Sunitinib malate

Crizotinib, sunitinib malate, cisplatin, carboplatin, doxorubicin and paclitaxel were purchased from Sigma-Aldrich^® (St. Louis, MO, USA). Imatinib and gefitinib were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions for the TKIs, doxorubicin, and paclitaxel were prepared in dimethyl sulfoxide (DMSO) with maximal final concentrations of 1%. The stock solutions for cisplatin and carboplatin were prepared in culture medium, since DMSO interacts with the platinum complexes resulting in inactivation (Hall et al. 2014 (link)).

Doxycycline (Dox), SU5416 (1, 3-Dihydro-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-2H-indol-2-one), Sunitinib (Sunitinib malate) and ML228 (N-([1, 1′Biphenyl]-4-ylmethyl)-6-phenyl-3-(2-pyridinyl)-1, 2, 4-triazin-5-amine) were purchased from Sigma-Aldrich. Chemical treatments were conducted in 6-well plates for zebrafish larvae from 3 dpf to 7 dpf and in 1-L tanks for juvenile fish.

Mouse antibodies, α-3BP2 C5, α-3BP2 C11, α-Myo1f C5, α-KIT (clone Ab81), and rabbit α-Kit (H300) were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Mouse anti-CD29-APC (α-integrin β1) clone MAR4 from BD Pharmigen (BD Biosciences, San José, CA), mouse α-integrin β7-PE from Biolegend (San Diego, CA), goat α-mouse alexa-647, and goat α-rabbit alexa-488 were from Life Technologies (Carlsbad, CA), mouse α-human-FcεRI-PE from eBioscience (San Diego, CA). Mouse α-Rac1, α-RhoA, and α-Cdc42 antibodies were from Cytoskeleton (Cytoskeleton Inc., Denver, CO), mouse α-Rac2 antibody was from antibodies-online. Antiphosphotyrosine (pTyr) monoclonal was obtained from Zymed Laboratories (Invitrogen Life Technologies, Carlsbad, CA). Biotinylated human IgE (IgEB) was obtained from Abbiotec (San Diego, CA, USA). Anti-mouse peroxidase Ab was obtained from DAKO (Carpinteria, CA, USA). Streptavidin, the tyrosine kinase inhibitor sunitinib malate, puromycin, poly-lysine-D, fibronectin, doxycycline hyclate, mouse α-tubulin (DM1A), and mouse α-flag (m2Ab) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). α-pKIT Tyr703 was from Cell Signaling (Cell Signaling Technology, Danvers, MA) and α-GPF from Roche (Roche Molecular Biochemical, Pleasanton, CA). Goat α-rabbit-HRP was from Life Technologies (Life Technologies).

Sunitinib malate, hydrogen peroxide (H₂O₂), l-ascorbic acid (AA), hydrochloric acid (37% w/w), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydrogen carbonate, pepsin from porcine gastric mucosa, esterase from porcine liver, α-amylase from porcine pancreas, pancreatin from porcine pancreas, sodium cholate, bile extract porcine and l-α-phosphatidylcholine from egg yolk were purchased by Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO).
All solvents were reagent-grade or HPLC-grade and provided by Carlo Erba Reagents (Milan, Italy).
Dialysis tubes MWCO: 3500 Da and 12 000–14 000 Da were provided by Spectrum Laboratories Inc (Rancho Dominguez, CA).
IR spectra were recorded as films or KBr pellets on a Jasco FT-IR 4200 (Easton, MD). Absorption spectra were recorded with a Jasco V-530 UV/Vis spectrometer (Easton, MD).

Sunitinib malate (Sigma-Aldrich, St Louis, MO, USA), was resuspended in DMSO to a final concentration of 10 mM and stored at +4°C, according to the manufacturer’s instructions. Sorafenib (Bayer Pharmaceuticals, Leverkusen, Germany) was resuspended in DMSO to a stock concentration of 10 mM and stored at -20°C. Bicalutamide (Casodex) (Sigma-Aldrich, St Louis, MO, USA), was resuspended in DMSO to a stock concentration of 10 mM, according to the manufacturer’s instructions. Drugs were diluted into the culture medium shortly before performing the assays.

The photosensitizer (PS) TPCS_2a (PCI Biotech AS, Oslo, Norway) was dissolved at 0.4 mg/mL in 3% Tween 80, 2.8% mannitol, 50 mM Tris, pH 8.5 (all from Sigma-Aldrich), and kept protected from light at 4 °C. Sunitinib malate (PZ0012, Sigma-Aldrich) was for in vitro experiments dissolved in dimetylsulfoxid (DMSO) to a final concentration of 2.5 mM, stored as aliquots at −20 °C, and subjected to maximum two freeze-thaw cycles. Sunitinib malate for in vivo experiments was dissolved at a concentration of 8 mg/mL (20 mM) in a vehicle containing distilled water with 1.8% NaCl, 0.5% carboxymethylcellulose, 0.4% Tween 80 and 0.9% benzylalcohol. The pH of the solution was adjusted to 6.0. The sunitinib mixture was sonicated to achieve stable dispersion [45 (link)] and used within 24 h after sonication. All sunitinib reagents were stored protected from light. Recombinant gelonin (rGel) was generously provided by Dr. Michael Rosenblum’s laboratory at M.D. Anderson Cancer Center (Houston, TX, USA). Aliquots of rGel were stored in phosphate-buffered saline (PBS) at −20 °C. All experiments in vitro and in vivo with sunitinib and/or TPCS_2a were performed under subdued light.