In this study, we hypothesize that the direct intra-tumoral injection of zinc could be a safe and efficacious treatment for prostate cancer. To our knowledge, this is the first examination of intra-tumoral zinc delivery as a treatment strategy for prostate cancer, and we feel that these data form powerful preliminary evidence indicating that such a minimally invasive strategy could be efficacious. Furthermore, because of the preferential accumulation of Saracatinib price zinc in prostate tissue, it is conceivable that such a strategy could be entirely free of the debilitating and dose-limiting side effects typical of other cancer chemotherapeutics. Methods Cell lines

PC3, DU148, LNCaP cells were originally obtained from ATCC (Rockville, Maryland, USA). Cells were maintained at 37°C, 5% CO2 and 95% humidity in DMEM (CellGro, Herndon, Virginia, USA)

supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, Maryland, selleck chemicals USA), 2 mM L-glutamine and 100 units/ml penicillin and 1000 ug/ml streptomycin (Invitrogen, Carlsbad, California, USA). Animals NOD/SCID mice at 8 weeks of age were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA) and were housed at the Saint Louis University comparative medicine facility. Animals were allowed to acclimate for 2 weeks prior to experimentation. The animals were under the care of a staff veterinarian and managed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Xenografts PC3 cells grown to 70% confluence were harvested and injected in the dorsum of animals subcutaneously. Each inoculum consisted of 100 μL of cell suspension at a concentration of 107 cells/ml in phosphate-buffered saline. Tumors were allowed to grow to a size of 300 mm3 prior to intra-tumoral PD-1 antibody inhibitor injection. Tumors were injected with 200 μL of 3 mM zinc acetate solution every 48 hours. Tumors were measured every 2–3 days with digital calipers. Tumor volume was determined using the following formula: Volume = Length × Width2. Zinc Measurements

Zinc was quantified in serum and tissues using the TSQ fluorophore (Invitrogen, Carlsbad, California, USA). 50 mM TSQ was prepared in 10 mM Tris buffer (ph = 8.0). TSQ was added to samples and standard zinc solutions to a final concentration of 10 μM in black round-bottom 96 well plates. Endpoint fluorescence was read on a Spectfluor with excitation wavelength of 360 nm and emission wavelength of 535 nm. Tissue zinc levels were measured similarly, after weighing and homogenizing tissue in water by repeated freeze/thaw cycles. MTT Assay Cell viability was determined via MTT assay. Briefly, media was aspirated from cells grown in 6 well plates and 1 ml of MTT (1 mg/ml) solution was added. After 1 hour incubation, MTT solution was aspirated and 0.04 N HCL was added to solubilize the cells and absorbance at 540 nM was measured.