Curcumin

To conduct antitumor drug tests against the cancer cell spheroids, doxorubicin (5927S, Cell Signaling, Danvers, MA, USA), docetaxel (01885, Sigma-Aldrich), resveratrol (R5010, Sigma-Aldrich), 5-FU (F6627, Sigma-Aldrich), vincristine (V8879, Sigma-Aldrich), and curcumin (C7727, Sigma-Aldrich) were used as antitumor agents in the present study. After spheroid formation, the drug-free culture media was replaced with 1 μM doxorubicin, 0.5 μM docetaxel, 100 μM resveratrol, 5 μM 5-FU, 1 μM vincristine, 10 μM curcumin, 100 μM Tiam1/Rac1 inhibitor (2221, NSC23766, Tocris Bioscience, Bristol, UK), and 5 μM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (2634, DAPT, an inhibitor of the γ-secretase complex, Tocris Bioscience) in RPMI-1640. The efficacy of combination treatment of doxorubicin with either Tiam1 inhibitor or γ-secretase inhibitor, or with both agents was examined. Control groups of conventional 2D-cultured cells were treated with the same concentrations of these antitumor agents after reaching 60% to 80% confluence. After 24h treatment, cell viability was assessed using the EZ-Cytox assay (EZ-3000, Daeil Lab Service, Seoul, Korea) in 96-well microtiter plates by reading at an OD of 450 nm.

Human recombinant TGF-β1 was obtained from PeproTech, (Cranbury, NJ, USA). FICZ and kynurenine were purchased from Sigma (St. Louis, MO, USA). ITE and curcumin were purchased from TOCRIS (Bristol, UK). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA) and penicillin, streptomycin, L-glutamine from Dutscher (Brumath, France). MEM non-essential amino acid and Cellytic™ buffer were supplied by Sigma (St. Louis, MO, USA). Minimum Essential Medium Eagle was obtained from Eurobio (Montpellier, France).

Bis(heptyl)-cognitin was synthesized as previously described by us11 (link). The purity of bis(heptyl)-cognitin was evaluated by using liquid chromatography-mass spectrometry. Bis(heptyl)-cognitin was dissolved in Milli-Q water at a concentration of 1 mM and stored frozen at −20 °C. Before being used, bis(heptyl)-cognitin was further diluted with Milli-Q water. Donepezil, tacrine, methyllycaconitine (MLA) and hexafluoroisopropanol (HFIP) were purchased from Sigma (St Louis, MO, USA). Curcumin, KT5720, MG624 and H89 were purchased from Tocris (Bristol, UK). Curcumin, Donepezil, KT5720, MG624 and H89 were dissolved in dimethyl sulfoxide (DMSO) with a maximum final concentration of 0.1% (DMSO). Other chemicals were prepared in Milli-Q water. All media and supplements used for cell culture were from Invitrogen (Carlsbad, CA).