Chelerythrine Inhibits Stemness of Cancer Stem-Like Cells of Osteosarcoma and PI3K/AKT/mTOR Signal

Abstract

Chelerythrine (CHE) is widely found in many herbs and is the main alkaloid constituent of Toddalia asiatica (L.) LAM. It has been proved to exert remarkable antitumor, antifungal, anti-inflammatory, and antiparasitic effects. In osteosarcoma, CHE is reported to inhibit proliferation and promote apoptosis. However, the effect of CHE on cancer stem-like cells (CSCs), which contribute to metastasis and recurrence in osteosarcoma, is still largely unknown. In this study, we investigated the effects of CHE on the stemness and malignant behaviors of CSCs derived from osteosarcoma cells. CSCs were enriched by culturing in serum-free medium. The effects of CHE on stemness were measured by detecting stemness factors and sphere formation ability. The effects of CHE on chemosensitivity to doxorubicin and MTX were measured by Annexin V-FITC/PI double staining. The effects of CHE on CSC malignancy were measured by performing CCK-8, colony formation, tumor formation in soft agar, migration, and invasion assays. We first enriched CSCs from osteosarcoma cells, which were characterized by upregulated stemness markers, including Oct4, Nanog, and Nestin. The addition of CHE clearly decreased malignant behaviors, including colony formation, tumor formation in soft agar, migration, and invasion. CHE also inhibited stemness and thus induced the failure of sphere formation. Moreover, CHE promoted apoptosis induced by chemo agents, including doxorubicin (DOX) and methotrexate (MTX). After CHE treatment, the protein expression of MMP-2/9 was significantly decreased, potentially inhibiting invasion. CHE also exhibited an inhibitory effect on the phosphorylation of PI3K, AKT, and mTOR, which is an upstream regulatory signaling pathway of MMP-2/9. In summary, CSCs derived from U2OS and MG-63 cells, CHE could inhibit the stemness and malignant behaviors of CSCs potentially by inhibiting the PI3K/AKT/mTOR signaling pathway.