Human PROM1 (Prominin-1) ELISA Kit

CD133 (PROM1) is a potential marker for cancer stem cells (CSCs), including those found in brain tumors. Recently, medulloblastoma (MB)-derived CD133-positive cells were found to have CSC-like properties and were proposed to be important contributors to tumorigenicity, cancer progression, and chemoradioresistance. However, the biomolecular pathways and therapeutic targets specific to MB-derived CSCs remain unresolved. In the present study, we isolated CD133(+) cells from MB cell lines and determined that they showed increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared to CD133(-) cells. Bioinformatics analysis suggested that the STAT3 pathway might be important in MB and CD133(+) cells. To evaluate the effects of inhibiting the STAT3 pathway, MB-derived CD133(+/-) cells were treated with the potent STAT3 inhibitor, cucurbitacin I. Treatment with cucurbitacin I significantly suppressed the CSC-like properties and stemness gene signature of MB-derived CD133(+) cells. Furthermore, cucurbitacin I treatment increased the apoptotic sensitivity of MB-derived CD133(+) cells to radiation and chemotherapeutic drugs. Notably, cucurbitacin I demonstrated synergistic effects with ionizing radiation to inhibit tumorigenicity in MB-CD133(+)-inoculated mice. These results indicate that the STAT3 pathway plays a key role in mediating CSC properties in MB-derived CD133(+) cells. Targeting STAT3 with cucurbitacin I may therefore represent a novel therapeutic approach for treating malignant brain tumors.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.