Abstract
BackgroundThe gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine.MethodsMedullary thyroid carcinoma (MTC) TT cell line has been used to evaluate the effects of berberine on RET expression and its downstream signaling pathways. The specificity of berberine was demonstrated by using the papillary thyroid carcinoma TPC1 cell line, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement.ResultsBerberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells. Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells. The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.ConclusionOur data strongly suggest that the G-quadruplex forming region and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription.
Highlights
The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs)
Cell culture & media The human medullary thyroid carcinoma cell line (TT) was obtained from the American Type Culture Collection (Manassas, VA, USA) and it was cultured in DMEM 1640 medium (Cellgro, Manassas, VA, USA) supplemented with 15 % heat inactivated fetal bovine serum (FBS)
The human papillary thyroid carcinoma cell line (TPC1) was received from Dr Rebecca Schweppe (University of Colorado, Denver) and was cultured in RPMI-1640 medium supplemented with 9 % FBS
Summary
The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). The RET proto-oncogene encodes a tyrosine kinase receptor on the cell surface, which binds the members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands via the ligand binding site on the extra-cellular domain [4, 5]. Kumarasamy et al BMC Cancer (2015) 15:599 reported in MTC due to the mutations at gatekeeper residues, which render the receptor inaccessible to drug binding [17, 18] To overcome these challenges, we have been developing a new strategy to turn off the transcription of RET proto-oncogene using small molecules rather than directly inhibiting the kinase activity of the receptor [19]. The biological relevance of Gquadruplexes is well established based on their formation in the telomere region and in the promoter region of other oncogenes [22,23,24,25,26]
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