Gli1 Protein Expression Research Articles

MODL-10. COMPUTATIONAL DRUG SENSITIVITY PREDICTS TAILORED THERAPY FOR MEDULLOBLASTOMA SUBGROUPS

Abstract BACKGROUND Medulloblastoma is the most common malignant pediatric brain tumor. Tumors are typically characterized as SHH α- δ, WNT α-β, Group 3α- γ, or Group 4 α- γ. Current standard-of-care includes surgery, radiation, and chemotherapy; however, treatment response and prognosis vary widely between subgroups. Despite extensive characterization of medulloblastoma tumors into four molecular subgroups, few subgroup specific therapies have advanced to the clinic and a universal approach is unlikely to be effective due to the considerable intertumor heterogeneity of medulloblastoma. Hence, there is an urgent demand for innovative therapeutic strategies. METHODS To address heterogeneity between patients, we have developed a novel platform called DrugSeq, which predicts drug sensitivities in patients and is able to stratify drug response based on subgroup. We first calculated disease signatures for each patient by normalizing patient gene expression to median gene expression across the Cavalli dataset. Using previously developed transcriptional consensus signatures (TCSs), which contain genes that are consistently dysregulated following drug treatment, we then calculated the discordance between each patient disease signature and drug TCS. In doing so, we were able to identify drugs to target specific molecular subtypes based on Cavalli molecular classifications. RESULTS As a proof of concept we identified vismodegib and curcumin as selective for SHH-MB. Furthermore, we correlated predicted drug response with target protein expression. For example, we found that predicted sensitivity to vismodegib correlates closely with GLI1 protein expression. CONCLUSIONS Collectively, we show that DrugSeq may identify novel therapies and facilitate patient stratification in clinical trials, leading to more successful targeted medulloblastoma therapies.

Expression and significance of Shh, Gli1 and β-catenin in triple-negative breast cancer tissues

Objective: To detect the expression of Shh, Gli1 and β-catenin in triple negative breast cancer tissues and paracancer breast tissues by qRT-PCR and immunohistochemistry and to analyze their correlation with clinicopathological features.Methods: (1) qRT-PCR was used to detect the mRNA expression of Shh, Gli1 and β-catenin in 30 cases of triple negative breast cancer and their paracancer breast tissues, and the correlation among them was analyzed. (2) The expression of Shh, Gli1 and β-catenin proteins in 30 triple negative breast cancer and their paracancer breast tissues was detected by immunohistochemistry, and their correlation with clinicopathological features was analyzed.Results: (1) Shh mRNA expression (1.2334 ± 0.27867), Gli1 mRNA expression (1.2135 ± 0.20636) and β-catenin mRNA expression (1.1421 ± 0.32330) in triple negative breast cancer tissues were higher than that in paracancer breast tissues, i.e., Shh mRNA expression (1.0022 ± 0.06721), Gli1 mRNA expression (1.0003 ± 0.02420) and -catenin mRNA expression (1.0033 ± 0.07920) were significantly different (p < .05). There was a significantly positive correlation between the mRNA expression of Shh and Gli1 (r = .989, p < .001), and between the mRNA expression of Shh and β-catenin (r = .868, p < .001). There was a significantly positive correlation between the mRNA expression of Gli1 and β-catenin expression (r = .869, p < .001). (2) The positive expression rates of Shh, Gli1 and β-catenin in triple negative breast cancer tissues were 93.3% (28/30), 96.7% (29/30) and 93.3% (28/30), respectively, which were higher than those in paracancer tissues 60% (18/30), 73.3% (22/30) and 73.3% (22/30), the differences were statistically significant (p < .05). There was a significantly positive correlation between the mRNA expression of Shh and Gli1 (r = .958, p < .001), and between the mRNA expression of Shh and β-catenin (r = .952, p < .001). There was a significantly positive correlation between the mRNA expression of Gli1 and β-catenin expression (r = .927, p < .001). The expression of Shh, Gli1 and β-catenin protein in triple negative breast cancer was not correlated with age and tumor size (p > .05), but Shh was positively correlated with histological grade (G) (r = .774, p < .001). Furthermore, Gli1 was positively correlated with histological grade (r = .757, p < .001). β-catenin was positively correlated with histological grade (r = .739, p < .001). Shh was positively correlated with TNM staging (r = .460, p = .010). Gli1 was positively correlated with TNM staging (r = .414, p = .023). -catenin was positively correlated with TNM staging (r = .404, p = .027). Shh was positively correlated with lymph node metastasis (r = .540, p = .002). Gli1 was positively correlated with lymph node metastasis (r = .515, p = .004). β-catenin was positively correlated with lymph node metastasis (r = .559, p = .001).Conclusions: (1) The up-regulated expression of Shh, Gli1 and β-catenin proteins in triple negative breast cancer suggests that Shh, Gli1 and β-catenin proteins are involved in tumor genesis. The combined detection of the three proteins may provide a theoretical basis for the diagnosis and prognosis evaluation of triple negative breast cancer. (2) Shh was positively correlated with Gli1 protein expression and β-catenin protein expression. Gli1 was positively correlated with β-catenin protein expression, suggesting that the three types of proteins play a synergistic role in the occurrence and development of TNBC. There may be crosstalk in the Wnt/β-catenin and Hedgehog signaling pathways in TNBC, which may provide a new approach for the treatment of TNBC. (3) The expression of Shh, Gli1 and β-catenin proteins was correlated with the degree of differentiation, TNM staging and lymph node metastasis of triple negative breast cancer, but not correlated with age and tumor size. Therefore, it was predicted that the three types of proteins were related to the invasion, metastasis and prognosis of TNBC.

B13, a well-tolerated inhibitor of hedgehog pathway, exhibited potent anti-tumor effects against colorectal carcinoma in vitro and in vivo

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.

Abstract A031: Combination of hedgehog targeting and autophagy inhibitor potentiate cytotoxicity in 3D spheroids of cervical cancer: A promising therapeutic strategy in metastatic disease

Abstract Cervical cancer is the second most common malignancy in women and a major cause of morbidity and mortality worldwide. Statistics show that present biologic therapy has had little impact on survival in recurrent and metastatic disease. Consequently, there is an urgent need to explore the best treatment options for advanced cervical cancer. Hedgehog (Hh) signaling pathway has been shown to play an important role in metastasis, recurrence and drug resistance of cervical cancer. Therefore, inhibition of hedgehog signaling pathway is emerging as a new strategy for the treatment of advanced cervical cancer. Reports from various solid tumors show that efficacy of targeted therapy can be compromised due to the cytoprotective effect of autophagy upregulation in cancer cells. Therefore, the present study was designed to elucidate the effects of targeting Hh pathway on autophagic flux in cervical cancer spheroids (cervicospheroids). We developed 3D cervicospheroids as an in vitro model to study the Hh signaling and role of autophagy in cervical cancer. Characterisation of the cervicospheroids showed high expression of stemness genes like oct4, sox2 , nanog and CD 133 as well as epithelial to mesenchymal trasition genes like vimentin, snail and twist indicating that spheroids were enriched in cancer stem cell population. Acridine orange staining of 3D spheroids showed that autophagy was also enhanced in the cervicospheroids compared to monolayer culture. We observed a significant positive correlation between the expression of hedgehog mediator, Gli1 and autophagy protein, LC31 in immunohistochemistry data of archival sections of 45 patients of advanced cervical cancer. A higher autophagy flux, as indicated by presence of larger number autophagic vesicle compared to normal cervix, was also observed in these sections. These results further validated our in vitro observations in the 3D model of cervicospheres. MTT data, showed that autophagy help to evade the cytotoxicity of the chemotherapeutic drug, cisplatin in the cervicospheres. We further found an aberrantly activated Hh pathway in the cervicospheroids and observed that the effect of cisplatin and Hh inhibitor, GANT61 was potentiated when the autophagy inhibitor , chloroquine, was added to the 3D spheroid culture. These results led us to conclude that that 3-D spheroid grown in nonadherent conditions can be used as surrogate model to study molecular pathways involved in metastasis. Based on our present findings, we suggest a combinatorial approach targeting both cytoprotective autophagy and Hh pathway as a new therapeutic approach for targeting cancer stem cell population in cervical cancer . Citation Format: Shalmoli Bhattacharyya, Renaissa De, Radhika Srinivasan. Combination of hedgehog targeting and autophagy inhibitor potentiate cytotoxicity in 3D spheroids of cervical cancer: A promising therapeutic strategy in metastatic disease [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A031.

Carvacrol instigates intrinsic and extrinsic apoptosis with abrogation of cell cycle progression in cervical cancer cells: Inhibition of Hedgehog/GLI signaling cascade.

Recent times have seen a strong surge in therapeutically targeting the hedgehog (HH)/GLI signaling pathway in cervical cancer. HH signaling pathway is reported to be a crucial modulator of carcinogenesis in cervical cancer and is also associated with recurrence and development of chemoresistance. Moreover, our previous reports have established that carvacrol (CAR) inhibited the proliferation of prostate cancer cells via inhibiting the Notch signaling pathway and thus, it was rational to explore its antiproliferative effects in cervical cancer cell lines. Herein, the present study aimed to investigate the anticancer and apoptotic potential of CAR on C33A cervical cancer cells and further explore the underlying mechanisms. We found that CAR significantly suppressed the growth of C33Acells, induced cell cycle arrest, and enhanced programmed cell death along with augmentation in the level of ROS, dissipated mitochondrial membrane potential, activation of caspase cascade, and eventually inhibited the HH signaling cascade. In addition, CAR treatment increased the expression of pro-apoptotic proteins (Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c) and concomitantly reduced the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in C33Acells. CAR mediates the activation of caspase-9 and -3 (intrinsic pathway) and caspase-8 (extrinsic pathway) accompanied by the cleavage of PARP in cervical cancer cells. Thus, CAR induced apoptosis by both the intrinsic and extrinsic apoptotic pathways. CAR efficiently inhibited the growth of cervical cancer cells via arresting the cell cycle at G0/G1 phase and modulated the gene expression of related proteins (p21, p27, cyclin D1 and CDK4). Moreover, CAR inhibited the HH/GLI signaling pathway by down regulating the expression of SMO, PTCH and GLI1 proteins in cervical carcinoma cells. With evidence of the above results, our data revealed that CAR treatment suppressed the growth of HPV-C33A cervical cancer cells and further elucidated the mechanistic insights into the functioning of CAR.

High Expression of SMO and GLI1 Genes with Poor Prognosis in Malignant Mesothelioma.

To investigate the value of SMO and GLI1 genes in the hedgehog pathway in malignant mesothelioma specimens. Further study on the expression and prognosis of SMO and GLI1 in malignant mesothelioma tissues and the relationship between the two and the molecular mechanisms of mesothelioma immunity and to further investigate the prognostic value of mesothelioma expression. Immunohistochemistry and RT-qPCR were applied to detect the expression of SMO and GLI1 proteins and mRNA in biopsy specimens and plasma cavity effusion specimens from malignant mesothelioma (n = 130) and benign mesothelial tissues (n = 50) and to analyze the clinicopathological significance and survival risk factors of SMO and GLI1 protein expression in mesothelioma. The mechanisms of mesothelioma cell expression and immune cell infiltration were investigated using bioinformatics methods. SMO and GLI1 in mesothelioma tissues detected high concordance between the diagnostic results of mesothelioma biopsy specimens and plasma cavity effusion specimens. The expression levels of SMO and GLI1 protein and mRNA in mesothelioma tissues were higher than those in benign mesothelioma tissues. The expression levels of SMO and GLI1 protein were correlated with the age, site, and asbestos exposure history of patients with mesothelioma. The expression levels of SMO and GLI1 protein were correlated with the expressions of ki67 and p53 (P < 0.05). SMO and GLI1 gene expression levels were negatively correlated with good prognosis in mesothelioma patients (P < 0.05). Cox proportional risk model indicated that protein expressions of invasion, lymph node metastasis, distant metastasis, staging, and genes were independent prognostic factors of mesothelioma. The GEPIA database showed the overall survival rate and the disease-free survival rate of mesothelioma patients in the high SMO and GLI1 expression groups; the UALCAN database analysis showed lower SMO expression levels in mesothelioma patients with more pronounced TP53 mutations (P = 0.001); GLI1 gene expression levels were strongly correlated with lymph node metastasis in mesothelioma patients (P = 0.009). Timer database analysis showed that the mechanism of immune cell infiltration was closely related to SMO and GLI1 expression. The degree of immune cell infiltration was strongly correlated with the prognosis of mesothelioma patients (P < 0.05). The expression levels of both SMO and GLI1 proteins were higher than those of normal mesothelial tissues, and the mRNA expression levels also changed in the same direction. SMO and GLI1 gene expressions in mesothelioma were negatively correlated with age, site of occurrence, and history of asbestos exposure. Positive expression of SMO and GLI1 was negatively correlated with patient survival. The Cox proportional risk model showed that gender, history of asbestos exposure, site of occurrence, SMO, and GLI1 were independent prognostic factors for mesothelioma. The mechanism of immune cell infiltration in mesothelioma is closely related to the gene expression of both and the survival prognosis of mesothelioma patients.

Neuroprotective Effect of Sonic Hedgehog Mediated PI3K/AKT Pathway in Amyotrophic Lateral Sclerosis Model Mice

The Sonic Hedgehog (SHH) signaling pathway is related to the progression of various tumors and nervous system diseases. Still, its specific role in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), remains studied. This research investigates the role of SHH and PI3K/AKT signaling pathway proteins on ALS development in a SOD1-G93A transgenic mouse model. After injection of SHH and PI3K/AKT signaling pathway inhibitors or agonists in hSOD1-G93A (9 weeks of age) transgenic mice, we studied skeletal muscle pathology using immunohistochemical staining and Western blot methods. In addition, recorded data on rotation time, weight, and survival were analyzed for these mice. Our study showed that the expression of SHH, Gli-1 and p-AKT in ALS mice decreased with the progression of the disease. The expression of p-AKT changed together with Gli-1 while injecting PI3K/AKT signaling pathway inhibitor or agonist; SHH and Gli-1 protein expression remained unchanged; p-AKT protein expression significantly decreased while injecting PI3K/AKT signaling pathway inhibitor. These results indicate that SHH has a regulatory effect on PI3K/AKT signaling pathway. In behavioral experiments, we found that the survival time of hSOD1-G93A mice was prolonged by injection of SHH agonist while shortened by injection of SHH inhibitor. In conclusion, we confirmed that the SHH pathway played a neuroprotective role in ALS by mediating PI3K/AKT signaling pathway.

Inhibitory Effects of Rhein on Renal Interstitial Fibrosis via the SHH-Gli1 Signal Pathway.

Background Rhein is the main extract of Rheum palmatum L., which has been proved to improve the renal function of chronic kidney disease, but its mechanism is not clear. Therefore, this experiment explored the potential pharmacological effect of rhein on renal interstitial fibrosis rats. Methods This study explores the potential pharmacological action of rhein. In this work, we investigate the potential pharmacological action of rhein in unilateral urethral obstruction (UUO) rats. Thirty Sprague Dawley rats were randomly divided into three groups: sham, UUO, and rhein (rhein-treated UUO rats) groups. The left ureters of the UUO group rats were exposed and bluntly dissected. The rhein group rats were administered an intragastric gavage of rhein (2 mg·kg−1·d−1) for 14 d. Kidney function-related indicators were monitored in these rats, while indexes of pathologic aspects were determined histologically. The expression of α-SMA, TGF-β1, SHH, Gli1, and Snail was quantified using real-time polymerase chain reaction and western blotting. The NRK-49F cells were incubated with and without SHH (100 ng·ml−1) for 48 hours. The SHH-activated NRK-49F cells were incubated with cyclopamine (CNP, 20 umol L−1) or rhein (1 ng·ml−1). The Gli1 and Snail mRNA and protein level were detected. Results In the in vivo experiment, the results exhibited that UUO caused renal pathological damages. However, these changes could be significantly reversed by the administration of rhein. Compared with the untreated UUO group, the rhein group showed reduced kidney tubular atrophy and necrosis, interstitial fibrosis, hyperplasia, and abnormal deposition of extracellular matrix. Rhein reduced the RNA and protein expression of SHH, Gli1, and Snail of the UUO rats. In the in vitro experiment, CNP or rhein treatment decreased the expression of Gli1 and Snail on mRNA and protein levels in SHH-induced NRK-49F cells, suggesting that CNP or rhein suppresses SHH-induced NRK-49F activation. Taken together, these results demonstrated that rhein suppresses SHH-Gli1-Snail signal pathway activation, with potential implications for the treatment of renal fibrosis. Conclusions Treatment with rhein remarkably ameliorated renal interstitial fibrosis in UUO rats by regulating the SHH-Gli1-Snail signal pathway.

Metformin exerts anti-tumor effects via Sonic hedgehog signaling pathway by targeting AMPK in HepG2 cells.

Metformin, a traditional first-line pharmacological treatment for type 2 diabetes, has recently been shown to have anti-cancer effects on hepatocellular carcinoma (HCC). However, the molecular mechanism underlying the anti-tumor activity of metformin remains unclear. The Sonic hedgehog (Shh) signaling pathway is closely associated with the initiation and progression of HCC. Therefore, the aim of the current study was to investigate the effects of metformin on the biological behavior of HCC and the underlying functional mechanism of metformin in the Shh pathway. HCC was induced in HepG2 cells using recombinant human Shh (rhShh). The effects of metformin on proliferation and metastasis were evaluated using in vitro proliferation, wound healing, and invasion assays. The mRNA and protein expression levels of proteins related to the Shh pathway were measured using western blotting, quantitative PCR, and immunofluorescence staining. Metformin inhibited rhShh-induced proliferation and metastasis. Furthermore, metformin decreased the mRNA and protein expression of Shh pathway components, including Shh, Ptch, Smo, and Gli-1. Silencing of AMPK in the presence of metformin revealed that metformin exerted its inhibitory effects via AMPK. Our findings demonstrate that metformin suppresses the migration and invasion of HepG2 cells via AMPK-mediated inhibition of the Shh pathway.

Expression of Shh, Gli1, and Cyr61 in Gastric Cancer Predicts Overall Survival of Patients: A Retrospective Study.

This study aimed to evaluate the expression levels of Shh, Gli1, and Cyr61 proteins in gastric cancer tissues and analyze the relationship between these three proteins and the clinicopathological factors and prognosis of patients. This was a retrospective study. Four hundred gastric cancer tissue specimens from patients who underwent radical gastrectomy in Zhangye People's Hospital affiliated to Hexi University between February 2013 and February 2021 underwent immunohistochemical analysis. The positive expression rates of Shh, Gli1, and Cyr61 in gastric cancer tissues were 55.5%, 56.5%, and 64.5%, respectively. The expressions of Shh, Gli1, and Cyr61 in gastric cancer tissues were significantly correlated with tumor size, depth of invasion, and degree of differentiation (P < .05). The expression of Shh protein was positively correlated with the expression of Gli1 protein (P < .01), and the expression of Gli1 protein was positively correlated with the expression of Cyr61 protein (P < .01). Univariate and multivariate analyses showed that the expression of Shh, Gli1, and Cyr61 could predict the prognosis of patients (P < .05). Receiver operating characteristic curve analysis combined with TNM staging could better predict the three-year overall survival of patients (P < .05). Shh, Gli1, and Cyr61 proteins are significantly expressed in gastric cancer tissues and are risk factors for the prognosis of patients with gastric cancer.

The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.

Propofol Inhibits Thyroid Cancer Cell Proliferation, Migration, and Invasion by Suppressing SHH and PI3K/AKT Signaling Pathways via the miR-141-3p/BRD4 Axis.

Objective This study explores the effect and mechanism of propofol for thyroid tumor. Methods Culture human normal thyroid cells Nthy-ori 3-1 and thyroid cancer cell line TPC-1. TPC-1 cells were divided into the propofol group (treated with propofol), miR-141-3p group (transfected with the miR-141-3p mimic), negative control group (transfected with miR-NC), miR-141-3p + pcDNA-BRD4 group (transfected with the miR-141-3p mimic and pcDNA-BRD4), miR-141-3p + pcDNA group (transfected with the miR-141-3p mimic and pcDNA), siBRD4 group (transfected with siBRD4), and si-control group (transfected with si-control). The detection of miR-141-3p and BRD4 expression in cells was done by RT-qPCR, and the dual-luciferase reporter gene method and western blotting were used to verify the targeting relationship between miR-141-3p and BRD4. MTT method was used to test cell proliferation, transwell method was used to test cell migration and invasion, and western blotting was used to test SHH, GLI1, p-PI3K, and p-AKT protein expression. Results Compared with Nthy-ori 3-1 cells, the expression of miR-141-3p in TPC-1 cells was markedly decreased. Propofol treatment and excessive expression of miR-141-3p could influence the phenotype of TPC-1 cells. BRD4 is one of the target genes of miR-141-3p, and its expression is negatively regulated by miR-141-3p. Overexpression of BRD4 can partially reverse the restraining effect of miR-141-3p on the TPC-1 cell phenotype. Both miR-141-3p and BRD4 can regulate the activity of SHH and PI3K/AKT signaling pathways. Conclusion Propofol can inhibit the activity of SHH and PI3K/AKT pathways by targeting downregulating BRD4 through miR-141-3p, thereby inhibiting the phenotype of TPC-1 cells.

Electroacupuncture of "Shuigou"(GV26) improves neurological function by promoting angioge-nesis and Shh signaling in ischemic cerebral tissue of rats with cerebral infarction

To observe the effect of electroacupuncture (EA) of "Shuigou" (GV26) on the activities of sonic hedgehog(Shh) signaling molecules (Shh, Ptch, Smo，Gli and Gli2) in ischemic brain tissues in rats with cerebral ischemia (CI), so as to explore its mechanisms underlying improvement of CI. Male Wistar rats were randomly divided into blank control group(n=10), sham operation group (n=90), model group(n=90) and EA group (n=90). The CI model was established by occlusion of the right middle cerebral artery (MCAO). According to the postoperative time points of MCAO, the later three groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups, with 10 rats in each subgroup. EA (15 Hz, 2 mA) was applied to GV26 for 20 min. The 1 h to 24 h subgroups were treated immediately after modeling, the 3-12 d subgroups treated one time a day. The neurological severity score (NSS, 0 to 18 points) was used to evaluate the rats' neurological function, and TTC staining was employed to assess the cerebral ischemic volume (percentage of cerebral infarct volume, CIV). Western blot was employed to detect the expression of Shh, Ptch, Smo, Gli1 and Gli2 proteins in the ischemic cerebral tissue. Compared with the sham operation group, the NSS scores of the model group increased at all time points (P<0.01). The percentages of CIV of the model group from 3 h to 12 d were obviously higher than those of the sham operation group (P<0.01). The NSS scores at 3, 7 and 12 d and the percentages of CIV at 1, 3, 7 and 12 d after MCAO were significant lower in the EA group than in the model group (P<0.05). The protein expression levels of Shh from 12 h to 12 d (i.e. 12 h, 24 h, 3, 7 and 12 d), Ptch from 6 h to 12 d, Smo from 9 h to 12 d, Gli1 at 9 h, 12 h, and from 3 d to 12 d, Gli2 at 6, 9 and 12 h, and 3 d were significantly higher in the model group than in the sham operation group (P<0.05, P<0.01), while those of Shh at 3, 7 and 12 d, Ptch from 24 h to 7 d, Smo from 12 h to 7 d, Gli1 from 24 h to 7 d, Gli2 at 12 h, 3 and 7 d were significantly higher in the EA group than in the model group (P<0.05, P<0.01). No statistical significances were found between the sham operation and the blank control groups in all the indexes mentioned above (P>0.05). EA of GV26 can improve neurological function and reduce infarct volume in MCAO rats, which may be related to its function in up-regulating the activities of Shh signaling pathway in the ischemic cerebral tissues.

Inoscavin A, a pyrone compound isolated from a Sanghuangporus vaninii extract, inhibits colon cancer cell growth and induces cell apoptosis via the hedgehog signaling pathway

BackgroundSanghuangporus vaninii, a large precious medicinal fungus called Sanghuang in China, has significant antitumor activity. We previously reported that a Sanghuangporus vaninii extract could lead to apoptosis in HT-29 cells through the intrinsic apoptotic pathway. We further found that Inoscavin A exhibited anti-colon cancer activity, but its specific mechanisms have not been fully elucidated. MethodsInoscavin A was obtained from Sanghuangporus vaninii by the classic phytochemical separation technology. The male BALB/c nude mice were injected with HT-29 colon cancer cells as animal model. In order to observe the pathological changes of tumor section, the hematoxylin-eosin(H&E) staining was applied in the histological analysis. Metabolomics was utilized for the investigation of the overall changes of serum metabolites in animal model, and the potential targets of Inoscavin A were analyzed by Ingenuity Pathway Analysis (IPA). We further employed a molecular docking approach to predict the degree of combination of Inoscavin A and Smo. Then we further performed Western blotting and immunofluorescence analysis to investigate the expression of proteins involved in Hh-related pathways in tumor tissues. In addition, the colony formation assay, scratch-wound assay and transwell migration and invasion assay were conducted to evaluate the anti-colon-cancer activity of Inoscavin A. Concurrently, the mitochondrial membrane potential assay and TUNEL apoptosis assay were detected to demonstrate the effect of Inoscavin A on promoting HT-29 cells apoptosis. Western blot experiments verified the anti-tumor effects of Inoscavin A were modulated the protein expression of Shh, Ptch1, Smo and Gli1 in HT-29 cells. ResultsWe showed that Inoscavin A, a pyrone compound isolated from the Sanghuangporus vaninii extract, exerted its antitumor activity in an HT-29 colon cancer cell xenograft mouse model. Subsequently, we first time prove that the antitumor effects of Inoscavin A were related to the hedgehog (Hh) signaling pathway. Furthermore, we demonstrated that Smo, the core receptor of the Hh pathway, was critical for the induction of apoptosis of Inoscavin A and that overexpression of this target could significantly rescue cell apoptosis induced by Inoscavin A treatment. ConclusionThus, our studies first propose that the natural outgrowth Inoscavin A exerted its anti-cancer effects by inhibiting Smo to suppress the activity of the Hh pathway though inhibiting cell proliferation and promoting apoptosis. These findings further indicate that Inoscavin A will be expected to be a prospective remedical compound for the treatment of colon cancer.

20(S)-Ginsenoside Rg3 regulates the Hedgehog signaling pathway to inhibit proliferation and epithelial-mesenchymal transition of lung cancer cells.

20(S)-Ginsenoside Rg3 (S-Rg3) has good antitumor activity and has been used in clinical oral antitumor therapy. However, the effect of S-Rg3 on the Hedgehog (Hh) signaling pathway has not been reported. In this study, we used CCK8, cell wound healing, Transwell, and western blotting assays as well as small interfering RNA to decrease GLI1 protein expression to investigate the effect of S-Rg3 on the Hh pathway in A549 cells. The results showed that S-Rg3 substantially inhibited the proliferation, migration, and invasion of A549 cells in a concentration-dependent manner. Furthermore, S-Rg3 had significant regulatory effects on PTCH1 and GLI1, key proteins in the Hh pathway, causing significant upregulation of PTCH1 levels and downregulation of GLI1 expression. After silencing the Hh signaling pathway, the inhibitory effect of S-Rg3 administration on the expression of epithelial mesenchymal transition-related proteins was further enhanced. Molecular dynamics simulations showed that Rg3 molecules could bind stably to PTCH1 protein through hydrophobic interactions, hydrogen bonds, and π-π stacking forces. Thus, S-Rg3 can regulate Hh signaling pathway transduction in A549 cells to inhibit lung cancer cell proliferation, migration, invasion, and epithelial mesenchymal transition.

Mediators of human ureteral smooth muscle contraction-a role for erythropoietin, tamsulosin and Gli effectors.

BackgroundUreteral contractility is a poorly understood process. Contractions have been demonstrated to occur in the smooth muscle layers of the ureter. Previous work suggests the involvement of Gli family proteins and erythropoietin (EPO) in regulating mammalian ureteral smooth muscle contraction. We sought to devise a method by which the effects of these proteins and tamsulosin on distal human ureteral tissue contractility could be investigated to better understand mechanisms regulating human ureteral function.MethodsIRB approval was obtained to procure portions of extraneous distal ureteral tissue from living donor renal transplants. Contractility was measured by placing the tissue in Krebs buffer and stimulating via a uniform electric current. Contractile force was recorded with each stimulation with and without the presence of a Gli inhibitor (GANT61) or EPO. Each ureteral specimen was subsequently fixed and tested by immunohistochemistry to determine Gli, EPO and alpha-adrenergic receptor activity.ResultsElectrical field stimulation successfully elicited contractions in the ureteral tissue. Administering tamsulosin decreased force and duration of ureteral contractions. Inhibiting Gli signaling decreased contractility and EPO decreased ureteral contractile forces within 5 minutes of administration versus untreated controls. Staining confirmed Gli1 protein and α-adrenergic receptor expression in ureteral smooth muscle and epithelial tissue with EPO receptor expression confined to the epithelial layer.ConclusionsDistal ureteral contractile forces are decreased by inhibition of Gli family proteins and the α-adrenergic receptor. EPO acts within five minutes, suggesting ion channel involvement instead of changes in gene expression. Continuing work will elucidate the role of these proteins in coordinating ureteral contractions. This has implications for the use of pharmacologic methods to address ureteral contractility and dysfunctional peristalsis during stone passage, ureteroscopy, in transplant patients and potentially to reduce symptoms from ureteral stents.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

Silencing glioma-associated oncogene homolog 1 suppresses the migration and invasion of hepatocellular carcinoma in vitro.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-associated death worldwide. Glioma-associated oncogene homolog 1 (Gli1) is a key component and functions as a reliable marker of Hedgehog signaling pathway activation. Previous studies have demonstrated that Gli1 serves important roles in the progression of various types of cancer, including HCC. However, its effect on HCC invasion and metastasis and the underlying mechanism remain to be elucidated. Small interference RNA was employed to silence the Gli1 gene in liver cancer cells. Reverse transcription-quantitative PCR and western blot analysis were performed to evaluate the mRNA and protein expression of Gli1, respectively. A series of assays, including Cell Counting Kit-8, adhesion, wound healing and Matrigel invasion were performed to investigate cell viability, adhesive, migratory and invasive capabilities of liver cancer cells, respectively. In addition, immunofluorescence staining was performed to determine the cellular localization of focal adhesion kinase (FAK), phosphorylated (p-)FAK and p-AKT. The mRNA and protein expression of Gli1 in liver cancer cells (HepG2 and SK-Hep1) were markedly decreased in a dose-dependent manner following Gli1-knockdown. Gli1 silencing significantly inhibited the adhesion, migration and invasion of SK-Hep1 cells. Additionally, knockdown of Gli1 markedly suppressed the expression of metalloproteinase (MMP)-2 and MMP-9. Furthermore, downregulation of Gli1 blocked the FAK/AKT signaling pathway. Gli1 serves significant roles in the migration and invasion of HCC cells through activation of the FAK/AKT signaling pathway and subsequent upregulation of MMP-2 and MMP-9 expression. Thus, Gli1 may be a potential protein target for the regulation of HCC migration and invasion.

Sodium Butyrate Combined with Docetaxel for the Treatment of Lung Adenocarcinoma A549 Cells by Targeting Gli1.

PurposeThis study is aimed to investigate the combined treating efficacy of sodium butyrate and docetaxel on proliferation and apoptosis of the lung adenocarcinoma A549 cell line based on Gli1 regulation in vitro and in vivo.Materials and MethodsRNA interference method was used to overexpress Gli1 in A549 cells. Cells were treated with varying concentrations of sodium butyrate, docetaxel or both in combination. CCK-8, colony formation assay, Hoechst 33258 staining, flow cytometry and TUNEL assay were employed to detect proliferation, cell cycle and apoptosis. qRT-PCR and Western blot analysis were applied to detect the mRNA and protein expression of Gli1. In vivo tumorigenicity was detected by tumor transplantation in nude mice. Downstream protein levels of Gli1 were detected using Western blot assay.ResultsIt was found that sodium butyrate or docetaxel alone, respectively, inhibited proliferation and promoted apoptosis of A549 cells in vitro and in vivo, while the combination of the two generated significantly higher responses, which were also effective in another lung adenocarcinoma cell line H1299. Furthermore, the combined therapy had an additive effect in suppressing Gli1 expression and regulating the expression of its downstream proteins that involve in proliferation, cell cycle and apoptosis of A549 cells in vitro and in vivo, including decreased protein expression of Ki-67, CDK1, CDK2, Cyclin D1, Bcl-2 and Survivin, and increased protein expression of Cyclin A, p21, Bax and cleaved-Caspase 3. On the other hand, Gli1 overexpression perceptibly reversed the above-mentioned additive effect in vitro and in vivo.ConclusionThis study demonstrates that the combined therapy of sodium butyrate and docetaxel additively inhibits proliferation and promotes apoptosis of A549 lung adenocarcinoma cells via suppressing Gli1 expression in vitro and in vivo. Targeting Gli1 by the combined therapy may provide new insights into the therapeutic management of patients with lung adenocarcinoma.

A Novel Therapeutic siRNA Nanoparticle Designed for Dual-Targeting CD44 and Gli1 of Gastric Cancer Stem Cells.

PurposeGastric cancer stem cells (CSCs) are important for the initiation, growth, recurrence, and metastasis of gastric cancer, due to their chemo-resistance and indefinite proliferation. Herein, to eliminate gastric CSCs, we developed novel CSC-targeting glioma-associated oncogene homolog 1 (Gli1) small interfering RNA (siRNA) nanoparticles that are specifically guided by a di-stearoyl-phosphatidyl-ethanolamine- hyaluronic-acid (DSPE-HA) single-point conjugate, as an intrinsic ligand of the CD44 receptor. We refer to these as targeting Gli1 siRNA nanoparticles.MethodsWe used the reductive amination reaction method for attaching amine groups of DSPE to aldehydic group of hyaluronic acid (HA) at the reducing end, to synthesize the DSPE-HA single-point conjugate. Next, targeting Gli1 siRNA nanoparticles were prepared using the layer-by-layer assembly method. We characterized the stem cellular features of targeting Gli1 siRNA nanoparticles, including their targeting efficiency, self-renewal capacity, the migration and invasion capacity of gastric CSCs, and the penetration ability of 3D tumor spheroids. Next, we evaluated the therapeutic efficacy of the targeting Gli1 siRNA nanoparticles by using in vivo relapsed tumor models of gastric CSCs.ResultsCompared with the multipoint conjugates, DSPE-HA single-point conjugates on the surface of nanoparticles showed significantly higher binding affinities with CD44. The targeting Gli1 siRNA nanoparticles significantly decreased Gli1 protein expression, inhibited CSC tumor spheroid and colony formation, and suppressed cell migration and invasion. Furthermore, in vivo imaging demonstrated that targeting Gli1 siRNA nanoparticles accumulated in tumor tissues, showing significant antitumor recurrence efficacy in vivo.ConclusionIn summary, our targeting Gli1 siRNA nanoparticles significantly inhibited CSC malignancy features by specifically blocking Hedgehog (Hh) signaling both in vitro and in vivo, suggesting that this novel siRNA delivery system that specifically eliminates gastric CSCs provides a promising targeted therapeutic strategy for gastric cancer treatment.