Cl-amidine

Cell lines

HCT 116 wild-type (WT) colon cancer cells, HCT 116 p53−/− colon carcinoma cells, LS-180 colon cancer cells

Preparation Method

HCT 116 WT and p53−/− cells were cultured in McCoy's medium supplemented with 10% Newborn Calf Serum, 2 mM glutamine, penicillin (10 U/ml) and streptomycin (10 μg/ml). LS-180 colon cancer cells were cultured in DMEM Medium supplemented with 10% Newborn Calf Serum, 2 mM glutamine, penicillin (10 U/mL) and streptomycin (10 μg/mL).

Reaction Conditions

1×10⁶ were incubated in 6 well plates in McCoy's 5A medium 1 day before treatment and then treated with 50 µg/mL Cl-amidine. The cells were harvested and fixed with ice cold ethanol. After washing with 1x PBS containing 0.5% BSA twice, the cells were incubated with DNA staining solution consisting of Propidium Iodide (PI; 10 µg/mL) and RNase (0.1 mg/mL) for 30 min at room temperature in the dark.

Applications

Cl-amidine could lead to a cell cycle arrest at the G1 phase through a p53-mediated mechanism. Cl-amidine could cause miRNA-16 induction in a p53-dependent manner.

Animal models

Male C57BL/6J mice (18–26 g)

Preparation Method

Mice were injected with intra-peritoneal Cl-amidine dissolved in DMSO, or vehicle DMSO 1 h after cecal ligation and puncture (CLP). DMSO was also injected into mice that were not subjected to CLP (n = 12/group). Mortality was monitored for 10 days after surgeries.

Dosage form

40 mg/kg

Applications

Cl-amidine protects mice from sepsis-induced lethality. The inhibition of PAD by Cl-amidine significantly suppresses the CitH3 production, while the CitH3 neutralization markedly improves the survival in septic mice.

References:

[1]. Cui X, et al. The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest. PLoS One. 2013;8(1):e53791.

[2]. Ting Z, et al. Protective effect of Cl-amidine against CLP-induced lethal septic shock in mice. Sci Rep. 2016; 6: 36696.