The Shh/Gli1 signaling pathway regulates regeneration via transcription factor Olig1 expression after focal cerebral ischemia in rats.

OBJECTIVE: Ischemic stroke is a major cause of death in the global population, with a high disability and mortality rate. Lack of regenerative ability is considered to be the fundamental cause. This study aims to determine the effect of Shh pathway, which mediates regenerative signaling in response to CNS injury, on myelin repair and Olig1 expression in focal ischemic lesions in the rat. METHODS: A model of middle cerebral artery occlusion (MCAO) was established using the intraluminal suture method where the middle cerebral artery (MCA) was restricted for 120 min. Cyclopamine, a specific inhibitor of Shh, or saline was administered 12h after MCAO surgery and lasted for 7d. After MCA occlusion, male Sprague-Dawley rats were randomly allocated to cyclopamine- or saline-treated groups. A group of no-injection animals after MCAO were used as control. The Shh signaling pathway, myelinogenesis-related factor MBP and Olig1 were tested using immunohistochemistry and RT-PCR assay. RESULTS: The levels of Shh and its component Gli1 were elevated from 1d up to 14d following ischemia, indicating that the Shh-Gli1 axis was broadly reactivated. Treatment with cyclopamine can partially block the Shh signaling pathway, prevent myelin repair, and decrease the Olig1 expression following ischemic stroke. CONCLUSION: That blockade of Shh signaling concurrently with the creation of a lesion aggravated ischemic myelin damage, probably via its downstream effects on Olig1 transcription. Shh plays a contributory role during regeneration in the CNS, thereby providing promising new therapeutic strategies to assist in recovery from ischemic stroke.

AIM2 inhibits colorectal cancer cell proliferation and migration through suppression of Gli1.

Colorectal cancer (CRC) is a common malignant tumor and is one of the leading causes of cancer-related deaths worldwide. Absent in melanoma 2 (AIM2), as a member of the pyrin-HIN family proteins, plays contentious roles in different types of cancers. In the present work, we provide evidence that AIM2 was commonly downregulated in human CRC and loss of AIM2 significantly correlated with tumor size, depth of invasion, lymph node metastasis (LNM) and TNM (Tumor, Node, Metastases) stage in patients suffering from CRC. AIM2 knockdown promoted CRC cell proliferation, migration and epithelial-mesenchymal transition (EMT) progress, whereas AIM2 overexpression did the opposite. AIM2 inhibited glioma-associated oncogene-1 (Gli1) expression through Smoothened homolog (SMO)-independent pathway and regulated CRC cell proliferation and migration in a Gli1-dependent manner. Moreover, AIM2 could modulate Protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway and the increased Gli1 expression and EMT progress induced by AIM2 depletion was reversed after incubation with AKT inhibitor Ly294002 in CRC cells. In conclusion, our results define AIM2 as a novel regulator of Gli1 in CRC cell growth and metastasis, and suggest that the AIM2/AKT/mTOR/Gli1 signaling axis may serve as a potential target for treatment of CRC.

Cyclopamine Suppresses Human Esophageal Carcinoma Cell Growth by Inhibiting Glioma-Associated Oncogene Protein-1, a Marker of Human Esophageal Carcinoma Progression.

BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. MATERIAL AND METHODS Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. RESULTS Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 µM, (13.37±1.42)% at 5.0 µM, (22.3±2.92)% at 10.0 µM, and (33.57±1.75)% at 20.0 µM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent. CONCLUSIONS Gli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.

Discovery of Novel Macrocyclic Hedgehog Pathway Inhibitors Acting by Suppressing the Gli-Mediated Transcription.

A systemic medicinal chemistry campaign was conducted based on a literature hit compound 5 bearing the 4,5-dihydro-2H-benzo[b][1,5]oxazocin-6(3H)-one core through cyclization of two side substituents of the bicyclic skeleton combined with N-atom walking or ring walking and the central ring expansion or extraction approaches, leading to several series of structurally unique tricyclic compounds. Among these, compound 29a was identified as the most potent against the Hedgehog (Hh) signaling pathway showing an IC50 value of 23 nM. Mechanism studies indicated that compound 29a inhibited the Hh signaling pathway by suppressing the expression of the transcriptional factors Gli rather than by interrupting the binding of Gli with DNA. We further observed that 29a was equally potent against both Smo wild type and the two major resistant mutants (Smo D473H and Smo W535L). It potently inhibited the proliferation of medulloblastoma cells and showed significant tumor growth inhibition in the ptch± ;p53-/- medulloblastoma allograft mice model. Though more studies are needed to clarify the precise interaction pattern of 29a with Gli, its promising in vitro and in vivo properties encourage further profiling as a new-generation Hh signaling inhibitor to treat tumors primarily or secondarily resistant to current Smo inhibitors.

Nanoquinacrine caused apoptosis in oral cancer stem cells by disrupting the interaction between GLI1 and ß catenin through activation of GSK3ß.

Presences of cancer stem cells (CSCs) in a bulk of cancer cells are responsible for tumor relapse, metastasis and drug resistance in oral cancer. Due to high drug efflux, DNA repair and self-renewable capacity of CSCs, the conventional chemotherapeutic agents are unable to kill the CSCs. CSCs utilizes Hedgehog (HH-GLI), WNT-ß catenin signalling for its growth and development. GSK3ß negatively regulates both the pathways in CSCs. Here, we have shown that a nano-formulated bioactive small molecule inhibitor Quinacrine (NQC) caused apoptosis in oral cancer stem cells (OCSCs; isolated from different oral cancer cells and oral cancer patient derived primary cells) by down regulating WNT-ß catenin and HH-GLI components through activation of GSK3ß. NQC activates GSK3ß in transcriptional and translational level and reduces ß catenin and GLI1 as well as downstream target gene of both the pathways Cyclin D1, C-Myc. The transcription factor activity of both the pathways was also reduced by NQC treatment. GSK3ß, ß catenin and GLI1 interacts with each other and NQC disrupts the co-localization and interaction between ß catenin and GLI1 in OCSCs in a dose dependent manner through activation of GSK3ß. Thus, data suggest NQC caused OCSCs death by disrupting the crosstalk between ß catenin and GLI1 by activation of GSK3ß.

Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice.

BACKGROUND: While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb. METHODS: Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression. RESULTS: The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated. CONCLUSION: Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research 109:49-54, 2017. © 2016 Wiley Periodicals, Inc.

MiR-873-5P controls gastric cancer progression by targeting hedgehog-GLI signaling.

Gastric cancer is one of the most common human malignancies. Thus, it is important to explore the specific mechanism in which gastric cancer is induced. The level of miR-873-5p was determined using real time PCR. The expression of Gli1 was determined using western blot and immunohistochemistry. A specific siRNA targeting Gli1 was selected. The role of Gli1 and miR-873-5p on gastric cancer cell viability, apoptosis and cell cycle was determined using MTT assay and flow cytometry, respectively. The 3'untranslated region (3'UTR) of Gli1 was cloned into the pmirGLO plasmid. Dual luciferase reporter assay was applied to determine the target gene of miR-873-5p. The expression of miR-873-5p was decreased in gastric cancer, while the expression of Gli1 was significantly enhanced. Overexpression of miR-873-5P decreased cell viability, increased cell apoptosis and cell cycle arrest. Meanwhile, knockdown of Gli1 obviously induced SGC-7901 cell apoptosis and cell cycle arrest. Dual luciferase reporter assay showed that Gli1 was the target gene of miR-873-5p. More importantly, inhibition of miR-873-5p obviously decreased the protein expression of CyclinB1 and Bcl2 even in cells transfected with si-Gli1. To conclude, MiR-873-5p functions as a tumor suppressor in gastric cancer mainly by targeting Gli1.