Activation of Shh/Smo is sufficient to maintain oligodendrocyte precursor cells in an undifferentiated state and is not necessary for myelin formation and (re)myelination.

Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function), and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (Smoothened Agonist (SAG) or cyclopamine treatment, respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting oligodendrocyte precursor cells (OPCs) in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.

Hedgehog signaling is required for larval muscle development and larval metamorphosis of the mussel Mytilus coruscus.

Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh, McPtc, McSmo, and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates.

Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

Veratramine inhibits porcine epidemic diarrhea virus entry through macropinocytosis by suppressing PI3K/Akt pathway.

Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC50) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.

Hydroxylated cyclopamine analogues from Veratrum californicum and their hedgehog pathway inhibiting activity.

Cyclopamine (1), the teratogenic steroidal alkaloid isolated from corn lily (Veratrum californicum), has recently gained renewed interest due to its anticancer potential, that has been translated into the FDA approval of three Hedgehog (Hh) pathway inhibiting antitumor drugs. A chemical analysis of mother liquors obtained from crystallization of cyclopamine, extracted from roots and rhizomes of V. californicum, resulted in the isolation of two unprecedented cyclopamine analogues, 18-hydroxycyclopamine (2) and 24R-hydroxycyclopamine (3), the first compounds of this class to show modifications on rings D-F. The stereostructures of these new natural compounds have been established based on a detailed MS and 1D/2D NMR investigation. The isolated compounds were evaluated with the dual-luciferase bioassay for their inhibition of the hedgehog pathway in comparison to cyclopamine, providing new insights into the structure-activity relationships for this class of compounds.

Insights into the Cardiotoxic Effects of Veratrum Lobelianum Alkaloids: Pilot Study.

Jervine, protoveratrine A (proA), and protoveratrine B (proB) are Veratrum alkaloids that are presented in some remedies obtained from Veratrum lobelianum, such as Veratrum aqua. This paper reports on a single-center pilot cardiotoxic mechanism study of jervine, proA, and proB in case series. The molecular aspects were studied via molecular dynamic simulation, molecular docking with cardiac sodium channel NaV1.5, and machine learning-based structure-activity relationship modeling. HPLC-MS/MS method in combination with clinical events were used to analyze Veratrum alkaloid cardiotoxicity in patients. Jervine demonstrates the highest docking score (-10.8 kcal/mol), logP value (4.188), and pKa value (9.64) compared with proA and proB. Also, this compound is characterized by the lowest calculated IC50. In general, all three analyzed alkaloids show the affinity to NaV1.5 that highly likely results in cardiotoxic action. The clinical data of seven cases of intoxication by Veratrum aqua confirms the results of molecular modeling. Patients exhibited nausea, muscle weakness, bradycardia, and arterial hypotension. The association between alkaloid concentrations in blood and urine and severity of patient condition is described. These experiments, while primary, confirmed that jervine, proA, and proB contribute to cardiotoxicity by NaV1.5 inhibition.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU­SKI43, cyclopamine and GLI­antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786­O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription­quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma­associated zinc finger protein (GLI)1­3, BCL2 apoptosis regulator (BCL2), MYC proto­oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786­O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU­SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU­SKI43 appear to be potential anti­renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

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

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

Anticancer effects of veratramine via the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin and its downstream signaling pathways in human glioblastoma cell lines.

AIMS: Antitumor effects of veratramine in prostate and liver cancers has been investigated, but it is still unclear whether veratramine can be used as an effective therapeutic agent for glioma. The aim of this study was to evaluate the potential pharmacological mechanism of veratramine in glioma. MAIN METHODS: Using four types of human glioblastoma cell lines, including A172, HS-683, T98G, and U-373-MG the dose-dependent antitumor effect of veratramine was evaluated. The cytotoxicity and cell proliferation were examined by CCK-8, and cell proliferation was further confirmed by anchorage-independent colony formation assay. The cell cycle distribution and apoptotic rate was assessed by flow cytometry, and apoptosis was further evaluated by apoptosis assay. The migration and invasiveness capacity were analyzed by using transwell. Protein and mRNA levels of related factors were determined by western blotting and RT-qPCR, respectively. KEY FINDINGS: Veratramine markedly induced apoptosis, suppressed the cell proliferation via the cell cycle G0/G1 phase arrest, and reduced the capacity for the migration and invasion in human glioblastoma multiforme cell lines. Moreover, veratramine was sufficient to affect the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin signaling pathway and its downstream Mdm2/p53/p21 pathway in human glioblastoma cell lines. SIGNIFICANCE: Antitumor effects of veratramine in suppression of glioma progression was mediated by the regulation of PI3K/Akt/mTOR and Mdm2/p53/p21 signaling pathway.

Sensitive timing of undifferentiation in oligodendrocyte progenitor cells and their enhanced maturation in primary visual cortex of binocularly enucleated mice.

Sensory experience modulates proliferation, differentiation, and migration of oligodendrocyte progenitor cells (OPCs). In the mouse primary visual cortex (V1), visual deprivation-dependent modulation of OPCs has not been demonstrated. Here, we demonstrate that undifferentiated OPCs developmentally peaked around postnatal day (P) 25, and binocular enucleation (BE) from the time of eye opening (P14-15) elevated symmetrically-divided undifferentiated OPCs in a reversible G0/G1 state even more at the bottom lamina of the cortex by reducing maturing oligodendrocyte (OL) lineage cells. Experiments using the sonic hedgehog (Shh) signaling inhibitor cyclopamine in vivo suggested that Shh signaling pathway was involved in the BE-induced undifferentiation process. The undifferentiated OPCs then differentiated within 5 days, independent of the experience, becoming mostly quiescent cells in control mice, while altering the mode of sister cell symmetry and forming quiescent as well as maturing cells in the enucleated mice. At P50, BE increased mature OLs via symmetric and asymmetric modes of cell segregation, resulting in more populated mature OLs at the bottom layer of the cortex. These data suggest that fourth postnatal week, corresponding to the early critical period of ocular dominance plasticity, is a developmentally sensitive period for OPC state changes. Overall, the visual loss promoted undifferentiation at the early period, but later increased the formation of mature OLs via a change in the mode of cell type symmetry at the bottom layer of mouse V1.

Eriodictyol attenuates dextran sodium sulphate-induced colitis in mice by regulating the sonic hedgehog signalling pathway.

CONTEXT: Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties. OBJECTIVE: To investigate the protective effect and mechanism of EDT in ulcerative colitis (UC). MATERIALS AND METHODS: UC model was induced by 3% dextran sulphate sodium (DSS) solution for 7 days, meanwhile, EDT and Smoothened (Smo) inhibitor cyclopamine (Cyc) were intraperitoneally injected. In the first experiment, C57BL/6 mice divided into blank control, DSS, DSS + EDT (20 or 40 mg/kg) groups. In second experiment, added Cyc (5 mg/kg) and EDT + Cyc groups. All mice were sacrificed on day 8. Disease activity index (DAI), colon length and colon histology as well as MDA levels, SOD, and GSH-Px activities were measured. The expression of Sonic hedgehog (Shh), Patched, Smo, glioblastoma-1, zonula occludens-1 (ZO-1), occludin, cleaved caspase 3, Bax and Bcl-2 in colon was detected using RT-PCR and Western blotting. RESULTS: After EDT treatment, compared with the DSS group, DAI (2.33 ± 0.516 vs. 3.67 ± 0.516), colon shortening (5.27 ± 0.476 vs. 4.53 ± 0.528 cm) and histological score (6.67 ± 1.211 vs. 12 ± 1.265) was significantly decreased. EDT also reduced inflammation, oxidative stress and apoptosis in colon. Additionally, EDT increased the expression of the tight junction proteins ZO-1 (35%) and occludin (66.3%). Mechanistically, EDT upregulated the Shh signalling pathway. However, Cyc-mediated inhibition of the Shh pathway partially abolished the effects of EDT. DISCUSSION AND CONCLUSIONS: These results indicate EDT attenuates DSS-induced colitis by activating the Shh pathway. Further clinical trials are needed to demonstrate its efficacy on UC.

Inactivation of Hedgehog signal transduction in adult astrocytes results in region-specific blood-brain barrier defects.

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.

A condensate-hardening drug blocks RSV replication in vivo.

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.

Synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine can target Hedgehog signaling pathway to inhibit myelodysplastic syndrome cell line.

OBJECTIVE: Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine in MUTZ-1 cell lines remains lacking. METHODS: We used a CCK-8 assay to detect the in-vitro proliferation rate of MUTZ-1 cell lines. Besides, the Annexin V-FITC/PI double staining flow cytometry was utilized to detect the apoptosis rate and cell cycle changes. The expression levels of mRNA were quantified by using qRT-PCR, and the western blot was employed to detect the expression of proteins. RESULTS: We found that the single-agent jervine or decitabine can significantly inhibit the proliferation rate of MUTZ-1 cell lines, and this inhibitory effect is time-dependent and concentration-dependent. The combined intervention of the jervine and decitabine can more significantly inhibit cell proliferation, induce cell apoptosis, and block the G1 phase of the cell cycle. The combined intervention of the two drugs significantly reduced Smo and G1i-1 mRNA expression in MUTZ-1 cells. Furthermore, after combining both of the drug treatments, the proteins levels of Smo, G1i-1, PI3K, p-AKT, Bcl2, and Cyclin Dl were significantly downregulated, and Caspase-3 is upregulated, indicating that jervine with its combination of decitabine might be effective for controlling the proliferation, apoptosis, and cell cycle. CONCLUSION: The Smo inhibitor jervine and its combination with decitabine have a synergistic effect on the proliferation, cell cycle, and apoptosis of MUTZ-1 cells, and its mechanism may be achieved by interfering with the Shh signaling pathway.

The Sonic Hedgehog signaling pathway regulates autophagy and migration in ovarian cancer.

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.

Down-regulation of astrocytic sonic hedgehog by activation of endothelin ETB receptors: Involvement in traumatic brain injury-induced disruption of blood brain barrier in a mouse model.

In the adult brain, sonic hedgehog acts on cerebral microvascular endothelial cells to stabilize the blood-brain barrier. The expression of sonic hedgehog by astrocytes is altered during brain injury, and this change has been shown to affect permeability of blood-brain barrier. However, much remains unknown about the regulation of astrocytic sonic hedgehog production. Our results showed that endothelin-1 reduced sonic hedgehog mRNA expression and extracellular protein release in mouse cerebral cultured astrocytes, but had no effect in bEnd.3, a mouse brain microvascular endothelial-derived cell line. The effect of endothelin-1 on astrocyte sonic hedgehog expression was suppressed by an ETB antagonist BQ788, but was unchanged by the ETA antagonist FR139317. In cultured astrocytes and bEnd.3, endothelin-1 did not affect the expression of the sonic hedgehog receptor-related molecules, patched-1 and smoothened. In an animal model of traumatic brain injury, fluid percussion injury on the mouse cerebrum increased the expression of sonic hedgehog, patched-1, and smoothened. Repeated administration of BQ788 enhanced sonic hedgehog expression at 5 days after fluid percussion injury. Histochemical examination revealed sonic hedgehog expression in glial fibrillary acidic protein-positive astrocytes in the cerebrum after fluid percussion injury. Administration of exogenous sonic hedgehog and BQ788 suppressed Evans blue extravasation, an indicator of blood vessel permeability, induced by fluid percussion injury. The effects of BQ788 on fluid percussion injury-induced Evans blue extravasation were reduced by the administration of jervine, a sonic hedgehog inhibitor. Altogether, these results suggest that endothelin-1 down-regulates astrocytic sonic hedgehog to promote disruption of the blood-brain barrier during traumatic brain injury.

Exovesicular-Shh confers Imatinib resistance by upregulating Bcl2 expression in chronic myeloid leukemia with variant chromosomes.

Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor outcomes on the current therapeutic regimes using Imatinib and its variants. It has been derived so far that leukemic stem cells (LSCs) are responsible for Imatinib resistance and CML progression. Sonic hedgehog (Shh) signaling has been implicated in proliferation of this Imatinib-resistant CD34(+) LSCs. Our work here identifies the molecular mechanism of Shh-mediated mutation-independent Imatinib resistance that is most relevant for treating CML-variants and non-compliant patients. Our results elucidate that while Shh can impart stemness, it also upregulates expression of anti-apoptotic protein-Bcl2. It is the upregulation of Bcl2 that is involved in conferring Imatinib resistance to the CD34(+) LSCs. Sub-toxic doses of Bcl2 inhibitor or Shh inhibitor (<

Knockdown of E-cadherin induces cancer stem-cell-like phenotype and drug resistance in cervical cancer cells.

Cervical cancer is one of the leading causes of mortality amongst women in developing countries, and resistance to therapy is the main reason for treatment failure. Recent advances suggest that cancer stem cells (CSCs) are critically involved in regulating the chemo-resistant behavior of cervical cancer cells. In our study, cells with the CSC phenotype were isolated, and we examined the expression levels of stem cell markers and genes associated with epithelial-mesenchymal transition (EMT) using different assays. However, the cells with the CSC phenotype could not be cultured for further cytotoxicity studies, so we established a model of CSC in cervical cancer cells. We performed siRNA-mediated knockdown of E-cadherin in these cells, and studied them for EMT-associated stem-cell-like properties. We also performed dose-dependent cell viability assays using clinically relevant drugs such as cisplatin, cyclopamine, and GANT58 to analyze the drug resistant behavior of these cancer cells. We found that knockdown of E-cadherin induces EMT in cervical cancer cells, imparting stem-cell like characteristics along with enhanced tumorsphere formation, cell migration, invasiveness, and drug resistance. This is the first study to establish a CSC model in cervical cancer cells by knockdown of E-cadherin, which can be used to develop anti-cancer therapies.

Curcumin- and Cyclopamine-Loaded Liposomes to Enhance Therapeutic Efficacy Against Hepatic Fibrosis.

BACKGROUND 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.