OBJECTIVE: To explore the regulatory role of SOX2-OT in migration of lung squamous cell carcinoma H520 cells and the underlying mechanisms. METHODS: Wound- healing and Transwell migration assays were performed to examine the changes in migration and invasion capacity of lung squamous cell line H520, which expressed higher levels of SOX2-OT than other lung cancer cell lines, following RNA interference-mediated SOX2-OT knockdown. The transcription levels of epithelial-mesenchymal transition (EMT)-related components was detected by qRT-PCR and immunoblotting. Gli1 gain-of-function analysis was performed in H520 cells with SOX2-OT knockdown and the changes in EMT phenotype of the cells were examined. miR-200c mimic and inhibitor were used to analyze the mechanism by which SOX2-OT positively regulates Gli1 and the mediating role of SOX2. RESULTS: SOX2-OT knockdown significantly lowered the invasiveness and migration capacity of H520 cells and caused changes in EMT phenotype of the cells. Overexpression of Gli1, which was positively regulated by SOX2-OT, reversed the inhibitory effect of SOX2-OT knockdown on migration of H520 cells. Transfection of the cells with miR-200c inhibitor effectively reversed SOX2-OT knockdown-induced down-regulation of SOX2. CONCLUSION The SOX2-OT/SOX2 axis positively regulates migration of lung squamous H520 cells via Gli1-mediated EMT.
Humans ; Epithelial-Mesenchymal Transition/genetics* ; Zinc Finger Protein GLI1/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Lung Neoplasms/genetics* ; MicroRNAs/metabolism* ; Gene Expression Regulation, Neoplastic ; Cell Proliferation/genetics* ; Neoplasm Invasiveness/genetics* ; SOXB1 Transcription Factors/metabolism*

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

OBJECTIVETo investigate the effects of bisdemethoxycurcumin (BDMC) on non-small cell lung cancer (NSCLC) cell line, A549, and the highly metastatic lung cancer 95D cells.

METHODSCCK-8 assay was used to assess the effect of BDMC on cytotoxicity. Flow cytometry was used to evaluate apoptosis. Western blot analysis, electron microscopy, and quantification of GFP-LC3 punctuates were used to test the effect of BDMC on autophagy and apoptosis of lung cancer cells.

RESULTSBDMC inhibited the viability of NSCLC cells, but had no cytotoxic effects on lung small airway epithelial cells (SAECs). The apoptotic cell death induced by BDMC was accompanied with the induction of autophagy in NSCLC cells. Blockage of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) repressed the growth inhibitory effects and induction of apoptosis by BDMC. In addition, BDMC treatment significantly decreased smoothened (SMO) and the transcription factor glioma-associated oncogene 1 (Gli1) expression. Furthermore, depletion of Gli1 by siRNA and cyclopamine (a specific SMO inhibitor) induced autophagy.

CONCLUSIONAberrant activation of Hedgehog (Hh) signaling has been implicated in several human cancers, including lung cancers. The present findings provide direct evidence that BDMC-induced autophagy plays a pro-death role in NSCLC, in part, by inhibiting Hedgehog signaling.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cell Line, Tumor ; Curcumin ; analogs & derivatives ; chemistry ; pharmacology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Hedgehog Proteins ; genetics ; metabolism ; Humans ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Signal Transduction ; drug effects ; Zinc Finger Protein GLI1

OBJECTIVETo study the role and possible mechanism of glioma-associated oncogene-1 (Gli1) in regulating the cell invasion and migration of pancreatic cancer cells.

METHODSQuantitative real-time (qRT) -PCR was used to detect the effect of siRNA interference on Gli1, murine double minute 2 (MDM2) and p53 genes. Cell invasion and migration assays were used to observe the effect of Gli1, MDM2 and p53 silence on cell invasion and migration in p53 wild-type Capan-2 pancreatic cancer cells, respectively. Meanwhile, immunoblotting (IB) was used to detect the protein level of matrix metalloproteinase (MMP) -9, phospho-excelluar signal-regulated kinase (pERK) and phosphorylation protein kinase B (pAKT) in Gli1-silencing Capan-2 cells. The data were analyzed by paired t-test.

RESULTSqRT-PCR showed that the expression of Gli1, MDM2 and p53 is down-regulated 70.5% and 74.5%, 61.8% and 65.3%, and 73.8% and 78.2% after siRNA interference, compared with the mock and siRNA control groups, respectively. Cell invasion (94 ± 8) and migration (143 ± 8) in p53 wild-type Capan-2 cells transfected with Gli1siRNA were significantly decreased, compared with the siRNA control group (150 ± 7, 190 ± 10) (t = 6.584, P = 0.022; t = 8.266, P = 0.014) , while MDM2 silence inhibited cell invasion (experiment group:85 ± 12, control group: 138 ± 6) and migration (experiment group: 127 ± 9, control group:180 ± 10) in the same cells, respectively (t = 5.097, P = 0.036;t = 4.860, P = 0.040). However, cell invasion (experiment group: 153 ± 11, control group: 106 ± 7) and migration (experiment group: 209 ± 13, control group: 164 ± 8) in p53-silencing Capan-2 cells were significantly enhanced (t = 4.669, P = 0.043; t = 4.990, P = 0.038). IB showed that Gli1 silence down-regulated MMP-9 but not pERK and pAKT protein expression.

CONCLUSIONGli1 might contribute to the cell invasion and migration in pancreatic cancer via the regulation of MDM2, p53 and MMP-9 expression.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Neoplasm Invasiveness ; Oncogene Proteins ; genetics ; metabolism ; Pancreas ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; RNA, Small Interfering ; genetics ; Trans-Activators ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism.

METHODSTo establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.

RESULTSThe fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group.

CONCLUSIONSThe overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.

Alanine Transaminase ; analysis ; Animals ; Aspartate Aminotransferases ; analysis ; Dimethyl Sulfoxide ; pharmacology ; Disease Models, Animal ; Fluoride Poisoning ; drug therapy ; metabolism ; Fluorosis, Dental ; diagnosis ; Hedgehog Proteins ; antagonists & inhibitors ; metabolism ; Hepatocytes ; metabolism ; Kruppel-Like Transcription Factors ; metabolism ; Liver ; metabolism ; Liver Diseases ; drug therapy ; metabolism ; RNA, Messenger ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Veratrum Alkaloids ; pharmacology ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.

METHODSHuman SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.

RESULTSIn vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).

CONCLUSIONThe shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.

Animals ; Cell Proliferation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyaluronan Receptors ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Octamer Transcription Factor-3 ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Smoothened Receptor ; Transcription Factors ; metabolism ; Transfection ; Tumor Burden ; Zinc Finger Protein GLI1

OBJECTIVETo study the clinicopathological significance and relationship of Gli1, MDM2 and p53 expression in human pancreatic cancer.

METHODSThe expression of Gli1, MDM2 and p53 proteins in 57 paired paraffin embedded pancreatic ductal adenocarcinoma (PDAC) specimens and adjacent non-cancerous pancreatic tissues was detected by immunohistochemistry. The relationship between their expression and clinicopathological characters was analyzed. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of Gli1 mRNA level in 14 paired fresh PDAC specimens and adjacent non-cancerous pancreatic tissues. siRNA interference were used to further detect the close relationship among them.

RESULTSIHC showed the expression of Gli1 (50.9%), MDM2 (57.9%) and p53 (56.1%) was increased in 57 cases of pancreatic cancer compared to that in paired normal pancreatic tissues (33.3%, 26.3% and 17.5% respectively, t = 2.413, 2.848 and 2.960, all P < 0.05). Gli1 expression was positively associated with tumor TNM stage (χ(2) = 8.211, P = 0.004), invasion depth (χ(2) = 4.247, P = 0.039) and MDM2 expression (r = 0.299, χ(2) = 5.105, P = 0.024), while expression of MDM2 and p53 was associated with tumor invasion depth (χ(2) = 5.182, P = 0.023) and TNM stage (χ(2) = 5.696, P = 0.017), respectively. Univariate and multivariate analysis revealed that Gli1 was an independent adverse prognostic indicator for patients with PDAC (RR = 2.290, 95%CI: 1.051-4.992, P = 0.037), and patients with Gli1 and MDM2 co-expression had a significantly poorer overall survival than patients with their negative expression (P = 0.034). Gli1 mRNA expression was much higher in 14 cases of PDAC than that in adjacent normal pancreatic tissues (t = 2.926, P = 0.012). In p53 mutant AsPC-1 cells, Gli1 knockdown down regulated MDM2, but had no effect on p53 expression, whereas Gli1 knockdown down regulated MDM2 and up regulated p53 protein levels in p53 wild-type Capan-2 cells.

CONCLUSIONSGli1, MDM2 and p53 are overexpressed in PDAC, and are benefit for predicting patients' prognosis. Gli1can regulate MDM2 and wild-type p53 expression. Their co-expression might coordinately contribute to the development and progression of PDAC.

Adult ; Aged ; Carcinoma, Pancreatic Ductal ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Oncogene Proteins ; metabolism ; Pancreatic Neoplasms ; metabolism ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; RNA, Messenger ; metabolism ; Trans-Activators ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the effect of Helicobacter Pylori lipopolysaccharide (Hp-LPS) on expression of Gli and Ptch-1 proteins in sonic hedgehog (Shh) signaling pathway of gastric mucosa GES-1 cells.

METHODSThe LPS was extracted from Hp by hot phenol water method, and then the concentration of LPS was detected by the kinetic turbidimetric assay. GES-1 cells were stimulated by different concentrations of Hp-LPS (0, 1, 10, 20, 30 and 40 μg/ml). The inhibition rates of cell growth were measured by MTT assay after treated with Hp-LPS for 24 h. The expression of Gli and Ptch-1 proteins were determined by Western Blot.

RESULTSMTT assay showed that the inhibition rates of GES-1 cell growth after treatment by different concentrations of Hp-LPS (1, 10, 20, 30 and 40μg/ml) were 25.8% ± 2.7%, 34.2% ± 3.1 %, 46.3% 3.4%, 60.8% ± 2.1% and 82.9% ± 2.8% respectively (r=0.985, P<0.001). Western blot showed that the expressions of Gli and Ptch-1 proteins were decreased after Hp-LPS treatment (0, 1, 10, 20, 30 and 40 μg/ml): the relative expression values of Gli were 1.286 ± 0.180, 0.963 ± 0.067, 0.850 ± 0.085, 0.566 ± 0.058, 0.549 ± 0.056 and 0.377 ± 0.047, respectively (r=-0.945, P<0.001); those of Ptch-1 were 1.688 ± 0.088, 1.466 ± 0.061, 1.170 ± 0.065, 1.042 ± 0.064, 0.648 ± 0.057 and 0.482 ± 0.074, respectively (r=-0.985, P<0.001).

CONCLUSIONHp-LPS can decrease the related protein expression of Shh signaling pathway, which indicates that Hp may interfere with the function of Shh signaling pathway in gastric mucosa via the effect of its LPS.

Cells, Cultured ; Epithelial Cells ; drug effects ; Gastric Mucosa ; cytology ; Hedgehog Proteins ; metabolism ; Humans ; Lipopolysaccharides ; administration & dosage ; pharmacology ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; metabolism ; Signal Transduction ; Transcription Factors ; metabolism ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the effect of cyclopamine on metastatic ability of human esophageal cancer EC109 cells and explore the possible mechanism.

METHODSTranswell chamber assay and angiogenesis assay were used to examine the metastatic ability, invasiveness and angiogenesis of EC109 cells treated with cyclopamine for 48 h. The expression of Gli-1 mRNA was detected using RT-PCR, and Western blotting was used to examine the protein expressions of Gli-1, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF).

RESULTSInhibition of the hedgehog signaling pathway by cyclopamine suppressed the migration, invasion, and angiogenesis of EC109 cells. Cyclopamine treatment significantly lowered the expression of Gli-1 mRNA (P<0.05) and the protein expressions of Gli-1, MMP-9 and VEGF (P<0.05).

CONCLUSIONCyclopamine can significantly inhibit the metastatic capacity of EC109 cells possibly by down-regulating MMP-9 and VEGF expression as a result of Gli-1 inhibition.

Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Transcription Factors ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Veratrum Alkaloids ; pharmacology ; Zinc Finger Protein GLI1

OBJECTIVETo investigate the role of the hedgehog (HH) signaling pathway transcription factor glioma-associated oncogene hoinolog 1 (GLI-1) in EGF-regulated enhancement of the invasiveness of the prostate cancer ARCaP(E) cell line in vitro.

METHODSThe expressions of EGFR and GLI-1 in prostate cancer ARCaP(E) cells were analyzed by immunofluorescence staining. ARCaP(E) cells were treated with EGF at 100 ng/ml, followed by detection of the changes in cell morphology and invasiveness, as well as in the expressions of p-ERK, ERK and GLI-1. Migration transwell assay was used to determine the effects of 100 ng/ml EGF and GLI-1 antagonist GANT61 on the invasiveness of the ARCaP(E) cells.

RESULTSBoth EGFR and GLI-1 were expressed in the ARCaP(E) cells. EGF induced morphological transition of epithelial-like ARCaP(E) cells to mesenchymal-like cells, increased their in vitro invasiveness, and significantly upregulated the expressions of p-ERK and GLI-1 in the ARCaP(E) cells (P<0.05). GANT61 significantly inhibited the in vitro invasiveness of the ARCaP(E) cells and reduced the enhancing effect of EGF on their invasiveness (P<0.05).

CONCLUSIONThe results from ARCaP(E) cells shed light on the cross-talk of the HH pathway with the EGF/ERK signaling pathway. GLI-1 might be responsible for EGF-regulated enhancement of the invasiveness of ARCaP(E) cells in vitro.

Cell Line, Tumor ; Epidermal Growth Factor ; metabolism ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Signal Transduction ; Transcription Factors ; genetics ; metabolism ; Zinc Finger Protein GLI1