Establishment of Ameloblastoma Cell Population for the Study of Therapeutics

Ameloblastoma is a benign very aggressive tumor of odontogenic epithelium origin with a high rate of recurrence and the potential to metastasize, requiring extensive and debilitating surgery. Cyclopamine is an inhibitor of the hedgehog (Hh) pathway activation by directly binding to smoothened (Smo). Several studies have tested the response of cyclopamine on breast and gastric cancers as well as oral squamous cell carcinoma showing it is effective in reducing tumor cell viability with promising therapeutic potential. Objectives: 1) To establish an ameloblastoma-derived cell population (AB-1); 2) characterize the selective gene profile of AB-1 cells; and 3) test cyclopamine as a potential therapeutic compound in inhibiting the Hh pathway in ameloblastomas. Methods: Fresh ameloblastoma tissue was harvested with IRB approval and signed consent. Primary explant cultures were grwon in DMEM supplemented with 10% fetal bovine serum and 100 units/mL penicillin and streptomycin to established a cell population (AB-1). Cells were initially screened using cytokeratin (CK19) and amelogenin (Amel). Expression of the members of the Hh pathway sonic hedgehog (SHH), patched (Ptch), SMO and Gli-1 were then tested using RT-PCR and immunohistochemistry. AB-1 cells were grown on 96 well plates for 24 hrs, cyclopamine (0, 5, 10 and 20 NM) was added with or without SHH with tomatidine used as a positive control. A MTT cell proliferation viability assay was performed on these cultures 96 hours later. Results: AB-1 cells tested positive for CK19 and Amel and the expression of SHH, Ptch, Smo and Gli-1 was confirmed. Cyclopamine arrested the growth of AB-1 cells in a dose response manner by interrupting the Hh signaling pathway.Conclusion: We have established successfully an ameloblastoma derived cell population capable of proliferating and being maintained in vitro for the purpose of testing potential therapeutic compounds such as cyclopamine. Support: UAB Department of Pathology and SOD/IOHR.