Host-guest inclusion for enhancing anticancer activity of pemetrexed against lung carcinoma and decreasing cytotoxicity to normal cells

Article abstract of DOI:10.1016/j.cclet.2021.03.079

Advanced chemotherapy strategies are in urgent demand for improving anticancer efficacy. Herein, a water-soluble pillar[6]arene (WP6A) was used to load chemotherapeutic agent pemetrexed (PMX) by forming direct host-guest inclusion, which is beneficial for

Full text of DOI:10.1016/j.cclet.2021.03.079

G Model
CCLET-6275; No. of Pages 5
Chinese Chemical Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Host-guest inclusion for enhancing anticancer activity of pemetrexed
against lung carcinoma and decreasing cytotoxicity to normal cells
a,1
b,1
b
c
b
c
Junyi Chen , Yahan Zhang , Yadan Zhang , Liang Zhao , Longming Chen , Yao Chai ,
b
a,c,
b,
a,c,
Zhao Meng , Xueshun Jia *, Qingbin Meng **, Chunju Li
*
a
College of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
Department of Chemistry, Center for Supramolecular Chemistry and Catalysis, Shanghai University, Shanghai 200444, China
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Advanced chemotherapy strategies are in urgent demand for improving anticancer efficacy. Herein, a
water-soluble pillar[6]arene (WP6A) was used to load chemotherapeutic agent pemetrexed (PMX) by
forming direct host-guest inclusion, which is beneficial for decreasing cytotoxicity of PMX on BEAS-2B
cells. NMR and florescence titration served to confirm the complexation between WP6A and ATP with
Received 9 March 2021
Received in revised form 26 March 2021
Accepted 29 March 2021
Available online xxx
5
higher affinity [(5.67 Æ 0.31) Â 10 L/mol], favoring competitive replacement of PMX. Complexation ATP
by WP6A effectively prevented ATP from being hydrolyzed in presence of alkaline phosphatase. The
formed host-guest complex was further used to block the efflux pump by cutting off energy source from
ATP hydrolysis, which was accompanied with releasing PMX to produce synergistic enhancement of
anticancer performance towards A549 cells. This supramolecular strategy would also be extended to
other clinical chemotherapeutic agents and it was expected to provide salutary profits for cancer
patients.
Keywords:
Supramolecular chemotherapy
Host-guest complexation
Pemetrexed
ATP
Competitive binding
©
2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Cancer signals a leading cause of mortality over the world and
the patient number is forecast to reach over 21 million by 2030 [1].
Though considerable efforts have been made for treatment of
cancer, chemotherapy is still the most important approach in clinic
[11–14]. Adenosine 5'-triphosphate (ATP), one of the most
important biomolecules in living organisms, is seen as direct
source of energy for various life activities [15,16]. Cells produce ATP
through two related mechanisms, namely that oxidative phos-
phorylation in mitochondria and glycolysis in the cytosol [17–19].
Compared with normal cells, cancerous cells are characterized by a
marked increase in glycolytic metabolism, leading to significant
elevation in ATP level [20,21]. Efflux transporter, like P-glycopro-
tein (P-gp) can utilize the energy from ATP hydrolysis to decrease
intracellular levels of anticancer agents below their effective dose,
even leading to multidrug resistance [22–24]. Cutting off the
energy source by host-guest encapsulation using artificial recep-
tors was expected to block the efflux pump [25,26]. Given that, ATP
can be identified as a feasible biomarker in this work.
[
2–4]. However, traditional chemotherapy faces lots of challenges
in practical application, such as serious side effects and ambiguous
anticancer efficacy [5–7]. Supramolecular chemotherapy originat-
ed from the marriage between host-guest chemistry and
chemotherapy, provides an effective and readily generalizable
strategy for enhancing anticancer bioactivity of chemotherapy
agents against cancer cells and decreasing cytotoxicity to normal
cells [8–10]. The key to this strategy lies in one of the cancer
biomarkers, which is overexpressed in cancerous microenviron-
ments. Biomarker-triggered release of anticancer agents and
simultaneous consumption of biomarker via noncovalent inter-
actions can achieve synergistic improvement of therapeutic effect
As a proof of concept, pemetrexed (PMX), the first-line
chemotherapeutic agent for lung adenocarcinoma, served as
model anticancer agent [27–29]. A water-soluble pillar[6]arene
(
WP6A) containing multiple pyridinium moieties was prepared as
*
Corresponding authors at: College of Environmental and Chemical Engineering,
macrocyclic receptor, affording strong binding to PMX as well as
ATP (Fig. 1) [30–42]. Strong binding to PMX is prerequisite to avoid
its serious damage against normal cells. Then considerable binding
to ATP is necessary to trigger the PMX release. The formation of a
stable host-guest inclusion complex ATP/WP6A was found to
Shanghai University, Shanghai 200444, China.
** Corresponding author.
E-mail addresses: xsjia@mail.shu.edu.cn (X. Jia), nankaimqb@sina.com
(
Q. Meng), cjli@shu.edu.cn (C. Li).
1
These two authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2021.03.079
001-8417/© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
1
Please cite this article as: J. Chen, Y. Zhang, Y. Zhang et al., Host-guest inclusion for enhancing anticancer activity of pemetrexed against lung
carcinoma and decreasing cytotoxicity to normal cells, Chin. Chem. Lett., https://doi.org/10.1016/j.cclet.2021.03.079

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Fig. 1. (a) Chemical structures of PMX, ATP, macrocycle monomer (WM) and WP6A. (b) Schematic illustration of chemotherapy strategy based on host-guest complexation
between PMX and WP6A and release of PMX from WP6A via competitive binding to ATP.
efficiently inhibit the hydrolysis of ATP in the presence of alkaline
phosphatase. High concentration of ATP in cancerous micro-
environments was conducive to replacing of PMX from PMX/
WP6A. As a consequence, the efficacy of chemotherapy of PMX was
significantly improved against human lung adenocarcinoma
epithelia cells and cytotoxicity toward human normal pulmonary
epithelial cells has receded.
1H NMR spectroscopy was employed first to verify host-guest
complexation between host (WP6A) and guestes (PMX and ATP).
1
Figs. 2a-c show the H NMR spectra of PMX in deuterated
phosphate buffer (pD 7.4) in the absence and presence of WP6A.
Upon addition of one equivalent of WP6A, the resonance peaks
a b e f
related to protons H , H , H and H of PMX exhibited remarkable
upfield shifts and broadening effects comparing to free PMX (Dd
À
0.22 ꢀ À0.31) suggesting that these protons were engulfed by the
cavity of WP6A and were shielded by the electron-rich cyclic
structure by forming an inclusion complex. In additon, the host
shows similar binding behaviors toward ATP (Fig. S9 in Supporting
a b c
information). The proton signals for H , H and H of ATP
underwent substantial upfield shift and broadening effects,
indicating that ATP was partially located in the cavity of WP6A.
1
For comparison, controlled H NMR experiments of mixtures of
macrocycle monomer (WM, 6 equv.) and two guests (PMX and
ATP) were also measured (Figs. S10 and S11 in Supporting
information), showing that PMX and ATP could not interact with
WM.
The binding affinities of WP6A with two guests (PMX and ATP)
were determined using a fluorescent indicator dispalcement assay
[
43–45] with Eosin Y (EY) as the optimal reporter dye. The
complexation stoichiometry between WP6A and EY was verified to
be 1:1 by the continuous variation method (Job’s plot method)
result using the fluorescence emission values at 538 nm (Fig. S12 in
Supporting information). EY was strongly complexed by WP6A
Fig. 2. 1H NMR spectra (400 MHz, 298 K) of (a) PMX (2.0 mmol/L), (b) PMX
(
2.0 mmol/L) + WP6A (2.0 mmol/L) and (c) WP6A (2.0 mmol/L) in the 20 mmol/L
phosphate buffer solution (pD 7.4).
2

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Fig. 3. (a) Direct fluorescence titration of EY (1.0
m
mol/L) with WP6A in 10 mmol/L HEPES buffer at pH 7.4,
l
ex =517 nm. (b) The associated titration curve at
mol/L) and WP6A
em =538 nm and fit according a 1:1 competitive binding
lem =538 nm and
fit according a 1:1 binding stoichiometry based on the concentration of WP6A. (c) Competitive fluorescence titration of PMX in the presence of EY (1.0
2.5 mol/L) in 10 mmol/L HEPES buffer at pH 7.4, ex =517 nm. (d) The associated competitive titration curve at
m
(
m
l
l
model. Data are from n = 3 independent experiments and are presented as mean Æ SD.
6
with a binding constant of K
a
= (3.43 Æ 0.07) Â 10 L/mol, which
studied using high-performance liquid chromatography (HPLC).
First, appropriate calibration curve was derived (Fig. S14 in
Supporting information). As shown in Fig. 4a, unpon exposure
to CIAP for 120 min, free ATP underwent rapid hydrolysis and
became undetectable. In the sharp contrast, ATP/WP6A retained
(20.73 Æ 0.82)% of original amount for even 240 min. The above
phenomenon occurred because ATP was engulfed in the undis-
turbed cavity of WP6A preventing ATP from being hydrolyzed.
The ATP-triggered release was then investigated via an
indicator displacement assay. Fluorescein (FI) was screened as
was ideal for the projected competitive titrations (Figs. 3a and b).
The displacement of EY/WP6A by gradual addition of PMX resulted
in regeneration of the instrinsic emission of EY (Fig. 3c). The data
were well fitted by a 1:1 competitive binding model, giving a K
a
4
value of (8.32 Æ 0.27) Â 10 L/mol (Fig. 3d). Robust binding to PMX
would be prerequisite to avoid unwarranted off-target leaking
a
during delivery process. By comparison with the K value of ATP/
5
WP6A [(5.67 Æ 0.31) Â 10 L/mol, Fig. S13 in Supporting informa-
tion], such a high binding constant would endow ATP with an
advantage to competitively bind to WP6A for thoroughly releasing
PMX from the PMX/WP6A complex.
Previous studies have shown that the hydrolysis of ATP could be
inhibited by host-guest inclusion using macrocyclic receptors
a
reporter dye since K for FI/WP6A was comparative to that of PMX/
WP6A at the same order of magnitude (Fig. S16 in Supporting
information). Adding WP6A to FI solution gave rise to the
fluorescence quenching (Fig. 4b). ATP was severely overexpressed
[
25,26]. Drug efflux pump of cancer cells, like P-gp protein, can
in cancer microenvironment ([ATP] < 100
tissues ([ATP] < 10 mol/L) [46–48]. Therefore 10 nmol/L and
100 mol/L ATP were employed to reflect those of normal
physiological environment and cancer microenvironment respec-
tively. The intrinsic emission of FI was almost completely
mmol/L) than in normal
utilize the energy from ATP hydrolysis to transport anticancer
agents out resulting in multidrug resistence [22–24]. The design of
this chemotherapy strategy was expected to provide a benefit in
therapeutic effect via competitive binding between ATP and WP6A.
To test this hypothesis, the hydrolysis rate of ATP and 1:1 mixture
of ATP/WP6A in presence of alkaline phosphatase (CIAP) was
m
m
regenerated at 100
mmol/L ATP, while negligible change of
fluorescence at 10 nmol/L ATP was observed (Fig. 4b). We thus
Fig. 4. (a) ATP (1.0
mmol/L) hydrolysis in the presence of alkaline phosphatase (CIAP,10 mg/mL). (b) Fluorescence spectra of FI in the absence (black) and presence of 4.0 mmol/
L WP6A (red), and upon addition of 100
mmol/L ATP (blue) and 10 nmol/L ATP (green) in 10 mmol/L HEPES buffer at pH 7.4.
3

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Fig. 5. Cytotoxicity seen in the (a) A549 and (b) BEAS-2B cell lines incubated with the indicated agents for 48 h. Cell death was measured using CCK-8 assays (mean Æ SD,
n = 5).
suggested that PMX/WP6A underwent ATP-triggered competitive
release of PMX within ATP-overexpressed cancer microenviron-
ment.
interestingly, PMX/WP6A exhibited better anticancer activity
because the thorough release of PMX by overexpressed ATP and
simultaneous comsumption of ATP by WP6A can produce
synergistic chemotherapeutic efficacy. This research not only
demonstrates that host-guest inclusion is a promising approach to
improve treatment effect on cancer but also manifests that water-
soluble pillararenes can serve as an ideal platform to endow other
clinical agents with benefits.
To verify the above chemotherapy strategy could work at the
cellular level, the cytotoxicity of WP6A in the human lung
adenocarcinoma epithelia cells (A549) and human normal
pulmonary epithelial cells (BEAS-2B) were assessed using a Cell
Counting Kit-8 (CCK-8) assay. WP6A displayed minimal cytotoxic-
ity against these two cell lines over the testing range (Fig. S17 in
Supporting information). These same A549 and BEAS-2B cell lines
were then used test the in vitro cell inhibitory effect of PMX/WP6A
and two controls (Figs. 5a and b). Concentration-dependent cell
death could be seen in all formulations. The half-maximum
inhibitory concentration (IC50) value of free PMX for A549 cell line
Declaration of competing interest
The authors report no declarations of interest.
Acknowledgments
(
3.07 mmol/L) matched that recorded in the literature [49–51] and
the cytotoxic activity of PMX + WM was nearly equal to that of free
PMX over a range of concentrations. While PMX/WP6A exhibited
higher anticancer bioactivity than PMX alone. The significant
anticancer efficacy improvement was likely attributed to selected
We acknowledge the National Natural Science Foundation of
China (Nos. 21772118, 21971192, 81573354), and the Natural
Science Foundation of Tianjin City (No. 20JCZDJC00200).
release of PMX from complex upon encountering
a
high
Appendix A. Supplementary data
concentration ATP inside cancer cells and the energy source of
efflux pump was cut off by forming stable inclusion complex ATP/
WP6A. In addition, we found interestingly that, via host-guest
complexation, the cytotoxicity of PMX/WP6A to BEAS-2B pre-
sented significant reduce. The reason was that ATP level in normal
cells was frequently lower than that in cancer cells and lower
concentration of ATP would be insufficient to cause complete PMX
release via competitive replacement.
Moreover, from the morphology of living cells we could further
confirmed that PMX/WP6A could improve anticancer bioactivity of
PMX against A549 cells and decrease its cytotoxicity to BEAS-2B
cells (Fig. S18 in Supporting information). In addition, to
investigate the apoptosis of A549 cells and BEAS-2B cells, FITC-
Annexin V/propidium iodide (PI) method was employed. As shown
in Fig. S19 (Supporting information), negligible apoptosis hap-
pened in the PBS group in A549 cell line. The percentage of
apoptotic cells (including early and late apoptotic cells) in free PMX
group was about 24.24%, while that in the presence of PMX/WP6A
was significantly higher at 41.59%. For BEAS-2B cell line, the total
apoptotic ratios of PMX/WP6A (14.53%) was lower than that of free
PMX (19.20%) (Fig. S20 in Supporting information). Taken in
concert, the cellular results provided support for the conclusion
that inclusion PMX within WP6A could improve the anticancer
bioactivity of PMX and decrease its toxic side effects to normal
cells.
Supplementary material related to this article can be found, in
the
online
version,
at
doi:https://doi.org/10.1016/j.
cclet.2021.03.079.
References
[
1] C. Allemani, T. Matsuda, V.D. Carlo, et al., Lancet 391 (2018) 1023–1075.
[2] J.J. Shi, P.W. Kantoff, R. Wooster, O.C. Farokhzad, Nat. Rev. Cancer 17 (2017) 20–
7.
3
[
[
3] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646–674.
4] S. Kummar, H.X. Chen, J. Wright, et al., Nat. Rev. Drug Discov. 9 (2010) 843–856.
[5] R. Meel, E. Sulheim, Y. Shi, et al., Nat. Nanotechnol. 14 (2019) 1007–1017.
6] P.N. Sliwinska, A. Weiss, X. Ding, et al., Nat. Protoc. 11 (2016) 302–315.
7] R. Kumar, W.S. Shin, K. Sunwoo, et al., Chem. Soc. Rev. 44 (2015) 6670–6683.
8] H.B. Chen, Z.J. Gu, H.W. An, et al., Sci. China Chem. 61 (2018) 1503–1552.
[9] J. Yang, D.H. Dai, L.J. Ma, Y.W. Yang, Chin. Chem. Lett. (2020), doi:http://dx.doi.
org/10.1016/j.cclet.2020.08.035.
10] J. Zhou, G.C. Yu, F.H. Huang, Chem. Soc. Rev. 46 (2017) 7021–7053.
[
[
[
[
[
11] Y.Y. Chen, Z.H. Huang, J.F. Xu, et al., ACS Appl. Mater. Interfaces 8 (2016) 22780–
22784.
[12] Q. Hao, Y.Y. Chen, Z.H. Huang, et al., ACS Appl. Mater. Interfaces 10 (2018) 5365–
372.
13] C. Sun, H.P. Zhang, S.K. Li, et al., ACS Appl. Mater. Interfaces 10 (2018) 25090–
5098.
[14] B. Yang, X.D. Zhang, J. Li, et al., CCS Chem. 1 (2019) 156–165.
5
[
2
[
[
[
15] S.K. Kim, D.H. Lee, J.I. Hong, J. Yoon, Acc. Chem. Res. 42 (2009) 23–31.
16] M.B. Yaffe, Nat. Rev. Mol. Cell Biol. 3 (2002) 177–186.
17] R.A. Cairns, I.S. Harris, T.W. Mak, Nat. Rev. Cancer 11 (2011) 85-85.
[18] W.G. Kaelin Jr, C.B. Thompson, Nature 465 (2010) 562–564.
19] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646–674.
20] T.W. Traut, Mol. Cell. Biochem. 140 (1994) 1–22.
[
[
[
In summary, we have demonstrated an effective and feasible
chemotherapy strategy that involved the direct encapsulation of an
anion chemotherapeutic agent, PMX by an appropriate macrocy-
clic host WP6A. Host-guest complexation served to decrease
cytotoxicity of PMX to normal cells. The higher binding affinity of
ATP/WP6A is conducive to replacing PMX from its complex. More
21] R.J. DeBerardinis, J.J. Lum, G. Hatzivassiliou, C.B. Thompson, Cell Metab. 7
(2008) 11–20.
[
[
[
22] C.F. Higgins, K.J. Linton, Nat. Struct. Mol. Biol. 11 (2004) 918–926.
23] E.V. Batrakova, A.V. Kabanov, J. Control. Release 130 (2008) 98–106.
24] M.M. Gottesman, Annu. Rev. Med. 53 (2002) 615–627.
[25] G.C. Yu, J. Zhou, J. Shen, G.P. Tang, F.H. Huang, Chem. Sci. 7 (2016) 4073–4078.
4

G Model
CCLET-6275; No. of Pages 5
J. Chen, Y. Zhang, Y. Zhang et al.
Chinese Chemical Letters xxx (xxxx) xxx–xxx
[
[
26] J. Zhou, M. Chen, G.W. Diao, Chem. Commun. 50 (2014) 11954–11956.
27] T. Ciuleanu, T. Brodowicz, C. Zielinski, J.H. Kim, C.P. Belani, Lancet 374 (2009)
[38] T.X. Xiao, L. Zhou, L.X. Xu, et al., Chin. Chem. Lett. 30 (2019) 271–276.
[39] X. Zhang, X.Y. Wang, B. Wang, et al., Chin. Chem. Lett. 31 (2020) 3230–3232.
[40] T.X. Xiao, W.W. Zhong, L. Zhou, et al., Chin. Chem. Lett. 30 (2019) 31–36.
[41] Y. Hang, D. Bardelang, R.B. Wang, Trends Chem. 3 (2021) 1–4.
[42] S.W. Guo, Q.X. Huang, Y. Chem, et al., Angew. Chem. Int. Ed. 60 (2021) 618–623.
[43] G. Ghale, W. Nau, Acc. Chem. Res. 47 (2014) 947–955.
1432–1440.
[28] G. Scagliotti, N. Hanna, F. Fossella, et al., Oncologist 14 (2009) 253–263.
[29] N. Hanna, F.A. Shepherd, F.V. Fossela, et al., J. Clin. Oncol. 22 (2004) 1589–1597.
[30] T. Ogoshi, S. Kanai, S. Fujinami, T. Yamagishi, Y. Nakamoto, J. Am. Chem. Soc.130
(
2008) 5022–5023.
[44] R. Dsouza, U. Pischel, W. Nau, Chem. Rev. 111 (2011) 7941–7980.
[45] A. Henning, H. Bakirci, W. Nau, Nat. Methods 4 (2007) 629–632.
[46] F. Mimoto, K. Tatsumi, S. Shimizu, et al., Cell Rep. 33 (2020)108542.
[47] J. Gao, J. Li, W.C. Geng, et al., J. Am. Chem. Soc. 140 (2018) 4945–4953.
[48] R. Mo, T.Y. Jiang, R. DiSanto, W.Y. Tai, Z. Gu, Nat. Commun. 5 (2014) 3364.
[49] J.D. Cai, J.N. Huang, W.L. Wang, J. Zeng, P. Wang, Cancer Manag. Res. 12 (2020)
1597–1609.
[
31] T. Ogoshi, T. Yamagishi, Y. Nakamoto, Chem. Rev. 116 (2016) 7937–8002.
[
[
[
[
[
32] J.Y. Chen, H.Z. Ni, Z. Meng, et al., Nat. Commun. 10 (2019) 3546.
33] L.L. Tan, H.W. Li, Y.C. Qiu, et al., Chem. Sci. 6 (2015) 1640–1644.
34] M.F. Li, N. Zhang, W. Xia, et al., J. Am. Chem. Soc. 138 (2016) 6643–6649.
35] Y.C. Chang, K. Yang, P. Wei, et al., Angew. Chem. Int. Ed. 53 (2014) 13126–13130.
36] B. Li, Z. Meng, Q.Q. Li, et al., Chem. Sci. 8 (2017) 4458–4464.
[
37] M. Zuo, W. Qian, M. Hao, et al., Chin. Chem. Lett. (2020), doi:http://dx.doi.org/
0.1016/j.cclet.2020.09.033.
[50] Y. Zhang, X.L. Feng, T. Li, E.P. Li, Y. Li, Cancer Med. 6 (2017) 1965–1975.
[51] J. Tian, C. Yao, W.L. Yang, Chin. Chem. Lett. 28 (2017) 798–806.
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