| Background and significanceChronic lymphocytic leukemia is a kind of lymphocyte clone proliferation of tumor diseases.A large number of small lymphocytes infiltrate in bone marrow,peripheral blood,lymph nodes,liver,spleen and other lymphoid organs,eventually lead to normal hematopoietic function failure.It has been identified as one of the most common malignant hematopoietic diseases in western countries.In recent years,along with the development of molecular biology,the diagnostic rate of chronic lymphocytic leukemia has increased.However,the pathogenesis is still unclear.Emerging data indicates that innumerable signaling pathways constituting information transmission network is considered to account for malignant proliferation and metastasis.The study of interaction between signaling pathways has gradually become the frontier of tumor research.Previous studies have conformed the interaction of EGER,Notch,Hedgehog with Wnt signaling pahtway regulate tumor cell proliferation and differentiation,such as lung,breast,colon and liver cancers.Hedgehog signaling pathway is activated through protein PTCH and Smoothened?Smo?.In the absence of Hh,PTCH is a suppressor or inhibitory protein and forms an inactive complex with downstream protein SMO.When Hedgehog signaling pathway is activated,Hh ligand anchoring to PTCH results in endocytosis of the PTCH-ligand complex,followed by migration of activated SMO to the cytoplasm and association with gloma-associated oncogene homolog?GLI?protein.The GLI proteins is then involved in promoting the transcription of target genes.The Wnt signaling pathway is conducted mainly through three ways,canonical Wnt/?-catenin signaling pathway is considered as the most important pathway,which accumulates ?-catenin to nucleus and initiate the expression of target gene,such as c-myc,cyclinD1,COX-2,MMP,etc.Therefore,abnormal accumaulation and localization in cell nucleus of ?-catenin is considered as important symbol of canonical Wnt pathway.Inappropriate activation of the Wnt signaling pathway is associated with a variety of epithelial cancers,such as carcinoma of gallbladder,lung cancer and breast carcinoma,etc.However,more conflicting data have been reported that Hedgehog and Wnt signaling pathway have functional antagonism in vertebrates and invertebrates.The two signaling pathways resulted in opposite proliferative outcomes in neural stem/progenior cells in the ciliary marginal zones,and limb regeneration of amphibian.Hedgehog negative regulates Wnt activity through transcriptional regulation of SFRP1,and the Wnt/p-catenin pathway downregulates Hedgehog activity through Gli3 transcriptional regulation.Further researches indicate that Wnt might be the downstream signaling pathway of Hedgehog signaling pathway,the "crosstalk"mechanism also be demonstrated wih gastric caner and cervical cancer.Recent researches have shown that there are many types of Hedgehog pathway blockers,such as Cyclopamine,GANT-58 and GANT-61.GANT-61 acts directly on the transcription factor Glil/2,specifically blocks Glil/2 mediated target gene activation,eventually inhibits cell proliferation and differentiation.Further study of the crosstalk of different signaling pathways could overcome the limitation of single pathway,and more comprehensive reveal the importance of signaling pathway in the development of cancer process,and the interaction between hedgehog and Wnt signaling pathway in malignancies might provide a hope for potential therapy of such cancers.In order to further explore the antagonism between Hedgehog and Wnt signaling pathways and the relationship of chronic lymphocytic leukemia,our study focuses on the two main aspects:? To detect the expression of Glil,SFRP-1,?-catenin,Gli3 in CLL patients,and analyze the prognostic significance with clinical data ?We deal with CLL cell lines MEC-1 with different concentrations of GANT-61,cell survival rate was detected by Flow cytometry,to explore the antagonism between Hedgehog and Wnt signaling pathways through different expression of Glil,SFRP-1,?-catenin,Gli3 proteins.Part I the abnormal activation of Hedgehog and Wnt signaling pathway and prognosis in chronic lymphocytic leukemiaObjective:To detect the expression of Glil,SFRP-1,?-catenin,Gli3 in CLL patients,and analyze the prognostic significance.Methods and materials:43 patients diagnosed with chronic lymphocytic leukemia from December 21,2010 to October 31,2015 at the Department of Heamatology,Shandong Provincial Hospital,were enrolled in the study.Western blot was performed to detect the expression of Glil,SFRP-1,?-catenin,Gli3.All statistical analyses were performed with SPSS 19.0 statistical software.We used the Spearman analysis to examine the association of Gli1,SFRP-1,p-catenin,Gli3 and clinicopathogic factors,Kaplan-Meier method to calculate the survival curves,and log-rank test was used to compare the statistical significance between the survivals of patients subgroups.Multivariate COX regression analysis was used to analyses prognosis.Results:The expression of Gli1,SFRP-1,?-catenin,Gli3 in CLL patients was significantly higher than control group?P<0.05.?,the difference was statistically significant.Multivariate COX regression analysis showed that high expression of Gli1,?-catenin,age,Rai stage and Benit stage were significantly related to prognosis?P<0.05?,however,sex,level of LDH and ?2-microglobin,WBC count,B symptoms,SFRP-1,and Gli3 have no significant correlation with survival?P>0.05?.Conclusions:The high expression of Gli1,SFRP-1,?-catenin,Gli3 indicated the important significance in chronic lymphocytic leukemia,and the high expression of Gli3 indicated that the abnormal activation of Wnt signaling pathway might activate the negative feedback of Hedgehog pathway.The high expression of Gli1 and 3-catenin related to poor prognosis.Part ? Hedgehog inhibiting Wnt signaling pathway influences the proliferation of MEC-1Objective:?To detect the protein expression of Glil,SFRP-1,?-catenin,Gli3 in CLL cell line MEC-1,and analyze the significance in the proliferation of MEC-1.? Dealing with CLL cell lines MEC-1 with different concentrations of GANT-61,cell survival rate was detected by Flow cytometry and CCK-8 method,to explore the antagonism between Hedgehog and Wnt signaling pathways through different expression of Glil,SFRP-1,?-catenin,Gli3.Methods and materials:Western blot was performed to detect the expression of Gli1,SFRP-1,?-catenin,Gli3 in MEC-1.Dealing with different concentrations of GANT-61,CCK-8 method was performed to decect the survival rate in different concentrations?10-150umol/L?and different time?24-72hours?.Flow cytometry was performed to detect the cell survival rate.The antagonism between Hedgehog and Wnt signaling pathways was identified through comparing the different expression of Glil,SFRP-1,?-catenin,Gli3.Results:The expression of Glil,SFRP-1,?-catenin,Gli3 in MEC-1 was significantly higher than control group?P<005?,the difference was statistically significant.GANT-61 and proliferation of MEC-1 have time-concentration dependence.The expression of Glil was significantly reduced in 50umol/L concentration and 24 hours?P<0.05?.The expression of SFRP1 was significantly reduced in 50umol/L concentration and 24 hours?P<0.05?.The expression of?-catenin was significantly increased in 50umol/L concentration and 24 hours?P<0.05?.And the expression of Gli3 was significantly increased in 48 hours?P<0.05?.Conclusions:The high expression of Gli1,SFRP-1,?-catenin,Gli3 indicated the important significance in MEC-1 cell lines.Hedgehog works as the upstream of Wnt signaling pathway,inhibits the activation of Wnt signaling pathway,and promotes the proliferation of MEC-1.GANT-61 and proliferation of MEC-1 have time-concentration dependence.Furthermore,the up-regulation of ?-catenin promotes the expression of Gli3 protein,the abnormal activation of Wnt signaling pathways may initiate the negative feedback inhibition of Hedgehog pathway.The above conclusions may indicate the "crosstalk" mechanism between Hedgehog and Wnt signaling pathways. |
PDF Full Text Request