Folinic acid

Myrislignan ([erythro-(1R,2S)-2-(4-allyl-2,6-dimethoxyphenoxyl)-1-(4-hydroxy-3-methoxyphenyl) propan-1-ol], batch number DST180502-043) was purchased from Desite Biotechnology Co., Ltd. (Chengdu, China) and had a purity greater than 99%. For the in vitro experiments, myrislignan was dissolved in dimethyl sulfoxide (DMSO, Sigma, USA) to a concentration of 25 mg/ml and diluted in DMEM containing 1% FBS to different concentrations. Sulfadiazine (Sigma, USA), which was used as positive control drug, was dissolved in DMEM with 1% FBS to a concentration of 0.4 μg/ml. For the in vivo experiments, myrislignan was dissolved in solvent 1 (isotonic saline containing 5% ethanol and 5% Cremophor EL) to concentrations of 5 or 10 mg/ml. Sulfadiazine (10 mg/ml), pyrimethamine (5 mg/ml, Sigma, USA), and folinic acid (1.5 mg/ml, Sigma, USA) were suspended in physiological saline containing 1% CMC-Na (solvent 2) and used as a positive control.

Cells were depleted of endogenous folate cofactors by culturing for 10 days in folate-free depletion medium consisting of folic acid free RPMI (Life Technologies) with 10% dialysed FCS (Life Technologies) and 2mM L-glutamine, supplemented with 10 μM thymidine (Sigma-Aldrich, St Louis, MO), and 100 μM adenosine (Sigma-Aldrich) as previously described [37 ]. For experiments, cells were trypsinized then washed in folate-free assay medium consisting of folic acid free RPMI with 10% dialysed FCS and 2mM L-glutamine. Cells were diluted in folate-free assay medium and plated in 6-well plates at the following cell numbers: CHP-134: 600 cells/well; Kelly: 400 cells/well; NB69: 400 cells/well; SHEP: 150 cells/well supplemented with folinic acid (Sigma-Aldrich) at 0, 0.5nM, 1nM, 2.5nM, 5nM and 10nM. After 9 days (CHP-134, NB69 and SH-EP) or 12 days (Kelly), colonies were stained with 0.5% crystal violet in 50% methanol. Plates were scanned, and colonies counted. Colony numbers are normalised to 10 nM folinic acid at 100%, and comparisons made to the 5 nM folinic acid values. Results are from triplicate experiments.

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).

Methotrexate (MTX), hydroxychloroquine (HCQ), lipopolysaccharide (LPS), caffeine (CAFF), theophylline (THEO), folinic acid (FA) and parthenolide (PAR) were from Sigma-Aldrich (St Louis, MO, USA). Secreted interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in culture supernatants were quantitated using enzyme-linked immunosorbent assay (ELISA) kits from Abcam (Cambridge, MA, USA) and results were expressed in standardized concentrations using reagents provided with these kits.

10 mg/mL 5-fluorouracil (F6627, Sigma-Aldrich) and 1 mg/mL SN38 (29112, MedChem Express, Monmouth Junction, NJ, USA) were dissolved in sterile DMSO (Sigma-Aldrich), and 20 mg/mL folinic acid (F787, Sigma-Aldrich) and 5 mg/mL oxaliplatin (O9512, Sigma-Aldrich) in UltraPure distilled sterile water. Aliquots were stored at −80 °C and thawed before each experiment for one-time use. A maximal concentration of 0.08% DMSO in cell culture media was used as control (sham). Cells were seeded in 96-well plates at different densities (2500 cells/well for HCT116 and DLD1, 3500 cells/well for LS174T and 5000 cells/well for SW620). 24 h post-seeding single drugs or pre-mixed drug combinations were incubated for 72 h. Cell metabolic activity (ATP) was measured using the bioluminescent-based CellTiter-Glo^® assay (G7572, Promega) according to the manufacturer’s instructions. The intensity of the luminescence signal was detected via the BioTek Cytation 3 imaging reader with corresponding Gen5 Image software version 3.04.

Folinic acid, Trolox, salubrinal, succinylacetone, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). Fluorouracil was from LKT Laboratories (St. Paul, MN). Oxaliplatin was from LC Laboratories (Woburn, MA). Antibodies directed toward full-length (FL)/cleaved caspase-3, FL/cleaved PARP, phospho-eIF2α (P-eIF2α), total eIF2α (T-eIF2α), P-PERK, and total-PERK were from Cell Signaling Technology (Beverly, MA). Anti-CHOP10 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH antibody and GSK2606414 were obtained from EMD Millipore (Burlington, MA). Anti-ferritin antibody was purchased from Abcam (Cambridge, MA). Anti-rabbit 800CW and anti-mouse 680RD secondary antibody IRDyes were from LI-COR Biosciences (Lincoln, NE). The heme oxygenase inhibitor, QC-308, was purchased from AsisChem Inc. (Waltham, MA).