Journal: Cell Death & Disease

Article Title: Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

doi: 10.1038/cddis.2016.195

Figure Lengend Snippet: Compound 12 inhibition of Hh-dependent MB tumor cell growth. ( a – c ) Ex vivo cell cultures from Ptch1 +/− mice MBs were treated with compound 12 , Cyclopamine, Vismodegib, LDE-225 or DMSO only. ( a and b ) After the indicated times, a trypan blue count was performed to determine the growth rate of viable cells. ( c ) Gli1 mRNA expression levels were determined by qRT-PCR normalized to endogenous control ( β2-microglobulin and HPRT ). ( d – f ) Compound 12 inhibits MB-SCs self-renewal. ( d ) Suspension of single MB-SCs isolated from Ptch1 +/− mice were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of compound 12, Cyclopamine or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self-renewal MB-SCs' capability is expressed as percentage of neurosphere-forming cells. ( e ) Representative bright-field images of tumor neurospheres after compound 12 or Cyclopamine treatment are also shown. ( f ) MB-SCs isolated from Ptch1 +/− mice MBs were treated for 48 h with compound 12 or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA. For qRT-PCR, results were normalized to endogenous control ( β2-microglobulin and HPRT ). All data show the mean±S.D. of three independent experiments. * P <0.05; ** P <0.01 versus DMSO (CTR)

Article Snippet: Cells were treated with SAG (200 nM, Alexis Biochemicals Farmingdale, NY, USA), Bodipy-Cyclopamine (5 nM, BioVision Inc., San Francisco, CA, USA), Cyclopamine (Calbiochem, Nottingham, UK), Vismodegib (Selleckchem, Munich, Germany), LDE-225 (Selleckchem, Munich, Germany).

Techniques: Inhibition, Ex Vivo, Expressing, Quantitative RT-PCR, Control, Suspension, Isolation, Cell Culture, Derivative Assay

Journal: Cell Death & Disease

Article Title: Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

doi: 10.1038/cddis.2016.195

Figure Lengend Snippet: Compound 12 inhibition of Hh-dependent BCC cell growth in vitro and in vivo . ( a – c ) Compound 12 inhibition of Hh-dependent BCC tumor cell growth. ASZ001 BCC cells were treated with compound 12 , Cyclopamine, Vismodegib, LDE-225 or DMSO only ( a and b ). After the indicated times, a trypan blue count was performed to determine the growth rate. Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with compound 12 , Cyclopamine, Vismodegib, LDE-225 or DMSO only ( c ). Results were normalized to endogenous control ( β2-microglobulin and HPRT ). All data show the mean±S.D. of three independent experiments. * P <0.05; ** P <0.01 versus DMSO (CTR). ( d – g ) ASZ001 BCC allografts. Change of tumor volume during compound 12 or vehicle treatment period ( d ). Representative flank allografts average volumes ( e ). H&E and immunohistochemical staining of Ki67 of allograft tumor samples ( f ). Scale bars represent 250 μ m for H&E and Ki67. Quantification of Ki67 staining from immunohistochemistry shown in ( f ). ( g ) Shown is the mean±S.D. of tumor ( n =6) for each treatment. * P <0.05 versus CTR

Article Snippet: Cells were treated with SAG (200 nM, Alexis Biochemicals Farmingdale, NY, USA), Bodipy-Cyclopamine (5 nM, BioVision Inc., San Francisco, CA, USA), Cyclopamine (Calbiochem, Nottingham, UK), Vismodegib (Selleckchem, Munich, Germany), LDE-225 (Selleckchem, Munich, Germany).

Techniques: Inhibition, In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Control, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

doi: 10.1038/cddis.2016.195

Figure Lengend Snippet: Compound 12 inhibition of human Hh-dependent tumor cell growth. Human medulloblastoma DAOY ( a and b ) or human prostate carcinoma epithelial 22Rv1 cells ( d and e ) were treated with compound 12 , Cyclopamine, Vismodegib, LDE-225 or DMSO only. After the indicated times, a trypan blue count was performed to determine the growth rate. Gli1 mRNA expression levels were determined by qRT-PCR after treatment of DAOY ( c ) or 22Rv1 ( f ) cells with compound 12 , Cyclopamine, Vismodegib, LDE-225 or DMSO only. Results were normalized to endogenous control ( β-actin and HPRT ). All data show the mean±S.D. of three independent experiments. * P <0.05; ** P <0.01 versus DMSO (CTR)

Article Snippet: Cells were treated with SAG (200 nM, Alexis Biochemicals Farmingdale, NY, USA), Bodipy-Cyclopamine (5 nM, BioVision Inc., San Francisco, CA, USA), Cyclopamine (Calbiochem, Nottingham, UK), Vismodegib (Selleckchem, Munich, Germany), LDE-225 (Selleckchem, Munich, Germany).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Effect of rapamycin, vismodegib, and both on BTC cell survival and proliferation. ( A ) Mz-ChA-1 and ( B ) Sk-ChA-1 cells were treated for 72 h at serial concentrations (0.25–50 μ mol l −1 ). Rapamycin, vismodegib, and both caused a dose-dependent decrease in BTC cell proliferation, as measured by the CellTiter-Glo assay. ( C ) Clonogenic assay. Cells were treated with DMSO (control), 1 μ mol l −1 rapamycin, 10 μ mol l −1 vismodegib, or both for72 h, cells stained with 0.5% crystal violet. ( D ) Colony numbers as determined by clonogenic assay. The number of colonies with ⩾50 cells was counted. Values are mean±s.d. Results are representative of three independent experiments. * P <0.05, ** P <0.01.

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Glo Assay, Clonogenic Assay, Control, Staining

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Synergistic effect of combination treatment with rapamycin and vismodegib on BTC cells

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques:

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Cell-cycle analysis. ( A ) Mz-ChA-1 and ( B ) Sk-ChA-1 cells were treated with 1 μ mol l −1 rapamycin, 10 μ mol l −1 vismodegib, or both for 72 h. Cell-cycle distribution was analyzed by flow cytometry.

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Cell Cycle Assay, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Effect of rapamycin, vismodegib, or both on stem cell sphere formation. Effects of vismodegib, rapamycin or both on tumoursphere formation and Nanog, Oct-4, and E-Cadherin gene expression. Mz-ChA-1 ( A ) and Sk-ChA-1 ( B ) tumour spheres. Third passage single cells were cultured for 72 h, and then treated with vehicle, rapamycin (1 μ mol l −1 ), vismodegib (10 μ mol l −1 ), or both for 7 days. Tumourspheres were imaged by Olympus microscope. Compared with control group, the combination treatment inhibited Nanog, Oct-4, and E-Cadherin expression in Mz-ChA-1 ( C ) (** P <0.01) and Sk-ChA-1 ( D ) (* P <0.05).

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Gene Expression, Cell Culture, Microscopy, Control, Expressing

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: ALDH activity measured by FACS analysis. Aldefluor analysis of Mz-ChA-1 cells ( A ) and Sk-ChA-1 cells ( B ) treated with vehicle, rapamycin (1 μ mol l −1 ), vismodegib (10 μ mol l −1 ), or both for 72 h. ALDH + Mz-ChA-1 cells were significantly decreased in the combined treatment group (compared with control and vismodegib, P <0.01 and P <0.05, respectively).

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Activity Assay, Control

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Effect of rapamycin, vismodegib, or both on protein expression. Mz-ChA-1 cells ( A ) and Sk-ChA-1 cells ( B ) were treated with vehicle, rapamycin (1 μ mol l −1 ), vismodegib (10 μ mol l −1 ), or both for 24 h. Actin was used as a loading control (* P <0.05, ** P <0.01).

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Expressing, Control

Journal: British Journal of Cancer

Article Title: Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

doi: 10.1038/bjc.2014.625

Figure Lengend Snippet: Effect of rapamycin, vismodegib, or both on Mz-ChA-1 cell xenograft tumors. ( A ) Effects on xenograft growth. Mice treated with vehicle only, rapamycin (1 mg kg −1 , b.i.d.), vismodegib (100 mg kg −1 , b.i.d.), or both when tumour volume reached 100 mm 3 . Values are expressed as mean±s.d. ( n =8 mice per treatment group). ( B ) Nude mouse weight. The mice were weighed every 3 days and there was no significant change in nude mice weight. Values are expressed as mean±s.d. ( C ) Immunohistochemical analysis of xenograft tumour tissue. Tumour tissues were subjected to immunostaining for p-p70S6K and Gli1 protein expression. Quantified p-p70S6K and Gli1 protein expression levels in a representative xenograft tumour from each treatment group are shown at right. * P <0.05, ** P <0.01.

Article Snippet: Rapamycin was purchased from LC laboratories (Woburn, MA, USA) and vismodegib was obtained from Genentech (South San Francisco, CA, USA).

Techniques: Immunohistochemical staining, Immunostaining, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance

doi: 10.1158/1078-0432.CCR-14-0011

Figure Lengend Snippet: Hedgehog pathway inhibition with Smo shRNA or vismodegib blocks spheroid formation. A. Western blot demonstrating knockdown of Smo in gastric cancer cell lines AGS, MKN-45, and N87 following transduction with Smo shRNA (Smo.shRNA) lentivirus compared to scrambled shRNA control (Scr.shRNA) lentivirus. B. Immunofluorescence photos for CD44 (green) and nuclei (blue) of gastric cancer cell lines treated with Smo.shRNA or Scr.shRNA and grown in spheroid formation conditions or treated with vismodegib (10 μM) or carrier (DMSO) and grown in spheroid formation conditions. C. Single cell assay of spheroid cells showing diameter of spheroids at selected time points following treatment with Smo.shRNA vs. Scr.shRNA or vismodegib (Vis, 10 μM) vs. carrier (DMSO). Bars represent standard deviation.

Article Snippet: Vismodegib was purchased from LC Laboratories (Woburn, MA).

Techniques: Inhibition, shRNA, Western Blot, Transduction, Immunofluorescence, Standard Deviation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance

doi: 10.1158/1078-0432.CCR-14-0011

Figure Lengend Snippet: CD44(+) gastric cancer cells demonstrate chemotherapy resistance, which can be reversed with Hedgehog pathway inhibition. A. Immunofluorescence images of CD44 (green) and nuclei (blue) for CD44(+) and CD44(−) cells following magnetic bead sorting of AGS, MKN-45, and N87 gastric cancer cell lines. B. Western blot showing expression of Hedgehog pathway proteins Sonic hedgehog (Shh), Patch 1 (Ptch1), Smoothened (Smo), and Gli1 in CD44(+) and CD44(−) cells. Proliferation assay for CD44(+) and CD44(−) cells (C) and spheroid cells and monolayer cells (D) following treatment with 5-fluorouracil (5-FU) or cisplatin chemotherapy and vismodegib (Vis, 10 μM) compared to DMSO. Bars represent standard deviation.

Article Snippet: Vismodegib was purchased from LC Laboratories (Woburn, MA).

Techniques: Inhibition, Immunofluorescence, Western Blot, Expressing, Proliferation Assay, Standard Deviation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance

doi: 10.1158/1078-0432.CCR-14-0011

Figure Lengend Snippet: Gastric cancer spheroid cells demonstrate increased migration, colony formation, and anchorage independent growth, which can be attenuated by Hedgehog pathway inhibition. A. Photos and graphs of migration assay of gastric cancer monolayer cells and spheroid cells treated with Smo shRNA (Smo.shRNA) compared to control (Scr.shRNA) or vismodegib (10 μM) compared to DMSO. B. Colony formation assay of gastric cancer monolayer cells and spheroid cells treated with Smo shRNA (Smo.shRNA) compared to control (Scr.shRNA) or vismodegib (10 μM) compared to DMSO. C. Photos and graph of soft agar assay of gastric cancer monolayer cells and spheroid cells treated with Smo shRNA (Smo.shRNA) compared to control (Scr.shRNA). Bars represent standard deviation.

Article Snippet: Vismodegib was purchased from LC Laboratories (Woburn, MA).

Techniques: Migration, Inhibition, shRNA, Colony Assay, Soft Agar Assay, Standard Deviation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance

doi: 10.1158/1078-0432.CCR-14-0011

Figure Lengend Snippet: CD44 may be a response biomarker in advanced gastric and gastroesophageal cancer patients treated with chemotherapy with or without vismodegib. A. Schema of the clinical trial. B. CD44 immunohistochemistry of patient tumor samples showing low, intermediate, and high CD44 expression. C. Kaplan-Meier overall survival curves for patients receiving chemotherapy alone and patients receiving chemotherapy plus vismodegib stratified by low and high CD44 score.

Article Snippet: Vismodegib was purchased from LC Laboratories (Woburn, MA).

Techniques: Biomarker Assay, Immunohistochemistry, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance

doi: 10.1158/1078-0432.CCR-14-0011

Figure Lengend Snippet: CD44 score, progression, and survival

Article Snippet: Vismodegib was purchased from LC Laboratories (Woburn, MA).

Techniques:

Journal: PLoS ONE

Article Title: Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

doi: 10.1371/journal.pone.0120517

Figure Lengend Snippet: Single doses of vismodegib were administered at discrete time points indicated by tick marks on the x-axis, including: GD7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.625, 8.75, 8.875, 9.0, 9.25, 9.5, 9.75, and 10.0. Cyclopamine was administered by subcutaneous infusion from GD8.25 to ~9.375. Representative examples of distinct face and palate phenotypes are shown, including apparently normal (Normal), HPE, CL/P, and CPO. Note that lateral lip clefts resulting from acute vismodegib exposure typically extended into the primary palate (D’), while those resulting from cyclopamine exposure extended into both the primary and secondary palate (F’). The penetrance of HPE, CL/P, and CPO phenotypes resulting from stage-specific vismodegib exposure is shown in the graph. 5–7 litters were examined for each exposure permutation.

Article Snippet: Vismodegib (Toronto Research Chemicals) was suspended as 3mg/ml in 0.5% methyl cellulose (Sigma) with 0.2% Tween (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

doi: 10.1371/journal.pone.0120517

Figure Lengend Snippet: Superior views of dissected brains are shown for a vehicle-exposed normal animal (A) and for representative examples of animals with vismodegib-induced HPE (B), cyclopamine-induced CL/P (C), and vismodegib-induced CPO (D). Severe hypoplasia of the cerebral cortices (cc) and olfactory bulb (ofb) absence is apparent in the animal with HPE. In animals with CL/P and CPO, the cerebral cortices appear to be of approximately normal size but the olfactory bulbs are hypoplastic. Serial coronal sections of comparably classified animals are shown in E-P. Notable HPE-associated features include a single central nasal passage with nasal septum (ns) cartilage absence (black arrow), olfactory bulb agenesis (J), and a single telencephalic vesicle (N). Grossly normal division of the olfactory bulbs (K, L) and cerebral cortices (O, P), and apparent forebrain septal (s) region hyperplasia (white arrows) is observed in animals with CL/P and CPO. (t) Tongue, (e) eye, (sp) secondary palate.

Article Snippet: Vismodegib (Toronto Research Chemicals) was suspended as 3mg/ml in 0.5% methyl cellulose (Sigma) with 0.2% Tween (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

doi: 10.1371/journal.pone.0120517

Figure Lengend Snippet: In vehicle-exposed embryos at GD14.5 (A) the secondary palatal shelves have approximated and made contact at the midline. In affected cyclopamine-exposed embryos with cleft lip (B), palatal shelves are widely spaced and deficient in width. In vismodegib-exposed embryos (C), secondary palatal shelves have also elevated but are deficient in both length and width. Length (D) and width (E) measurements (arbitrary units), as depicted by the dashed calipers, were made on light microscopy images . Shelf width was determined at 1/3 shelf length from the most rostral aspect. *** p<0.001, **** p<0.0001

Article Snippet: Vismodegib (Toronto Research Chemicals) was suspended as 3mg/ml in 0.5% methyl cellulose (Sigma) with 0.2% Tween (Sigma).

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

doi: 10.1371/journal.pone.0120517

Figure Lengend Snippet: Along with a vehicle-exposed control (A), representative examples of phenotypic outcomes are shown with numbers indicating the gestational stage of acute vismodegib administration. Later exposure was associated with forelimb ectrodactyly, as exhibited bilaterally in fetuses exposed from 9.25 to 9.75 (arrows point to absent fifth digits on the right limb). Kinked tail phenotypes were caused by exposure between GD9.5 and 10.0 (arrowheads). Edema is also apparent in fetuses exposed at GD9.75 and 10.0. For each treatment group the number of litters and fetuses examined, mean litter size and crown-rump length, and the incidence of edema, forelimb ectrodactyly, and kinked tail defects are presented in .

Article Snippet: Vismodegib (Toronto Research Chemicals) was suspended as 3mg/ml in 0.5% methyl cellulose (Sigma) with 0.2% Tween (Sigma).

Techniques: Control