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

Summary

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

Abstract

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-1α-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-1α-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-1α, 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-1α-FoxO1 pathway.

Introduction

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 nodes^1,2. 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 deaths

Protocol

The MCF-7 cells used for the present study were obtained from a commercial source (see the Table of Materials).

1. Cell culture

1. Culture the MCF-7 cells in a humidified 5% CO₂ atmosphere at 37 °C with DMEM containing 10% FBS and 1% penicillin (10,000 U/mL)/streptomycin (10,000 µg/mL) (see the Table of Materials).
   ​NOTE: The cells covering 90% of the bottom of the dish were employed for the experimen.......

Discussion

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 treatments^4,5. Promisingly, a large body of evidence has revealed the anti-cancer effects of Sal²⁴

Materials

Name	Company	Catalog Number	Comments
1% penicillin/streptomycin	HyClone	SV30010
AKT antibody	ImmunoWay Biotechnology Company	YT0185
Annexin V-FITC/PI kit	MultiSciences Biotech Co., Ltd.	AP101
Automatic microplate reader	Molecular Devices	SpectraMax iD5
Bax antibody	Cell Signaling Technology, Inc.	#5023
BCA kit	Biosharp Life Sciences	BL521A
Bcl-2 antibody	Cell Signaling Technology, Inc.	#15071
Bim antibody	Cell Signaling Technology, Inc.	#2933
Ca2+–ATPase assay kit	Nanjing Jiancheng Bioengineering Institute	A070-4-2
Cell counting kit-8	Biosharp Life Sciences	BS350B
Cell cycle staining kit	MultiSciences Biotech Co., Ltd.	CCS012
cleaved caspase-3	Cell Signaling Technology, Inc.	#9661
cleaved caspase-7	Cell Signaling Technology, Inc.	#8438
cleaved caspase-9	Cell Signaling Technology, Inc.	#20750
Crystal violet solution	Beyotime Biotechnology	C0121
DMEM high glucose culture medium	Servicebio Technology Co., Ltd.	G4510
Doxorubicin hydrochloride	MedChemExpress	HY-15142
ECL chemiluminescent solution	Biosharp Life Sciences	BL520B
Fetal bovine serum	Procell Life Science & Technology Co., Ltd.	164210
Flow cytometer	BD	FACSCanto
Fluo-4 AM	Beyotime Biotechnology	S1060
FoxO1 antibody	ImmunoWay Biotechnology Company	YT1758
Goat anti-rabbit IgG secondary antibody	MultiSciences Biotech Co., Ltd.	70-GAR0072
GraphPad Prism software	La Jolla	Version 6.0
HIF-1α antibody	Affinity Biosciences	BF8002
Human breast cancer cell line MCF-7	Procell Life Science & Technology Co., Ltd.	CL-0149
Loading buffer	Biosharp Life Sciences	BL502B
LY294002	MedChemExpress	HY-10108
Matrigel	Thermo	356234
mTOR antibody	Servicebio Technology Co., Ltd.	GB11405
Na+–K+–ATPase assay kit	Nanjing Jiancheng Bioengineering Institute	A070-2-2
Optical microscope	Olympus	IX71PH
p-AKT antibody	ImmunoWay Biotechnology Company	YP0006
PI3K antibody	Servicebio Technology Co., Ltd.	GB11525
p-PI3K antibody	Affinity Biosciences	AF3241
Quantitative western blot imaging system	Touch Image Pro	eBlot
Reverse transcription first strand cDNA synthesis kit	Servicebio Technology Co., Ltd.	G3330-100
ROS assay kit	Beyotime Biotechnology	S0033S	DCFH-DA fluorescence probe is included here
Salidroside	Chengdu Herbpurify Co., Ltd.	H-040
SDS-PAGE kit	Servicebio Technology Co., Ltd.	G2003-50T
Total RNA isolation kit	Foregene	RE-03014
Trypsin	HyClone	SH30042.01
β-actin	Affinity Biosciences	AF7018