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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describesa comprehensive strategy for evaluating the pharmacological action and mechanism of salidroside in inhibiting MCF-7 cell proliferation and migration.

Abstract

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer mechanisms have been only incompletely elucidated. Hence, this protocol intended to decode the potential of Sal in regulating the PI3K-AKT-HIF-1α-FoxO1 pathway in the malignant proliferation of human breast cancer MCF-7 cells. First, the pharmacological activity of Sal against MCF-7 was evaluated by CCK-8 and cell scratch assays. Moreover, the resistance of MCF-7 cells was measured by migration and Matrigel invasion assays. For cell apoptosis and cycle assays, MCF-7 cells were processed in steps with annexin V-FITC/PI and cell cycle-staining detection kits for flow cytometry analyses, respectively. The levels of reactive oxygen species (ROS) and Ca2+ were examined by DCFH-DA and Fluo-4 AM immunofluorescence staining. The activities of Na+-K+-ATPase and Ca2+-ATPase were determined using the corresponding commercial kits. The protein and gene expression levels in apoptosis and the PI3K-AKT-HIF-1α-FoxO1 pathway were further determined using western blot and qRT-PCR analyses, respectively. We found that Sal treatment significantly restricted the proliferation, migration, and invasion of MCF-7 cells with dose-dependent effects. Meanwhile, Sal administration also dramatically forced MCF-7 cells to undergo apoptosis and cell cycle arrest. The immunofluorescence tests showed that Sal observably stimulated ROS and Ca2+ production in MCF-7 cells. Further data confirmed that Sal promoted the expression levels of pro-apoptotic proteins, Bax, Bim, cleaved caspase-9/7/3, and their corresponding genes. Consistently, Sal intervention prominently reduced the expression of the Bcl-2, p-PI3K/PI3K, p-AKT/AKT, mTOR, HIF-1α, and FoxO1 proteins and their corresponding genes. In conclusion, Sal can be used as a potential herb-derived compound for treating breast cancer, as it may reduce the malignant proliferation, migration, and invasion of MCF-7 cells by inhibiting the PI3K-AKT-HIF-1α-FoxO1 pathway.

Introduction

As one of the most commonly diagnosed cancers and most common malignancies, the latest statistics indicate that 2.3 million cases of breast cancer emerged around the world by 2020, accounting for 11.7% of all cancer cases1. Common symptoms of breast cancer include breast tenderness and tingling, breast lumps and pain, nipple discharge, erosion or sunken skin, and enlarged axillary lymph nodes1,2. Even more alarming, the number of new cases and the overall incidence of breast cancer continues to increase at an overwhelming rate each year, accounting for 6.9% of cancer-related deaths1. At present, breast cancer intervention still mainly involves chemotherapy, surgery, radiotherapy, and comprehensive treatment. Although treatment can effectively reduce the recurrence rate and mortality rate of patients, long-term treatment application often causes produce multidrug resistance, large-area hair loss, nausea and vomiting, and serious mental and psychological burden2,3. Notably, the potential risk of multiple organ metastases from breast cancer also forces people to seek novel herbal sources of drug therapy4,5.

Phosphoinositide 3 kinase (PI3K)-mediated signaling is implicated in the growth, proliferation, and survival of breast cancer through splicing that affects the expression of multiple genes6. As a downstream signal-sensing protein of PI3K, numerous evidence suggests that protein kinase B (AKT) could couple with the mammalian target of rapamycin (mTOR) protein to further increase breast cancer7,8,9. Moreover, the deactivation of PI3K/AKT/mTOR signaling has also been claimed to be a key component in drugs inhibiting malignant proliferation and stimulating apoptosis in breast cancer10,11,12. It is well known that extreme hypoxia in the tumor microenvironment forces a massive surge in hypoxia-inducible factor 1 alpha (HIF-1α), which further worsens the progression of breast cancer13,14,15. In parallel, AKT stimulation also leads to excessive accumulation of HIF-1α, limiting apoptosis in breast cancer samples16,17. Interestingly, the activation of PI3K-AKT-HIF-1α signaling has been confirmed to be involved in pathologic progression and metastasis in a variety of cancers, including lung cancer18, colorectal cancer19, ovarian cancer20, and prostate cancer21. In addition to being orchestrated by HIF-1α, forked head transcription factor 1 (FoxO1) overexpression is also triggered by AKT signaling stimulation, which promotes cycle arrest and the inhibition of apoptosis in breast cancer cells22,23. Together, the above solid evidence suggests that the inhibition of the cascade of PI3K-AKT-HIF-1α-FoxO1 signaling may be a potential novel target for drug therapy in breast cancer.

Salidroside (Sal) has been widely demonstrated to exert anti-cancer24,25, anti-hypoxia26,27,28,29, and immune-enhancing pharmacological activities30. It is a light brown or brown powder that is easily soluble in water, is a type of phenylethanoid glycoside, and has a chemical structure formula of C14H20O7 and a molecular weight of 300.331,32. Modern pharmacological investigations have demonstrated that Sal can promote the apoptosis of gastric cancer cells by restraining PI3K-AKT-mTOR signaling24. Further evidence also suggests that the suppression of PI3K-AKT-HIF-1α signaling by Sal treatment may contribute to the apoptosis of cancer cells by enhancing their sensitivity to chemotherapeutic agents25. Evidence also suggests that Sal restricts cell migration and invasion and causes cycle arrest by promoting apoptosis in the human breast cancer MCF-7 cells33,34. However, it remains to be seen whether Sal can regulate PI3K-AKT-HIF-1α-FoxO1 signaling and inhibit the malignant proliferation of MCF-7 cells. Therefore, this protocol aimed to explore the effects of Sal on MCF-7 cell migration, invasion, cell cycle, and apoptosis via the PI3K-AKT-HIF-1α-FoxO1 pathway. The integrated research strategies comprising conventional, low-cost, and independent experiments, such as cell migration and invasion assessments, apoptosis and cell cycle detection by flow cytometry, reactive oxygen species (ROS) and Ca2+ fluorescence determination, etc., can provide a reference for the overall design of experiments for anti-cancer research with traditional herbal medicine. The experimental process of this study is shown in Figure 1.

Protocol

The MCF-7 cells used for the present study were obtained from a commercial source (see the Table of Materials).

1. Cell culture

  1. Culture the MCF-7 cells in a humidified 5% CO2 atmosphere at 37 °C with DMEM containing 10% FBS and 1% penicillin (10,000 U/mL)/streptomycin (10,000 µg/mL) (see the Table of Materials).
    ​NOTE: The cells covering 90% of the bottom of the dish were employed for the experiment and assigned into the following groups: control group, doxorubicin hydrochloride group (DXR, 5 µM), and Sal groups (20 µM, 40 µM, and 80 µM), or control group, LY294002 (10 µM, an inhibitor of PI3K) group, Sal (80 µM) group, and LY294002 (10 µM) + Sal (80 µM) group (see the Table of Materials).

2. Cell viability assay

NOTE: For details on this procedure, please refer to a previous report27.

  1. Seed MCF-7 cells with a density of 8 x 104 cells/well in 96-well plates, and incubate with Sal (10 µM, 20 µM, 40 µM, 80 µM, 160 µM, and 320 µM) for 24 h or Sal (20, 40, 80 µM) for 12 h/24 h/36 h/48 h overnight until the cells are adherent.
  2. After treatment, co-incubate the MCF-7 cells with 10 µL/well of CCK-8 solution (see the Table of Materials) for 2 h. Then, measure the optical density at 450 nm (OD450) with an automatic microplate reader.

3. Cell migration and invasion

NOTE: For details on this procedure, please refer to a previous report35.

  1. Incubate 2 mL of 5 x 106 cells/mL cells seeded in 6-well plates to cover 90% of the bottom of the dish.
  2. Perform a linear scratch wound along the center of the cell monolayer with a sterile pipette tip. Acquire images using an optical microscope at 0 h, 24 h, and 48 h.
  3. Use Image J software to measure the width of the scratch wounds.
  4. For the transwell assay, suspend MCF-7 cells with no serum medium, and seed in the upper transwell chamber pre-coated with or without Matrigel (see the Table of Materials).
  5. In the bottom of the transwell chamber, use DMEM complete medium as a chemical inducer. After 24 h, remove the cells in the upper chamber, and fix the remaining invasive and migrant cells in methanol35.
  6. Stain the MCF-7 cells with crystal violet solution (see the Table of Materials). Capture the images using an optical microscope.

4. Activity evaluation of Na+-K+-ATPase and Ca2+-Mg2+-ATPase

  1. Use a bicinchoninic acid (BCA) kit to measure the protein concentration in lysed MCF-7 cells, following the manufacturer's instructions (see the Table of Materials).
    1. Add samples of the lysed cells in their corresponding groups into the 96-well plates. After that, add the working solution to further incubate at 37 °C for 5 min. Finally, measure the values of OD636 with a multifunctional microplate reader.

5. Flow cytometry analysis of apoptosis and the cell cycle

NOTE: For details on this procedure, please refer to a previous report31.

  1. Digest the cells, and harvest to resuspend in phosphate-buffered saline (PBS) for a 20 min staining with annexin V-FITC and propidium iodide (PI) (see the Table of Materials), and then determine the number of apoptotic cells using a flow cytometer.
  2. Mix the resuspended sample solution with PI solution for a 30 min incubation, followed by detecting with a flow cytometer.

6. DCFH-DA and Fluo-4 AM fluorescence staining

NOTE: For details on this procedure, please refer to a previous report29.

  1. Incubate the MCF-7 cells for 20 min with a 10 µM DCFH-DA fluorescence probe (see the Table of Materials) at 37 °C. After thoroughly removing the surplus DCFH-DA by washing three times with PBS, test the fluorescence intensity at excitation and emission wavelengths of 488 nm and 525 nm, respectively, using a fluorescence microscope.
  2. Incubate the MCF-7 cells with a 5 µM Fluo-4 AM fluorescent probe solution (see the Table of Materials) for 45 min at 37 °C. After washing with PBS three times, determine the fluorescence intensity values at excitation and emission wavelengths of 488 nm and 516 nm, respectively, using a fluorescence microscope.

7. Western blot

  1. Add 2 mL of 5 x 106 cells/mL cell suspensions containing different drugs into 6-well plates, and culture in a 37 °C, 5% CO2 incubator for 24 h.
    NOTE: The groups for the western blot analysis were set as follows: control group, LY294002 (10 µM) group, Sal (80 µM) group, and LY294002 (10 µM) + Sal (80 µM) group (see the Table of Materials).
  2. Collect the cells and supernatant, and centrifuge at 560 × g at 4 °C for 3 min. Discard the supernatant, and wash twice with pre-cooled PBS. After each wash, centrifuge the cells at 560 × g at 4 °C for 3 min.
  3. Add 50 µL lysis buffer to the cell sample from step 7.2 for a 15 min ice bath. Centrifuge at 8550 × g at 4 °C for 10 min to collect the supernatant protein sample.
  4. After detecting the protein concentration using a BCA method, mix the protein sample in step 7.3 with loading buffer in a ratio of 4:1, denature at 100 °C for 10 min in a metal bath, and cool to room temperature.
  5. Add 20 µg of the protein sample from step 7.4, separate the proteins with different molecular weights35using 10% SDS-PAGE (see the Table of Materials), and then transfer to 0.22 µm PVDF membranes. After blockage with 5% BSA, incubate the membranes with the corresponding primary antibodies overnight at 4 °C.
    NOTE: The dilute concentration of the following primary antibodies is 1:1,000: cleaved caspase-9/7/3 (CC-9/7/3), Bim, Bax, Bcl-2, p-PI3K/PI3K, p-AKT/AKT, mTOR, HIF-1α, FoxO1, and β-actin (see the Table of Materials).
  6. The next day, incubate the membranes with goat anti-rabbit IgG secondary antibody for 2 h at 37 °C. Develop the membranes using an ECL chemiluminescent solution, and capture images using a contactless quantitative western blot imaging system (see the Table of Materials).

8. qRT-PCR

  1. Perform centrifugation to collect MCF-7 cells at 560 x g at 4 °C for 3 min, and add 500 µL buffer RL1 into 5 × 106 cells. Blow and mix repeatedly with a pipette until no cell masses are visible.
    NOTE: Buffer RL1 is one of the components in the total RNA isolation kit (see the Table of Materials).
  2. Transfer the cell homogenates in step 8.1 to the DNA-cleaning column embedded in the collection tube, and centrifuge at 8,550 × g at 4 °C for 2 min. Remove the DNA-cleaning column, and keep the supernatant in the collection tube.
    NOTE: The DNA-cleaning column and the collection tube are two of the components in the total RNA isolation kit (see the Table of Materials).
  3. Add 800 µL buffer RL2 to 500 µL of the supernatant from step 8.2, and mix gently.
    NOTE: Buffer RL2 is one of the components in the total RNA isolation kit (see the Table of Materials). Add 120 mL of anhydrous ethanol to 60 mL of buffer RL2 before use.
  4. Transfer 700 µL of the mixture from step 8.3 into the RNA-only column embedded in the collection tube, and centrifuge at 8,550 × g at 4 °C for 1 min. Discard the waste liquid in the collection tube.
    NOTE: The RNA-only column is one of the components in the total RNA isolation kit (see the Table of Materials).
  5. Repeat step 8.4 to process the remaining mixture from step 8.3.
  6. Add 500 µL of buffer RW1 to the RNA-only column, and centrifuge at 8,550 × g at 4 °C for 1 min. Discard the waste liquid in the collection tube.
    NOTE: Buffer RW1 is one of the components in the total RNA isolation kit (see the Table of Materials).
  7. Add 700 µL of buffer RW2 to the RNA-only column, and centrifuge at 8,550 × g at 4 ◦C for 1 min. Discard the waste liquid in the collection tube. Repeat step 8.7 once.
    NOTE: Buffer RW2 is one of the components in the total RNA isolation kit (see the Table of Materials).
  8. Place the RNA-only column back into the collection tube, and centrifuge at 8550 × g at 4 °C for 2 min to remove the remaining buffer RW2.
  9. Transfer the RNA-only column to a new collection tube, and drip 100 µL of RNase-free ddH2O preheated at 65 °C into the center of the membrane for the RNA-only column. Place at 25 °C for 2 min, and collect the RNA solution by centrifugation at 8,550 × g at 4 °C for 1 min.
    NOTE: RNase-free ddH2O is one of the components in the total RNA isolation kit (see the Table of Materials).
  10. Add 4 µL of 5x reaction buffer, 1 µL of oligo (dT)18 primer, 1 µL of random hexamer primer, 1 µL of gene-specific primer (see Table 1), 1 µL of enzyme mix, and 5 µg of the RNA solution from step 8.9 into a 100 µL 8-tube strip. Supplement the reaction system with RNase-free water to 20 µL.
  11. Set the reaction conditions of the system as follows: (1): 95 °C, 30 s, 1 cycle; (2): 95 °C, 5 s, 50 cycles, 60 °C, 34 s; (3): 95 °C, 5 s, 1 cycle, 65 °C, 60 s, 97 °C, 1 s; and (4): 42 °C, 30 s, 1 cycle. Execute the qRT-PCR procedure.
    NOTE: The relative expression levels of the target genes were quantitatively analyzed by the 2−ΔΔCT method36,37. The primer information of each gene used for the qRT-PCR analysis is listed in Table 1.

9. Statistical analysis

  1. Express the data as the mean ± standard deviation of three independent experiments.
  2. Analyze the comparisons between multiple groups using graphing and analysis software (see the Table of Materials) with a one-way analysis of variance (ANOVA) followed by a Tukey's test. In this work, P < 0.05 was defined as statistically significant.

Results

Effects of Sal on inhibiting excess proliferation and delaying wound healing in MCF-7 cells
To probe the potential of Sal against breast cancer, we first tested its anticancer properties using cell proliferation toxicity and scratch assays of the human breast cancer MCF-7 cell line. These cells were co-incubated with a concentration series of Sal (5-320 µM) for 24 h, and the cell proliferation was evaluated using a CCK-8 assay. A dose-dependent inhibitory effect of Sal on cell proliferation wa...

Discussion

Breast cancer affects individuals of all ages and causes incalculable physical and mental burden and great economic pressure1. Breast cancer, with its increasing morbidity and mortality each year, has also attracted worldwide attention in terms of seeking effective herbal-based compound therapies beyond conventional treatments4,5. Promisingly, a large body of evidence has revealed the anti-cancer effects of Sal24

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Health Commission of Sichuan Province (120025).

Materials

NameCompanyCatalog NumberComments
1% penicillin/streptomycinHyCloneSV30010
AKT antibodyImmunoWay Biotechnology CompanyYT0185
Annexin V-FITC/PI kitMultiSciences Biotech Co., Ltd.AP101
Automatic microplate readerMolecular DevicesSpectraMax iD5
Bax antibodyCell Signaling Technology, Inc.#5023
BCA kitBiosharp Life SciencesBL521A
Bcl-2 antibodyCell Signaling Technology, Inc.#15071
Bim antibodyCell Signaling Technology, Inc.#2933
Ca2+–ATPase assay kitNanjing Jiancheng Bioengineering InstituteA070-4-2
Cell counting kit-8Biosharp Life SciencesBS350B
Cell cycle staining kitMultiSciences Biotech Co., Ltd.CCS012
cleaved caspase-3Cell Signaling Technology, Inc.#9661
cleaved caspase-7Cell Signaling Technology, Inc.#8438
cleaved caspase-9Cell Signaling Technology, Inc.#20750
Crystal violet solutionBeyotime BiotechnologyC0121
DMEM high glucose culture mediumServicebio Technology Co., Ltd.G4510
Doxorubicin hydrochlorideMedChemExpressHY-15142
ECL chemiluminescent solutionBiosharp Life SciencesBL520B
Fetal bovine serumProcell Life Science & Technology Co., Ltd.164210
Flow cytometerBDFACSCanto figure-materials-2474
Fluo-4 AMBeyotime BiotechnologyS1060
FoxO1 antibodyImmunoWay Biotechnology CompanyYT1758
Goat anti-rabbit IgG secondary antibodyMultiSciences Biotech Co., Ltd.70-GAR0072
GraphPad Prism softwareLa JollaVersion 6.0
HIF-1α antibodyAffinity BiosciencesBF8002
Human breast cancer cell line MCF-7Procell Life Science & Technology Co., Ltd.CL-0149
Loading bufferBiosharp Life SciencesBL502B
LY294002MedChemExpressHY-10108
MatrigelThermo 356234
mTOR antibodyServicebio Technology Co., Ltd.GB11405
Na+–K+–ATPase assay kitNanjing Jiancheng Bioengineering InstituteA070-2-2
Optical microscopeOlympusIX71PH
p-AKT antibodyImmunoWay Biotechnology CompanyYP0006
PI3K antibodyServicebio Technology Co., Ltd.GB11525
p-PI3K antibodyAffinity BiosciencesAF3241
Quantitative western blot imaging systemTouch Image ProeBlot
Reverse transcription first strand cDNA synthesis kitServicebio Technology Co., Ltd.G3330-100
ROS assay kitBeyotime BiotechnologyS0033SDCFH-DA fluorescence probe is included here
SalidrosideChengdu Herbpurify Co., Ltd.H-040
SDS-PAGE kitServicebio Technology Co., Ltd.G2003-50T
Total RNA isolation kitForegeneRE-03014
TrypsinHyCloneSH30042.01
β-actinAffinity BiosciencesAF7018

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