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'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><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>Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;ndash;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 &amp;amp; Technology Co., Ltd.</td><td>164210</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>FACSCanto &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65634/65634eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</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&amp;alpha; 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 &amp;amp; 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&amp;nbsp;</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&lt;sup&gt;+&lt;/sup&gt;&amp;ndash;K&lt;sup&gt;+&lt;/sup&gt;&amp;ndash;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>&amp;beta;-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.

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

Author Spotlight: Exploring Salidroside's Molecular Mechanisms in Breast Cancer Treatment 

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

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 ...

exploring-pharmacological-action-molecular-mechanism-salidroside

Research

JoVE Journal

Medicine

1.5K Views.  Suining Central Hospital. We richten ons op het onderzoeken van actieve stoffen die zijn afgeleid van natuurlijke producten en hun potentieel bij de behandeling van borstkanker. Daarom willen we specifiek de farmacologische effecten van salidroside en zijn moleculaire mechanismen bij het combineren van borstkanker ophelderen. Momenteel ontbreken betrouwbare experimentele technieken om de moleculaire doelwitten van salidroside bij de behandeling van borstkanker te valideren.Een belangrijke bevinding in het veld ...

Video: Auteur in de schijnwerpers: onderzoek naar de moleculaire mechanismen van salidroside bij de behandeling van borstkanker 

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Cancer Research

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Bioengineering

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Biology

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...

Virtual Screening and Validation of UCHL3 Inhibitors Using Molecular Docking

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately involved in a myriad of physiological and pathological processes. Therefore, developing small molecule inhibitors targeting Ubiquitin C-Terminal Hydrolase L3 (UCHL3) is of great significance. This protocol aims to establish a process for virtual screening and in vitro validation of small molecule inhibitors of cysteine DUB represented by UCHL3. Firstly, potential inhibitors of UCHL3 are virtually screened using molecular docking technology, and the interaction between drugs and protein targets is visualized. Subsequently, the effectiveness of the screened drug, Danshensu, is verified through in vitro activity inhibition assays. Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and hemagglutinin-ubiquitin-vinyl sulfone (HA-Ub-VS) are used as probes for in vitro activity testing, as they can competitively bind to DUB with small molecule inhibitors to assess the activity of UCHL3. The results indicate that Danshensu has a good binding affinity with UCHL3 in molecular docking, and it can competitively inhibit the activity of UCHL3 with HA-Ub-VS. These findings provide important references for further research and development of therapeutic drugs targeting UCHL3.

The experiment used here shows a method of molecular docking combined with probe technologies to predict and validate the interaction between small molecules of traditional Chinese medicine and protein targets.

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately ...

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

Samenvatting

Meer video's verkennen