| Chapter OneA study of the Hedgehog/Gli1 pathway in chemotherapy resistance of gliomasObjectives:This study aimed at investigating roles of the Hedgehog/Gli1 pathway in human primitive and recurrent gliomas and preliminarily exploring mechanisms which affected chemotherapy.Methods:(1)Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of SHH and Gli1 protein using immunohistochemistry and explored relationship between the Hedgehog/Gli1 pathway and glioma recurrence.(2)Received the four glioma cell lines including U87, U251, SHG44, A172 and analyzed the active level of their Hedgehog/Gli1 pathway.(3)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could promote resistance of tumor cells to chemotherapeutic agents.(4) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Gli1 pathway in U87, SHG44, U251, and A172 cells, explored whether this pathway repressed could promote sensitivity of tumor cells to chemotherapeutic agents.(5)Explored roles of the Hedgehog/Gli1 pathway regulating MDR-related genes including MDR1 and MRP1 etc. (6)To confirm that effects of Cyclopamine was achieved through Hedgehog/Glil pathway regulating MDR-related genes, we performed LV-shGlil conduction in Glil level in U87, SHG44, and U251.Results:(1)In the primary glioma group,20 samples had Glil expression, and 23 samples had Glil expression in the in recurrent glioma group. These 43 samples belonged to 23 patients, every patient had Glil expression in the recurrent glioma group, and 3 didn't have Glil expression in the primary glioma group. Glil-positive rate in the recurrent group (n=23) was 48.9±20.3%, and also Glil-positive rate in the primary group (n=23) was 34.4±19.8%. There was significant difference between these two groups (P<0.01). Moreover, there was significant difference of Glil expression between the SHH-positive and the SHH-negative group (P<0.01).(2)Among the four lines, Glil was more highly expressed in U87 and SHG44 cells. In U251 lines, Glil expression was modest. Glil expression in A172 cells was quite low. Baseline levels of Glil expression in A172 and U251 were relatively low.(3) Activation of the Hedgehog/Gli1 pathway supported resistance of U251 and A172 cells to chemotherapeutic agents.(4)Repression of Glil expression promoted the response of U87, SHG44, and U251 cells to chemotherapeutic agents and contributed to a further increase in cell apoptosis.(5)The effects of LV-shGli1 and Cyclopamine on the expression of downstream MDR-related genes in glioma cells with the Hedgehog/Glil pathway activityConclusions:(1)There is the existence of Hedgehog/Glil pathway activated in a subset of gliomas, and overactivation of the Hedgehog/Gli1 pathway correlates with glioma recurrence.(2)Elevated active levels of the Hedgehog/Gli1 pathway promote resistance of glioma cells to chemotherapeutic agents. Conversely, inhibiting this pathway may enhance the effects of chemotherapeutic agents on glioma cell death.(3)There is certain correlation between activation of the Hedgehog/Gli1 pathway and expression of MDR-related genes including MDR1,MRP1,LRP,MGMT,Bcl-2,Survivin in gliomas. Blockade of the Hedgehog/Glil pathway can downregulate the expressions of MDR1, MRP1, LRP, MGMT, Bcl-2 and Survivin genes.(4)Interfering the Hedgehog/Gli1 pathway in combination with current chemotherapy regimens may provide a promising approach to improving chemotherapeutic response. Chapter TwoA study of the Hedgehog/Glil pathway regulating expression of the angiogenesis-related genes in gliomasObjectives:This study aimed at investigating relationship between the aberrant active Hedgehog/Glil pathway in glioma recurrence and angiogensis, and roles of this pathway regulating VEGF, MMP2 and MMP9.Methods:(1) Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of CD34 and Glil protein using immunohistochemistry and explored relationship between the Hedgehog/Gli 1 pathway and MVD.(2)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could upregulate expression of VEGF, MMP2 and MMP9.(3) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Glil pathway in U87 and SHG44 cells, explored whether this pathway repressed could downregulate expression of VEGF, MMP2 and MMP9.(5)At the Gli1 level, using RNAi further confirmed that expression of VEGF, MMP2 and MMP9 downregulated was achieved through Hedgehog/Gli1 pathway inhibited.Results:(1)MVD in gliomas with Hedgehog/Glil activaton (n=43) was 27.4±4.6, and MVD in gliomas without Hedgehog/Gli1 activaton (n=17) was 20.9±3.5, there was significant difference between these two groups (P<0.01).(2)MVD in gliomas with high level expression of Gli1 was 23.7±3.5, MVD in gliomas with moderate level expression of Glil was 27.3±3.4, and MVD in gliomas with low level expression of Gli1 was 33.2±2.9. There was significant difference in these three groups. What'more, Glil expression was positively related with MVD (r=0.756, P<0.01).(3)Overactivation of the Hedgehog/Glil pathway effectively upregulated VEGF, MMP 2 and MMP 9 expression.(4)Repression of the Hedgehog/Glil pathway activity promoted downregulating expression of VEGF, MMP 2 and MMP 9.(5)In order to make our results more reliable, we performed RNAi at direct GLi1 level in U87 and SHG44. The data showed that downregulation of Gli1 can indeed inhibit mRNA and protein expression of VEGF, MMP2 and MMP9.Conclusions:(1)We show for the first time that status of the Hedgehog/Glil pathway activated is correlated with angiogenesis in gliomas.(2)The Hedgehog/Glil pathway can effectively regulate expression of VEGF, MMP2 and MMP9.(3)Inhibiting Hedgehog/Glil pathway, and further downregulating expression of VEGF, MMP2, MMP9 may be a valid therapeutic strategy which represses angiogenesis of gliomas.
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