Kong. In vivo tumor-forming ability of cells after treatment
with compound 2 was evaluated. Paclitaxel was used as a negative
control. For this experiment, HEYA8 CSCs were pretreated with 2
(4 µM) or paclitaxel (0.1 µM) for 2 days in a suspension medium,
respectively. Then cells were allowed to proliferate in full medium
in the absence of drugs for 10 days. After that, 106 cells were sub-
cutaneously (s.c.) injected into the flank of athymic nude mice bi-
laterally. The length and width of a tumor were measured with a
caliper for 25 days after injection and tumor volume was calculated
as Tumor volume V = (π × L × W2)/6, where L represents the larg-
est tumor diameter and W represents the perpendicular tumor di-
ameter.
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ASSOCIATED CONTENT
Supporting Information. Full experimental details are given in
the Supplementary Information.
AUTHOR INFORMATION
Corresponding Author
Dan Yang ‒ Morningside Laboratory for Chemical Biology,
Department of Chemistry, The University of Hong Kong, Pok-
fulam Road, Hong Kong, China; Email: yangdan@hku.hk
Authors
Fang-Fang Shen ‒ Morningside Laboratory for Chemical Bi-
ology, Department of Chemistry, The University of Hong Kong,
Pokfulam Road, Hong Kong, China
Sheng-Yao Dai ‒ Morningside Laboratory for Chemical Biol-
ogy, Department of Chemistry, The University of Hong Kong,
Pokfulam Road, Hong Kong, China
Nai-Kei Wong ‒ Morningside Laboratory for Chemical Biol-
ogy, Department of Chemistry, The University of Hong Kong,
Pokfulam Road, Hong Kong, China; Department of Infectious
Diseases, Shenzhen Third People’s Hospital, The Second Hos-
pital Affiliated to Southern University of Science and Technol-
ogy, Shenzhen 518112, China
Shan Deng ‒ School of Biological Sciences, The University of
Hong Kong, Pokfulam Road, Hong Kong, China; Morningside
Laboratory for Chemical Biology, Department of Chemistry,
The University of Hong Kong, Pokfulam Road, Hong Kong,
China
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ACKNOWLEDGMENT
We acknowledge support from The University of Hong Kong,
Morningside Foundation, and the Hong Kong Research Grants
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