Tanshinone II A improves the chemosensitivity of breast cancer cells to doxorubicin by inhibiting β‐catenin nuclear translocation

Shizheng Li1 | Chunxia Wu2 | Chenxing Fan2 | Puwei Zhang2 | Guifa Yu2 | Kun Li2
1Department of Emergency Surgery, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
2Department of Clinical Laboratory, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China

Correspondence
Kun Li, Department of Clinical Laboratory, The First Affiliated Hospital of Jinzhou Medical University, 121001 Jinzhou, China.
Email: [email protected]

Funding information
National Natural Science Foundation of China, Grant/Award Number: 81303255; Foundation of Educational Department of Liaoning Province, Grant/Award Number: JYTQN201722

1 | INTRODUCTION

Chemotherapy is one of the most important treatments for breast cancer. As the most commonly used drug for breast cancer chemother- apy, Dox is often combined with one or two other drugs with different mechanisms of action to treat breast cancer. However, many patients after undergoing the initial effective treatment, breast cancer cells are
gradually resistant to Dox, and even cross‐resistant to other drugs with
different mechanisms of action, leading to chemotherapy failure.[1,2]

Drug resistance is the self‐protection of breast cancer cells to avoid drug attacks during chemotherapy, in which numerous
mechanisms are involved. With the deepening understanding of the mechanisms of breast cancer resistance, some strategies based on the corresponding mechanism have been carried out to reverse breast cancer resistance, and among the total, chemo- sensitizer is the earliest and the most studied reversal strategy. However, most of the chemosensitizers either require a larger dose, either interfere with the metabolism of chemotherapeutic

Shizheng Li and Chunxia Wu contributed equally to this study.

J Biochem Mol Toxicol. 2020;e22620. wileyonlinelibrary.com/journal/jbt © 2020 Wiley Periodicals LLC | 1 of 10

drugs, or produce strong toxic side effects while improving the chemosensitivity and, therefore, cannot be widely used in the clinic.[3] The ideal chemosensitizer should possess synergism and attenuation effects on chemotherapeutic drugs, but such che- mosensitizers are rarely available in clinic.
Tanshinone II A (Tan II A) is one of the main lipid components of salvia miltiorrhiza, which is commonly used in the treatment of cardiovascular diseases in clinic.[4] Dox, as the most commonly used drug for breast cancer chemotherapy, when killing breast cancer cells, will also produce strong cytotoxicity to nontumor tissues, especially heart and kidney. Previous studies have shown
that Tan II A can alleviate the toxic side effects of Dox‐induced
cardiotoxicity and nephrotoxicity,[5–7] in addition, it also has antitumor activity.[8–13] In view of the above‐mentioned phar- macological actions of Tan II A, we chose it as the research object
to evaluate its feasibility as a chemosensitizer in combination with Dox for breast cancer chemotherapy.
The development of breast cancer is the result of the
cross‐regulation of various factors. In the complex regulatory network, β‐catenin signaling has been receiving much attention. Previous studies have shown that aberrant nuclear β‐catenin accumulation is an important factor contributing to breast
cancer, moreover, growing evidence indicates that aberrant nuclear β‐catenin accumulation plays an important role in breast
cancer resistance.[14] Therefore, targeted inhibition of β‐catenin
nuclear translocation may effectively improve the chemosensi-
tivity of breast cancer. MCF‐7/dox cells, a Dox‐resistant human breast cancer cell line, were constructed by long‐term continuous exposure of its parental Dox‐sensitive MCF‐7 cells to Dox. Here, we employed MCF‐7 and MCF‐7/dox cells to evaluate the effect of Tan II A intervention on the localization of β‐catenin in these cells treated by Dox, meanwhile, to determine whether the effect
would affect the chemosensitivity of breast cancer cells to Dox.

2 | MATERIAL AND METHODS

2.1 | Materials

Dox was purchased from Shenzhen Main Luck Pharmaceuticals Inc,
China; Tan II A was purchased from Shanghai Jianglai Biotech Co Ltd, China; antibody against β‐catenin were purchased from Affinity Bios-
ciences (OH); antibodies against c‐Myc, E‐cadherin, MMP‐2, and
MMP‐9 were purchased from Shenyang Wanlei Biotech Co Ltd (China).

2.2 | Cell culture

Breast cancer MCF‐7 cell line and its corresponding Dox‐ resistant MCF‐7/dox cell line were purchased from Keygen

Biotech. The above cells were cultured in RPMI‐1640 medium containing 10% fetal bovine serum. Additionally, the medium for MCF‐7/dox cells culture was further supplemented with 2 μg/mL Dox to maintain the cell’s resistance to Dox. Before being used in experiments, MCF‐7/dox cells were cultured in Dox‐free medium for 1 week.

2.3 | 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐ carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐ tetrazolium (MTS) assay

To select a proper concentration of Tan II A for the study, we
first assessed the effect of Tan II A on the proliferation of MCF‐7 and MCF‐7/dox cells using MTS assay. The results showed that Tan II A could inhibit the proliferation of MCF‐7 and MCF‐7/dox cells in a dose‐dependent manner, but it had little effect on the proliferation of the above cells at concentrations no higher than
20 µg/mL ( 4A). Since Tan II A has antitumor activity, to exclude the antitumor activity of Tan II A after the combination of Dox and Tan II A, so that the effect of Tan II A on the che- mosensitivity of Dox could be better evaluated, we chose the
above‐mentioned critical concentration of Tan II A (20 µg/mL) for
the following experiments. MCF‐7 and MCF‐7/dox cells were
respectively cultured in the medium containing Dox, Dox plus 20 µg/mL Tan II A, or Dox plus 20 µg/mL Tan II A and 0.1 µM
WAY‐262611 for 24 hours, and then cell proliferation were
measured. In our experiments, cell proliferation rate = (mean optical density [OD] of experimental group − mean OD of blank)/ (mean OD of control group − mean OD of blank) × 100%, sensi- tizing fold = IC50 (Dox before combination)/IC50 (Dox after combination).
2.4 | Scratch assay

MCF‐7 and MCF‐7/dox cells were replated in six‐well plates at a density of 1 × 106 cells/well and incubated in the complete
medium to form monolayers. The monolayers were scratched with 200 μL pipette tips and then washed thoroughly. The wounded cell monolayers were respectively cultured in serum‐free medium containing Dox, Dox plus 20 µg/mL Tan II A
or Dox plus 20 µg/mL Tan II A plus 0.1 µM WAY‐262611
for 48 hours. To avoid mass cell death caused by high doses
of Dox, so that the cell monolayers could be maintained, we se- lected 0.1 µg/mL Dox for MCF‐7 cells and 5 µg/mL Dox for
MCF‐7/dox cells. In the process, images were captured using the
phase‐contrast microscope. In our experiments, wound clo- sure = (wound area of T0 − wound area of Tt)/wound area of
T0 × 100%.

2.5 | Western blot analysis

MCF‐7 and MCF‐7/dox cells were respectively cultured in the medium containing Dox, Dox plus 20 µg/mL Tan II A or Dox plus 20 µg/mL Tan II A plus 0.1 µM WAY‐262611. Since the medium used
for culturing MCF‐7/dox cells needed to be further supplemented
with 2 μg/mL Dox to maintain the cell’s resistance to Dox, we used to choose 2 μg/mL Dox for our study.[15] Here, we still chose 2 μg/mL Dox. and the cells were then collected to extract total, nuclear, and
cytoplasmic protein. Each of the above protein content was de- termined by the bicinchoninic acid assay. Equal proteins (30 µg) were
loaded on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with the corresponding primary anti- bodies overnight at 4°C according to different detection purposes,
among the total, the membranes for detecting nuclear protein ex- pression were incubated with antibodies β‐catenin and LaminB1, the membranes for detecting cytoplasmic protein expression were in- cubated with antibodies β‐catenin and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and the membranes for detecting total
protein expression were incubated with antibodies β‐catenin, c‐Myc, E‐cadherin, MMP‐2, MMP‐9, and GAPDH. After incubation, the membranes were incubated with the secondary antibodies for
2 hours at room temperature, then developed with the chemilumi- nescence detection kit, and finally detected by a chemiluminescence detection system.

2.6 | Statistical analysis

Each experiment was repeated at least three times. The experimental data were analyzed by SPSS 13.0 software. All data were presented as mean ± SD. P < .05 was considered to have a significant statistical difference between different groups.

3 | RESULTS

3.1 | Nuclear β‐catenin accumulation in MCF‐7/dox cells were much higher than that of in MCF‐7 cells

Previous studies have indicated that aberrant nuclear β‐catenin accumulation plays an important role in breast cancer re-
sistance.[14] MCF‐7/dox is a Dox‐resistant cell line corresponding to breast cancer MCF‐7 cell line. Our results showed that the expressions of total and nuclear β‐catenin and the ratio of nuclear to cytoplasmic β‐catenin in MCF‐7/dox cells were sig- nificantly higher than those in MCF‐7 cells ( 1A), which suggested that β‐catenin in MCF‐7/dox cells took place significant nuclear translocation, thereby resulting in the aberrant
nuclear β‐catenin accumulation. Adenomatous polyposis coli (APC) is a negative regulator of β‐catenin. It has been reported that there is low or lack expression for APC in some cancers, therefore, we
also detected the expression of APC. The results showed that the

 1 Characterization of MCF‐7 and MCF‐7/dox cells. A, The expressions of total, nuclear, and cytoplasmic β‐catenin in MCF‐7 and MCF‐7/dox cells were evaluated via Western blot analysis. *P < .05, significantly different from MCF‐7 cells. B, The expressions of APC in MCF‐7 and MCF‐7/dox cells were evaluated using Western blot analysis. *P < .05, significantly different from MCF‐7 cells. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase, MMP, matrix metallopeptidase; dox, doxorubicinexpression of APC in MCF‐7/dox cells was significantly lower than that in MCF‐7 cells (1B), which indicated that the aberrant nuclear β‐catenin accumulation in MCF‐7/dox cells might arise out of the decreased APC expression.
3.2 | Tan II A decreased the accumulation of β‐catenin in the nucleus by inhibiting β‐catenin nuclear translocation

To evaluate whether Tan II A could affect the sensitivity of breast cancer cells to Dox by targeting regulation of β‐catenin’s locali- zation in the cells, we evaluated the effect of Tan II A interven-
tion on the expression of β‐catenin in Dox‐treated MCF‐7 and MCF‐7/dox cells. The results showed that after Tan II A inter- vention, the expressions of total and nuclear β‐catenin in Dox‐ treated MCF‐7 and MCF‐7/dox cells, especially in Dox‐treated MCF‐7/dox cells, were significantly decreased, and the ratio of nuclear to cytoplasmic β‐catenin was also obviously decreased
. The above results suggested that Tan II A could de- crease the nuclear β‐catenin accumulation in Dox‐treated MCF‐7
and MCF‐7/dox cells by inhibiting β‐catenin nuclear transloca-
tion, and the inhibition effect was better for Dox‐resistant MCF‐7/dox cells.
3.3 | Tan II A intervention decreased the expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9

Previous studies have indicated that the famous downstream targets of β‐catenin including c‐Myc, E‐cadherin, MMP‐2, and MMP‐9 are closely related to breast cancer distant metastasis and poor
prognosis.[16,17] Therefore, while evaluating the effect of Tan II A
intervention on the expression of β‐catenin in Dox‐treated MCF‐7 and MCF‐7/dox cells, we also evaluated the effect of Tan II A in- tervention on the expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9. The results showed that after Tan II A intervention, the

 2 Effect of Tan II A intervention on the expression of β‐catenin. MCF‐7 and MCF‐7/dox cells were treated with 2 µg/mL Dox or 2 µg/mL Dox plus 20 µg/mL Tan II A for 24 hours. Western blot analysis was used to analyze the expressions of total, nuclear, and cytoplasmic β‐catenin in MCF‐7 cells (A) and MCF‐7/dox cells (B). Dox, doxorubicin; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase,

MMP, matrix metallopeptidase; Tan, tanshinone. #P < .05, significantly different from Tan II A alone; *P < .05, significantly different from Dox aloneexpression of c‐Myc which mediates cell proliferation was sig- nificantly decreased, the expression of E‐cadherin which promotes cell adhesion and yet inhibits cell migration was significantly in-

creased, and the expressions of MMP‐2 and MMP‐9 which inhibit cell
adhesion and yet promote cell migration were significantly decreased ( 3).
3.4 | Tan II A enhanced the inhibition effect of Dox on cell proliferation, but WAY‐262611 intervention reduced the aforementioned effect

The aforementioned western blot results led us to speculate that Tan
II A might enhance the inhibition effect of Dox on MCF‐7 and MCF‐7/ dox cells proliferation by inhibiting β‐catenin nuclear translocation and
thereby downregulating the expression of its downstream target c‐Myc. To further corroborate our speculation, we analyzed the pro- liferation of the above cells using MTS assay. The results showed that
when Dox was combined with different concentrations of Tan II A, with the increase of Tan II A concentrations, the inhibition effects of
Dox and Tan II A on MCF‐7 and MCF‐7/dox cell proliferation became more and more obvious ( 4B). Even the nontoxic dose of Tan II A
also enhanced the proliferation inhibition effect of DOX on the above cells, however, after WAY‐262611 intervention, the enhancement effect was significantly reduced

3.5 | Tan II A enhanced the inhibition effect of Dox on cell migration, but WAY‐262611 intervention reduced the aforementioned effectIn addition, we also employed scratch assay to further corroborate that Tan II A could enhance the inhibition effect of Dox on MCF‐7

and MCF‐7/dox cells migration by inhibiting β‐catenin nuclear
translocation and thereby downregulating the expressions of its downstream targets E‐cadherin, MMP‐2, and MMP‐9. The results showed that Tan II A could significantly enhance the inhibition effect
of Dox on MCF‐7 and MCF‐7/dox cells migration, however, after WAY‐262611 intervention, the enhancement effect was significantly reduced ( 5).

3 Effect of Tan II A intervention on the expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9. MCF‐7 and MCF‐7/dox cells were treated with 2 µg/mL Dox or 2 µg/mL Dox plus 20 µg/mL Tan II A for 24 hours. Western blot analysis was used to analyze the expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9 in MCF‐7 cells (A) and MCF‐7/dox cells (B). Dox, doxorubicin; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase, MMP, matrix metallopeptidase; Tan, tanshinone. #P < .05, significantly different from Tan II A alone; *P < .05, significantly

different from Dox alone

 4 β‐Catenin agonist WAY‐262611 intervention reduced the enhancement effect of tanshinone (Tan) II A on doxorubicin (Dox) in inhibiting cell proliferation. A, Effect of Tan II A intervention at different concentrations (ranging from 0 to 1 mg/L) on the proliferation of MCF‐7 and MCF‐7/dox cells. B, Effect of Tan II A intervention at different concentrations (ranging from 0 to 1 mg/L) on the proliferation of MCF‐7 and MCF‐7/dox cells treated by 2 µg/mL Dox. C, Effect of β‐catenin agonist WAY‐262611 intervention on the proliferation of MCF‐7 and MCF‐7/dox cells treated by Dox combined with Tan II A. (a) Effect on the proliferation of MCF‐7 cells. (b) Effect on the proliferation of MCF‐7/dox cells. (c) Effect on the sensitizing fold. #P < .05, significantly different from Dox alone; *P < .05, significantly different from the combination of Dox and Tan II A

3.6 | WAY‐262611 intervention mediated the re‐nuclear translocation of β‐catenin and thereby upregulated the expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9WAY‐262611 is a β‐catenin agonist. To further corroborate our speculation, that was, it was because of the re‐nuclear transloca- tion of β‐catenin mediated by WAY‐262611 that the sensitizationeffect of Tan II A on Dox was significantly reduced after WAY‐262611 intervention, we next evaluated the expression of β‐catenin in MCF‐7 and MCF‐7/dox cells treated by Dox combined with Tan II A after WAY‐262611 intervention. The results showed that after WAY‐262611 intervention, the expressions of total and nuclear β‐catenin in the above cells were significantly increased, and the ratio of nuclear to cytoplasmic β‐catenin was also ob- viously increased ( 6A). Meanwhile, after WAY‐262611 in- tervention, with the re‐nuclear translocation of β‐catenin, the expression levels of its downstream targets also changed accord-

ingly, such as the expression of c‐Myc which mediates cell pro- liferation was significantly increased, the expression of E‐cadherin

5β‐catenin agonist WAY‐262611 intervention reduced the enhancement effect of tanshinone (Tan) II A on doxorubicin (Dox) in inhibiting cell migration. After scratching the monolayers of MCF‐7 and MCF‐7/dox cells with 200 μL pipette tips, the cells were treated by WAY‐262611, Tan II A, Dox, Dox combined with Tan II A, or Dox combined with Tan II A and WAY‐262611, and images were captured at 24 and 48 hours, respectively. #P < .05, significantly different from Tan II A alone; *P < .05, significantly different from Dox alone; &P < .05,

significantly different from combination of Dox and Tan II A which promotes cell adhesion and yet inhibits cell migration was significantly decreased, and the expressions of MMP‐2 and MMP‐9 which inhibit cell adhesion and yet promote cell migration were

significantly increased (6B).

4 | DISCUSSION

β‐Catenin is a multifunctional protein, which widely exists in various types of cells and plays an important role in regulating the proliferation,

 6 β‐catenin agonist WAY‐262611 intervention mediated the re‐nuclear translocation of β‐catenin in MCF‐7 and MCF‐7/dox cells treated by Dox combined with Tan II A. MCF‐7 and MCF‐7/dox cells were treated by a combination of Dox and Tan II A with or without WAY‐262611 intervention 24 hours. Western blot was used to analyze the expressions of β‐catenin and its downstream targets. A, The expressions of total, nuclear, and cytoplasmic β‐catenin in MCF‐7 cells (a) and MCF‐7/dox cells (b). *P < .05, significantly different from combination of Dox and Tan II A. B, The expressions of c‐Myc, E‐cadherin, MMP‐2, and MMP‐9 in MCF‐7 cells (a) and MCF‐7/dox cells (b).

*P < .05, significantly different from combination of Dox and Tan II A. Dox, doxorubicin; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase,
MMP, matrix metallopeptidase; Tan, tanshinone differentiation, adhesion, and migration of these cells.[18] β‐Catenin can exist in the cell membrane, cytoplasm, and nucleus, and its biological function varies with its different localizations. β‐Catenin in normal

mature cells mainly locates on the cell membrane. The β‐catenin on
the cell membrane mostly binds with the membrane surface adhesion molecule E‐cadherin, and then connect to the actin cytoskeleton
through α‐catenin to form a catenin‐cadherin‐actin complex, which
adheres to the cell membrane and mediates cell adhesion and migration.[19,20] Only a small amount of β‐catenin in normal mature
cells locates in the cytoplasm. The β‐catenin in the cytoplasm is phos-
phorylated by the synergy of CK1 and APC/Axin/GSK‐3β complex, and the phosphorylated β‐catenin is recognized by β‐TrCP, resulting in its ubiquitination and degradation.[21,22] If anyone of the above processes

is abnormal, β‐catenin degradation will be inhibited and transported to the nucleus, resulting in aberrant nuclear β‐catenin accumulation.[23] The β‐catenin in the nucleus binds with the T‐cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to form a transcription
factor complex and then initiates the transcription of its target genes.[14] Many of these genes can encode proteins capable of accel-
erating cell proliferation and promoting cell migration, such as c‐Myc,
E‐cadherin, MMP‐2, MMP‐9, and others, eventually leading to abnor- mal cell proliferation and migration.[16,17] Besides its clear role in tumorigenesis, the nuclear translocation of β‐catenin and its accumulation in the nucleus also plays an important role in cancer
chemoresistance.[14] Previous studies have shown that the accumula- tion of β‐catenin in the nucleus and its subsequent binding with

TCF/LEF transcription factors can trigger the expression of efflux ABC transporter proteins to mediate cancer chemoresistance.[24,25] As an
important driver of epithelial‐mesenchymal transition (EMT), the ac-
cumulation of β‐catenin in the nucleus and its subsequent binding with
TCF/LEF transcription factors can also induce the expression of genes responsible for the EMT process.[26] It has been shown that EMT sig- nificantly contributes to cancer chemoresistance.[27,28] And the study
has showed that the nuclear translocation of β‐catenin and its accu-
mulation in the nucleus can induce the expression of ZEB1, which, in
turn, regulated DNA damage repair and chemoresistance in cancer cells.[24] Therefore, targeted inhibition of β‐catenin nuclear transloca- tion may effectively improve the chemosensitivity of breast cancer. MCF‐7/dox is a Dox‐resistant cell line corresponding to breast cancer
MCF‐7 cell line, and we found that the expressions of total and nuclear
β‐catenin and the ratio of nuclear to cytoplasmic β‐catenin in MCF‐7/ dox cells were significantly higher than those in MCF‐7 cells, while the expression of APC was significantly lower than that in MCF‐7 cells. The findings suggested that β‐catenin in MCF‐7/dox cells took place sig- nificant nuclear translocation and the nuclear translocation of β‐catenin arose out of the decreased APC express, thereby resulting in the
aberrant nuclear β‐catenin accumulation. Here, we evaluated the effect of Tan II A intervention on the localization of β‐catenin in MCF‐7 and MCF‐7/dox cells treated by Dox, and determined whether the effect would affect the chemosensitivity of breast cancer cells to Dox. Our
results showed that Tan II A could significantly decrease the expres- sions of total and nuclear β‐catenin and the ratio of nuclear to cyto-
plasmic β‐catenin in Dox‐treated MCF‐7 and MCF‐7/dox cells,
especially in Dox‐treated MCF‐7/dox cells, which suggested that Tan II A could inhibit Dox‐mediated β‐catenin nuclear translocation, and the inhibition effect was better for Dox‐resistant MCF‐7/dox cells. Mean- while, as the inhibition of β‐catenin nuclear translocation, the expres- sion levels of β‐catenin target genes also changed accordingly, such as the expression of c‐Myc which mediates cell proliferation was de- creased, the expression of E‐cadherin which promotes cell adhesion and yet inhibits cell migration was increased, and the expressions of
MMP‐2 and MMP‐9 which inhibit cell adhesion and yet promote cell migration were decreased. This could also explain why our later ex- perimental results showed that Tan II A could enhance the inhibition
effect of Dox on cell proliferation and migration. Therefore, we speculated that Tan II A could improve the chemosensitivity of breast
cancer cells to Dox by inhibiting β‐catenin nuclear translocation. Our
subsequent studies further confirmed our speculation, that was when
MCF‐7 and MCF‐7/dox treated by Dox combined with Tan II A were intervened with β‐catenin agonist WAY‐262611, with the re‐nuclear translocation of β‐catenin in the above cells, the sensitization WAY-262611  effect of Tan II A on Dox breast cancer chemotherapy was greatly reduced.

5 | CONCLUSION

In conclusion, our data demonstrated that Tan II A could improve the chemosensitivity of breast cancer cells to Dox by inhibiting β‐catenin nuclear translocation. Since Tan II A could also alleviate the toxic side

effects of Dox, such as cardiotoxicity and nephrotoxicity, therefore, we believed that Tan II A could be used as a potential chemosensi- tizer in combination with Dox for breast cancer chemotherapy.

ACKNOWLEDGMENTS
We sincerely thank Prof Huang Jianhua for providing us with his technical guidance for this study. The study was supported by the National Natural Science Foundation of China (81303255) and the Foundation of Educational Department of Liaoning Province (JYTQN201722).

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

ORCID
Kun Li http://orcid.org/0000-0002-8668-9405

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