gant-61 and cyclopamine

Important steps in embryonic development are governed by the Hedgehog (Hh) signaling pathway, an evolutionary conserved signal transduction cascade. However, Hh activity not only is crucial during embryo formation but also is involved in adult tissue repair and in several malignancies. Particularly due to its link to cancer, small molecule Hh pathway inhibitors have been developed and the first compounds have been approved for use in Hh-driven basal cell carcinoma. Almost all advanced Hh inhibitors target the critical signaling component Smoothened (SMO), but preclinical research has identified additional compounds that can block the Hh pathway along its entire signaling cascade, which, in light of emerging drug resistance occurring with SMO inhibitors, is of high importance. Herein we give an overview on currently known Hh pathway inhibitors, delineating their respective strengths and weaknesses and describing potential drug targeting strategies to interfere with Hh signaling in different cancer settings.

Topics: Animals; Antineoplastic Agents; Carcinoma, Basal Cell; Drug Approval; Drug Design; Hedgehog Proteins; Humans; Ligands; Molecular Targeted Therapy; Neoplasms; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

Since its discovery by C. Nüsslein-Volhard and E. F. Wieschaus, hedgehog (hh) signaling has come a long way. Today it is regarded as a key regulator in embryogenesis where it governs processes like cell proliferation, differentiation, and tissue patterning. Furthermore, in adults it is involved in the maintenance of stem cells, and in tissue repair and regeneration. But hh signaling has a second-much darker-face: it plays an important role in several types of human cancers where it promotes growth and enables proliferation of tumor stem cells. The etiology of medulloblastoma and basal cell carcinoma is tightly linked to aberrant hh activity, but also cancers of the prostate, the pancreas, the colon, the breasts, rhabdomyosarcoma, and leukemia, are dependent on irregular hh activity. Recent clinical studies have shown that hh signaling can be the basis of an important new class of therapeutic agents with far-reaching implications in oncology. Thus, modulation of hh signaling by means of small molecules has emerged as a valuable tool in combating these hh-dependent cancers. Cyclopamine, a unique natural product with a fascinating history, was the first identified inhibitor of hh signaling and its story is closely linked to the progress in the whole field. In this review we will trace the story of cyclopamine, give an overview on the biological modes of hh signaling both in untransformed and malignant cells, and finally present potent modulators of the hh pathway-many of them already in clinical studies. For more than 30 years now the knowledge on hh signaling has grown steadily-an end to this development is far from being conceivable.

Topics: Antineoplastic Agents; Hedgehog Proteins; Humans; Neoplasms; Signal Transduction; Veratrum Alkaloids

Topics: Aging; Animals; Cell Survival; Cells, Cultured; Gene Expression Regulation, Developmental; Hedgehog Proteins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoclasts; Osteogenesis; Osteoporosis; Pyridines; Pyrimidines; Receptor, Macrophage Colony-Stimulating Factor; Signal Transduction; Smoothened Receptor; Thiophenes; Up-Regulation; Veratrum Alkaloids; X-Ray Microtomography; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Transitional cell carcinoma (TCC) is the most commonly diagnosed tumor of the canine urinary system. Hedgehog (HH) signaling represents one possible novel therapeutic target, based on its recently identified central role in human urothelial carcinoma. The purpose of this study was to determine if HH mediators are expressed in canine TCC and the effect of inhibition of this pathway on cell growth and survival. HH pathway mediators were found to be expressed in five canine TCC cell lines. Indian HH was expressed in tumor cells in five canine bladder tumor tissues, but not in normal canine bladder tissue. Inhibition of HH signaling with cyclopamine and GANT61 led to significantly decreased cell proliferation but had a smaller effect on apoptosis. These results support future investigation of inhibitors of HH signaling in the treatment of canine TCC.

Topics: Animals; Apoptosis; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Polymerase Chain Reaction; Pyridines; Pyrimidines; Urinary Bladder Neoplasms; Veratrum Alkaloids

The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Chromones; Enzyme Activation; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Morpholines; Neoplasm Invasiveness; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pyridines; Pyrimidines; Signal Transduction; Snail Family Transcription Factors; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Veratrum Alkaloids; Vimentin; Zinc Finger Protein GLI1

Rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB).. We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches.. The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3β, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts.. GDNF/RET/AKT/GSK3β signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth.. Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor; Glycogen Synthase Kinase 3 beta; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Neuroblastoma; Nuclear Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-ret; Pyridines; Pyrimidines; RNA, Small Interfering; Signal Transduction; Transfection; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Hedgehog (Hh) signalling contributes to carcinogenesis and represents a valid druggable target in human cancers, possibly also in biliary tract cancer (BTC). We analysed the expression of Hh components in BTC using eight heterogeneously differentiated cell lines, xenograft tumours and a human tissue microarray. The dose-, time- and cell line-dependent effects of two Hh inhibitors (cyclopamine and Gant-61) were analysed in vitro for survival, apoptosis, cell cycle distribution and possible synergism with conventional chemotherapeutic agents. In human BTC samples, the sonic Hh ligand and the Gli1 transcription factor showed increased expression in tumours compared to normal adjacent tissue and were significantly associated with high tumour grade and positive lymph node status. In BTC cell lines, we could confirm the Hh component expression at varying extent within the employed cell lines in vitro and in vivo indicating non-canonical signalling. Both Hh inhibitors showed dose-dependent cytotoxicity above 5 µM with a stronger effect for Gant-61 inducing apoptosis whereas cyclopamine rather inhibited proliferation. Cytotoxicity was associated with low cytokeratin expression and higher mesenchymal marker expression such as vimentin. Additionally, drug combinations of Gant-61 with conventional chemotherapy (cisplatin) exerted synergistic effects. In conclusion, Hh pathway is significantly activated in human BTC tissue compared to normal adjacent tissue. The current data demonstrate for the first time an effective anticancer activity of especially Gant-61 in BTC and suggest second generation Hh pathway inhibitors as a potential novel treatment strategy in BTC.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biliary Tract Neoplasms; Cell Line, Tumor; Cisplatin; Dose-Response Relationship, Drug; Hedgehog Proteins; Humans; Mice, Mutant Strains; Molecular Targeted Therapy; Pyridines; Pyrimidines; Signal Transduction; Tissue Array Analysis; Transcription Factors; Veratrum Alkaloids; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Female; Gene Amplification; Hedgehog Proteins; Humans; In Vitro Techniques; Luciferases; Mice; Mice, Nude; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Survival; Flavins; Humans; Keratinocytes; Male; Melanoma; Mice; Mice, Inbred C57BL; Pyridines; Pyrimidines; Riboflavin; Veratrum Alkaloids

The molecular responses of two small molecules, SANT-2 and GANT-61, potentially interfering with the sonic hedgehog pathway (Shh) have been studied in zebrafish embryos by microarray analysis. For both compounds and the positive reference cyclopamine previous reporter gene assays for the transcription factor Gli1 have indicated an inhibition of the hedgehog signaling pathway. In zebrafish embryos a typical phenotype (cyclopia) associated with Shh interference was only observed for cyclopamine. Furthermore, only cyclopamine led to the repression of genes specifically associated with hedgehog signaling and confirmed published microarray data. In contrast to these data hspb11 was additionally identified as the most pronounced down-regulated genes for exposure to cyclopamine. No or different effects on gene expression patterns were provoked by SANT-2 or GANT-61, respectively. Reasons for the discrepancies between cellular reporter and the zebrafish embryo assay and potential implications for the identification of compounds interfering with specific developmental pathways are discussed.

Topics: Animals; Benzamides; Benzimidazoles; Embryo, Nonmammalian; Gene Expression Profiling; Gene Expression Regulation, Developmental; Hedgehog Proteins; Pyridines; Pyrimidines; Transcription, Genetic; Veratrum Alkaloids; Zebrafish; Zebrafish Proteins

Aberrant reactivation of Gli signaling has been described in a wide variety of human cancers and rapamycin can down-regulate Gli pathway in some solid tumors. In this study, we attempt to define the cytotoxic effect of Gli inhibitor on AML cells. And the regulation action of rapamycin on Gli in AML cells also has been assessed. Gli inhibitor GANT61 caused growth arrest and apoptosis in AML cells. Rapamycin decreased not only the Gli protein and mRNA expressions but also expression of the Gli-luciferase reporter in AML cells. Synergism effect between GANT61 and rapamycin was found in Kasumi-1, HL-60 and U937 cell lines. The results suggest that aberrant Gli activation is a feature of some myeloid leukemic cells and Gli activiation can be down-regulated by rapamycin.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Evaluation, Preclinical; Drug Synergism; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Pyridines; Pyrimidines; RNA, Small Interfering; Sirolimus; Transcription Factors; U937 Cells; Veratrum Alkaloids; Zinc Finger Protein GLI1

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.

Topics: Apoptosis; beta Catenin; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Butyrates; Cell Survival; Chemokine CCL2; Colonic Neoplasms; Cytokines; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Hedgehog Proteins; HT29 Cells; Humans; Interleukin-8; Kruppel-Like Transcription Factors; Oncogene Proteins; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.

Topics: Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Fas-Associated Death Domain Protein; HCT116 Cells; Hedgehog Proteins; HT29 Cells; Humans; Kruppel-Like Transcription Factors; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Appearance of mouse fetal Leydig cells requires activation of the Hedgehog pathway. Upon binding to the membrane-bound receptor patched, Hedgehog ligands induce intracellular responses via a combined effect of Gli transcription factors. Szczepny et al. (Biol Reprod 2009; 80:258-263) found that Gli1, one of the three Gli transcription factors, is present in the fetal testis and that its expression is suppressed by the Hedgehog inhibitor cyclopamine. In this study, we investigated the involvement of the Gli1 and Gli2 factors in mouse fetal Leydig cell differentiation. The Gli1 and Gli2 transcription factors showed an overlapping expression pattern in the testis interstitium at the time when fetal Leydig cells appear. Despite their similar expression, Gli1 and Gli2 patterns were differentially regulated. Initial Gli1 and Gli2 expression depends upon an active Hedgehog pathway; however, maintenance of only Gli1, but not Gli2, expression requires activation of the pathway. Inactivation of either the Gli1 or Gli2 gene did not affect fetal Leydig cell development and testis morphology, suggesting a functional redundancy. When the transcriptional activity of both GLI1 and GLI2 was suppressed by a selective inhibitor, GANT61, in cultured fetal testes before the appearance of fetal Leydig cells, Gli1 and Gli2 expression and steroidogenic marker activity were completely abolished. However at later stages when Leydig cells were already present, GANT61 treatment inhibited Gli1 expression but had no effects on Gli2 expression and fetal Leydig cell appearance. Our results reveal overlapping and redundant Gli1 and Gli2 roles in fetal Leydig cell differentiation and a novel regulation of Gli2 expression in the fetal testis.

Topics: Animals; Cell Differentiation; Cholesterol Side-Chain Cleavage Enzyme; Down-Regulation; Embryo, Mammalian; Genes, Reporter; Hedgehog Proteins; In Situ Hybridization; Kruppel-Like Transcription Factors; Leydig Cells; Male; Mice; Mice, Knockout; Organ Culture Techniques; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; RNA, Messenger; Smoothened Receptor; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Aberrant activation of HH/GLI has recently been reported in multiple cancer types, yet its role in oral squamous cell carcinoma (OSCC) has not been investigated. In this study, we aimed to determine the role of HH/GLI in OSCC. Expression of GLI1 and GLI2 was examined in OSCC samples from 136 patients by immunohistochemistry and correlated with clinicopathology parameters and clinical outcomes of the patients. Two HH/GLI specific small molecule inhibitors cyclopamine and GANT61, were used to test the potential role of HH/GLI in OSCC. We found that GLI2, one of the main transcriptional activators of HH/GLI signalling, was expressed in 60 (44%) of the 136 OSCC samples and the expression was significantly associated with poor clinical outcomes. Only 44% of the patients whose tumours expressed GLI2 survived at 5years after surgery compared to 77% of those whose tumours lacked the GLI2 expression (P<0.0001). Both cyclopamine and GANT61 effectively inhibited GLI expression, slowed cell growth, promoted G1 arrest, increased apoptosis and inhibited migration of OSCC cells. Our results demonstrate that activation of HH/GLI pathway plays an important role in OSCC progression. Together with the finding that expression of GLI2 is strongly associated with a poor clinic outcome of OSCC patients, the data suggest that a subset of OSCC patients may benefit from anti-HH/GLI therapies.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Immunohistochemistry; Male; Middle Aged; Molecular Targeted Therapy; Mouth Neoplasms; Neoplasm Proteins; Prognosis; Pyridines; Pyrimidines; Signal Transduction; Transcription Factors; Veratrum Alkaloids; Young Adult; Zinc Finger Protein GLI1

The Hedgehog pathway functions as an organizer in embryonic development. Recent studies have shown that mutation of the PTCH1 gene involved in the Hedgehog pathway affects rhabdomyosarcoma development. However, the expression of Hedgehog pathway molecules in human rhabdomyosarcoma cells has not been well clarified. In addition, the effect of pharmacological inhibition of the Hedgehog pathway is not known. We investigated the expression of the genes involved in the Hedgehog pathway using human rhabdomyosarcoma cell lines and biopsy specimens. Further, we evaluated the effect of pharmacological inhibition of the Hedgehog pathway using cyclopamine or GANT61 by WST assay, cell proliferation assay and cell death detection assay. Real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens overexpressed the following genes: Sonic hedgehog, Indian hedgehog, Desert hedgehog, PTCH1, SMO, GLI1, GLI2 and ULK3. Immunohistochemistry revealed that rhabdomyosarcoma cell lines and biopsy specimens expressed SMO and GLI2. Inhibition of SMO by cyclopamine slowed the growth of human rhabdomyosarcoma cell lines. Similarly, inhibition of GLI by GANT61 slowed the growth of human rhabdomyosarcoma cell lines. Inhibition of cell proliferation and apoptotic cell death together prevented the growth of rhabdomyosarcoma cells by cyclopamine and GANT61 treatment. Our findings suggest that pharmacological inhibition of the Hedgehog pathway may be a useful approach for treating rhabdomyosarcoma patients.

Topics: Cell Death; Cell Growth Processes; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Nuclear Proteins; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; Rhabdomyosarcoma; Signal Transduction; Smoothened Receptor; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein Gli2

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; DNA Damage; Gene Expression; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Pyridines; Pyrimidines; Signal Transduction; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

Hedgehog signaling regulates Gli transcription factors. Aberrant hedgehog signaling can be oncogenic and drugs that block hedgehog are being tested as anticancer agents. We considered whether hedgehog/Gli signaling may be involved in human bladder transitional cell carcinoma proliferative or invasive behavior.. We stratified the human bladder transitional cell carcinoma lines RT4 (ATCC), 253JP, 253BV, UMUC6 and UMUC3 for relative growth rate by cell counting and for in vitro invasiveness by Matrigel invasion assay. Cells were tested for growth inhibition by the hedgehog blocking drug cyclopamine or the inactive mimic tomatidine. Cell RNA was characterized for hedgehog signaling component expression, including ligands, receptors and signaling mediators, by quantitative reverse transcriptase-polymerase chain reaction. Gli2 expression or activity was modified by Gli2 expression lentiviruses or the Gli inhibitor GANT61. We measured effects on proliferation and invasiveness.. Cell growth rates and invasiveness were stratified into an equivalent order (RT4 <243JP <253BV

Topics: Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Dioxoles; Gene Expression Profiling; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Linear Models; Neoplasm Invasiveness; Nuclear Proteins; Piperazines; Pyridines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tomatine; Urinary Bladder Neoplasms; Veratrum Alkaloids; Zinc Finger Protein Gli2

The Hedgehog (Hh) pathway regulates cell proliferation and survival and contributes to tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and in healthy B lymphocytes. Profiling of cognate Hh pathway members revealed reduced expression of two key Hh signaling effectors, Smoothened (SMOH) and GLI, in CLL cells, whereas transcription levels of other investigated members resembled normal B-lymphocyte levels. Examining the functional role of SMOH and GLI in cell survival, we found that CLL cells were hardly sensitive toward specific SMOH inhibition, but showed an unspecific decline in cell viability in response to high concentrations of the SMOH antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT61 reduced expression of the target gene Patched and preferentially decreased the viability of malignant cells. Specific RNA interference knockdown experiments in a CLL-derived cell line confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemic cells was partly attenuated by protective stromal cells, but not soluble sonic hedgehog ligand. In summary, our data show a downregulation of the classical Hh pathway in CLL and suggest an intrinsic SMOH-independent role of GLI in the ex vivo survival of CLL cells.

Topics: Animals; Apoptosis; B-Lymphocytes; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Hedgehog Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Oncogene Proteins; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; RNA, Messenger; Smoothened Receptor; Trans-Activators; Veratrum Alkaloids; Zinc Finger Protein GLI1