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

The MCF-7 cells used for the present study were obtained from a commercial source (see the Table of Materials).
1. Cell culture
<ol>
	<li>Culture the MCF-7 cells in a humidified 5% CO2 atmosphere at 37 &#176;C with DMEM containing 10% FBS and 1% penicillin (10,000 U/mL)/streptomycin (10,000 &#181;g/mL) (see the Table of Materials). 
	&#8203;NOTE: The cells covering 90% of the bottom of the dish were employed for the experimen...

Salidroside&#039;s Mechanism in Suppressing MCF-7 Cell Migration and Proliferation

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<su...

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-1&#945;), which further worsens the progression of breast cancer13,14,15. In parallel, AKT stimulation also leads to excessive accumulation of HIF-1&#945;, limiting apoptosis in breast cancer samples16,17. Interestingly, the activation of PI3K-AKT-HIF-1&#945; 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-1&#945;, 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-1&#945;-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-1&#945; 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-1&#945;-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-1&#945;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% penicillin/streptomycin</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>AKT antibody</td>
			<td>ImmunoWay Biotechnology Company</td>
			<td>YT0185</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V-FITC/PI kit</td>
			<td>MultiSciences Biotech Co., Ltd.</td>
			<td>AP101</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax iD5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bax antibody</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#5023</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA kit</td>
			<td>Biosharp Life Sciences</td>
			<td>BL521A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bcl-2 antibody</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#15071</td>
			<td></td>
		</tr>
		<tr>
			<td>Bim antibody</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#2933</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+&#8211;ATPase assay kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A070-4-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting kit-8</td>
			<td>Biosharp Life Sciences</td>
			<td>BS350B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell cycle staining kit</td>
			<td>MultiSciences Biotech Co., Ltd.</td>
			<td>CCS012</td>
			<td></td>
		</tr>
		<tr>
			<td>cleaved caspase-3</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#9661</td>
			<td></td>
		</tr>
		<tr>
			<td>cleaved caspase-7</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#8438</td>
			<td></td>
		</tr>
		<tr>
			<td>cleaved caspase-9</td>
			<td>Cell Signaling Technology, Inc.</td>
			<td>#20750</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet solution</td>
			<td>Beyotime Biotechnology</td>
			<td>C0121</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose culture medium</td>
			<td>Servicebio Technology Co., Ltd.</td>
			<td>G4510</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>MedChemExpress</td>
			<td>HY-15142</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL chemiluminescent solution</td>
			<td>Biosharp Life Sciences</td>
			<td>BL520B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Procell Life Science &#38; Technology Co., Ltd.</td>
			<td>164210</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>FACSCanto <img alt="Equation 1" src="/files/ftp_upload/65634/65634eq01.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Beyotime Biotechnology</td>
			<td>S1060</td>
			<td></td>
		</tr>
		<tr>
			<td>FoxO1 antibody</td>
			<td>ImmunoWay Biotechnology Company</td>
			<td>YT1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG secondary antibody</td>
			<td>MultiSciences Biotech Co., Ltd.</td>
			<td>70-GAR0072</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software</td>
			<td>La Jolla</td>
			<td>Version 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>HIF-1&#945; antibody</td>
			<td>Affinity Biosciences</td>
			<td>BF8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Human breast cancer cell line MCF-7</td>
			<td>Procell Life Science &#38; Technology Co., Ltd.</td>
			<td>CL-0149</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading buffer</td>
			<td>Biosharp Life Sciences</td>
			<td>BL502B</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002</td>
			<td>MedChemExpress</td>
			<td>HY-10108</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Thermo&#160;</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTOR antibody</td>
			<td>Servicebio Technology Co., Ltd.</td>
			<td>GB11405</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+&#8211;K+&#8211;ATPase assay kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A070-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus</td>
			<td>IX71PH</td>
			<td></td>
		</tr>
		<tr>
			<td>p-AKT antibody</td>
			<td>ImmunoWay Biotechnology Company</td>
			<td>YP0006</td>
			<td></td>
		</tr>
		<tr>
			<td>PI3K antibody</td>
			<td>Servicebio Technology Co., Ltd.</td>
			<td>GB11525</td>
			<td></td>
		</tr>
		<tr>
			<td>p-PI3K antibody</td>
			<td>Affinity Biosciences</td>
			<td>AF3241</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantitative western blot imaging system</td>
			<td>Touch Image Pro</td>
			<td>eBlot</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcription first strand cDNA synthesis kit</td>
			<td>Servicebio Technology Co., Ltd.</td>
			<td>G3330-100</td>
			<td></td>
		</tr>
		<tr>
			<td>ROS assay kit</td>
			<td>Beyotime Biotechnology</td>
			<td>S0033S</td>
			<td>DCFH-DA fluorescence probe is included here</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>Chengdu Herbpurify Co., Ltd.</td>
			<td>H-040</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE kit</td>
			<td>Servicebio Technology Co., Ltd.</td>
			<td>G2003-50T</td>
			<td></td>
		</tr>
		<tr>
			<td>Total RNA isolation kit</td>
			<td>Foregene</td>
			<td>RE-03014</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Affinity Biosciences</td>
			<td>AF7018</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring the pharmacological action and molecular mechanism of salidroside in inhibiting mcf-7 cell proliferation and migration

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-1&#945;-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-1&#945;-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-1&#945;, 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-1&#945;-FoxO1 pathway.

Author Spotlight: Exploring Salidroside&#039;s Molecular Mechanisms in Breast Cancer Treatment 

Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration

We focus on exploring active compounds derived from natural products and their potential in treating breast cancer. So specifically we aim to elucidate the pharmacologic effects of salidroside and its molecular mechanisms in combining breast cancer. Currently, reliable experimental techniques are lacking to validate salidroside's molecular targets in treating breast cancer.One significant finding in the field is a potential molecular mechanism of salidroside in enhancing the proliferation and migration of MCF7 cells through the regulation of PI3K/AKT/HIF-1 alpha/FoxO1 signaling. The laboratory's future research directly is to explore the pathophysiological mechanisms underlying breast cancer onset, development, prognosis and outcome. Additionally, we screen for active small molecular drugs derived from natural sources that may add in breast cancer prevention and treatment.

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 &#181;M) 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...

Watch this Scientific Journal Video about Exploring Salidroside's Molecular Mechanisms in Breast Cancer Treatment at JoVE.com

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

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer ...

exploring-pharmacological-action-molecular-mechanism-salidroside