MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial-mesenchymal transition in A549/Taxol cells

Article abstract of DOI:10.1016/j.cbi.2020.109074

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.

Full text of DOI:10.1016/j.cbi.2020.109074

Chemico-Biological Interactions 323 (2020) 109074
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/
Taxol cells and inhibits epithelial-mesenchymal transition in A549/Taxol
cells
a
a
a
b
a
b
a
Jianan Du , Jingnan Li , Minghuan Gao , Qi Guan , Tong Liu , Yue Wu , Zengqiang Li ,
a,∗
b,∗∗
a,∗∗∗
Daiying Zuo , Weige Zhang , Yingliang Wu
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
a
b
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang, 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Apoptosis
Multidrug resistance
Epithelial-mesenchymal transition
P-glycoprotein
MAY
Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR)
and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer
drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-
(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on
A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase
arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial
pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a
substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the
expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by
targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and
A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a
promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.
1. Introduction
Epithelial-mesenchymal transition (EMT) is a reversible biological
process and plays an important role in the malignant development of
Non-small-cell lung cancer (NSCLC) has increasing rates of in-
cidence and has become one of the most prevalent malignant tumors
1–4]. Chemotherapy is one of the most valid methods and paclitaxel is
the most commonly used drug for the clinical treatment of NSCLC
5–7]. However, resistance to paclitaxel reduces the efficacy of che-
tumors [15,16]. EMT can not only strengthen the motility of tumor
cells, but also give them stem cell characteristics [17]. Studies have
shown that EMT can lead to increase of the expression and function of
ABC transporters, causing chemoresistance in cancers [16,18,19]. Many
studies show that EMT transcription factors (EMT-TFs) regulate the
pumping function of ABC transporters and EMT process in a variety of
tumor cells [20–23]. Therefore, it is of great important to develop
chemotherapeutic agent that can inhibit EMT and drug resistance.
A series of microtubule inhibitors have been synthesized by our
group. Among them, 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)
piperazine-1-carbonyl)-2H- 1,2,3-triazole (MAY) (Fig. 1a) was identi-
fied as a colchicine binding site inhibitor of tubulin and can strongly
suppress microtubule polymerization in SGC-7901 cells [24]. In the
present study, MAY exhibited cytotoxic effect on various cancer cells,
[
[
motherapy and limits its clinical application.
Many studies on multidrug resistance (MDR) have revealed the re-
sistant mechanisms of drugs, including change of drug target structure,
repair of DNA damage, and the most important one is enhancing the
function of ATP-binding cassette transporters (ABC transporters)
[
8–11]. P-glycoprotein (P-gp), an ABC transporter, can decrease the
concentration of drugs by pumping them out of the cells and inhibit the
efficacy of drugs [12–14]. Thus, suppressing the function of P-gp is the
main method to overcome chemotherapy resistance.
∗
Corresponding author.
Corresponding author.
∗
∗
∗
∗∗
Corresponding author.
E-mail addresses: zuodaiying@163.com (D. Zuo), zhangweige2000@sina.com (W. Zhang), yingliang_1016@163.com (Y. Wu).
https://doi.org/10.1016/j.cbi.2020.109074
Received 13 January 2020; Received in revised form 9 March 2020; Accepted 21 March 2020
Available online 23 March 2020
0009-2797/ © 2020 Elsevier B.V. All rights reserved.

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
Fig. 1. Effects of MAY on A549 and A549/Taxol cell viability and proliferation. (a) The chemical structure of MAY. (b) Synthetic route of MAY. Reagents and
conditions: a. 1) n-BuLi (2.5 M in hexane), THF, −40 °C, 30 min; 2) DMF, −40 °C, 3) r.t., 1 h; b. NaN3, DMSO, r.t., 30 min; c. H , KOH, MeOH, 30 min; d.
2 2
O
arylpiperazine, EDCI, HOBt, ethyl acetate, r.t., overnight. (c) Cell viabilities of A549 and A549/Taxol cells were detected by the MTT assay after treatment with MAY
for 24, 48 and 72 h. (d) The growth inhibition effects of MAY on A549 and A549/Taxol cells were measured by colony formation assay.
particularly taxol-resistant A549 cells (A549/Taxol cells). MAY induced
apoptosis in A549 and A549/Taxol cells and inhibited EMT in A549/
Taxol cells. However, the detailed mechanism has not been described.
In this study, we intend to clarify the mechanisms by which MAY in-
duces apoptosis and inhibits EMT. This will provide an evidence for
MAY as a potential agent to treat paclitaxel-resistant NSCLC.
chlorophenyl)-2H-1,2,3-triazole-4-carbaldehyde (3), followed by oxi-
dation to 5-(2-chlorophenyl)-2H-1,2,3-triazole-4-carboxylic acid (4)
with hydrogen peroxide. Finally, amidation was carried by 1-Ethyl-3-
(3-dimethyl laminopropyl)carbodiie hydrochlide (EDCI) and Nhydrox-
ybenzotrizole (HOBt) to generate the desired compound 5-(2-chlor-
ophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H
−
1,2,3-triazole (5, MAY). The MAY was purified by column chroma-
tography, and the purity was more than 98% by HPLC method. The
data of HRMS and NMR spectroscopy are in agreement with our pre-
vious study [24]. Combretastain A-4 (CA-4), Taxol, doxorubicin (ADM)
and verapamil (VRP) were purchased from Sigma Chemical (St. Louis,
MO, USA). MTT and propidium iodide (PI) were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Hoechst 33342, BCA protein assay kit,
crystal violet and Rhodamine123 (Rh123) were purchased from Beyo-
time Biotechnology (Nanjing, P.R.C). Annexin V-FITC/PI staining kit
was purchased from KeyGEN BioTECH (Nanjing, China). Primary
2
. Materials and methods
2.1. Materials
MAY was synthesized by our laboratory. The synthetic route was
outlined in Fig. 1b. 1-Chloro-2-ethynylbenzene (1) was used as the
starting material, which was converted into 3-(2-chlorophenyl)propio-
laldehyde (2) by the formylation reaction. Then, a thermal azide-alkyne
1,3-dipolar cycloaddition was carried out for obtaining 5-(2-
2

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
Table 2
The resistance index (RI) for various compounds in A549/Taxol cells
Table 1
IC50 of MAY against various human cancer cell lines (mean ± SD, n = 3).
(mean ± SD, n = 3).
Tumor type
Cell line
IC50 (nM)
Compounds
IC50 (nM)
A549
RI
Stomach
Liver
SGC-7901
HepG2
BEL-7402
HeLa
MCF-7
MCF-7/ADR
A549
95.5 ± 1.5
149.5 ± 3.5
89.0 ± 1.5
81.0 ± 7.5
96.5 ± 1.5
295.7 ± 3.7
67.0 ± 2.5
52.0 ± 1.0
A549/Taxol
Cervix
Breast
Taxol
ADM
CA-4
MAY
20.3 ± 3.1
135.5 ± 21.2
24.2 ± 1.8
67.0 ± 2.5
6992.4 ± 266.2
968.7 ± 193.3
988.4 ± 95.9
52.0 ± 1.0
344.5
7.2
40.8
0.7
Lung
A549/Taxol
2
.6. Cell cycle analysis
antibodies against α-tubulin, CyclinB1, Cdc2, CDK7 and HRP-con-
jugated and FITC-conjugated secondary antibodies were purchased
from Santa Cruz Biotechnology (Delaware Ave Santa Cruz, CA, USA). P-
gp, matrix metalloprotein 2 (MMP2) and β-actin antibodies were pur-
chased from Bioss (Beijing, P.R.C). Bax, Bcl-2, PARP, caspase 3, caspase
Cells were incubated with MAY or medium for the different times,
then the cells were collected and fixed in 70% ice ethanol. Next, the
cells were stained with PI for 30 min. Finally, the cells were detected by
FACS (BD Biosciences, NY, USA).
8, caspase 9, E-cadherin, N-cadherin, Vimentin, Snail1, Twist1 and
matrix metalloprotein 9 (MMP9) antibodies were obtained from Pro-
teintech (Chicago, IL, USA). Primary antibodies against FAS and FASL
were obtained from Wanleibio (Shenyang, China).
2
.7. Hoechst staining
4
Cells were seeded in six-well plates at a density of 1.5 × 10 cells/
well and incubated overnight. And cells were treated with MAY or CA-4
for different times. Then the cells were stained with Hoechst 33342.
Images were photographed using fluorescence microscope (Olympus,
Tokyo, Japan).
2
.2. Cell culture
A549, HepG2, SGC-7901, HeLa, BEL-7402, MCF-7 and adriamycin-
resistant MCF-7 (MCF-7/ADR) cells were purchased from the Shanghai
Institute of Cell Resource Center Life Science (Shanghai, China). Taxol-
resistant A549 (A549/Taxol) cells were obtained from Professor Yang
2
.8. Cell apoptosis analysis
Cell apoptosis was detected by Annexin V-FITC/PI staining kit. Cells
(
Shenyang Pharmaceutical University). All cells were cultured in RPMI
640 or DMEM containing 10% fetal bovine serum at 37 °C and 5%
CO
were treated with MAY or CA-4 for the indicated times and collected.
Then the cells were stained based on the kit instructions. Samples were
detected by FACS (BD Biosciences, NY, USA).
1
2
.
2.9. Mitochondrial membrane potential analysis
2.3. Cell viability
Changes in mitochondrial transmembrane potential as a result of
mitochondrial perturbation were measured after staining with Rh123
26]. Cells were incubated with MAY for different times, then the cells
were stained with Rh123 in the dark for 30 min at 37 °C. Samples were
detected by FACS (BD Biosciences, NY, USA).
Growth inhibition of tumor cells was examined by the MTT assays
25]. Briefly, cells were seeded in 96-well plates at a density of
000–5000 cells/well and incubated overnight. The cells were treated
with compounds or medium for different times and 20 μl MTT was
added to per well. After incubation for 4 h, the supernatant was re-
moved and 150 μl DMSO was added. Finally, the absorbance was
measured at 420 nM by a microplate reader (MK3, Thermo, Germany).
The resistance index (RI) was the ratio of the IC50 of the resistant cells
to that of parental cells.
[
3
[
2
.10. Wound healing assay
Wound healing assay was conducted based on previous description
17]. Concisely, cells were seeded in six-well plates and incubated
[
overnight. Wounds were formed by scraping with pipette tips, then the
cells were washed with PBS and the medium with or without MAY was
added. Images were photographed at 48 h by a microscope (Olympus,
Tokyo, Japan).
2
.4. Colony formation assay
Cells were seeded in six-well plates at a density of 6000 cells/well
and incubated overnight. After treatment with different concentrations
of MAY or medium for 48 h, the original medium was removed and
replaced with the drug-free medium. After incubation for 7 days, cells
were fixed with 4% paraformaldehyde for 20 min and stained with
crystal violet for 15 min, then photographed by a camera (Canon,
Tokyo, Japan).
2
.11. Transwell migration and invasion assays
Cells seeded in the upper chamber (Corning, NY, USA) were in-
cubated with serum-free medium and medium with 10% FBS was added
to the lower chamber. After being incubated with MAY or medium for
4
8 h, the cells migrated on the lower membrane were fixed and stained.
Invasion assay was similar to the description except adding the matrigel
(BD Bioscience, NY, USA) to the upper chamber.
2.5. Immunofluorescence staining
Cells were seeded in six-well plates with glass coverslips and treated
2.12. Intracellular Rh123 accumulation assay
with MAY or medium for 24 h. Then the cells were incubated overnight
at 4 °C with the primary antibodies and incubated with FITC-conjugated
secondary antibodies at room temperature for 3 h. Nuclei were stained
with DAPI. Images were photographed using confocal microscope
4
Cells were seeded in six-well plates at a density of 1.0 × 10 cells/
well and incubated overnight. Cells were treated with or without MAY
and co-incubated with 15 mM Rh123 in the dark for 48 h. Samples were
detected by FACS (BD Biosciences, NY, USA).
(
Nikon C2, Tokyo, Japan).
3

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
Fig. 2. MAY inhibits microtubule polymerization and induces G2/M phase arrest. (a) Immunofluorescence analysis showed microtubule distribution in A549 and
A549/Taxol cells with MAY (280 and 200 nM, respectively) treatment for 24 h. Scale bar = 10 μm. (b) FACS analysis showed the cell cycle distribution in A549 and
A549/Taxol cells after treatment with MAY (280 and 200 nM, respectively) for the indicated times. (c) and (d) Western blot showed the expression changes of cell
cycle-related proteins in MAY-treated A549 and A549/Taxol cells. *p < 0.05, **p < 0.01 vs control.
2.13. Western blot analysis
2.14. Statistical analysis
Western blot was conducted according to the previous description
25]. Concisely, the protein samples were separated on polyacrylamide
All experiments were performed three times and the data were ex-
pressed as mean ± SD. All statistical analysis was performed by one-
way analysis of variance (ANVOA) and LSD tests using SPSS 22.0
software. p-values < 0.05 were considered to be statistically significant.
[
gels and transferred to PVDF membranes. Then the membranes were
incubated with the primary antibodies at 4 °C overnight and the HRP-
conjugated secondary antibodies at room temperature for 3 h. Proteins
were visualized using enhanced chemiluminescence (ECL) reagent.
Densitometry analysis was performed using Image J software.
3. Results
3.1. MAY shows effective cytotoxic effect on multiple cancer cells
The MTT assays revealed that MAY inhibited the growth of multiple
4

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
Fig. 3. MAY induces apoptosis in A549 and A549/Taxol cells. (a) The changes of nuclei were detected by Hoechst 33342 staining. Scale bar = 10 μm. (b) FACS
analysis showed MAY (280 and 200 nM, respectively) induced apoptosis in A549 and A549/Taxol cells. *p < 0.05, **p < 0.01 vs control.
cancer cells, such as A549, A549/Taxol, HepG2, SGC-7901, HeLa, BEL-
402, MCF-7 and MCF-7/ADR cells, indicated that MAY had broad
3.2. MAY inhibits microtubule polymerization and induces G2/M phase
arrest in A549 and A549/Taxol cells
7
spectrum anti-tumor activity (Table 1). Interestingly, ADM, paclitaxel
and CA-4 significantly inhibited A549 cells but weakly inhibited A549/
Taxol cells. However, MAY had a stronger inhibitory effect on A549/
Taxol cells compared with A549 cells, and the RI was 0.7 (Table 2). The
MTT assays revealed that MAY suppressed the growth of A549 and
A549/Taxol cells in time- and concentration-dependent manners
We observed the changes of intracellular microtubule network by
immunofluorescence assay. The results showed that the microtubules
were obviously depolymerized and the fragments were dispersed in the
cytoplasm after MAY treatment for 24 h, whereas the untreated cells
exhibited intact and clear microtubules in both A549 and A549/Taxol
cells (Fig. 2a). Studies have shown that microtubule inhibitors usually
cause cell cycle arrest [27,28]. Cell cycle assays showed that MAY
treatment for 12 h led to cell accumulation in the G2/M phase in both
A549 and A549/Taxol cells (Fig. 2b). Next, we examined the key mo-
lecules during the G2/M phase by western blot. The results indicated
that MAY significantly increased the expression of CyclinB1, CDK7 and
Cdc2 at 12 h in A549 and A549/Taxol cells (Fig. 2c and d). Above data
indicated that MAY induced G2/M phase arrest in both A549 and
(
Fig. 1c). Next, we detected cell proliferation by colony formation
assay. The results indicated that MAY inhibited the ability of colony
formation in concentration-dependent manners in A549 and A549/
Taxol cells (Fig. 1d).
5

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Chemico-Biological Interactions 323 (2020) 109074
Fig. 4. MAY induces apoptosis through the intrinsic pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. (a) Western blot showed the
expression of apoptosis-related protein in MAY (280 nM)-treated A549 cells. (b) FACS analysis showed the mitochondrial membrane potential of A549 cells after
treatment with MAY (280 nM) for 24, 48 and 72 h. (c) Western blot showed the expression of apoptosis-related protein in MAY (200 nM)-treated A549/Taxol cells.
*
p < 0.05, **p < 0.01 vs control.
A549/Taxol cells.
.3. MAY induces apoptosis through the mitochondrial pathway in
in time-dependent manners. Furthermore, the results showed that the
effect of MAY was significantly stronger than that of CA-4 at same
concentration in A549/Taxol cells. Next, in order to explore the way of
apoptosis in A549 and A549/Taxol cells, we conducted the following
experiments. As shown in Fig. 4a, MAY decreased the expression of Bcl-
3
A549 cells and through the extrinsic pathway in A549/Taxol cells
2
, caspase 9, caspase 3 and PARP and strengthened the expression of
The above data showed that MAY suppressed the growth of NSCLC
cells and induced G2/M phase arrest, so we explored whether MAY
induced apoptosis. First of all, we observed the changes of nuclei by
Hoechst 33342 staining. A549 and A549/Taxol cells were stained
evenly and the nucleus edge was neat and round in the control group.
After treatment with MAY, the cells were stained in bright blue, the
nuclei were expanded and apoptotic bodies were obvious (Fig. 3a). As
shown in Fig. 3b, MAY induced apoptosis in A549 and A549/Taxol cells
Bax, cleaved-caspase 9, cleaved-caspase 3 and cleaved-PARP in time-
dependence manners in A549 cells. However, the expression of caspase
8
expression. In A549 cells, after treatment with MAY, the extracellular
Rh123 fluorescence intensity was increased, indicating that the in-
tracellular mitochondrial membrane potential was decreased (Fig. 4b).
Above results revealed that MAY induced apoptosis in A549 cells
had almost no change and there was no detectable cleaved-caspase 8
6

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Chemico-Biological Interactions 323 (2020) 109074
Fig. 5. MAY inhibits P-gp expression and function in A549/Taxol cells. (a) Western blot showed the expression of P-gp in A549 and A549/Taxol cells. (b) Cell
viabilities of A549/Taxol cells were detected by the MTT assays after treatment with different concentrations of compounds alone or combined with VRP (10 mM) for
72 h. (c) Western blot showed the expression of P-gp in MAY (200 nM)-treated A549/Taxol cells. (d) The accumulation of Rh123 was measured by FACS analysis.
*
p < 0.05, **p < 0.01 vs control.
through the intrinsic pathway. Then, we examined the expression of
apoptosis-related proteins in A549/Taxol cells by western blot. The
results showed that MAY decreased the expression of caspase 8, caspase
3.4. MAY inhibits P-gp expression and function in A549/Taxol cells
P-gp pumping drugs out of cells to reduce drug concentration is one
of the main causes of chemotherapy resistance in tumor cells [29].
Immunoblotting analysis showed that P-gp was highly expressed in
A549/Taxol cells compared with A549 cells (Fig. 5a). Most drugs tar-
geting tubulins are substrates of P-gp, which are pumped out of cells by
P-gp, resulting in drug resistance [30]. To investigate whether MAY was
a substrate of P-gp, we examined the effects of several drugs on A549/
Taxol cell viabilities by co-incubating these drugs with VRP. The
growth inhibition of A549/Taxol cells showed that VRP enhanced the
inhibition effect of taxol and ADM, which are typical substrates of P-gp,
3
and PARP and up-regulated the expression of FAS, FASL, cleaved-
caspase 8, cleaved-caspase 3 and cleaved-PARP in time-dependence
manners in A549/Taxol cells. However, there was little change in the
expression of caspase 9 and there was no detectable cleaved-caspase 9
expression (Fig. 4c). These results revealed that MAY induced apoptosis
in A549/Taxol cells through the extrinsic pathway.
7

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Chemico-Biological Interactions 323 (2020) 109074
Fig. 6. EMT features in A549/Taxol cells. (a) Wounding healing assay showed the motility of A549 and A549/Taxol cells. (b) Transwell assays showed crystal violet
stained A549 and A549/Taxol cells on the insert membranes after incubation for 48 h. (c) Immunofluorescence showed the expression of epithelial marker (E-
cadherin) and mesenchymal marker (N-cadherin) in A549 and A549/Taxol cells. Scale bars = 50 μm. (d) Western blot showed the expression levels of Ecadherin, N-
cadherin and Vimentin in A549 and A549/Taxol cells. *p < 0.05, **p < 0.01 vs A549 cells.
whereas VRP did not affect the inhibitory effect of CA-4 and MAY
Fig. 5b), which indicates that MAY is not a substrate for P-gp. In ad-
exhibit EMT-like changes. Because of the relationship between EMT and
cell motility, we analyzed the ability of metastasis in A549/Taxol cells.
The wound healing assay indicated that drug resistance enhanced cell
motility (Fig. 6a). The migration and invasion assays showed the same
results (Fig. 6b). Moreover, in order to demonstrate whether A549/
Taxol cells occur EMT, we investigated the expression of EMT-related
markers. Immunofluorescence indicated that the mesenchymal marker
N-cadherin was increased and the epithelial marker E-cadherin was
reduced in A549/Taxol cells compared with A549 cells (Fig. 6c). As
shown in Fig. 6d, E-cadherin in A549/Taxol cells was significantly re-
duced compared with A549 cells. However, N-cadherin and Vimentin
were increased significantly in A549/Taxol cells. Together, above re-
sults showed that A549/Taxol cells underwent EMT.
(
dition, MAY significantly reduced the expression of P-gp in time-de-
pendence manners in A549/Taxol cells (Fig. 5c). Next, we detected the
intracellular accumulation of P-gp fluorescent substrate Rh123. As
shown in Fig. 5d, Rh123 levels in A549 cells were much higher than
those in A549/Taxol cells. Interestingly, MAY could lead to intracellular
Rh123 accumulation in A549/Taxol cells, which indicates that MAY
can inhibit P-gp function in A549/Taxol cells (Fig. 5d).
3.5. EMT is distinct in A549/Taxol cells
We observed that A549 cells were in a flat form, while A549/Taxol
cells were transformed into elongated shape, suggesting that cells may
8

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Chemico-Biological Interactions 323 (2020) 109074
Fig. 7. MAY inhibits EMT by targeting Twist1 in A549/Taxol cells. (a) Wounding healing assay showed the motility of A549/Taxol cells treated with MAY for 48 h.
b) Transwell assays showed crystal violet stained A549/Taxol cells treated with MAY treatment for 48 h. (C) Western blot showed the expression of EMT-related
proteins in MAY- treated A549/Taxol cells. *p < 0.05, **p < 0.01 vs control.
(
3.6. MAY inhibits EMT by targeting Twist1 in A549/Taxol cells
MAY inhibited EMT through targeting Twist1 in A549/Taxol cells.
Drugs targeting microtubules affect the ability of cell motility [31].
4
. Discussion
Therefore, we explored the influence of MAY on A549/Taxol cell mo-
tility. When the inhibition rate of a drug on cells is less than 10%, it
indicates that there is no significant cytotoxic effect on the cells [32].
The inhibition rate of A549/Taxol cells by MAY (100 nM) treatment for
Lung cancer has the highest morbidity and mortality among ma-
lignant tumors. Among them, more than 80% of lung cancer patients
are NSCLC [33]. Although paclitaxel is the commonly used drug for
NSCLC treatment, increased drug-resistance limits its clinical use [34].
Drug-resistant NSCLC leads to poor outcome due to lack of further ef-
fective drugs [35]. In this study, we found that MAY inhibited micro-
tubule polymerization and induced G2/M phase arrest in A549 and
A549/Taxol cells. Furthermore, MAY induced apoptosis through the
mitochondrial pathway in A549 cells and through the extrinsic pathway
in A549/Taxol cells. Our research shows that MAY is a promising an-
ticancer drug for NSCLC.
4
8 h was about 10%, so we chose 100 nM as the maximum con-
centration for the following experiments. The results indicated that
MAY suppressed the migration and invasion of A549/Taxol cells in
concentration-dependent manners (Fig. 7a and b). Western blot in-
dicated that MAY enhanced the expression of E-cadherin and inhibited
the expression of N-cadherin and Vimentin (Fig. 7c). These results in-
dicated that MAY inhibited EMT in A549/Taxol cells. To further in-
vestigate the potential molecular mechanisms, we examined the ex-
pression of EMT-TFs by western blot. The results revealed that MAY
significantly reduced the expression of Twist1 and almost had no effect
on MMP2, MMP9 and Snail1 (Fig. 7c). Therefore, we speculated that
Due to the complex mechanism of MDR, understanding its me-
chanism is necessary to overcome chemotherapy resistance in NSCLC
cells. ABC transporters overexpress in most drug-resistant cancers and
9

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
protect tumor cells by reducing intracellular drug concentration [36].
P-gp is a typical ABC transporter in drug-resistant tumor cells and has
multiple functions and substrates [37]. In this study, we found that
highly expressed P-gp was the main factor of chemoresistance in A549/
Taxol cells. In addition, MAY was not a substrate for P-gp and inhibited
the expression and function of P-gp.
EMT is an important process in the malignant development of tu-
mors and can promote tumors metastasis [38]. Studies have shown that
EMT can be regulated by a variety of cytokines and growth factors [15].
In the presence of EMT, epithelial cells gradually obtain mesenchymal
phenotype and enhance migration and invasion abilities [39]. In this
study, we discovered that the resistant NSCLC cells acquired me-
senchymal-like phenotype and displayed greater motility. Simulta-
neously, the expression levels of epithelial markers were decreased and
the expression levels of mesenchymal markers were increased in A549/
Taxol cells compared with A549 cells.
doi.org/10.1016/j.cbi.2020.109074.
References
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA A Cancer J. Clin. 69
(
1) (2019) 7–34.
2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA A Cancer J. Clin. 68
1) (2018) 7–30.
[3] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA A Cancer J. Clin. 67
1) (2017) 7–30.
4] L.A. Torre, R.L. Siegel, A. Jemal, Lung cancer statistics, Adv. Exp. Med. Biol. 893
2016) 1–19.
[
(
(
[
(
[5] Z. Bai, et al., BZML, a novel colchicine binding site inhibitor, overcomes multidrug
resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic
catastrophe, Canc. Lett. 402 (2017) 81–92.
[
6] F. Wu, et al., The role of Axl in drug resistance and epithelial-to-mesenchymal
transition of non-small cell lung carcinoma, Int. J. Clin. Exp. Pathol. 7 (10) (2014)
6653–6661.
[
7] Y.J. Wang, et al., Regorafenib overcomes chemotherapeutic multidrug resistance
mediated by ABCB1 transporter in colorectal cancer: in vitro and in vivo study,
Canc. Lett. 396 (2017) 145–154.
Studies have reported that a variety of transcription factors were
participated in the regulation of EMT, including Twist1, Snail1 and Slug
[8] M.P. Ceballos, et al., ABC transporters: regulation and association with multidrug
resistance in hepatocellular carcinoma and colorectal carcinoma, Curr. Med. Chem.
[
29,40]. Twist1, a member of zinc-finger transcription factor, is the
2
6 (7) (2019) 1224–1250.
most important adjustment factor of EMT [41]. Many studies have
shown that Twist1 inhibits E-cadherin transcription through binding to
the E-box site in the E-cadherin promoter [42,43]. In addition, Twist1
not only promotes the EMT process, but also enhances drug resistance
[
9] M. Zhou, et al., Overcoming chemotherapy resistance via simultaneous drug-efflux
circumvention and mitochondrial targeting, Acta Pharm. Sin. B 9 (3) (2019)
6
15–625.
[
10] A.K. Nanayakkara, P.D. Vogel, J.G. Wise, Prolonged inhibition of P-glycoprotein
after exposure to chemotherapeutics increases cell mortality in multidrug resistant
cultured cancer cells, PloS One 14 (6) (2019) e0217940.
[
44]. In this study, MAY inhibited the expression of Twist1 and led to
[
11] H. Yin, et al., Design, synthesis and biological evaluation of chalcones as reversers
of P-glycoprotein-mediated multidrug resistance, Eur. J. Med. Chem. 180 (2019)
the inhibition of migration and invasion in A549/Taxol cells.
In conclusion, MAY inhibits microtubule polymerization and in-
duces G2/M phase arrest in A549 and A549/Taxol cells. Furthermore,
MAY induces apoptosis in A549 and A549/Taxol cells. These results
indicate that MAY is a promising anticancer drug for NSCLC. In addi-
tion, we find that highly expressed P-gp is the main factor of che-
moresistance in A549/Taxol cells. Importantly, MAY is not a P-gp
substrate, and it can inhibit P-gp expression and function in A549/
Taxol cells. Besides, MAY inhibits EMT by targeting Twist1 in A549/
Taxol cells. Our findings suggest that MAY could provide a promising
method for the treatment of NSCLC, especially for the treatment of
resistant NSCLC.
3
50–366.
[12] W. Guo, et al., Mitochondria P-glycoprotein confers paclitaxel resistance on ovarian
cancer cells, OncoTargets Ther. 12 (2019) 3881–3891.
[
13] H.Y. Liu, et al., DJ-1 overexpression confers the multidrug resistance phenotype to
SGC7901cells by upregulating P-gp and Bcl-2, Biochem. Biophys. Res. Commun.
5
19 (1) (2019) 73–80.
[14] M. Levi, et al., P-glycoprotein and breast cancer resistance protein in canine in-
flammatory and noninflammatory grade III mammary carcinomas, Vet. Pathol. 56
(6) (2019) 840–847 300985819868647.
[
15] J. Wu, et al., RCCD1 depletion attenuates TGF-beta-induced EMT and cell migration
by stabilizing cytoskeletal microtubules in NSCLC cells, Canc. Lett. 400 (2017)
1
8–29.
[
[
16] V. Odero-Marah, et al., Epithelial-mesenchymal transition (EMT) and prostate
cancer, Adv. Exp. Med. Biol. 1095 (2018) 101–110.
17] W. Gou, et al., CD74-ROS1 G2032R mutation transcriptionally up-regulates Twist1
in non-small cell lung cancer cells leading to increased migration, invasion, and
resistance to crizotinib, Canc. Lett. 422 (2018) 19–28.
CRediT authorship contribution statement
[
18] Y. Hu, et al., Sabutoclax, pan-active BCL-2 protein family antagonist, overcomes
drug resistance and eliminates cancer stem cells in breast cancer, Canc. Lett. 423
Jianan Du: Methodology, Formal analysis, Investigation, Writing -
original draft, Writing - review & editing. Jingnan Li: Methodology,
Resources, Formal analysis. Minghuan Gao: Methodology, Resources,
Formal analysis. Qi Guan: Methodology, Resources. Tong Liu:
Methodology, Resources, Formal analysis. Yue Wu: Methodology,
Resources. Zengqiang Li: Software, Resources. Daiying Zuo:
Conceptualization, Project administration, Supervision, Writing - re-
view & editing. Weige Zhang: Project administration, Supervision.
Yingliang Wu: Project administration, Supervision.
(
2018) 47–59.
[
19] M. Lim, et al., Targeting metabolic flexibility via angiopoietin-like 4 protein sen-
sitizes metastatic cancer cells to chemotherapy drugs, Mol. Canc. 17 (1) (2018) 152.
[20] M. Saxena, et al., Transcription factors that mediate epithelial-mesenchymal tran-
sition lead to multidrug resistance by upregulating ABC transporters, Cell Death Dis.
2
(2011) e179.
[
[
[
[
21] W. Li, et al., Overexpression of Snail accelerates adriamycin induction of multidrug
resistance in breast cancer cells, Asian Pac. J. Cancer Prev. APJCP 12 (10) (2011)
2
575–2580.
22] K. Zhu, et al., Short hairpin RNA targeting Twist1 suppresses cell proliferation and
improves chemosensitivity to cisplatin in HeLa human cervical cancer cells, Oncol.
Rep. 27 (4) (2012) 1027–1034.
23] E. Mato, et al., ABCG2/BCRP gene expression is related to epithelial-mesenchymal
transition inducer genes in a papillary thyroid carcinoma cell line (TPC-1), J. Mol.
Endocrinol. 52 (3) (2014) 289–300.
Declaration of competing interest
24] Y. Wu, et al., Design and synthesis of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-2H-
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
1
,2,3-triazole derivatives as colchicine binding site inhibitors, Sci. Rep. 7 (1) (2017)
17120.
[25] M. Han, et al., 5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-(4-((4-
methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)pyrimidine-2,4-diamine
(WY-135), a novel ALK inhibitor, induces cell cycle arrest and apoptosis through
Acknowledgement
inhibiting ALK and its downstream pathways in Karpas299 and H2228cells, Chem.
Biol. Interact. 284 (2018) 24–31.
[
[
[
[
26] A. Nahata, et al., Sphaeranthus indicus induces apoptosis through mitochondrial-
dependent pathway in HL-60 cells and exerts cytotoxic potential on several human
cancer cell lines, Integr. Canc. Ther. 12 (3) (2013) 236–247.
We gratefully acknowledge the National Natural Science
Foundation of China (81872394 and 81673293) and Liaoning
Revitalization Talents Program of China (XLYC1802072) for the gen-
erous financial support.
27] F. Liu, et al., The novel nature microtubule inhibitor ivalin induces G2/M arrest and
apoptosis in human hepatocellular carcinoma SMMC-7721 cells in vitro, Medicina
5
5 (8) (2019).
28] W. Li, et al., Synthesis, molecular properties prediction and biological evaluation of
indole-vinyl sulfone derivatives as novel tubulin polymerization inhibitors targeting
the colchicine binding site, Bioorg. Chem. 85 (2019) 49–59.
29] T. Tomono, K. Yano, T. Ogihara, Snail-induced epithelial-to-mesenchymal transi-
tion enhances P-gp-Mediated multidrug resistance in HCC827 cells, J. Pharmacol.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
10

J. Du, et al.
Chemico-Biological Interactions 323 (2020) 109074
Sci. 106 (9) (2017) 2642–2649.
30] Z.A. Yochum, et al., Targeting the EMT transcription factor TWIST1 overcomes
resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer, Oncogene
8 (5) (2019) 656–670.
31] I.T. Tsai, et al., Novel microtubule inhibitor MPT0B098 inhibit shypoxia-induced
epithelial-to- mesenchymal transition in head and neck squamous cell carcinoma, J.
Biomed. Sci. 25 (1) (2018) 28.
32] H. Lu, et al., Novel ADAM-17 inhibitor ZLDI-8 inhibits the proliferation and me-
tastasis of chemo-resistant non-small-cell lung cancer by reversing Notch and epi-
thelial mesenchymal transition in vitro and in vivo, Pharmacol. Res. 148 (2019)
resistant K562/A02 cells, Bioorg. Med. Chem. 27 (15) (2019) 3347–3357.
[37] S. Mollazadeh, et al., Synthesis, in silico and in vitro studies of new 1,4-dihy-
dropiridine derivatives for antitumor and P-glycoprotein inhibitory activity, Bioorg.
Chem. 91 (2019) 103156.
[38] J. Lee, J.H. Choi, C.K. Joo, TGF-beta1 regulates cell fate during epithelial-me-
senchymal transition by upregulating survivin, Cell Death Dis. 4 (2013) e714.
[39] J.J. Deng, et al., Twist mediates an aggressive phenotype in human colorectal
cancer cells, Int. J. Oncol. 48 (3) (2016) 1117–1124.
[
[
[
3
[40] J. Yao, et al., ABCB5-ZEB1 Axis promotes invasion and metastasis in breast cancer
cells, Oncol. Res. 25 (3) (2017) 305–316.
1
04406.
[41] C.H. Kwon, et al., TWIST mediates resistance to paclitaxel by regulating Akt and
Bcl-2 expression in gastric cancer cells, Tumour Biol 39 (10) (2017)
1010428317722070.
[
33] H. Lu, et al., Novel ADAM-17 inhibitor ZLDI-8 inhibits the proliferation and me-
tastasis of chemo-resistant non-small-cell lung cancer by reversing Notch and epi-
thelial mesenchymal transition in vitro and in vivo, Pharmacol. Res. 148 (2019)
[42] Y. Li, et al., CASC15 promotes epithelial to mesenchymal transition and facilitates
malignancy of hepatocellular carcinoma cells by increasing TWIST1 gene expres-
sion via miR-33a-5p sponging, Eur. J. Pharmacol. 860 (2019) 172589.
[43] C.Y. Yu, et al., HR23A-knockdown lung cancer cells exhibit epithelial-to-me-
senchymal transition and gain stemness properties through increased Twist1 sta-
bility, Biochim. Biophys. Acta Mol. Cell Res. 1866 (12) (2019) 118537.
[44] W.J. Chen, et al., Multidrug resistance in breast cancer cells during epithelial-me-
senchymal transition is modulated by breast cancer resistant protein, Chin. J. Canc.
29 (2) (2010) 151–157.
1
04406.
[
34] D.S. Metzinger, D.D. Taylor, C. Gercel-Taylor, Induction of p53 and drug resistance
following treatment with cisplatin or paclitaxel in ovarian cancer cell lines, Canc.
Lett. 236 (2) (2006) 302–308.
35] X. Xie, et al., Overcoming drug-resistant lung cancer by paclitaxel loaded tetra-
hedral DNA nanostructures, Nanoscale 10 (12) (2018) 5457–5465.
36] M. Kairuki, et al., Designed P-glycoprotein inhibitors with triazol-tetra-
hydroisoquinoline-core increase doxorubicin-induced mortality in multidrug
[
[
11