PRP-1 significantly decreases the [ALDH.sup.high] cancer stem cell population and regulates the aberrant Wnt/[beta]-catenin pathway in human chondrosarcoma JJ012 cells

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline-rich poly-peptide 1 (PRP-1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP-1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/[beta]-catenin signaling. A significant decrease in [ALDH.sup.high] CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP-1. For [RT.sup.2] profiler PCR array analysis of Wnt/[beta]-catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) [ALDH.sup.high] cells sorted from untreated JJ012 cells ([ALDH.sup.high-untreated]); and iii) [ALDH.sup.low] cells sorted from PRP-1-treated JJ012 cells ([ALDH.sup.low-PRP-1]). The expression levels of Wnt/[beta]-catenin signaling genes were determined to be downregulated in the [ALDH.sup.high-untreated] cells and upregulated in [ALDH.sup.low-PRP-1] cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be down-regulated in the [ALDH.sup.low-PRP-1] cells. Immunocytochemistry demonstrated the localization of [beta]-catenin in the nuclei of the PRP-1-treated cells. Western blotting indicated increased [beta] -catenin expression in the [ALDH.sup.low-PRP-1] cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP-1 and [beta]-catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of [beta]-catenin following PRP-1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP-1 resulted in a significant decrease in cytoplasmic [beta]-catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of [beta]-catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/[beta]-catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.Academi