Gli1 Protein Expression Research Articles

Abstract 3729: Inhibition of non small-cell lung cancer by stress reduction

Abstract Non small-cell lung cancer (NSCLC) is the leading type of lung cancer with a poor prognosis. Smoking is a risk factor but NSCLC develops in a significant number of nonsmokers. We have reported that social stress significantly promoted the growth of NSCLC xenografts, a response mediated by multiple cAMP-driven signaling cascades downstream of Gαi-coupled beta-adrenergic receptors activated by the stress neurotransmitters norepinephrine and epinephrine and reversed by γ-aminobutyric acid (GABA) via Gαi-coupled GABA-B-receptor signaling. In addition, improved clinical outcomes in NSCLC patients with incidental beta-blocker therapy have been reported. These findings suggest that psychological stress with the associated increase in systemic stress neurotransmitter levels and suppression of physiological agonists for Gαi-coupled receptors may significantly contribute to the high incidence and poor therapeutic response of NSCLC. On the other hand, these findings suggest that stress reduction may have significant inhibitory effects on NSCLC. To test this hypothesis, we achieved stress reduction in athymic nude mice by maintaining the animals in larger cages and by providing them with several enrichment items, resulting in reduced systemic levels of corticosterone, norepinephrine and epinephrine while the levels of GABA and the physiological agonists for Gαi-coupled opioid receptors, dynorphin A and B and met-enkephalin increased. We found that stress reduction significantly inhibited the establishment of xenografts and significantly reduced xenograft sizes. Xenografts in the stress reduction group expressed lower levels of cAMP, VEGF and sonic hedgehog (SHH) accompanied by reduced protein expression of p-ERK, p-AKT, p-CREB, p-SRc and Gli1 while cleaved caspase-3 and p53 proteins were induced. Based on the observed reduction of the cancer stem cell markers SHH and Gli1 in xenograft tissues, we conducted additional mechanistic experiments with cancer stem cells enriched from three NSCLC cell lines under selective sphere formation conditions over 21 days with subcultures every seven days. We found that epinephrine significantly increased stem cell self renewal associated with increased intracellular cAMP, increased levels of the stem cell markers SHH and aldehyde dehydrogenase-1 and increased expression of Gli1. Simultaneous treatment of the enriched cancer stem cells with GABA or dynorphin B completely reversed all of these effects. We conclude that stress reduction can act as a powerful inhibitor of NSCLC by restoring cAMP homeostasis and should be incorporated as important component into existing NSCLC prevention and therapy protocols to improve clinical outcomes. Supported in part by 5RC1CA144640 and State of Tennessee Center of Excellence for Human and Livestock Disease Citation Format: Jheelam Banerjee, Arokya M. S Papu John, Hildegard M. Schuller. Inhibition of non small-cell lung cancer by stress reduction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3729. doi:10.1158/1538-7445.AM2015-3729

Abstract 3245: Targeted inhibition of Hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS) reduces cell survival in vitro and tumor growth in vivo

Abstract RMS is the most common sarcoma of childhood. Approximately 60% of embryonal RMS (ERMS) and more rarely alveolar RMS (ARMS) express HH pathway components. However, roles for the HH signal transduction pathway in RMS biology and as a therapeutic target in RMS have been incompletely characterized. Therefore, we determined if RMS cells lines have HH signaling pathways that are responsive to pathway activation or inhibition in vitro. We demonstrated expression of HH pathway genes (Indian HH, Desert HH, PTCH1, SMO, GLI1, GLI2, and GLI3) by qRT PCR in ERMS (Rh18, Ruch2, SMS-CTR, and RD) and ARMS (Rh 30 and Rh41) cells. We showed high GLI1 protein expression, the downstream mediator of HH signaling, by Western blot in Rh18, Rh30, and Rh41 cells but not in RD or Rh28 cells. Exposure of Rh41 cells to Sonic HH-containing conditioned media caused a 12-fold increase in PTCH1 expression and exposure of Rh28 cells to Sonic HH peptide increased GLI1 expression 1.5-fold, suggesting intact HH signal transduction in some RMS cell lines. Down-regulation of HH pathway activity with the GLI1 inhibitor GANT61 reduced Rh41 and RD cell viability by MTT assays in a dose dependent manner. Reduced cell viability appeared to result at least in part from down-regulation of the anti-apoptotic GLI1 target gene, BCL2. These results suggest a role for HH signaling in RMS cell survival. Next, we used subcutaneous xenografts (with either Rh30 cells, n = 4; Rh41cells, n = 3; or RD cells, n = 3) to assess whether the HH pathway represents a potentially important therapeutic target in RMS in vivo. Mice were treated with either cyclopamine (subcutaneous SMO inhibitor), GANT61 (subcutaneous GLI1 inhibitor), or Vismodegib (oral SMO inhibitor) for 16 days once the tumor volume was 250 mm3. The study endpoints were HH pathway inhibition (qRT PCR), tumor volume (mm3), proliferation (BrdU immunostain), and apoptosis (Caspase immunostain) on day 17 of treatment. Cyclopamine and GANT61 reduced GLI1, PTCH1, and BCL2 expression in 7/10 and 4/10 mice, respectively. We observed partial responses to cyclopamine and GANT61 in Rh41 xenograft mice; GANT61 caused significant regression in tumor volume from baseline (p = 0.03). We did not detect changes in proliferation or apoptosis post-therapy by immunohistochemistry. Our results suggest that targeted inhibition of the HH signaling pathway with small-molecule inhibitors sometimes inhibits RMS xenograft tumor growth in vivo and support further RMS xenograft studies, assessing HH pathway inhibitors in combination with chemotherapeutic agents. Citation Format: Joon Won Yoon, Christopher Chandler, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Gregory Taborn, Philip Iannaccone, David Walterhouse. Targeted inhibition of Hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS) reduces cell survival in vitro and tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3245. doi:10.1158/1538-7445.AM2015-3245

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.

Synergistic inhibition of colon carcinoma cell growth by Hedgehog-Gli1 inhibitor arsenic trioxide and phosphoinositide 3-kinase inhibitor LY294002.

The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer.

Background:Activation of the PI3K/mTOR and Hedgehog (Hh) signalling pathways occurs frequently in biliary tract cancer (BTC). Crosstalk between these pathways occurs in other gastrointestinal cancers. The respective signalling inhibitors rapamycin and vismodegib may inhibit BTC synergistically and suppress cancer stem cells (CSCs).Methods:Gene expression profiling for p70S6k and Gli1 was performed with BTC cell lines. Tumour and pathway inhibitory effects of rapamycin and vismodegib were investigated in BTC preclinical models and CSCs.Results:Rapamycin and vismodegib synergistically reduced BTC cell viability and proliferation. This drug combination arrested BTC Mz-ChA-1 cells in the G1 phase but had no significant effect on the cell cycle of BTC Sk-ChA-1 cells. Combined treatment inhibited the proliferation of CSCs and ALDH-positive cells. Nanog and Oct-4 expression in CSCs was decreased by the combination treatment. Western blotting results showed the p-p70S6K, p-Gli1, p-mTOR, and p-AKT protein expression were inhibited by the combination treatment in BTC cells. In an Mz-ChA-1 xenograft model, combination treatment resulted in 80% inhibition of tumour growth and prolonged tumour doubling time. In 4 of 10 human BTC specimens, tumour p-p70S6K and Gli1 protein expression levels were decreased with the combination treatment.Conclusions:Targeted inhibition of the PI3K/mTOR and Hhpathways indicates a new avenue for BTC treatment with combination therapy.

Sonic Hedgehog Signaling Mediates Resveratrol to Increase Proliferation of Neural Stem Cells After Oxygen-Glucose Deprivation/Reoxygenation Injury in Vitro

Background/Aims: There is interest in drugs and rehabilitation methods to enhance neurogenesis and improve neurological function after brain injury or degeneration. Resveratrol may enhance hippocampal neurogenesis and improve hippocampal atrophy in chronic fatigue mice and prenatally stressed rats. However, its effect and mechanism of neurogenesis after stroke is less well understood. Sonic hedgehog (Shh) signaling is crucial for neurogenesis in the embryonic and adult brain, but relatively little is known about the role of Shh signaling in resveratrol-enhanced neurogenesis after stroke. Methods: Neural stem cells (NSCs) before oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were pretreated with resveratrol with or without cyclopamine. Survival and proliferation of NSCs was assessed by the CCK8 assay and BrdU immunocytochemical staining. The expressions and activity of signaling proteins and mRNAs were detected by immunocytochemistry, Western blotting, and RT-PCR analysis. Results: Resveratrol significantly increased NSCs survival and proliferation in a concentration-dependent manner after OGD/R injury in vitro. At the same time, the expression of Patched-1, Smoothened (Smo), and Gli-1 proteins and mRNAs was upregulated, and Gli-1 entered the nucleus, which was inhibited by cyclopamine, a Smo inhibitor. Conclusion: Shh signaling mediates resveratrol to increase NSCs proliferation after OGD/R injury in vitro.

Apigenin inhibits the self-renewal capacity of human ovarian cancer SKOV3‑derived sphere-forming cells.

Casein kinase 2 (CK2) is a protein kinase which is frequently activated in cancer. The Hedgehog (Hh) signaling pathway is involved in the stimulation of cancer stem cell growth. Its aberrant activation has been validated in several types of cancer, including ovarian cancer. In the present study, the sphere‑forming cells (SFCs) of the human ovarian cancer SKOV3 cell line were observed to have self‑renewal capacity, indicating the possession of ovarian cancer stem‑like cell properties. SKOV3‑derived SFCs had higher levels of CK2α and glioma‑associated oncogene 1 (Gli1) proteins compared with those of parental cells. Apigenin, a common flavonoid, significantly inhibited the self‑renewal capacity and the protein expression of CK2α and Gli1 proteins in the SKOV3‑derived SFCs, which occurred in a concentration‑dependent manner. In addition, CK2α small interfering RNA downregulated the protein expression of CK2α and Gli1 and synergistically inhibited the self‑renewal capacity of the SKOV3‑derived SFCs with apigenin. However, forced overexpression of CK2α resulted in an increase in the expression of CK2α and Gli1 and attenuated the apigenin‑inhibited self‑renewal effect in the SKOV3‑derived SFCs. These results suggested that apigenin inhibited the self‑renewal capacity of SKOV3‑derived SFCs and was involved in downregulating the expression of Gli1 by the inhibition of CK2α.

Analysis of oncogene GLI1 protein expression levels between differing grades of astrocytoma

Aberrant expression of oncogene GLI1 has been linked to malignancies in many types of tissues including brain, pancreas, skin and breast. It has been found that GLI1 expression is important for tumor progression and has been linked to tumor grade. In this study, GLI1 expression has been analyzed in different malignancy grade astrocytomas. GLI1 protein expression has been evaluated in 87 clinical tumor samples by using Western blot analysis. Associations between GLI1 expression and tumor grade were analyzed by applying Mann-Whitney or Kruskal-Wallis tests. KaplanMeier survival analysis was performed to evaluate the prognosis of patients. Western blot analysis did not show statistically significant correlation between GLI1 protein expression and astrocytoma tumor grade (P = 0.294). Kaplan-Meier survival also did not show correlation between survival rates and GLI1 expression (P = 0.081). Our results show that GLI1 protein expression levels in astrocytomas have higher variability than previously shown. Our results indicate that GLI1 protein expression is not an absolute requirement for the process of gliomagenesis. Yet, a relatively high (52%) occurrence of GLI1 expression in astrocytomas suggests the need for further analysis on the involvement of GLI1-mediated Hedgehog signaling pathway in glioma tumorogenesis.

Abstract 121: Sulforaphene inhibited migration through down-regulating the Hedgehog signaling in SUM159 human breast cancer cells

Abstract Accumulating evidences have demonstrated the antineoplastic properties of sulforaphane, a major isothiocyanates present in cruciferous vegetables. However, the natural analogue of sulforaphane, sulforaphene, rich in radish, has yet not been extensively studied for its potential in preventing cancer. In this study, sulforaphene exerted inhibiting effect on migration of SUM159 human breast cancer cells, the migrated cells were reduced from 72.3% in control group to 17.5% in sulforaphene(10uM) treated group. The Hedgehog signaling, known to be involved in cancer invasiveness, was found to be regulated by sulforaphene. The mRNA and protein expression of Gli1 and Smoothened(Smo) which were Hedgehog signaling activating mediators were significantly suppressed by sulforaphene, but the expression of Patched1(Ptch1) an inhibiting mediator, was induced. In addition, the Hedgehog signaling inhibitors, cyclopamine and vismodegib, strongly inhibited the migration of SUM159 breast cancer cells, suggesting the association of Hedgehog signaling with the migration of breast cancer cells. Taken together, sulforaphene significantly inhibited the migration via down-regulating the Hedgehog signaling pathway and suggest that sulforaphene may be considered as a potent phytochemical in suppressing the mammary carcinogenesis. Citation Format: Cheng Bao, Jaehoo Lee, Jiwon Ko, Hyun-Chang Park, Hong jin Lee. Sulforaphene inhibited migration through down-regulating the Hedgehog signaling in SUM159 human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 121. doi:10.1158/1538-7445.AM2014-121

Co-Activation of Hedgehog and Met Pathways in Mediating Resistance to Gefitinib in Non-Small Cell Lung Cancer Cell Line Harboring Activating Mutation of Egfr

ABSTRACT Aim: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective treatments in the management of patients harboring EGFR activating mutations. However, all initially responsive patients experience relapse as a result of acquired drug resistance. The epithelial-to-mesencymal transition (EMT) is a critical event in the development of metastases and represents an important mechanism of acquired resistance to molecularly targeted agents in non-small cel lung cancer (NSCLC). Among the various molecular pathways, the Hedgehog (Hh) signaling cascade has emerged as an important mediator of EMT. Methods: We studied the activation of Hh signaling in a NSCLC model of acquired resistance to gefitinib (HCC827-GR) obtained from a cell line harboring an activating mutation (del exon 19) of EGFR (HCC827). Results: HCC827-GR cells displayed an EMT phenotype, along with higher expression aand increased activation of MET, MAPK and AKT. Asignificantly increased expression of Shh, Smo and Gli1 mRNAs and proteins were evidenced as compared to parental HCC827 cells. Combined inhibition of MET and Hh pathways strongly inhibited cell proliferation, migration, invasion, reverted the mesenchymal phenotype and induced apoptosis at higher level than single inhibition of each pathway. MET and Smo proteins co-precipitated in HCC827-GR cells suggesting a reciprocal activation with a canonical activation of Gli1. Furthermore, MET inhibition strongly reduced Gli1 trascriptional activity, indicating a direct activity of MET-activated downstream pathways on Gli1 activation suggetsing also a cMET-mediated non canonical activation of Gli1. Conclusions: Our data suggest that both MET and Hh pathways provide alternative proliferation and survival mechanisms for NSCLC cancer cells harboring EGFR activating mutations in which EGFR is blocked by gefitinib. Disclosure: All authors have declared no conflicts of interest.

Prognostic impact of the expression of Hedgehog proteins in cervical carcinoma FIGO stages I–IV treated with radiotherapy or chemoradiotherapy

ObjectiveHedgehog signaling proteins were assessed in patients with cervical carcinoma receiving chemoradiation. Associations between five Hedgehog proteins and prognosis were studied. MethodsIn all, 131 cases of cervical carcinomas (FIGO stages I–IV) were immunohistochemically (IHC) analyzed for Patched (PTCH), Smoothened (SMO), and GLI1, GLI2 and GLI3 protein expression. Associations between Hedgehog protein expressions, clinicopathological factors, and clinical outcome data were examined. ResultsPositive IHC staining for the five Hedgehog proteins was recorded in 8% to 37% of the tumor cells. The highest frequency was noted for SMO and the lowest for GLI1. There was a significant association between low SMO- and GLI2-expression and KRAS-mutation. Tumors with overexpressed SMO had a higher frequency of residual tumor or local recurrences than tumors with low SMO expression. Patients with tumors expressing PTCH in more than 75% of the cells had significantly (P=0.023) better recurrence-free survival than patients with tumors with low expression. The opposite situation was true for SMO. For GLI2, there was a statistically significant difference with regard to overall (P=0.004) and distant (P=0.015) relapse rate for groups with expression of GLI2 in the range of 5–25% compared to higher rates. ConclusionsA predictive and prognostic value was found for PTCH, SMO, and GLI2 with regard to residual carcinoma, local recurrences, and for GLI2 distant relapses. The Hedgehog signaling pathway also seems to play an important role in cervical carcinogenesis together with HPV16-infection and KRAS-mutation.

The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling

BackgroundThe crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma.MethodssiRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response.ResultsAlthough knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined.ConclusionOur results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-600) contains supplementary material, which is available to authorized users.

Expressions of sonic hedgehog, smoothened and Glioblastoma transcriptional factor in human pancreatic cancer and their correlation with clinicopathologic characteristics

Objective To investigate the differential expression of sonic hedgehog (Shh),Smoothened (Smo) and Glioblastoma transcriptional factor (Gli1) in the pancreatic cancers and their adjacent tissues,and their correlation with pathogenesis of pancreatic cancer.Methods Reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blotting were used to determine the mRNA and protein expression of Shh,Smo and Gli1 in 50 cases of pancreatic cancers and their adjacent tissues.The relationship between the expression of Shh,Smo and Gli1 and the clinicopathological features of pancreatic cancer was analyzed.Results The expression of Shh,Smo and Gli1 mRNA in pancreatic cancer was 0.309 ± 0.162,0.327 ±0.264 and 0.341 ±0.132,and that in the para-cancerous adjacent tissue was 0.184 ± 0.227,0.148 ±0.276 and 0.105 ±0.351 respectively (P ＜0.05).The expression of Shh,Smo and Gli1 protein in pancreatic cancer was 0.624 ±0.139,0.547 ±0.418 and 0.563 ±0.271,and that in paracancerous adjacent tissue was 0.388 ± 0.294,0.294 ± 0.152 and 0.328 ± 0.129 respectively (P ＜ 0.05).The expression levels of Shh,Smo and Gli1 mRNA and protein in pancreatic cancer tissue were higher than in paracancerous adjacent tissue.The overexpression of Shh,Smo and Gli1 was correlated with differentitation of pancreatic cancer cells (P ＜ 0.05),but not with age,tumor size,TNM grade,invasion to nerves and vascellum,lymph node metastasis and distant metastasis (P ＞ 0.05).Conclusion The expression level of Shh,Smo and Gli1 is increased in human pancreatic cancer cells.The overexpression of hedgehog signaling pathway might play an important role in carcinogenesis and development of pancreatic cancer. Key words: Pancreatic cancer; Sonic hedgehog; Smoothened ; Glioblastoma transcriptional factor ; Hedgehog pathway

Evaluation of Jagged2 and Gli1 expression and their correlation with prognosis in human hepatocellular carcinoma.

Jagged2 is closely associated with numerous congenital diseases and has an important role in multiple malignancies. It has been identified thatJagged2 is a sonichedgehog‑regulated factor. However, its expression and correlation with Gli1 in hepatocellular carcinoma (HCC) remain unknown. A total of 58 samples of surgically resected paired HCC and normal tumor‑adjacent tissues were collected, and the Jagged2 and Gli1 expression was detected in the samples using immunohistochemical staining. The correlation between Jagged2 and Gli1 protein expression, and their correlation with the clinicopathological features of HCC were analyzed. The protein expression of Jagged2 and Gli1 were significantly upregulated in HCC tissues compared with the normal tumor‑adjacent tissues (P<0.001, respectively), and Jagged2 expression was positively correlated with Gli1 protein in HCC tissues (r=0.643, P<0.001). Jagged2 and Gli1 protein were expressed at significantly higher levels in patients with intrahepatic metastasis, high histological grade and advanced tumor-node-metastasis stage (TNM) stage (P<0.05, respectively). With the Cox proportional hazard regression mode, the independent factors predictive of poor long‑term HCC survival following radical liver resection included high expression of Jagged2, advanced TNM stage and high histological grade (P<0.05). In HCC, high expression of Jagged2 was closely correlated with poor clinicopathological features, and it may therefore be a potential prognosis predictor for patients with HCC.

Umbilical cord blood cells regulate the differentiation of endogenous neural stem cells in hypoxic ischemic neonatal rats via the hedgehog signaling pathway

Transplantation of umbilical cord blood mononuclear cells (UCBMC) promotes the proliferation of endogenous neural stem cells (NSCs), but it has been unclear whether the proliferating NSCs can differentiate into mature neural cells. Therefore, we explored the effects of UCBMC transplantation on the differentiation of endogenous NSCs and their underlying mechanisms. Seven-day-old Sprague-Dawley rats underwent left carotid ligation followed by hypoxic stress. UCBMC were transplanted 24h after hypoxia ischemia (HI). BrdU/β-tubulin/HNA/DAPI, BrdU/GFAP/HNA/DAPI, Ngn1/DAPI, and BMP4/DAPI were measured by immunofluorescence staining; Shh, Gli1, Ngn1, and BMP4 proteins were measured by western-blot analysis 28 days after transplantation. More newborn neurons and fewer astrocytes were observed in the HI+UCBMC group, its neuronal percentage was higher, and glial percentage was lower compared with the N+UCBMC (P<0.05) and HI+PBS groups (P<0.01), while fewer newborn neurons and more newborn astrocytes were found in the HI+cyclopamine (an antagonist of the hedgehog protein)+UCBMC group compared with the HI+UCBMC group (P<0.01). The expression of Shh, Gli1, and Ngn1 proteins was higher and BMP4 protein was lower in the HI+UCBMC compared with the HI+PBS group (P<0.01) and the HI+cyclopamine+UCBMC group (P<0.01). Linear regression analysis showed that the differentiation of NSCs correlated with expression of Ngn1 and BMP4 proteins (P<0.01). In conclusion, UCBMC promote neuronal differentiation and reduce glial differentiation in hypoxic ischemic neonatal rats via the hedgehog signaling pathway.

MiR-133b is frequently decreased in gastric cancer and its overexpression reduces the metastatic potential of gastric cancer cells

BackgroundEmerging evidence has shown that microRNAs are involved in gastric cancer development and progression. Here we examine the role of miR-133b in gastric cancer.MethodsQuantitative real-time PCR analysis was performed in 140 patient gastric cancer tissues and 8 gastric cancer cell lines. The effects of miR-133b in gastric cancer cells metastasis were examined by scratch assay, transwell migration and matrigel invasion. In vivo effects of miR-133b were examined in an intraperitoneal mouse tumor model. Targets of miR-133b were predicted by bioinformatics tools and validated by luciferase reporter analyses, western blot, and quantitative real-time PCR.ResultsMiR-133b was significantly downregulated in 70% (98/140) of gastric cancer patients. Expression of miR-133b was negatively correlated with lymph node metastasis of gastric cancer in patients. Similarly, the expression of miR-133b was significantly lower in seven tested gastric cancer cell lines than in the immortalized non-cancerous GES-1 gastric epithelial cells. Overexpression of miR-133b markedly inhibited metastasis of gastric cancer cells in vitro and in vivo. Moreover, the transcriptional factor Gli1 was identified as a direct target for miR-133b. Level of Gli1 protein but not mRNA was decreased by miR-133b. Activity of luciferase with Gli1 3′-untranslated region was markedly decreased by miR-133b in gastric cancer cells. Gli1 target genes, OPN and Zeb2, were also inhibited by miR133b.ConclusionsMiR-133b is frequently decreased in gastric cancer. Overexpression of miR-133b inhibits cell metastasis in vitro and in vivo partly by directly suppressing expression of Gli1 protein. These results suggested that miR-133b plays an important role in gastric cancer metastasis.

Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.

Protein and mRNA expression of Shh, Smo and Gli1 and inhibition by cyclopamine in hepatocytes of rats with chronic fluorosis

In order to investigate the Sonic hedgehog (Shh) signaling pathway and the effect of cyclopamine in rat hepatocytes with chronic fluorosis, 48 Wistar rats were randomly divided into 4 groups. The control group was provided with tap water in which the fluorine concentration was <1mg/L, while the remaining three groups were provided with water containing sodium fluoride (NaF) at a concentration of 50mg/L. After 6 months, the blocking and blocking control groups were injected intraperitoneally once every 2 days for 6 days with 10mg/kg cyclopamine or dimethyl sulfoxide, respectively. The urinary and skeletal fluoride contents were determined by the ion selective electrode method. Levels of aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP) and albumin (Alb) in the serum were determined by using autobiochemical machine. Histological changes in liver tissue were evaluated with Hematoxylin & Eeosin (H&E) staining using light microscopy. The protein and mRNA expression of Shh, Smo and Gli1 in hepatocytes of experimental animals was determined by immunohistochemistry (IHC), Western blotting (Wb) and Real-time quantitative PCR (RT-qPCR). Fluoride content of the urine and bone was increased in the fluorosis and blocking groups compared to those in the control group (P<0.05), while fluoride content in the blocking group was decreased compared to the fluorosis and blocking control groups (P<0.05). The expression of Shh, Smo and Gli1 at the mRNA and protein levels was significantly increased in hepatocytes from the fluorosis and blocking control groups compared with the control group, and expression in the blocking group was lower than that of the fluorosis and blocking control groups. The difference between any two groups was considered to be statistically significant (P<0.05). Taken together, our study indicates that the expression of Shh, Smo and Gli1 at the protein and mRNA level in hepatocytes of rats with chronic fluorosis can be increased by fluoride and may be inhibited by cyclopamine and that the Shh signaling pathway plays an important role in the liver pathogenesis caused by fluorosis.

Genistein attenuates cancer stem cell characteristics in gastric cancer through the downregulation of Gli1

Genistein is an isoflavone from soy with multiple action targets in cellular processes. Hedgehog signaling and its activator Gli1 are involved not only in oncogenesis, but also in cancer stemness and overexpression of CD44, a typical cancer stem cell surface marker. It has been shown that levels of Gli1 and CD44 expression are downregulated by genistein. Genistein may modulate distinctive cellular characteristics in cancer stem cells by inhibiting Gli1-related signaling pathways. In the present study, we sorted cells from MKN45, a human gastric cancer cell line, according to CD44 expression. CD44(+) cells showed properties of cancer stem-like cells and formed sphere colonies. In addition, sonic hedgehog (Shh) signaling genes were upregulated in CD44(+) cells when compared with these levels in CD44(-) cells. When CD44(+) cancer stem-like cells were treated with genistein, Gli1 and CD44 mRNA and protein expression was significantly reduced. Moreover, other stem cell markers were downregulated by genistein. Gli1 siRNA was used to confirm the action of genistein in inhibiting Gli1 expression. The high cell migration capacity of CD44(+) cells was blocked by genistein. in conclusion, genistein inhibits Gli1 gene expression, resulting in the attenuation of cancer stem-like properties in gastric cancer cells. In addition, genistein suppresses the cell invasive capacity that is required for tumor growth and metastasis. Our data showed that genistein can be an effective agent for gastric cancer therapy by targeting cancer stem-like characteristics.

Establishment of human pancreatic cancer cell line with stable knockdown of GLI1 gene

Objective To establish a human pancreatic cancer cell line stably transfected with siRNA expression vector targeting GLI1 gene and examine the interference efficiency.MethodsThe expression of GLI1 gene in five human pancreatic cancer cell lines was detected by quantitative real-time PCR(qRT-PCR);the one with the highest expression level of GLI1 was selected as the target cell line and was transfected with three recombinant plasmids pGCsi-U6-GLI1 siRNA-1,-2,and-3.The positive clones were screened by G418,and the transfection rate was observed by fluorescence microscope.The expression of GLI1 mRNA and protein was analyzed by qRT-PCR and Western blotting analysis,respectively.ResultsPanc-1 cell line was found to have the highest GLI1 expression and was selected as the target cell line for transfection.Plasmids pGCsi-U6-GLI1 siRNA-1,-2,and-3 were successfully transfected into Panc-1 cells separately.After 4 weeks of G418 screening,three stably transfected cell lines named Panc-1/ GLI1 siRNA-1,-2,and-3 were obtained,with the transfection rates all higher than 80%.qRT-PCR and Western blotting analysis showed that the expression levels of GLI1 in Panc-1/ GLI1 siRNA-1,-2,and-3 cells were all significantly lower than those in Panc-1/siControl cells and the blank control cells(P 0.05),with the lowest expression found in Panc-1/ GLI1 siRNA-1 cells.ConclusionWe have successfully constructed a cell line Panc-1/ GLI1 siRNA-1 with GLI1 gene stably silenced,which paving a way for future research.