Study On Mechanism Of Sonic Hedgehog Signal Pathway In Development Of Pancreatic Carcinoma

Study on Mechanism of Sonic Hedgehog Signal Pathway in Development of Pancreatic CarcinomaObjective To investigate the role of Sonic hedgehog(SHH) signal pathway and its clinical significance in human pancreatic cancer. Explore the correlation between SHH,WNT/β-catenin and EGFR signal pathways. To assess the inhibitory effects through the blockade of the SHH signaling pathways by cyclopamine.Methods Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, Immunofluorescence and immunohistochemistry assay were used to determine the mRNA and protein expression of SHH in human pancreatic cancer tissue and pancreatic cancer cell lines. Analysis the correlation between SHH,WNT/β-catenin and EGFR signal pathways. Detect the EGFR expression level after cyclopamine treatment, or transfected with the wild-type PTC1 plasmid. Pancreatic cancer cells were treated with different concentrations of cyclopamine, alone or in combination with iressa, the antiproliferative effect of pancreatic cancer cells was analysed by methyl thiazolyl tetrazolium(MTT) assays. Flow cytometry analysis was used to detect the cellular cycle distribution and apoptosis of pancreatic cancer cells. RT-PCR and Western blot were used to detect the expression of Bcl-2 and p21.Results The SHH mRNA and protein expression was high in pancreatic cancer, compared with normal tissue adjacent to cancer(P <0.05). The relationship between SHH andβ-catenin or EGFR protein was positive(P<0.05), and these expressions have no correlation with age, tumor size, pathological type and tumor site(P >0.05), but has a relationship with lymph node metastasis and TNM stage(P <0.05). The EGFR expression level was downregulated after cyclopamine treatment, or transfected with the wild-type PTC1 plasmid. Cyclopamine and Iressa induced a growth inhibitory effect in a dose-dependent manner. Moreover, the combined use of 2.5μM cyclopamine and 1μM Iressa induced an additive inhibitory effect, which was much more than that of 5μM cyclopamine or 5μM Iressa alone. The percentage of cell population of the G0/G1 and sub-G1 phase was significantly increased, while that in S and G2/M phase decreased, along with the increasing dose of cyclopamine and/or Iressa. Moreover, 2.5μM cyclopamine plus 1μM Iressa induced greater apoptosis rate than any agent alone. The expression of Bcl-2 and p21 were downregulated by Cyclopamine.Conclusion The SHH and WNT/β-catenin signaling pathways were active in human pancreatic cancer. The crosstalk between these pathways may play an important role in the carcinogenesis and development of pancreatic carcinoma. Sonic hedgehog and EGFR signal pathways are active in pancreatic cancer cells. Blockade of SHH signaling could downregulate EGFR expression. Cyclopamine could downregulate the expression of Bcl-2 and p21, and induced cell cycle arrest and apoptosis.The simultaneous blockade of SHH and EGFR signaling represents possible targets of new treatment strategies for pancreatic carcinoma. PART ONE The expression of sonic hedgehog(SHH) in human pancreatic cancer Objective To investigate the expression of Sonic hedgehog(SHH)and its clinical significance in human pancreatic cancer.Methods Reverse transcription-polymerase chain reaction (RT-PCR) assay and immunohistochemistry were used to determine the mRNA and protein expression of SHH,SMO and Gli1 in human pancreatic cancer tissue and normal tissues adjacent to cancer.Results The SHH mRNA expression was detected in 78.9%(30/38)of pancreatic cancer, while only 18.4%(7/38) of normal tissue adjacent to cancer. Immunohistochemical analysis showed that the SHH protein expression was 84.2% (32/38) in pancreatic cancer tissues, and 21.1%(8/38)in normal tissues. There was a significant difference between them(P <0.01). Overexpression of SHH protein in pancreatic cancer has no correlation with age, tumor size, pathological type and tumor site(P >0.05), but the difference of lymph node metastasis,TNM stage were significant(P <0.05). The SMO mRNA expression in cancer tissue was 2.38 stronger than that in normal tissue. The Gli1 mRNA expression in cancer tissue was 2.21 stronger than that in normal tissue. The SMO protein expression was detected in 92.1%(35/38)of pancreatic cancer, while only 18.4%(7/38) of normal tissue adjacent to cancer. The Gli1 protein expression was detected in 86.8%(33/38)of pancreatic cancer, while only 13.2%(5/38) of normal tissue adjacent to cancer.Conclusion The expression of SHH signal molecular was increased in human pancreatic cancer. The overexpression of SHH signaling pathway may play an important role in the carcinogenesis and development of pancreatic carcinoma. PART TWO The expression of sonic hedgehog andβ-catenin in human pancreatic cancerObjective To investigate the expression of Sonic hedgehog(SHH)and WNT/β-catenin signaling pathways in human pancreatic cancer.Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were used to determine the mRNA and protein expression of SHH andβ-catenin in human pancreatic cancer tissue and normal tissues adjacent to cancer.Results The SHH mRNA and protein expression was detected in 81.6% and 79.6% of pancreatic cancer, respectively. Theβ-catenin protein expression was 71.4% in pancreatic cancer tissues. These were significantly different from that of normal tissue adjacent to cancer(P <0.05). But the expression level ofβ-catenin mRNA was low in both pancreatic cancer tissues and normal tissues. There was no significant difference between them(P >0.05). The expression of SHH andβ-catenin protein in pancreatic cancer has no correlation with age, tumor size, pathological type and tumor site(P >0.05), but has a relationship with lymph node metastasis and TNM stage(P <0.05). The relation between SHH andβ-catenin protein was positive(γ=0.352, P<0.05).Conclusions The SHH and WNT/β-catenin signaling pathways were active in human pancreatic cancer. The crosstalk between these pathways may play an important role in the carcinogenesis and development of pancreatic carcinoma. PART THREE The correlation between sonic hedgehog and EGFR signal pathwaysPAPER 1 The expression of sonic hedgehog and EGFR in pancreatic cancer Objective To investigate the expression of Sonic hedgehog(SHH)and EGFR signaling pathways in human pancreatic cancer.Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay ,immunohistochemistry were used to determine the mRNA and protein expression of SHH and EGFR in human pancreatic cancer tissue and normal tissues adjacent to cancer. Results The SHH mRNA and protein expression was detected in 81.6% and 79.6% of pancreatic cancer, respectively. The EGFR mRNA and protein expression were both 73.5% in pancreatic cancer tissues. These were significantly different from that of normal tissue adjacent to cancer(P <0.05). The expression of SHH and EGFR protein in pancreatic cancer has no correlation with age, tumor size, pathological type and tumor site(P >0.05), but has a relationship with lymph node metastasis and TNM stage(P <0.05). The relationship between SHH and EGFR protein was positive(γ=0.232, P<0.05).Conclusion The SHH and EGFR signaling pathways were active in human pancreatic cancer. The crosstalk between these pathways may play an important role in the carcinogenesis and development of pancreatic carcinoma. PAPER 2 The expression of SHH/SMO in human pancreatic cancer cells and correlation with EGFRObjective To investigate the expression of sonic hedgehog (SHH) and epidermal growth factor receptor (EGFR) signal molecules in pancreatic cancer cells, and to assess the inhibitory effects through the blockade of the SHH signaling pathways by cyclopamine.Methods The expression of SHH, SMO and EGFR in pancreatic cancer cell lines (PANC-1, SUIT-2, and ASPC-1) was detected by RT–PCR,Immunofluorescence and Western blot analysis. Detect the expression of EGFR before and after cyclopamine treatment.Results All of the 3 pancreatic cancer cell lines expressed SHH, Smoothened (SMO), and EGFR, but the expression level was higher in SUIT-2 cells than the other two cell lines; SHH was located in cell plasma, and EGFR was located on cell membrane. Cyclopamine could downregulate the expression of EGFR in all cell lines.Conclusion Sonic hedgehog and EGFR signal pathways are active in pancreatic cancer cells. Blockade of SHH signaling could downregulate EGFR expression. PAPER 3 The EGFR expression in pancreatic cancer cells transfected with PTC1 plasmid in vitroObjective: To investigate the effects of the tumor suppressor gene PTC1 on the growth inhibition and down-regulation of EGFR in pancreatic cancer cells.Method:SUIT-2 cells were transfected with wild-type PTC1 plasmids and mutant-PTC1 plasmids in vitro. After transfection, the expression of the PTC1 and EGFR were detected by western blot. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results:After transtected with wild-type PTC1, the growth rates of pancreatic cells were slow down, but the other groups of cells have no change. Compared with the control, the expression of Ptc1 were increased when transfected with the wild-type PTC1 and EGFR were down-regulated respectively. The apoptosis rates in wild-type PTC1 transtected group was 24.5%, and the mutant-PTC1 transtected group was 8.3%(P<0.01). But the apoptosis rate of blank plasmid group has no change.Conclusion:Wild-type PTC1 could induce a cell cycle arrest in G1 phase. Wild-type PTC1 could decrease the EGFR expression level and induce cells apoptosis. PART FOUR Blockade of sonic hedgehog signal pathway induce antiproliferative effect and apoptosis in pancreatic cancer cellsObjective To investigate the inhibitory effects through the blockade of SHH and EGFR signaling pathways by cyclopamine and Iressa, respectively.Methods The PANC-1, SUIT-2, ASPC-1 cells were treated with different concentrations of cyclopamine, alone or in combination with iressa, the antiproliferative effect of pancreatic cancer cells was analysed by methyl thiazolyl tetrazolium(MTT) assays. Flow cytometry analysis was used to detect the cellular cycle distribution and apoptosis of pancreatic cancer cells. RT-PCR and western blot were used to detect Bcl-2 and p21 expression.Results Cyclopamine and Iressa induced a growth inhibitory effect in a dose-dependent manner. Moreover, the combined use of 2.5μM cyclopamine and 1μM Iressa induced an additive inhibitory effect, which was much more than that of 5μM cyclopamine or 5μM Iressa alone. The percentage of cell population of the G0/G1 and sub-G1 phase was significantly increased, while that in S and G2/M phase decreased, along with the increasing dose of cyclopamine and/or Iressa. Moreover, 2.5μM cyclopamine plus 1μM Iressa induced greater apoptosis rate than any agent alone. Cyclopamine could decrease the Bcl-2 expression and increase the p21 expression.Conclusion Cyclopamine could induce cell cycle arrest and apoptosis in pancreatic cancer cells. The simultaneous blockade of SHH and EGFR signaling represents possible targets of new treatment strategies for pancreatic carcinoma.