Elsevier

Gynecologic Oncology

Volume 129, Issue 3, June 2013, Pages 598-605
Gynecologic Oncology

The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells

https://doi.org/10.1016/j.ygyno.2013.03.005Get rights and content

Highlights

  • Salinomycin reduced the level of fibronectin expression, which was enhanced in endometrial cancer stem cells.

  • Salinomycin induced apoptosis and inhibited Wnt signaling.

  • Salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of endometrial cancer stem cells.

Abstract

Goals

We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial–mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells.

Method

We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin.

Results

We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells.

Conclusion

This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Introduction

Stem cells have a marked self-renewal capacity and are pluripotent. They can be identified in embryonic tissues as well as in normal adult tissues. The existence of cancer stem-like cells (CSCs) has been proposed and CSCs have been identified in leukemia and several solid tumors [1], [2], [3]. The properties of CSCs are as follows: i) they possess self-renewal capacity, ii) they can produce progeny cells, iii) they constitute a small minority of neoplastic cells within a tumor, and iv) they possess the developmental potential for expression of multiple specific markers [4]. CSCs are resistant to current cancer treatment, resulting in an increased risk of recurrence.

Side-population (SP) cells are enriched in stem cells and have been isolated and characterized, using fluorescence-activated cell sorting (FACS). The methodology is based on the cells' ability to reduce the intracellular concentration of the fluorescent dye Hoechst 33342 [5]. The identification of SP cells is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/BCRP1. The ATP-binding-cassette (ABC) transporters represent the largest family of transmembrane proteins capable of exporting a wide variety of molecules and structurally unrelated chemotherapeutic drugs from the cytosol. These proteins confer multidrug resistance to CSCs [6], [7].

We isolated and characterized SP cells present in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells). The SP cells showed reduced expression levels of differentiation markers, long-term proliferative capacity of the cell cultures, self-renewal capacity, enhanced tumorigenicity, and enhanced migration. These findings demonstrate that SP cells have features of CSCs, including the potential to differentiate into the mesenchymal cell lineage [8].

The epithelial–mesenchymal transition (EMT) occurs during normal early embryonic development. This program allows a polarized epithelial cell, which normally interacts with the basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity and invasiveness [9]. EMT is also a key developmental program that is often activated during cancer invasion and metastasis. The EMT program enables cancer cells to disseminate from a primary tumor by losing epithelial characteristics and acquiring a mesenchymal phenotype. Mani et al. have reported a direct link between EMT and the gain of epithelial stem cell properties [10]. Human breast cancer stem cells express Snail and Twist, EMT-inducing transcription factors. When their expression is elevated, the size of the stem cell population is increased and expression levels of mesenchymal markers fibronectin and vimentin are enhanced [10]. Thus, EMT promotes the generation of cancer stem cells.

Salinomycin is a selective inhibitor of CSCs [11]. Salinomycin is an antibacterial and coccidiostatic therapeutic drug. It has been shown to kill breast cancer stem cells in mice at least 100-times more effectively than the commonly used anti-cancer drug paclitaxel. Although several studies demonstrate that salinomycin has an inhibitory effect on proliferation of CSCs, the effects of salinomycin on the properties of endometrial CSCs are unclear.

In this study, we used Hec1-SP cells and RK12V-SP cells to analyze the association of EMT with the properties of endometrial CSCs and the effects of salinomycin on the proliferative capacity, migration and invasiveness of endometrial CSCs.

Section snippets

Cell lines and cell culture

An endometrial cancer cell line (Hec-1) and a rat endometrial cell line (RENT4) were used in the present study. The Hec-1 cell line was established by Kuramoto et al. from explants of adenocarcinoma of human endometrium [12] and it was a gift from Dr. Kuramoto. RENT4 cells were established by Wiehle et al. [13] and obtained from the European Collection of Cell Cultures (ECACC). Both cell lines were authenticated by Takara Bio Inc. using the short tandem repeat (STR) DNA profiling. The STR

EMT: an important characteristic of endometrial CSCs

We previously demonstrated that both Hec1-SP cells and RK12V-SP cells have features of CSCs [8], [14]. We found that the quality of mRNA derived from RK12V-SP cells isolated by flow cytometry was better than that from Hec1-SP cells. Thus, we performed microarray expression analysis to screen for up-regulated genes in CSCs on a set of RK12V-SP cells and -non-SP (NSP) cells. We identified 450 genes that were up-regulated more than two-fold in RK12V-SP cells compared with those in RK12V-NSP cells.

Discussion

Previous studies have shown that CSCs are resistant to chemo- or radio-therapy [7], [16], [17], [18]. Current cancer treatment may fail to eliminate CSCs and surviving CSCs can regenerate new tumors, leading to relapse and metastasis. Mani et al. showed that EMT in normal or neoplastic mammary epithelial cell populations results in the enrichment of stem-like cells [10]. Gupta et al. demonstrated that normal and cancer cell populations engaged in the EMT process exhibit increased resistance to

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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