Bioorganic & Medicinal Chemistry

Sinomenine inhibits hypoXia induced breast cancer side population cells metastasis by PI3K/Akt/mTOR pathway

Lingqin Song a,*, Haifeng Zhang b, Min Hu c, Chengcheng Liu d, Yang Zhao a, Shuqun Zhang a,
Di Liu a
a Department of Oncology, the Second Affiliated Hospital, Xi’an Jiaotong University, No. 157, Xiwu Road, Xi’an, Shaanxi 710004, PR China
b Department of Pathology, Xi’an Jiaotong University Health Science Center, No. 76, Yanta West Road, Xi’an, Shaanxi 710061, PR China
c Department of Applied Chemistry, School of Science, Xi’an Jiaotong University, 28 Xianning West Road, Xi’an, Shaanxi 710049, PR China
d Department of Pathogenic Microbiology & Immunology, Xi’an Jiaotong University Health Science Center, No. 76, Yanta West Road, Xi’an, Shaanxi 710061, PR China

A R T I C L E I N F O

Keywords: Sinomenine Breast cancer
Side population cell Metastasis, hypoXia

A B S T R A C T

Sinomenine is an alkaloid derived from Chinese medicinal plant Sinomenium acutum. Our previous studies sug- gested that sinomenine can inhibit the metastasis of breast cancer. However, whether sinomenine can inhibit the metastasis characteristics of breast cancer side population (SP) cells is still unknown. In present study, we iso- lated the side population (SP) cells from MDA-MB-231 cells by fluorescence-activated cell sorting (FACS). MDA- MB-231 SP cells were treated with different concentrations of sinomenine at the absence or presence of hypoXia, and cell viability were measured by CCK-8 assay. The transwell invasive assay were conducted to assess of the effect of sinomenine on the invasion of hypoXic MDA-MB-231 SP cells. The protein expression was detected by Western blot assay. Sinomenine inhibited the cell viability and invasion of hypoXic MDA-MB-231 SP cells. Western blot assay results showed that the upregulation of MMP-2 and MMP-9 by hypoXia was inversed by sinomenine. Additionally, it was found that sinomenine suppressed the activation of PI3K/Akt/mTOR pathway under hypoXia in MDA-MB-231 SP cells. Moreover, the inhibiton of sinomenine on metastasis of hypoXic MDA- MB-231SP cells and PI3K/Akt/mTOR pathway could be rescued by PI3K activator IGF-1. Our study suggested that sinomenine inhibits invasion of breast cancer SP cells under hypoXia through PI3K/Akt/mTOR pathway.

1. Introduction

Breast cancer is the most commonly malignant tumors threatening to female health around the world, with 1.67 million new cases and 0.52 million cases resulting in death worldwide in 2012.1 The main reason for
breast cancer-related deaths is tumor metastasis. The 5-year survival
rate of early breast cancer was higher, reaching 94–99%, while that of metastatic breast cancer was significantly reduced, only 17–27%.2 Thus, the prevention and treatment of breast cancer metastasis is the key to
improve the survival rate of patients.
Growing evidence suggests that cancer stem-like side populations (SP) has the potential of rapid proliferation and multidirectional dif- ferentiation of stem cells,3 and is related to the chemoradiotherapy
resistance, invasion and metastasis of various tumors, and is regarded as the “seed” of tumor recurrence and metastasis.4–7 Therefore, SP cells are the preferred cells for the study of tumor recurrence and metastasis. The
limitations of previous cancer therapies may be due to the fact that
traditional therapies only target tumor host cells and ignore SP cells. The research on the mechanism of invasion and metastasis of SP cells will help to reduce tumor recurrence and metastasis, and lay a foundation for the development of new anti-tumor drugs in clinical.
Sinomenine (SIN), an alkaloid derived from Chinese medicinal plant
Sinomenium acutum, has obvious therapeutic effect on neuralgia and rheumatoid arthritis.8–10 In recent, its role in the occurrence and
development of cancer has attracted the attention. It has been suggested that sinomenine can inhibit the invasion of human lung cancer cells by mediating the STAT3 signaling pathway.11 In esophageal, sinomenine
enhanced the sensitivity of cancer cells to 5-fluorouracil without increasing the side effects of chemotherapy.12 Chen et al. has reported that sinomenine could alleviate cancer-induced bone pain in rats.13 Sinomenine promoted apoptosis of malignant glioma cells14 and renal cell carcinoma.15 Our previous studies confirmed that sinomenine can
inhibit the proliferation, migration and invasion of breast cancer MDA- MB-231 cells, suggesting that sinomenine may play an important role in

Corresponding author.
E-mail address: [email protected] (L. Song).

https://doi.org/10.1016/j.bmc.2020.115986

Received 2 July 2020; Received in revised form 18 December 2020; Accepted 22 December 2020
Available online 30 December 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.
Fig. 1. Identification of SP cells in human breast cancer cells. (A) SP sorting of MDA-MB-231 cells. Cells were stained with Hoechst Red and Hoechst Blue in the absence or presence of Verapamil and then analyzed by flow cytometry. (B) SP sorting of MCF-7 cells.

breast cancer metastasis.16,17 However, whether sinomenine can inhibit the metastasis characteristics of breast cancer SP cells is still unknown. HypoXia, one of the basic characteristics of the solid tumor micro-
environment, is associated with the survival of tumor cells, invasion and metastasis.18,19 It has been reported that hypoXia is related to breast cancer metastasis20 and regulates breast cancer MDA-MB-231 cells stemness.21 This study aims to explore the effect of sinomenine on
hypoXic breast cancer SP cells metastasis and its mechanism, providing preclinical experimental data and theoretical basis for sinomenine, this low price of traditional Chinese medicine monomer, in the treatment of breast cancer. And the elucidation of molecular mechanism of sinome- nine will be conducive to the modernization and internationalization of sinomenine preparation in the future.
2. Materials and methods
2.1. Cell culture

The human breast cancer cell lines MDA-MB-231 and MCF-7 from the Cell Bank of the Chinese Academy of Sciences (Beijing, China) were
cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 ◦C with 5% CO2.

2.2. Side population sorting
Side population isolation was conducted as previous.22 Briefly, 1 106 cells/ml of MDA-MB-231 or MCF-7 cells were suspended in DMEM with 2% FBS and 10 mM HEPES (Invitrogen) for 10 min. Cells were labeled for 1.5 h with Hoechst Red and Hoechst Blue (5 µg/ml; Sigma, St. Louis, MO, USA) in the presence or absence of verapamil (5 µmol/L; Sigma) and placed on ice for 10 min to terminate the staining. After resuspension in HBSS, cells were stained with 1 µg/ml propidium iodide.

Finally, cells were sorted on FACSAria (Becton Dickinson, Mountain View, CA, USA). UV laser at 375 nm was used to excite the Hoechst dye, and its fluorescence was measured using 450/20 BP filter (Blue) and 675 BP optical filter (Hoechst Red). Propidium iodide (PI) was excited at 488 nm (red, 575/26).
2.3. Hypoxia treatment

For the hypoXia experiments, SP cells were cultured under hypoXia with 5% CO2, 1% O2 and 94% N2 for 24 h.21
2.4. CCK-8 assay
The sorted SP cells were seeded into a 96-well plate (2 106 cells/ well) and subjected to hypoXia condition or treated with the indicated concentrations of sinomenine (Selleck Chemicals, Shanghai, China) for 24 h. Each well was supplemented with cell counting kit-8 (CCK-8) so- lution (Dojindo Laboratories, Kumamoto, Japan) for 2 h and the absorbance at a wavelength of 450 nm was determined by microplate reader.
2.5. Transwell assay

Transwell invasion assay was conducted to measure the invasive ability of cells. 100 μl of diluted Martrigel was evenly spread on the bottom of Transwell chamber in the upper chamber. 5 104 cells were
plated in serum-free medium the top side of transwell filter. The lower chamber was full of media with 10% FBS. After incubation for 24 h, cotton was used to carefully swab any no membrane-penetrating cells in the chamber, followed with 4% paraformaldehyde fiXation and 2% crystal violet staining. SiX fields were randomly selected for each well, and the number of invading cells was detected under the microscope.

Fig. 2. Sinomenine suppresses invasion of hyp- oXic SP cells from MDA-MB-231 and MCF-7 cells.
(A) and (B) Effects of different conditions of sinomenine on cell viability of SP cells from MDA- MB-231 and MCF-7 cells under normoXia or hyp- oXia environment. Cells were treated with sino- menine (0, 0.25, 0.5, 1, 2, and 4 mM) for 24 with
or without hypoXia. Then cell viability was
measured by CCK-8 assay. (C) and (D) Transwell assay to assess cell invasion of SP cells from MDA- MB-231 and MCF-7 cells. Cells were observed
under a light microscope (×200). *P < 0.05 vs control group. #P < 0.05 vs hypoXia + 0 group.

2.6. Western blot

Protein was extracted from cells using RIPA lysis buffer and its concentration was detected with BCA assay (Bio-Rad, Hercules, CA, USA). Afterward, 20 μg of protein was separated by 12% SDS-PAGE and electroblotted on to PVDF membranes (Millipore Corporation, Billerica, MA, USA). Subsequently, the membrane was blocked in 5% non-fat milk, followed by 12 h’s incubation with primary antibodies from Abcam: MMP-2 (1:2000; ab92536), MMP-9 (1:1000; ab38898), PI3K
p85 (1:1000; ab86714), p-PI3K p85 (phospho Y607) antibody (1:1000; ab182651), p-Akt (phospho T308) (1:1000; ab38449), Akt (1:500; ab8805), mTOR (1:2000; ab32028), p-mTOR (phospho S2448),
(1:2000; ab109268), and loading control β-actin (1:5000; ab179467).
After HRP-conjugated secondary antibody (1:5000; abcam) incubation, detection was performed with ECL system (Millipore) and quantified by Image J software.
2.7. Statistical analysis

Data are expressed as mean SD and analyzed using SPSS 20.0 software (SPSS Inc., Chicago, IL,USA). Significance of differences was determined by Student’s t test. P < 0.05 was considered significant.

3. Results
3.1. Isolation and identification of SP cells in breast cancer cell lines

First, SP cells in MDA-MB-231 cells or MCF-7 cells was detected and sorted by flow cytometry with fluorescent DNA-binding dye Hoechst Red and Hoechst Blue. The results showed that SP cells accounted for (2.12 0.15)% of MDA-MB-231 cells (Fig. 1A) and (1.88 0.12)% of
MCF-7 cells (Fig. 1B). After the addition of ABC transporter inhibitors Verapamil, the proportion of SP cells decreased to (0.08 ± 0.04)% (Fig. 1A) and (0.09 ± 0.01)% of MCF-7 cells (Fig. 1B).

3.2. Sinomenine inhibited invasion of breast cancer SP cells under hypoxia
Effects of sinomenine (0.25, 0.5, 1, 2, 4 mM) on cell viability of non- side population (non-SP) and SP from MDA-MB-231 cells and MCF-7 cells were detected by CCK-8 assay. As shown in Supplementary Fig. 1A and B, cell viability at 1, 2 and 4 mM of sinomenine under normoXia in non-SP cells from MDA-MB-231 and MCF-7 cells were
significantly decreased as compared to the control cells (P < 0.05).
Under hypoXia, 0.25, 0.5 and 1 mM of sinomenine did not exert sig- nificant inhibition on the cell viability in both non-SP cells from MDA-
MB-231 and MCF-7 cells (P > 0.05). Data from Fig. 2A and B showed
that there was no obvious difference in cell viability at 0.25, 0.5 and 1 mM of sinomenine in the presence or absence of hypoXia in SP cells from
Fig. 3. Effect of sinomenine on protein levels of E-cadherin, N-cadherin, vimentin, MMP-2 and MMP-9 in MDA-MB-231 SP cells. Protein levels of E-
cadherin, N-cadherin, vimentin, MMP-2 and MMP-9 in MDA-MB-231 SP cells were measured by western blot. *P < 0.05 vs control group. #P < 0.05 vs hypoXia + 0 group.
MDA-MB-231 and MCF-7 cells (P > 0.05). Whereas 2 and 4 mM of sinomenine significantly inhibited the cell viability of MDA-MB-231 and MCF-7 SP cells both under normoXia and hypoXia conditions (P < 0.05).
In order to exclude that the effect of sinomenine on the invasion of SP cells was caused by the inhibition of cell viability, 0.25, 0.5, and 1 mM of sinomenine were selected in the following experiments.
To explore the effect of sinomenine on the invasion of hypoXic SP cells, transwell assay were conducted. Results showed that sinomenine obviously decreased the promotion of cell invasion by hypoXia in SP

cells from both MDA-MB-231 and MCF-7 cells (Fig. 2C and D; P < 0.05). Sinomenine also inhibited the elevation of cell invasion induced by hypoXia in non-SP cells from both MDA-MB-231 and MCF-7 cells (Sup-
plementary Fig. 1C and D; P < 0.05).
Also, hypoXia induced the upregulation of N-cadherin, vimentin, MMP-2 and MMP-9 was suppressed by sinomenine (Fig. 3; P < 0.05). The inhibitory effect of hypoXia on the expression of E-cadherin was reversed by sinomenine (Fig. 3; P < 0.05). This effect of sinomenine on the protein expression of E-cadherin, N-cadherin, vimentin, MMP-2 and
MMP-9 in MDA-MB-231 non-SP cells was also confirmed (Supplemen- tary Fig. 2). The above results suggest that sinomenine treatment significantly inhibited hypoXia induced invasion of breast cancer SP cells. MDA-MB-231 SP cells were used in the next experiments.

3.3. Sinomenine inhibited the activation of PI3K/Akt/mTOR pathway under hypoxia in breast cancer SP cells
Since biological functions of breast cancer cells is mediated by the phosphoinositide-3 kinase/protein kinase B/mammalian target of
rapamycin (PI3K/AKT/mTOR) signaling pathway.23 To illustrate the underlying mechanisms of sinomenine, the level of proteins involved in the PI3K/AKT/mTOR pathways were assessed by western blot. Fig. 4 showed that, phosphorylated levels of PI3K, Akt, and mTOR were all
significantly increased by hypoXia (P < 0.05). Meanwhile, that increase was observably decreased by sinomenine (P < 0.05). These data indi-
cated that sinomenine suppressed hypoXia-induced PI3K/Akt/mTOR pathway activation in breast cancer SP cells.

3.4. Sinomenine inhibited invasion of hypoxic breast cancer SP cells through PI3K/Akt/mTOR pathway
To further determine whether PI3K/Akt/mTOR pathway was involved in sinomenine exerted anti-tumor effects in hypoXic breast cancer SP cells, MDA-MB-231 SP cells were incubated with PI3K agonist
Fig. 4. Sinomenine inhibited PI3K/Akt/mTOR pathway activation in MDA-MB-231 SP cells under hypoXia. Cells were treated with sinomenine (0, 0.25, 0.5 and 1 mM) for 24 h. (A) The protein expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR detected by Western blot. (B) The normalized ratio of p-PI3K to PI3K. (C) The normalized ratio of p-Akt to Akt. (D) The normalized ratio of p-mTOR to mTOR. *P < 0.05 vs control group. #P < 0.05 vs hypoXia + 0 group.

 

Fig. 5. Sinomenine inhibited invasion of hypoXic MDA-MB-231 SP cells through PI3K/Akt/mTOR pathway. MDA-MB-231 SP cells were incubated with sinomenine (1 nM) with or without PI3K activator IGF-1 (10 nmol/L), and cultured under hypoXia for 24 h. (A) The protein expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR detected by Western blot. (B) The normalized ratio of p-PI3K to PI3K. (C) The normalized ratio of p-Akt to Akt. (D) The normalized ratio of p-mTOR to mTOR.
(E) The normalized ratio of MMP-2 and MMP-9 to β-actin. (F) Transwell assay to assess cell invasion. Cells were observed under a light microscope (×200). (G) Quantitative assay of cell invasion. N = 3, *P < 0.05 vs. control group; #P < 0.05 vs. hypoXia + 0 group; &P < 0.05 vs. hypoXia + 1 group.

IGF-1 (10 nmol/L). As shown in Fig.5A–D, IGF-1 treatment activated PI3K/Akt/mTOR signaling and counteracted the suppression of PI3K/ Akt/mTOR signaling by sinomenine in hypoXic MDA-MB-231 SP cells. The suppressed expression of MMP-2 and MMP-9 were also elevated by IGF-1 treatment (Fig. 5E). Furthermore, the suppressed invasiveness (Fig.5F and G) of hypoXic MDA-MB-231 SP cells by sinomenine was abolished by IGF-1. Taken together, we concluded that sinomenine inhibited invasion of hypoXic breast cancer SP cells through PI3K/Akt/ mTOR pathway.
4. Discussion

In the present study, the suppressive effect of sinomenine on breast cancer SP cells and PI3K/Akt/mTOR pathway was investigated. It was found that sinomenine at noncytotoXic concentrations prominently inhibited the invasive abilities of MDA-MB-231 SP cells under hypoXia in vitro through PI3K/Akt/mTOR pathway. These results indicated that sinomenine may be a candidate for therapy of breast cancer.
Traditional Chinese medicine has the overall treatment advantages of multiple pathways and multiple targets. In recent years, searching for new anti-cancer drugs from natural resources has become a research hotspot. Sinomenine, which is extracted from the traditional Chinese medicinal plant Sinomenium acutum, is widely used in rheumatoid arthritis treatment in China. In recent years, the anticancer activity of sinomenine has been gradually revealed. It has been found that sino-
menine exerted a significant inhibitory effect on hepatocellular carci- noma,24 lung cancer,11 prostate cancer,25 et al. Our previous work also
found the repression of sinomenine on breast cancer.17,25 Intervention of stem cell function with traditional Chinese medicine has great poten- tial.26,27 HypoXia, an important environment of breast cancer, affects cancer progression and metastasis.28 In this study, we investigated the effect of sinomenine on breast cancer SP cells under hypoXia. Consist with previous study in MDA-MB-231 cells,29 it was also found that
hypoXia increased the invasive ability of MDA-MB-231 SP cells. This increased invasion of MDA-MB-231 SP cells could be inhibited by sinomenine.
Metalloprotease (MMPs), a family of zinc-containing endopepti- dases, can almost degradation of extracellular matriX of various proteins
and destroy the tumor cell migration of histology barrier. They play a key role in progression and metastasis of cancers.30,31 MMP-2 and MMP- 9 are crucial biomarkers for cancer migration and invasion.32 E-cad-
herin, N-cadherin and vimentin related to the epithelial-mesenchymal transition were also important for cell invasion. In the present study, it was demonstrated that protein levels of E-cadherin, N-cadherin, vimentin, MMP-2 and MMP-9 were affected in MDA-MB-231 SP cells under hypoXia, and were reversed by sinomenine.
PI3K/AKT/mTOR pathway is involved in the modulation of various cellular processes including cell survival and metastasis.33 The aberrant
activation of this pathway has been observed in various tumors, including breast cancer34 and this pathway is regarded as a therapeutic target for effective management of breast cancer.35 It was reported that hypoXia could activate PI3K/Akt/mTOR pathway.36 There are few
studies focus on the effects of PI3K/Akt/mTOR pathway on the invasion of breast cancer SP cells, especially under hypoXia. To investigate the pivotal role of PI3K/Akt/mTOR signaling in the effect of sinomenine on breast cancer SP cells, the proteins in PI3K/Akt/mTOR signaling was detected. It was found that sinomenine inhibited the PI3K/Akt/mTOR pathway in hypoXic MDA-MB-231 SP cells. Subsequently, to verified the crucial role of PI3K/Akt/mTOR pathway in the effect of sinomenine on the invasion of breast cancer SP cells under hypoXia, PI3K activator IGF-1 was used to treated MDA-MB-231 SP cells. IGF-1 partly counteracted the inhibitory effect of sinomenine on invasion of MDA-MB-231 SP cells

12 Wang J, Yang ZR, Dong WG, et al. Cooperative inhibitory effect of sinomenine combined with 5-fluorouracil on esophageal carcinoma. World J Gastroenterol. 2013;under hypoXia. Overall, these experiments stated that sinomenine19:8292–8300.

13 Chen SP, Sun J, Zhou YQ, et al. Sinomenine attenuates cancer-induced bone pain via affected invasion of MDA-MB-231 SP cells under hypoXia possibly throughPI3K/Akt/mTOR pathway.

5. Conclusion

Our study demonstrated that sinomenine could alleviate the invasion of breast cancer SP cells under hypoXia, possibly through PI3K/Akt/ mTOR pathway. This study provides that sinomenine might be a po- tential drug for breast cancer treatments.

Funding

This study was supported by the funding of Social Development Plans of Shaanxi Province (No. 2019SF-096).
Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared Compound 19  to influence the work reported in this paper.
Acknowledgements

We gratefully acknowledge OXford Edit for their language help.
Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi. org/10.1016/j.bmc.2020.115986.
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