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Objective:To explore the potential mechanism and feasibility of sonic hedgehog (SHH) signal pathway inhibitor NVP-LDE225 combined with radiotherapy in the treatment of melanoma.Methods:The Gli1 mRNA expression of the melanoma cells (Melan-A and SK-MEL-2) was detected by qPCR. After treatment with NVP-LDE225, GDC-0449 or combined with radiation for 24 hours, the number and activity of Melan-A and SK-MEL-2 were detected by cell counting and CCK-8 kit. The survival and proliferation ratio of Melan-A and SK-MEL-2 were detected by cell cloning. The changes of cell cycle of melanoma Melan-A cells were determined by flow cytometry. The levels of the Bax and Caspase3 of Melan-A apoptosis protein in melanoma cells were detected by Western blot.Results:NVP-LDE225, an inhibitor of Smo, decreased the mRNA expression of Gli1 in the melanoma cells in a dose-dependent manner, inhibited the proliferation ratio of the melanoma cells, induced apoptosis, and arrested melanoma cells in G 0 / G 1 phase. Gamma ray irradiation after NVP-LDE225 treatment further inhibited the SHH signal pathway, arrested the melanoma cells in G 2 / M phase, and further increased the inhibitory effect on melanoma cell proliferation. Conclusions:NVP-LDE225, an inhibitor of Smo, can inhibit SHH signal pathway in melanocytes, suppress cell proliferation, and further increases the effect of inhibiting cell proliferation after combining with irradiation. It can be used as a potential drug in combination with radiotherapy in the treatment of melanoma, which is worthy of further study.
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Objective: To investigate the effect of Gli inhibitor GANT61 on epithelial-mesenchymal transition (EMT) of the human esophageal adenocarcinoma OE19 and OE33 cells, and to explore its possible mechanism. and recombinant sonic hedgehog (Shh) protein (0.5 mg/mL) (as positive control) for 24 h. The mRNA and protein expression levels of Gli1, Gli2, N-cadherin (N-cad), E-cadherin (E-cad) and vimentin were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The migration of OE19 and OE33 cells was measured by wound-healing assay. Results: The results of real-time fluorescent quantitative PCR and Western blotting showed that treatment with GANT61 for 24 h, the mRNA and protein expression levels of Gli1, Gli2, vimentin and N-cad in OE19 and OE33 cells were significantly decreased, but the expression levels of E-cad mRNA and protein in OE19 and OE33 cells were significantly increased (all P < 0.01). After stimulation of Hedgehog pathway with recombinant Shh protein, the mRNA and protein expression levels of Gli1, Gli2, vimentin and N-cad in OE19 and OE33 cells were significantly increased, but the mRNA and protein expression levels of E-cad in OE19 and OE33 cells were significantly decreased (all P < 0.01). The result of wound-healing assay showed that GANT61 could inhibit the migration abilities of OE19 and OE33 cells, but recombinant Shh could increase the migration abilities of OE19 and OE33 cells (all P < 0.01). Conclusion: GANT61 can effectively inhibit EMT of esophageal adenocarcinoma by down-regulating the expressions of Gli1 and Gli2, which lead to reduce migration abilities of esophageal adenocarcinoma cells. Gli may be a new and potential target for treatment of esophageal adenocarcinoma.
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Objective To study the relationship between expression of Shh and Gli1 in Hedgehog(Hh) signal pathway and esophageal squamous cell carcinoma (ESCC).Methods Expressions of Shh andGli1 were detected in 64 cases primary tumor tissues and 24 cases normal esophageal mucosa with SP immunohistochemistal method.Results There were significant differences of Shh and Gli1 expressions between esophageal carcinoma and normal mucosa epithelium (67.19％ (43/64) vs.4.17％ (1/24), 60.94％ (39/64) vs.4.17％ (1/24);x2 =27.729,22.689;P＜0.01).There were significant differences of Shh expression between different degrees of differentiation in esophageal squamous cell carcinoma, metastasis of lymph node and different clinical staging group (x2 =3.873, 11.349,6.429;P ＜ 0.05 or P＜ 0.01).The positive expression rate of Gli1 showed significant differences between different degrees of differentiation in esophageal squamous cell carcinoma, metastasis of lymph node and different clinical staging group (x2=12.598, 9.741,26.341;P ＜0.01).There was a positive relationship between Shh and Gli1 expression(r =0.259, P＜0.05).Conclusion Hh signaling pathway is abnormally activated in ESCC, so that Shh and Gli1 play important roles in carcinogenesis and progression ofesophageal carcinoma.The Hh signaling pathway may be a useful target in ESCC.
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Objective To investigate the regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia.Methods The IEC-6 cells of rats were divided into 3 groups:the normoxia group (21％ oxygen concentration),the hypoxia group (2％ oxygen concentration) and the hypoxia + cyclopamine group (cells pretreated by 5 mmol/L of cyclopamine,and then exposed in an atmosphere with 2％ oxygen concentration).The mRNA expressions of IHH,PTCH and GLI-1 were detected,and the transepithelial electrical resistance (TER) was determined.The protein expressions of tight junction proteins (ZO-1,Occludin,Claudin-1) and IHH were assayed by using the Western blot.All data were analyzed using the one-way analysis of variance or LSD-t test.Results The relative mRNA expressions of IHH,PTCH and GLI-1 were 0.056 ± 0.009,0.459 ± 0.087,0.142 ± 0.023 in the normoxia group,and 0.303 ± 0.052,0.678 ± 0.073,0.483 ± 0.061 in the hypoxia group,with significant difference between the 2 groups (t =-14.05,-11.85,-6.52,P ＜ 0.05).The relative protein expressions of IHH in the normoxia group and the hypoxia group were 0.39 ±0.06 and 0.91 ±0.15,with a significant difference between the 2 groups (t =-8.08,P ＜ 0.05).The TERs of the normoxia group,the hypoxia group and the hypoxia + cyclopamine group were (134 ± 5) Ohm/cm3,(100 ± 6) Ohm/cm3 and (118 ± 5) Ohm/cm3,with significant difference between the 3 groups (F =1.04,P ＜ 0.05).Compared with the normoxia group,the TER of the hypoxia group was decreased by 27.7％ (t =7.84,P ＜ 0.05) ; compared with the hypoxia group,the TER of the hypoxia + cyclopamine group were increased by 16.4％,but it was still significantly lower than the normoxia group (t =4.23,P ＜ 0.05).The expressions of ZO-1,Occludin and Claudin-1 were 1.18 ± 0.24,0.80 ±0.13 and 0.90 ±0.09 in the normoxia group,and 0.58 ±0.08,0.32 ±0.05 and 0.50 ±0.09 in the hypoxia group,and 0.92 ± 0.21,0.43 ± 0.10 and 0.82 ± 0.11 in the hypoxia + cyclopamine group,with significant difference between the 3 groups (F =4.95,2.88,10.09,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in hypoxia group were decreased by 48.7％,40.0％ and 55.6％ when compared with the normoxia group (t =12.86,9.35,18.90,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in the hypoxia + cyclopamine group were increased by 59.9％,35.2％ and 65.1％ when compared with the hypoxia group (t =5.63,2.92,6.66,P ＜ 0.05).Conclusion Hedgehog signal pathway could be activated under hypoxia,and then the expressions of tight junction proteins are decreased,which finally induces the injury of intestinal epithelial barrier function.
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Objective To investigate the expression of Hedgehog signaling pathway members,Shh,Gli1,Sufu and TAK1 as well as phosphorylation-TAK1 (p-TAK1) protein in human pancreatic carcinoma tissues and to explore its relationship between the expression of proteins and the clinicopathologic parameters.Methods The expressions of Shh,Glil,Sufu,TAK1,p-TAK1 proteins in 38 samples of pancreatic cancer tissues and pairing adjacent normal pancreatic tissues were detected by immunohistochemical method.The relationship among the proteins and the relationship between the expression of the proteins and the clinicopathologic parameters were examined.Results The positive expression rates of Shh,Glil,Sufu,TAK1,p-TAK1 protein in human pancreatic carcinoma were 86.8％ (33/38),52.6％ (22/38),68.4％(26/38),55.3％ (21/38),52.6％ (20/38),but they were not expressed in adjacent normal pancreatic tissues.Gli1 expression was positively related to distant metastasis and clinical staging (r =0.524,0.361,P ＜0.05),Sufu expression was positively related to patient's gender (r =-0.378,P ＜0.05),TAK1 expression was positively related to clinical staging (r =0.468,P ＜ 0.05),p-TAK1 expression was positively related to clinical staging and distant metastasis (r =0.418,0.361,P ＜ 0.05).Gli1 expression was positively related to TAK1 and p-TAK1 expression in pancreatic cancer tissues (P ＜ 0.05).Conclusions Hedgehog signaling pathway and p-TAK1 may play a role in the pathogenesis of pancreatic cancer,and the two pathways may interact with each other.
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As one of the most major disease,malignant tumor is harmful to human health.Althought it' s exact pathogenesis is still not clear,it is certainly that the formation of cancer is typical of containing many factors and having gone through many steps.Resulting from the abnormal activation of a series of proto-oncogene as well as the inactivation caused by many anti-oncogene mutation,some signal pathways work abnormally.In recent years,the study of Hedgehog ( Hh),as the signal pathway,shows that Hh encodes a series of secreted signaling protein,having an important effect on the differentiation and proliferation of cells in the process of embryogenesis.The occurrence of abnormal activation of Hh signaling pathway plays a significant role in the process of tumorigenesis.Signal paths of Hh signaling protein include as much as following at least:patched(Ptch),smoothened (Smo),fused (Fu),suppressor of fused (SuFu),costaⅠ-2 (Cos)-2 and cubitus interruptus (Ci) and so on.In this paper,the function of Hh genetic family and the mechanism of its relation to many kinds of human cancers will be givien a brief overview.
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Objective To investigate the effects on self-renewal of pancreatic cancer stem cells by inhibiting hedgehog signaling pathway through cyclopamine. Methods PANC1 stem cells, PANC1 adherent cells and immortalized pancreatic ductal epithelial H6C7 cells were treated with 0.5, 1, 2, 5, 10 mol/L of cyclopamine for 24, 48, 72 h. The expression of Smo mRNA and Gli1 mRNA were detected by real-time PCR.Cell growth viability was measured by CCK 8. Cell cycle and apoptosis were determined by flow cytometry.Results Seventy-two hours after cyclopamine treatment, the Smo mRNA expressions of PANC1 stem cells,PANC1 adherent cells and H6C7 cells were 1,0.83 and 2.61; the expressions of Gli mRNA were 57.27,26.35,1; the inhibitory rates were ( 37.85 ± 13.69 ) %, ( 8.53 ± 4.43 ) %, (43.55 ± 28.98 ) %. Compared with PANC1, the expressions of Smo mRNA, Gli1 mRNA and the inhibitory rate of PANC1 stem cells significantly increased ( P ＜ 0.05 ). The proportion of G1 stage of PANC1 stem cells significantly decreased from (67.41 ±6.35)% to (36.53 ±6.03)% (P ＜0.05), and the apoptosis decreased from (10.95 ±5.68) % to ( 5.73 ± 1.42 ) % ( P ＞ 0.05 ). The proportion of G1 stage of PANC1 cells significantly decreased from ( 67.64 ± 6.88 ) % to ( 53.13 ± 1.10 ) % ( P ＜ 0.05 ); the apoptosis decreased from ( 12.08 ±4.12)% to (5.66 ± 1.33)% (P ＞0.05). While both the proportion of G1 stage and apoptosis of H6C7 cells was not significantly different. Conclusions Cyclopamine can inhibit the proliferation of PANC1 stem cells via blocking hedgehog signal pathway, and the mechanism may not be associated with cell apoptosis.
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Objective: To study the effect of PTCH1 methylation on gastric carcinogenesis and the therapeutic effect of methylation inhibitor, 5-aza-2′-deoxyeytidine (5-aza-dC), for treatment of gastric cancer. Methods: The total RNAs were extracted from 10 gastric cancer tissues, their corresponding adjacent normal tissues, and gastric cancer cell line AGS. The PTCH1 mRNA expression was detected by Quantitative real-time PCR (QRT-PCR) and the methylation of the promoter was examined by methylation specific PCR (MSP). AGS cells were treated by 5-Aza-dC; the cell cycle and apoptosis were examined by flow cytometry, and the methylation level was also observed. Results: PTCH1 expression was negatively correlated with promoter methylation in gastric cancer tissues, their corresponding adjacent normal tissues, and gastric cancer cell line AGS (r = -0.591, P = 0.006). 5-Aza-dC treatment caused apoptosis and G 0/G1 phase arrest of AGS cells, and also induced demethylation of PTCH1 and increased its expression. Conclusion: Hypermethylation of PTCH1 gene promoter region is one of the main causes of low PTCH1 expression in AGS cells. Demethylation agent 5-Aza-dC can reverse this methylation status of PTCH1 and regulate the expression of PTCH1, suggesting a role for it in gastric cancer treatment.
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Objective To investigate the expression of SUFU protein in human pancreatic carcinoma tissues and to explore the relationship between the expression of SUFU protein and the clinicopathologic parameters.Methods The expression of SUFU protein in 28 samples of pancreatic cancer tissues and 20 adjacent normal pancreatic tissues and 4 normal pancreatic tissues was detected by immunohistochemical method.And the relationship between the expression of SUFU protein and the clinicopathologic parameters were determined.Results SUFU protein was positively expressed in cytoplasm and nucleus of pancreatic carcinoma cells, while it was not expressed in the duct, acinar and islet of tumor-adjacent tissues and normal pancreatic tissues, and difference was statistically significant (P ＜ 0.05).The high SUFU protein expression was related to the clinical stage ( P ＜ 0.05 ), but not the age, gender, tumor location , tumor size, lymph node metastasis and liver metastasis.Conclusions SUFU protein was highly expressed in pancreatic cancer, and SUFU may play certain role in the pathogenesis of pancreatic cancer.