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 experiment and assigned into the following groups: control group, doxorubicin hydrochloride group (DXR, 5 &#181;M), and Sal groups (20 &#181;M, 40 &#181;M, and 80 &#181;M), or control group, LY294002 (10 &#181;M, an inhibitor of PI3K) group, Sal (80 &#181;M) group, and LY294002 (10 &#181;M) + Sal (80 &#181;M) group (see the Table of Materials).</li>
</ol>
2. Cell viability assay
NOTE: For details on this procedure, please refer to a previous report27.
<ol>
	<li>Seed MCF-7 cells with a density of 8 x 104 cells/well in 96-well plates, and incubate with Sal (10 &#181;M, 20 &#181;M, 40 &#181;M, 80 &#181;M, 160 &#181;M, and 320 &#181;M) for 24 h or Sal (20, 40, 80 &#181;M) for 12 h/24 h/36 h/48 h overnight until the cells are adherent.</li>
	<li>After treatment, co-incubate the MCF-7 cells with 10 &#181;L/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.</li>
</ol>
3. Cell migration and invasion
NOTE: For details on this procedure, please refer to a previous report35.
<ol>
	<li>Incubate 2 mL of 5 x 106 cells/mL cells seeded in 6-well plates to cover 90% of the bottom of the dish.</li>
	<li>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.</li>
	<li>Use Image J software to measure the width of the scratch wounds.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Stain the MCF-7 cells with crystal violet solution (see the Table of Materials). Capture the images using an optical microscope.</li>
</ol>
4. Activity evaluation of Na+-K+-ATPase and Ca2+-Mg2+-ATPase
<ol>
	<li>Use a bicinchoninic acid (BCA) kit to measure the protein concentration in lysed MCF-7 cells, following the manufacturer&#39;s instructions (see the Table of Materials).
	<ol>
		<li>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 &#176;C for 5 min. Finally, measure the values of OD636 with a multifunctional microplate reader.</li>
	</ol>
	</li>
</ol>
5. Flow cytometry analysis of apoptosis and the cell cycle
NOTE: For details on this procedure, please refer to a previous report31.
<ol>
	<li>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.</li>
	<li>Mix the resuspended sample solution with PI solution for a 30 min incubation, followed by detecting with a flow cytometer.</li>
</ol>
6. DCFH-DA and Fluo-4 AM fluorescence staining
NOTE: For details on this procedure, please refer to a previous report29.
<ol>
	<li>Incubate the MCF-7 cells for 20 min with a 10 &#181;M DCFH-DA fluorescence probe (see the Table of Materials) at 37 &#176;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.</li>
	<li>Incubate the MCF-7 cells with a 5 &#181;M Fluo-4 AM fluorescent probe solution (see the Table of Materials) for 45 min at 37 &#176;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.</li>
</ol>
7. Western blot
<ol>
	<li>Add 2 mL of 5 x 106 cells/mL cell suspensions containing different drugs into 6-well plates, and culture in a 37 &#176;C, 5% CO2 incubator for 24 h. 
	NOTE: The groups for the western blot analysis were set as follows: control group, LY294002 (10 &#181;M) group, Sal (80 &#181;M) group, and LY294002 (10 &#181;M) + Sal (80 &#181;M) group (see the Table of Materials).</li>
	<li>Collect the cells and supernatant, and centrifuge at 560 &#215; g at 4 &#176;C for 3 min. Discard the supernatant, and wash twice with pre-cooled PBS. After each wash, centrifuge the cells at 560 &#215; g at 4 &#176;C for 3 min.</li>
	<li>Add 50 &#181;L lysis buffer to the cell sample from step 7.2 for a 15 min ice bath. Centrifuge at 8550 &#215; g at 4 &#176;C for 10 min to collect the supernatant protein sample.</li>
	<li>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 &#176;C for 10 min in a metal bath, and cool to room temperature.</li>
	<li>Add 20 &#181;g 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 &#181;m PVDF membranes. After blockage with 5% BSA, incubate the membranes with the corresponding primary antibodies overnight at 4 &#176;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-1&#945;, FoxO1, and &#946;-actin (see the Table of Materials).</li>
	<li>The next day, incubate the membranes with goat anti-rabbit IgG secondary antibody for 2 h at 37 &#176;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).</li>
</ol>
8. qRT-PCR
<ol>
	<li>Perform centrifugation to collect MCF-7 cells at 560 x g at 4 &#176;C for 3 min, and add 500 &#181;L buffer RL1 into 5 &#215; 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).</li>
	<li>Transfer the cell homogenates in step 8.1 to the DNA-cleaning column embedded in the collection tube, and centrifuge at 8,550 &#215; g at 4 &#176;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).</li>
	<li>Add 800 &#181;L buffer RL2 to 500 &#181;L 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.</li>
	<li>Transfer 700 &#181;L of the mixture from step 8.3 into the RNA-only column embedded in the collection tube, and centrifuge at 8,550 &#215; g at 4 &#176;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).</li>
	<li>Repeat step 8.4 to process the remaining mixture from step 8.3.</li>
	<li>Add 500 &#181;L of buffer RW1 to the RNA-only column, and centrifuge at 8,550 &#215; g at 4 &#176;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).</li>
	<li>Add 700 &#181;L of buffer RW2 to the RNA-only column, and centrifuge at 8,550 &#215; g at 4 &#9702;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).</li>
	<li>Place the RNA-only column back into the collection tube, and centrifuge at 8550 &#215; g at 4 &#176;C for 2 min to remove the remaining buffer RW2.</li>
	<li>Transfer the RNA-only column to a new collection tube, and drip 100 &#181;L of RNase-free ddH2O preheated at 65 &#176;C into the center of the membrane for the RNA-only column. Place at 25 &#176;C for 2 min, and collect the RNA solution by centrifugation at 8,550 &#215; g at 4 &#176;C for 1 min. 
	NOTE: RNase-free ddH2O is one of the components in the total RNA isolation kit (see the Table of Materials).</li>
	<li>Add 4 &#181;L of 5x reaction buffer, 1 &#181;L of oligo (dT)18 primer, 1 &#181;L of random hexamer primer, 1 &#181;L of gene-specific primer (see Table 1), 1 &#181;L of enzyme mix, and 5 &#181;g of the RNA solution from step 8.9 into a 100 &#181;L 8-tube strip. Supplement the reaction system with RNase-free water to 20 &#181;L.</li>
	<li>Set the reaction conditions of the system as follows: (1): 95 &#176;C, 30 s, 1 cycle; (2): 95 &#176;C, 5 s, 50 cycles, 60 &#176;C, 34 s; (3): 95 &#176;C, 5 s, 1 cycle, 65 &#176;C, 60 s, 97 &#176;C, 1 s; and (4): 42 &#176;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&#8722;&#916;&#916;CT method36,37. The primer information of each gene used for the qRT-PCR analysis is listed in Table 1.</li>
</ol>
9. Statistical analysis
<ol>
	<li>Express the data as the mean &#177; standard deviation of three independent experiments.</li>
	<li>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&#39;s test. In this work, P &#60; 0.05 was defined as statistically significant.</li>
</ol>

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

The authors have nothing to disclose.

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 ...

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Medicine

Evaluating the Migration and Invasion of Salidroside Treated MCF-7 Cells in a Wound Scratch Assay

To begin, incubate two milliliters of the cultured MCF-7 cells in a six-well plate until 90%confluency. In the grown cell monolayer, create a linear scratch wound down the center using a sterile pipette tip. Then capture the images with an optical microscope at different time intervals.For the transwell assay, suspend MCF-7 cells in a serum-free medium. Then see the cells in the upper transwell chamber, either pre-coded or non-coded with Matrigel. Add DMEM complete medium at the bottom of the transwell chamber to act as a chemical inducer.After 24 hours, remove the cells in the upper chamber and fix the remaining invasive and migrant cells using methanol. Stain the MCF-7 cells using crystal violet solution. Next, image the stained cells with an optical microscope.A dose dependent inhibitory effect of salidroside on cell proliferation was observed, with a 50%decline in cell vitality at 40 micromolar. Further, salidroside could inhibit the vitality of MCF-7 cells over time, with a 50%decrease in MCF-7 cell vitality after 24 hours of co-incubation. The wound-scratch assay showed an inhibitory effect for salidroside treatment on MCF-7 cells.Further, salidroside treatment significantly reduced the undesirable migration and invasion of MCF-7 cells.

Determining Na+-K+-ATPase and Ca2+-Mg2+-ATPase Activity in Salidroside Treated MCF-7 Cells

Using a bicinchoninic acid kit, measure the protein concentration of lysed MCF-7 cells following the manufacturer's instructions. Add the lysed cell samples into the corresponding groups in a 96-well plate. Then, add the working solution and incubate at 37 degrees Celsius for five minutes.Finally, measure the optical density values at 636 nanometers in a functional microplate reader. Salidroside restricted the enzyme vitalities of sodium potassium ATPases and calcium ATPase, indicating its potential role in promoting mitochondrial dysfunction.

Flow Cytometry Analysis of Apoptosis and the Cell Cycle in Salidroside Treated MCF-7 Cells

To begin, harvest the trypsinized MCF7 cells. Next, stain the PBS suspended cells with an X in five of ITC, and propidium iodide, for 20 minutes. Then determine the number of apoptotic cells using a flow cytometer.For cell cycle analysis, mix the re-suspended cells with propidium iodide solution in a fresh tube, and incubate the mixture for 30 minutes. Then detect the sample using a flow cytometer. Salidroside increased the number of MCF7 cells in the early and late apoptotic stages.Meanwhile, compared with the control group, salidroside treatment also sharply increased the number of cells in the G0 G1 phase, while reducing the proportion of S phase cells.

DCFH-DA and Fluo-4 AM Fluorescence Staining of Salidroside Treated MCF-7 Cells

Add 10 micromolar of Dcfh-DA fluorescence probe to MCF7 cells and incubate at 37 degrees Celsius for 20 minutes. After incubation, remove the excess Dcfh-DA by washing the cells three times with PBS. Next, test the fluorescence intensity using a fluorescence microscope and an excitation wavelength of 488 nanometers, and an emission wavelength of 525 nanometers.Incubate the MC7 cells with a five micromolar Fluo-4 AM fluorescent probe solution for 45 minutes at 37 degrees Celsius. After washing the cells three times with PBS, determine the fluorescence intensity using a fluorescence microscope with excitation at 488 nanometers and emission at 516 nanometers. Salidroside significantly enhanced ROS fluorescence signals shown by Dcfh-DA immunofluorescent staining.Consistently, Salidroside intervention also distinctly elevated calcium production, evidenced by Fluo-4 AM immunofluorescent staining.

Analyzing Protein Expression in Salidroside Treated MCF-7 Cells by Western Blotting

Begin by adding two milliliters of MCF-7 cells containing different drugs into the wells of a six-well plate. Incubate the cell cultures for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Harvest the cells by centrifugation at 560 x g for three minutes at four degrees Celsius.Then wash the cells twice with pre-cool PBS, centrifuging at 560 x g for three minutes between the washes. Add 50 microliters of lysis buffer to the washed cells and place the sample in an ice bath for 15 minutes. Then centrifuge the sample at 8, 550 x g for 10 minutes at four degrees Celsius, and collect the supernatant protein sample.Combine the protein sample with the loading buffer in a four to one ratio. Next, denature the mixture at 100 degrees Celsius for 10 minutes in a metal bath. Then cool the mixture at room temperature.Separate the protein sample using 10%SDS polyacrylamide gel electrophoresis. Transfer the proteins onto a 0.22 micrometer PVDF membrane. After blocking the membrane with 5%BSA, incubate it with the corresponding primary antibodies overnight at four degrees Celsius.The following day, incubate the membrane with goat anti-rabbit IgG secondary antibody at 37 degrees Celsius for two hours. Then develop the membranes using an ECL chemiluminescence solution and capture the images using a contactless quantitative Western blot imaging system. Western blot showed that salidroside treatment promoted the protein expression of the pro-apoptotic factors CC-9, CC-7, CC-3, Bim, and Bax while it inhibited the protein expression of anti-apoptotic BCL-2.Salidroside prominently limited the ratios of p-PI3K to PI3K and p-AKT to AKT. Meanwhile, the protein expression of mTOR, HIF-1 alpha, and Fox01 was also notably suppressed.

Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR

Harvest the MCF-7 cells by centrifugalization at 560-G for three minutes, at four degrees Celsius. Add 500 microliters of buffered RL-1 to five times 10 to the six cells and mix thoroughly until no cell masses are visible. Next, transfer the cell homogenates to a DNA cleaning column embedded in the collection tube.Centrifuge the samples at 85-50-G for two minutes, at four degrees Celsius. After centrifuging, remove the DNA cleaning column, retaining the supernatant in the collection tube. Add 800 microliters of buffer RL-2 and 500 microliters of the supernatant, and mix gently.Next, transfer 700 microliters of the mixture into an RNA-only column embedded in the collection tube. Centrifuge the tube at 8, 550-G for one minute, at four degrees Celsius. Then discard the flow-through in the collection tube.Wash the RNA-only column first with 500 milliliters of buffer RW-1, and then with 700 milliliters of buffer RW-2, by centrifuging for one minute at 8, 550-G, and four degrees Celsius at each wash. After removing the residual RW-2 by centrifuging again, transfer the RNA only column to a new collection tube. Add 100 microliters of RNAs-free deionized water, preheated at 65 degrees Celsius to the center of the membrane in the RNA-only column, and wait for two minutes.Then centrifuge at 8, 550-G for one minute, at four degrees Celsius to collect the RNA solution. Set up the reaction mixture for PCR and then set the PCR reaction conditions of the system. Execute the QRT PCR procedure.QRT PCR showed that cylindricity treatment promoted the gene expression of the pro-apoptotic factors, CC-9, CC-7, CC-3, vim, and vax, while it inhibited the gene expression of anti-apoptotic BCL-2. Further, cylindricide administration reduced the gene expression levels of PI-3K, AKT, M-tor, HIF1-Alpha and Fox-01.

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

Salidroside's Mechanism in Suppressing MCF-7 Cell Migration and Proliferation

Summary