Hydralazine

Matrigel was from BD Biosciences (Le-Pont-de-Claix, France). Calcein-AM bioreagent, trolox, diphenylene iodonium (DPI), GW4869, Vas2870, and hydralazine were from Sigma, and 4-HNE was from Calbiochem, [methyl-¹⁴C]choline-sphingomyelin was from Perkin-Elmer. 5- (and 6-)Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), was from Molecular Probes (Invitrogen France). The anti-CD31 was from Abcam, the anti-4-HNE Michael adducts were from Calbiochem, the anti-LOX-1 antibody (aLox1 Ab) was from R&D Systems, and the anti-CD68 was from Thermo Fisher Scientific. Alexa Fluor 488-conjugated and Alexa Fluor 546-conjugated secondary antibodies were from Invitrogen. Cell culture reagents and other materials were from WWR or Sigma. Bisvanillin (BV) and bisvanillyl-hydralazone (BVH) were synthesized as reported [39 (link)].

PCa cell lines LNCaP and PC-3 were kindly provided by Prof. Ragnhild A. Lothe from the Department of Cancer Prevention at the Institute for Cancer Research, Oslo, Norway, whereas DU145 was kindly provided by Professor Fátima Baltazar at ICVS, University of Minho, Braga, Portugal and 22Rv1 cells were kindly provided by Dr. David Sidransky at the Johns Hopkins University School of Medicine, Baltimore, MD, USA. LNCaP and 22Rv1 cells were grown in RPMI 1640, DU145 cells were maintained in MEM and PC-3 cells were grown in 50% RPMI-50% F-12 medium (GIBCO, Invitrogen, Carlsbad, CA, USA). All basal culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (GIBCO, Invitrogen, Carlsbad, CA, USA). Cells were maintained in an incubator at 37ºC with 5% CO₂. All PCa cell lines were karyotyped by G-banding (for validation purposes) and routinely tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set, Clontech Laboratories). Hydralazine was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS (GIBCO, Invitrogen, Carlsbad, CA, USA). For control purposes, cell lines were exposed to the vehicle of the drug (PBS).

Eight to twelve week old CD57BL/6 mice were anesthetized with isoflurane inhalation, analgesia was performed by subcutaneous Buprenorphine injection. The ureter was separated from the surrounding tissues and two ligatures were placed about 5 mm apart in the upper two-thirds of the ureter of the left kidney to obtain reliable obstruction. On the same day after surgery and recovery, mice were treated with either vehicle buffer PBS, 5′-Azacytidine (10 mg/kg/day, Sigma, St. Louis, USA), low-dose, or high-dose Hydralazine (5 mg/kg/day or 50 mg/kg/day, respectively, Sigma, St. Louis, USA) given intraperitoneally every other day. Mice were sacrificed at indicated time points after ureter ligation (Tampe et al., 2014 (link)).

Astrocyte cells (C6, rat astrocyte cell line) and endothelial cells (bEnd.3, mouse endothelial cell line) were from ATCC (Manassas, VA, USA). Cell culture inserts for 24-well plates (transparent PET membrane transwells, 0.4 µm pore size) were obtained from Dominique Dutscher (Alsace, France). Imaging Plate FC, 24-well plates, TC-surface was purchased from Ozyme (Saint Quentin Yvelines, France). The EVOM voltohmmeter system was from World Precision Instruments (Hertfordshire, UK). ZO-1 antibodies were from Life Technologies (cat 40-2200; Saint Aubin, France) and claudin-5 antibodies were from ABCAM (ab15106; Paris, France). P-gp and MRP-1 antibodies were from GeneTex (GTX23364; San Antonio, TX, USA) and Santa Cruz Biotechnology (sc-13960; Dallas, TX, USA), respectively. Standard protein and all compounds for Ringer HEPES buffer, BCECF-AM, probenicid, verapamil, sodium fluorescein (Na-Fl), were from Sigma-Aldrich (St. Quentin Fallavier, France). Rhodamine-123 was from Thermo Fisher Scientific (Eugene, OR, USA). Hydralazine, MTT kit and Dulbecco’s Modified Eagle’s Medium (DMEM) were also from Sigma-Aldrich. Antibodies and reagents for the detection of HIF-1α were products of R&D systems (Lille, France). YC-1 was purchased from VWR International (Fontenay-sous-Bois, France).