Cell experiment:

One million SCC25 cells are seeded in 10-cm plates and incubated overnight to allow for attachment and recovery. The following day, cells are pretreated with 0, 1, or 5 μM Monensin sodium salt for 24 hours then treated with 10 μM erlotinib alone or in combination with Monensin sodium salt for a further 24 hours. Adherent and cells in suspension are collected by centrifugation and fixed in 3 mL of cold 80% ethanol overnight at -20℃. Before analysis, cell pellets are washed with PBS resuspended in staining buffer containing 25 μg/mL propidium iodide and 40 μg/mL RNase A and incubated for a minimum of 1 hour in the dark at room temperature[1].

Animal experiment:

Multiple intestinal neoplasia (Min) mice are used in this study. Four-week-old pups are weaned, genotyped, and randomized. The animals are divided into two groups and treated with Monensin sodium salt (10 mg/kg) or vehicle (DMSO). Daily oral applications continue for 6 weeks. In addition, six pairs of Apc+/Min mice age 7, 10, 13, 16, 19, and 22 weeks are treated with Monensin sodium salt or vehicle for 5 weeks. The mice are sacrificed and the intestines are dissected, washed in PBS, and fixed in 4% formaldehyde (v/v) in PBS for 3 days. Fixed intestines are embedded in paraffin, sectioned and stained. The number and size of the neoplastic lesions are quantified using Ellipse software[2].

Monensin sodium salt is an antibiotic secreted by the bacteria Streptomyces cinnamonensis.

Monensin sodium salt is an antibiotic secreted by the bacteria Streptomyces cinnamonensis. Untreated cells display 2.5% apoptosis; 48 hours treatment with 1 μM Monensin sodium salt shows 4.5% apoptosis whereas 5 μM Monensin sodium salt for 48 hours induces a greater apoptotic response (16.4%). Pretreatment with either 1 or 5 μM Monensin sodium salt for 24 hours followed by 10 μM erlotinib treatment for another 24 hours results in a marked increases in apoptotic events (14.6% and 38.7%, respectively) when compare with either Monensin sodium salt or erlotinib treatments alone. Combination of 5 μM Monensin sodium salt with 10 μM erlotinib shows the highest percentage of apoptosis (38.7%)[1].

Although the numbers of tumors do not change substantially, a significant (P=0.0144) reduction in the average size of lesions is observed in Monensin sodium salt-treated Apc+/Min mice when compare with control animals (mean 0.199 mm2 vs. 0.299 mm2). The total tumor area estimated in one animal is decreased in individuals receiving Monensin sodium salt (mean 10.16 mm2 vs. 16.46 mm2; P=0.0125). Monensin sodium salt treatment increases the numbers of apoptotic cells and cells expressing the p21 cell-cycle inhibitor at the surface area of the neoplastic outgrowths. No changes in the cell proliferation, differentiation, and tissue architecture in the healthy parts of mucosa are noted after exposure to Monensin sodium salt[2].

References:
[1]. Dayekh K, et al. Monensin inhibits epidermal growth factor receptor trafficking and activation: synergistic cytotoxicity in combination with EGFR inhibitors. Mol Cancer Ther. 2014 Nov;13(11):2559-71.
[2]. Tumova L, et al. Monensin inhibits canonical Wnt signaling in human colorectal cancer cells and suppresses tumor growth in multiple intestinal neoplasia mice. Mol Cancer Ther. 2014 Apr;13(4):812-22.