flow cytometry analysis of apoptosis and the cell cycle in salidroside treated mcf-7 cells

Mécanisme du salidroside dans la suppression de la migration et de la prolifération des cellules MCF-7

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.

Pleins feux sur l’auteur : Explorer les mécanismes moléculaires du salidroside dans le traitement du cancer du sein 

Exploration de l’action pharmacologique et du mécanisme moléculaire du salidroside dans l’inhibition de la prolifération et de la migration des cellules MCF-7

Watch this Scientific Journal Video about Flow Cytometry Analysis of Apoptosis and the Cell Cycle in Salidroside Treated MCF-7 Cells at JoVE.com

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

Analyse par cytométrie en flux de l’apoptose et du cycle cellulaire dans les cellules MCF-7 traitées au salidroside

Pour commencer, récoltez les cellules MCF7 trypsinisées. Ensuite, colorez les cellules en suspension PBS avec un X dans cinq de l’ITC et de l’iodure de propidium pendant 20 minutes. Déterminez ensuite le nombre de cellules apoptotiques à l’aide d’un cytomètre en flux.Pour l’analyse du cycle cellulaire, mélangez les cellules remises en suspension avec une solution d’iodure de propidium dans un tube frais et incubez le mélange pendant 30 minutes. Détectez ensuite l’échantillon à l’aide d’un cytomètre en flux. Le salidroside a augmenté le nombre de cellules MCF7 aux stades précoces et tardifs de l’apoptose.Pendant ce temps, par rapport au groupe témoin, le traitement par salidroside a également fortement augmenté le nombre de cellules dans la phase G0 G1, tout en réduisant la proportion de cellules en phase S.

flow-cytometry-analysis-apoptosis-cell-cycle-salidroside-treated-mcf

Research

JoVE Journal

Medicine

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

184 vues. Pour commencer, récoltez les cellules MCF7 trypsinisées. Ensuite, colorez les cellules en suspension PBS avec un X dans cinq de l’ITC et de l’iodure de propidium pendant 20 minutes. Déterminez ensuite le nombre de cellules apoptotiques à l’aide d’un cytomètre en flux.Pour l’analyse du cycle cellulaire, mélangez les cellules remises en suspension avec une solution d’iodure de propidium dans un tube frais et incubez le mélange pendant 30 minutes. Détectez ensuite l’...

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

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.

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 ...

Recherche

Médecine

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

Évaluation de la migration et de l’invasion des cellules MCF-7 traitées par salidroside dans un test de grattage de plaie

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

Détermination de l’activité de la Na+-K+-ATPase et de laCa 2+-Mg2+-ATPase dans les cellules MCF-7 traitées au salidroside

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

Coloration par fluorescence DCFH-DA et Fluo-4 AM de cellules MCF-7 traitées au salidroside

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

Analyse de l’expression des protéines dans les cellules MCF-7 traitées au salidroside par Western Blot

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

Détermination de l’expression génique dans les cellules MCF-7 traitées au salidroside à l’aide de la qRT-PCR