Docetaxel

[³H]-Paclitaxel (37.9 Ci/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin, and trypsin 0.25% were purchased from Hyclone (Waltham, MA). Monoclonal antibody against GAPDH was purchased from Cell Signaling Technologies (Beverly, MA). The polyclonal antibody PA5-23652 for ABCC10 was obtained from Thermo scientific (Rockford, IL). Monoclonal antibody 3D7G8 against EphB4 was obtained from Life technologies, Invitrogen (New York, NY). NVP-BHG712 was a gift from Novartis (Basel, Switzerland). Cepharanthine was generously donated by Kakenshoyaku Co. (Tokyo, Japan). PAK-104P was a gift from Nissan Chemical Industries (Tokyo, Japan). Paclitaxel, docetaxel, colchicine, mitoxantrone, vincristine, vinblastine and cisplatin were purchased from Tocris Bioscience (Ellisville, MO). 3-(4, 5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO) and verapamil were obtained from Sigma-Aldrich Co. (St. Louis, MO).

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.

Liquid paclitaxel (6 mg/mL) was purchased from Sagent Pharmaceuticals (Schaumburg, IL, United States). Powdered paclitaxel was purchased from LC Laboratories (Woburn, MA, United States). Sterile (2 MRad gamma irradiation), lyophilized keratose was obtained from KeraNetics LLC (Winston-Salem, NC, United States) and used without further modification and under aseptic conditions. Iohexol (OMNIPAQUE^TM [300 mgI/mL]) was purchased from GE Healthcare (Little Chalfont, United Kingdom). Docetaxel was purchased from Tocris Bioscience (Bristol, United Kingdom). DMSO, methanol, water, acetonitrile, and formic acid were procured through Sigma-Aldrich (St. Louis, MO, United States). Absolute ethanol 200-proof was purchased from Fisher Scientific (Hampton, NH, United States).

Antibody against BCO2 (Catalog number [Cat#] 14324-1-AP, 1:1000) was acquired from ProteinTech Group (Rosemont, IL, USA). Antibodies against cleaved caspase-3 (Cat# 9662, 1:1000), p53 (Cat# 2524, 1:1000), Bax (Cat#2772, 1:1000) and Bcl-2 (Cat#15071, 1:1000) were obtained from Cell Signaling (Danvers, MA, USA). Antibody to β-actin (Cat# A5441, 1:10,000) was obtained from Sigma Aldrich (St Louis, MO, USA). The 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCF-DA) and N-acetyl cystenine (NAC) were purchased from Sigma Aldrich. Lutein was obtained from Kemin Health (Des Moines, IA, USA; FloraGLO Lutein 10% VG TabGrade). Beta-carotene and astaxanthin were obtained from Sigma-Aldrich. Working solutions of carotenoids were freshly prepared with dimethyl sulfoxide (DMSO) and tetrahydrofurlan (THF) in a ratio 2:1 immediately before use. Docetaxel and paclitaxel were obtained from Tocris Bioscience (Minneapolis, MN, USA).

Cisplatin was purchased from Sigma and reconstituted in PBS (2 g/L). Docetaxel was purchased from Tocris Bioscience and reconstituted in DMSO (5 g/L stock dilution at -20°C; further dilution in fresh culture medium) for in vitro assays, or reconstituted in ethanol/polysorbate 80 (1:1; 5 g/L stock dilution at -20°C; further dilution in PBS) for in vivo assays. GDC0449 was purchased from Selleck Chemicals and reconstituted in DMSO (30 mM stock dilution). Shh human recombinant protein was obtained from eBioscience and used at 1,200 ng/mL.