Effects of Icaritin on the physiological activities of esophageal cancer stem cells

https://doi.org/10.1016/j.bbrc.2018.08.060Get rights and content

Highlights

  • CSCs were used instead of using cancer cells.

  • The effect of Icaritin on the Hedgehog and Wnt signaling pathway was investigated.

  • Our study found that Icaritin down-regulated the level of Hedgehog, Smo and Gli in Hedgehog pathway, up-regulated GSK3β and down-regulated Wnt and β-catenin in Wnt pathway, indicating that Icaritin may inhibit the activity of esophageal CSCs through regulating the expression of proteins in important pathways related to esophageal cancer.

Abstract

Icaritin is a compound extracted from herb, recent study have found it is able to influence the activity of various types of cancer. Our aim was to investigate the effects of Icaritin on the physiological activities of esophageal cancer stem cells (CSCs). In this study, esophageal cancer cells were cultured and CD133 positive esophageal CSCs were sorted by flow cytometry. Changes in the physiological activity of esophageal CSCs following treatment with different concentrations of Icaritin (0, 12.5, 25, 50, and 100 μmol/L) were evaluated. The CCK-8 method and Transwell assay were used to determine the effects of Icaritin on the proliferation, migration, and invasion of esophageal CSCs. Flow cytometery was used to investigate its effect on the apoptosis of CSCs. The effect of Icaritin on the expression of proteins in Wnt and Hedgehog signaling pathways were determined using western blot test. Consequently, Icaritin inhibited the proliferation, migration, and invasion of esophageal CSCs in a dose-dependent manner. It promoted cell apoptosis, and influenced the levels of proteins in Wnt and Hedgehog signaling pathways. It may act as a promising drug in the therapy of esophageal cancer.

Introduction

Esophageal cancer refers to malignancies that occur in the esophageal epithelium. Based on its histology, esophageal cancer can be divided into esophageal adenocarcinomas and esophageal squamous cell carcinomas. According to the World Health Organization, esophageal cancer is a common malignancy whose incidence ranks the eighth highest among malignant tumors, accounting for 3.2% of all cancer cases worldwide in 2012 [1]. The outcomes of patients with esophageal cancer are often poor, and the mortality due to esophageal cancer ranks the sixth highest among all malignant tumors [2]. The onset of esophageal cancer is insidious with a lack of early symptoms, numerous patients are therefore diagnosed in the middle and late stages of the disease and require chemoradiotherapy [3]. Cancer stem cells (CSCs) are a part of cells in tumor tissue, they are responsible for tumor initiation and play critical roles in the persistence, development and migration of tumor tissue [4]. Icaritin is an alkaloid extracted from Herba epimedii, which has antioxidant, immunoregulatory, anti-arteriosclerosis, and anti-angiogenic effects [5]. Studies have shown that it has inhibitory effect on hepatocellular carcinoma, breast cancer, ovarian cancer and other cancers [[5], [6], [7]]. However, no studies have investigated its role and possible mechanismin esophageal cancer. Therefore, in this study, we aimed to provide novel insight into the potential treatments for esophageal cancer by exploring the effects of Icaritin on the physiological activities of esophageal cancer stem cells.

Section snippets

Cell line

Esophageal cancer cell line ECA109 were stored in the central laboratory of Gansu Provincial People's Hospital.

Reagent and equipment

Icaritin was purchased from Sigma-Aldrich company (St. Louis, USA). RPMI-1640 medium and fetal bovine serum (FBS) were bought from Gibco Company (Gaithersburg, USA). Transwell chambers were purchased from Corning Company (New York, USA), and reverse transcription kits were purchased from Takara Company (Ostsu, Japan).

Cell sorting and culture

Cells were subcultured in RPMI-1640 complete medium containing 10%

Selection of esophageal cancer stem cells

Stem cells were sorted by flow cytometry, CD133 positive cells were selected. Results are shown in Fig. 1.

Icaritin inhibited the proliferation of cancer stem cells

The CCK8 method was used to examine the inhibitory effects of Icaritin on the proliferation of stem cells treated with different concentrations of Icaritin (0, 12.5, 25, 50, and 100 μmol/L). The results showed that Icaritin significantly inhibited the in vitro proliferation of stem cells. Cell survival decreased with increasing Icaritin concentrations (Fig. 2A).

Icaritin inhibited the migration and invasion of cancer stem cells

The Transwell method was used

Discussion

The results of our experiment showed that Icaritin inhibited the physiological activities of esophageal CSCs. Following treatment with different concentrations of Icaritin, the proliferation, migration, and invasion of esophageal CSCs were inhibited and apoptosis of these stem cells was promoted. In addition, Icaritin down-regulated the level of Hedgehog, Smo and Gli in Hedgehog pathway, up-regulated GSK3β and down-regulated Wnt and β-catenin in Wnt pathway.

The CSC hypothesis refers to the

References (20)

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These authors contributed equally.

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