1,1-Dimethylbiguanide hydrochloride (metformin) was purchased from Sigma-Aldrich (St. Louis, MO). S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was obtained from Macrocyclics (Plano, TX). DOTA-(Tyr³)-octreotide was purchased from Bachem (Torrance, CA). Amino acid derivatives were purchased from Novabiochem (San Diego, CA) or Chem-Impex International (Wood Dale, IL). All other chemical reagents and solvents were purchased from Sigma-Aldrich and were used as received unless otherwise specified. 68GaCl3 was obtained from elution using a 68Ge/68Ga generator (Eckert & Ziegler, Berlin, Germany). 111InCl3 was obtained from Triad Isotopes (Houston, TX). 64CuCl2 was supplied by the Cyclotron Radiochemistry Facility at The University of Texas MD Anderson Cancer Center (Houston, TX).
Metformin 1 1 dimethylbiguanide hydrochloride
Manufactured by Merck Group
Sourced in United States
Metformin (1,1-dimethylbiguanide hydrochloride) is a white, crystalline powder. It is a synthetic biguanide compound used as an active pharmaceutical ingredient.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
68ge 68ga generator, by Eckert & Ziegler (1 mentions)
S 2 4 isothiocyanatobenzyl 1 4 7 triazacyclononane 1 4 7 triacetic acid p scn bn nota, by Macrocyclics (1 mentions)
Atp assay kit, by Abcam (1 mentions)
Glut4, by Cell Signaling Technology (1 mentions)
Anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
D glucose glucose, by Merck Group (1 mentions)
Fluo 4 direct calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
D glucose, by Merck Group (1 mentions)
T25 flask, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
X rad 320, by Precision X-Ray (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Hoechst staining, by Beyotime (1 mentions)
Phospho c jun, by Cell Signaling Technology (1 mentions)
Sp600125, by Selleck Chemicals (1 mentions)
30 protocols using metformin 1 1 dimethylbiguanide hydrochloride
1
Radiolabeling of Chelated Compounds
2
DPHC Assay for Calcium Signaling
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Diphlorethohydroxycarmalol (DPHC) was isolated as previously described by Heo et al. [19 (link)]. Fluo-4 Direct™ Calcium Assay kit was purchased from Invitrogen by Thermo Fisher Scientific (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT); 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM); D-(+)-glucose (glucose); 2,4,5,6(1H,3H)-pyrimidinetetrone (alloxan monohydrate); and 1,1-dimethylbiguanide hydrochloride (metformin) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against phospho-AMP-activated protein kinase (Thr172), AMPK and p-Akt (Ser473), Akt, Glut4, and GAPDH were obtained from Cell Signaling Technology (Bedford, MA, USA), and anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ATP Assay Kit (colorimetric/fluorometric) was purchased from Abcam (Abcam Inc., Cambridge, USA).
3
Clonogenic Assay for Cell Survival
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We performed clonogenic assays to evaluate treatment effects on cell survival. Low passage cells were plated in T25 flasks (Becton, Dickinson, Heidelberg, Germany) with 5 ml medium as described previously.13 (link) TMZ was obtained by Schering-Plough (Kenilworth, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Deisenhofen, Germany) at concentrations < 0.5%. Cell samples were incubated with 10 μM or 50 μM TMZ for 4 hours prior to irradiation. 1,1-Dimethylbiguanide hydrochloride (metformin; 97%) was provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and applied in concentrations of 1 mM or 20 mM for 24 hours prior to irradiation. Immediately prior to irradiation, all samples were rinsed twice with phosphate buffered saline and fresh medium was added. Adherent cells were irradiated using a 6MV photon linear accelerator (XRAD 320 Precision X-ray Inc., N.Bradford, USA) with single photon doses up to 6 Gy. Cells were fixed with 70% ethanol and stained with methylene blue. Colonies of > 50 were counted. Experiments were repeated in triplicate at least three times.
4
Metformin-Induced Apoptosis and Autophagy
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1,1-Dimethylbiguanide hydrochloride (metformin) (#D150959) was obtained from Sigma Aldrich, then was dissolved in distilled water at 1M concentration and finally was stored at -20°C before use. 3-MA (M9281) was purchased from Sigma-Aldrich. SP600125 (#1460) was purchased from Selleckchem (Houston, TX, USA). NAC, Hoechst staining as well as JC-1 were bought from Beyotime Institute of Biotechnology (Shanghai, China). The antibodies to cleaved-PARP (#5652), cleaved-caspase3 (#9661), Bcl-2 (#15071), LC3II/I (#12743), p62 (#8025), Beclin-1 (#3495), JNK (#9252), phospho-JNK (#4668), c-Jun (#9165), phospho-c-Jun (#3270), Cyclin D1 (#2978), and secondary antibodies (anti-rabbit and anti-mouse) were bought from Cell Signaling Technology. The antibody to GAPDH (#I121209) was bought from TransGen.
5
Gallium Maltolate Pharmacokinetics Assay
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Gallium maltolate was obtained from Titan Pharmaceuticals (South San Francisco, CA). Human transferrin (Tf), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), oligomycin, carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and 1.1-dimethylbiguanide hydrochloride (metformin) were obtained from Sigma Chemical Company (St. Louis, MO). Antibodies to transferrin receptor1 (TfR1), heme oxygenase-1 (HO-1), and beta-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), while 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di-acetoxymethyl ester (6-H2DCF-AM) was purchased from Invitrogen (Carlsbad CA). Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor drug [30 (link)], was obtained from Howard L. Elford, PhD (Molecules for Health, Richmond, VA) and was prepared as previously described [43 (link)]. 55FeCl3 was purchased from Perkin Elmer (Richmond, CA) and 55Fe-Tf was prepared as previously described [44 (link)].
6
Palmitate-induced Metabolic Dysfunction in NIT-1 Cells
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NIT-1 cells were purchased from ATCC (Cat# CRL-2055, RRID: CVCL_3561; Manassas, VA, USA) and cultured in RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 100 U/mL penicillin, and 100 µg/mL streptomycin. Palmitate was dissolved at 100 mM in ethanol to make stock solution. Palmitate stock solution was incubated at 37℃ for an hour and diluted in the culture medium to which fatty acid-free bovine serum albumin (BSA) had been added in a 1:3 molar ratio, to prepare BSA-conjugated palmitate. Cells were incubated with the BSA-conjugated palmitate (0.2 to 0.5 mM) for the indicated time periods. The compounds 1,1-dimethylbiguanide hydrochloride (metformin) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) used as an AMPK agonist as well as compound C as an AMPK antagonist were purchased from Sigma (St. Louis, MO, USA).
7
Evaluating AMPK and Metabolic Regulators
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Phenformin hydrochloride, 1,1-dimethylbiguanide hydrochloride (metformin), echinomycin, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for AMPKα, phospho-AMPKα (Thr172), phosphoAMPKα1 (Ser485), acetyl-CoA carboxylase, and phospho-acetyl-CoA carboxylase (Ser79) were purchased from Cell Signaling Technology (Beverly, MA, USA), and antibodies for Cav-1 and Bcl-2 as well as AMPKα1/2 small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α antibody was obtained from R&D Systems (Minneapolis, MN, USA).
8
Cytotoxicity of Metformin in KRAS/LKB1 Lung Cancer
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Mouse cell lines were generated from lung nodules of KRASG12D/LKB1wt (K) and KRASG12D/LKB1del (KL) transgenic mice as described in [34 (link)], and cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 2 mM of L-glutamine (Microgem) and 10% (v/v) fetal bovine serum (FBS, Euroclone). Cells were grown at 37°C in a humidified atmosphere supplemented with 5% (v/v) CO2.
For cytotoxicity experiments, K and KL cell lines were seeded at respectively 6500 c/mL and 7000 c/mL, in 96-well plates and treated after 24 h with 2 mM of metformin (1,1-Dimethylbiguanide hydrochloride, #D150959 Sigma-Aldrich) and a 50% reduction of glucose, glutamine and FBS in the medium. Cell viability was examined after 72 h with MTS assay (Promega) and absorbance was acquired using a plate reader (GloMax Discover, Promega). The mean and SD of at least three independent experiments, each consisting of six replicates, are presented.
The cell lines are routinely tested by polymerase chain reaction for mycoplasma contamination.
For cytotoxicity experiments, K and KL cell lines were seeded at respectively 6500 c/mL and 7000 c/mL, in 96-well plates and treated after 24 h with 2 mM of metformin (1,1-Dimethylbiguanide hydrochloride, #D150959 Sigma-Aldrich) and a 50% reduction of glucose, glutamine and FBS in the medium. Cell viability was examined after 72 h with MTS assay (Promega) and absorbance was acquired using a plate reader (GloMax Discover, Promega). The mean and SD of at least three independent experiments, each consisting of six replicates, are presented.
The cell lines are routinely tested by polymerase chain reaction for mycoplasma contamination.
9
Synthesizing ENOblock for Cell Studies
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ENOblock(N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-4-[[4-[(cyclohexylmethyl)amino]-6-[[(4-fluorophenyl)methyl]amino]-1,3,5-triazin-2-yl]amino]-benzeneacetamide hydrochloride) was designed and synthesized by Professor Young-Tae Chang, Pohang University of Science and Technology, Republic of Korea and synthesized Professor Jin Hee Ahn, Gwangju Institute of Science and Technology, Republic of Korea, in accordance with the published protocol6 (link). Dexamethasone, forskolin, rapamycin and rosiglitazone were purchased from Santa Cruz Biotechnology (CA, USA). Metformin (1,1-dimethylbiguanide hydrochloride), orlistat, OSMI-1, 3-isobutyl-1-methylxanthine (IBMX), sodium fluoride (NaF), oil red O and an antibody for α–smooth muscle actin (catalogue number A5228) were purchased from Sigma-Aldrich (MO, USA). Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) was purchased from Thermo Fisher Scientific (MA, USA).
10
Exploring AMPK Signaling in VEGF Response
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Metformin (1,1-dimethylbiguanide hydrochloride) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma–Aldrich (St Louis, MO). 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (compound C) was obtained from Calbiochem (Darmstadt, Germany). VEGF was obtained from Peprotech (Rocky Hill, NJ). The ON-TARGET plus SMART pool siRNAS targeting AMPKα1 (PRKAA1 L-005027-00) and α2 (PRKAA2 L-005361-00) and ON-TARGET plus Nontargeting Pool negative control siRNA (D-001810) were purchased from Dharmacon (Lafayette, CO). Lipofectamine RNAiMAX (catalog number 13778-075) was purchased from Invitrogen (Eugene, OR). Amicon Ultra-4 centrifugal Filter Units were purchased from Millipore Corporation (Billerica, MA).
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