N-Carbamyl-L-glutamic acid

Kinase experiment:

Caspase activity is measured by using a fluorimetric caspase-3 assay kit. In brief, cells that are treated with Carglumic Acid or that are left untreated are lysed in a lysis buffer, and 50 μg of protein lysate is incubated with Ac-DEVD-AMC substrate in the assay buffer for 1 h. The resultant fluorescence signals are read by using a fluorometer (excitation 360 nm, emission 460 nm), and the results are tabulated as fold changes relative to the untreated control cells[1].

Cell experiment:

Cell viability is evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In brief, various cancer cell lines are seeded (1×104 cells/well) in a 96-well plate and treated with different doses of Carglumic Acid. After 48 h, 50 μL of MTT solution per well (stock solution concentration 5 mg/mL) is added to each well, and the cells are incubated for 2 h more, followed by addition of 100 μL of dimethyl sulfoxide to each well. Absorbance at 570 nm is measured immediately using a multiwell scanner[1].

Animal experiment:

For orthotopic cancer models, AsPC1/luc human pancreatic cancer cells (1×106) are injected into the pancreas of nude mice or MDA-MB-231 human triple-negative breast cancer cells (3×106) are injected into the mammary fat pad of nude mice. Carglumic acid is administered to mice 5 days after tumor inoculation in the pancreatic cancer model and 7 days after tumor inoculation in the triple-negative breast cancer model. Tumor-bearing mice receive a Carglumic acid dose of 120 mg/kg orally every day for 10 days, 60 mg/kg orally three times per week for 2 weeks, or 60 mg/kg intravenously three times per week for 2 weeks. Tumor volume is determined by measuring luciferase signals using the in vivo imaging system in the pancreatic cancer model[1].

References:

[1]. Chen CT, et al. Carglumic acid promotes apoptosis and suppresses cancer cell proliferation in vitro and in vivo. Am J Cancer Res. 2015 Nov 15;5(12):3560-9.