Sonic Hedgehog mRNA Expression Research Articles

Expression of Hedgehog signalling molecules inmicrocystic adnexal carcinoma.

Microcystic adnexal carcinoma (MAC) is a rare skin neoplasm that has not been characterized on a molecular basis. To assess expression profiles of Hedgehog (HH) signalling molecules in MAC and control tumours. Immunohistochemistry was performed for Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched 1 (PTCH1) and Smoothened (SMO) on patient MAC tissue (n=26) and control tumour tissue, including syringoma (SyG; n=11), trichoepithelioma (TE; n=11) and basal cell carcinoma (BCC; n=12) tissues. Patched 1 and SMO immunoreactivity was significantly higher in BCC than in SyG, TE or MAC (P<0.001 and P<0.03, respectively). The highest IHH expression was observed in BCC and TE compared with SyG and MAC (P<0.04). Notably, the highest SHH protein expression was observed in SyG compared with MAC, TE and even BCC (P<0.001). In patients with MAC, SMO immunoreactivity significantly (r=0.51; P<0.01) correlated with PTCH1 expression. Further correlation studies did not show significant associations between the HH expression markers assessed (P>0.05). Our results indicate that alterations of the HH signalling are unlikely to play a major role in the pathogenesis of MAC, which is in contrast to the morphologically similar BCC and TE. Our observation provides additional information to the limited molecular pathology knowledge on this rare tumour.

Berberine inhibits colorectal tumor growth by suppressing SHH secretion.

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 μM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 μM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

IL-4/IL-13 upregulates Sonic hedgehog expression to induce allergic airway epithelial remodeling.

We have previously demonstrated that upregulation of Sonic hedgehog (SHH) expression in allergic airway epithelia essentially contributes to the goblet cell metaplasia and mucous hypersecretion. However, the mechanism underlying the upregulation of SHH expression remains completely unknown. In cultured human airway epithelial cells, IL-4/IL-13 but not IL-5 robustly induces the mRNA and protein expression of SHH and in turn activates SHH signaling by promoting the JAK/STAT6-controlling transcription of SHH gene. Moreover, intratracheal instillation of IL-4 and/or IL-13 robustly activates STAT6 and concomitantly upregulates SHH expression in mouse airway epithelia, whereas, in Club cell 10-kDa protein (CC10)-positive airway epithelial cells of children with asthma, activated STAT6 closely correlates with the increased expression of SHH and high activity of SHH signaling. Finally, intratracheal inhibition of STAT6 by AS-1517499 significantly diminished the allergen-induced upregulation of SHH expression, goblet cell phenotypes, and airway hyperresponsiveness, in an ovalbumin- or house dust mite-induced mouse model with allergic airway inflammation,. Together, upregulation of SHH expression by IL-4/IL-13-induced JAK/STAT6 signaling contributes to allergic airway epithelial remodeling, and this study thus provides insight into how morphogen signaling is coordinated with Th2 cytokine pathways to regulate tissue remodeling in chronic airway diseases.

Compound Phyllanthus urinaria L Inhibits HBV-Related HCC through HBx-SHH Pathway Axis Inactivation.

Compound Phyllanthus urinaria L (CP) is a traditional formula widely used in clinical practice for hepatocellular carcinoma (HCC), especially HBV-related HCC. HBx, HBV X gene encoded X protein, has positive correlation with the abnormal SHH pathway in HBV-related HCC. So, we predicted that CP has the capability of anti-HBV-related HCC maybe via inactivating the HBx-Hedgehog pathway axis. HepG2-HBx cells, HBx overexpression, were treated with CP (70μg/ml and 35 μg/ml, respectively) for 48 hours and the mice which received the HepG2-HBx cells were treated with CP (625mg/kg and 300 mg/kg, respectively) for 17 days to evaluate the effect of CP on HBV-related HCC. HBx could accelerate HepG2 cells proliferation, clone formation, and migration in vitro and also could strengthen tumor growth in mice. However, CP could significantly decrease HepG2-HBx cells proliferation, clone formation, and migration in vitro and also could inhibit tumors growth in mice in a dose-dependent manner. Mechanism studies suggested that HBx upregulated the mRNA and proteins expression of Sonic hedgehog (SHH), transmembrane receptor patched (PTCH-1), smoothened (SMO), oncogene homolog transcription factors-1 (GLI-1), and oncogene homolog transcription factors-2 (GLI-2), which are compositions of the SHH pathway. CP could inhibit the mRNA and proteins expression of SHH, PTCH-1, GLI-1, and HBx. It may be one of the underlying mechanisms of CP to delay the HBV-related HCC development through the HBx-SHH pathway axis inactivation.

Sonic Hedgehog Signaling Regulates Hematopoietic Stem/Progenitor Cell Activation during the Granulopoietic Response to Systemic Bacterial Infection.

Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 107 CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2–SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition of increase in blood granulocytes following bacteremia. Our results indicate that SHH signaling is critically important in the regulation of hematopoietic stem/progenitor cell activation and reprogramming during the granulopoietic response to serious bacterial infection.

Role of Hedgehog-Gli1 signaling in the enhanced proliferation and differentiation of MG63 cells enabled by hierarchical micro-/nanotextured topography.

Hedgehog–Gli1 signaling is evolutionarily conserved and plays an essential role in osteoblast proliferation and differentiation as well as bone formation. To evaluate the role of the Hedgehog–Gli1 pathway in the response of osteoblasts to hierarchical biomaterial topographies, human MG63 osteoblasts were seeded onto smooth, microstructured, and micro-/nanotextured topography (MNT) titanium to assess osteoblast proliferation and differentiation in terms of proliferative activity, alkaline phosphatase (ALP) production, and osteogenesis-related gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of Sonic hedgehog (Shh), Smoothened (Smo), and Gli1, and the protein levels were assayed by Western blotting. MG63 cells treated with the Smo inhibitor cyclopamine were seeded onto the titanium specimens, and the cell proliferation and differentiation were studied in the presence or absence of cyclopamine. Our results showed that compared to the smooth and microstructured surfaces, the MNTs induced a higher gene expression and protein production of Shh, Smo, and Gli1 as well as the activation of Hedgehog signaling. The enhanced proliferative activity, ALP production, and expression of the osteogenesis-related genes (bone morphogenetic protein-2, ALP, and runt-related transcription factor 2) enabled by the MNTs were significantly downregulated by the presence of cyclopamine to a similar level as those on the smooth and acid-etched microstructured surfaces in the absence of cyclopamine. This evidence explicitly demonstrates pivotal roles of Hedgehog–Gli1 signaling pathway in mediating the enhanced effect of MNTs on MG63 proliferation and differentiation, which greatly advances our understanding of the mechanism involved in the biological responsiveness of biomaterial topographies. These findings may aid in the optimization of hierarchical biomaterial topographies targeting Hedgehog–Gli1 signaling.

Autocrine Sonic hedgehog signaling promotes gastric cancer proliferation through induction of phospholipase Cγ1 and the ERK1/2 pathway.

BackgroundSonic hedgehog (SHH) plays critical roles in cell growth and development. Tumor cells express SHH, which can promote cell proliferation and epithelial-to-mesenchymal transition. However, the autocrine SHH pathway has not been described in gastric cancer. The aim of this study was to explore molecular mechanisms underlying autocrine SHH signaling in gastric cancer cells.MethodsSHH expression was assessed using immunohistochemistry and the results were compared with clinicopathologic parameters, including survival. Using gastric cancer cell lines, we measured SHH mRNA and protein expression, and studied the effects of SHH signaling on cell proliferation and SHH secretion. We also studied the effects of an inhibitor of PLC-γ1 on phosphorylation of phospholipase Cγ1 and extracellular signal-regulated kinases (ERK)1/2.ResultsSHH protein expression in gastric cancer tissue was significantly higher compared with that in normal gastric tissue (P < 0.001), and the increased expression was significantly associated with pT staging (P = 0.004), pN staging (P = 0.018), pM staging (P = 0.006), and pTNM staging (P < 0.001). In multivariate analyses, overall survival in gastric cancer was significantly shorter in cases with high SHH expression (HR = 1.734, 95 % CI: 1.109–2.713, P = 0.016). The AGS and SGC-7901 gastric cancer cell lines expressed SHH mRNA and protein. In these cell lines, SHH promoted carcinogenesis through activation of the PLCγ1-ERK1/2 pathway, resulting in increased cell proliferation and survival.ConclusionsIncreased SHH expression is associated with shorter survival in gastric cancer patients, and SHH could represent a useful biomarker or therapeutic target for this disease.

The Effects of Sonic Hedgehog on Retinal Müller Cells Under High-Glucose Stress.

To investigate sonic hedgehog (SHH) expression and its effects on retinal Müller cells in diabetic rats and in vitro culture systems under high-glucose stress. Diabetic rats were induced by intraperitoneal injection of streptozotocin. Primary rat retinal Müller cells were grown in medium containing 5.5 or 35 mM glucose with SHH and/or cyclopamine. Retinas' and primary Müller cells' expression of SHH pathway components protein and mRNA were determined by Western blot analysis and real-time PCR. The effects of exogenous SHH and its inhibitor cyclopamine on retinal ganglion cells (RGCs) survival after 3-month diabetes were examined by the counting of Brn-3a-labeled RGCs. Phosphoinositide 3-kinase (PI3K), extracellular signal regulated kinases 1 and 2 (ERK1/2), and P38 were detected by Western blot. Both mRNA and protein expression of SHH, SMO, GLI1, and glial fibrillary acidic protein (GFAP) in the retinas of diabetic rats and high-glucose cultured Müller cells increased in a time-dependent manner. Exogenous SHH increased the mRNA and protein expression of SHH, SMO, and GLI1 and cyclopamine reversed that effect. Three months after onset of diabetes, administration of SHH inhibited gliosis significantly and promoted RGC survival. Exogenous SHH upregulated the phosphorylation of PI3K and downregulated ERK1/2, but did not affect the expression of P38. Sonic hedgehog signaling pathway was upregulated in diabetic rat retina and high-glucose cultured Müller cells, and SHH exerted neuroprotective effects on damaged RGCs in a rat diabetes model. The neuroprotective effects of SHH may act indirectly, via Müller cells, through PI3K or ERK1/2 pathways.

Sonic Hedgehog pathway is upregulated in adamantinomatous craniopharyngiomas.

Pituitary stem cells play a role in the oncogenesis of human adamantinomatous craniopharyngiomas (aCPs). We hypothesized that crosstalk between the Wnt/β-catenin and Sonic Hedgehog (SHH) pathways, both of which are important in normal pituitary development, would contribute to the pathogenesis of aCPs. To explore the mRNA and protein expression of components of the SHH signaling pathway in aCPs and their relationship with the identification of CTNNB1/β-catenin mutations and patients outcomes. In 18 aCP samples, CTNNB1 was sequenced, and the mRNA expression levels of SHH pathway members (SHH, PTCH1, SMO, GLI1, GLI2, GLI3, and SUFU) and SMO, GLI1, GLI3, SUFU, β-catenin, and Ki67 proteins were evaluated by quantitative real-time PCR and immunohistochemistry respectively. Anterior normal pituitaries were used as controls. Associations between molecular findings and clinical data were analyzed. The aCPs presented higher mRNA expression of SHH (+400-fold change (FC); P<0.01), GLI1 (+102-FC; P<0.001), and GLI3 (+5.1-FC; P<0.01) than normal anterior pituitaries. Longer disease-free survival was associated with low SMO and SUFU mRNA expression (P<0.01 and P=0.02 respectively). CTNNB1/β-catenin mutations were found in 47% of the samples. aCPs with identified mutations presented with higher mRNA expression of SMO and GLI1 (+4.3-FC; P=0.02 and +10.2-FC; P=0.03 respectively). SMO, GLI1, GLI3, and SUFU staining was found in 85, 67, 93, and 64% of the samples respectively. Strong GLI1 and GLI3 staining was detected in palisade cells, which also labeled Ki67, a marker of cell proliferation. The upregulation of SHH signaling occurs in aCPs. Thus, activation of Wnt/β-catenin and SHH pathways, both of which are important in pituitary embryogenesis, appears to contribute to the pathogenesis of aCP.

Hypermethylation of SHH in the pathogenesis of congenital anorectal malformations

ObjectiveThis study sought to examine promoter methylation and expression of the identified sonic hedgehog (SHH) gene in terminal rectal tissues of children with congenital anorectal malformations (ARMs). MethodsTissue samples from the terminal rectum of pediatric patients with ARMs (five cases each of high and intermediate malformation — two cases of rectovesical fistula, two cases of rectourethral prostatic fistula, one case of cloaca with >3cm common channel, four cases of rectourethral bulbar fistula and one case of imperforate anus without fistula, respectively, and ten cases of low malformation — five cases of perineal fistula and five cases of vestibular fistula, respectively), and patients with non-gastrointestinal tract malformation (six cases, anal fistula) were collected and divided into three groups: high-intermediate ARM (ARMhi-int), low ARM (ARMlo), and control (Cont.). Real-time RT-PCR was used to detect mRNA expression levels of the verified differentially methylated gene SHH, and bisulfite genomic sequencing was performed to evaluate DNA methylation in the SHH promoter region. ResultsThe average methylation levels of the SHH promoter were significantly higher in ARMhi-int (0.850±0.030, P=0.0036) and ARMlo (0.540±0.053, P=0.0087) groups than in Cont. group (0.280±0.032). SHH mRNA expression levels were lower in ARMhi-int (0.340±0.015, P=0.0065) and ARMlo (0.530±0.042, P=0.0156) groups than in Cont. group (0.870±0.046). The average methylation levels of the SHH promoter were higher in ARMhi-int group than in ARMlo group (0.850±0.030 vs. 0.540±0.053, P=0.0095), while SHH expression was significantly reduced in ARMhi-int group compared to ARMlo group (0.340±0.15 vs. 0.530±0.042, P=0.0252). The methylation levels of the SHH promoter in ARMhi-int group were negatively correlated with SHH gene expression (r=−0.89, P<0.01). ConclusionsThe SHH gene, which plays a major role in the development of the anorectum and enteric nervous system, is hypermethylated at its promoter, and this is correlated with low levels of SHH gene expression. This epigenetic modification may therefore be responsible for the observed changes in SHH expression, which could in turn underlie the pathogenesis of congenital ARMs.

Sonic Hedgehog Signaling Is Active in Human Adrenal Cortex Development and Deregulated in Adrenocortical Tumors

The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability. The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

Hedgehog-signaling is upregulated in non-producing human adrenal adenomas and antagonism of hedgehog-signaling inhibits proliferation of NCI-H295R cells and an immortalized primary human adrenal cell line

Hedgehog (Hh)-signaling pathway is important in embryonic development. Activation of Hh-signaling is associated with tumorigenesis. Recent studies demonstrate that Hh-signaling is involved in the development of the adrenal gland in mice and is important in regulating adrenal proliferation.We studied the expression of Sonic hedgehog (SHH), Smoothened (SMO), Patched1 (PTCH1) and GLI family zinc finger 1 (GLI1) in human adrenal and in adrenocortical tumors using immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Modulation of GLI1 and SMO messenger ribonucleic acid (mRNA) expression was investigated with forskolin. The role of Hh-signaling was studied in NCI-H295R cells and in an immortalized primary cell line using the Hh-agonist smoothened agonist (SAG) and the Hh-antagonist cyclopamine.The Hh-pathway components SHH, GLI1, PTCH1 and SMO were detectable in all adrenal glands. While in cortisol-producing adenomas (CPA), Hh-signaling expression levels were comparable to that in normal adrenal cortex, a much higher mRNA expression of GLI1, SMO and SHH was observed in non-producing adenomas (NPA). Interestingly, stimulation of cultured adrenal cells with forskolin led to a decrease in expression of GLI1 and SMO mRNAs. Antagonism of Hh-signaling resulted in a lower proliferation rate of adrenocortical cells, while Hh-agonism had no significant effect on adrenal cell proliferation.Our data show Hh-signaling activity in adult adrenal glands. Activation of the PKA pathway results in lower expression of Hh-signaling proteins. This might explain the lower expression of the Hh components GLI1 and SMO in CPA in comparison to the higher expression in NPA. Hh-signaling might be involved in the tumorigenesis of NPA.

Eradication of H. pylori Did Not Improve Abnormal Sonic Hedgehog Expression in the High Risk Group for Gastric Cancer

Sonic hedgehog (SHH) acts as a proliferation factor in both the normal mucosa and in malignant lesions. Helicobacter pylori-associated atrophic gastritis is characterized by loss of SHH. The purpose of this study was to investigate the effects of H. pylori eradication on SHH mRNA and methylation levels in the patients at high risk for gastric cancer comparing to those in the controls. Gastric corpus biopsies taken from 20 patients with endoscopic resection for early gastric cancer and 14 sex- and age-matched controls before and 1 year after eradication were examined for SHH and downstream regulators mRNA expression using whole biopsy specimens and microdissected gastric glands. Methylation of SHH promoter was evaluated using quantitative methylation-specific PCR. SHH mRNA levels eradication were significantly lower (2.75 × 10(-2) vs. 7.37 × 10(-2), P = 0.004) in the cancer group than in the controls. PTCH and BMP4 mRNA levels as well as MUC5AC were significantly increased only in the control group and were significantly higher in the controls than those in the cancer group after eradication. After eradication, SHH methylation levels in the non-metaplastic glands were significantly higher (86.4% vs. 22.2%, P < 0.001) in the cancer group than in the controls. H. pylori eradication can enhance SHH and its downstream regulators expression diminishing SHH methylation and reverse gastric phenotype, but not in the patients with high risk for gastric cancer.

Paradox of sonic hedgehog (SHH) transcriptional regulation: Alternative transcription initiation overrides the effect of downstream promoter DNA methylation

Recently, DNA methylation has been suggested as a potential mechanism involved in the transcriptional regulation of SHH gene expression in cancer. However, detailed analyses on the underlying transcriptional mechanisms of SHH expression have not been presented so far and were therefore the focus of this study. We found that the genomic region of SHH contains two different transcriptional start sites and four CpG islands spread from the 5' promoter region to the 3' end of the SHH gene. Based on this CpG island topology we analyzed the influence of DNA methylation within the promoter region as well as in exon 2 and exon 3 on SHH mRNA expression in a large set (n = 14) of benign and malignant human cell lines, and further elucidated the functionality of the two identified SHH transcription initiation sites. Methylation-specific PCR (MSP) clearly showed that SHH is expressed independently of DNA methylation within exon 2 and exon 3 of its genomic region, while methylation of the promoter region is able to abrogate SHH expression. Most interesting, we found activation of the upstream SHH promoter in several breast cancer cell lines when the downstream SHH promoter is methylated. These observations lead us to propose a transcriptional model for the SHH gene, in which combined mechanisms of DNA methylation and alternative promoter usage coordinate the transcriptional activity of this important developmental gene.

Expression of sonic hedgehog and β-catenin in human pancreatic cancer

Objective To investigate the expression of Sonic hedgehog(SHH)and WNT/β-cate-nin in human pancreatic cancer and explore its clinical significance.Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were used to determine the mRNA and protein expression of SHH and β-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.The β-catenin protein expression was 71.4% in pancreatic cancer tis-sues.These were significantly different from those of normal tissue adjacent to cancer (P＜0.05).But the expression level of β-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 and β-catenin protein in pancreatic cancer had no correlation with age, tumor size, pathological type and tumor site (P＞0.05), but had a relationship with lymph node metastasis and TNM stage (P＜0.05).The cor-relation between SHH and β-catenin protein was positive (r=0.352, P＜0.05).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 pancreat-ic carcinoma. Key words: Pancreatic neoplasms; Tumor; Sonic hedgehog; WNT; β-catenin

Distinct expression patterns of hedgehog ligands between cultured and primary colorectal cancers are associated with aberrant methylation of their promoters

Ligand-dependent activation of the Hedgehog (Hh) pathway in colorectal cancers (CRCs) is controversial, and the regulation mechanism of Hh ligands expression remains to be determined. In the present study, the mRNA expressions of Sonic hedgehog (SHH), Indian hedgehog (IHH), Patched, Smoothened, and Gli1 were examined in four cultured colon cancer cell lines by reverse transcription-PCR. Moreover, ligands mRNA expression (SHH and IHH) were examined by reverse transcription-PCR, and SHH protein expression by immunohistochemistry in 25 primary CRCs. The methylation status of SHH and IHH was also investigated by bisulfate sequencing or methylation specific PCR. IHH mRNA was completely absent in cell lines studied, and expressed at a very low level or not expressed at all in primary CRCs. Methylation analysis revealed that IHH promoter was hypermethylated in colon cancer cell lines. Absence of SHH mRNA expression and hypermethylation of its promoter was also observed in colon cancer cell lines. However, high level expression of SHH and hypomethylation of its promoter was detected in primary CRCs. In conclusion, ligand dependent activity of Hh pathway is inactive in cultured colon cancer cells correlating to ligands hypermethylation. In contrast, SHH overexpression, possible consequence of promoter hypomethylation, could play a role in the carcinogenesis of primary CRCs.

Expression and significance of sonic hedgehog in human pancreatic cancer

Objective To investigate the expression of sonic hedgehog(SHH) and its clinical significance in human pancreatic cancer(hPC). Methods RT-PCR and immunohistochemistry were used to determine the mRNA and protein expression of SHH in hPC tissue and normal tissues adjacent to the cancer. Results The SHH mRNA expression was detected in 78. 9%(30/38) of the samples of pancreatic cancer, while in only 18. 4%(7/38) of those of normal tissues adjacent to the cancer. Immunohistochemical analysis showed that the SH H protein expression rate was 84.2%(32/38) in pancreatic cancer tissue and 21.1%(8/38) in normal tissues. There was significant difference between them(P 0. 05). Conclusion The expression level of SHH signal molecule is increased in hPC. The overexpression of SHH signaling pathway might play an important role in carcinogenesis and development of pancreatic cancer. Key words: Pancreatic neoplasms; Tumor; Sonic hedgehog

Expression of sonic hedgehog and EGFR in human pancreatic cancer

Objective To investigate the expression of Sonic hedgehog(SHH)and EGFR signaling pathways in human pancreatic cancer.Methods Reveme transcription-polymerase chain reaction (RT-PCR)and Western blot assay were used to detect the mRNA and protein expression of SHH and EGFR in human pancreatic cancer tissue and normal tissues adjacent to cancer.Resuits The SHH mRNA and protein expression was detectable in 81.6%and 79.6%of pancreatic cancer.respectively.The EGFR mRNA and protein expression was both 73.5%in pancreatic cancer tissues.These was significant difference from that of normal tissue adjacent to cancer(P＜0.05).The expression of SHH and EGFR protein in pancreatic cancer had no correlation with age,tumor size,pathological type and tumor site(P＞0.05),but had a relationship with lymph node metastasis and TNM stage(P＜0.05).The relationship between SHH and EGFR protein was positive(r=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. Key words: Pancreas; Tumor; EGFR

Sonic hedgehog mRNA expression by real-time quantitative PCR in normal and tumor tissues from colorectal cancer patients

Shh is expressed in the early stages of embryogenesis and in the foregut development. Although Shh has been shown to be overexpressed in brain, pancreas, gastric and lung cancers, its role in the development of colorectal cancer has not been examined. We used real-time quantitative PCR to assess Shh mRNA expression levels in tumor and matched normal tissue from 57 colorectal cancer patients and correlated the results with patient clinicopathological characteristics. Shh expression levels were higher in tumor tissue than in normal tissue from the same patient ( P=0.00001). Higher levels of Shh expression were associated with early stage disease ( P=0.02). Shh overexpression may influence the development of colorectal cancer.