OBJECTIVE: To investigate the self-renewal mechanism of CD133+ cancer stem cells from Hep-2 cell line. METHOD: The CD133+ cells were sorted by flow cytometry from Hep-2 cell line. Then the sorted CD133+ cells were cultured in RPMI1640. The ability of self-renewal of CD133+ cells were tested by MTT assay. mRNA and protein expression of self-renewal related genes were detected by western blot and RT- PCR. RESULT: (3.10 ± 0.21)% of Hep-2 cells expressed the membrane antigen CD133. CD133+ fraction was raised to (90.20 ± 5.51)% by flow cytometry. In vitro culture and growth curve showed CD133+ cells had more active proliferation ability than CD133- cells, which showed statistically significant difference between these two group (P < 0.01). RT- PCR and western blot results showed upregulated mRNA and protein expression of Fas, c-myc, survivin in CD133+ group (P < 0.01). In the same time, the ratio of Bcl-2/Bax gene expression was obviously increased in CD133+ group. Self-renewal related gene such as β-catenin, SHH, SMOH and Bmi-1,Gli-1 were all up-regulated in CD133+ group both in mRNA and protein. On the contrary, PTCH gene was down-regulated. CONCLUSION CD133 positive cells are a small proportion of a Hep-2 cell line. The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells. Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133+ cells. Cancer stem cells related signal pathways such as Hedgehog, Wnt and Bmi-1 pathway are in state of activation. The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
AC133 Antigen ; Antigens, CD ; Apoptosis ; Cell Physiological Phenomena ; physiology ; Down-Regulation ; Flow Cytometry ; Glycoproteins ; Humans ; Laryngeal Neoplasms ; Neoplastic Stem Cells ; physiology ; Patched Receptors ; Patched-1 Receptor ; Peptides ; Receptors, Cell Surface ; genetics ; metabolism ; Signal Transduction ; beta Catenin ; genetics

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

BACKGROUNDStudies have shown that abnormal activation of the sonic hedgehog pathway is closely related to tumorigenesis in central nervous system. This study aimed to investigate the role of the sonic hedgehog signaling pathway in the occurrence of brainstem and supratentorial glioma.

METHODSReal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to detect the expression of sonic hedgehog-related components in 5 specimens of normal brain tissue, 10 of grade II brainstem glioma, and 10 of grade II supratentorial glioma. The significance of differences between two groups was determined using the Mann-Whitney U test or the two-sample test according to the results of normality distribution tests.

RESULTSThe mRNA expression levels of sonic hedgehog-related genes were higher in brainstem astrocytomas than in supratentorial astrocytomas and normal brain tissue. The level of protein patched homolog 1 (PTCH1) was significantly higher in brainstem astrocytomas than in supratentorial astrocytomas and normal brain tissue (P < 0.01). Immunohistochemistry semi-quantitative analysis was consistent with the qRT-PCR result that PTCH1 expression was increased significantly in brainstem astrocytomas at the protein level (P < 0.05).

CONCLUSIONSEnhanced PTCH1 expression and activation of the sonic hedgehog pathway are involved in brainstem glioma. This may be related to the difference in malignant biological behavior between brainstem and hemispheric glioma, and could be an ideal therapeutic target in brainstem glioma.

Adolescent ; Adult ; Astrocytoma ; genetics ; metabolism ; Brain Stem Neoplasms ; genetics ; metabolism ; Female ; Glioma ; genetics ; metabolism ; Hedgehog Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Patched Receptors ; Patched-1 Receptor ; Real-Time Polymerase Chain Reaction ; Receptors, Cell Surface ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology ; Supratentorial Neoplasms ; genetics ; metabolism ; Young Adult

Medulloblastoma is the most common malignant pediatric brain tumor. Despite its prevalence and importance in pediatric neuro-oncology, the genes and pathways responsible for its initiation, maintenance, and progression remain poorly understood. Genetically engineered mouse models are an essential tool for uncovering the molecular and cellular basis of human diseases, including cancer, and serve a valuable role as preclinical models for testing targeted therapies. In this review, we summarize how such models have been successfully applied to the study of medulloblastoma over the past decade and what we might expect in the coming years.
Animals ; Cerebellar Neoplasms ; genetics ; metabolism ; pathology ; Disease Models, Animal ; Genetic Engineering ; Hedgehog Proteins ; metabolism ; Humans ; Medulloblastoma ; genetics ; metabolism ; pathology ; Mice ; Mice, Transgenic ; Mutation ; Patched Receptors ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptors, Cell Surface ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Smoothened Receptor ; Tumor Suppressor Protein p53 ; genetics ; metabolism

Hedgehog was first described in Drosophila melanogaster by the Nobel laureates Eric Wieschaus and Christiane Nüsslein-Volhard. The hedgehog (Hh) pathway is a major regulator of cell differentiation, proliferation, tissue polarity, stem cell maintenance, and carcinogenesis. The first link of Hh signaling to cancer was established through studies of a rare familial disease, Gorlin syndrome, in 1996. Follow-up studies revealed activation of this pathway in basal cell carcinoma, medulloblastoma and, leukemia as well as in gastrointestinal, lung, ovarian, breast, and prostate cancer. Targeted inhibition of Hh signaling is now believed to be effective in the treatment and prevention of human cancer. The discovery and synthesis of specific inhibitors for this pathway are even more exciting. In this review, we summarize major advances in the understanding of Hh signaling pathway activation in human cancer, mouse models for studying Hh-mediated carcinogenesis, the roles of Hh signaling in tumor development and metastasis, antagonists for Hh signaling and their clinical implications.
Animals ; Antineoplastic Agents ; therapeutic use ; Basal Cell Nevus Syndrome ; drug therapy ; metabolism ; Carcinoma, Basal Cell ; drug therapy ; metabolism ; Cell Differentiation ; Cerebellar Neoplasms ; drug therapy ; metabolism ; Hedgehog Proteins ; antagonists & inhibitors ; metabolism ; Humans ; Medulloblastoma ; drug therapy ; metabolism ; Models, Animal ; Neoplasms ; drug therapy ; metabolism ; Patched Receptors ; Receptors, Cell Surface ; genetics ; metabolism ; Signal Transduction ; drug effects ; Skin Neoplasms ; drug therapy ; metabolism

AIMTo clarify the role of PTCH in patients with NBCCS-related and non-sydromic keratocystic odontogenic tumors.

METHODOLOGYMutation analysis was undertaken in 8 sporadic and 4 NBCCS-associated KCOTs.

RESULTSFour novel and two known mutations were identified in 2 sporadic and 3 syndromic cases, two of which being germline mutations (c.2179delT, c.2824delC) and 4 somatic mutations (c.3162dupG, c.1362-1374dup, c.1012 C>T, c.403C>T).

CONCLUSIONOur findings suggest that defects of PTCH are associated with the pathogenesis of syndromic as well as a subset of non-syndromic KCOTs.

Adolescent ; Adult ; Amino Acid Sequence ; Basal Cell Nevus Syndrome ; genetics ; Chromatography, High Pressure Liquid ; Codon, Nonsense ; genetics ; Codon, Terminator ; genetics ; Conserved Sequence ; genetics ; Cytosine ; Exons ; genetics ; Female ; Frameshift Mutation ; genetics ; Gene Duplication ; Germ-Line Mutation ; genetics ; Guanine ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Mutation, Missense ; genetics ; Odontogenic Tumors ; genetics ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Deletion ; genetics ; Syndrome ; Threonine ; genetics ; Thymine

OBJECTIVETo investigate the significance of sonic hedgehog (Shh), indian hedgehog (Ihh), smoothened (Smo) and patched (Ptch) expressions in uterine cervical lesions and their relationships with HPV type 16 infection.

METHODSTotally 183 cases of cervical lesions, including 32 non-neoplastic cervix, 71 cervical intraepithelial neoplasia (28 CINI, 18 CINII, and 25 CINIII) and 80 squamous cell carcinomas (SCC) were selected from the Department of Pathology, Yanbian University Hospital, Yanbian Women Hospital, and Yanbian Tumor Hospital. Shh, Ihh, Ptch and Smo proteins expression were investigated by immunohistochemistry using tissue microarry platform, and the presence of HPV type 16 was detected by PCR method.

RESULTSImmunohistochemical staining showed that the frequencies of Shh, Ihh, Ptch and Smo expression were rare in normal cervical epithelium, but were strongly expressed in cervical cancer and its precursor lesions (CINII/III) (P < 0.01, P < 0.01, P < 0.05, P < 0.05, respectively). In cervical cancer, the expression rate of Shh (95%) was higher than that of CIN (CINI to CINIII) (46.4%, 61.1%, 80.0%, respectively, P < 0.05). HPV16 was positive in 77.5% of SCC. In cervical cancer, the expression of Shh was related with HPV16 infection (P < 0.05), and the expression of Smo was correlated with lymph node metastasis (P < 0.05).

CONCLUSIONSShh, Ihh, Ptch, and Smo genes may play important roles in the development of cervical cancer. Detection of Hedgehog signaling pathway molecules seems helpful for the early diagnosis of cervical cancer and its precursor lesions, and are potentially therapeutic targets as well.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Female ; Hedgehog Proteins ; metabolism ; Human papillomavirus 16 ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Papillomavirus Infections ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Smoothened Receptor ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology ; Young Adult

OBJECTIVETo investigate the effects of the tumor suppressor gene PTC on the growth inhibition and down-regulation of epidermal growth factor receptors (EGFR) in pulmonary adenocarcinoma cell A549.

METHODSPulmonary adenocarcinoma cell A549 were divided into wild type group, mutant type group, blank group and control group. They were transfected with wild-type PTC1 plasmids, mutant-PTC1 plasmids and blank plasmids, respectively. After transfection, the cell growth curve was drown every day for a week. The expression of PTC1 and EGFR were detected by western blot. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.

RESULTSAfter transfected with wild-type PTC1, the growth rates of A549 cells were slow down, but the other groups of cells had no change. Compared with the control group, the expression of EGFR were down-regulated. The apoptosis rates in wild type group was 24.5%, and the mutant type group was 8.3% (P < 0.01). But the apoptosis rate of blank group has no change.

CONCLUSIONWild-type PTC1 could induce apoptosis and inhibitory effect on A549 cells.

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Patched Receptors ; Plasmids ; genetics ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Transfection

BACKGROUNDNevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disease characterized by a combination of development anomalies and a predisposition to tumour formation. Mutation of patched gene (PTCH), considered the molecular defect of NBCCS, in a Chinese NBCCS family was investigated in this study.

METHODSGenomic DNA was isolated from blood samples of all 12 members of this family. The mutated PTCH gene was screened by polymerase chain reaction amplification and direct sequencing.

RESULTSA new mutation of 3 bp (GAT deletion) was found in all seven affected members of this family. This mutation caused one aspartate deletion in the fourth transmembrane domain of the PTCH protein located within the sterol sensing domain (SSD). This deletion was not found in any unaffected members of this family nor in 200 control samples.

CONCLUSIONSOur findings suggest that one 3-bp deletion in PTCH gene was the cause of nevoid basal cell carcinoma in a Chinese family through affecting the conformation and function of PTCH protein.

Basal Cell Nevus Syndrome ; genetics ; Humans ; Mutation ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; genetics

OBJECTIVETo investigate whether the sonic hedgehog signaling pathway is involved in the development of human fetal prostate, and to evaluate the changing staining patterns of its molecules, sonic hedgehog (SHH), patchedl (PTC1), smoothened (SMO), and GLI1, in the human fetal prostate at various gestation stages.

METHODSFifteen human fetal prostate specimens at various developmental stages (10 - 39 weeks) were included in this study. SHH, PTC1, SMO and GLI1 were detected in all the specimens by immunohistochemical technique. All the slides were observed and assessed under the light microscope.

RESULTSSHH, PTC1, SMO and GLI1 could be detected in human fetal prostate tissues, and their expression formed two surges, the former at week 16, and the latter at week 28. The staining of SHH and SMO was distributed only in the ductal epithelium but not in the stroma. The expression of PTC1 and GLI1 could be found mainly in the epithelium, with minimal staining in the stroma.

CONCLUSIONThe sonic hedgehog signaling pathway is involved in the development of the human fetal prostate. The high expression of its molecules at early gestation stages might be associated with the induction of prostatic buds, while their abundant expression at later gestation stages might be related to the prostate ductal branching, growth, differentiation and morphogenesis.

Gene Expression Regulation, Developmental ; physiology ; Hedgehog Proteins ; biosynthesis ; Humans ; Male ; Oncogene Proteins ; biosynthesis ; Patched Receptors ; Prostate ; embryology ; metabolism ; Receptors, Cell Surface ; biosynthesis ; Receptors, G-Protein-Coupled ; biosynthesis ; Signal Transduction ; physiology ; Smoothened Receptor ; Trans-Activators ; biosynthesis ; Zinc Finger Protein GLI1