RESUMEN

Medulloblastoma (MB) is a frequently occurring malignant brain tumor in children, and many of these tumors are identified by the abnormal activation of the Sonic Hedgehog (SHH) pathway. Although the Shh inhibitor GDC0449 initially shows some effectiveness in certain tumors, they eventually recur due to drug resistance mechanisms, highlighting the need for new treatment options. In this study, we explore whether GDC0449 induces autophagy in the human MB cell lines. To investigate the ultrastructural pathology changes of GDC0449-treated Daoy and D283 cells, we employed Transmission Electron Microscopy (TEM) technology to identify the expression of autophagic vacuoles. Our results indicate that GDC0449 only increases autophagy in Daoy cells by increasing the LC3-II/LC3-I ratio and autophagosome formation.We also analyzed Beclin1, LC3, Bax, and Cleaved-caspase3 protein and mRNA expression levels of autophagic and apoptotic markers using fluorescence confocal microscopy, RT-PCR, and Western blot. We found that cell autophagy and apoptosis increased in a dose-dependent manner with GDC0449 treatment. Additionally, we observed increased mammalian target of rapamycin (mTOR) phosphorylation and decreased protein kinase B (AKT/PKB), Ribosomal Protein S6, eIF4E-binding protein (4EBP1) phosphorylation in GDC0449-treated Daoy cells. It was observed that inhibiting autophagy using Beclin1 siRNA significantly blocked the apoptosis-inducing effects of GDC0449, suggesting that GDC0449 mediates its apoptotic effects by inducing autophagy.Our data suggests that GDC0449 inhibits the growth of human MB Daoy cells by autophagy-mediated apoptosis. The mechanism of GDC0449-induced autophagy in Daoy cells may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.

Asunto(s)

Neoplasias Cerebelosas , Meduloblastoma , Niño , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/farmacología , Proteínas Hedgehog/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Beclina-1/farmacología , Meduloblastoma/tratamiento farmacológico , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/farmacología , Apoptosis , Autofagia , Neoplasias Cerebelosas/tratamiento farmacológico , Línea Celular Tumoral

RESUMEN

PURPOSE: Gliomagenesis and resistance of glioblastoma (GBM) are believed to be mediated by glioma stem cells (GSC). Evidence suggests that SHH signaling promotes GSC proliferation and self-renewal. METHODS: ABTC-0904 was a two-arm, multicenter phase 0/II study of GDC-0449, an oral inhibitor of Smoothened (SMO) in patients undergoing resection for recurrent GBM. All patients (Arms I and II) had surgery and received drug post-operatively. Only patients in Arm I received drug prior to surgery. The primary objective was to determine 6-month progression free survival (PFS-6). Secondary endpoints include median PFS (mPFS) and overall survival (mOS), response rate, and toxicity. Correlative studies included bioanalysis of GDC-0449, and inhibition of SHH signaling, GSC proliferation and self-renewal. RESULTS: Forty-one patients were enrolled. Pharmacokinetics of GDC-0449 in plasma demonstrated levels within expected therapeutic range in 75% of patients. The proportion of tumorcells producing CD133+ neurospheres, neurosphere proliferation, self-renewal, and expression of the SHh downstream signaling was significantly decreased in Arm I following GDC-0449 treatment (p < 0.005; p < 0.001 respectively) compared to Arm II (no drug pre-op). Treatment was well tolerated. There were no objective responders in either arm. Overall PFS-6 was 2.4% (95% CI 0.9-11.1%). Median PFS was 2.3 months (95% CI 1.9-2.6) and mOS was 7.8 months (95% CI 5.4-10.1). CONCLUSIONS: GDC-0449 was well tolerated, reached tumor, and inhibited CD133+ neurosphere formation, but had little clinical efficacy as a single agent in rGBM. This suggests growth and maintenance of rGBM is not solely dependent on the SHH pathway thus targeting SMO may require combined approaches.

Asunto(s)

Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/patología , Proteínas Hedgehog/metabolismo , Recurrencia Local de Neoplasia/patología , Glioma/patología , Antineoplásicos/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Encefálicas/patología

RESUMEN

Epilepsy is one of the common encephalopathies caused by sudden abnormal discharges of neurons in the brain. About 30% of patients with epilepsy are insensitive and refractory to existing antiseizure medications. The sonic hedgehog signaling pathway is essential to the development and homeostasis of brain. Aberrant sonic hedgehog signaling is increased in refractory epileptic lesions and may involve the etiology of epilepsy. Thus, new inhibitors of Smoothened, a key signal transducer of this signaling pathway are urgently need for refractory epilepsy. We have established a high-throughput screening platform and discovered several active small molecules targeting Smoothened including TT22. Here we show that the novel Smoothened inhibitor TT22 could block the translocation of ßarrestin2-GFP to Smoothened, reduce the accumulation of Smoothened on primary cilia, displace Bodipy-cyclopamine binding to Smoothened, and inhibit the expression of downstream Gli transcription factor. Moreover, TT22 inhibits the abnormal seizure-like activity in neurons. Furthermore, we demonstrated that FDA-approved Smoothened inhibitor GDC-0449 and LDE-225 are able to inhibit abnormal seizure-like activity in neurons. Thus, our study suggests that targeting the sonic hedgehog signaling with new small-molecule Smoothened inhibitors might provide a potential new therapeutic avenue for refractory epilepsy.

Asunto(s)

Epilepsia Refractaria , Proteínas Hedgehog , Receptor Smoothened , Humanos , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Convulsiones , Transducción de Señal/fisiología , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo

RESUMEN

Non-small cell lung cancer (NSCLC) is one of the most common and deadly cancers worldwide. Among NSCLC patients, almost half have wild-type epidermal growth factor receptor (EGFR WT). The primary therapeutic option for these EGFR WT NSCLC patients is chemotherapy, while NSCLC patients with EGFR mutations have more diverse therapeutic options, including EGFR tyrosine kinase inhibitors. Moreover, NSCLC patients with EGFR WT have worse chemotherapy response than EGFR mutant NSCLC patients. Thus, an urgent need exists for novel therapeutic strategies to improve chemotherapy response in EGFR WT NSCLC patients. Hedgehog signaling is known to be highly active in NSCLC; however, its potential role in chemoresistance is not fully understood. In the present study, we found that paclitaxel (PTX) treatment induces hedgehog signaling in EGFR WT NSCLC cells, and inhibition of hedgehog signaling with GDC-0449 (Vismodegib) increases sensitivity to PTX-stimulated apoptosis. Furthermore, GDC-0449 potentiates PTX-induced reactive oxygen species and mitochondrial dysfunction. In contrast, a hedgehog agonist, Hh-Ag1.5, attenuates PTX-induced apoptosis. Mechanistic experiments revealed that hedgehog induces phosphorylation of Akt at Ser473. Akt then phosphorylates Bax at Ser184, which can switch its activity from pro-apoptosis to anti-apoptosis. Taken together, our findings suggest that inhibition of hedgehog signaling might be a promising therapeutic strategy to improve PTX response in EGFR WT NSCLC.

RESUMEN

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 µM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 µM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

Asunto(s)

Antineoplásicos/uso terapéutico , Berberina/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Hedgehog/metabolismo , Animales , Línea Celular Tumoral , Proteínas Hedgehog/genética , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , ARN/metabolismo , Estabilidad del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The Hedgehog/GLI (HH/GLI) signaling pathway plays a critical role in human oncogenesis. Unfortunately, the clinical use of HH inhibitor(s) has been associated with serious adverse effects and mutation-related drug resistance. Since the efficacy of SMO (Smoothened) and GLI inhibitors is limited in clinical trials, there remains a critical need for the HH/GLI pathway inhibitors with different mechanisms of action. Here, we show that esophageal adenocarcinoma (EAC) cell lines are insensitive to vismodegib (SMO inhibitor) but respond to GANT61 (GLI1 inhibitor). Furthermore, we examine the role of GLI1 in tumorigenicity of EAC and how a selective bromodomain inhibitor IBET-151 downregulates transcriptional activity of the GLI1 transcription factor in EAC. Our study demonstrates that GLI1 plays an important role in tumorigenicity of EAC and that elevated GLI1 expression in patients' ultrasound-assisted endoscopic biopsy may predict the response to neoadjuvant chemotherapy (NAC) FOLFOX. Importantly, IBET-151 abrogates the growth of vismodegib-resistant EAC cells and downregulates HH/GLI by reducing the occupancy of BRD4 at the GLI1 locus. IBET-151 also attenuates tumor growth of EAC-PDXs and does so in an on-target manner as it reduces the expression of GLI1. We identify HH/GLI signaling as a novel druggable pathway in EAC as well as validate an ability of clinically relevant GLI inhibitor to attenuate the viability of vismodegib-resistant EAC cells. Therefore, we propose that selective bromodomain inhibitors, such as IBET-151, could be used as novel therapeutic agents for EAC patients harboring GLI-dependent tumors.

RESUMEN

Rationale: Hedgehog (Hh) pathway plays an essential role in liver fibrosis by promoting the proliferation of hepatic stellate cells (HSCs) by enhancing their metabolism via yes-associated protein 1 (YAP1). Despite the presence of several inhibitors, Hh signaling cannot be controlled exclusively due to their poor efficacy and the lack of a suitable delivery system to the injury site. Therefore, it is rationale to develop new potent Hh inhibitors and suitable delivery carriers. Methods: Based on the structure and activity of Hh inhibitor GDC-0449, we replaced its sulfonamide group with two methylpyridine-2yl at amide nitrogen to synthesize MDB5. We compared the Hh pathway inhibition and anti-fibrotic effect of MDB5 with GDC-0449 in vitro. Next, we developed MDB5 loaded micelles using our methoxy poly(ethylene glycol)-blockpoly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (PEG-PCC-g-DC) copolymer and characterized for physicochemical properties. We evaluated the therapeutic efficacy of MDB5 loaded micelles in common bile duct ligation (CBDL) induced liver fibrosis, mouse model. We also determined the intrahepatic distribution of fluorescently labeled micelles after MDB5 treatment. Results: Our results show that MDB5 was more potent in inhibiting Hh pathway components and HSC proliferation in vitro. We successfully developed MDB5 loaded micelles with particle size of 40 ± 10 nm and drug loading up to 10% w/w. MDB5 loaded micelles at the dose of 10 mg/kg were well tolerated by mice, without visible sign of toxicity. The serum enzyme activities elevated by CBDL was significantly decreased by MDB5 loaded micelles compared to GDC-0449 loaded micelles. MDB5 loaded micelles further decreased collagen deposition, HSC activation, and Hh activity and its target genes in the liver. MDB5 loaded micelles also prevented liver sinusoidal endothelial capillarization (LSEC) and therefore restored perfusion between blood and liver cells. Conclusions: Our study provides evidence that MDB5 was more potent in inhibiting Hh pathway in HSC-T6 cells and showed better hepatoprotection in CBDL mice compared to GDC-0449.

Asunto(s)

Derivados del Benceno/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Cirrosis Hepática/tratamiento farmacológico , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anilidas/farmacología , Animales , Línea Celular , Modelos Animales de Enfermedad , Dodecanol/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Ratones , Ratones Endogámicos C57BL , Micelas , Poliésteres/química , Polietilenglicoles/química , Ratas

RESUMEN

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.

Asunto(s)

Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Transducción de Señal , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Proteínas Hedgehog/metabolismo , Humanos , Receptor Smoothened/metabolismo , Relación Estructura-Actividad

RESUMEN

Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation. Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment. Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC 0-∞ ) by 7-fold, while t1/2,e and AUC 0-∞ of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation. Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.

Asunto(s)

Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Portadores de Fármacos/administración & dosificación , Micelas , MicroARNs/farmacología , MicroARNs/farmacocinética , Neoplasias Pancreáticas/tratamiento farmacológico , Anilidas/farmacocinética , Anilidas/farmacología , Estructuras Animales/química , Animales , Carbocianinas/farmacocinética , Cromatografía Liquida , Modelos Animales de Enfermedad , Fluorometría , Ratones , Imagen Óptica , Neoplasias Pancreáticas/patología , Plasma/química , Piridinas/farmacocinética , Piridinas/farmacología , Espectrometría de Masas en Tándem , Resultado del Tratamiento , Imagen de Cuerpo Entero

RESUMEN

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the radiation sensitizing effects of vismodegib by analyzing viability, cell cycle distribution, cell death, DNA damage repair and clonogenic survival in three-dimensional cultures of a BCC and a head and neck squamous cell carcinoma (HNSCC) cell line. We found that vismodegib decreases expression of the Hedgehog target genes glioma-associated oncogene homologue (GLI1) and the inhibitor of apoptosis protein (IAP) Survivin in a cell line- and irradiation-dependent manner, most pronounced in squamous cell carcinoma (SCC) cells. Furthermore, vismodegib significantly reduced proliferation in both cell lines, while additional irradiation only slightly further impacted on viability. Analyses of cell cycle distribution and cell death induction indicated a G1 arrest in BCC and a G2 arrest in HNSCC cells and an increased fraction of cells in SubG1 phase following combined treatment. Moreover, a significant rise in the number of phosphorylated histone-2AX/p53-binding protein 1 (γH2AX/53BP1) foci in vismodegib- and radiation-treated cells was associated with a significant radiosensitization of both cell lines. In summary, these findings indicate that inhibition of the Hedgehog signaling pathway may increase cellular radiation response in BCC and HNSCC cells.

Asunto(s)

Anilidas/farmacología , Antineoplásicos/farmacología , Rayos gamma/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patología , Carcinoma Basocelular/terapia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Terapia Combinada/métodos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/terapia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Especificidad de Órganos , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Survivin/genética , Survivin/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo

RESUMEN

Aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway promotes prostate cancer (PC) growth and progression by regulating cancer-related genes through its downstream effectors GLI1 and GLI2. Therefore, targeting the SHH-GLI pathway provides an alternative approach to avoid cancer progression. The aim of this study was to delineate the underlying molecular mechanisms by which GDC-0449 (a SMO receptor inhibitor) and GANT-61 (a GLI transcription factor inhibitor) regulate cellular proliferation and self-renewal in human PC stem cells (ProCSCs). Inhibition of the SHH signaling pathway by GANT-61 induced apoptosis with more efficacy than by GDC-0449 in ProCSCs and PC cell lines. GLI1 and GLI2 expression, promoter-binding activity and GLI-responsive luciferase reporter activity were all decreased with either GDC-0449 or GANT-61 treatment. Expression of Fas, DR4, DR5, and cleavage of caspase-3 and PARP were increased, whereas levels of PDGFR-α and Bcl-2 were reduced. Double knockout of GLI1 and GLI2 using shRNA abolished the effects observed with either GDC-0449 or GANT-61 treatment. Collectively, our results showed that GANT-61 and GDC-0449 induced ProCSC apoptosis by directly or indirectly inhibiting the activities of the GLI family transcription factors, may enhance the efficacy of PC treatment.

Asunto(s)

Anilidas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína Gli2 con Dedos de Zinc/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Humanos , Masculino , Células Madre Neoplásicas/citología , Proteínas Nucleares/genética , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína Gli2 con Dedos de Zinc/genética

RESUMEN

OBJECTIVE: The present study was been designed to investigate the role and pharmacological potential of hedgehog in oestrogen-deficient rat heart. METHODS: Oestrogen deficiency was produced in female Wistar rats by the surgical removal of both ovaries and these animals were used four weeks later. Isolated rat heart was subjected to 30 min ischaemia followed by 120 min of reperfusion (I/R). The heart was subjected to pharmacological preconditioning with the hedgehog agonist purmorphamine (1µM) and GDC-0449, a hedgehog antagonist, in the last episode of reperfusion before I/R. Myocardial infarction was assessed in terms of the increase in lactate dehydrogenase (LDH), creatinine kinase-MB (CK-MB), myeloperoxidase (MPO) level and infarct size (triphenyltetrazolium chloride staining). Immunohistochemistry analysis was done for the assessment of tumour necrosis factor (TNF)-α level in cardiac tissue. eNOS expression was estimated by rt-PCR. RESULTS: Pharmacological preconditioning with purmorphamine significantly attenuated I/R-induced myocardial infarction, TNF-α, MPO level and release of LDH and CK-MB compared to the I/R control group. However, GDC-0449 prevented the ameliorative preconditioning effect of estradiol. CONCLUSION: It may be concluded that the hedgehog agonist purmorphamine prevents the ovariectomised heart from ischaemic reperfusion injury.

Asunto(s)

Cardiotónicos/uso terapéutico , Corazón/efectos de los fármacos , Proteínas Hedgehog/agonistas , Morfolinas/uso terapéutico , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Purinas/uso terapéutico , Animales , Femenino , Corazón/fisiopatología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Ovariectomía , Ratas Wistar

RESUMEN

The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo. Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo.

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dismal overall prognosis mainly unchanged over the past decades. PDAC is generally refractory to conventional treatments, and thus novel therapies are urgently needed. Recently, accumulating evidence has indicated that human pancreatic stellate cells (PSCs) facilitate PDAC development and drug resistance through paracrine activation of hedgehog pathway. Here, we report that smart SN38 (active metabolite of irinotecan) polymeric prodrug-based nanoparticles effectively encapsulate the commercial hedgehog pathway inhibitor GDC-0449 for co-delivery. More intriguingly, we obtained size-tunable nanoparticles with increased GDC-0449 loading efficiency by simply extending the chain length of the hydrophobic SN38 block. To better evaluate the efficacy and investigate the synergistic mechanisms, we immortalized human PSCs and established fibroblast-containing models in vitro and in vivo. In PSCs, BxPC-3 cells and MIA PaCa-2 cells, GDC-0449 suppressed the co-culture induced up-regulations of the two drug resistance contributors: sonic hedgehog transcription factor glioma-associated protein1 (GLI-1) and UGT1A glucuronosyltransferase. Importantly, the nanoparticle-mediated co-delivery system exhibited potent antitumor efficacy with enhanced apoptosis and reduced collagen, α-SMA and GLI-1 expression in tumor tissues. These findings reveal a potential strategy to utilize nanoparticle-mediated drug co-delivery platform as an effective combination therapy for fibroblast-enriched PDAC.

Asunto(s)

Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Camptotecina/análogos & derivados , Fibroblastos/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Piridinas/uso terapéutico , Anilidas/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Camptotecina/administración & dosificación , Camptotecina/uso terapéutico , Técnicas de Cocultivo , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas Hedgehog/metabolismo , Humanos , Irinotecán , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones Desnudos , Nanopartículas/administración & dosificación , Nanopartículas/uso terapéutico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Profármacos/administración & dosificación , Profármacos/uso terapéutico , Piridinas/administración & dosificación , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos

RESUMEN

The sonic hedgehog (Shh) pathway has been proven to be involved in embryonic development and cancer growth. GDC-0449, an antagonist of the hedgehog signaling receptor Smoothened (Smo), was recently approved by the US Food and Drug Administration as a prescription for skin basal cell carcinoma. However, the efficacy of GDC-0449 in the treatment of colon cancer and other malignancies, such as basal cell carcinoma and pancreatic cancer, has remained to be proven. The present study assessed the effect of GDC-0449 on the colon cancer cell lines Caco-2 and Ht-29. A Cell Counting Kit-8 assay was applied to assess the cell proliferation rate and apoptosis was tested by flow cytometry. Reverse-transcription quantitative PCR and western blot analysis were used for analyzing expression levels of target genes. Cell proliferation was inhibited, while apoptosis was increased by GDC-0449, whereas the expression of B-cell lymphoma 2 (Bcl-2), a downstream target of Shh signaling, was decreased. Consistent with the inhibition of Gli1 expression, the cancer stem cell markers CD44 and ALDH were decreased in the presence of GDC-0449. In conclusion, GDC-0449 was shown to inhibit the replication of colon cancer cells and trigger apoptosis through downregulating Bcl-2. This may also influence the stemness of cancer stem cells as indicated by the decreased stem cell surface markers.

RESUMEN

Liver fibrosis is a pathological process complicating schistosomiasis. It is an active process of continuous extracellular matrix accumulation. In Egypt, schistosomiasis re-infection is a continuing problem especially in rural areas. In this study we examined the antifibrotic effect of GDC-0449 (Vismodegib), a hedgehog-pathway inhibitor as a new molecular target for Schistosoma-induced liver fibrosis, in addition to exploring its effect as antischistosomal drug. The effect of GDC-0449 alone or combined with Praziquantel was tried experimentally in infected mice with Schistosoma mansoni. Fifty CD-1 Swiss female albino mice were used, forty mice were infected with Schistosoma mansoni cercariae. Animals were grouped into five groups; uninfected control, infected untreated, infected treated with Praziquantel (500mg/kg/day) for two days, infected treated with GDC-0449 (40mg/kg/day) for seven days, and infected treated with combined Praziquantel and GDC-0449. Parasitological and chemical parameters, hydroxyproline level and liver granuloma were assessed. Liver fibrosis was reduced significantly evidenced by reduced hydroxyproline levels [P<0.01 for combined (Praziquantel/GDC-0449) treatment groups, P<0.001 for GDC-0449-treated group]. Also, histopathological examination of liver tissues revealed that the mean diameter of granulomas was statistically reduced (P=0.001) with a reduction rate of 24.4% on treatment with GDC-0449. In GDC-0449/Praziquantel combined treatment group, number and mean diameter of the granulomas were reduced significantly P<0.001, and P=0.001 respectively. No antischistosomal effect was recorded for GDC-0449 in this study.

Asunto(s)

Anilidas/uso terapéutico , Cirrosis Hepática/tratamiento farmacológico , Hígado/efectos de los fármacos , Piridinas/uso terapéutico , Schistosoma mansoni/efectos de los fármacos , Esquistosomiasis mansoni/tratamiento farmacológico , Anilidas/administración & dosificación , Animales , Cercarias/efectos de los fármacos , Modelos Animales de Enfermedad , Quimioterapia Combinada , Egipto/epidemiología , Femenino , Proteínas Hedgehog/antagonistas & inhibidores , Hidroxiprolina/sangre , Hígado/parasitología , Hígado/patología , Cirrosis Hepática/parasitología , Ratones , Recuento de Huevos de Parásitos , Praziquantel/uso terapéutico , Piridinas/administración & dosificación , Esquistosomiasis mansoni/epidemiología

RESUMEN

PURPOSES: To determine the pharmacokinetic parameters and biodistribution of GDC-0449 loaded polymeric micelles after systemic administration into common bile duct ligation (CBDL) induced liver fibrotic mice. METHODS: We used GDC-0449 encapsulated methoxy poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (PEG-PCD) non-targeted polymeric micelles for GDC-0449 delivery to normal and liver fibrotic mice. To maximize GDC-0449 delivery to hepatic stellate cells (HSCs), mixed micelles formulations with 10, 20 and 30% w/w mannose-6-phosphate (M6P)-conjugated micelles were administered to normal and liver fibrotic mice for targeting M6P/IGF-IIR overexpressed on activated HSCs and biodistribution of GDC-0449 was determined at 30 and 120 min post systemic administration. RESULTS: GDC-0449 distributed to all major organs after systemic administration of drug loaded micelles, with higher accumulation in the liver of both normal and fibrotic mice. The plasma concentration versus time profiles suggest rapid clearance of GDC-0449 after systemic administration of drug loaded micelles in both normal and fibrotic mice, with similar plasma clearance (CL), area under the curve (AUCint) and volume of distribution at steady state (Vss). However, there is significant increase in GDC-0449 accumulation in the liver when M6P-conjugated mixed micelles were injected, with the highest GDC-0449 concentration in the liver with mixed micelles carrying 30% M6P-conjugated polymer. HSCs accounted for 14.19% of GDC-0449 accumulation for M6P-targeted micelles in fibrotic mice compared to 5.62% of non-targeted micelles in the liver uptake study. CONCLUSION: M6P-conjugated GDC-0449 loaded mixed micelles may be used as a potential drug delivery vehicle for treating liver fibrosis.

Asunto(s)

Anilidas/farmacocinética , Cirrosis Hepática/metabolismo , Piridinas/farmacocinética , Anilidas/administración & dosificación , Anilidas/química , Animales , Química Farmacéutica , Portadores de Fármacos , Liberación de Fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Masculino , Manosafosfatos/química , Ratones Endogámicos C57BL , Micelas , Poliésteres/química , Polietilenglicoles/química , Piridinas/administración & dosificación , Piridinas/química , Propiedades de Superficie , Distribución Tisular

RESUMEN

Hedgehog (Hh) signaling plays an important role in the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). Although gemcitabine (GEM) has been used as a first-line therapy for PDAC, its rapid metabolism and short plasma half-life restrict its use as a single chemotherapy. Combination therapy with more than one drug is a promising approach for treating cancer. Herein, we report the use of methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (mPEG-b-PCC-g-DC) copolymer for conjugating GEM and encapsulating a Hh inhibitor, vismodegib (GDC-0449), into its hydrophobic core for treating PDAC. Our objective was to determine whether the micelle mixtures of these two drugs could show better response in inhibiting Hh signaling pathway and restraining the proliferation and metastasis of pancreatic cancer. The in vivo stability of GEM significantly increased after conjugation, which resulted in its increased antitumor efficacy. Almost 80% of encapsulated GDC-0449 and 19% conjugated GEM were released in vitro at pH 5.5 in 48 h in a sustained manner. The invasion, migration, and colony forming features of MIA PaCa-2 cells were significantly inhibited by micelle mixture carrying GEM and GDC-0449. Remarkable increase in PARP cleavage and Bax proved increased apoptosis by this combination formulation compared to individual micelles. This combination therapy efficiently inhibited tumor growth, increased apoptosis, reduced Hh ligands PTCH-1 and Gli-1, and lowered EMT-activator ZEB-1 when injected to athymic nude mice bearing subcutaneous tumor generated using MIA PaCa-2 cells compared to monotherapy as observed from immunohistochemical analysis. In conclusion, micelle mixtures carrying GEM and GDC-0449 have the potential to treat pancreatic cancer.

Asunto(s)

Anilidas/administración & dosificación , Anilidas/química , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Piridinas/administración & dosificación , Piridinas/química , Adenocarcinoma/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Dodecanol/química , Humanos , Masculino , Ratones , Ratones Desnudos , Micelas , Polietilenglicoles/química , Polímeros/química , Propano/análogos & derivados , Propano/química , Transducción de Señal/efectos de los fármacos , Gemcitabina

RESUMEN

In liver fibrosis, secretion of growth factors and hedgehog (Hh) ligands by hepatic parenchyma upon repeated insults results in transdifferentiation of quiescent hepatic stellate cells (HSCs) into active myofibroblasts which secrete excessive amounts of extracellular matrix (ECM) proteins. An Hh inhibitor GDC-0449 and miR-29b1 can play an important role in treating liver fibrosis by inhibiting several pro-fibrotic genes. Our in-silico analysis indicate that miR-29b1 targets several profibrotic genes like collagen type I & IV, c-MYC, PDGF-ß and PI3K/AKT which are upregulated in liver fibrosis. Common bile duct ligation (CBDL) resulted in an increase in Ptch-1, Shh and Gli-1 expression. miR-29b1 and GDC-0449 were co-formulated into micelles using methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) copolymer, and injected systemically into CBDL mice. High concentrations of GDC-0449 and miR-29b1 were delivered to liver cells as determined by in situ liver perfusion at 30 min post systemic administration of their micelle formulation. There was a significant decrease in collagen deposition in the liver and serum injury markers, leading to improvement in liver morphology. Combination therapy was more effective in providing hepatoprotection, lowering liver injury related serum enzyme levels, reducing fibrotic protein markers such as collagen, α-SMA, FN-1 and p-AKT compared to monotherapy. In conclusion, inhibition of Hh pathway and restoration of miR-29b1 have the potential to act synergistically in treating CBDL-induced liver fibrosis in mice.

Asunto(s)

Modelos Animales de Enfermedad , Proteínas Hedgehog/antagonistas & inhibidores , Cirrosis Hepática/tratamiento farmacológico , MicroARNs/administración & dosificación , Animales , Línea Celular , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/uso terapéutico , Ratas

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of death in Asian countries. Sonic hedgehog (Shh) pathway plays a role in hepatocarcinogenesis. We investigated the treatment effect of mouse HCC with Shh inhibitor GDC-0449. METHODS: Mouse hepatoma ML-1 cells were implanted in B6 mice. Fifteen days later, GDC-0449 (vismodegib), antagonist of smoothened, was used to treat HCC-bearing mice. The tumor size and liver histopathological features were analyzed, as well as gene expression in Shh pathways. RESULTS: GDC-0449 treatment effectively reduced tumor size and cell infiltration of the HCC in mice. Gene expression of Shh pathway molecules was altered, including upregulated Shh expression and downregulated smoothened expression in tumor fractions after GDC-0449 treatment. CONCLUSION: GDC-0449 could effectively mitigate HCC growth in vivo.

Asunto(s)

Anilidas/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/patología , Piridinas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Regulación hacia Abajo , Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Ratones Endogámicos C57BL , Receptores Patched , Receptor Patched-1 , ARN Mensajero/metabolismo , Distribución Aleatoria , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteína con Dedos de Zinc GLI1