| Background:Glioma is the most common primary malignant tumor of the nervous system. Traditional treatment includes resection and radiation therapy, but as the high invasiveness of the tumor cells, the postoperative recurrence rate is very high. With more studies in stem cells, people have found out that only a relatively small fraction of tumor cells, termed cancer stem cells (CSCs), possess the stem cell-like characteristics, such as the ability to self-renew, unlimited proliferation, and the potentiality of multi-directional differentiation. In recent years, researchers have successfully isolated brain glioma stem cells(BGSCs) from the glioma tissues and cell lines, and considered them playing a decisive role in glioma's occurrence, development and recurrence. Because of the BGSCs'theory, we have a new perspective about gliomas'occurrence and development mechanisms. Also it offers the hope for promoting a new type of molecular targeted therapy.Hedgehog-Gli (Hh-Gli or HH) signaling pathway is composed by the HH protein, membrane receptors, nuclear transcription factor Gli, and downstream target genes. In the development of nervous system, HH signaling pathway plays a key role on neural stem cells. Recently, with the deep study, we found that the anomaly of HH signaling pathway has a close relationship with human tumorigenesis. The abnormal activation of HH signaling pathway has been found in many kinds of tumors. Using the specific inhibitors block the HH signaling pathway can inhibit tumor growth. Some research in the brain tumor showed that HH signaling pathway may be involved in glioma's start. At present, brain tumor stem cells might be considered to originate in mutational neural stem cells, so HH signaling pathway may also play an important role in the proliferation and differentiation of BGSCs.Current studies of specific inhibitors mainly focus on a plant steroidal alkaloid-Cyclopamine, which blocks the activity of HH signaling pathway by antagonizing the membrane receptor Smo. It promotes glioma cell apoptosis and inhibits the growth of xenograft tumor both in vitro and ex vivo tests. However, theoretically, blocking pathway activity at the most downstream Gli transcription factor is the best choice for many Gli-dependent tumors, and has a broader application.Objectives:This study aims to separate and identify the BGSCs in the human glioma cell line U87-MG. Gli-1 is the marker of HH signaling pathway activation. We tested its expression in BGSCs'proliferation, differentiation and normal brain tissues. By using GANT-61, the specific inhibitor of Gli-1, to block the pathway in differentiation of BGSCs, we can identify its influence on the pathway's activity, apoptosis and tumorigenicity.Methods:The BGSCs were isolated from human glioma cell line U87-MG by magnetic activated cell sorting, and then cultured these cells in serum-free medium. We identified the characteristics of BGSCs by testing the expression of CD 133+through flow cytometer, Single-cell clonal culture and xenograft tumor formation in nude mice. We got the expression of Gli-1 in BGSCs'proliferation, differentiation and normal brain tissues by RT-PCR and Western Blot.Using MTT assay, we studied the influence of different GANT-61 concentrations on cytoactive, and identified its effective concentration. We used GANT-61 to affect the BGSCs, which cultured in serum-containing medium. DMSO was added to the control group. We compared the changed expression of Gli-1 with anti-apoptosis gene Bcl-2 by real-time PCR and Western Blot. Flow cytometer was used to test the drug's influence on cell cycle and apoptosis as well as to identify whether the tumorigenicity in nude mice was lost or not.Results:The isolated glioma cells, with a high expression of the stem cells'marker CD133+, can form floating cloning-sphere in serum-free medium, and are consistent with the ultrastructural characteristics of stem cells. After transplanting these cells to nude mice, we discovered that they can form xenograft tumor with typical characteristics of glioma cells. There was a high expression of Gli-1 in BGSCs'proliferation. The differentiation of BGSCs had a lower expression of Gli-1, but still significantly higher than normal brain tissues.We used GANT-61 in differentiation of BGSCs, which has abnormally high expression of Gli-1. It reached the maximum inhibition after 48h administration, and the IC50 was about 10uM. After adding GANT-61 to block HH signaling pathway in differentiation of BGSCs, the transcriptional activity of Gli-1 was visibly inhibited; the expression of Gli-1 and Bcl-2 was reduced; the cell activity was inhibited; the number of viable apoptotic cells significantly increased. The treated cells lost tumorigenicity. The control group had no marked changes. The differences between groups were significant.Conclusions:There was a small part of human glioma cells, termed BGSCs, possess the ability of self-renew, unlimited proliferation, and the potentiality of multi-directional differentiation. HH signaling pathway has abnormal activation both in proliferation and differentiation of BGSCs, more obvious in proliferation. In differentiation of BGSCs, GANT-61 can block HH pathway through the inhibition of Gli-1 transcriptional activity. In differentiation of BGSCs, GANT-61 can induce apoptosis significantly. It might be related with its effect on cell cycle factor Cylin D2 and anti-apoptotic factor Bcl-2.
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