Metformin hcl

The following cell lines were included in the study and were maintained in a standard tissue culture incubator set to 37 °C and 5% CO₂: HL-60, U937, and Jurkat (hematological cancers); LNCaP, DU145, and PC3 (prostate cancer); HepG2, HLE, and SK-HEP-1 (liver cancer); AsPC-1, BxPC-3, and MIA PaCa-2 (pancreatic cancer); MDA-MB-231 and MCF-7 (breast cancer); HCT116 and HT-29 (colorectal cancer). Cell lines representing hematological cancers were cultured in RPMI 1640-GlutaMAX™ medium (Gibco, Darmstadt, Germany) supplemented with 10% FCS (v/v) (Gibco), and 1% penicillin/streptomycin (v/v) (Gibco). Other cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-GlutaMAX™ (Gibco), supplemented with 10% FCS (v/v) (Gibco), 1% penicillin/streptomycin (v/v) (Gibco). Treatments with 1,25(OH)₂D₃ (Cayman Chemicals—Biomol GmbH, Hamburg, Germany) were performed in standard medium for different time points. 2-deoxyglucose (Fluka-Sigma-Aldrich, Steinheim, Germany), MG-132 (Sigma-Aldrich, Steinheim, Germany), CHX (Fluka-Sigma-Aldrich), metformin HCl (Sigma-Aldrich), and phloretin (Sigma-Aldrich) were used as indicated. For glucose deprivation experiments, RPMI 1640 without glucose medium (Gibco) was used.

High-methoxy citrus pectin and metformin HCl were purchased from Sigma-Aldrich Chemical Co., UK, and Tokyo Chemical Industry Ltd., UK, respectively. Sulphuric acid, phenol, Span 80, hexane and light liquid paraffin (dynamic viscosity 25–80 mPa s at 20°C) were purchased from Fisher Scientific Ltd., UK. All the chemicals were of analytical grade and used as received without any further modification and/or purification.

AF cells are plated onto FlexCell plates or cell culture chamber slides and split into four treatment groups: control, metformin, IL‐1β, and metformin + IL‐1β (Figure 1). Cultures were pretreated with either 100 mM metformin HCl (Sigma‐Aldrich) or F‐12 (1% FBS/1% PS) medium for 4 h in a hypoxic incubator (5% CO₂, 5% O₂, 37.5°C). Then, each of the metformin and medium pretreated cells were further treated with 1 ng/ml of IL‐1β (R&D Systems) for 30 min, 4 h or 24 h, respectively. Metformin was added as a pretreatment to allow cells to incorporate metformin sufficiently into their metabolism before other treatments which is a typical study design for studying inhibitors. In addition, pretreatment with metformin is consistent with the typically chronic, not episodic, clinical use of metformin.

The FDA Pharmakon compound library was purchased from MicroSource Discovery Systems, Inc. (USA). Compounds (10 mM, dissolved in DMSO) were stored at -80 °C until use. Levamisole and ivermectin, used for the in vitro controls, were purchased from Fluka and Sigma-Aldrich, respectively (Buchs, Switzerland), dissolved at 10 mM stock solutions in DMSO, and stored at -20 °C until further use. For the in vivo studies, bitoscanate, chlorcyclizine HCl, clemastine, dicyclomine HCl, drofenine HCl, ethopropazine HCl, lansoprazole, metformin HCl, morantel, natamycin, trichlorfon, and trimipramine maleate were purchased from Sigma-Aldrich.