Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine

Abstract

Background: 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). 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.

Methods: Medullary 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.

Results: Berberine 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.

Conclusion: Our 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.

Fig. 1

Taq DNA polymerase stop assay showing the stabilization of the RET G-quadruplex with berberine and its structural analogues. a Structures of berberine and its analogues. b Sequence of the single-stranded template DNA annealed with the 5′-[³²P] labelled primer used in DNA polymerase stop assay. c DNA polymerase stop assay at increasing concentrations of berberine (0, 0.75, 1.5, 15 μg/ml) and its analogues (0, 0.75, 1.5,3, 15 μg/ml). Lanes A, G, T & C represent the di-deoxy sequencing reactions with the same template, which serve as the marker to identify the exact stop site. Lane P represents the position of the free primer on the gel

Fig. 2

CD spectroscopic studies to validate the stabilization of RET G-quadruplex with berberine. a CD titration spectra for the RET-WT (5 μM) in the absence and presence of increasing concentrations of berberine (b) CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of berberine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of berberine (5 Equivalents)

Fig. 3

Effect of berberine on RET expression in both the TT and TPC1 cell lines. a Effect of berberine on the RET mRNA expression in TT cells after 24 and 48 h treatments at various concentrations. b RET protein expression in TT cells were determined by western blotting after the 24 and 48 h exposure to increasing concentrations of berberine. c Effect of berberine on the RET/PTC1 mRNA expression in TPC1 cells was determined following 24 h exposure with various concentrations of berberine. d Protein expression levels of VEGF and c-MYC in TT cells in the presence of berberine at various concentrations and exposed up to 48 h

Fig. 4

Effect of berberine on the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)

Fig. 5

Effect of canadine on the G-quadruplex stabilization and on the RET promoter activity. a Structures of berberine and canadine. b CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of canadine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of canadine (5 Equivalents). d Effect of canadine on the luciferase expression level in HEK293-WT cell line at various concentrations after 24 h treatment. e Effect on the RET protein expression in TT cells after 48 h incubation at an increasing concentrations of canadine

Fig. 6

Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/−s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells