R. J. Ryan1, P. Ramamoorthy3, D. Subramaniam3, P. DiPasco1,3, S. Weir2, S. Anant3 1University Of Kansas Medical Center,Medicine,Kansas City, KS, USA 2University of Kansas Medical Center,Pharmacology, Toxicology And Therapeutics,Kansas City, KS, USA 3University of Kansas Medical Center,Cancer Biology,Kansas City, KS, USA
Introduction:
Esophageal cancer continues to have poor outcomes despite the strides made in cancer treatment. While the overall 5-year survival is 30%, patients with advanced disease make up one third of new diagnoses and have a 5-year survival of only 5%. Better therapies are needed to improve these outcomes. Drug repositioningis as an efficient strategy to transition promising agents from bench to bedside. Ciclopirox olamine (CPX) is an FDA-approved anti-fungal agent that has demonstrated anti-tumor effects. Our work demonstrates the inhibitory effects of CPX on esopahgeal tumor cells in vitro and in vivo with preliminary evidence of mechanisms of inhibition.
Methods:
We maintained four esophageal cell lines, TE10, SKGT4, FLO1, and ESO1, in standard culture flasks with RMPI or DMEM media. Cell lines were exposed to increasing concentrations of CPX (0-50μM) for increasing periods of time (12-48 hr) to obtain reported results. Hexosaminidase assay was performed to assess cell proliferation. Clonogenicity assay was performed by plating cells (500 cells/mL), treating with CPX for 24 and 48 hours, and susequently culturing in standard media for 5-10 days. Cells were then fixed and stained with crystal violet dye. Spheroid assay was performed on TE10 and SKGT4 cells grown in ultra-low binding round-bottom plates and photographed at 3, 5, and 7 days after treatment. For cell cycle analysis, flow cytometry was performed in permeabilized cells stained with propidium iodide. Western blot was performed on protein lysates after treatment for 24 and 48 hours. For in vivo analysis, 2×106 ESO1 cells were injected into flanks of nude mice. Mice were treated with either IP injection of 300 mg/m2 CPX or no treatment. Tumor volumes were measured every 3-5 days and mice were euthanized at 28 days.
Results:
In each of the four esophageal tumor cell lines, CPX demonstrated growth inhibition in a time and concentration-dependent manner. CPX also inhibited growth of SKGT4 and TE10 spheroids. Cell cycle analysis showed arrest at G0/G1 in all cell lines. Protein analysis of proteins invovled in cell cycle progession demonstrated decreased expression of CDK4, CDK6, and cyclinD1. Analysis of the WNT/β-catenin pathway revealed a decrease in expression of two transcriptionally active forms of phosphorylated β-catenin (Ser675, Ser552). Tumor xenografts had significantly decreased growth in mice treated with CPX.
Conclusion:
We have shown that CPX inhibits growth of esophageal tumor cells in vitro and in vivo. Our current data suggests that CPX interferes with cell cycle progression and the WNT/β-catenin pathway in these cells. Given our current findings and its established safety in humans, CPX shows promise as a potential adjunct treatment for esopahgeal cancer.
