Targeting Therapy Using A Small Molecule Antagonist Against Hedgehog Signal Pathway In Human Malignant Glioma

[Background]Glioma is the most common malignancy in brain. Malignant glioma,which stands for WHO gradeⅢ-Ⅳ, is always associated with rapid progression, quick recurrence and poor prognosis. Currently radiotherapy and chemotherapy combined with surgical resection is the classic therapeutic strategy. However, due to inevitable relapse, the overall prognosis of patients with malignant glioma remains poor. Up to now,the effect of adjuvant chemotherapy is so limited, mainly because of the non-specificity, cytotoxicity of the chemical agents and the drug resistance of the tumor cells. Constitutive activation of some signal pathways has recently been implicated in the growth of a number of human malignancies. As a result, therapies which are targeting on such signal pathways, key genes and regulatory elements may have a good prospect. The cancer stem cell hypothesis implies that a small fraction of cells, called cancer stem cells (CSCs), present the stem cell ability such as indefinite proliferation, extensive self-renewal, and multi-linkage differentiation. They are the sources of tumor initiation, metastasis and relapse. The CD133 positive cells have been identified as brain tumor stem cells (BTSCs) in glioma. They display strong capability of drug resistance, anti-apoptosis, highly invasive thus represent novel targets for therapeutics. As BTSCs may originate from the transformation of normal neural stem cells (NSCs). similar signalling pathways, such as Hedrehog (Hh), Wnt and Notch pathways, may regulate self-renewal, proliferation and differenation in NSCs as well as in BTSCs. It has become clear that constitutive Hh signalling is an etiologic factor in many malignancies and application of specific Hh-pathway inhibitors has proven effective in preventing growth of many of these tumours in vitro and in vivo. However, the mechanism is not completely known. A detailed understanding of the role of Hh in the control of BTSCs behavior is critical for future therapeutic strategies.Current strategies focus on the use of small molecule inhibitors of Smo, such as cyclopamine et al. However, the approach is limited because only tumors where pathway activation has occurred at the level of Smo or upstream can be treated. Tumors with inactivating mutations in SuFu or other downstream components will not respond to these antagonists. In that case, downstream Hh pathway blocker is needed and antagonists against GLI transcription factors would be a better choice.[Method]Part 1:To identify whether Hh signaling pathway is active in glioma cell line or primary glioma cells. Rt-PCR and cytoimmunofluorescence were used to detect the Gli1 level in all of the samples. Real-time PCR and western blot were used to determine the influence of a GLI transcription antagonist GANT61 on Glil mRNA and Ptchl protein respectively. Then in order to determine whether GANT61 could inhibit glioma cell viability in a Gli1 level dependent way. MTS trials were performed in all 7 samples. Finally, cell cycle test and Annexin V/PI double staining were used to explore the mechanism leading to cell viability inhibition.Part 2:CD133 positive cells were isolated through MACS. These cells were verified as BTSCs using Single-cell clonal culture, cytoimmunofluorescence staining and xenograft tumor formation in nude mice. The Glil mRNA level was evaluated in both CD133 positive and CD133 negative subgroup using real-time PCR. After being cultured with GANT61 the Gli1 mRNA level. CD133 positive rate, the capacity of self-renewal and in vivo tumorigenecity were re-determinated to find out whether BTSCs can be depleted in vitro.Part 3:in order to evaluate the effect of GANT61 against glioma in vivo the drug was injected subcutaneously every other day for 2 weeks after establishment of xenograft tumors. Tumor volumes were calculated and BRDU as well as TUNEL staining were performed on the tissue section after remove of tumor. Finally, secondary tumorsphere formation ability and in vivo tumorigenecity were determined to evaluate whether all of the BTSCs have been depleded after treatment in vivo.[Results:]Seven samples were included in the experiment with 3 belonging to WHO gradeⅢand 4 belonging to WHO gradeⅣ. The Gli1 level was highly expressed in 2 of the primary glioma cells and 1 of the glioma cell lines. However. Gli1 level was not correlated with tumor grade.The Glil mRNA level and Ptchl protein level of the glioma cells were significantly decreased 48h after 5μM GANT61 administration. It suggested that Gli-mediated transcription in glioma cells was inhibited by GANT61 in vitro.Treatment of GANT61 inhibited glioma cell viability in a time and dose dependent way. The IC50 was about 10μM and the maxmal effect exhibited 48h after administration. The proportion of cells in sub-G1 and G0/G1 phase both increased significantly 48 h after 10μM GANT61 administration. It implied the agent suppressed glioma cell proliferation by means of cell-cycle arrest as well as increasing cell death.The proportion of early stage apoptosis increased significantly 48 h after 10μM GANT61 administration.CD133 positive cells were isolated through MACS. These cells were characterized as BTSCs because they hold the capacity of self-renewal. multi-linkage differentiation and in vivo tumorigenecity.The Gli1 mRNA level of the CD133 positive group was significantly higher than that of the CD133 negtive group. Treatment of 10μM GANT61 for 48h can significantly suppress the Gli1 mRNA.Neither 10μM GANT61 for 2 days nor 20μM GANT61 for 7 days could increase the proportion of early stage apoptosis in CD133 positive cells. It implies that inhibit Hh pathway only may not be enough to deprive the high capacity of anti-apoptosis in BTSCs.After blocking of Hh signaling for 7 days in vitro the CD133 positive rate as well as capacity of Single-cell tumorsphere formation and in vivo tumorigenecity of BTSCs dropped dramaticly. It implied that the self-renewal capacity of BTSCs may have been depleted by inhibition of Gli1 transcription in vitro.Treatment with GANT61 resulted in the inhibition of xenograft tumor growth and limited the increase in tumor size.The Ptchl mRNA level and protein level of the xenograft tumor were significantly suppressed after GANT61 administration. It suggested that Gli1-mediated transcription in glioma cells was inhibited by GANT61 in vivo.The TUNEL staining positive rate in GANT61-treated samples were significantly higher than that of the control while the BRDU positive rate was much lower. This result might indicate that GLI transcription inhibition favors induction of apoptosis and suppress tumor proliferation in vivo.Secondary tumorspheres originating from xenograft tumor of the treatment group can be found in culture favoring stem cell. It implies that despite the tumor shrinking some BTSCs can still survive after GANT61 administration in vivo.Conclusion1. Hh pathway was activated in a subset of malignant glioma including primary glioma samples and glioma cell lines. However,Gli1 level was not correlated with tumor grade. 2. Treatment of GANT61 inhibited glioma cell viability in a time and dose dependent way. This agent suppressed glioma cell proliferation by means of G1/S cell-cycle arrest as well as increasing cell apoptosis in vitro.3. Hh pathway activity was much higher in BTSCs than in CD133 negtive glioma cells. The self-renewal capacity and in vivo tumorigenecity but not anti-apoptosis capacity could be abolished by treatment of GANT61 in vitro.4. Subcutaneously administration of GANT61 could decrease glioma cell proliferation and increase apoptosis of xenograft tumor in nude mice. However, some BTSCs can still survive after treatment.