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Both triptolide (TP) and cyclopamine (CPA) exhibit strong anticancer effects in vitro with adverse reactions and low bioavailabilities because of their water insolubility and poor liposolubility. The preparation of functional polymer materials into micelles not only improved the apparent solubility of the drug, changed the release behavior of the drug, but also made the drug targeted. All-atom molecular dynamics (MD) methods were used to simulate the states of both TP and CPA in PLA-PEG block copolymer micelles. MD results showed that TP and CPA were concentrated in the core layer of micelles formed by PLA-PEG block copolymers. The polymers PLLA-PEG-COOH were synthesized using HO-PEG-COOH and PLA and characterized by FTIR, 1H NMR, 13 C NMR, and gel chromatography. The dual drug-loaded polymeric micelle ((TP + CPA)-PM) was prepared by solvent evaporation, and the encapsulation rates of TP and CPA were 65.44% and 78.16% using the HPLC method, respectively. The polymer micelles were nearly spherical and did not aggregate under transmission electron microscopy (TEM), and the particle size was about 75.32 ± 0.20 nm through dynamic light scattering (DLS). (TP + CPA)-PM showed similar inhibitory effects to free drugs on A2780, A549, HepG2, and SKOV3 cells, which was proved by the MTT proliferation inhibition experiment, Hoechst33258 fluorescence staining experiment, and flow cytometry experiment. These results appeared to support the assumption that polymeric micelles encapsulation did not affect drug activity. PLA-PEG block copolymer may be a promising drug delivery vehicle for loading TP and CPA in antitumor therapy. [ABSTRACT FROM AUTHOR]

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules. [ABSTRACT FROM AUTHOR]

Abstract Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening. [ABSTRACT FROM AUTHOR]

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

Osteosarcoma (OSA) is the most common malignant bone cancer in dogs. Canine and human OSA share several features, including tumour environments, response to traditional treatment, and several molecular pathways. Hedgehog (Hh) signalling is known to contribute to tumorigenesis and progression of various cancers, including human OSA. This study aimed to identify the role of the Hh signalling pathway in canine OSA cell lines, including Abrams, D17, and Moresco, focusing on the signal transducer Smoothened (SMO). mRNA and protein levels of Hh pathway components, including SHH, IHH, SMO, and PTCH1, were aberrant in all examined OSA cell lines compared with canine osteoblast cells. The SMO inhibitor cyclopamine significantly decreased cell viability and colony‐forming ability in the canine OSA cell lines in a dose‐dependent manner. Moresco cells, which expressed the highest level of SMO protein, were the most sensitive to the anticancer effect of cyclopamine among the three canine OSA cell lines tested. Hh downstream target gene and protein expression in canine OSA cell lines were downregulated after cyclopamine treatment. In addition, cyclopamine significantly increased apoptotic cell death in Abrams and Moresco cells. The findings that Hh/SMO is activated in canine OSA cell lines and cyclopamine suppresses OSA cell survival via inhibition of SMO suggest that the Hh/SMO signalling pathway might be a novel therapeutic target for canine OSA. [ABSTRACT FROM AUTHOR]

The Veratrum genus is composed of plants containing a diverse set of steroidal alkaloids. Veratrum plant material has been utilized for centuries as herbal medicines, however the alkaloids have such a low therapeutic index that they are not used in modern medicine. Here we report an incident of inadvertent ingestion of V. parviflorum by hikers in Georgia that allowed detection, and in several instances identification of alkaloids from the plant, and correlated their presence within patient blood and breast milk specimens. Eight patients, three male and five female, presented in the spring of 2020 and 2021 with symptoms requiring emergent medical attention after ingestion of Veratrum parviflorum. All patients believed the plants to be a local native species of wild leek, Allium tricoccum, locally known as ramps. Plants were identified using photographs as well as fresh and cooked plant material provided by patients, in consultation with botanists at the University of Georgia Herbarium. Written consent was obtained from all patients for collection of blood and breast milk specimens for laboratory identification of Veratrum alkaloids. V. parviflorum plant material, and patient serum and breast milk were analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF) to identify steroidal alkaloids. The V. parviflorum extract was confirmed to contain cyclopamine, veratramine, jervine, and muldamine. Two out of the eight patients had detectable concentrations of Veratrum alkaloids. Of the alkaloids identified in the plant, cyclopamine and jervine were detected within patient serum, and cyclopamine and veratramine were observed to be present in breast milk. Toxicity resulting from Veratrum steroidal alkaloids has primarily been reported from V. album and V. viride. This is the second report of V. parviflorum poisoning. The present work reports for the first time the presence of muldamine and jervine within V. parviflorum. This work provides the first instance of identification of Veratrum alkaloids in breast milk. Thus, the findings presented herein add to literature record causative agents contributing to the toxicity of V. parviflorum when ingested and potential for secondary poisoning through breastfeeding. V. parviflorum toxicity was observed to cause nausea, vomiting, hypotension, bradycardia, abdominal pain, light-headedness, blurred vision, and tingling in the arms. Patients experiencing mild symptoms improved with supportive care, IV fluids, and antiemetics, but hemodynamically unstable patients required atropine and vasopressors. This study demonstrated that more lipophilic Veratrum alkaloids can be passed along in breast milk, which suggests additional precautions may be critical to limit further poisonings. [ABSTRACT FROM AUTHOR]

Amaç: Bu çalışmada, glioblastoma hücre hattında (U87) siklopamin ve temozolomid (TMZ) tedavisinin ayrı ayrı ve kombine olarak uygulanmasından sonra miR-20a ekspresyonunun araştırılması ve bu miRNA’nın glioblastoma tedavisindeki etkinliğinin değerlendirilmesi amaçlanmıştır. Gereç ve Yöntem: U87 MG, tedaviden önce 35 mm'lik kültür kabı üzerine yerleştirildi ve çoğaltıldı. Çalışma kapsamında 7 grup oluşturuldu: Grup 1: Kontrol grubu, Grup 2: Sham grubu (Dimetil sülfoksit), Grup 3: 2 Gün TMZ, Grup 4: 5 Gün TMZ, Grup 5: 3 saat siklopamin, Grup 6: 2 Gün TMZ + 3 saat siklopamin, Grup 7: 5 Gün TMZ + 3 saat siklopamin. Tedaviden sonra total miRNA izolasyonu ve qRT-PCR yapıldı. Bulgular: Glioblastoma hücre hattında miR-20a ekspresyonunun grup 3, grup 4, grup 5, grup 6 ve grup 7’de belirgin olarak azaldığı tespit edildi. Ancak bu azalmanın en fazla grup 7 de olduğu saptandı . Ek olarak grup 7’de hücre sayısının azaldığı görüldü. Sonuç: Glioblastoma hücrelerinde miR-20a’nın yüksek oranda eksprese olduğu; ancak siklopamin ve TMZ tedavilerinden sonra ekspresyonun azaldığı tespit edildi. miR-20a’nın glioblastoma için yeni bir terapötik hedef ve biyobelirteç olarak kullanılabileceği, siklopamin ve TMZ’nin glioblastomada tedavi amaçlı kullanılabileceği ancak bu konuda daha ileri çalışmaların yapılması gerektiği sonucuna varıldı.

Tuberculous meningitis, a severe complication of Mycobacterium tuberculosis (M. tb) infection, involves the dissemination of bacilli in the brain. This study explored the role of the sonic hedgehog (SHH) signaling pathway in regulating blood-brain barrier (BBB) integrity, M. tb invasion into the central nervous system (CNS), and disease progression of Central Nervous System Tuberculosis (CNS-TB) in a Balb/c mouse model. The modulation of the SHH pathway using agonist Purmorphamine (PUR) and antagonist Cyclopamine (CYC) revealed that CYC treatment led to a rapid and extensive invasion of M. tb in the brain, with bacterial loads increasing by 99 % compared to the untreated-infected group. In contrast, PUR reduced M. tb loads by 50 % and delayed disease progression. Histopathological analysis showed that CYC exacerbated inflammation and immune cell infiltration, while PUR mitigated these responses. Immunohistochemistry demonstrated that CYC caused severe BBB breakdown and reactive gliosis, while PUR partially attenuated this response. Further analysis revealed that CYC upregulated Matrix Metalloproteinase-9 (MMP-9) secretion, a key contributor to BBB disruption. These findings highlight the critical role of the SHH pathway in maintaining BBB integrity and regulating the immunopathological response during CNS-TB, opening up future scope for drug discovery. This Cyclopamine-induced model of rapid M. tb invasion and chronic inflammation provides a new tool for studying CNS-TB pathogenesis and evaluating potential therapeutic interventions targeting the SHH signaling axis. SIGNIFICANCE STATEMENT: Understanding how tuberculosis (TB) infection can spread to the brain is crucial, as this "central nervous system TB" (CNS-TB) is a serious and potentially life-threatening health complication. However, studying CNS-TB in humans is very difficult. Animal models are needed to better understand how TB gets into the brain and the resulting damage. This study in mice showed that blocking a signaling pathway called Sonic Hedgehog (SHH) allowed TB to rapidly spread to the brain, damaging the blood-brain barrier and causing severe inflammation. In contrast, activating the SHH pathway helped protect the brain from TB. These findings provide important insights that could lead to new ways to prevent or treat this dangerous form of TB., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024. Published by Elsevier B.V.)

Abstract Background Breast cancer is the fastest-growing cancer among females and the second leading cause of female death. At present, targeted antibodies combined with hyperthermia locally in tumor has been identified as a potential combination therapy to combat tumors. But in fact, the uniformly deep distribution of photosensitizer in tumor sites is still an urgent problem, which limited the clinical application. We reported an HER2-modified thermosensitive liposome (immunoliposome)-assisted complex by reducing gold nanocluster on the surface (GTSL-CYC-HER2) to obtain a new type of bioplasma resonance structured carrier. The HER2 decoration on the surface enhanced targeting to the breast cancer tumor site and forming irregular, dense, "petal-like" shells of gold nanoclusters. Due to the good photothermal conversion ability under near-infrared light (NIR) irradiation, the thermosensitive liposome released the antitumor Chinese traditional medicine, cyclopamine, accompanied with the degradation of gold clusters into 3–5 nm nanoparticles which can accelerate renal metabolism of the gold clusters. With the help of cyclopamine to degrade the tumor associated matrix, this size-tunable gold wrapped immunoliposome was more likely to penetrate the deeper layers of the tumor, while the presence of gold nanoparticles makes GTSL-CYC-HER2 multimodal imaging feasible. Results The prepared GTSL-CYC-HER2 had a size of 113.5 nm and displayed excellent colloidal stability, photo-thermal conversion ability and NIR-sensitive drug release. These GTSL-CYC-HER2 were taken up selectively by cancer cells in vitro and accumulated at tumour sites in vivo. As for the in vivo experiments, compared to the other groups, under near-infrared laser irradiation, the temperature of GTSL-CYC-HER2 rises rapidly to the phase transition temperature, and released the cyclopamine locally in the tumor. Then, the released cyclopamine destroyed the stroma of the tumor tissue while killing the tumor cells, which in turn increased the penetration of the liposomes in deep tumor tissues. Moreover, the GTSL-CYC-HER2 enhanced the performance of multimodal computed tomography (CT) and photothermal (PT) imaging and enabled chemo-thermal combination therapy. Conclusions This optically controlled biodegradable plasmonic resonance structures not only improves the safety of the inorganic carrier application in vivo, but also greatly improves the anti-tumor efficiency through the visibility of in vivo CT and PT imaging, as well as chemotherapy combined with hyperthermia, and provides a synergistic treatment strategy that can broaden the conventional treatment alone. Graphic Abstract

Plants of the Veratrum genus have been used throughout history for their emetic properties, rheumatism, and for the treatment of high blood pressure. However, inadvertent consumption of these plants, which resemble wild ramps, induces life-threatening side effects attributable to an abundance of steroidal alkaloids. Several of the steroidal alkaloids from Veratrum spp. have been investigated for their ability to antagonize the Hedgehog (Hh) signaling pathway, a key pathway for embryonic development and cell proliferation. Uncontrolled activation of this pathway is linked to the development of various cancers; most notably, basal cell carcinoma and acute myeloid leukemia. Additional investigation of Veratrum spp. may lead to the identification of novel alkaloids with the potential to serve as chemotherapeutics. V. parviflorum is a relatively uncommon species of Veratrum that resides in the southeastern regions of North America. The phytochemical profile of this plant remains largely unexplored; however, bioactive steroidal alkaloids, including cyclopamine, veratramine, veratridine, and verazine were identified in its extract. The structural elucidation and bioactivity assessment of steroidal alkaloids in lesser abundance within the extract of V. parviflorum may yield potent Hh pathway inhibitors. This review seeks to consolidate the botanical and phytochemical information regarding V. parviflorum. [ABSTRACT FROM AUTHOR]

Abstract Background The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood. Methods We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well‐known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK‐8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT‐PCR and Western Blot. Results We found up‐regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low‐dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells. Conclusions Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer.

TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis.

The development of amoebic liver abscess (ALA) leads to liver necrosis, accompanied by an exacerbated inflammatory response and the formation of multiple granulomas. Adequate management of the infection through the administration of treatment and the timely response of the organ to the damage allows the injury to heal with optimal regeneration without leaving scar tissue, which does not occur in other types of damage such as viral hepatitis that may conducts to fibrosis or cirrhosis. The Hedgehog signaling pathway (Hh) is crucial in the embryonic stage, while in adults it is usually reactivated in response to acute or chronic injuries, regeneration, and wound healing. In this work, we characterized Hh in experimental hepatic amoebiasis model, with the administration of treatment with metronidazole, as well as a pathway inhibitor (cyclopamine), through histological and immunohistochemical analyses including an ultrastructure analysis through transmission electron microscopy. The results showed an increase in the percentage of lesions obtained, a decrease in the presence of newly formed hepatocytes, a generalized inflammatory response, irregular distribution of type I collagen accompanied by the presence of fibroblast-type cells and a decrease in effector cells of this pathway. These results constitute the first evidence of the association of the activation of Hh with the liver regeneration process in experimental amebiasis. [Display omitted] • The hedgehog signaling pathway (Hh) is activated in response to repair liver damage caused by a parasite. • Inhibition of the Hedgehog signaling pathway (Hh) results in larger lesions. • Inhibition of the Hedgehog signaling pathway (Hh) results in histopathological changes. [ABSTRACT FROM AUTHOR]

Ting Zhang,1 Yanping Li,2 Yi Song,1 Xiaoshuang Chen,1 Jing Li,1 Qiang Peng,3 Jinhan He,1,2 Xiaofan Fei1 1Department of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China; 2Laboratory of Clinical Pharmacy and Adverse Drug Reaction, West China Hospital of Sichuan University, Chengdu, People’s Republic of China; 3State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, People’s Republic of ChinaCorrespondence: Xiaofan Fei Tel + 86-28-85423475Email 1145342867@qq.comBackground and Purpose: Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix (ECM). Inhibition of HSC activation may be an effective treatment. Since various pathways control HSC activation, a combination of drugs with different mechanisms may be more effective than monotherapy.Methods: Here, we prepared liposomes loaded with curcumin and cyclopamine to inhibit HSC activation. We systematically analyzed the physicochemical characteristics of liposomes loaded with the two drugs, as well as their effects on HSC proliferation, activation and collagen production on gene, protein and cellular levels.Results: The prepared liposomes helped solubilize both drugs, contributing to their uptake by cells. Liposomes loaded with both drugs inhibited cell proliferation, migration and invasion, as well as induced more apoptosis and perturbed the cell cycle more than the free combination of both drugs in solution or liposomes loaded with either drug alone. Liposomes loaded with both drugs strongly suppressed HSC activation and collagen secretion.Conclusion: Our results suggest that liposome encapsulation can increase the uptake of curcumin and cyclopamine as well as the synergism between them in anti-fibrosis. This approach shows potential for treating hepatic fibrosis.Keywords: hepatic stellate cells, HSCs, hepatic fibrosis, cyclopamine, curcumin, synergistic effect

Odontogenic keratocysts (OKC) are benign, developmental, locally-aggressive odontogenic cystic lesions with a high risk of recurrence. As such, the most effective treatment modalities remain controversial. The mainstay of treatment remains enucleation with or without decompression. The use of adjunctive therapies is widely reported. Our aim was to review our experience of OKCs and therefore identify the treatment modality, if there is any single one, with the lowest rate of recurrence. We also aimed to identify any common themes linking those patients experiencing cystic recurrence. Data were collected on 50 patients treated at UHCW NHS Trust over a 14-year period (2005-2018) via an anonymised database. Surgical pathways were analysed, including details of the location of the cysts and the use of adjunctive therapies, namely; mechanical debridement, cryotherapy, and the use of Carnoy's solution. Fifty-six keratocysts, both primary (91%, n = 51) and recurrent (9%, n = 5) were included. A total of 6% of patients had a pre-existing diagnosis of Gorlin-Goltz Syndrome (n = 3). Enucleation was performed in an approximately 3:1 ratio to decompression with secondary enucleation (n = 41:15). Twenty-seven percent of patients had adjunctive therapies (n = 15). There was a 12% recurrence rate (n = 6) found only within the group of primary cysts that had been enucleated only. Notably, there were no recurrences in those cysts that had undergone adjunctive therapy. None of the cysts that underwent initial decompression or marsupialisation recurred. Following surgical intervention, no tertiary recurrent cysts were detected postoperatively. This study demonstrated the advantage of establishing a correct diagnosis prior to definitive treatment. Initial decompression in selected patients followed by enucleation, along with adjunctive therapies showed a benefit in reducing recurrences. However, in the absence of high-quality evidence for the most effective management of odontogenic keratocysts, finding a common approach will remain controversial. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is the fastest-growing cancer among females and the second leading cause of female death. At present, targeted antibodies combined with hyperthermia locally in tumor has been identified as a potential combination therapy to combat tumors. But in fact, the uniformly deep distribution of photosensitizer in tumor sites is still an urgent problem, which limited the clinical application. We reported an HER2-modified thermosensitive liposome (immunoliposome)-assisted complex by reducing gold nanocluster on the surface (GTSL-CYC-HER2) to obtain a new type of bioplasma resonance structured carrier. The HER2 decoration on the surface enhanced targeting to the breast cancer tumor site and forming irregular, dense, "petal-like" shells of gold nanoclusters. Due to the good photothermal conversion ability under near-infrared light (NIR) irradiation, the thermosensitive liposome released the antitumor Chinese traditional medicine, cyclopamine, accompanied with the degradation of gold clusters into 3–5 nm nanoparticles which can accelerate renal metabolism of the gold clusters. With the help of cyclopamine to degrade the tumor associated matrix, this size-tunable gold wrapped immunoliposome was more likely to penetrate the deeper layers of the tumor, while the presence of gold nanoparticles makes GTSL-CYC-HER2 multimodal imaging feasible. Results: The prepared GTSL-CYC-HER2 had a size of 113.5 nm and displayed excellent colloidal stability, photo-thermal conversion ability and NIR-sensitive drug release. These GTSL-CYC-HER2 were taken up selectively by cancer cells in vitro and accumulated at tumour sites in vivo. As for the in vivo experiments, compared to the other groups, under near-infrared laser irradiation, the temperature of GTSL-CYC-HER2 rises rapidly to the phase transition temperature, and released the cyclopamine locally in the tumor. Then, the released cyclopamine destroyed the stroma of the tumor tissue while killing the tumor cells, which in turn increased the penetration of the liposomes in deep tumor tissues. Moreover, the GTSL-CYC-HER2 enhanced the performance of multimodal computed tomography (CT) and photothermal (PT) imaging and enabled chemo-thermal combination therapy. Conclusions: This optically controlled biodegradable plasmonic resonance structures not only improves the safety of the inorganic carrier application in vivo, but also greatly improves the anti-tumor efficiency through the visibility of in vivo CT and PT imaging, as well as chemotherapy combined with hyperthermia, and provides a synergistic treatment strategy that can broaden the conventional treatment alone. [ABSTRACT FROM AUTHOR]

Background: The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood. Methods: We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well‐known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK‐8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT‐PCR and Western Blot. Results: We found up‐regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low‐dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells. Conclusions: Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Plants of the Veratrum genus have been used throughout history for their emetic properties, rheumatism, and for the treatment of high blood pressure. However, inadvertent consumption of these plants, which resemble wild ramps, induces life-threatening side effects attributable to an abundance of steroidal alkaloids. Several of the steroidal alkaloids from Veratrum spp. have been investigated for their ability to antagonize the Hedgehog (Hh) signaling pathway, a key pathway for embryonic development and cell proliferation. Uncontrolled activation of this pathway is linked to the development of various cancers; most notably, basal cell carcinoma and acute myeloid leukemia. Additional investigation of Veratrum spp. may lead to the identification of novel alkaloids with the potential to serve as chemotherapeutics. V. parviflorum is a relatively uncommon species of Veratrum that resides in the southeastern regions of North America. The phytochemical profile of this plant remains largely unexplored; however, bioactive steroidal alkaloids, including cyclopamine, veratramine, veratridine, and verazine were identified in its extract. The structural elucidation and bioactivity assessment of steroidal alkaloids in lesser abundance within the extract of V. parviflorum may yield potent Hh pathway inhibitors. This review seeks to consolidate the botanical and phytochemical information regarding V. parviflorum.

Hedgehog (Hh) pathway plays a key role in development from invertebrate to vertebrate. It is known to be involved in cell differentiation, polarity, proliferation, including the development of vertebrate limb and the establishment of flies’ body plan. To investigate how the regulation of Hh pathway affects the development of parthenogenetic murine embryos, the parthenogenetically activated murine embryos were treated with either cyclopamine (Cyc), an antagonist of Hh pathway, or purmorphamine, an agonist of Hh pathway. While Cyc did not affect the blastocyst formation and its total cell number, the chemical reduced the hatching rate of embryos and the expression levels of Fn1 mRNA. The results of the present study show the possibility that Cyc may affect the development of embryos at blastocyst stage by blocking Hh pathway and this may cause detrimental effect to the embryos at peri-, and post-implantation stages.

Background: Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective therapy. Blood-brain barrier (BBB) damage is a hallmark of ICH-induced brain injury that leads to edema formation, leukocytes infiltration, influx of blood components into the perihematomal (PHE) region, and eventually brain injury. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted molecules that contribute to the association between these cells. Sonic hedgehog (SHH) derived from astrocytes promotes the maturity and integrity of the BBB by upregulating tight junctions (TJs) in brain capillary endothelial cells (ECs). However, the effect of SHH on BBB in ICH has not been investigated.Methods: Cyclopamine (CYC) is a potent, selective inhibitor that specifically blocks the SHH signaling pathway. Here, we used pharmacological inhibitions (CYC and its derivatives) to determine a critical role of the SHH signaling pathway in promoting BBB integrity after ICH by mechanisms of regulating the TJ proteins in vivo and in vitro.Results: The expression of astrocytic SHH was upregulated in mouse brains after ICH. Compared with the vehicle-treated group, inhibition of the SHH signaling pathway with CYC and its derivatives treatments aggravated neurological function deficits, brain edema, hematoma volume, and BBB impairment by downregulating TJs in ECs through the SHH-Gli-1 axis in vivo and in vitro.Conclusions: SHH signaling pathway at the level of the BBB provides a barrier-promoting effect, suggesting that the SHH signaling pathway may function as a potential therapeutic target for restoring BBB function in ICH.

TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis. [ABSTRACT FROM AUTHOR]

Background : Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective therapy. Blood-brain barrier (BBB) damage is a hallmark of ICH-induced brain injury that leads to edema formation, leukocytes infiltration, influx of blood components into the perihematomal (PHE) region, and eventually brain injury. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted molecules that contribute to the association between these cells. Sonic hedgehog (SHH) derived from astrocytes promotes the maturity and integrity of the BBB by upregulating tight junctions (TJs) in brain capillary endothelial cells (ECs). However, the effect of SHH on BBB in ICH has not been investigated. Methods : Cyclopamine (CYC) is a potent, selective inhibitor that specifically blocks the SHH signaling pathway. Here, we used pharmacological inhibitions (CYC and its derivatives) to determine a critical role of the SHH signaling pathway in promoting BBB integrity after ICH by mechanisms of regulating the TJ proteins in vivo and in vitro. Results : The expression of astrocytic SHH was upregulated in mouse brains after ICH. Compared with the vehicle-treated group, inhibition of the SHH signaling pathway with CYC and its derivatives treatments aggravated neurological function deficits, brain edema, hematoma volume, and BBB impairment by downregulating TJs in ECs through the SHH-Gli-1 axis in vivo and in vitro. Conclusions : SHH signaling pathway at the level of the BBB provides a barrier-promoting effect, suggesting that the SHH signaling pathway may function as a potential therapeutic target for restoring BBB function in ICH. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) has shown great promise in breast cancer treatment. However, simplex target ligand modification or stimuli release cannot meet the requirement of effective drug delivery to solid tumor tissue. To overcome continuous bio-barriers existing in the tumor microenvironment, multi-stage response drug delivery was desirable. Herein, we developed a unique tumor microenvironment tailored nanoplatform for chlorin e6 (Ce6) delivery. We chose bovine serum albumin (BSA) as “mother ships” material for effective tumor periphery resident, cyclopamine (CYC) as extracellular matrix (ECM) inhibitor and synergistic anti-tumor agent, and diselenide containing amphiphilic hyaluronic acid-chlorin e6 polymers (HA-SeSe-Ce6) synthesized as “small bombs” for internal tissue destruction. The above three distinct function compositions were integrated into an independent CYC and HA-SeSe-Ce6 co-delivery albumin nano-system (ABN@HA-SeSe-Ce6/CYC). The obtained nano-system presents good biocompatible, long circulation and effective tumor accumulation. After entering tumor microenvironment, CYC gradually releases to disrupt the ECM barrier to open the way for further penetration of HA-SeSe-Ce6. Subsequently, targeted tumor cell internalization and intracellular redox response release of Ce6 would achieve. Moreover, CYC could also make up the deficiency of Ce6 in hypoxia area, owing to its anti-tumor effect. Improved therapeutic efficacy was verified in a breast cancer cell line and tumor-bearing mice model.

Meilin Liu, Xiaolong Chen, Henan Liu, Yu Di Department of Ophthalmology, Shengjing Affiliated Hospital, China Medical University, Shenyang, Liaoning, People’s Republic of China Aim: The aim of the study was to investigate the signal transduction mechanism of Hedgehog–vascular endothelial growth factor in oxygen-induced retinopathy (OIR) and the effects of cyclopamine on OIR. Methods: An OIR model was established in C57BL/6J mice exposed to hyperoxia. Two hundred mice were randomly divided into a control group, an OIR group, an OIR-control group (treated with isometric phosphate-buffered saline by intravitreal injection), and a cyclopamine group (treated with cyclopamine by intravitreal injection), with 50 mice in each group. The retinal vascular morphology was observed using adenosine diphosphatase and number counting using hematoxylin and eosin-stained image. Quantitative real-time quantitative polymerase chain reaction was used to detect mRNA expression. Protein location and expression were evaluated using immunohistochemistry and Western blot. Results: The OIR group and OIR-control group demonstrated large-area pathological neovascularization and nonperfused area when compared with the control group (both P

The Hedgehog (Hh) signaling pathway is one of the major regulators of embryonic development and tissue homeostasis in multicellular organisms. However, the role of this pathway in the silkworm, especially in the silkworm midgut, remains poorly understood. Here, we report that Bombyx mori Hedgehog (BmHh) is expressed in most tissues of silkworm larvae and that its functions are well‐conserved throughout evolution. We further demonstrate that the messenger RNA of four Hh signaling components, BmHh ligand, BmPtch receptor, signal transducer BmSmo and transcription factor BmCi, are all upregulated following Escherichia coli or Bacillus thuringiensis infection, indicating the activation of the Hh pathway. Simultaneously, midgut cell proliferation is strongly promoted. Conversely, the repression of Hh signal transduction with double‐stranded RNA or cyclopamine inhibits the expression of BmHh and BmCi and reduces cell proliferation. Overall, these findings provide new insights into the Hh signaling pathway in the silkworm, B. mori. [ABSTRACT FROM AUTHOR]

Veratrum spp. grow throughout the world and are especially prevalent in high mountain meadows of North America. All parts of Veratrum plants have been used for the treatment of ailments including injuries, hypertension, and rheumatic pain since as far back as the 1600s. Of the 17–45 Veratrum spp., Veratrum californicum alkaloids have been proven to possess favorable medicinal properties associated with inhibition of hedgehog (Hh) pathway signaling. Aberrant Hh signaling leads to proliferation of over 20 cancers, including basal cell carcinoma, prostate and colon among others. Six of the most well-studied V. californicum alkaloids are cyclopamine (1), veratramine (2), isorubijervine (3), muldamine (4), cycloposine (5), and veratrosine (6). Recent inspection of the ethanolic extract from V. californicum root and rhizome via liquid chromatography–mass spectrometry has detected up to five additional alkaloids that are proposed to be verazine (7), etioline (8), tetrahydrojervine (9), dihydrojervine (10), 22-keto-26-aminocholesterol (11). For each alkaloid identified or proposed in V. californicum, this review surveys literature precedents for extraction methods, isolation, identification, characterization and bioactivity to guide natural product drug discovery associated with this medicinal plant.

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.

Hedgehog signaling pathway participates in a chain of necessary physiological activities and dysregulation of the hedgehog signaling has been implicated in birth defects and diseases. Although substantial studies have uncovered that the hedgehog pathway is both sufficient and necessary for patterning vertebrate muscle differentiation, limited knowledge is available about its role in molluscan myogenesis. Here, the present study firstly identified and characterized the key genes (CgHh, CgPtc, CgSmo, CgGli) in the hedgehog pathway of the Pacific oyster Crassostrea gigas, and investigated the function of this pathway in embryonic myogenesis of C. gigas. Bioinformatics analysis revealed that the functional domains of the key genes were highly conserved among species. Quantitative analysis indicated that CgHh, CgPtc, CgGli mRNA began to accumulate during the blastula to gastrulation stages and accumulated throughout trochophore and into the D-shaped stage. RNA localization patterns by whole-mount in situ hybridization revealed that the key genes own the strongest specific staining in gastrulation, trochophore, and D-shaped stage. Hedgehog pathway genes showed a high expression level in myogenesis stage including trochophore and D-shaped stages, suggesting that the hedgehog pathway would be involved in myogenesis of C. gigas. In adult oysters, the key genes were expressed at various tissues, indicating that hedgehog pathway governed a series of development events. To further examine the role of hedgehog signaling in C. gigas myogenesis, we used cyclopamine treatment in C. gigas larvae to inhibit the signaling pathway. The quantification of the expression of the key genes in hedgehog pathway showed that expressions of key genes were severely down-regulated in treated larvae compared with normal larvae. The velum retractors, ventral retractors, anterior adductor, and posterior adductor muscles of larvae treated with cyclopamine at 4–6 μM for 6–12 h were severely destroyed, suggesting that the hedgehog pathway took part in the myogenesis of C. gigas. These findings provide a foundation for uncovering the molecular mechanisms of hedgehog signaling in molluscan physiological activity and enable us to better understand the signaling pathway involving in molluscan physiological activity.

The aim of this paper is to investigate the potential beneficial effects of taurine in cochlear neural stem cell (NSC) transplantation and elucidate the underlying molecular mechanism. The NSC cells were isolated from neonatal Balb/c mice and an auditory neuropathy gerbil model was established by microinjection of ouabain. The spiral ganglion neurons (SGN) were characterized with immunofluorescence stained with Tuj1 antibody. Cell proliferation was determined by BrdU incorporation assay and the morphologic index was measured under the light microscope. The relative protein level was determined by immunoblotting. The hearing of the animal model was scored by click- and tone burst-evoked auditory brainstem response (ABR). Here we consolidated our previous finding that taurine stimulated SGN density and the proliferation index, which were completely abolished by Shh inhibitor, cyclopamine. Transplantation of cochlear NSCs combined with taurine significantly improved ouabain-induced auditory neuropathy in gerbils. In addition, cyclopamine antagonized taurine's effect on glutamatergic and GABAergic neuron population via suppression of VGLUT1 and GAT1 expression. Mechanistically, taurine evidently activated the Sonic HedgeHog pathway and upregulated Shh, Ptc-1, Smo and Gli-1 proteins, which were specifically blockaded by cyclopamine. Here, for the first time demonstrated we that co-administration with taurine significantly improved NSC transplantation and the Shh pathway was identified in this beneficial effect. [ABSTRACT FROM AUTHOR]

In this Account we present the total syntheses of artemisinin (an important antimalarial agent) and cyclopamine (the first natural inhibitor of the hedgehog signaling pathway). Furthermore, we describe the design and development of a γ-butyrolactone as an inhibitor of Gcn5 (a histone N-acetyltransferase), discuss the discovery of hyperforin and guttiferon G as the first natural products acting as inhibitors of sirtuins, and present the design of inhibitors of sialic acid biosynthesis. The biomedical background and importance are also discussed. Finally, we present the discovery and development of methods for transesterification, IBX-mediated oxidations, reduction of several functional groups with LiBH4 /Me3 SiCl, as well as a process enabling the synthesis of kilogram amounts of D-isocitric acid and its transformation to valuable chiral derivatives. 1 Artemisinin and Malaria 2 Cyclopamine and Cyclops 3 Sunflowers, Krebs Cycle, and Isocitric acid 4 Histones and N-Acetyltransferase Gcn5 5 St. John's Wort, Hippocrates, and Sirtuins 6 Sialic Acid and Inhibitors of Its Biosynthesis 7 Transesterification 8 IBX Reloaded 9 And Finally: A Small Mistake with a Big Impact 10 Epilogue [ABSTRACT FROM AUTHOR]

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood‐brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up‐regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL‐induced up‐regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin‐5 and zonula occludens‐1 (ZO‐1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli‐1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL–up‐regulated expression of occludin, claudin‐5 and ZO‐1. Moreover, TXL up‐regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli‐1, and thus activated the Shh pathway. And cyclopamine inhibited TXL‐induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL‐induced protection against BBB disruption after ischaemic stroke. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure‐activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine‐based delivery approaches for Hh pathway inhibitors are also discussed concisely. Small molecule based inhibitors of Hh signaling pathway for cancer treatment [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of the hedgehog (Hh) pathway inhibitor, cyclopamine, and activator purmorphamine on articular cartilage cell proliferation. Methods: Rats were subjected to AA and CIA models. Secondary paw swelling was measured at 12, 15, 18, 21, 24, 27, and 30 days. The rats were sacrificed on day 30. Tissues from the cartilage and knee joints were collected. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay while cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide assay. Protein expression levels of Shh, Ptch1 and Gli1 were determined by Western blotting. Results: Compared with the control group, arthritis index and secondary foot swelling of the adjuvant arthritis (AA) and collagen-induced arthritis (CIA) groups deteriorated significantly (p < 0.05). MTT data revealed that cyclopamine promoted articular cartilage cell proliferation of the AA and CIA groups. The cell proliferation rates of AA and CIA groups were significantly higher than that of control group (p < 0.05). Flow cytometry showed that the cell apoptosis rates of AA and CIA groups were significantly lower than that of control group (p < 0.05). Compared with control, cyclopamine decreased the protein expression levels of sonic Hh, patched homologue 1 and glioma-associated oncogene homologue, but the effect of purmorphamine was the reverse. Conclusion: Hh pathway inhibitor (cyclopamine) and activator (purmorphamine) affect the expression of Hh pathway. Disruption of the Hh pathway may be of potential therapeutic significance in protecting articular cartilage from rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Abstract Hedgehog (Hh) pathway hyperactivation has been observed in various tumors, including breast cancer, and Hh pathway inhibitors have demonstrated antitumor activity in breast cancer. The tumor microenvironment (TME) has been shown to play an important role in modulating cancer cell drug sensitivity, but the TME response to Hh pathway inhibitors is unclear. In the current study, we observed increased TME infiltration of macrophages in breast cancer tissue, and specifically, M2 polarized macrophages after neoadjuvant chemotherapy. Furthermore, we observed an enhanced tolerance to Hh pathway inhibitors in MDA-MB-231 cells after co-culturing with M2 macrophages. In addition, we demonstrated that Hh pathway inhibition significantly induced IL6 expression, and validated that the tolerance to Hh pathway inhibitors was IL6-dependent. This study demonstrates a role of macrophages in Hh pathway inhibition resistance and a role of macrophage-derived IL6 in this resistance of breast cancer cells to Hh inhibition. These data indicate that antagonizing IL6 together with Hh pathway inhibitors may be a novel therapeutic strategy for breast cancer. Highlights • Breast cancer tissue has higher M1 and M2 macrophage infiltration. • Neoadjuvant chemotherapy increases breast cancer tissue M2 macrophage infiltration. • M2 macrophages attenuate the efficacy of a Hh inhibitor in breast cancer cells. • Hh pathway inhibition induces polarization of macrophages toward an M2 phenotype. • M2 macrophage-derived IL6 enhances cyclopamine resistance in breast cancer cells. [ABSTRACT FROM AUTHOR]

Hedgehog signaling pathway participates in a chain of necessary physiological activities and dysregulation of the hedgehog signaling has been implicated in birth defects and diseases. Although substantial studies have uncovered that the hedgehog pathway is both sufficient and necessary for patterning vertebrate muscle differentiation, limited knowledge is available about its role in molluscan myogenesis. Here, the present study firstly identified and characterized the key genes (CgHh, CgPtc, CgSmo, CgGli) in the hedgehog pathway of the Pacific oyster Crassostrea gigas , and investigated the function of this pathway in embryonic myogenesis of C. gigas. Bioinformatics analysis revealed that the functional domains of the key genes were highly conserved among species. Quantitative analysis indicated that CgHh, CgPtc, CgGli mRNA began to accumulate during the blastula to gastrulation stages and accumulated throughout trochophore and into the D-shaped stage. RNA localization patterns by whole-mount in situ hybridization revealed that the key genes own the strongest specific staining in gastrulation, trochophore, and D-shaped stage. Hedgehog pathway genes showed a high expression level in myogenesis stage including trochophore and D-shaped stages, suggesting that the hedgehog pathway would be involved in myogenesis of C. gigas. In adult oysters, the key genes were expressed at various tissues, indicating that hedgehog pathway governed a series of development events. To further examine the role of hedgehog signaling in C. gigas myogenesis, we used cyclopamine treatment in C. gigas larvae to inhibit the signaling pathway. The quantification of the expression of the key genes in hedgehog pathway showed that expressions of key genes were severely down-regulated in treated larvae compared with normal larvae. The velum retractors, ventral retractors, anterior adductor, and posterior adductor muscles of larvae treated with cyclopamine at 4–6 μM for 6–12 h were severely destroyed, suggesting that the hedgehog pathway took part in the myogenesis of C. gigas. These findings provide a foundation for uncovering the molecular mechanisms of hedgehog signaling in molluscan physiological activity and enable us to better understand the signaling pathway involving in molluscan physiological activity. [ABSTRACT FROM AUTHOR]

The unique tumour microenvironment (TM) of pancreatic ductal adenocarcinoma (PDA) including highly desmoplastic ECM and low tumour perfusion supports a considerable barrier for effective delivery of nanomedicines. Effectively modulating PDA microenvironment to enhance tumour drug delivery represents a pinpoint in the field of PDA treatment. In this study, it was the first time that biomimetic nanoparticles, which were designed in the form of erythrocyte membrane-camouflaged PLGA nanoparticles (MNP), were utilized for PDA microenvironment modulation. Cyclopamine (CYC), an inhibitor of Hedgehog pathway that contributed a lot to desmoplastic ECM of PDA, was selected as the model drug and successfully encapsulated into MNP. Advantages of CYC-loaded MNP (CMNP) included favourable biocompatibility, long circulation time, and powerful TM modulation effect. CMNP could effectively deliver CYC to the tumour site, disrupt tumour ECM, increase functional vessels, and improve tumour perfusion significantly. The combination treatment with CMNP and PTX-loaded MNP (PMNP) successfully improved PTX delivery to tumour, resulting in remarkable tumour growth inhibition in vivo. Therefore, biomimetic nanoparticles provide a new strategy for modulating PDA TM and will have great potential to improve the therapeutic effects of nanomedicines for PDA patients. [ABSTRACT FROM AUTHOR]

As one of the most intractable tumours, pancreatic ductal adenocarcinoma (PDA) has a dense extracellular matrix (ECM) which could increase solid stress within tumours to compress tumour vessels, reduce tumour perfusion and compromise nanomedicine delivery for PDA. Thus, alleviating solid stress represents a potential therapeutic target for PDA treatment. In this study, cyclopamine, a special inhibitor of the hedgehog signalling pathway which contributes a lot to ECM formation of PDA, was exploited to alleviate solid stress and improve nanomedicine delivery to PDA. Results demonstrated that cyclopamine successfully disrupted ECM and lowered solid stress within PDA, which increased functional tumour vessels and resulted in enhanced tumour perfusion as well as improved tumour nanomedicine delivery in PDA-bearing animal models. Therefore, solid stress within PDA represents a new therapeutic target for PDA treatment. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) relapse and metastasis are the leading cause of death and, together with drug resistance, keep mortality still high. The Hedgehog (Hh) pathway is expressed during embryogenesis, organogenesis and in adult tissue homeostasis and its aberrant activation is often associated with cancer. Carbonic anhydrase (CA) enzymes are important during development; they play a key role in controlling several cellular mechanisms, such as pH regulation, survival, and migration, and they are aberrantly expressed in cancer. The goal of this study was to investigate the interplay between the Hh pathway and CAXII in terms of BC cell migration. We here demonstrated that smoothened (SMO) silencing resulted in a reduction of CAXII expression at mRNA and protein level. This led to a decrease in cell migration, which was restored when cells were treated with an SMO agonist, Sag dihydrochloride (SAG), but not when cells were cotreated with SAG and the CAs inhibitor Acetazolamide. This suggested that the ability of SAG to promote cell migration was impaired when CAXII was inhibited. The reduction was also confirmed within hypoxic and inflammatory microenvironment, typical of BC, indicating a key role of the Hh pathway in controlling CAXII expression. Our results may contribute to further understand the physiology of BC cells and indicate that the Hh pathway controls BC cell migration and cell invasion also through CAXII, with important implications in identifying novel therapeutic targets. We here demonstrated that Smoothened (SMO) silencing resulted in a reduction of CAXII expression at messenger RNA (mRNA) and protein level in breast cancer (BC) cell lines. This led to a decrease in cell migration, which was restored when cells were treated with a SMO agonist. The reduction was confirmed also within hypoxic and inflammatory microenvironment, typical of BC, indicating a key role of the Hedgehog (Hh) pathway in controlling CAXII expression. Our results indicate that the Hh pathway controls BC cell migration and cell invasion through CAXII, with important implications in identifying novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds- HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells. • Hedgehog (EsHH) and Smoothened (EsSMO) homolog were identified from Chinese mitten crab. • Expression of EsHH was up-regulated at the basal growth phase, and down-regulated at the proecdysial growth stage. • Blockage of Hedgehog pathway with Smoothened inhibitor or RNAi (ds-HH) repressed limb regeneration in E. sinensis. • Hedgehog pathway regulates limb regeneration in through promoting proliferation and myogenic differentiation of blastema cells. [ABSTRACT FROM AUTHOR]

Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3) that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine), an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1) transcription and sonic hedgehog (Shh) target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO) agonist (SAG) or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway.

In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.

Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.

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.

Veratrum californicum is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the alkaloid composition of V. californicum by plant part through quantitative analysis of cyclopamine, veratramine, muldamine and isorubijervine in the leaf, stem and root/rhizome of the plant. To determine whether additional alkaloids in the extracts contribute to Hh signaling inhibition, the concentrations of these four alkaloids present in extracts were replicated using commercially available standards, followed by comparison of extracts to alkaloid standard mixtures for inhibition of Hh signaling using Shh-Light II cells. Alkaloid combinations enhanced Hh signaling pathway antagonism compared to cyclopamine alone, and significant differences were observed in the Hh pathway inhibition between the stem and root/rhizome extracts and their corresponding alkaloid standard mixtures, indicating that additional alkaloids present in these extracts are capable of inhibiting Hh signaling. [ABSTRACT FROM AUTHOR]