Cancer Letters

Cancer Letters

Volume 311, Issue 1, 1 December 2011, Pages 113-121
Cancer Letters

Salinomycin inhibits osteosarcoma by targeting its tumor stem cells

https://doi.org/10.1016/j.canlet.2011.07.016Get rights and content

Abstract

Osteosarcoma is the most common primary bone tumor in children and adolescents and is typically associated with a poor prognosis. Tumor stem cells (TSCs) are presumed to drive tumor initiation and tumor relapse or metastasis. Hence, the poor prognosis of osteosarcoma likely results from a failure to target the osteosarcoma stem cells. Here, we have utilized three different methods to enrich TSCs in osteosarcoma and further evaluated whether salinomycin could selectively target TSCs in osteosarcoma. Our results indicated that sarcosphere selection, chemotherapy selection and stem cell marker OCT4 or SOX2 over-expression are all effective in the enrichment of TSCs from osteosarcoma cell lines. Further investigation found that salinomycin inhibited osteosarcoma by selectively targeting its stem cells both in vitro and in vivo without severe side effects, and the Wnt/β-catenin signaling pathway may be involved in this inhibition of salinomycin. Taken together, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells, supporting the use of salinomycin for elimination of osteosarcoma stem cells and implying a need for further clinical evaluation.

Highlights

► We have utilized three different methods to enrich TSCs in osteosarcoma. ► Salinomycin could selectively target TSCs in osteosarcoma both in vitro and in vivo. ► Wnt/β-catenin signaling pathway may be involved in this inhibition of salinomycin. ► Salinomycin is an effective inhibitor of osteosarcoma stem cells.

Introduction

Osteosarcoma (OS) is the most common primary bone tumor, mainly affecting children and adolescents [1]. The standard treatment for patients with osteosarcoma is neoadjuvant chemotherapy, followed by surgical resection and post-operative chemotherapy. Despite aggressive chemotherapy and improved surgery, the 5-year survival rate remains at 60–70% for localized disease and only 20% for metastatic disease. Therapeutic failure is mainly due to tumor recurrence or lung metastasis [2], [3], which probably results from the inability to eliminate the residual tumor cells after chemotherapy and surgery [4], [5]. This dismal situation for osteosarcoma patients necessitates further understanding of the biological features of osteosarcoma and also calls for novel approaches to eliminate the therapy-resistant tumor cells [5], [6].

Recent studies indicate that solid tumors of different origins, including osteosarcoma, are driven by a population of tumor stem cells (TSCs) or tumor initiating cells (TICs) [7], [8]. It is believed that TSCs fuel tumor growth and seed metastasis. After conventional chemotherapy targets proliferating tumor cells, it is common to find tumor regression. However, some patients with osteosarcoma will develop a tumor relapse or metastasis after chemotherapy and surgery. This clinical clue leads us to presume that current approaches are not efficient to target TSCs, according to the concept of TSCs [5], [9].

Although osteosarcoma stem cells can be experimentally enriched by several methods [5], drugs or compounds that selectively target TSCs in osteosarcoma have not been explored thus far. Recently, salinomycin has been identified as a selective inhibitor of breast stem cells [10] and leukemia stem cells [11], but its role in the inhibition of TSCs of osteosarcoma remains to be determined. In this study, we successfully utilized three different methods to enrich osteosarcoma stem cells, and our results suggested that salinomycin was able to target osteosarcoma stem cells both in vitro and in vivo.

Section snippets

Reagents and antibodies

Methotrexate (MTX), adriamycin (ADM), cisplatin (DDP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and salinomycin were purchased from Sigma (St. Louis, MO). Antibodies against OCT4, Sox2, GSK-3β, phosphorylated-GSK-3β (Ser 9) and β-catenin were from Cell Signaling Technology (Danvers, MA). Antibodies against Cyclin D1, GAPDH and HSP70 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells and culture conditions

The human osteosarcoma cell lines (U2OS, MG63, SAOS2) were gifts from Dr. M. Serra

TSCs can be enriched by sarcosphere selection

An important feature of TSCs is that they have the capacity to form spheres under serum-free conditions. Therefore, we speculated that sphere selection should be an effective approach to enrich TSCs. Our results show that sarcosphere formation could be observed in all the osteosarcoma cell lines, including the two commercial cell lines U2OS and MG63, as well as the primary cell culture line, ZOS (Fig. 1A). Next, we performed MTT assay, cell counting and caspase-3 activity assay, the results

Discussion

Accumulating evidence supports the TSC theory, which hypothesizes that tumors of various origins are driven and sustained by a small proportion of TSCs [19]. Recent studies show that tumor stem cells, including osteosarcoma stem cells, are responsible for tumor chemoresistance, relapse and metastasis [16], [20], [21]. Current chemodrugs for the treatment of osteosarcoma mainly target proliferating tumor cells, which can dramatically reduce tumor bulk but have minimal cytotoxicity on

Conclusion

In the present study, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells both in vitro and in vivo without severe side effects, at least partially through Wnt/β-catenin self-renewal pathway. Our study supports the use of salinomycin for elimination of osteosarcoma stem cells and implies a need for further clinical evaluation.

Conflicts of interest

None declared.

Authorship contribution

Conceived and designed the experiments: Jin Wang, Tiebang Kang. Performed the experiments: Qing-Lian Tang, Zhi-Qiang Zhao, Jin-chun Li, Yi Liang. Analyzed the data: Jun-Qiang Yin, Xian-Biao Xie, Chang-Ye Zou. Wrote the manuscript: Qing-Lian Tang, Yi Liang. Provided discussion and revision of critically important Intellectual content: Yi-Xin Zeng, Jing-Nan Shen.

Acknowledgments

We thank Duanqing Pei (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China) for kindly providing the OCT4/GFP and SOX2/GFP plasmids and the members of the laboratory for their helpful comments on the manuscript. This work was supported by grants from National Nature Science Foundation of China (Nos. 30872967 and 81072193 to Jin Wang, No. 30930045 to Tiebang Kang), from the 973 project (No. 2010CB912201 to Tiebang Kang) and from Nature Science Foundation of

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