Garlic is reported to reduce cisplatin-induced nephrotoxicity and oxidative stress [53], which could explain the better performance of these animals. activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in ?20 C. We found that Orexin 2 Receptor Agonist GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data show that the reduced proliferation of the malignancy cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress. = 16). This dose is equivalent to 15 mL/day in humans, based upon surface area calculations. The vehicle group (= 16) was injected daily with 200 L of a solution of 0.90% of NaCl, containing 6.5 L 20% ethanol. When the tumours were palpable, the tumour sizes were measured by electronic Vernier Calipers three times a week. The body excess weight was measured twice a week throughout the experiment. Tumour volumes were calculated using the formula for Orexin 2 Receptor Agonist any spheroid: is the tumor width and 2is the tumor height. After 28 days, the mice were euthanized using carbon dioxide (2 L per min). In an additional experiment, cisplatin (3 mg/kg) and gemcitabine (0.5 mg/kg) in 200 L 0.90% of NaCl solution were injected i.p. (= 15) (FOTS application 7133), alone or in combination with GE (6.5 L in 200 L 0.90% of NaCl solution) (= 15). The vehicle group (= 14) was injected daily with 200 L of a solution of 0.90% of NaCl, containing 6.5 L 20% ethanol. Data from this experiment is shown until day 27. Three animals from the vehicle group were terminated on day 27, because of the tumor sizes exceeded the limit. The remaining animals were terminated on day 29. 2.7. Preparation of Cell Extracts and Western Analysis The 67NR cells were treated with GE at given concentrations. The cells were harvested after 4 and 24 h, the cell pellet was re-suspended in 1 packed cell volume of buffer 1 (10 mM Tris-HCl pH 8.0, 200 mM KCl), and diluted in the same volume (packed cell volume + buffer 1) of buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM KCl, 10 mM EGTA, 10 mM MgCl2, 40% glycerol, 0.5% NP40, 1 mM DTT, 1% phosphatase inhibitor cocktails 1 and 3 (Sigma-Aldrich), 2% Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland), and 2 L/mL Omnicleave (Epicentre Technologies, Madison, WI, United States). After incubation for 1.5 h at 4 C, the cell Orexin 2 Receptor Agonist extracts were centrifuged at 14,000 rpm for 10 min. Supernatants were collected and separated on 10% Bis-Tris gels (NuPAGE, Invitrogen). After gel electrophoresis, the polyvinylidene fluoride membranes (Immobilion, Millipore, Burlington, MA, USA) were blocked in 50% Odyssey blocking buffer (LI-COR Bioscience) in TBS (Tris-buffered saline). The primary antibodies against AKT (phospho-Ser473), ERK1/2 (phospho-Thr202/Tyr204/phospho-Thr185/Tyr187), p70 S6 kinase (phospho-Thr389) (Cell Signaling, Danvers, MA, USA), and -tubulin (Abcam, Cambridge, UK), as well as the fluorescently-labelled secondary antibodies, goat anti-rabbit 680RD and goat anti-mouse 800CW (LI-COR Bioscience) were diluted in 20% Odyssey blocking buffer in TBST (TBS with 0.1% Tween 20). The proteins were visualized with the Odyssey infrared imaging system (LI-COR Bioscience) and quantified using Odyssey Image Studio V2. Protein levels were compared to the protein level in untreated cells, which was set to 100%. -tubulin was used as reference for data normalization. 2.8. Multiplexed Inhibitor Assay and Mass Spectrometry Analysis Three different kinase inhibitors (Purvalanol B (Tocris Bioscience), Bisindolmaleimide X (Activate Scientific), and SB6-060-05 [30]) were immobilized using ECH Sepharose 4B and EAH Sepharose 4B Rabbit polyclonal to KATNA1 (GE Healthcare) beads, according to the manufacturers instructions and as published elsewhere [31]. The following actions were performed as explained [32], using 100 L (0.1 mg) of cell extract per column (50 L of mixed inhibitor beads). 2.9. Fractionation and Purification of Garlic Extract GE (1 mL) was diluted 1:10 with distilled water and loaded on a Orexin 2 Receptor Agonist SepPac SPE tC18 1cc 100 mg cartridge (Waters, Milford, MA, USA), preconditioned with ethanol and.