determining gene expression in salidroside treated mcf-7 cells using qrt-pcr

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

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.

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.

Watch this Scientific Journal Video about Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR at JoVE.com

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.

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71 Views.  Suining Central Hospital. 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 tu...