Docetaxel

For cytotoxicity assay, the MTS assay (Promega, Beijing, China) was used to test the viability of 22RV1 and DU145 cells treated with docetaxel (Selleck, Shanghai, China). In brief, cells (1,000 for DU145 and 2,000 for 22RV1) were seeded in 96-well plates with different concentrations of docetaxel and cultured for 72 h. Then, we calculated the IC50 according to the absorbance at 492 nm. For colony formation assay, 1,500 DU145 cells were seeded in six-well plates and cultured in incubator for 10 days to form macroscopic clones. After staining with 0.1% crystal violet, we counted the number of colonies in different groups.
The 24-well Transwell chamber (8 μM, 353097; Corning, Glendale, AZ, United States) was used for the migration assay. In brief, 40,000 cells in 200 μL of 1% FBS medium were seeded in the top insert chamber, and 600 μL of medium containing 10% FBS was added into the lower chamber. The top chamber was fixed with 4% paraformaldehyde and stained with 0.2% crystal violet after 12 h incubation. The migrated cells on the lower membrane surface of the top chamber were detected under a microscope (Nikon, Tokyo, Japan).

Drug concentrations were as follows except where otherwise specified: GSK-126 (5 μM, Selleckchem, S7061), vorinostat (2 μM, Selleckchem, #S1047), panobinostat (50 nM, Selleckchem, #S1030), MAK683 (5 μM, Selleckchem, S8983), docetaxel (10 nM, Selleckchem, S7787).

All materials for tissue culture were purchased from the Euroclone group (Milan, Italy). Antibodies including anti-human E-selectin H-300 [sc-14011] were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-human E-selectin lidands (sLe^a and sLe^a/sialyl Lewis X) moAb HECA-452 was purchased from BD Biosciences (BD Italia, Milan, Itlay). The dual E-selectin/CXCR4 antagonist GMI-1359 and the E-selectin specific antagonist were designed and synthesized by GlycoMimetics, Inc. (Rockville, MD, USA). CTCE-9908 was kindly provided by Chemokine Therapeutics, Inc. (Vancouver, BC, Canada). Docetaxel was purchased from Selleck Chemicals (Aurogene, Roma, Italy). ELH-Eselectin1 (Soluble) Human ELISA Kit was purchased from Ray Biotech through the Italian distributor (Prodotti Gianni S.p.A, Milan, Italy). Phospho-CXCR4 (Ser339) Colorimetric Cell-Based ELISA Kit (OKAG01771) was purchased from Aviva Systems Biology, Corp (San Diego, CA, USA).

After pretreatment with 100 nM docetaxel (catalog No. T1034, TargetMol, Wellesley Hills, MA, USA) for 20 h, MDA-MB-468 cells were treated with 50 nM of either hesperadin (Catalog No. S1529, Selleckchem, Houston, TA, USA) or DMSO for different times in the continual presence of 100 nM docetaxel. Single-cell suspensions fixed with ice-cold ethanol were stained with propidium iodide staining solution and analyzed by flow cytometry.

Several cell-viability assays were performed with all the three prostate-cancer cell lines in order to study the cytotoxic effect of Ocoxin alone or combined with chemotherapy. First, 5 × 10⁴ cells/mL were cultured in 96-well plates in complete medium for 24 h in the case of RM-1 and 22Rv1 cells and 72 h for LNCaP. Then, cells were treated with different dilutions of Ocoxin ranging from 1:500 to 1:50 (V/V_f) (Catalysis S.L., Toledo, Spain), Docetaxel (2.5–12 nM), Enzalutamide (12.5–50 µM) and Olaparib (2.5–10 µM) (Selleckchem, Houston, TX, USA) for 48 and 72 h in 1% FBS supplemented medium. Cell viability was measured by using PrestoBlue™ Cell Viability Reagent (Invitrogen, Waltham, MA, USA) for 2 h following manufacturer’s indications so as to assess the most effective doses. Afterwards, the effect of combinations of Docetaxel, Enzalutamide and Olaparib with Ocoxin were tested following the same protocol.