Journal: Cancer Discovery

Article Title: A Drug Repositioning Approach Identifies Tricyclic Antidepressants as Inhibitors of Small Cell Lung Cancer and Other Neuroendocrine Tumors

doi: 10.1158/2159-8290.cd-13-0183

Figure Lengend Snippet: Figure 5. The candidate drugs inhibit the expansion of SCLC cells via several GPCRs. A and B, MTT viability assays of cells cultured at 2% serum ( n ≥ 3 independent experiments) and treated with the ADRA1 antagonist doxazosin mesylate in comparison with treatment with imipramine (Imip) for 48 hours (A) and with increasing doses of epinephrine (Epi) in the absence or presence of 50 μmol/L imipramine or 30 μmol/L promethazine (Prom; B). The paired t test was used to calculate the P values of epinephrine-, imipramine-, and promethazine-treated cells versus control cells and of imipramine- and promethazine-treated cells versus epinephrine-treated cells combined with imipramine or promethazine. *, P < 0.05; **, P < 0.01; ns, not signifi cant. C, MTT viability assay for mSCLC (Kp1) and mNSCLC (LKR13) cells following 48 hours of treatment with increasing doses of the PKC inhibitor GF109203X. Values from three independent experiments are shown as the mean ± SEM. An unpaired t test was used to calculate the P values of the drug-treated cells versus control cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not signifi cant. D, MTT viability assay for mSCLC (Kp1) cells following 24 hours of treatment with 50 μmol/L imipramine alone and with increasing doses of PMA in the absence or presence of 50 μmol/L imipramine. An unpaired t test was used to calculate the P values of dimethyl sulfoxide (DMSO)–treated cells versus PMA-treated cells and of imipramine-treated cells versus PMA-treated cells combined with imipramine. ns, not signifi cant. E, representative immunoblotting of p-PKC, total PKC, p-CREB, and total CREB in mSCLC cells (Kp1) untreated and treated with 50 μmol/L imipramine for 30 minutes. Tubulin was used as a loading control. F and G, MTT viability assay for mSCLC (Kp1) and mNSCLC (LKR13) cells following 48 hours of treatment with increasing doses of the adenyl cyclase inhibitor KH7 (F) and the PKA inhibitor H89 dihydro- chloride (G). Values from three independent experiments are shown as the mean ± SEM. An unpaired t test was used to calculate the P values of the drug- treated cells versus control cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not signifi cant. H, MTT viability assay for mSCLC (Kp1) and hSCLC (H187) cells following 24 hours of treatment with 50 μmol/L forskolin (FSK), 100 μmol/L IBMX, or both drugs combined. An unpaired t test was used to calculate the P values of the drug-treated cells versus control DMSO-treated cells. ns, not signifi cant. I, effects of the combined treatment of 50 μmol/L imipramine and 50 μmol/L FSK alone, 100 μmol/L IBMX alone, or FSK and IBMX together, as measured by the MTT viability assay. Values from at least three inde- pendent experiments are shown as the mean ± SEM. An unpaired t test was used to calculate the P values of imipramine-treated cells versus control DMSO-treated cells and of imipramine-treated cells versus FSK-, IBMX-, and FSK + IBMX–treated cells combined with imipramine. J, representative immunoblotting of p-c-Jun and total c-Jun in mSCLC cells (Kp1) untreated, treated with 50 μmol/L imipramine for 30 minutes in the absence or presence of 50 μmol/L forskolin (FSK) and 100 μmol/L IBMX, and treated with DMSO. Tubulin was used as a loading control. The black bars in all the MTT assays represent the vehicle-treated cells normalized to 100%. IBMX, 3-isobutyl-1-methylxanthine.

Article Snippet: Z-VAD-FMK, ritanserin, 4-DAMP, doxazosin mesylate, H89 dihydrochloride, KH7, and GF109203X were all purchased from Tocris Bioscience.

Techniques: Cell Culture, Comparison, Control, MTT Viability Assay, Western Blot

Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: PKA activity correlates with loss of GLP-1R from the cell surface . MIN6 cells were transfected with a GFP-tagged GLP-1R (GLP-1R-GFP) expressed under the control of the constitutive CMV promoter. (A) GLP-1R-GFP transfected MIN6 cells were cultured at low glucose (3 mM; LG) or high glucose (25 mM; HG) for 4 h in the presence or the absence of the PKA inhibitor, H89 (HG + H89). GFP was visualized in green, nuclei stained with dapi (blue) and β-catenin immune-stained to mark the plasma membrane (red). (B) GLP-1R-GFP transfected MIN6 cells were infected with a recombinant adenovirus expressing a constitutively active PKA catalytic subunit (caPKA). Cells were cultured at low glucose (LG), and the localization of the GLP-1R-GFP was determined by fluorescence microscopy. Cells infected with the caPKA adenovirus were identified using an antibody against the FLAG-tag of the caPKA (red). (C) GLP-1R-GFP transfected MIN6 cells were cultured for 4 h at high glucose (HG) with: forskolin (Fsk), to raise cAMP levels through the activation of adenylyl cyclases; H89, to inhibit PKA; or expression of a dominantly negative PKA regulatory subunit (dnPKA). GLP-1-GFP was visualized in green, nuclei by staining with dapi (blue), and the FLAG-tag epitope on the dnPKA in red.

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Activity Assay, Transfection, Control, Cell Culture, Staining, Clinical Proteomics, Membrane, Infection, Recombinant, Expressing, Fluorescence, Microscopy, FLAG-tag, Activation Assay

Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: Chronic exposure of β-cells to the GLP-1R agonist exendin-4 . (A) MIN6 cells were chronically cultured (20 h) at low glucose in the absence (LG) or the presence (LG + Ex4) of the GLP-1R agonist exendin-4, fixed and incubated with fluorescein-tagged exendin-4 to bind cell surface (but not internal) GLP-1R. (B) MIN6 cells transfected with GLP-1R-GFP were cultured for 4 h at high glucose with exendin-4 in the absence (HG + Ex) or the presence of H89 (HG + Ex + H89), and GLP-1R-GFP localization was determined by fluorescence microscopy (green). Cells were stained for β-catenin to mark membranes (red) and dapi to identify nuclei (blue). (C) To determine the in vivo effects of chronic exendin-4 exposure, male mice were administered exendin-4 at 4–6 h intervals for 24 h or were administered saline at each time-point (controls). Mice were then given a 5 μg/kg body exendin-4 dose and an hour later a 3 g/kg intraperitoneal glucose bolus. Plasma insulin levels were measured for 15 min following the glucose challenge (C) and blood glucose levels for 120 min (D). Data were analyzed by 2-way ANOVA with Bonferroni post hoc tests. *, P < 0.05; ***, P < 0.001. n = 6–8 mice for both C and D).

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Cell Culture, Incubation, Transfection, Fluorescence, Microscopy, Staining, In Vivo, Saline, Clinical Proteomics

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 reverses ABCB1-mediated MDR in CRC cells. ( A ) The chemical structure of H89. ( B , C ) Cells were treated with the indicated drugs for 72 h and detected by MTT assay. Representative cell viability curves are shown. ** p < 0.01 compared to the corresponding control groups. Data are presented as mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: MTT Assay, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: The combination of H89 with doxorubicin or vincristine induces cell cycle arrest. Cells were treated with the indicated reagents for 24 h, and the cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining. The concentrations of each agent were as follows: 10 μM H89, 0.03 μM doxorubicin and 0.003 μM vincristine for HCT-8 cells; 10 μM H89, 0.1 μM doxorubicin and 0.1 μM vincristine for HCT-8/V cells. ( A ) Representative histograms. Bright blue indicates Sub G1 period, and red indicates G0/G1-G2/M period. ( B ) Representative quantitative data. ** p < 0.01 compared to the corresponding control groups. Data are presented as the mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Flow Cytometry, Staining, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 enhances the intracellular accumulation of substrate drugs within HCT-8/V cells. After preincubatingHCT-8 and HCT-8/V cells with different concentrations of H89 or 10 μM verapamil for 1 h, 10 μM doxorubicin (red) or rhodamine 123 (green) was added and incubated together for 2 h. Subsequently, the cells were observed and imaged under a microscope, and quantitative analysis was performed using flow cytometry. Higher intracellular fluorescence intensity indicates a greater intracellular drug accumulation. Representative images ( A , D ), histograms ( B , E ), and quantitative data ( C , F ) are shown. The scale bar is 200 μm. “+” indicates the combination of two drugs. *** p < 0.001 compared to the corresponding control groups. Data are presented as the mean ± SD ( n = 3).

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Incubation, Microscopy, Flow Cytometry, Fluorescence, Control

Journal: Biomedicines

Article Title: H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

doi: 10.3390/biomedicines13122869

Figure Lengend Snippet: H89 inhibits the ATPase activity of ABCB1 and occupies its ATP-binding pocket. ( A ) ABCB1 expression levels in HCT-8/V cells treated with 10 μM and 30 μM H89 for the indicated time points were measured by Western blot. ( B ) The effect of H89 on the ATPase activity of ABCB1 was determined using the ABCB1 ATPase assay kit. Data are presented as the mean ± SD ( n = 3). ( C ) The best-scoring pose of H89 in the ATP-binding domain of ABCB1. ABCB1 is displayed as a silver ribbon, and H89 is shown as a sphere model, predominantly in yellow, where yellow represents C (carbon), blue represents N (nitrogen), bright red represents O (oxygen), dark red represents Br (bromine), and tan represents S (sulfur). ( D ) The best-scoring binding conformation of ABCB1 with ATP. ABCB1 is displayed as a silver ribbon, and ATP is shown as a sphere model, predominantly in orange, where orange represents S (sulfur), blue represents N (nitrogen), cyan represents C (carbon), and bright red represents O (oxygen). ( E ) Detailed illustration of the interactions between H89 and the ATP-binding pocket of ABCB1. ABCB1 is displayed as a translucent silver ribbon, and the key amino acids are shown as orange-red sticks. H89 is presented as yellow sticks. Hydrophobic interactions are indicated by gray dashed lines, π-π stacking interactions by gray solid lineswith bond lengths of 2.9 Å, and hydrogen bonds by blue solid lines. The bond lengths between H89 and GLY-521, LYS-532, LEU-475, and ASN-899 are 3.4 Å, 3.6 Å, 3.0 Å, and 3.1 Å, respectively. ( F ) Detailed illustration of the interaction between ABCB1 and ATP. ABCB1 is displayed as a translucent silver ribbon, with key amino acids shown as orange-red sticks. ATP is presented as bright blue sticks. Gray dashed lines indicate hydrophobic interactions; yellow dashed lines represent salt bridges; gray solid lines denote π-π stacking interactions with bond lengths of 3.9 Å and 4.2 Å; blue solid lines indicate hydrogen bonds. The bond lengths between ATP and GLY-521, GLN-526, GLU-522, LYS-532, LEU-527, and ALA-525 are 3.1 Å, 2.7 Å, 2.3 Å, 3.9 Å, 3.0 Å, and 2.8 Å, respectively.

Article Snippet: H89 dihydrochloride (#460618), 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (#Q108115), doxorubicin hydrochloride (#A603456-0025), verapamil hydrochloride (#V4629), vincristine sulfate (#2068-78-2), oxaliplatin (#A8648), and rhodamine 123 (#83702) were purchased from TargetMol Chemicals Inc. (Shanghai, China), Dibo Biotechnology Co. (Shanghai, China), Bio Basic Inc. (Shanghai, China), Sigma-Aldrich (Shanghai, China), Aikon Biopharmaceutical R&D Co. (Nanjing, Jiangsu, China), APExBIO Technology LLC (Houston, TX, USA), and Sigma-Aldrich Trading Co. (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot, ATPase Assay