Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: Astemizole functions as a better PRC2 inhibitor by degrading PRC2 and AR. (a)C4–2 cells were treated with GSK126 (2 μM), EPZ5687 (5 and 10 μM), EPZ6438 (20 and 40 μM), EED226 (10 and 20 μM), astemizole (10 μM) as well as vehicle and lysed for immunoblot analysis 72 hr after drug treatment. (b-d)C4–2, LNCaP and VCaP cells treated with astemizole at dose gradients and lysed for immunoblot analysis 72 hr after treatment.

Article Snippet: GSK126 (406,228, MedKoo), EPZ5687 (S7004, Selleckchem), EPZ6438 (S7128, Selleckchem), EED226 (S8496, Selleckchem) and astemizole (3,489, Tocris) were dissolved in 100% ethanol or DMSO for cell treatment.

Techniques: Western Blot

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 knockdown and astemizole treatment demonstrate similar inhibition patterns of AR signaling blockage.(a) EZH2 knockdown and astemizole-treated samples cluster together based on log expression of 1,571 (top 10%) high variation genes. (b) Heat maps for the expression level of genes down- or up-regulated by EZH2 knockdown, GSK126 and astemizole treatment. (c) The number of overlapped differential genes in each paired group is significantly larger than the number of genes overlapped by chance. (d) 426 AR-induced genes were compared and the expression is similar between EZH2 knockdown and astemizole-treated samples. (e) Comparison of PSA gene track between groups. (f) Comparison of PSA gene track between groups. (g) GSEA shows that AR target genes are significantly enriched (Q value = 0.0429) in downregulated genes due to EZH2 knockdown. (h) GSEA shows that AR target genes are significantly enriched (Q value = 0.0413) in downregulated genes due to astemizole treatment.

Article Snippet: GSK126 (406,228, MedKoo), EPZ5687 (S7004, Selleckchem), EPZ6438 (S7128, Selleckchem), EED226 (S8496, Selleckchem) and astemizole (3,489, Tocris) were dissolved in 100% ethanol or DMSO for cell treatment.

Techniques: Knockdown, Inhibition, Expressing, Comparison

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: Astemizole has potent therapeutic effects on prostate cancer. (a) Astemizole critically thwarts cell proliferation in C4–2 and other AR-positive prostate cancer cell lines. (b) The wound healing assay indicates that astemizole compromises the migration of C4–2 cells. (c) Astemizole decreases the invasive abilities of C4–2 cells compared to vehicle treatment. Cell count was analyzed and the difference was statistically significant. (d)C4–2 cells were treated with 2.5, 5 and 7.5 μM of astemizole. Cells were lysed 48 hr after treatment and blotted with anti-LC3-A/B antibody. The ratio of LC3-A/B-II/I to GAPDH was elevated as dose increased, which indicates that astemizole induces autophagy in prostate cancer cells. (e) Castration-resistant VCaP xenograft mouse models were generated. Castrated mice bearing CPRC xenografts received vehicle or astemizole treatment (50 mg kg−1) daily (5 days per week). Caliper measurements were taken every 4 days to determine tumor volume. Mean tumor volume SEM, *p < 0.05, **p < 0.01 vs. vehicle was marked. (f) Kaplan–Meier survival plot compares progression-free survival. (g) Upper panel: Proteins were blotted and quantitated to compare the protein levels of EZH2 and AR in astemizole-treated group (n = 8) compared to vehicle-treated group (n = 12). Lower panel: The expression of EZH2 and AR was decreased in response to astemizole treatment. (h) The proportion of the cells stained with EZH2/AR/PSA in astemizole-treated group (n = 6) were significantly lower than that in vehicle-treated group (n = 6).

Article Snippet: GSK126 (406,228, MedKoo), EPZ5687 (S7004, Selleckchem), EPZ6438 (S7128, Selleckchem), EED226 (S8496, Selleckchem) and astemizole (3,489, Tocris) were dissolved in 100% ethanol or DMSO for cell treatment.

Techniques: Wound Healing Assay, Migration, Cell Counting, Generated, Expressing, Staining

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 1. Representative IC50 plots for telmisartan (A) and flunarizine (B) inhibition of astemizole O-demethylation using recombinant CYP2J2 with astemizole concen- trations of 0.1–20 mM.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition, Recombinant

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 2. Disappearance of astemizole (A), telmisartan (B), and flunarizine (C), measured from incubation with recombinant CYP2J2 in the presence of NADPH at different time points (n = 2).

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Incubation, Recombinant

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 3. IC50 determination of inhibition of CYP2J2-mediated astemizole O- demethylation by telmisartan (A) and flunarizine (B) in the presence and absence of NADPH. The inhibitors were preincubated with CYP2J2 for 30 minutes. The IC50 shift was calculated as IC50 in the absence of NADPH over IC50 in the presence of NADPH, to evaluate time-dependent inhibition.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 4. Inhibition assay against the enzymatic activity of recombinant CYP2J2. Nonlinear regression of the initial velocity at various substrate concentrations in the presence of telmisartan (A) and flunarizine (B) as the inhibitor with concentrations of 0.1–2 mM and 0.2–5 mM, respectively. Dixon plots with amplified insets for the enzyme kinetic study of CYP2J2-mediated astemizole O-demethylation in the presence of different concentrations of telmisartan (C) and flunarizine (D) as the inhibitor. Astemizole concentrations used were 0.05 (d), 0.1 (u), 0.15 (m), 0.3 ()), and 0.45 mM (,) (n = 4). The replots of the slope of Dixon plot versus reciprocal of substrate concentration for telmisartan (E) and flunarizine (F).

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition, Activity Assay, Recombinant, Amplification, Concentration Assay

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 7. Comparison of measured compound CYP2J2 inhibitory activities (IC50) with those reported in the literature (percentage activity remaining). Astemizole O- demethylation was used for evaluating the metabolic activity of CYP2J2; compounds with a measured IC50 value higher than 50 mM in our laboratory were treated as IC50 of 50 mM in the comparison; percentage activity remaining was obtained at single inhibitor concentration of 30 mM as reported in the literature, and only compounds with activity remaining less than 100% were included. Compounds included were amodiaquine, nicardipine, haloperidol, clozapine, lansoprazole, verapamil, fluoxetine, and omeprazole.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Comparison, Activity Assay, Concentration Assay

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 8. Overlay of substrate (astemizole) and the inhibitor within the binding pocket of CYP2J2 for telmisartan (A) and flunarizine (B), respectively. The CYP2J2 protein is in cartoon representation and colored in rainbow spectrum; the heme is in stick and colored in orange; telmisartan, flunarizine, and astemizole are in stick and colored in yellow, magenta, and green, respectively.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Identification of astemizole and ikarugamycin as immunostimulatory agents. (A–D) De-ini dendritic cells (DC) were cultured with the compounds of the indicated chemical libraries for 16 hours, then washed twice and pulsed with 500 µg/mL ovalbumin (OVA) protein for 4 hours, washed again twice, and then co-cultured with B3Z hybridoma cells for 16 hours. Then, the supernatant of cells was harvested and the production of interleukin 2 (IL2), as an indicator for antigen cross-presentation capacity, was measured by ELISA. IL2 concentration was normalized according to dimethyl sulfoxide (DMSO)-treated controls. (E–F) De-iniDCs and bone marrow-derived DCs (BMDCs) were treated with DMSO or ikarugamycin (0.1, 0.2, 0.5, 1 µM) (E), or astemizole (0.2, 0.5, 1, 2, 5 µM) (F) for 16 hours, then washed twice and pulsed with OVA protein or 10 ng/mL OVA257-264 peptide (SL8, as a positive control) for 4 hours, washed again twice, and then co-cultured with B3Z hybridoma cells. IL-2 secretion was measured by ELISA as an indication for antigen cross-presentation capacity. Data are reported as the mean±SD of three replicates, and statistical analyses were performed with a two-tailed unpaired Student’s t test. de-iniDCs, de-induced/de-immortalized DCs.

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay, Positive Control, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Validation of ikarugamycin as a dendritic cell (DC) stimulator. (A) Bone marrow-derived dendritic cells (BMDCs) were treated with ikarugamycin (IKA; 1 µM) or astemizole (AST; 1 µM) for 16 hours then DC activation and maturation was analyzed by flow cytometry, 1 µg/mL of lipopolysaccharide (LPS) was used as positive control. (B, C) BMDCs were treated with IKA (0.2, 0.5, 1, 2 µM) for 16 hours, then cells were harvested, and proteins were detected by western blot. (D–E) Representative western blot images (D) and quantification (E) of control (Ctr) or hexokinase 2 (Hk2) knockdown BMDC cells. (F) Ctr or Hk2 knockdown BMDCs were pulsed with 500 µg/mL ovalbumin (OVA) protein for 4 hours, washed twice, and then co-cultured with B3Z hybridoma cells for 16 hours. Interleukin 2 (IL2) secretion was measured by ELISA as an indicator for antigen cross-presentation capacity. (G) Control (Ctr) or Hk2 knockdown BMDCs were collected for cytofluorometric detection of DC activation and maturation markers. (H) Schematic overview of the treatment of murine MCA205 fibrosarcoma with IKA (0.2 mg/kg) and oxaliplatin (OXA; 10 mg/kg), alone or in combination. (I, J) The tumor growth curves (I, mean±SEM) and Kaplan-Meier overall survival (J) of murine fibrosarcoma MCA205 cells in C57BL/6 mice (n=8–10 mice /group). (K) Schematic overview of the treatment of murine MCA205 fibrosarcoma with IKA plus OXA (Comb) and neutralizing antibodies to CD4 and CD8 (αCD4/CD8), alone or in combination. (L, M) The tumor growth curves (L, mean±SEM) and Kaplan-Meier overall survival (M) of murine fibrosarcoma MCA205 cells in immunocompetent and antibody-neutralized C57BL/6 mice (n=8–10 mice/group). Data are reported as the mean±SD of three replicates, and statistical analyses were performed with two-tailed unpaired Student’s t test (A, C, E, F, G), and two-way analysis of variance followed by the Holm-Šídák post hoc test (I, L). Log-rank test was used for survival comparison.

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Biomarker Discovery, Derivative Assay, Activation Assay, Flow Cytometry, Positive Control, Western Blot, Control, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Validation of astemizole as a T-cell stimulator. (A) Schematic overview of different protocols for astemizole treatment. (B, C) De-ini dendritic cells (DC)s (B) and bone marrow-derived DCs (C) were treated with dimethyl sulfoxide (DMSO) or astemizole (AST; 0.2, 0.5, 1 µM) in different protocol, and then co-cultured with B3Z hybridoma cells. Interleukin 2 (IL2) secretion was measured by ELISA as an indicator for antigen cross-presentation capacity. (D) B3Z hybridoma cells were treated with DMSO or AST (0.1, 0.2, 0.5, 1 µM) for 16 hours. IL2 secretion was measured by ELISA as an indicator for antigen cross-presentation capacity. (E, F) Mouse primary CD8 + T cells were treated with DMSO, or AST (0.1, 0.2, 0.5, 1 µM) or phorbol myristate acetate (PMA; 50 ng/mL) for 16 hours, followed by 5-hour treatment with brefeldin A (5 µg/mL), and analyzed for expression of the indicated proteins or cytokines using flow cytometry. (I–W) AST was injected at 1 mg/kg body weight into the hind footpad either once (I), twice (N) or four times (S). The draining (popliteal) lymph nodes were collected and dissociated into single cell suspensions. Then the cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of the indicated proteins or cytokines using flow cytometry (n=5 mice /group). Each dot represents one lymph node. Data are reported as the mean±SD, and statistical analyses were performed with two-tailed unpaired Student’s t test (B, C, E, F, J, K, M, O, R, T, U), and Mann-Whitney U test (D, L, P, Q, V, W). de-iniDCs, de-induced/de-immortalized DCs.

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Biomarker Discovery, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Non-specific T-cell stimulation by astemizole. (A–D) Jurkat T cells were treated with astemizole (AST; 1 µM) and human CD3/CD28 dynabeads (10 6 cells per 25 µL) for 3 min and were then analyzed by western blotting. Representative western blot images (A) and quantification of p-LCK (tyr 394) to LCK (B), p-Zap-70 (tyr 319) to Zap-70 (C), and p-LAT (tyr 220) to LAT (D). (E) Jurkat-LCK T cells were treated with dimethyl sulfoxide (DMSO), AST (1 µM), AST (1 µM) plus LCK inhibitor (LCKi; 1 µM), and human T Cell TransAct nanobeads (10 5 cells per µL) in different time points and were measured by flow cytometry. (F) Jurkat T cells were pre-treated with DMSO or LCKi (1 µM) for 30 min, and treated with DMSO or AST (1 µM) for 3 min, then cells were harvested, and proteins were detected by western blot. (G–J) B3Z hybridoma cells were treated with AST (1 µM) alone or in combination with LCKi (0.2, 1 µM) for 16 hours, the cell supernatant was collected for the quantification of interleukin 2 (IL-2) and interferon gamma (IFN-γ) secretion by ELISA (I, J). While the remaining cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of CD69 or IFN-γ using flow cytometry (G, H). (K–M) Mouse primary CD8 + T cells were treated with AST (1 µM) alone or in combination with LCKi (0.2, 1 µM) for 16 hours, the cell supernatant was collected for the quantification of IFN-γ secretion by ELISA (M). While the remaining cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of CD69 or IFN-γ using flow cytometry (K, L). Data are reported as the mean±SD, statistical analyses were performed with two-tailed unpaired Student’s t test (B), Mann-Whitney U test (C, D), one-way analysis of variance (ANOVA) followed by the Holm-Šídák post hoc test (G–M), and two-way ANOVA followed by the Holm-Šídák post hoc test (E).

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Cell Stimulation, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Antihistaminergic mode of action of astemizole. (A, B) B3Z hybridoma cells were treated with astemizole (AST; 1 µM) alone or in combination with histamine (0.5, 1, 2, 5 mM) for 16 hours, the cell supernatants were collected for the quantification of interleukin 2 (IL2) secretion by ELISA (A). While the remaining cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of interferon gamma (IFN-γ) using flow cytometry (B). (C) Mouse primary CD8 + T cells were treated with AST (1 µM) alone or in combination with histamine (0.5, 1, 2, 5 mM) for 16 hours, followed by 5 hours of brefeldin A (5 µg/mL), and analyzed for expression of IFN-γ using flow cytometry. (D, E) B3Z hybridoma cells were treated with astemizole (1 µM) alone or in combination with HTMT dimaleate (0.5, 1, 2, 5 µM) for 16 hours, the cell supernatants were collected for the quantification of IL2 secretion by ELISA (D). While the remaining cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of IFN-γ using flow cytometry (E). (F) Mouse primary CD8 + T cells were treated with AST (1 µM) alone or in combination with HTMT dimaleate (0.5, 1, 2, 5 µM) for 16 hours, followed by 5 hours of brefeldin A (5 µg/mL), and analyzed for expression of IFN-γ using flow cytometry. (G) Jurkat T cells were pretreated with DMSO, histamine (5 mM) or HTMT dimaleate (5 µM) for 30 min, and treated with DMSO or AST (1 µM) for 3 min, then cells were harvested, and proteins were detected by western blot. (H) Jurkat T cells treated with AST, tefernadine (Tefer), ketotifen fumarate (Keto), promethazine (Prome), and desloratadine (Deslo) all at 1 µM for 3 min were analyzed by western blotting. (I) Mouse primary CD8 + T cells isolated from wild type or Hrh1 knockout C57BL/6 mice were treated with DMSO or AST (1 µM) for 16 hours, followed by 5 hours of brefeldin A (5 µg/mL), and analyzed for expression of IFN-γ using flow cytometry (n=4 mice /group, Data are reported as the mean±SEM). (J–L) Spleens from wild-type C57BL/6 or Hrh1 − / − mice were harvested dissociated into single cell suspensions, then the cells were treated with brefeldin A (5 µg/mL) for 5 hours, and analyzed for expression of the indicated proteins or cytokines using flow cytometry (n=4 mice /group, Data are reported as the mean±SEM). Data are reported as the mean±SD, and statistical analyses were performed with two-tailed unpaired Student’s t test (I–L), and one-way analysis of variance followed by the Holm-Šídák post hoc test (A–F).

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot, Isolation, Knock-Out, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Astemizole enhances antitumor effect of immunogenic chemotherapy. (A) Schematic overview of the treatment of murine MCA205 fibrosarcoma with astemizole (AST) and oxaliplatin (OXA), alone or in combination (Comb). (B, C) The tumor growth curves (B, mean±SEM) and Kaplan-Meier overall survival (C) of murine fibrosarcoma MCA205 cells in C57BL/6 mice (n=9–10 mice /group). (D) The tumor growth curves (mean±SEM) of murine fibrosarcoma MCA205 cells in nu/nu mice (n=10 mice /group). (E–H) MCA205 fibrosarcoma bearing mice were treated as in (A) and were euthanized on day 15 after tumor injection, tumors were harvested and dissociated into single cell suspensions, and analyzed for expression of the indicated proteins or cytokines using flow cytometry. Data are reported as the mean±SD, and statistical analyses were performed with one-way analysis of variance followed by the Holm-Šídák post hoc test (E–H), and two-way analysis of variance followed by the Holm-Šídák post hoc test (B, D). Log-rank test was used for survival comparison (C).

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Injection, Expressing, Flow Cytometry, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses

doi: 10.1136/jitc-2023-006785

Figure Lengend Snippet: Astemizole induces immune-dependent anticancer efficacy in orthotopic TC-1-luc lung cancer model. (A) Schematic overview of the treatment of murine orthotopic TC-1-luc lung cancer with astemizole (AST) and oxaliplatin (OXA), alone or in combination (Comb). (B–D) Representative bioluminescence images of tumor development under different treatment conditions are shown in (B), the quantification of bioluminescence signals was used to indicate tumor size and is reported as tumor growth curves (C, mean±SEM) and Kaplan-Meier overall survival (D) of murine orthotopic TC-1-luc lung cancer cells in C57BL/6 mice (n=7–13 mice/group). (E, F) Cured immunocompetent mice were rechallenged with living cancer cells of the same type (TC-1-luc), as well as of a different type (MCA205), the percentage of TC-1-luc tumor growth on cured mice is shown in (E) and the percentage of MCA205 tumor growth on cured mice is shown in (F). (G) Schematic overview of the treatment of murine orthotopic TC-1-luc lung cancer with AST plus OXA (Comb) in combination with neutralizing antibodies to CD4 and/or CD8 or IFN-γ,or the isotype (ISO) control antibodies (n=9–11 mice /group). (H–K) The quantification of bioluminescence signals was used to indicate tumor size and reported as tumor growth curves (H, J, mean±SEM) and Kaplan-Meier overall survival (I, K) of murine orthotopic TC-1-luc lung cancer in immunocompetent and antibody-neutralized C57BL/6 mice. Statistical analyses were performed with two-way analysis of variance followed by the Holm-Šídák post hoc test (C)(, H, J). Log-rank test was used for survival comparison (D, I, K).

Article Snippet: Astemizole (sc-201088), 2-Pyridylethylamine dihydrochloride (sc-203467) and 4-methylhistamine dihydrochloride (sc-203475A) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Control, Comparison

Journal: Genes

Article Title: Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and Its Regulation by pRb in HeLa Cells

doi: 10.3390/genes11020119

Figure Lengend Snippet: Eag1 was differentially expressed in human retinoblastoma and astemizole inhibited cell proliferation. ( A ) In comparison with the control (pediatric normal brain obtained postmortem, where the Eag1 mRNA level was set to the value of 1, Eag1 mRNA expression was very heterogeneous between the retinoblastoma samples. Many samples showed a lower expression, while other samples had clearly higher Eag1 mRNA levels. No differences in Eag1 expression versus the control was observed when separating the samples into unilateral ( n = 15) and bilateral ( n = 15) tumors ( B ). Examples of heterogeneous Eag1 protein expression studied using immunohistochemistry in three cases. Positive Eag1 expression in a zone of normal retina from an enucleated retinoblastoma case ( C ); weak Eag1 expression in retina and in tumor, upper right zone ( D ) and negative Eag1 expression ( E ). Magnification: 100×. Astemizole decreased the cell proliferation (assayed by metabolic activity) in retinoblastoma primary cultures ( F ) ( n = 10). Each cell proliferation experiment was performed with sextuplicates. Panel (F) shows mean ± s.d. * p < 0.05 versus control.

Article Snippet: Astemizole was kindly provided by Liomont Laboratories (Mexico City, Mexico).

Techniques: Comparison, Control, Expressing, Immunohistochemistry, Activity Assay