Cell experiment:

The effect of Salinomycin on HUVEC growth is determined by MTT assay. Briefly, HUVECs (6,000 cells/well) are seeded in 96-well culture plates for 24 h and incubated with different concentrations of Salinomycin. In the preliminary experiments, Salinomycin treatment for 12, 24, 48 and 72 h shows time-dependent effects on cell growth inhibition. However, treatment for 48 h is the optimal time and is selected for further mechanism evaluation. After Salinomycin treatment for 48 h, 20 μL/well of MTT solution (5 mg/mL) is added and incubated for 5 h. The medium is aspirated and replaced with 200 μL/well of DMSO to dissolve the formazan Salinomycint formed. The color intensity of the formazan solution is measured at 570 nm by a microplate spectrophotometer. The cell viability is expressed as % of the control (as 100%).

Animal experiment:

Human glioma U251 cells (1×107) suspended in 100 μL PBS are injected into the right lower hind flank of each 6-week-old male nude mouse. The mice are then randomly assigned into three groups of 10 mice in each group. After one week, Salinomycin (5 and 10 mg/kg) is administered into the caudal vein every other day for 16 days. Control mice receive an equal volume of vehicle (Salinomycinine) only. Body weight and tumor volume are monitored every two days. At the end of the experiments, tumors are excised, photographed, and weighed. Tumors from each group are used for western blotting and immunohistochemical (IHC) assay.

IC50: 7.7, 13.7 and 10.4 μM for HepG2, SMMC-7721 and BEL-7402 cell line, respectively (after 24h treatment)

Salinomycin (Sal) sodium salt, which is a polyether ionophore antibiotic from Streptomyces albus, has been proven to be able to kill different types of human cancer cells, most likely via interfering with ABC drug transporters, the Wnt/β-catenin signaling pathway, or other pathways.

In vitro: Several hepatocellular carcinoma (HCC) cell lines were treated with Sal. Results showed that Sal inhibited proliferation and decreased PCNA levels. Cell cycle analysis showed that Sal caused cell cycle arrest in different phases. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Compared to control, β-catenin expression was down-regulated by Sal treatment significantly. The Ca2+ concentration in HCC cells was examined by flow cytometry and it was found that higher Ca2+ concentrations were observed in Sal treatment groups [1].

In vivo: The in vivo anti-tumor effect of Sal was verified using the hepatoma orthotopic tumor model and results showed that the liver tumor size in Sal-treated groups decreased. Immunohistochemistry and TUNEL staining also demonstrated that Sal could in vivo inhibit proliferation and induced apoptosis [1].

Clinical trial: N/A

Reference:
[1] Wang F,He L,Dai WQ,Xu YP,Wu D,Lin CL,Wu SM,Cheng P,Zhang Y,Shen M,Wang CF,Lu J,Zhou YQ,Xu XF,Xu L,Guo CY.  Salinomycin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo. PLoS One.2012;7(12):e50638.