Name: Analyzing Protein Expression in Salidroside Treated MCF-7 Cells by Western Blotting
Uploaded: 2023-06-09T12:20:00
Description: Watch this Scientific Journal Video about Analyzing Protein Expression in Salidroside Treated MCF-7 Cells by Western Blotting at JoVE.com

analyzing protein expression in salidroside treated mcf-7 cells by western blotting

Salidroside'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'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 Analyzing Protein Expression in Salidroside Treated MCF-7 Cells by Western Blotting at JoVE.com

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.

Research

JoVE Journal

Medicine

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